,\ A T I O^ L EYE INSTITUTE » 1 9 9 Cover Photo Immunofluorescence showing the Purkinje cells of a transgenic mouse brain reacting with a chloramphenicol acetyltransferase antibody. The transgene was driven by the promoter and enhancer of the chicken 62- crystallin/argininosuccinate lyase gene. The photograph was taken by Dr. Steven Bassnet (Department of Anatomy and Cell Biology, Uniformed Services University of the Health Sciences) and is a 3-dimensional reconstruction using the Voxel View program on a Silicon Graphics Workstation taken with a confocal microscope. The photograph was originally published on the cover of Developmental Dynamics ^%■. 1993. Copynght ®1993, Wiley-Liss, a Division of John Wiley and Sons, Inc. and is described in Piatigorsky J: Puzzle of crystallin diversity in eye lenses. Developmental Dynamics ^%■.2Q7, 1993. Reprinted by permission of Wiley-Liss, a |~ii\':^inn of John Wiley and Sons, Inc. NATIONS. rilfSTmiTES OF NiHLIBRARy ifTHESDA, MO 20892-1150 National Eye Institute Annual Report Fiscal Year 1993 U.S. Department of Health and Human Services Public Health Service National Institutes of Health Re I ■•• :T?v*» i'-y Table of Contents Statement of the Institute Director i Carl Kupfer, M.D. Extramural Research 5 Report of the Associate Director 7 Jack A. McLaughlin, Ph.D. Division of Basic Vision Research 7 Peter Dudley, Ph.D. Retinal and Choroidal Diseases 7 Corneal Diseases 9 Lens and Cataract . . . 10 Glaucoma j j Strabismus, Amblyopia, and Visual Processing 12 Division of Collaborative Clinical Research 14 Richard Mowery, Ph.D. Research Results 14 Division of Biometry and Epidemiology 19 Report of the Acting Director 21 Roy C. Milton, Ph.D. Research Highlights 21 Research Activities 22 Professional Activities 24 Publications 25 International Program Activities 27 Report of the Acting Assistant Director 29 Terrence Gillen, M.A., M.B.A. Research Activities 29 Activities With International and Multinational Organizations 32 Science Policy and Legislation 33 Report of the Associate Director 35 Michael P. Davis, M.S. Policy, Legislation, Planning, and Evaluation Branch 35 Carmen P. Moten, Ph.D. Management Information Systems Branch 37 David Scheim, Ph.D. Scientific Reporting Branch 30 Judith A. Stein, M.A. Office of the Scientific Director 41 Report of the Scientific Director 43 Robert B. Nussenblatt, M.D. Office of the Scientific Director Francisco M. de Monasterio, M.D., D.Sc. Physiological Studies of the Primate Visual System 47 Anatomical Studies of the Primate Visual System 49 Helen H. Hess, M.D. Biochemistry of Retina and Pigmented Epithelium in Health and Disease 51 Laboratory of Immunology 55 Report of the Chief 57 Robert B. Nussenblatt, M.D. Section on Clinical Immunology Frangois G. Roberge, M.D. Study of Immunosuppressants for the Treatment of Uveitis in Animal Models 60 Section on Experimental Immunology Charles E. Egwuagu, Ph.D., M.P.H. Analysis of T Lymphocytes and Cytokines Involved in Experimental Autoimmune Uveoretinitis 63 Ectopic Expression of Interferon-Gamma in the Eyes of Transgenic Mice and Rats Induces Ocular Pathology and MHC Class II Gene Expression 66 Igal Gery, Ph.D. Immune Responses to Ocular Antigens 68 Section on Genetics and Molecular Immunology Moncef Jendoubi, Ph.D. Gene Therapy for Ocular Genetic Disease 72 Section on Lnmunology and Virology John J. Hooks, Ph.D. Interferon System in Cellular Function and Disease 75 Smdies on the Bioregulatory Aspects of the Retinal Pigment Epithelial Cell 78 Virus Infections in the Eye 81 Toxoplasmosis Infections in the Eye 86 Chandrasekharam N. Nagineni, Ph.D. Role of Retinal Pigment Epithelium in Retinal Disorders 88 Section on Immunopathology Chi-Chao Chan, M.D. Immunopathology in Eyes With Experimental and Clinical Ocular Diseases 91 Immunopathology of Ocular Diseases in Humans 96 Cytokines and Ocular Antigens in the Eye 97 Scott M. Whitcup, M.D. The Diagnosis and Treatment of Human Uveitis 98 Ocular Toxicity of 2',3'-Dideoxyinosine (ddl) 101 Cell Adhesion Molecules in Ocular Inflammation 103 Section on Immunoregulation Rachel R. Caspi, Ph.D. Cellular and Immunogenetic Mechanisms in Uveitis 107 Marc D. de Smet, M.D. Ocular Manifestations of the Acquired Immune Deficiency Syndrome 112 Characterization of Immune Responses to S- Antigen 115 Surgical Management of Uveitis 118 Robert B. Nussenblatt, M.D. Cyclosporine Therapy in Uveitis 122 Oral Administration of Antigen and tiie Ocular Immune Response 125 Laboratory of Mechanisms of Ocular Diseases 129 Report of the Acting Chief 131 J. Samuel Zigler, Jr., Ph.D. Section on Cataracts Deborah Carper, PhD. Structure and Expression of Polyol Pathway Enzymes 132 Donita L Garland, Ph.D Oxidation of Proteins in Cataractogenesis 134 James Fielding Hejmancik, M.D., Ph.D. Inherited Ocular Diseases 137 Paul Russell, Ph.D. Characterization of the Lens 142 Cataract in the Philly Mouse Strain 145 J. Samuel Zigler, Jr., Ph.D. Structure and Composition of Lens Crystallins with Respea to Cataractogenesis ........ 146 Section on Pathophysiology W. Gerald Robinson, Jr., Ph.D. Ultrastructure and Function of the CelLs and Tissues of the Eye 149 Laboratory of Molecular and Developmental Biology 153 Report of the Chief 155 Joram Piatigorsky, Ph.D. Section on Cellular Differentiation Peggy S. Zelenka, Ph.D. Proto-Oncogene Expression During Lens Differentiation and Development 157 Section on Molecular Genetics Joram Piatigorsky, Ph.D. Crystallin Genes: Structure, Organization, Expression, and Evolution 161 Molecular Biology of the Cornea 167 Section on Molecular Structure and Function Graeme J. Wistow, Ph.D. Molecular Biology and Functions of Lens Proteins 169 Section on Regulation of Gene Expression Ana B. Chepelinsky, Ph.D. Genetically Engineering the Eye with the aA-Crystallin Promoter 173 Regulation of Expression of Lens Fiber Membrane Genes 176 Section on Transgenic Animal and Genome Manipulation Eric Wawrousek, Ph.D. NET Central Transgenic Animal Production Facility 179 a-Crystallin Gene Disruption in the Mouse 182 Laboratory of Ocular Therapeutics 185 Report of the Chief 187 Peter F. Kador, Ph.D. Peter F. Kador, Ph.D. Pharmacology of Ocular Complications 188 Sanai Sato, M.D., Ph.D. Role of NADPH-Dependent Reductases in Ocular Complications 193 ui Laboratory of Retinal Cell and Molecular Biology 197 Report of the Chief 199 Gerald J. Chader. Ph.D. Section on Biochemistry Barbara Wiggert, Ph.D. Vitamin A and Ocular Tissues 201 Section on Gene Regulation Diane E. Borst, Ph.D. Molecular Genetics of the Eye and Ocular Diseases 204 Gerald J. Chader, Ph.D. Metabolism of the Retina and Pigment Epithelium 207 Visual Control Mechanisms 210 T. Michael Redmond, Ph.D. Molecular Biology of Outer Retina-Specific Proteins 213 Section on Molecular Biology Toshimichi Shinohara, Ph.D. Molecular Biology of Phototransduction 216 Molecular Biology of Experimental Autoimmune Uveitis 219 Laboratory of Sensorimotor Research 223 Report of the Chief 225 Robert H. Wurtz, Ph.D. Section on Neural Modeling Lance M. Optican, Ph.D. Information Processing by Visual System Neurons 228 Section on Neuro-Ophthalmologic Mechanisms Michael E. Goldberg, M.D. Cerebral Cortical Mechanisms for Eye Movements and Visual Attention 232 Section on Oculomotor Control Frederick A. Miles, D.Phil. Visual Motion and the Stabilization of Gaze 235 Section on Visual Behavior David Lee Robinson, Ph.D. Visuomotor Properties of Neurons in the Thalamus 238 Section on Visuomotor Integration Robert H. Wurtz, Ph.D. Visuomotor Processing in the Primate Brain , 241 Ophthalmic Genetics and Clinical Services Branch 245 Report of the Acting Chief 247 Muriel I. Kaiser-Kupfer, M.D. Section on Cataract and Corneal Diseases Manuel B. Datiles, M.D. The Effects of Corneal Contact Lenses on the Cornea 250 Documentation and Monitoring of Opacities in the Human Lens , 252 Use of Human Lens Material for Determining Possible Causes of Cataracts 255 Muriel I. Kaiser-Kupfer, M.D. Addendum to Use of Human Lens Material for Determining Possible Causes of Cataracts 257 Carl Kupfer, M.D. Anterior Chamber Anomalies Associated With Glaucoma or Ocular Hypertension ....... 259 iv Section on Eye Services Rafael Caruso, M.D. Clinical Psychophysics of the Visual System 261 Clinical Electrophysiology of the Visual System 264 Visual Function Diagnosis Service 267 Section on Ophthalmic Genetics Muriel I. Kaiser-Kupfer, M.D. Pigment Dispersion With and Without Glaucoma 269 Visual Function and Ocular Pigmentation in Albinism 271 Gyrate Atrophy of the Choroid and Retina and Other Retinal Degenerations 273 NIH Interinstitute Genetics Program: The Genetics Clinic 276 A Double-Masked Controlled Randomized Clinical Trail of Topical Cysteamine 279 Usher's Syndrome — Clinical and Molecular Studies 281 A Double-Masked Controlled Randomized Clinical Trial of Topical Cysteamine 283 Mark H. Scott, M.D. Characteristics of Macular Scotomas in Patients With Primary Monofixation Syndrome 285 Index 287 statement of the Institute Director statement of the Institute Director Carl Kupfer, M.D. With the publication this year of our newest long-range plan Vision Research — A National Plan: 1994-1998 we have taken stock of the ac- complishments and current status of vision research and have focused once again on the exciting research opportunities that lie ahead. Our extramural and our intramural laboratories and clinical scientists have helped us make excellent progress in accomplishing our mission of conducting and supporting research, training, health information dissemination, and other programs relevant to eye diseases and vision disor- ders. About $235 million went to extramural resear- chers in the form of grant support and $5 million was expended to support research and development contracts. Another $7.2 million was used to support training awards. This funding has led to a number of important findings this year. National Eye Instimte (NEI)-supported researchers have demonstrated that mutations in several retina- specific genes cause photoreceptor degeneration in humans and mice. Although the mechanism by which these gene mutations lead to photoreceptor degeneration is unknown, scientists have suggested that there is a common pathway in the disease process. Apoptosis, or programmed cell death, appears to play a role in all of these retinal degenerations through fragmentation of the deoxyribonucleic acid (DNA) by intracellular en- zymes at specific sites in the genome. If this is the case then there is the exciting possibility of an intervention for a variety of retinal degenerations based on inhibition of these DNA-cutting enzymes. Our knowledge of the genetic loci for some of the macular degenerations has also been expanded. The genes for at least three forms of macular degeneration have been localized to specific chromosomes by NEI-sponsored investigators. A juvenile form of macular degeneration known as Best's disease has been mapped to chromosome 11, and two other forms of hereditary macular disease have been linked to chromosome 6. Results from a prospective, double-masked clinical trial designed to assess the effectiveness of vitamin A and/or vitamin E supplements in halting or slowing the progression of retinitis pigmentosa (RP) were released. They showed that adults who sup- plemented their diets with 15,000 lU of vitamin A daily had on average about a 20 percent slower annual decline of remaining retinal function than those not taking this dose. Last year, clinical trial results released by the Herpetic Eye Disease Smdy (HEDS) investigators showed that oral acyclovir was no better than a placebo in treating active herpes simplex stromal keratitis. Another part of the HEDS examined the effect of steroid eye drops as a treatment for this disease. This year, researchers conducting the smdy reported rapid improvement of stromal keratitis with immediate steroid ther^y, but for those patients having their first episode of stromal keratitis, topical steroids could be safely deferred. Progress has also been made in further under- standing of the chaperon functions of a-crystallins. In vitro experiments have demonstrated that a- crystallin efficiently suppresses the aggregation of p- and Y-crystallins. This suggests that a biological role of a-crystallins is to prevent posttranslational chan- ges in the interactions between lens crystallins and hence to maintain the transparent state of the lens. NEI-supported scientists have been studying affected families in Michigan, the New England states, and Iowa in an attempt to identify a "glaucoma" gene and ultimately to characterize the protein encoded by this gene. Recently, the disease- associated gene has been mjqiped to chromosome 1. Although the link between juvenile onset glaucoma and the more prevalent primary open-angle glaucoma is unclear, finding the gene responsible for one form of glaucoma is a beginning in the quest for iden- tification of at least one of this disease's causative factors. The scientists conducting the Opuc Neuritis Treat- ment Trial (ONTT) reported that a three-day, high- dose treatment with intravenous corticosteroids followed by a short course of oral corticosteroid reduced the rate at which smdy participants Statement of the Institute Director NEI Annual Report— FY 1993 developed multiple sclerosis (MS). Last year the ONTT scientists reported that this treatment enabled patients to recover their vision about two weeks sooner than would be the case without treatment but that oral prednisone, when used alone, was ineffec- tive and actually increased a person's risk for future attacks. Intramural scientists have continued their research efforts to understand the systems within the brain that process visual information and produce eye movements as well as to understand what h^pens when disease or trauma lead these systems to fail. Through the use of a superb animal model, these researchers have found that the animals respond to simulations of motion projected on a screen with postural changes similar to those reported in humans. This iirformation will allow researchers to investigate further the regions of the brain that are known to process this type of visual motion information and to see whether alterations of these regions affect pos- tiu-e. They were also able to locate the approximate region of the frontal eye fields in humans and alter certain eye movements after first locating this region in monkeys. By using information obtained from animal models to locate areas in the himian brain that perform similar functions, we may gain a better understanding of how the intricate mechanisms that guide the visual process operate normally and how they might be impaired by disease or injury. In studies of the regulatory elements required for expression of genes in the eye and other tissues, intramural scientists have found that these elements are quite diverse with each having its own special properties. The elements may also be functionally redundant, that is, removing one does not necessarily eliminate the expression of the gene. A more complete understanding of gene expression in the eye may one day allow treatments to be directed to specific eye tissues. Intramural researchers have continued their leadership role in the study of gyrate atrophy. Dietary intervention studies are continuing in families with two affected children. These studies have demonstrated a marked decrease in the retinal progression of this disorder in the children who began the dietary intervention at an early age. It is anticipated that this original work will lead to gene therapy aimed at preventing this disease. Intramural epidemiologists investigated the effect of vitamin and/or mineral supplements on the risk of developing age-related cataracts in conjunction with two National Cancer Institute (NCI) trials using the same vitamin and/or mineral interventions in a population with chronic nutritional problems and high rates of esophageal and stomach cancer. In these highly cost-effective studies of populations with chronic deficiencies of multiple nutrients, NEI investigators and their colleagues found that use of the supplements was associated with a decreased risk of nuclear cataract. Additional research is underway to determine whether these findings apply to less nutritionally deficient populations. These are only a few highlights of the important accomplishments of vision researchers in fiscal year (FY) 1993, a year in which we marked the 25 th anniversary of the establishment of the NEI. An- niversary activities were organized around the theme A Celebration of Vision Research and were designed to provide the American public with a report on its investment in vision research. A traveling science museum has been developed that demonstrates the progress and accomplishments of vision research during these past 25 years. It is with great sadness that we must also note the passing of our friend and colleague Julian M. Morris. Much of what we have accomplished, since his selection as the NEI's first information officer in 1970, we owe to him and his dedication to the field of vision research. In recognition of his many contributions, we have dedicated Vision Research— A National Plan: 1994-1998 to his memory. We will miss him. Extramural Research Report of the Associate Director for Extramural Research Jack A. McLaughlin, Ph.D. Research activities supported by tiie Exti-amural Vision Research Program address the leading causes of bUndness and impaired vision in the United States, including retinal diseases, corneal diseases, cataract, glaucoma, strabismus, and amblyopia. The program seeks to increase understanding of the normal development and function of the visual system; to understand the causes of and better diagnose, prevent, and treat sight-threatening con- ditions; and to enhance the rehabilitation, training, and quality of life of individuals who are partially sighted or blind. In working to tliis end, the Vision Research Program supports vision research tlirough grants, cooperative agreements, and research and develop- ment contracts; encourages high-quality clinical research, including clinical trials and other epidemiologic studies; encourages research training and career development in the sciences related to vision; sponsors scientific workshops in high-priority research areas to encourage exchange of information among scientists; and carries out a construction, alteration, and instrumentation program of grants for public and private nonprofit vision research facilities. For FY 1993, an estimated total of $235,005,000 was expended for NEI extramural grants, cooperative agreements, and research and development contracts in the following categories and amounts: Research Grants Research Training Awards Research and Development Contracts Total Extramural Support $222,735,000 $7,226,000 $5.044.000 $235,005,000 Concurrent with the reorganization of the NEI, a number of personnel changes occurred in the Extramural Program during this fiscal year. Among these were the appointments of Drs. Lor6 Anne McNicol, Peter A. Dudley, and Richard L. Mowery, as director of the division of extramural activities, as director of the division of basic vision research, and as director of the division of collaborative clinical research, respectively. The following sections highlight some of the recent accomplishments of the NEI-supported investigators. Division of Basic Vision Research Peter Dudley, Ph.D., Director Retinal and Choroidal Diseases Retinal Degeneration and Apoptosis Cell death is an important part of normal develop- mental programs. For example, the balance of cell survival with neuronal cell death is thought to be an important mechanism whereby an organism controls the interconnections between populations of developing cells. Apoptosis, or programmed cell death, seems to be an important mechanism used by the retina during its development into a functional layered tissue. Mutations in several retina-specific genes have been shown to cause photoreceptor degeneration. Mutations in the rd, rds/peripherin, and rhodopsin genes cause retinal degenerations in humans and mice. The mechanism by which these gene mutations lead to photoreceptor degeneration is unknown. Recent work by Fulton Wong at Duke University shows that although the phenotypes of these degenerations are different, there appears to be a common pathway in the disease process. Apop- tosis appears to play a role in all of these retinal degenerations ttirough fragmentation of DNA by cleavage at specific sites in the genome. Apoptosis has been demonstrated to occur in retinal degenerations by observing a characteristic "ladder" of DNA fragments using gel electrophoresis. The Extramurai Research NEI Annual Report— FY 1993 DNA ladder gel pattern results from a predictable fragmentation of genomic DNA at internucleosomal sites during the degeneration process. Although apoptosis may be a common pathway that leads to cell death, the mechanism triggering this process is unknown. It seems likely that intracellular enzymes called endonucleases are activated to cut DNA into small fragments. If this is the case, then there is the exciting possibility of an intervention for a variety of retinal degenerations based on inhibition of these DNA-cutting enzymes. Cancer Associated Retinopathy Disorders of the retina leading to a loss in vision can result from the remote effects of cancer. Certain types of tumors at a distant site can set up an im- munological reaction that can manifest itself in the retina as well as in other tissues of the nervous system. When the retina is involved in this paraneoplastic syndrome, it is called cancer- associated retinopathy or CAR. This syndrome develops mainly as a result of a primary tumor in the lung, although other tissues may be implicated. Metastasis is not involved, but rather molecules of the immune system called autoantibodies appear in the serum of patients. This immimological reaction appears to be, in part, a response of the host or patient to the tumor. Thus, the patient appears to be producing antibodies as a defense against the tumor in the hopes of limiting its growth. Vision loss is frequently the first sign of illness leading to subse- quent clinical examinations that identify the causal cancer. Patients experiencing CAR report a sudden loss of vision resulting from inactivation of important proteins in the retina by the autoantibodies generated in response to the tumor. Patients with other types of retinopathies do not produce antibodies that react with the CAR protein or antigen in the retina, in- dicating a high degree of specificity of the im- munological reaction. Several NEI-supported investigators are looking into the role of specific retinal antigens in the development of CAR in patients, and further work hopefully will uncover the basis of this disease. Recent work has shown that one protein involved in phototransduction, called recoverin, may be present in cancer cells. It is the immune response to this protein that results in retinal involvement. Further, although recoverin has been found only in rod cells, antisera to recoverin label both rods and cones. The reason may be that there is a molecule in cones that has some sequence homology to recoverin and that reacts with antirecoverin antibody. Current inves- tigations are focusing on the isolation and cloning of the human recoverin gene. The mouse gene has recently been cloned, and the deduced amino acid sequence is identical to that of human recoverin protein. Segregation analysis shows close linkage to the tumor suppressor gene p53. The current hypothesis being tested is that CAR is the result of a single mutational event in a cell that deletes the tissue-specific regulatory elements of the recoverin gene while joining its coding sequence to an active gene. This would delete the function of the p53 cancer suppressor gene and simultaneously turn on synthesis of recoverin. The cell becomes cancerous because the p53 protein product is either absent or inactive and no longer functions as a cancer suppressor. These kinds of studies could lead to a method for early diagnosis of cancer and the opportunity to institute treatment at an early stage. Molecular Genetics of Macular Degeneration Macular degeneration is the most common cause of severe visual impairment in older persons in the United States. It robs otheirwise healthy older Americans of useful vision, depriving them of the ability to read, drive, and enjoy leisure activities. Currently, there is no effective tteatment for the vast majority of individuals with this condition because the basis for the disease is not understood. The genes for at least two forms of macular degeneration have been localized to specific chromosomes by Dr. Richard Stone at the University of Iowa. Best's disease is an autosomal dominant condition characterized by the accumulation of lipofuscin within and beneath the retinal pigment epithelium (RPE). It has an earlier age of onset than the more prevalent age-related macular degeneration (AMD), and there is an absence of drusen. Drusen are deposits of extracellular material lying between the RPE and Bruch's membraiie. The Best's gene has been localized to chromosome llqlS. Dominant macular dystrophy with flecks (DMDF) is an autosomal recessive degeneration characterized by severe vision loss with macular lesions ringed with NEI Annual Report— FY 1993 Extramural Research fleck-like deposits of yellow pigment Preliminary evidence indicates that the gene for DMDF is localized to chromosome 6q. Linkage analysis offers the opportunity to map human disease genes when the causative agent is unknown, as is the case for macular degenerations. Gene mapping can lead to actually cloning the gene responsible for specific eye diseases, for example, AMD, by application of reverse genetics. Retinitis Pigmentosa RP is a group of hereditary eye diseases with an overall incidence of about 1 in 3^00 births in the United States. The emotional and economic costs of the disease to society are enormous, particularly because no effective treatments are known for most types of retinal degeneration. RP is genetically heterogeneous and can be transmitted as a dominant, recessive, or X-linked trait NEI-supported research has led to significant advances in identifying the molecular defects in different forms of RP. Scientists reasoned that a gene coding for a structural or a functional protein, important in the physiology of the pigment epithelial and the photoreceptor cells of the retina, might be defective in patients with retinal degenerations. With this candidate gene ^proach, it was discovered that 20 to 30 percent of individuals with autosomal dominant RP have a mutation in the rhodopsin gene. In autosomal recessive RP, a different rhodopsin gene mutation was shown to be present. Mutations have also been found in the human homologue of the murine rds locus, the photoreceptor-specific peripherin/RDS gene, in some families with autosomal dominant RP. Although the function of this protein is not known, it may serve as an ad- hesion molecule, stabilizing the outer segment discs through interactions across the intradiscal space. Most recentiy. Dr. Ted Dryja at the Harvard Medical School has found that a third photoreceptor- specific gene is defective in patients with another form of autosomal recessive RP. Mutations in the gene encoding the beta-subunit of the cGMP phosphodiesterase, a key molecule of the visual transduction pathway, are present. This is of par- ticular interest because the murine homologue of this gene is defective in the rd strain of mice with retinal degeneration. In related research, a two-base pair deletion was found in the human peripherin/RDS gene in a family with autosomal dominant retinitis punctata albescens, an uncommon form of retinal degeneration clinically related to RP. A defect in the rhodopsin gene was also found in congenital stationary night blindness. In studying flie molecular mechanism of this genetic defect Dr. Dryja's group found that the mutant rhodopsin protein activated transducin without binding its natural chromophore, retinal. This appears to be caused by abnormal constitutive activation of transducin in the phototransduction pathway. The identification of genetic defects in retinal degenerations and dystrophies is an important step in developing effective ther^eutic strategies. With this information in hand, scientists will now be able to explore the molecular mechanisms responsible for these diseases and translate this information into rational and effective diagnosis, treatment, and prevention strategies. Corneal Diseases The corneal stroma is unique among the col- lagenous connective tissues in being transparent Understanding the molecular basis of transparency requires a detailed knowledge of the regulation of the collagen types, proteoglycans, and glycoproteins expressed in this organ. The Corneal Diseases Program is supporting several laboratories engaged in studies of the development and synthesis of specific collagen genes. Dr. Bjom R. Olsen at Harvard Medical School has been investigating the synthesis of type Vin collagen, one of the short-chain collagen species. This protein had been reported as a component of the intima layer of vascular endothelium and as the major structural component of Descemet's membrane in the cornea. Dr. Olsen has cloned and sequenced the two genes encoding type Vlll collagen and has found that it exists as a heterotrimer of composition [al(VIII)]2[0(2(Vin)]. In vitro hybridization studies revealed the surprising observation that the al gene is expressed in the lens as well as in the cornea. Chromosomal localization studies have shown that the a2 gene is located at the region of the defect dysgenetic lens (dyl) gene. This is a recessive hereditary disorder that shows a persistent coimection between the lens and the corneal epithehum as well Extramural Research NEI Annual Report— FY 1993 as various degrees of corneal opacity. Dr. Olsen has prepared transgenic mice carrying defects in both the al and a2 genes. This should permit a more detailed examination of the unexpected role of type VIII collagen in the embryonic development of the cornea and lens. Lens and Cataract Lens Biochemistry The a-crystallins are major structural proteins of the vertebrate lens and contribute to its refractive mass and transparency. During the past decade, it has been shown that in some species "housekeeping" enzymes that are found in nonlenticular tissue are recruited to serve as structural crystallins in the lens. This has led to the concept of "gene-sharing" implying that a single gene encodes a protein with dual functions. Evidence for a dual function for a- crystallins has come from two Unes of investigation. First, the a-crystalhns are found in nonlenticular tissues, including the heart, lung, spinal chord, brain, kidney, and retina oB-crystallin specifically ac- cumulates in many neurological disorders. Second, the a-crystallins are structurally related to the family of heat-shock proteins that can be induced by heat or hypertonic stress and accumulate in a number of pathological conditions. These two lines of evidence have converged with the recent finding by Dr. Joseph Horwitz from the University of California at Los Angeles that a- crystallins function in vitro as molecular chaperons. These are a subset of heat-shock proteins that are overproduced in response to physiologic "stress" and that act by affecting protein-protein interactions. They stabilize native protein conformations, mediate the folding and correct oligomeric assembly of nascent proteins, catalyze the membrane transloctions of secretory proteins, and prevent protein aggregation under conditions of heat denaturation. In vitro experiments have demonstrated that a-crystallin efficiently suppresses the aggregation of P- and y- crystallins. This suggests that a biological role of a- crystallins is to prevent posttranslational changes in the interactions between lens crystallins and hence maintain the transparent state of the lens. Developmental Biology In many vertebrate species, proper iris and cornea development appears to be coupled to lens growth and viability. A new tool in the study of early lens development has been the small eye (Sey) mutation in mice in which abnormalities in lens development are accompanied by other anterior chamber defects. This mutation has resulted in a potentially useful animal model of aniridia. This condition results from defects in PAX-6, a gene encoding paired-box and homeobox motifs that are expressed in the developing eye. It shares homology with paired box genes of Drosophila that conttol the development of body segmentation. The homeobox encodes the helix-tum-helix motif seen in DNA-binding proteins. Aniridia is a human developmental disorder closely related to the Sey mutation and is charac- terized by hypoplasia of the iris and is conmionly associated with other clinical anomalies such as cataracts and lens dislocation. The disease is in- herited in an autosomal dominant fashion. It is frequently cofransmitted with Wilms' nephroblas- toma, genitourinary abnormalities, and mental retar- dation (termed WAGR complex), demonstrating a close linkage with the genes responsible for these anomalies. This has greatly facilitated the mapping and identification of the human aniridia gene. The WAGR complex has been mapped to a large interstitial deletion on the short arm of human chromosome 11. Dr. Lisa Davis at the Applied Genetics Laboratory in Melbourne, Florida and Dr. Richard Maas of Harvard University have been working independently to fine map and characterize the gene. This area is homologous to the region of the mouse chromosome 2, which contains the Sey gene. Using a mouse PAX-6 clone as a probe, the aniridia region has been identified in human cDNA libraries. Physical mapping of the aniridia gene using DNA isolated from patients with aniridia will provide us with new insights into ocular develop- ment. 10 NEI Annual Report— FY 1993 Extramural Research Glaucoma Molecular Genetics Glaucoma is a potentially blinding condition as- sociated with increased intraocular pressure (lOP) and gradual destruction of the optic nerve. Little is known about the underlying causes of glaucoma and the accompanying nerve degeneration that leads to loss of vision. Furthermore, the relative influence of genetic and environmental characteristics is poorly understood. Fortuitously, juvenile onset glaucoma, a form of the disease characterized by early adulthood onset and elevated lOP, displays an autosomal dominant pattern of inheritance. The unambiguous phenotype, high degree of penetrance and early age of onset, makes a genetic approach ideal for the study of this form of the disease. Dr. Julia Richards at tiie University of Michigan has been working with a number of families that have sufGcient meiotic events to perform genetic linkage studies. To date, family histories have been collected from families in Michigan, the New England states, and Iowa. The ultimate goal is to identify a "glaucoma" gene using positional cloning, followed by sequencing analysis to characterize the encoded protein. Recently, the disease-associated gene has been mapped to chromosome 1. Cor- roborating data from different laboratories using different families have confirmed this locus. In one of the Michigan families, linkage analysis has placed the gene within a 14 centimorgan region of the chromosome, at Iq21-q31. The link between juvenile onset glaucoma and primary open-angle glaucoma is unclear, but finding the gene responsible for one form of glaucoma is a beginning in the quest for identification of at least one causative factor. Ganglion Cell Function Currently, there is controversy about whether glaucomatous damage is selective for a subset of ganglion cells. Each area of the retina has several functionally distinct types of ganglion cells serving the same photoreceptor cell in parallel pathways. The majority of ganglion cells have large cell bodies and large dendritic fields and are classified as M or magnocellular. The P or parvocellular ganglion cells are more numerous, have small cell bodies, restricted dendritic fields, and are involved in color vision. Early investigations seemed to indicate that glaucoma initially damages the large diameter nerve fibers that are prevalent in the M pathway. More recently, psychophysical studies carried out by Dr. Chris Johnson from the University of Califor- nia at Davis indicate that early neuropathy involves both P and M pathways. Using longitudinal studies, he has shown a link between abnormalities detected using blue-on-yellow perimetry and temporal modulation perimetry. Temporal modulation perimetry is a noninvasive psychophysical technique used to assess visual sensitivity at various frequen- cies of flickering light Short wavelength light responses are diagnostic of the P pathway that processes spatial and finely detailed information. Flicker sensitivity at low frequencies is a good monitor of the M pathway. Blue-on-yellow perimetry has application for psychophysically isolating and measuring the sensitivity of the short- wavelength or blue cone pathway. Johnson's work suggests tiiat there may be a lack of selectivity for any particular ganglionic cell subset in glaucoma. Patients tested with blue-on-yellow perimetry show deficits that precede standard visual field defects. Using temporal modulation perimetry there was an overall loss of flicker contrast sensitivity in patients with early glaucomatous visual field loss, but this deficit was not selective for high frequencies. The decrease in sensitivity demonsfrated in these tests occurs at the same time that early visual field defects are seen. This is consistent with the idea that early glaucomatous damage is not limited to a specific subset of ganglion cells. Ultimately, understanding optic nerve pathology can lead to more sensitive predictors for the risk of glaucomatous nerve fiber and hence vision loss. Aqueous Humor Dynamics An important role of the aqueous humor in the anterior portion of the eye is to maintain the proper lOP. Aqueous humor is derived from plasma in the capillaries that feed the anterior portion of the eye via a specific tissue — ^the ciliary body epithelium. Fluid produced by the epithelial cells (inflow) leaves the eye via the frabecular meshwork and Schlemm's canal (outflow), reentering the vascular system 11 Extramural Research NEI Annual Report— FY 1993 through the venous route. It is the balance between inflow and outflow that maintains the lOP. A major aim of current glaucoma research is to gain a better understanding of the mechanisms that regulate these two pathways. Our understanding of outflow biology has been enhanced by a recent discovery by the laboratory of Dr. James Nathanson at Massachusetts General Hospital, showing that nitrovasodilators lower lOP. Nitrovasodilators are a class of compounds produced in response to increased levels of nitric oxide in the cell. Nitric oxide has been shown to be an important mediator in many physiological functions, including muscle relaxation, vasodilation, and transmission of neural impulses. All these effects are mediated by the second messenger cGMP. This finding adds important information to our understanding of outflow regulation and opens the door to the pos- sibility of new therapeutic strategies. Strabismus, Amblyopia, and Visual Processing Development of Visual Pathways One of the primary characteristics of the visual system is the precise pattern of connections, a virtual map, that exist between the retina and the visual centers of the brain. This pattern is established during embryonic development and refined during early life. Activity mediated by chemical signals (neurotransmitters) at the contact points (synapses) between nerve cells is suspected to play an important role in this developmental process. Research by Dr. Steve McLoon at the University of Minnesota has shown that the presence of the chemical precursors for nitric oxide, a recently discovered neurotransmit- ter, in a visual center of the brain coincides with the timing of the ingrowing processes fi'om the retina in the chick embryo. In fact, the concentration of these chemicals reaches a peak just as the initial visual map is being established by the terminals of the retinal cells. Unlike the results from other studies that have implicated a number of chemical signals in the establishment of visual maps in the developing nervous system, these results demonstrate the presence of a chemical signal at the right time and the right place needed to establish a precise map. In related research, Dr. Carla Shatz from the University of California at Berkeley finds that when ganglion cells in the retina first make their connec- tions with nerve cells in the brain, the pattern is not nearly as precise as it is in the adult. Many extra connections are made that are later pruned away. How do cells "know" which connections to maintain and which ones to eliminate? Dr. Shatz examined all branches to determine if electrical signals were being transmitted from one nerve cell to the next. Branches to be eliminated were found to function while they were present. This result lead Dr. Shatz to envision that perhaps a branch from a nerve in the eye confirms that it is in the correct location by sending a signal to the nerve cell in the brain to verify its location, much like placing a telephone call to verify an address. Dr. Shatz found that by block- ing the signaling of the nerve cell it is possible to stop incorrect branches from being removed. Where do these signals originate? The coimections between the eye and the brain form very early in fetal life, even before vision begins. Dr. Shatz suspected that the nerve cells in the eye might be signaling spon- taneously to nerve cells in the brain. Dr. Shatz discovered that the retinal ganglion cells are spon- taneously and repeatedly signaling their target cells in the brain during the weeks before vision takes over. Thus, in the visual system, and very likely elsewhere in the developing brain, nerve cell sig- naling before birth plays a crucial role in establishing correct connections. Plasticity in the Visual Cortex The traditional view states that the overall ar- chitecture and the connections between nerve cells in the adult visual cortex of the brain are fixed fol- lowing a period in early postnatal life when these connections can be modified. This early period is called the critical period, and the ability of nerve cells to modify their connections is called plasticity. The connections between nerve cells carry visual information encoded in signals that are fransformed and integrated as they ascend sequentially through visual centers in the brain. The transformation that occurs in these centers is analyzed in terms of receptive fields. These are sets of nerve cells that encode features of a visual stimulus falling on the retina and connect with other sets of visual nerve cells along the visual pathway. In this way receptive 12 NEI Annual Report— FY 1993 Extramural Research fields form the building blocks that underlie visual p)erception. In the adult, the traditional view holds that the cortex processes the visual scene through a fixed set of receptive fields, handing on information to the next stage in the visual pathway. Clearly, our ability to store new memories in adulthood requires some form of cortical plasticity, but it was generally believed that this would only occur in high-order association. Experiments by Dr. Charles Gilbert at the Rock- efeller University have changed our view of cortical visual processing. Dr. Gilbert made small, focal retinal binocular lesions in monkeys and found that the area of cortex receiving input from those parts of the retina became initially silenced, as expected. However, to his surprise, in adult animals the initial- ly silent area of the cortex becomes remapped, responding instead to areas outside the lesion. Even more surprising was the finding that quite striking changes could be observed physiologically within minutes after making the lesion, and, finally. Dr. Gilbert found that a lesion is not necessary, certain patterns of visual stimulation can cause receptive fields to expand and contract over a time scale of minutes. The finding of this degree of cortical plasticity in adult animals presents a radically different idea of how the cortex works, and the functional implications of the findings are closely related to the time course over which the changes take place. Dynamic changes over a time scale of minutes are usefiil for adapting to changes in the sensory en- vironment. It is as if the cortex is constantly ex- panding and contracting its representation of various aspects of the sensory environment in response to the amount of input it receives from particular sets of stimuli. Future studies may imcover how these changes in visual cortex relate to learning and memory such as the representation of complex images occurring in higher cortical areas. Development of Myopia More than 25 percent of the adult population of the United States is near-sighted (myopic). This relractive error usually develops in the vast majority of people between the ages of six and 14 years. The relationship between accommodation and the development of myopia has been a controversial topic for a number of years. Animal models of myopia are beginning to shed some light on this issue. Recent experiments in the tree shrew (a mammal closely related to primates), the chicken, and the monkey have shown that a biological feed- back mechanism controls the shape of the eyeball. Under normal circumstances this effect causes the focal length of the visual image to fall on the retina (i.e., the eye is in good focus). Dr. Thomas Norton from the University of Alabama at Birmingham has done experiments that indicate that the presence of visual images on the retina produces a signal within the eye that, through a cascade of events, affects the structural nature of the sclera, the outer coat of the eye, without involving the central visual nervous system. Myopia occurs when this mechanism is disrupted, by visual deprivation in animals and by unknown perturbations in humans, causing the eye to become too long for its focal length. Work by Dr. Norton suggests that blurred images, or form deprivation, slow the accumulation of proteoglycans and collagen, two chemical components of the sclera. This in turn may cause the sclera to be less resistant to lOP and consequently causes the eye to become too long, producing myopia Low Vision AMD of the retina is a leading cause of low vision and poses a particularly difficult problem for vision testing because of the central field scotomas (blind areas) that commonly result from this disorder. Recent developments in eye monitoring technology have made it possible to position a target very precisely on known locations of the retina This has great potential for both documentation of visual loss and possible retraining of eye movements to enable use of remaining intact parts of the retina. Progress has been made in developing new devices that aid and assist visually impaired persons, e.g., more ergonomically satisfying magnifiers, cosmetically acceptable telescopic spectacles, and voice control and output for computers. Attention now is focused on devices to assist in changes in terrain, text navigation for both printed materials and computer screens, image processing, and route- finding. 13 Extramural Research NEI Annual Report— FY 1993 Division of Collaborative Clinical Research Richard Mowery, Ph.D., Director The Division plans and directs a program of grant, cooperative agreement, and contract support for applied clinical vision research, including clinical trials, natural history studies, surveys, cohort studies, and studies of cases and controls. The Division manages 21 clinical trials, 1 1 epidemiology studies, and three eye health education demonstration projects with an annual budget of $38.7 million. Research Results Retinitis Pigmentosa RP is a diverse group of hereditary retinal diseases that cause a progressive degeneration of the rod and cone photoreceptors and loss of visual function. RP affects approximately 100,000 people in the United States and approximately 1.5 million people worldwide. Individuals with RP typically begin to lose peripheral vision in adolescence and early adulthood, and most lose central vision later in life. Results from a prospective, double-masked clinical trial designed to assess the effectiveness of vitamin A and/or vitamin E supplements in halting or slowing the progression of RP showed that adults who supplemented their diets with 15,000 lU of vitamin A daily had on average about a 20 percent slower annual decline of remaining retinal function than those not taking this dose. Based on this finding, an average patient who started taking a 15,000 lU vitaniin A capsule at age 32 would retain some useful vision until age 70, whereas a patient not on this dose would lose useful vision at age 63. The study also found that in patients taking high- dose vitamin E supplements the disease appeared to progress faster on average than in patients taking a trace amount of the vitamin. Cytomegalovirus Retinitis Cytomegalovirus (CMV) retinitis is a potentially blinding disease of the retina that affects about 25 percent of people with acquired immunodeficiency virus (AIDS). NEI supports a network of inves- tigators with expertise in AIDS clinical research, retinal diseases, and clinical trial methodology to expedite the testing of treatments for CMV retinitis and other ocular complications seen in patients with AIDS. This network is called the Studies of Ocular CompUcations of AIDS (SOCA). The first clinical trial conducted under SOCA was designed to com- pare the efficacy and safety of foscarnet and gan- ciclovir. The investigators found that patients treated with foscarnet lived longer than those who received ganciclovir. Foscarnet patients lived an average of 12.6 months after starting treatment compared with 8.5 months for patients taking ganciclovir. The drugs appeared to be equally effective in halting the progression of CMV retinitis and preserving vision. VUreoretinopathy The most common cause of failure in retinal detachment surgery is the development of abnormal contractile tissue on the retinal surface. Mild forms of this condition can sometimes be treated by exter- nal surgery and the retina successfully reattached. However, in more severe forms intraocular surgery is required. The Silicone Oil Study, a multicenter clinical trial, was designed to evaluate the benefits and risks of using a long-acting gas or silicone oil as an aid in reattaching the retina. The study found that use of silicone oil is superior to use of long-acting gas, resulting in a higher rate of successfiil retinal reattachment. Retinopathy of Prematurity More than 4,000 infants weighing less than 1,251 grams at birth underwent sequential ophthalmic examinations to monitor the incidence and progres- sion of retinopathy of prematurity (ROP) in the multicenter Cryotherapy for Retinopathy of Prematurity Clinical Trial. Two-thirds of the infants developed some degree of ROP. The incidence and severity of ROP were higher in lower birth weight and gestational age categories. African-American infants appeared less susceptible to ROP than did Caucasian infants. Herpes Simplex About a one-half million Americans are affected by ocular herpes, which often begins as a relatively painful sore on the surface of the cornea. Like herpes cold sores, these ocular lesions may 14 NEI Annual Report— FY 1993 Extramural Research periodically recur, and the herpes virus can also over time cause an inflammation deep inside the cornea. This advanced infection is known as herpes simplex stromal keratitis, which can lead to severe corneal scarring, inflammation of the interior of the eye, and even blindness. A randomized, controlled clinical trial was conducted as part of the HEDS to evaluate whether oral acyclovir, when given to patients with steroid and antiviral eye drops, improved the treat- ment of active herpes simplex stromal keratitis. Researchers randomly assigned 104 patients to take either oral acyclovir or a placebo. After a 10-week treatment regimen and a six-month followup period, oral acyclovir was found to be no better than a placebo in successfully clearing the stromal keratitis. This indicates that the financial cost and minimal potential health risk associated with the use of this drug is not warranted. The role of acyclovir in the prevention of recur- rences of herpetic eye diseases during the course of one and one-half years and the role of acyclovir in preventing progression of superficial (epithelial) keratitis to the more severe stromal keratitis, or iritis, is currently being examined by the HEDS research group. Although many ophthalmologists use steroids to control the corneal inflammation associated with herpetic stromal keratitis, clinical research has yielded mixed results regarding their overall effect. For example, some clinicians have reported en- couraging results with this treatment, although others have indicated that steroid therapy worsens or prolongs the corneal lesions and predisposes patients to known complications such as glaucoma and cataract. A second randomized, clinical trial con- ducted as part of the HEDS examined the effect of steroid eye drops as a treatment for active herpetic stromal keratitis. After 10 weeks of treatment and six months of patient followup, corneal inflammation was held in check longer and corneal inflammation cleared faster in patients treated with steroids. However, delaying steroid therapy by one-to-three weeks did not significantly influence lesion recur- rence or affect visual acuity at six months. Thus, rapid improvement of stromal keratitis was achieved with immediate steroid therapy, but for those patients having their first episode of stromal keratitis topical steroids could be safely deferred. Corneal Transplantation More than 40,000 corneal transplant operations are performed annually in the United States. But about one in 10 patients receiving a corneal transplant is at high risk of rejecting the donor tissue or graft because: (1) they have previously rejected a corneal transplant or (2) new blood vessels have grown into their damaged cornea, introducing im- mune cells into this normally avascular region of the eye that may later recognize the graft as foreign and attack it The Collaborative Corneal Transplantation Study (CCTS) was designed to evaluate whether donor- recipient tissue typing, transplanting a donor cornea that has cell-surface proteins (human leukocyte antigens [HLA]) that closely resemble those on the recipients' s natural cornea, helps to prevent transplant rejection. These antigens serve as molecular "fingerprints" on every cell in the body and allow a person's immune system to distinguish its own cells from those belonging to another person. Previous studies had suggested that closely matching the donor's HLA with those of the recipient might increase the likelihood that the immune system would accept, rather than reject, the donor tissue. After three years of patient followup, CCTS researchers foimd that people who received corneal transplants with well-matched antigens did not fare significantiy better than those with a poor match. Each patient group had similar rates of initial im- mune reactions, graft rejection, and graft failure due to infection or other causes. These findings indicate that tissue typing was not an important factor in transplant survival. If donor-recipient tissue typing were to become standard practice in corneal transplantation, it would greatiy increase the cost and waiting period for this operation. The process of matching antigens is labor intensive and would add at least $1,(X)0 to the nearly $5,000 cost of a corneal transplant operation. Moreover, because there is already a national shortage of donor corneas, high-risk patients would likely have to wait even longer for a suitably matched donor cornea. 15 Extramural Research NEI Annual Report— FY 1993 Lens Opacities Case-Control Study Cataracts are a leading cause of visual disability and blindness, however, little information exists on the cause or progression of cataracts. Development of each cataract type (nuclear, cortical, mixed, and posterior subc^sular) could be influenced by dif- ferent risk factors. The Lens Opacities Case-Control Smdy (LOCS) evaluated medical, nutritional, demographic, familial, environmental, and ocular factors that could lead to cataract development. Of the 1,380 LOCS study participants, 435 were cataract free. Cases of cataract were as follows: 72 with posterior subcapsular cataract, 137 nuclear cataract, 290 cortical cataract, and 446 with mixed types of cataract. The results of the study indicate that development of all three cataract types was as- sociated with a lower educational level; and regular use of a multivitamin dietary supplement decreased the risk of cataract formation. Low dietary intakes of vitamins A, C, and E, riboflavin, niacin, thiamin, and iron were associated with development of cortical and mixed cataracts (odds ratios .31 to .56). Low intake of vitamins, low socioeconomic status, diabetes, race, use of some medications, smoking, and other factors were associated with development of specific types of cataracts. The rate of cataract progression and factors affecting this progression are currently being evaluated in the Natural History of Lens Opacities Study, where individuals will be examined aimually for five years. Linxian Eye Study The Linxian Cataract Studies sought to determine whether vitamin and mineral supplements were effective in preventing the development of lens opacities. In 1985, the NCI launched two nutrition intervention trials in Linxian, a county in north central China, whose population has chronic nutritional problems and high rates of esophageal and stomach cancer. Because the vitamins and minerals under study might have potential for preventing lens opacities, the NEI collaborated with the NCI to determine the effects of the supplements on the eye's lens. In 1991, the NEI provided support for eye examinations, including detailed lens evaluations, for participants in both trials. In the first trial, participants were randomly assigned to either a multivitamin and/or mineral supplement or a placebo. Examinations were con- ducted on 2,141 participants who were between the ages of 45 and 74. The smdy found a 36 percent reduction in nuclear opacities among the oldest participants (ages 65 to 74) who took a multivitamin and/or mineral supplement. In the second trial, participants were assigned randomly to various combinations of vitamins and minerals — a study design that allowed researchers to determine the effects of individual nutrients. Examinations were conducted on 3,249 participants who were also between the ages of 45 to 74. The results indicated a significantly lower prevalence of nuclear opacities in people taking riboflavin and niacin compared with those not taking these vitamins. Again, the oldest participants (ages 65 to 74) showed the greatest reduction, 44 percent, in nuclear cataracts. Optic Neuritis Optic neuritis is an acute debilitating inflam- mation of the optic nerve that affects more than 25,000 Americans each year, primarily women between the ages of 18 and 45. People with the disease usually have rapid vision loss and ocular pain. The ONTT compared oral corticosteroid, intravenous steroid followed by oral corticosteroid, and placebo for the treatment of new cases of optic neuritis. ONTT results showed that oral cor- ticosteroid, the most common treatment for the disease, when used alone is ineffective in treating the disease and actually increases a person's risk for future attacks. Strabismus Strabismus can result in amblyopia, which is a major cause of vision loss in the United States. The causes of strabismus are not well understood, but a defect in central nervous system control over the oculomotor system is thought to play a role. Mater- nal cigarette smoking during pregnancy as a risk factor for childhood strabismus was recentiy evaluated. A population-based, case-control study was conducted and evaluated all incident cases of strabismus diagnosed during a 21 -month period ft'om 1985 to 1986 in nine pediatric ophthalmology centers in Baltimore. Cigarette smoking was associated vwth esotropia but not exotropia for those women who smoked throughout pregnancy. The association between maternal smoking and esotropia was only seen in low-birth weight infants and infants in the upper-half of the birth weight distribution. The 16 NEI Annual Report— FY 1993 Extramural Research authors conclude that cigarette smoking may have a direct toxic effect on the developing nervous system, which can lead to abnormalities such as strabismus. 17 Division of Biometry and Epidemiology Report of the Acting Director, Division of Biometry and Epidemiology Roy C. Milton, Ph.D. The Division of Biometry and Epidemiology (DBE) comprises a Clinical Trials Branch, an Epidemiology Branch, and a Biometry Section. Dr. Roy Milton is the acting director for the Division. Drs. Frederick Ferris HI and Robert Sperduto serve as chiefs of the two Branches, respectively; Dr. Roy Milton is the head of the Biometry Section. The DBE has three main functions: research, education, and consultation. Research is the dominant function. It is the Division's mission to plan, develop, and conduct human population studies concerned with the cause, prevention, and treatment of eye disease and vision disorders, with emphasis on the major causes of blindness. This includes studies of incidence and prevalence in defined populations, prospective and retrospective studies of risk factors, natural history studies, clinical trials, genetic studies, and studies to evaluate diagnostic procedures. The DBE carries out a program of education in biometric and epidemiologic principles and methods for the vision research community. This program consists of courses, workshops, a fellowship program for ophthalmologists, publications, and consultation and collaboration on research. The Division provides biometric and epidemiologic assistance to NEI intramural and extramural staffs and to vision researchers in the pubhc and private sectors. The assistance ranges from consultation to collaboration as coinvestigator. Research Highlights The Krypton-Argon Regression Neovascularization Study This randomized multicenter clinical trial was de- signed to compare the efficacy of red krypton with blue-green argon laser photocoagulation for the management of high-risk proliferative diabetic retinopathy. Scatter laser photocoagulation with either argon or krypton appears to be equally effec- tive in arresting neovascularization of the disc. The Linxian Cataract Studies The Linxian Cataract Studies were two ran- domized clinical trials conducted in China that studied the effect of vitamin and/or mineral sup- plements on the risk of developing age-related cataracts. In these studies of populations with chronic deficiencies of multiple nutrients, use of the supplements was associated with a decreased risk of nuclear cataract. Additional research is underway to determine whether these findings ^ply to less nutritionally deficient populations. The Eye Disease Case-Control Study In a large epidemiologic study of neovascular AMD, an increased risk of disease was associated with cigarette smoking and higher levels of serum cholesterol. Decreased risk was associated with postmenopausal use of estrogens and higher serum levels of carotenoids. Results from the study are consistent with a hypothesis linking risk factors for cardiovascular disease with AMD. The hypothesis that higher serum levels of micronutrients with antioxidant c^abilities may be associated with a decreased risk of AMD was evaluated in the Eye Disease Case-Control Study. Persons with higher levels of carotenoids and those with higher levels of an antioxidant index derived from serum measurements of vitamins C and E, carotenoids, and selenium also showed a decreased risk of macular degeneration. Results from this observational study are now being tested in a cUnical trial. A study was conducted to evaluate the relative anatomic position of the crossing vessels at the site of occlusion in eyes with branch retinal vein oc- clusion. In 99% of eyes with a branch retinal vein occlusion, the artery was located anterior to the vein at the obstructed site. At comparable nonoccluded crossings, the artery was located anterior to the vein less than 65% of the time. The finding suggests a possible role for mechanical obstruction in the pathogenesis of branch retinal vein occlusion. 21 Division of Biometry and Epidemiology ISEI Annual Report— FY 1993 In a study designed to identify risk factors for idiopathic rhegmatogenous retinal detachment, only one clearly relevant risk factor, myopia, emerged from the analysis. An eye with a spherical equivalent refractive error of -1 to -3 diopters had a fourfold increased risk of retinal detachment com- pared with a nonmyopic eye. Data from the study suggest that almost 55% of nontraumatic detach- ments in eyes without previous surgery are at- tributable to myopia. Results from the study are consistent with a hypothesis suggesting that the etiology of retinal detachment is related to the architecture of the eye, rather than to systemic factors. A large epidemiologic study reported an increased risk of branch retinal vein occlusion in persons with a history of systemic hypertension, a history of cardiovascular disease, an increased body mass index at age 20, and a history of glaucoma. Risk of vein occlusion decreased with higher levels of alcohol consumption and high-density lipoprotein cholesterol. The data suggest a cardiovascular risk profile for patients with branch retinal vein occlusion and indicate that 50% of branch retinal vein occlusioas may be due to hypertension. The Sorbinil Retinopathy Trial This multicenter trial was designed to assess the ability of sorbinil, an aldose reductase inhibitor, to retard the development and progression of diabetic complications. Results for retinopathy have previously been published. The study now reports that no benefit was found from sorbinil in slowing the development of clinical diabetic polyneuropathy. The Early Treatment Diabetic Retinopathy Study This multicenter, randomized clinical trial of aspirin versus placebo among diabetics examined mortality and morbidity from all causes with special emphasis on cardiovascular events. There were no harmful effects of aspirin, and the suggestion of beneficial effects was similar to previous studies of mainly nondiabetic persons. Research Activities Clinical Trials The Early Treatment in Diabetic Retinopathy Study The Early Treatment in Diabetic Retinopathy Study (ETDRS) was designed to determine when to use photocoagulation for diabetic retinopathy. Patients with macular edema, preproliferative retinopathy, and mild or moderate proliferative retinopathy were studied. Three forms of photocoagulation treatment, ranging from restricted focal treatment to complete panretinal photocoagulation, were compared with no photocoagulation. In addition, the study evaluated the placebo-conttolled effects of daily administration of aspirin on the incidence of microvascular and macrovascular complications. The study also inves- tigated factors associated with the progression of disease. Recruitment was completed in March 1985 with the enrollment of 3,711 patients. In December 1985, the study reported that focal photocoagulation of clinically significant diabetic macular edema substan- tially reduces the risk of visual loss. It was further reported that focal tteatment increases the chances of visual improvement, decreases the frequency of persistent macular edema, and causes only minor visual field losses. Sixteen ETDRS reports have been published. Additional manuscripts are in preparation. Drs. Lloyd Aiello and Frederick L. Ferris, HI serve as cochairmen. Dr. Richard L. Mowery is project officer, and Dr. Emily Y. Chew serves as a member of the analysis plaiming group. The ETDRS results of aspirin effects on mortality and morbidity in patients with diabetes were analyzed and published. Analyses in progress include the effect of aspirin on vitreous hemorrhage, risk factors for severe visual loss, and risk factors for development of high-risk proliferative diabetic retinopathy. In addition, patients with mild to proliferative retinopathy are 22 NEI Annual Report— FY 1993 Division of Biometry and Epidemiology being followed with extensive psychophysical testing in the NEI Clinical Center to determine the mechanisms for loss of visual acuity in diabetic retinopathy. The Sorbinil Retinopathy Trial Dr. Daniel Seigel served as project officer for the Sorbinil Retinopathy Trial (SRT) until his retirement in November 1991. Sorbinil, a drug manufactured by Pfizer Laboratories, is an aldose reductase in- hibitor that has potential to prevent or retard diabetic neuropathy and retinopathy. The NEI provided scientific leadership for this multicenter clinical trial, which was funded by Pfizer. Approximately 500 patients were randomized to treatment and follov^oip, which ended in mid-1988. The results for retinopathy were published in 1990. No large benefit of treatment was observed. The effect of the treat- ment on neuropathy was summarized in a paper that was published this year. The Krypton-Argon Regression of Neovascularization Study The Clinical Trials Branch began the Krypton- Argon Regression of Neovascularization Study (KARNS) in three pilot clinics in December 1983. The major objective of this randomized clinical trial is to compare krypton laser with argon laser pan- retinal photocoagulation for treating neovas- cularization on the optic nerve head caused by diabetic retinopathy. Twenty-nine new clinics were enrolled in KARNS starting in August 1984. At the termination of the study in June 1990, a total of 1,063 patients had been randomized. This study is unique for the NEI because the functions for both the coordinating center and the fundus photography reading center are being handled by staff of the Clinical Trials Branch. Another feature of this multicenter trial is that the participating clinics receive no financial reimbursement from the NEI for their participation. Drs. Ferris and Chew direct this study along with Dr. Lawrence Singerman. Results of the KARNS were presented at the American Academy of Ophthalmology (AAO) in November 1992 and will appear in Ophthalmology. The Linxian Eye Study The NEI joined an ongoing NCI-supported clinical trial of nutrition and cancer in north central China in 1991 to determine whether the vitamin and/or mineral dietary supplements administered in the Linxian Cancer Trials for the preceding five years have affected the risk of age-related cataract and AMD. Eye examinations were conducted in 1991 on 5,390 members of the Linxian Study cohort. Dr. Sperduto is project officer, and the project team includes Drs. Milton and Chew from DBE and a Chinese ophthalmologist. Dr. Tian-Sheng Hu, from Beijing. Findings for cataract were published this year and are discussed in the research results section of the report of the Division of Collaborative Clinical Research. Intramural Program Clinical Trials Drs. Ferris and Chew are collaborating with Dr. Robert B. Nussenblatt on four additional randomized clinical trials in the NEI Intramural Program of the Clinical Center: (1) a trial of a sustained-release intraocular drug delivery system for gancyclovir therapy of CMV in patients with AIDS; (2) a trial to evaluate the efficacy of a heparin-surface modified intraocular lens in reducing the incidence and severity of postoperative inflammatory episodes following extracapsular surgery in uveitis patients with cataracts; (3) a trial of anti-inflammin, a pep- tide, in the treatment of anterior uveitis; and (4) a trial of S-antigen tablets in patients with uveitis. Other Dr. Seigel, as a special expert, continues to represent the NEI on the Data Monitoring Committee of the United Kingdom Prospective Diabetes Study, a clinical trial of alternative treatment regimens in the management of patients with diabetes. Followup is scheduled to continue in this study until 1994. Epidemiology The Age-Related Eye Disease Study The Age-Related Eye Disease Smdy (AREDS) is designed to collect natural history data of 4,600 patients between the ages of 55 and 78 years with bilateral drusen of different types or with unilateral advanced AMD. This study will evaluate the rates of development and progression of AMD, the rates of visual loss due to retinal lesions of AMD, and the risk factors associated with the development and progression of AMD. Evaluation of lens change during the 10-year AREDS study period will provide an opportunity to evaluate factors associated with the 23 Division of Biometry and Epidemiology NEI Annual Report— FY 1993 development of cataracts. In addition, a clinical trial will be perfonned to determine whether antioxidants (vitamins C, E, and beta-carotene) and zinc would prevent the development or retard the progression of AMD and cataract. There are 1 1 Clinical Centers, a Photographic Reading Center, a Central Laboratory, and a Coordinating Center. Identification of study participants began in September 1990. In November 1992, participants were evaluated with qualifying visits, and participants were randomly assigned to the study medications beginning in February 1993. Drs. Ferris (chairman), Sperduto (director of Leas Project), and Chew are directing the scientific aspects of the AREDS; Dr. Natalie Kurinij is the project officer. The Eye Disease Case-Control Study The Eye Disease Case-Control Study (EDCCS) is designed to identify risk factors for neovascular macular degeneration, idiopathic branch retinal vein occlusion, idiopathic central vein occlusion, rheg- matogenous retinal detachment, and idiopathic macular hole. Dr. Sperduto is study chairman, Ms. Rita Hiller is director of Data Analysis, and Dr. Chew is a member of the project team. All data have been collected. Five manuscripts were published this year: Risk Factors for Neovascular AMD, Antioxidant Status and Neovascular AMD, Arteriovenous Crossing Patterns in Branch Retinal Vein Occlusion, Risk Factors for Idiopathic Rheg- matogenous Retinal Detachment, and Risk Factors for Branch Retinal Vein Occlusion. The Diabetes in Early Pregnancy Study Dr. Emily Chew and Ms. Nancy Remaley, in col- laboration with Dr. James Mills of the National Institute of Child Health and Human Development (NICHD), are examining the effects of pregnancy on diabetic retinopathy in the Diabetes in Early Pregnan- cy Study (DEEPS). Data collection terminated in 1985. A manuscript has been prepared. The Italian-American Natural History Study of Age-Related Cataract In Parma, Italy, the Italian-American Natural History Study of Age-Related Cataract will estimate the rates of development and progression of the different types of lens opacities and the associated risk factors. Dr. Sperduto is the project officer; Dr. Milton and Ms. Remaley are on the project team. Data collection began in May 1989 with baseline data from the Italian-American Case-Control Study of Senile Cataract and was completed in May 1993. Analyses of development and progression of age- related cataract are under way. The Framingham Offspring Eye Study Dr. Sperduto is the project officer, Dr. Milton is the alternate project officer, and Drs. Marvin J. Podgor and Valeria Freidlin and Ms. Hiller are members of the project team for the Framingham Offspring Eye Smdy (FOES). This study is designed to examine familial relationships for age-related cataract and AMD among parents examined in the Framingham Eye Study (1973-1975) and their children examined between 1989 and 1991. Dr. Podgor has used generalized estimating equation methodology in the analyses of these data. A manuscript describing the study's findings for cataract has been prepared. Other A manuscript has been submitted for publication on risk factors for strabismus, using data fi-om the NICHD Collaborative Study and in collaboration witii Dr. Mark Klebanoff, NICHD. The DBE project team includes Drs. Chew, Tamboli, Zhao, Podgor, and Ms. Remaley. Statistical Methods Dr. Marvin Podgor and Dr. Joseph Gastwirth from the George Washington University collaborated in the investigation of various tests for the two- sample problem with location and scale change alternatives. Dr. Podgor presented some of these results at the NIH Conference on Current Topics in Biostatistics and at the 1993 Joint Statistical Meetings. A paper has been accepted for publication. Professional Activities Members of DBE are active in consultations and educational and professional activities, including referees for professional journals, associate editors or members of editorial boards, members of data and safety monitoring committees for clinical trials, training of staff fellows, invited and 24 NEI Annual Report— FY 1993 Division of Biometry and Epidemiology contributed presentations at professional society and other meetings, advisory committees for grant-sup- ported cooperative agreements, and technical advisors to the World Health Organization (WHO). Publications Bouzas EA, Freidlin V, Parry DM, Eldridge R, Kaiser-Kupfer MI: Lens opacities in neurofibromatosis 2: further significant cor- relations. BrJOphthalmomi6y.354-351, 1993. ETDRS Investigators: Aspirin effects on mortality and morbidity in patients with diabetes mellitus. ETDRS report No. 14. JAMA 268(10): 1292-300, 1992. Ferris HI FL: Diabetic retinopathy. Diabetes Care 16:322-323, 1993. Ferris HI FL: Issues in management of diabetic retinopathy. Hospital Practice 28(5):7, 1993. Ferris DI FL, Freidlin V, Kassof A, Green SB, Milton RC: Relative letter and position difficulty on ETDRS visual acuity charts. Am J Ophthal- mol 116(6):735-740, 1993. Glenn GM, Linehans WM, Hosoe S, et al.: Screening for von Hippel-Lindau disease by DNA polymorphism analysis. JAMA 267(9): 1226- 1231, 1992. Lasa MSM, Podgor MJ, Datiles MB, Caruso RC, Magno BV: Glare sensitivity in early cataracts. Br J Ophthalmol 77:489-491, 1993. Magno BV, Freidlin V, Datiles MB: Reproducability of the NEI Scheimpflug cataract imaging system. Invest Ophthalmol Vis Sci 35(7):3078-3084, 1994. Nussenblatt RB, de Smet MD, Rubin B, et al.: A masked, randomized, dose-response study bet- ween cyclosporine A and G in the treatment of sight-threatening uveitis of non-infectious origin. Am J Ophthalmol 115(5):583-591, 1993. Podgor MJ, Gastwirth JL: On nonparametric and generalized tests for the two-sample problem with location and scale change alternatives. Stat Med 13(5-7):747-758, 1994. Prior MJ, Prout T, Miller D, Ewart R, Kumar D and Early Treatment Diabetic Retinopathy study Research Group: C-peptide and the classification of diabetes mellitus patients in the Early Treat- ment Diabetic Retinopathy Study. ETDRS report No. 6. Ann Epidemiol 3:9-17, 1993. Rodgers GP, Walker EC, Podgor MJ: Is "relative" hypertension a risk factor for vaso-occlusive complications in sickle cell disease? Am J Med 5d 305:150-156, 1993. Sastry SM, Sperduto RD, Waring GO, Remaley NA, Lynn MJ, Blanco PE, Miller DN: Relationship of radial keratotomy and intraocular pressure. Refractive and Corneal Surgery 9(6):459^64, 1993. Singerman LJ, Chew EY, Ferris HI FL, Murphy RP, Brucker AJ, Remaley NA, and The Krypton Argon Regression Neovascularization Study (KARNS): Randomized comparison of krypton vs. argon scatter photocoagulation for diabetic disc neovascularization. KARNS report No. 1. Ophthalmology 100(11): 1655-1664, 1993. Sorbinil Retinopathy Trial Research Group: The Sorbinil Retinopathy Trial: neuropathy results. Neurology 43:1141 -\\49, 1993. Sperduto RD, Hu T-S, Milton RC, et al.: The Linxian Cataract Studies — Two nutrition interven- tion trials. Arch Ophthalmol 111:1246-1253, 1993. Sperduto RD: Epidemiologic aspects of age-related cataract. In: Tasman W, Jaeger EA (eds): Duane's clinical ophthalmology. Philadelphia, J.B. Lippincott Co, 1992, Chapter 73 A, pp 1-5. The Eye Disease Case-Control Study Group: Risk factors for neovascular age-related macular degeneration. Arch Ophthalmol 110:1701-1708, 1992. The Eye Disease Case-Control Study Group: An- tioxidant status and neovascular age-related macular degeneration. Arch Ophthalmol 111:104- 109, 1993. The Eye Disease Case-Control Study Group: Risk factors for idiopathic rhegmatogenous retinal detachment. Am J Epidemiol 137:749-757, 1993. 25 Division of Biometry and Epidemiology NEI Annual Report— FY 1993 Eye Disease Case-Control Study Group: Risk factors for branch retinal vein occlusion. Am J Ophthal- mol 116:286-296, 1993. Zhao J, Sastry SM, Sperduto RD, Chew EY, Remaley NA, and The Eye Disease Case-Control Study Group: Arteriovenous crossing patterns in branch retinal vein occlusion. Ophthalmol 100:423-428, 1993. 26 International Program Activities Report of the Acting Assistant Director for International Program Activities Terrence Gillen, M.A., M.B.A. The mission of the NEI includes the reduction of the prevalence of blindness, visual impairment, and eye disease worldwide through basic and applied research and training. Although excellent ophthalmic procedures and eye-care delivery systems are acces- sible in the developed world, adequate health care is not readily available in all parts of the developing world. This widening gap in visual health between developed and developing nations threatens to have ominous consequences. If present trends continue, the number of blind people — ^today estimated at 24 million — will more than quadruple during the next 40 years. Tragically, as many as 90% of these blind people will live in developing countries. This large-scale disablement caused by blindness is not only a costly obstacle to economic develop- ment, it is a catastrophic loss of human potential in the very parts of the world most desperately in need of a healthy workforce. In addition, because more than 80% of all cases of blindness can be considered avoidable — that is, they could have been prevented or could be cured using available and locally ap- propriate technology — such deprivation is a truly needless denial of a basic human right for millions and millions of people. Therefore, the NEI under- takes international activities to facilitate the develop- ment and application of effective prevention and intervention programs. These efforts are coordinated by the Institute's Office of International Program Activities (OBPA), which was aeated in February 1989. OIPA enhances NEI's international programs, which include: • Evaluating available health technologies, promoting the most cost-effective intervention and prevention programs, and encouraging their availability for affected populations, especially in developing countries. • Conducting collaborative applied research studies to develop preventive methods for treating specific eye diseases. • Conducting controlled clinical evaluations of promising research findings. • Exchanging information on recent scientific advances and their appropriate application to visual problems. NfEI currently supports international research on several blinding diseases that have a major worldwide impact: cataract, onchocerciasis, ocular toxoplasmosis, glaucoma, diabetic retinopathy, and vitamin A deficiency. During the past year, the NEI has continued to support investigations of important blinding eye diseases with worldwide impact. These studies are implemented through bilateral agreements with the U.S. Government, other types of country-to- country programs (such as those supported by the U.S. Agency for International Development [U.S. AID]), and through collaborative activities with the WHO, the Pan-American Health Organization, and with foundations and private and voluntary or- ganizations such as the International Association of Lions Clubs. Research Activities Cataract Because cataract is responsible for about one-half of the developing world's curable blindness and is a major problem for the United States as well, the NEI has developed a collaborative research program that includes projects to prevent blindness from cataract with collaborating groups in Italy, India, and Latin America. Additionally, health services research expertise from the NEI is made available to selected collaborating partners through training activities and the conduct of joint research projects. An NEI-supported randomized clinical trial to compare intracapsular cataract surgery plus aphakic spectacles with extracapsular cataract extraction plus implantation of an intraocular lens (lOL) is being conducted at the Aravind Eye Hospital in Madurai, India. The trial's primary comparison concerns operative and postoperative complications. 29 International Program Activities NEI Annual Report— FY 1993 Secondary evaluation endpoints include measurement of vision function assessed by interview using a multi-item questionnaire and appraisal of economic impact in terms of direct and indirect cost associated with blindness and cataract surgery. The Collaborative Italian-American Case Control Study of Age-Related Cataract, which was started in September 1986, as part of the program of cooperation in biomedical research between the United States and Italy, has now been completed. The objectives of the study were to: (1) identify risk factors for age-related cataract, (2) evaluate metiiods of in vivo cataract classification, and (3) compare the findings with those of parallel studies being con- ducted in the United States and India. Data were collected at the Institute of Ophthalmology at the University of Parma. The Laboratory for Epidemiology and Biostatistics at the Istituto Superiore di Sanita in Rome served as the study's coordinating center. Data collection ended in April 1989, after 1,477 subjects had been entered into the system. Four papers describing the study's findings have been published. Ihe Collaborative Italian-American Study of the Natural History of Age-Related Cataract has added a four-year follov^p component to the recently completed case-control study of age-related cataract. Approximately 1,000 subjects with cataracts and 300 subjects fi-ee of cataracts are being examined every six months for four years to collect data on the natural history of the various types of cataracts. Data collection began in May 1989. The objectives of the natural history study are to estimate the rates of development and progression of the various types of lens opacities, identify risk factors associated with the development and progression of cataracts, and determine whether the risk factors affecting rates of progression differ for the various types of leas opacities. Data collection ended in April 1993 at the Institute of Ophthalmology, University of Parma. The Laboratory for Epidemiology and Biostatistics, Istituto Superiore di Sanita in Rome serves as the Coordinating Center. Preliminary findings from the study were presented in Stresa, Italy, at the 1992 International Congress of Eye Research meeting. Preliminary findings from the study were presented at the meeting of the Association for Research in Vision and Ophthalmology in May 1993. A paper describing incidence and progression rates for specific cataract types has been prepared for publicatioa International collaborators have been established by scientists in the NEI's Laboratory of Mechanisms of Ocular Diseases, Section on Cataract to further our understanding of the relationship between en- zyme deficiency diseases and cataract For example, these NEI scientists have begun a candidate gene study to determine whether a deficiency in sorbitol dehydrogenase (SDH) in a family where several members have congenital cataracts is due to changes in SDH gene structure or expression. This study is possible through the cooperation of the Unidad de Investigacion Biomedica Hospital de Pediatria, Instituto Mexicano del Soguro Social, Guadalajara, Mexico. Vitamin A Deficiency Although not a major problem in the United States, vitamin A deficiency worldwide affects an estimated 14 million children annually. It is the world's major cause of childhood blindness, accoun- ting for 250,000 to 500,000 new cases of blindness per year. In addition, children die at higher rates from common childhood infections if they are deficient at any level of severity. The NEI supports basic research on the interaction of nutrients such as vitamins A, C, and E on retinal and other eye tissue development. Such investigations can lead to clinical interventions that may help alleviate morbidity from malnutrition eye disease. In addition, the NEI has provided technical consultation for a study in south India that has shown an impressive reduction in childhood mortality associated with improved vitamin A nutritional status, and other efforts to transfer this technology to alleviate world blindness are under way. Particularly in Asia, vitamin A deficiency is a public health problem and the leading cause of blindness among preschool-age children. The most effective way of providing affordable prevention programs is under study by the University of Michigan and the Nepal Netra Jyoti Sangh, Nepal's national society for the prevention and control of blindness. OIPA is providing technical oversight for this three-year project for the U.S. Department of Health and Human Services (DHHS) Office of International Health and the U.S.AID East Bureau. 30 NEI Annual Report— FY 1993 International Program Activities Glaucoma Open-angle glaucoma is the leading cause of blindness among African Americans and is a major cause of visual impairment and disability. The incidence of glaucoma has not been measured precisely in any population, and the risk factors related to its development are largely unknown. In the Barbados Eye Study more than 4,200 persons between the ages 40 and 86 years were examined from 1988 to 1992 as part of a population-based study to determine the prevalence and risk factors for glaucoma and other eye disorders such as diabetic retinopathy, AMD, cataract, and visual impairment. In 1992, the Barbados Incidence Study was initiated to estimate the incidence of glaucoma and other ocular disorders from individuals free of disease in the Barbados prevalence survey. Also, risk factor analysis will be conducted for associations with development of glaucoma and to characterize those who have progressive eye disease. The Early Manifest Glaucoma Trial is a ran- domized, controlled clinical trial designed to deter- mine whether and to what extent reduction of lOP influences the course of chronic open-angle glaucoma. Investigators at the University of Lund in Malmo, Sweden, collaborating with investigators at the State University of New York at Stony Brook, will study an estimated 300 patients with newly diagnosed disease. Participants will be randomized either to pressure-lowering treatment or to obser- vation without treatment Both groups will be followed closely with computerized perimetry and fundus photography. Recruitment of patients began in 1993 and will continue for an estimated two years. FoUowup of patients will be conducted for four years. Retinal Degenerations In collaboration with protein biochemists at the Karolinska Institute in Stockholm, Sweden, NEI cataract researchers are investigating the evolutionary relationships of i^-crystallin, an enzyme/crystallin of certain species, with other oxido-reductases. Es- tablishing such relationships with enzymes of known function should help in identifying the physiological roles of ^-crystallin both in the lens and in other tissues where it is present at low levels. Diabetic Retinopathy The United Kingdom Prospective Diabetes Study is a prospective randomized study of different therapies to determine whether improved blood glucose control or improved blood pressure control of noninsuhn-dependent diabetes will reduce mor- bidity and mortality. The study began in 1977 and has recruited 5,102 newly diagnosed diabetic patients. Patients who fail to respond to diet therapy are randomized to diet therapy or "active therapy" with sulfonylurea, insulin, or metformin. As part of the study, hypertensive diabetic patients have been randomized to "tight blood pressure" control with either an ACE inhibitor or beta-blocker to "less tight control." The development and progression of diabetic retinopathy in these patients is being as- sessed by retinal photography. The study is currently completing 10 years of patient followup. Management for Eye-Care Delivery Course As a WHO Collaborating Center for the Preven- tion of Blindness, the NEI offered a course at the Aravind Eye Hospital in Madurai, India, in January 1993, on management for eye-care delivery. The purpose of this five-day course was to enhance the effectiveness of mid- and senior-level managers of eye-care programs and facilities. The course broadened the management perspective of the students, extended their understanding of decision- making, and developed their problem-solving skills. Emphasis was placed on demonsfrating the ap- plication of operations research and management science to "real world" problems. Individually and in small discussion groups, participants analyzed each case by identifying the basic problems involved, characterizing the relevant background setting and facts, formulating an ^- propriate analytical framework or model, and generating alternative solutions. Subsequentiy, in a large-group classroom setting, all participants ex- changed views concerning the cases and tested their conclusions. Group discussion forced participants to examine critically their own assumptions and to narrow their thinking to a plan of action. Members of the faculty guided the classroom discussion and ensured that all significant issues were addressed and that there was full participation. 31 International Program Activities NEI Annual Report— FY 1993 Consultation to the World Bank The director, deputy director, and special advisor to the director, NEI, have participated as consultants to the World Bank in the development of a proposal by the Government of India for a major initiative in cataract blindness control. Technical meetings were held in New Delhi and Madurai to provide the knowledge base upon which training and surgical guidelines can be developed for a significant expan- sion of cataract surgery with explicit attention to the quality and extent of vision restoration. Activities With International and Multinational Organizations In FY 1993 NEI staff continued to provide tech- nical advice to Lions Clubs International in the development of their $100 million SightFirst initiative, a global sight-conservation program aimed at substantially reducing the prevalence and incidence of preventable and curable vision loss. Also in FY 1993, NEI continued its activities as a WHO Collaborating Center for the Prevention of Blindness. The NEI director continues to serve on the WHO'S Special Advisory Panel in the Prevention of Blindness, and the assistant director for Inter- national Program Activities serves on the Global Advisory Committee. Other NEI staff members have, on request, given consultations to the WHO program. In addition, an ophthalmologist from India visited the NEI, under the auspices of a WHO fellowship, to study cataract etiology and prevention. NEI continues working closely with non- governmental organizations in designing service and research programs to reduce the prevalence of blindness, regardless of its etiology, throughout the world. Extramural Programs In FY 1993, NEI granted 14 awards to foreign institutions in eight countries. Research and training projects were supported in lens and cataract, glaucoma, visual system development, photorecep- tors, phototransduction, visual cortex, visual abnor- malities, Leber disease, nutrition of the eye, ocular complications of diabetes, and the prevention of blindness. Awards covered both basic and clinical research projects. Intramural Programs NEI continues to serve as an international center for research and training on eye disease. In FY 1993, 18 visiting fellows, 22 visiting associates, 13 visiting scientists, 21 special volunteers, and eight guest researchers, from more than 20 countries conducted research in NEI's Bethesda, Maryland, facilities. Their work included basic laboratory investigations on the molecular structure and development of the visual system, sensory and motor disorders of vision, and the biochemical bases of retinal and corneal diseases and cataract develop- ment. In addition, visiting scientists collaborated with NEI investigators in clinical studies to define, treat, and prevent vision disorders such as genetic and developmental defects, ocular inflammatory disease, and ocular complications due to systemic conditions such as diabetes. 32 Science Policy and Legislation Report of the Associate Director for Science Policy and Legislation Michael P. Davis, M.S. The Office of Science Policy and Legislation (OSPL) is responsible for a broad and diverse range of management activities that support and further the NEI's mission. Among these are providing leadership and direction for program planning, analysis, evaluation, and legislative functions, including the development and main- tenance of a computerized management information system and public information and scientific reporting services in support of the NEI's research programs. This year has been a period of significant change and yet has also been a time of significant ac- comphshment. Mr. Julian Morris, who had served the NEI for more than 20 years in positions ranging from information officer to associate director for science policy and legislation, succumbed after a lengthy illness. As a tribute to his numerous contributions to the institute, the NEI staff and the National Advisory Eye Council (NAEC) honored his memory by dedicating the most recent long-range plan for vision research to him. During this past fiscal year, the three sections that make up the OSPL were elevated to branch level. Mr. Michael P. Davis, who had served as acting associate director during Mr. Morris' illness, was selected to fill the vacancy. Subsequently, Dr. Carmen P. Moten, a program analyst within the Policy, Legislation, Planning, and Evaluation Branch (PLPEB), was selected as chief of that branch. Also of great significance was the completion of Vision Research— A National Plan: J 994-] 998. After final updating by staff, the plan was sent to MERIT awardees for their comments and sugges- tions, prior to final review and approval by the NAEC. Development and publication of such a comprehensive plan by so small an office, especially during a period complicated by many external factors, was an enormous undertaking. Without the hard work and extra effort by Dr. Moten and Mr. Whitaker of the PLPEB, Dr. McLaughlin and tiie program directors firom tiie extramural research program, and a very talented group of publication experts at CSR, Inc., final publication would not have occurred. We are greatly indebted to them for their assistance in bringing this excellent document to completion. Policy, Legislation, Planning, and Evaluation Branch Carmen P. Moten, Ph.D., Chief The PLPEB advises the NEI director on program plaiming, analysis, evaluation, and legislation and serves as the focal point within the Institute for these functions. In addition, PLPEB develops and executes a comprehensive program planning strategy for the Institute, including the periodic development of a national five-year vision research plan in con- junction with the NAEC; plans, coordinates, carries out, and/or monitors NEI program evaluations; and prepares recurring and ad hoc program analyses in response to requests fi"om the NIH, the Public Health Service (PHS), and the Department During FY 1993, the principal activities for the PLPEB were: • Preparation of material for the 1993-1994 Bien- nial Report of the Director, NIH, describing research accomplishments, outiining future oppor- tunities, and assessing important policy issues. • Preparation of briefing materials for the confir- mation hearing of NIH Director-Designate Dr. Harold Varmus. • Support of the NEI extramural grant information retrieval system by assigning scientific, biotech- nology, diabetes, and other codes to awarded grants for the purpose of analyzing and reporting research activity in areas of interest to the NEI, NIH, DHHS, Congress, or non-governmental organizations and individuals. 35 Science Policy and Legislation NEI Annual Report— FY 1993 • Preparation of materials for the Congressional Appropriations Committee Report — Decade of the Brain. • Preparation of materials for NIH-coordinated activities with PHS agencies. • Preparation for publication by NIH The 1993- 1994 Prevention Annual Report. • Preparation of materials for the NIH director on supported research related to FDA-approved drugs. • Preparation for publication by NIH of The 1992 Annual Report on Rare Disease Research Ac- tivities. • Reparation of briefing materials for the General Accounting Office (GAO) on activities duplicated among PHS agencies. The PLPEB has also been involved in researching, writing, and editing a variety of reports requested by the NIH, PHS, DHHS, Congress, and non-governmental organizations and individuals that include the following: Report to Congress on sickle cell anemia research and the role of minority institutions in research. Review of the draft update of the Healthy People 2000: Public Health Service Action Report. NEI submission on fibromyalgia for the Congres- sional Appropriations Report. NEI submission for the Diabetes Mellitus Interagency Coordinating Committee (DMICC) Annual Report. Scientific advances for the NIH FY 1994 Congressional Justification. Report on breast cancer-related research during FY 1992, in response to an NCI request. Recommendations for the implementation of the NIH Strategic Plan, in response to an OSPL request. Review of the draft report — Public Version of the Strategic Plan. Support of the NIH Conference on Disease Prevention Research. Review of the Cross-Cutting for Healthy People 2000 Progress Report— American Indians and Alaskan Natives; Adolescents and Young Adults: Hispanic Americans; and Women. Recommendations for technical changes to the H.R.4, the NIH Revitalization Act of 1993. Review of the draft report — Clinician 's Handbook of Preventive Services 1993 and Clinical Preven- tive Services: What Works and What It Costs, supported by the National Coordinating Com- mittee on Chnical Preventive Services (NCCCPS). Report on space medicine-related research, in response to a NIH director request. Report on research use of FDA-approved drugs, in response to a Congressional request. Report on federally funded current research, training, and intervention projects related to injury control. Briefings for the GAO. — Breast cancer — Immunology — Diabetes — Environmental health Policy briefing materials for the director, NIH. Report on adolescent-related research, in response to a NICHD Office of Demographic and Behavioral Sciences request Report on trauma-related research, in response to an NIH director request. Submission of highlights of drug-related research for Congressman Waxman's request for infor- mation concerning NIH-supported research on drugs. Final review of the draft report — Implementation Plan on Health and Behavior Research. Report to the Office of the Inspector General for an on site discussion of the Institute's mission and program activities. For the NEI Scientific Reporting Branch, data on inti-amural and extramural research in the fol- lowing areas: — Dry eye — AMD — Glaucoma — Cataract — RP — Sjogren syndrome 36 NEI Annual Report— FY 1993 Science Policy and Legislation — Ocular implants — Corneas — Retinal transplants • For the NEI Financial Management Branch, information on intramural and extramural research costs for the following areas: — Biotechnology — Prevention — Immunology — Vaccine development — Decade of the Brain — Breast cancer — Tuberculosis — Aging — Nutrition — AIDS — Women's health issues — Diabetes — Cancer — Sexually transmitted diseases The PLPEB also provided editorial review of a variety of letters, reports, and other narrative materials for other offices within the NEI. Management Information Systems Branch David Scheira, Ph.D., Chief During the past fiscal year, the Management Information Systems Branch (MISB) has made significant changes to its local area network (LAN) configuration prompted by the move of the NEI's Extramural and Collaborative Program to Executive Plaza South (EPS) in April 1993. To accommodate this move, MSB configured a new LAN at EPS, connected to the NEI's Building 31 LAN via NIHnet using TCP/IP. MISB designed the star topology for the EPS LAN, based upon unshielded twisted pair UTP cabling. To support this distributed configuration of two LANs connected over NIHnet, the total number of network file, print, and database servers was increased from six to eight. The MISB procured the needed computer hardware, designed the network cabling and EPS machine room arrangement, and reassembled all 40 workstations at EPS after the move. During the fiscal year, MISB also configured additional Dell 486 computers to replace the last of the NEI's outmoded Zenith 286 PCs, yielding a total of 95 NEI workstations. MISB also upgraded the network cards in all workstations to Etherlink II or III for improved performance and reliability. During the past fiscal year, the MISB also upgraded all client network software to LAN Manager 2.1a. The network's database server was upgraded to a Dell 486/50 with 2.1 gigabytes of disk space to support greatly expanded database functionality. SQL Bridge was installed and con- figured to allow transparent access by Building 31 NEI staff to database systems, which used the NEI's SQL server located at EPS. Remote printing capability via BARRHASP was added to the EPS LAN and was enhanced to allow more flexible routing to different network printers at the Building 31 site. Daily network backup procedures using two digital/audiotape (DAT) drives were enhanced to ensure more reliable operation. An uninterrupted power supply unit (UPS) was procured and installed at the NEI's EPS site, providing con- tinued operation in the event of a temporary power loss and an automatic smooth network shutdown in the event of a protracted power outage. The MISB provided extensive enhancements to its grants information systems during FY 93. Microsoft SQL Server, the database server for all systems, was upgraded to version 4.2 to allow enhanced functionality. JAM and JAM/DBI, the client tools for these systems, were also upgraded to allow additional functionality. The existing NEI snapshot, council letter, and grants coding systems were upgraded for this new environment. Three new systems for, respectively, user-friendly grants queries, CRISP queries, and pay plan management were designed and programmed by MISB staff. Automated security and login functions integrated with LAN login were developed by MISB staff. These functions provide automatic login to grants information systems for NEI staff and also 37 Science Policy and Legislation NEI Annual Report— FY 1993 automatic tracking of system usage. Database system status information has been integrated into this automatic logon procedure, so that users receive messages concerning system shutdowns when they occur and estimated times of resumed operation upon logon to these systems. Automatic daily check and backup procedures have been implemented through custom programming by MISB for all active NEI databases. These procedures are automatically run overnight from an NEI LAN server with results automatically recorded in a log table that can be instantly checked by MISB staff. The NEI's weekly grants update batch procedures have been fine-tuned to provide virtually no weekday system downtime during FY 1993. These procedures continue to be nm each Sunday by MISB staff to allow full system availability during the work week. During the end of the fiscal year, these update procedures, now miming in Paradox, were completely reprogrammed by MISB staff in Microsoft SQL Server Transact SQL language. When tested and implemented in FY 1994, this reprogramming will allow Paradox operations to be fully superseded by the more reliable, state-of-the-art client-server architecture. This reprogramming effort will streamline the NEI's database configuration, provide enhanced reliability and maintainability, and establish a foundation for additional advanced system development for both grants and other NEI functions. The MISB has continued to provide custom information reports to NEI staff for internal use and public distribution, with 107 new requests logged for FY 1993 and rapid turnaround achieved in every case. Weekly and monthly reports, as well, continue to be provided. In addition to its own programnung efforts, the MISB has continued to support NEI staff in the use of information resources provided by the Division of Research Services (DRG), the National Library of Medicine (NLM), and other sources, including the DRG information system, CRISP, FOCUS, WYLBUR, MEDLINE, Gratefiil Med, Legislate, the electronic NIH library catalog. Gopher, and other specialized systems. During the past fiscal year, MISB has implemented upgrades to several of the software packages it operates while continuing to provide support for its full LAN software array, including Harvard Graphics, Quattro, Paradox, Calendar, FTP PC/TCP, Microsoft Mail, Virus scaimer, and other packages used for specialized functions. The MISB has continued to support all computer hardware in- house, with service calls made only to order parts not internally available or to handle unusual problems. This internal support has resulted in substantial savings to the NEI. The MISB has arranged for the services of a high school student intern during FY 1993, who assisted with its evaluation of a client-server gateway that the Division of Cancer Research and Treatment (DCRT) is now in the process of procuring for NIH-wide use. This gateway will allow NEI transparent access into personnel and administrative database systems whose data is stored in the DB2 mainframe database sys- tem. The MISB has continued to be a leader in the development of client-server database systems at the NIH, and MISB staff members have demonstrated NEI systems at various intercampus forums. The MISB has continued to handle a number of IRM functions for the NEI, including its environment and resources report, strategic plan, tactical plan, budget report, and security functions. The MISB staff members have continued to represent the NEI on a number of NIH-wide committees, including the Office Technical Coordinators and its network subcommittee, the ADP Extramural Programs Coor- dinating Committee and its steering committee, the Database Technology Task Force, the NIH lead users group, the Campus Users Research Exchange, and the Technical LAN Coordinators Committee. Scientific Reporting Branch Judith A. Stein, M.A., Chief This year, the Scientific Reporting Branch (SRB) provided numerous reporting and public infor- mation activities in support of NEI programs. Specific activities included the development of responses to inquiries received from the general public, professionals, and the media; the development and dissenunation of information and education materials; development of A Celebration of Vision Research, highlighting the NEI's 25th anniversary; planning, implementation, and coordination of the 38 NEI Annual Report— FY 1993 Science Policy and Legislation National Eye Health Education Program (NEHEP); maintenance of an NEI exhibit program; and preparation of reports to the Congress. Specific accomplishments in these areas are ouflined below. • The SRB responded to approximately 17,000 written, telephone, and in-person inquiries from the general public, patients and their families, students, health professionals, the media, and other professionals. This figure represents almost a threefold increase in inquiries from FY 1992. In addition, staff members responded to 15 pieces of controlled correspondence. These correspon- dences included responses to congressional in- quiries and Presidential greetings and proclamations. To support the public inquiry function of this office, the SRB staff developed and/or updated publications, including a new brochure for people at risk for AMD. • A new edition of the book. Clinical Trials Sup- ported by the National Eye Institute was produced. This book describes 16 nationwide extramural clinical trials supported by the NEI and, for the first time, five intramural studies conducted at the NIH. The book will be promoted to practitioners through exhibits at the upcoming meetings of the AAO and the American Academy of Optometry and through public service advertisements and announcements in various professional journals. • The results of the Retinitis Pigmentosa Vitamin Study were aimounced in May. This included the writing and dissemination of a press release to the print and electronic media A "Dear Colleague" letter was prepared and distributed to all members of the AAO and the American Optometric As- sociation. • To highlight the NEI's 25th aimiversary, A Celebration of Vision Research was initiated. The objectives of this activity are to provide the American public with a report on its investment in vision research, highlighting the achievements and firontiers of publicly funded vision research; increasing awareness of the benefits derived from vision research; and stimulating interest in biomedical research. A traveling science museum was developed that will present the progress and future of vision research by highlighting the anatomy and physiology of the eye and visual system through the use of interactive modules; artifacts firom the past such as eyeglasses, advertisements, equip- ments, etc; common eye diseases and disorders, focusing on both basic and clinical research; and predictions for the future of vision research. In addition, the development of a school program for grades four to eight was initiated. • The NEHEP continued to expand its efforts to reach people at risk for glaucoma or diabetic eye disease. This included the distribution of the three NEHEP education kits to individuals responsible for educating people at risk. Since the kits became available in the spring of 1992, more than 37,350 kits have been distributed. In addition, initial plans were developed to expand the diabetic eye disease program to reach Native Americans and Hispanics. This included con- ducting literature searches, identifying or- ganizations, and reviewing current materials designed for these audiences. The membership in the NEHEP Partnership grew from 43 to 50 organizations. A Partnership action plan was developed to monitor activities as well as to identify opportunities for future involvement in the NEHEP. Technical assistance was provided to many community-based or- ganizations to increase their participation in the NEHEP, particularly through the involvement of local Partnership members. Plans were made to hold the Third National Eye Health Education Conference in December 1993 in Pennsylvania. The purpose of this conference will be to foster the development of local eye health education programs. The conference will provide the NEHEP Partnership with the opportu- nity to work together and to learn new skills that can be used to implement programs at the local level. • SRB staff members represented the NEI at the meetings of several professional organizations, including the American Diabetes Association, the Association of State and Territorial Directors of Public Health Education, the American As- sociation of Diabetes Educators, and the National Association of Area Agencies on Aging. 39 Office of the Scientific Director Report of the Scientific Director Robert B. Nussenblatt, M.D. This past year has been one of change and achievement in the Intramural Research Program of the National Eye Institute (NEI). Work has progressed in both clinical and basic research spheres. There has continued to be a heavy empha- sis in the area of AIDS (acquired immune deficiency syndrome), with an ongoing clinical trial to evaluate the effectiveness of a sustained-release device for ganciclovir placed into the eye for the treatment of cytomegalovirus (CMV) retinitis. A randomized masked study to immunomodulate uveitis has con- tinued. This attempt at using oral tolerization is an attempt to down-regulate inflammatory disease in patients' eyes without the use of medications. In the area of basic research, there have been developments in exciting new concepts about gene regulation, as well as continued advancement toward the ultimate goal of the use of gene therapy to treat eye diseases. Following are a few highlights of research achievements by the NEI intramural scien- tists during Fiscal Year 1993. Laboratory of Mechanisms of Ocular Diseases (LMOD) LMOD investigators have continued studies on a broad range of topics relating to the biology of various tissues, looking in depth at the molecular mechanisms responsible for certain ocular disorders. The major emphases of this group continue to be cataract and the ocular complications of diabetes. This year the group has shown, using site-directed mutagenesis, that the histidine at position 1 10 of the enzyme aldose reductase appears critical for catalytic activity. Studies have been instituted to evaluate a family with congenital cataracts whose members have a probable defect in the sorbitol dehydrogenase gene. On the clinical level the group also has evaluated the protein composition of normal human lenses and cataracts. Dr. Fielding Hejtmancik and his col- leagues have been studying the structure and function as well as the relationships of p-crystallins. At the same time, they are conducting gene mapping studies on a variety of genetic disorders that have ocular manifestations, including Usher's syndrome type I. These researchers have mapped two independent genes to chromosome 2. Dr. W. Gerald Robison has continued to refine and better characterize the rat model for diabetic retinopathy. Rats with this disorder will develop striking angiopathy in the retina. Laboratory of Sensorimotor Research (LSR) This Laboratory of international standing has continued to concentrate its research efforts on the brain mechanisms underlying the visual sense and visually controlled movements. Skilled motor control is one of the tasks the LSR scientists have concentrated on, particularly in relation to the visual control of eye movements. They have integrated, in a superb fashion, the observations made in humans, using an excellent animal model, the Rhesus mon- key. Its use permits exploration of not only the exact behavioral mechanisms related to visual motor behavior but also the underlying brain mechanisms. One area of particular interest has been the genera- tion of rapid or saccadic eye movements. Dr. Fred Miles and his group have attempted to increase understanding of the problem solving that goes into generating very rapid saccades. Some work has centered on the evaluation of what happens when monkeys or humans are confronted with different-sized images that are presented before each eye. Building on past observations. Dr. Miles' group has found that humans immediately adjust the amplitude of their eye movements in ways that are appropriate to the size of the stimulus. These LSR researchers have conjectured that the horizontal disparity in the images detected by the visual system controls this movement. They were able to reproduce this phenomenon in the monkey, thereby 43 Office of the Scientific Director NEI Annual Report— FY 1993 permitting further evaluation of the brain mecha- nisms underlying this control. In parallel fashion, Dr. Wurtz and his group have evaluated the control of movement through the environment and the stabilization of posture. Dr. Lance Optican, who has an ongoing interest in the way neurons convey visual information, has demonstrated that nem"ons convey information about visual features by using a temporal code. This work may elucidate the fascinating concepts of how the brain processes information that ultimately leads to visual perception. Dr. Michael Goldberg and his collaborators have used a variety of techniques to evaluate the control of eye movements in the monkey. More recently they have applied these techniques to the human situation. In recent work, using PET scanning, they have identified in the human the approximate region in which frontal eye fields are located, which they had previously noted in the monkey. During this past year the whole LSR moved to the new Silvio O. Conte Building. This facility provides a state-of-the-art environment for nonhuman primate research. Laboratory of Ocular Therapeutics (LOT) This Laboratory has continued to focus on the development of new ophthalmic drugs — aldose reductase inhibitors as well as anticataract agents. This year more effective and less toxic aldose reductase inhibitors that are unrelated to previous aldose reductase inhibitors have been developed through the use of biochemical, pharmacological, and computer molecular design techniques. The Laboratory has continued long-range studies evaluating the effect of galactose on dogs. The retinal changes that have been seen are typical of diabetic retinopathy as it progresses to the prolifera- tive stage. This dog model is the first experimental model that demonstrates both clinical and histolog- ical changes found in all stages of diabetic retinopathy. Laboratory of Molecular and Developmental Biology (LMDB) LMDB investigators continue to focus on under- standing the fundamentals of gene expression and cellular differentiation in the eye. Major empha- sis is placed on the lens. On the basis of their earlier observations that lens crystallins ^pear to be multi- functional proteins that are expressed outside the lens and eye, they have broadened their research during the past year to include new areas of metabolism and gene expression in various tissues. Of interest is the fact that transcriptional factors involved in the expression of crystallin genes are present in many tissues and are used to control numerous biological processes. The implications of gene control in the eye go far beyond this organ. Of great importance this year has been the LMDB's identification of regulatory elements re- quired for expression of genes in the eye and other tissues. Regulatory elements may be functionally redundant, that is, removing a regulatory element will not necessarily eliminate expression of the gene. A great deal of emphasis has been placed on the expression of proto-oncogenes and cyclins, which have been linked to the normal processes of cellular differentiation in the lens, as well as to the cell cycle and growth control. The development of a transgenic facility within this Laboratory has expanded the NEI's ability to use transgenic animal models. An ongoing, fruitful interaction between the LMDB and the Laboratory of Immunology (LI) has resulted in genetic engineering experiments which have the potential to contribute animal models for research on autoimmune diseases of the eye. Ophthalmic Genetics and Clinical Services Branch (OGCSB) The OGCSB conducts clinical and laboratory research on gene expression and molecular interactions important to the eye. There is also an 44 NEI Annual Report— FY 1993 Office of the Scientific Director application of clinically relevant research findings for the prevention, diagnosis, and treatment of diseases affecting the eye and visual system. The Branch has continued using different systems to develop objec- tive and subjective methods of monitoring and documenting opacities in the human lens. Among various objective systems that have been utilized are the Scheimpflug camera and the retroillumination camera. Other subjective systems utilized include the LOCS-2 grading system, as well as contrast sensitivity and glare testing. The OGCSB has continued its ongoing cataract research, carefiilly documenting cataracts in patients using a variety of techniques. Small pieces of tissue from cataracts extracted extracapsularly are used for various laboratory tests. Abnormal proteins have been identified by immunoblotting techniques as well as protein sequencing. It has been shown that in aging there is an acidic shift of proteins and that an increased number of polypeptide species exist within the molecular weight range of the crystallins. The Branch also has been a leader in the study of gyrate atrophy. Dietary intervention studies will continue in families with two affected siblings. A marked decrease in the retinal progression of this disorder now can be seen in the children who began the dietary intervention at an early age. This original work will lead to the exciting area of gene therapy. These studies will be performed in collaboration with the Laboratory of Immunology (LI). Laboratory of Retinal Cell and Molecular Biology (LRCMB) The research focus of the LRCMB has been to elucidate new genes and biochemical mecha- nisms that deal in particular with the retinal pigment epithelial (RPE) complex, in both health and disease. The long-term goal of this group is to establish basic information that will permit the Intramural Program to apply rational methods of gene therapy to various disorders. Several retina-specific genes identified by subtractive cloning have been located on the short arm of the X chromosome. In a similar fashion, LRCMB scientists have cloned RPE-specific genes that appear unique to the RPE. A new 65-kD protein of potential immuno- logic importance has been isolated from the human RPE and its gene has been cloned. This is the first RPE-specific gene to be reported and characterized. Work also has centered around a pigment epitheli- um-derived factor, which in very low concentrations causes the extension of elaborate neuronal processes from cultured retinoblastoma cells. It is hoped that this work may ultimately lead to understanding cone neuron development. LRCMB researchers continue to collaborate with LI investigators in studies of the immunopathology of experimental autoimmune uveitis. Laboratory of Immunology (LI) The LI is dedicated to the evaluation, diagnosis, and treatment of ocular inflammatory diseases. The research carried out by this group is both clinical and basic. In the clinical arena, the group continues to have a major conamitment to the study of the ocular complications of AIDS. The group has been interested in establishing noninvasive methods for diagnosing the presence of CMV retinitis in a non- ophthalmic setting. The use of the cell flare meter has helped in this regard. It shows an increase in the protein content in the anterior chamber that appears in AIDS patients who have CMV retinitis. This machine also is being used for a variety of thera- peutic studies. A randomized study to evaluate the usefulness of an intraocular slow-release device containing ganci- clovir that releases the drug for an 8-month period was in progress this past year. Patient recruitment continues but will end shortly. Newer medications such as rapamycin have been studied extensively in the experimental uveitis model. In addition, planning has begun for the use of monoclonal antibody therapy. The initial target is the interleukin 2 (IL-2) receptor. The Laboratory has had a long-term interest in toxoplasmosis. During the past year LI researchers have developed a reliable ocular model for toxoplas- mosis in which retinal as well as brain cysts of this origin can be demonstrated. This model has been used to evaluate the virulence of various strains, including those obtained from southern Brazil. RPE transplants have been studied in some detail by the LI. One accomplishment is the establishment 45 Office of the Scientific Director NEI Annual Report— FY 1993 of a reliable method for studying rejection phenome- na of RPE cells. The group also has pursued its long-term interest in experimental uveitis models. The evaluation of various systems has resulted in further elucidation of the toleragenic state induced with the feeding of uveitogenic antigens. In parallel with these studies is an ongoing randomized masked study that will more fully evaluate the usefulness of S-antigen feeding in patients with intraocular inflam- matory disease. During the past year the group also has continued working on the development of a gene therapy approach for gyrate atrophy. The work to date has shown the feasibility of this approach; various studies have been designed to develop optimum ways by which this gene can be transfected into cells. 46 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00065-16 OSD PROJECT NUMBER PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Physiological Studies of the Primate Visual System PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Francisco M. de Monasterio M.D., D.Sc. Medical Officer OSD, NEI COOPERATING UNITS (if any} LAB/BRANCH Office of the Scientific Director SECTION INSTITUTE AND LOCATION NEI, MH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.2 PROFESSIONAL: 0.2 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (b) Human tissues [x] (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project involves study of the physiological organization of neurons of the visual system of primates, with emphasis on the chromatic properties of color-opponent ganglion cells and of cells from the lateral geniculate nucleus and the primary visual cortex of macaques. 47 PHS 6040 (Rev. 5/92) Office of the Scientific Director NEI Annual Report— FY 1993 Project Description Objectives The purpose of this project is to study the neural organization underlying the processing of visual information at different levels of the primate visual system. Methods This research includes intracellular and extracellular recordings from single neurons, extracellular record- ings of mass responses, computer video stimulation, and tangent screen chromatic and spatial stimulation. Major Findings The studies have been resumed only recently, after underground construction work ended both along the wall separating the laboratory rooms from the street and in a nearby building across the street. The construction work made microelectrode recordings from single neurons almost impossible due to the nearly continuous shaking of the ground, which was transmitted to the recording system despite attempts at vibration isolation. In addition, street digging by heavy machinery resulted in damage to the computer hard disks. The studies involve an attempt of simultaneous recordings from both ganglion cell axons and genicu- late cells receiving input from such axons to examine spectral property transformations between these two levels of the visual pathway. Significance to Biomedical Research and the Program of the Institute Numerous behavioral, psychophysical, and electro- physiological studies show that the visual perfor- mance and characteristics of macaques and humans are extremely similar to one another. An understand- ing of nonhuman primate physiology provides a useful animal model for human visual function. Proposed Course These studies will be continued. NEI Research Program Strabismus, Amblyopia, and Visual Processing — Visual Processing and Amblyopia (Structure and Function) 48 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00122-13 OSD PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit or) or>e lir\e betweer) the borders.) Anatomical Studies of the Primate Visual System PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Francisco M. de Monasterio M.D., D.Sc. Medical Officer OSD, NEI COOPERATING UNITS (if any) LAB/BRANCH Office of the Scientific Director SECTION INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.8 PROFESSIONAL: I OTHER: 0.8 I 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects [x] (b) Human tissues □ (c) Neither □ (a1) Minors D (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project involves study of the anatomical properties and organization of cells in the visual system of primates, with emphasis on the retina and the visual cortex. The studies include (1) the anatomical association of outer-retinal cells selectively stained with tissue-reactive dyes and (2) the pattern distribution of cones in the retinas of human donors. Systematic study of the anatomical association at the light-microscope level of blue cones and horizontal and bipolar cells selectively stained by several tissue-reactive dyes in the macaque retina continues. The results have provided information on the probable retinal circuitry of the blue-sensitive cone pathway of primate retina. Retinal cone studies of eyes from human donors also continue. Evidence of a cone population with a point pattern resembling that of blue-sensitive cones selectively stained by tissue-reactive dyes has been obtained in quickly fixed, well-preserved retinas of some donors with a clinical history of diabetes. Cone density studies are being conducted on the retinas of macaques and human donors of various ages to resolve issues on the degree of primate photoreceptor losses with aging. 49 PHS 6040 (Rev. 5/92) Office of the Scientific Director NEI Annual Report— FY 1993 Project Description Objectives This project was designed to study the anatomical properties and neural organization of the primate visual system. Methods This project involves retinal histological processing, intravitreal injection of dyes, computer modeling and spatial statistical analyses of point and area patterns, silver staining of cells and myelin, histological processing of the cerebral cortex, deoxyglucose labeling, autoradiogr^hy, and cytochrome oxidase labeling. Major Findings 1. Human donor retinas fixed within 3 hours or less of time of death show a cone population with a point pattern distribution resembling that of the cones identified as blue-sensitive ones (i.e., absence in the central most region of the fovea, a peak density in the parafovea, and a regular though slightly disor- dered spacing in which stained cones are separated by two to three unstained cones) in donors with a reported longstanding clinical history of diabetes. This finding is consistent with clinical and psycho- physical observations of a dysfunction of blue- sensitive cones in diabetic retinopathy. Fixation of the donor retinas in as short a time as possible after death continues to be a major obstacle in the obtain- ing of well-preserved material from a sizable number of cases. 2. Thin serial sections of macaque retinas, stained with a tissue-reactive dye that selectively stains blue-sensitive and some post receptoral cells, are being examined systematically by light microsco- py to trace the anatomical relationship between selectively stained blue cones, HI horizontal cells, and blue-cone bipolar cells of the more peripheral macaque retina. Data obtained so far provide evi- dence that two or three blue cones are commonly contacted by blue-cone bipolar cells in peripheral retina, whereas a single blue cone is typically con- tacted by a single blue-cone bipolar cell. These results indicate that the blue-sensitive cone system behaves in a manner similar to that of the so-called midget cell system, with increasing retinal eccen- tricity. 3. A modification of the tissue-reactive staining technique used to label blue-sensitive cones of primate retina has provided initial results of a sub- labeling of the "unstained" cones into two apparentiy distinct populations. Because these populations may represent the green-sensitive and red-sensitive cones, several j^proaches are being tried to enhance such a labeling, which, if successful, would pennit a direct observation of the point pattern of all three primate cone types. Significance to Biomedical Research and the Program of the Institute Information on the anatomical properties of blue- sensitive cones is important not only to the function- al prop)erties of these cones investigated in different basic disciplines but also to the clinical research and diagnosis of acquired retinal disease. The data obtained from the eyes of diabetic human donors are particularly promising in this respect. Proposed Course These studies will be continued. NEI Research Program Retinal and Choroidal Diseases — ^Fundamental Processes and Retinal Disorders (Retinal Organiza- tion, Neurotransmission, and Adaptation) 50 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00135-21 OSD PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on or\e line between the borders.) Biochemistry of Retina and Pigmented Epithelium in Health and Disease PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Helen H. Hess M.D. Medical Officer (Research) OSD, NET COOPERATING UNITS (if any) LAB/BRANCH Office of the Scientific Director SECTION INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.0 PROFESSIONAL: 1.0 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (b) Human tissues |x] (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Effects of nutrition, oxidation, and other environmental factors Oight intensity or darkness) on incidence and progress of posterior subcapsular opacities (PSO) associated with genetically influenced retinal degeneration are being studied in pink-eyed Royal College of Surgeons (RCS) rats, in which rod photoreceptor outer segment debris accumulates secondary to a phagocytic defect in retinal pigmented epithelium (RPE). Preoxidation in polyunsaturated fatty acids in debris led to water-soluble toxic aldehydes, detectable in the vitreous and toxic to lens cells and membranes. Dystrophic rats fed a natural ingredient diet (NIH-07) were highly sensitive to retina light damage, beginning at an intensity of 1-4 footcandles (PC), and 27% of the rats developed mature cataracts by 5-12 months. Rhodopsin bleaching is essential for retina light damage and PSO. In vitro, free retinaldehyde has been shown to be a photosensitizer to generate singlet oxygen, an extremely damaging oxidant for both lipids and proteins, and this also may occur in vivo. A study of effects of environmental lighting on incidence of bilateral mature cataracts in pink-eyed RCS rats fed the natural ingredient diet (NIH-07) has been completed. Incidence of bilateral cataracts was 5% in rats reared in 1-4 PC of cyclic light but was 25% in rats reared in 10 PC constant light, 70% in 25 PC constant light, and 100% in 22- to 28-day-old rats given high-intensity light (700 PC) for 48 hours. In RCS rats reared at 1-4 PC, a purified diet (AIN-76A) fortified with antioxidants (0.4% p-carotene + 0.01% BHT) prevented PSO and mature cataracts. Currently a diet containing additional antioxidants (1,000 mg/kg diet of vitamin C and 150 mg/kg vitamin E) retarded retinal degeneration during the time the cataracts would have had their onset (23-53 postnatal days) if NIH-07 had been fed. A study using higher concentrations of vitamin E has been completed, but the histopathological evidence in the retina has not been evaluated. 51 PHS 6040 (Rev. 5/92) Office of the Scientific Director NEI Annual Report— FY 1993 Project Description Additional Personnel J. Samuel Zigler, Jr. Ph.D. Joseph J. Knapka Ph.D. Dennis Bernard M.S. Chief, LMOD, NEI Nutrition Consul- tant, Veterinary Research Program (VRP), National Center for Research Resources (NCRR) Nutritionist, VRP, NCRR Objectives This project is designed to study the biochemical and bionutritional relationships between lens, retinal photoreceptors, retina, retinal pigment epithelium (RPE), and biological fluids in health and disease. It also involves exploring the possibilities for slowing the rate of retinal degeneration and preventing lens opacities and mature cataracts, which are often associated with retinal degeneration in rats and humans. Diseases in which the RPE may be in- volved are of particular interest. Methods The Royal College of Surgeons (RCS) rat is being studied as an animal model of hereditary retinal degeneration that results from a defect in the RPE as well as a type of cataract that is secondary to retinal degeneration. Bionutrition is being used as a tool to combat lipid peroxidation in the RCS rat retina and to prevent water-soluble toxic aldehyde byproducts from reaching and damaging the lens. The RCS cataract is not genetic, because the mutant gene is expressed not in the lens but in the RPE; it is instead an outcome of environmental risk factors of both internal and external origin. Thus, the RCS rat is a living laboratory, and the cataracts are susceptible to orchestration by varying risk factors and preventive measures. Defined diets are prepared and fed to congenic affected and unaffected RCS rats in controlled experiments. The diets are fed to young breeding pairs prior to producing their first offspring and to their offspring after weaning, so that the experimen- tal animals will have received their diets from conception to date of observation. Clinical findings are recorded after indirect ophthalmoscopic and biomicroscopic slit-lamp examination. Postmortem examinations of the eye include dissecting microsco- py and light microscopy of stained specimens. At appropriate times, photography is used to record in vitro or in vivo data. Analytical methods include flameless atomic absorption, standard biochemical assays by spectrophotometry and fluorometry, and separation procedures. Special environmental light- ing conditions are employed to determine their histopathological effects on the retina and the lens. Major Findings 1. Previous work had yielded data on the inci- dence of bilaterality of mature cataracts in the pink- eyed, tan-hooded retinal dystrophic RCS rat under different intensities and duration of light, except for the standard conditions of cyclic light at 1-4 foot- candles (FC) inside the cages. The omission occurred because the early data were obtained during a collaborative agreement in which any rat that developed a single cataract was sent for examination, and such rats could not be returned to observe bilateral occurrence. When our animal room was changed in location and in type of lighting (i.e., incandescent instead of fluorescent), it seemed wise to determine (1) whether incidence of cataract was the same and (2) the incidence of bilaterality. The diet in all these experiments on bilaterality was the standard natural ingredient diet (NIH-07). This diet contains all nutrients required by rats but permits the cataracts to occur. The results showed the same incidence of cataract in incandescent as in fluorescent lighting (27%); 5% of the rats had bilateral cataracts by 1 year of age. In the previous studies, the incidence of bilaterality was greater in rats exposed to constant light: (a) 10 FC from 3 weeks to 1 year (25%), (b) 25 FC from 3 weeks to 1 year (70%), and 700 FC in 65-day-old rats in a short-term experiment of 2 days (100% of 15 rats). It seems possible that the 48-hour exposure at 700 FC could be reduced by using a program of short alternating exposures to light and darkness to produce the same percentage of cataracts and bilater- ality, since this regimen has been shown to produce light damage in the retina Thus, such experiments might be completed within the veterinary require- ment of holding a rat for only 24 hours after remov- ing it from the animal facility and also reduce the stress to the animal. 52 NEI Annual Report— FY 1993 OfTice of the Scientific Director At 700 FC exposure for 48 hours, bilateral mature cataracts also were produced in congenic control RCS rats (15 of 15 rats), but the lag time before appearance of the cataracts was greater than in dystrophic rats (13-15 months, as compared to 9-12 months). Furthermore, cataracts only occurred if the light exposure was preceded by a long period of dark adaptation (18 days). If short exposures to light and darkness could be substituted in these experiments, this possible model of age-related cataract could be pursued further. The requirement for long dark adaptation for production of mature cataracts in control RCS rats is consistent with our hypothesis that the cataracts are secondary to retinal light damage in both dystrophic and control rats. Long dark ad^tation of normal retina increases the content of rhodopsin by 50%, to parallel the situation in dystrophic rats, in which the retinal content of rhodopsin is 70-100% greater than normal because of accumulation of rod outer seg- ment debris from failure in phagocytic activity of the mutant RPE. In vitro, retinaldehyde has been shown to act as a photosensitizer to generate singlet oxygen, a highly energetic oxidant for polyunsaturated lipids, as well as proteins. In retinas with a high rhodopsin content, the retinaldehyde released by bleaching may exist in the free state long enough to act as a sensitizer to generate singlet oxygen. In accord with the hypothe- sis, lens opacities would be initiated by water-soluble toxic lipid peroxidation products from degenerating retina, carried through the vitreous to attack mem- branes of lens cells and fibers. The prevention of cataracts by antioxidants would begin in the retina, with quenching of singlet oxygen by vitamin E and beta-carotene, and continue as these and other antioxidants combat secondary products of peroxida- tion. 2. Antioxidant diets that prevent cataracts in pink-eyed RCS dystrophic rats have the effect of retarding retinal degeneration. None of the diets we have tried stops the degeneration, but when a certain degree of retardation is achieved, the lens is pro- tected. Last year we studied the AIN-76A purified diet, which contains twice the normal concentration of all the minerals in the AIN mineral mix plus 0.4% beta-carotene and 0.01% BHT, as well as 1,000 mg/kg vitamin C and 150 mg/kg vitamin E. After the rats consumed this diet, histopathological exami- nation showed retarded retinal degeneration during the time the cataracts would have had their onset (23-53 postnatal days) if the NIH-07 diet had been fed. This year we fed that same diet containing increased concentrations of vitamin E to explore whether retinal degeneration can be delayed further. The eyes from rats of different ages have been fixed for histopathological study, but the results are not yet available. The dystrophic retina is extremely sensi- tive to light damage and to peroxidation of its lipids. This sensitivity, a fundamental part of the pathophys- iology in the RCS rat, makes it more vulnerable to the defect of the RPE; furthermore, it may provide a clue to the underlying disease. Significance to Biomedical Research and the Program of the Institute The program goal of preventing posterior subcapsular cataracts (PSC) in RCS rats has been achieved, and the goal of slowing the rate of retinal degeneration has been advanced. When the retinal degeneration is slowed through 55 postnatal days, the cataracts are prevented. These results were obtained using bio- nutrition with a purified diet supplemented with antioxidants. PSC occurs in many varieties of human hereditary retinal degeneration known as retinitis pigmentosa (RP), as well as in so-called age- related cataracts and in some persons treated with steroids or exposed to short-wave radiation. None of the well-known types of human RP appears to show the problem of RPE phagocytosis found in the RCS rat; however, the number of types of RP appears to be very great, and many have not been explored for this phenomenon. Cataract is a predominant cause of vision loss and blindness in the United States and in the worid. Effective prevention in humans has not been devel- oped; the only treatment is removal of the opaque lens. In the United States, the surgical treatment is safe, and intraocular lens replacement is highly successful. However, the annual cost of cataract surgery totals $2 billion and will continue to rise as tlie population ages. Cataract surgery, therefore, has been targeted for reduction as an important cost- saving plan, and prevention or slowing of cataract development has become imperative. Among the known risk factors for cataractogene- sis are ocular characteristics of the RPE and iris color, as well as exposure to sunlight and ultraviolet (UV) radiation. In humans, these and other risk factors are difficult to control and quantitate. In 53 Office of the Scientific Director NEI Annual Report— FY 1993 RCS rats, however, the existence of both pink-eyed and black-eyed dystrophic and congenic control strains provides for control of ocular characteristics, and artificial illumination in the animal room pro- vides the equivalent of sunlight and known UV radiation. In the RCS rat, the cataracts are secondary to the retinal degeneration, in which peroxidation of polyunsaturated fatty acids leads to water-soluble toxic aldehydes that are carried through the vitreous to the back of the lens, where they can damage the cell membranes of lens cells and fibers. Principles involved in this example of cataractogenesis may have relevance to some of the types of human cataract, including factors in slowing or preventing cataracts and retinal degeneration. An initiative in the National Institutes of Health (NIH) Strategic Plan is the prevention or delay of cataract through nutrient-specific dietary intervention. Vitamins E and C and beta-carotene, as well as the antioxidant-associated trace minerals zinc and seleni- um, are to be tried in populations where diets are considered to be inadequate. In an animal model, there is total control over the content and intake of nutrients, whereas this is difficult or impossible in a human setting. Our diets are fed to the parents prior to conception, to the female during pregnancy and in the lactation period, and to the experimental offspring during its entire life. In RCS rats, we employed a purified ingredient diet supplemented with twice the usual concentra- tions of normal minerals (including zinc and seleni- um), vitamins E and C, and beta-carotene. With this diet, the cataracts were totally prevented, and retinal degeneration was delayed through the time when the cataract would have had an onset if a standard natural ingredient rat diet had been fed. With the natural ingredient diet, cataract incidence was 27% in this rat strain under standard light conditions (cyclic light giving 1-4 FC intensity inside the cage). The percentage of cataracts seen with this diet increased with the light intensity, whereas rearing in darkness prevented cataract. Proposed Course Histopathological effects of feeding a high vitamin E antioxidant diet to the pink-eyed dystrophic rat will be evaluated to determine whether the retinal degen- eration has been slowed fiirther, beyond 55 postnatal days. Histopathological material firom all previous rats with mature cataracts will be examined to compare the populations of lens epithelial cells. As pink-eyed, tan-hooded dystrophic breeders become available fi-om the NIH foundation colony, lens epithelial cell whole mounts will be prepared to compare the numbers of cells in lenses with and without cataract. Until now, studies of the mutant autosomal recessive rdy gene on Chromosome 3 of the RCS rat have not been feasible because the rat genome had not been well smdied. However, knowledge of the rat genome has been proceeding apace, and a collab- orative study to locate this gene, which appears to be involved in RPE phagocytosis, may soon be possible. NEI Research Program Cataract — Pathogenetic Mechanisms Retina — ^Retinitis Pigmentosa and Other Inherited Disorders 54 Laboratory of Immunology Report of the Chief, Laboratory of Immunology Robert B. Nussenblatt, M.D. The Laboratory of Immunology has finished its seventh year. Sections of the Laboratory include Immunology and Virology, headed by Dr. John J. Hooks; Experimental Immunology, headed by Dr. Igal Gery, who is also the Deputy Chief of the Laboratory; Immunoregulation, whose acting head is Dr. Rachel Caspi; Experimental Immunopathology, whose head is Dr. Chi-Chao Chan, and Clinical Immunology, whose acting head is Dr. Marc de SmeL For the recently formed Section on Molecular Biology, I serve as the acting head of an interdisci- plinary group. Section on Clinical Immunology The Section on Clinical Immunology has contin- ued to focus on major questions of clinical relevance. Studies centering on the evaluation of the use of the Molteno glaucoma implant and 5-fluoro- uracil combined with trabeculectomy continue. Final results still have not been obtained in this random- ized long-term endeavor. On the basis of our obser- vations, we are rather optimistic about the outcome for the long-term efficacy of the Molteno implant, although scientific evaluation of the results is still needed. The Section continued its study of patients with AIDS (acquired immune deficiency syndrome), in collaboration with the National Institute of Allergy and Infectious Diseases. Evaluation of the safety of adnunistering anticytomegalovirus (anti-CMV) hyperimmunoglobulin to patients at risk for CMV retinitis was concluded. A randomized study to evaluate a slow-release implant filled with gancyclo- vir is being actively tested in AIDS patients with CMV retinitis. These implants, placed directiy into the eye through the porous plana, are calculated to release small but therapeutically effective amounts of gancyclovir over an 8-month period. This random- ized study may yield information about an important alternative to systemic anti-CMV therapy for patients who cannot tolerate the intravenous infusions or who possibly do not have a specific indication for system- ic therapy. Recruitment has been brisk, and we hope that results will be obtained within the next calendar year. Also continuing are pediatric AIDS studies in which children are evaluated for the incidence of ocular infection. This study is done in conjunction with Dr. Philip Pizzo and his National Cancer Institute laboratory. The Section also has been particularly interested in the development of immunosuppression. A randomized masked study to look at the effectiveness of oral tolerization has been in progress this past year. The research group is attempting to evaluate the usefulness of S-antigen (S-Ag) given per os in the induction of tolerance in uveitis patients. Initial observations in a pilot study have demonstrated a positive therapeutic response to feeding. The aim is to complete this randomized study within the next year and to have final results shortly thereafter. Section on Molecular Biology To acquire a better understanding of the approaches to gene therapy, this group has focused on regulation of the omithine-6-aminotrans- ferase (OAT) gene in vivo as well as on genetic modification of somatic cell Unes mediated by recombinant retroviruses. Dr. Moncef Jendoubi placed human OAT cDNA under the control of the enhancer-promoter regulatory elements derived fi'om the Moloney murine leukemia virus long-terminal repeat, then transfected this construct into a safe packaging cell line (GP-i-E-86) to produce provirus particles. Supernatant from the ecotropic OAT producers of cell lines were useid to transduce mouse embryonal fibroblasts as well as stem cells. The recombinant retrovirus transferred the OAT gene to the recipient cells, which produced an immunore- active OAT. Northern blot analysis confirmed the presence of an OAT transcript in the transduced cell fines, even after a long time in vitro. 57 Laboratory of Immunology NEI Annual Report— FY 1993 The Human Genome Group was established, and several individuals from various parts of the Labora- tory have participated. The major goal is the devel- opment of a particular form of gene ther^y in the treatment of gyrate atrophy. This gene therapy would entail use of the OAT gene. The work has shown that this gene can be transfected and has potential for use in such therapy. We hope that in the not too distant future this therapy will become a reality at the National Eye Institute. Section on Immunoregulation This Section has maintained interest in the devel- opment and study of animal models of experi- mental ocular autoimmune disease. The work has centered particularly on the characterization of the murine experimental autoimmune uveitis (EAU) model because the mouse offers some very important advantages over other rodent models of uveitis. In addition, the group has been actively involved in the establishment of antigen-specific T-cell lines and clones, which permit investigators to identify and characterize cells capable of inducing ocular immu- nomodulation. This year the group has shown that the severity of inflammation and tissue damage in athymic rats is correlated with the proportion of lymphocytes in the intraocular infiltrate while the infiltrate in euthymic rats was predominantly lymphocytic with fewer monocytes and even fewer neutrophils. The sparse infiltrate in athymic rats was largely monocytic and had a relatively high proportion of neutrophils and eosinophils. It is interesting that reconstituted animals had an intermediate histological picture. These results indicate that recruited nonspecific T cells play a very major role in the pathogenesis of disease. Collaboration with Dr. Charles Egwuagu contin- ues in studies of the T-cell receptor (TCR) genes of cell lines and clones at the molecular level. Data collected to date indicate that TCR (variable region gene) usage in uveitis differs from that reported for other autoimmune diseases; it also may be more heterogeneous. It is interesting that recent experi- ments confirm our group's initial observations that TCR Vp8.2 may be a pathogenic clonotype in S-Ag- induced uveitis, whereas TCR VP8.3 may be one of the pathogenic clonotypes in uveitis induced by interphotoreceptor retinoid-binding protein (IRBP). These findings are exceptionally important because of their impact on the development of therapeutic strategies that would specifically target pathogenic cells via the T-cell receptor. Using a BIO. A strain of mice, the group has developed a pathogenic T-cell line specific to whole IRBP. Such cell lines are interesting for multiple reasons, including the fact that the TCR profile of the line changes with time in culture. There appears to be progressive enrichment in the vp8.2 and Vp8.6 TCR-expressing cells. In the mouse this would suggest that VP8.2 and vp8.6 represent pathogenic clonotypes in IRBP-induced uveitis. Section on Experimental Immunology Continuing its long-term investigation of the pathogenesis of inflammatory eye diseases, this Section has found that the induction of EAU by bovine IRBP in the Lewis rat involves a unique immunologic relationship. Lymphocytes sensitized against the immunodominant and uveitogenic deter- minant of IRBP do not recognize the rat homolog of this determinant; rather, they are stimulated by other peptides of the rat IRBP. In this description of the phenomenon, a surrogate peptide determinant of an organ-specific antigen is used to initiate an autoim- mune pathogenic response. In addition, the group has been actively involved in the S-Ag feeding oral tolerance studies. They have overcome the inaccessibility of large amounts of human S-Ag by using recombinant DNA technol- ogy to produce human S-Ag in Escherichia coli bacteria. During the past year the group has evaluated the novel immunomodulator linomide, which was found effective in inhibiting EAU development in rats and mice actively immunized with S-Ag or IRBP. On the other hand, treatment with linomide had no effect on the development of EAU adoptively transferred by presensitized lymphocytes. These findings suggest that linomide would probably not be useful for the treatment of uveitis in humans. 58 NEI Annual Report— FY 1993 Laboratory of Immunology Section on Experimental Immunopathology group also has demonstrated that cell adhesion molecules are important for both antigen sensitization and inflammatory cell infiltration into the eye. This new Section had an extremely productive year. Immunohistochemistry and in situ hybrid- ization have been used to identify and topographical- ly localize immunocompetent cells and analyze the alteration of surface markers on ocular resident cells and their cytokines in experimental uveitis models as well as in ocular tissues. The T-cell dominance of the response remains one of the major factors that is seen again and again. Moreover, the migration of inflammatory cells from the vessels into the target appears to be directed by adhesion molecules that can be expressed on the vascular endothelium as well as other resident cells in the eye. The group also has evaluated specimens firom human ocular tissues with various diseases, including uveitis, retinal disease(s), conjunctival and corneal diseases, metabolic genetic diseases, and tumors. The group's demonstration of the presence of a major lens protein, oB-crystallin, in retinoblastoma suggests that oB-crystallin is involved in tumor growth and/or is a marker for general oncogenic stress in retinoblastoma. Cell adhesion studies remain an area of great interest. The studies performed this year demonstrate that monoclonal antibodies against intercellular adhesion molecule 1 (ICAM-1) and its counterrecep- tor, lymphocyte function-associated antigen 1 (LFA- 1), inhibit the development of EAU in mice. The Section on Immunology and Virology This Section has continued to emphasize its interest in the study of the retinal pigmented epithelial (RPE) cell. The use of monoclonal anti- bodies has helped in identifying a 67-kD protein that appears to be an RPE-specific epitope. Its sequence homology is similar to that of the intermediate filament of protein. The group also has demon- strated that inflanmiatory mediators such as lipopoly- saccharide, tumor neaosis factor (TNF-a), and interleukin 1 (IL-1) induce interleukin 6 (IL-6) gene expression and secretion by human RPE cells. This effect also is noted to be synergistic with interferon gamma (IFN-y). RPE cell transplantation continues to be a major area of interest for this group as well. During the past year the group developed a reproducible model for ocular toxoplasmosis. The induction of this model has provided the Laboratory with an opportunity to evaluate strain virulence. A strain of toxoplasmosis virulent in humans and isolated from Brazil was shown to be particularly virulent in a mouse model. Studies have begun to evaluate the mechanisms involved in toxoplasmosis cyst formation as well as attempts to counteract the infection. 59 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00279-02 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) Study of Immunosuppressants for the Treatment of Uveitis in Animal Models PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) LI, NEI LI, NEI PI: Francois G. Roberge M.D. Others: Chi-Chao Chan M.D. Marc D. de Smet M.D. Robert B. Nussenblatt M.D. Dan Martin M.D. Margaret Cheung M.D., Ph.D David Parks M.D. Visiting Scientist Head, Section on Immunopathology Visiting Scientist Scientific Director Senior Staff Fellow Senior Staff Fellow Senior Staff Fellow LLNEI NEI LI, NEI LI, NEI LLNEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Clinical Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.7 PROFESSIONAL: 0.7 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The goals of the project are to acquire a better understanding of the immunopathogenic mechanism of noninfectious intraocular inflanmiatory diseases (uveitis) and to develop treatment and prevent the complications associated with these diseases. This past year we studied two specific aspects of the application of the new noncytotoxic inmiunosuppressant rapamycin (RAPA) in the treatment of uveoretinitis: (1) evaluation of the synergistic effect of RAPA with cyclosporine A (CsA) or dexamethasone (Dex) and (2) evaluation of the effect of RAPA on complications of uveitis, fibrosis, and intraocular membrane formation. We also evaluated the role played by nitric oxide (NO) in anterior uveitis. We demonstrated that combining RAPA with CsA or Dex can inhibit experimental uveitis in vivo at greatly reduced doses of each drug, compared with the doses necessary when these three drugs are used singly. We showed that RAPA also inhibits proliferation of human fibroblast and retinal pigment epithelial (RPE) cells, indicating the advantage of using RAPA in the treatment of uveitis, in which fibrosis and membrane formation are common complications. We also demonstrated that NO is an important mediator of endotoxin-induced anterior uveitis. 60 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Additional Personnel Bruce Pfeffer Ph.D. Senior Staff Fellow, LRCMB, NEI Clinical Protocol Numbers 91-191 91-187 Objectives The immunosuppressive treatment of autoimmune disease such as uveoretinitis usually has to be sus- tained for a long period of time. To avoid treatment complications as much as possible, we try to develop treatment using noncytotoxic drugs. In particular, we seek to develop combination ther^y that minimizes the toxicity of each drug while maximizing the beneficial effects. Previously we had demonstrated by in vitro studies that the combinations of rapamy- cin (RAPA) with cyclosporine A (CsA) and RAPA with dexamethasone (Dex) had a synergistic effect on the inhibition of the proliferation of retinal antigen- primed lymphocytes. This year we tested these results in the in vivo rat model of experimental autoinunune uveoretinitis (EAU). We evaluated the effect that RAPA could have on the fibrosis and membrane formation in the eye following inflamma- tion. We also have studied the role of nitric oxide (NO) in a model of anterior uveitis induced with endotoxin (lipopolysaccharide). NO is the oxidation product of one of the guanidino nitrogens of L- arginine. The reaction is catalyzed by two different forms of the enzyme nitric oxide synthase, which have specific tissue distributions. NO is produced after stimulation with endotoxin. The synthesis of NO can be competidvely blocked with L-arginine analogues such as N°-nitro-L-arginine methyl ester (L-NAME). The inhibition is enantiomer restricted, the D form of analogues being inactive; it is also fully reversible with an increased concentration of L- arginine. We used the specificity of L-NAME inhibitory action to evaluate indirectly the role of NO in endotoxin-induced uveitis (EIU). nated on Day 28 after immunization. RAPA was delivered by continuous intravenous (i.v.) infusion by means of a miniosmotic pump implanted in the abdominal cavity. CsA or Dex was given by intra- muscular (im) injection once a day. The disease was evaluated by histopathology. Two human RPE lines, between passages 4 and 8, and human skin fibroblasts were used. Proliferation assays were done in quadruplicate in Dulbecco's modified Eagle's medium/F12 medium, in microtiter plates at 6 X 10^ cells per well. Proliferation was measured by the incorporation of ^H-thymidine, added after 24 hours for 18 hours of incubation. Stimulants were 5% fetal bovine serum; IGF-1 (50 ng/ml); and aPGF, bFGF, EGF, and PDGF (10 ng/ml each). RAPA, CsA, FK 506, or solvent alone was added at the initiation of culture in concentiations ranging from 10"* to 10"'* M. Toxicity of the drugs was assessed by a quantitative tetrazolium-based colorimetric assay. Uveitis was induced in rats with subcutaneous LPS. The animals were treated with L-NAME, an L- arginine analog acting as a specific inhibitor of NO synthesis. Ocular inflammation was evaluated by measuring the protein concentration and leukocyte number in the aqueous humor of one eye and by histopathology of the contralateral eye. Major Findings We found that we could effectively inhibit EAU by using a combination of RAPA with CsA in which the doses were reduced by factors of 10 and 5, respectively, compared with the doses necessary when the drugs were used alone. Similarly, in the RAPA with Dex combination, the doses were re- duced by fourfold and fivefold, respectively. We found that RAPA could significantiy inhibit the proliferation of human retinal pigment epithelial cells as well as human fibroblasts, thus RAPA could be indicated for patients in whom uveitis is compli- cated by fibrosis and retinal membrane formation. We demonstrated for the first time that NO is a crucial mediator of EIU and that its inhibition may, in the future, become an avenue of therapy for anterior uveitis. Methods EAU was induced by immunization with S-antigen in Hunter's adjuvant Treatment was given for 14 days, starting on Day 7, and the experiment termi- Significance to Biomedical Research and the Program of the Institute Our study on combination therapy for EAU has shown that the observed in vitro synergy between 61 Laboratory of Immunology NEI Annual Report— FY 1993 RAPA and CsA and between RAPA and Dex was effective in vivo. Such combination therapy, if applied to humans, could significantly reduce the toxic side effect of the treatment while allowing good control of the disease. In addition, RAPA could benefit patients for whom uveitis is complicated by intraocular fibrosis and membrane formation, two complications that are often responsible for a large part of vision loss. The newly discovered role for NO in anterior uveitis could lead to improved therapy by finding inhibitors of the production of NO in the eye. Proposed Course In the fiiture, we intend to develop a clinical trial for the treatment of uveitis with RAPA in humans. A protocol has been developed and will be submitted to the producer of the drug. The study concerning NO and uveitis will be developed in the direction of identifying the cells responsible for the effect found in the eye. In addition, different types of NO inhibitors will be tested to find the one(s) most appropriate for uveitis therapy. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Li Q, Lopez JS, Caspi RR, Roberge FG, Nussenblatt RB, Kador PF, Chan C-C: Suppression of S- antigen induced experimental autoimmune uveo- retinitis in Lewis rats by oral administration with COS- 13080, a thromboxane synthetase inhibitor. Exp Eye Res 57:601-608, 1993. Roberge FG, Kozhich A, Chan C-C, Martin DF, Nussenblatt RB, de Smet, MD: Inhibition of cellular transfer of experimental autoimmune uveoretinitis by rapamycin. Ocular Immunol Inflam 1:269-73, 1993. Roberge FG, Xu D, Chan C-C, de Smet MD, Nuss- enblatt RB, Chen H: Treatment of autoimmune uveoretinitis in the rat with rapamycin, an inhibi- tor of lymphocyte growth factor signal transduc- tion. Curr Eye Res 12:197-203, 1993. 62 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00262-04 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Analysis of T Lymphocytes and Cytokines Involved in Experimental Autoimmune Uveoretinitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Charles E. Egwuagu Ph.D., M.P.H. Senior Research Scientist LI, NEl Others: Igal Gery Robert B. Nussenblatt Rachel Caspi Rashid Mahdi Ph.D. M.D. Ph.D. Head, Section on LI, NEI Experimental Immunology Scientific Director NEI Visiting Associate LI, NEI Biologist LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Section on Experimental Immunology INSTITUTE AND LOCATION NEI, NTH, Bethesda, MP 20892 TOTAL STAFF YEARS: TpROFESSIONAL: 0.7 0.7 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects n (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Experimental autoimmune uveoretinitis (EAU) is a T-cell-mediated autoimmune disease that serves as a model of human intraocular inflammatory diseases (uveitis). It is initiated in susceptible animals by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein (IRBP) or S-antigen (S-Ag). We had shown previously that vpS-expressing T cells accumulate in the retina during EAU. Because the vpS-expressing T cells comprise cells with distinct functional activities, we sought to identify the uveitogenic subpopulation by analyzing the T-cell antigen receptor (TCR) genes and the cytokines they secrete in the retina during EAU. Our results indicate that the Vp8 response is highly dependent on the antigen used for disease induction: Whereas only Vp8.2* T cells were found in the retinas of animals with S-Ag EAU, both vp8.2* and Vp8.3* T cells were present in the retinas of rats with IRBP EAU. In terms of the pattern of cytokine production, both Thl- and Th2-like lymphokine profiles were observed, and the cytokine detected in the retina was found to be dependent on the antigen used for disease induction. Our current data suggest that the T-cell Vp8 response in the retina during EAU is heterogeneous. Thus, despite the fact that there is a bias toward • use of Vp8* cells in the retina during EAU, a single anti-Vpg antibody cocktail may not confer full protection from uveitis because several autoantigens and an unknown number of immunopathogenic T-cell clonotypes may be involved in the induction of EAU. 63 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report— FY 1993 Project Description Objectives This project is aimed at determining the clonality of the T lymphocytes that mediate ocular autoimmune diseases. Identification of the pathogenic T-cell subset in experimental autoimmune uveoretinitis (EAU) is relevant to our goal of developing anti-T- cell receptor (TCR) ther^ies for the treatment of uveitis. Our effort during Fiscal Year 1993 focused on analyses of T cells present at the autoinunune site and the lymphokines they produce. Methods In situ, reverse-transcribed polymerase chain reaction (RT/PCR) was used to examine T-cell populations in the retinas of athymic and euthymic Lewis rats at different stages of EAU. We used the cDNAs generated to amplify transcribed genes that code for rat VP TCR chains, T-cell subset markers (CD4 and CDS), and cytokines associated with autoimmune pathology — ^interferon-gamma (IFN-y), tumor necro- sis factor (TNF-a), transforming grovith factor (TGF- P), interleukin 2 (IL-2), and interleukin 6 (IL-6). Conventional recombinant DNA techniques such as Southern blot hybridization, PCR, cDNA construc- tion, densitometry, and DNA sequencing were used to analyze the resultant PCR products. Major Findings Analysis of TCR Vp5 repertoire induced by S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). — We analyzed TCR genes expressed in response to immunopathogenic epitopes of human S-Ag or bovine IRBP by the PCR assay. We found that expression of VP8 genes in the retinas of S-Ag- immunized rats was restricted to vp8.2 TCR, while both Vp8.2 and Vp8.3 TCR isoforms were detected in IRBP-immunized rats. vps.T T cells were not detectable in the retinas of rats with either IRBP- or S-Ag-induced EAU. Analysis of Vp5 repertoire in adoptively trans- ferred EAU. — In contrast to EAU induced by active immunization, all three vps subfamily members were detected in the retina in EAU induced by adoptive transfer of IRBP- or S-Ag-specific uveito- genic T cells. Skewing of the vps repertoire during EAU induced by active immunization points out a potential confounding factor in vaccination studies in which complete Freund's adjuvant is a component of the vaccine preparations. The earliest Vp8* T cells detected in the retina were of the VP8.2 clonotype. This subpopulation was followed by vps.B- and Vp8.1 -expressing clonotypes. Similar to the re- sponse to active immunization, the initial appearance of VP8.3* cells was subsequently followed by a rapid decline in the numbers of VP8.3* lymphocytes. Analysis of cytokine production in the retina during EAU. — In adoptively transferred EAU, the cytokines in which production in the retina could be correlated with EAU pathogenesis are TNF-a, EFN-y, and IL-6. However, the temporal appearance of these cytokines in the retina was dependent on the antigen used for eliciting the uveitogenic T cells. Significance to Biomedical Research and the Program of the Institute Our current data show selective accumulation of Vp8* cells in the retina during EAU. However, the T-cell response is not oligoclonal, and distinct Vp8 subfamilies were differentially activated by the autoantigens S-Ag and IRBP. Furthermore, the patterns of lymphokine production indicate that distinct T-cell subsets may initiate IRBP- and S-Ag- induced EAU. Taken together, these findings sug- gest that a single anti-Vp antibody cocktail may not confer full protection from uveitis because several autoantigens and an unknown number of immuno- pathogenic T-cell clonotypes may be involved in the pathogenesis of EAU. Proposed Course Fumre analyses of uveitogenic T-cell clonotypes and the lymphokines they produce during EAU will be performed in nude rats. Because these rats do not normally produce T cells, we expect the intraretinal T-cell repertoire in these rats to be limited. This would allow for a more concise analysis of the identity of the relevant autoaggressive T cells in- volved in uveitis. NEI Research Program Retinal and Choroidal Diseases — ^Inflammatory Disorders Publications Egwuagu CE, Bahmanyar S, Mahdi R, Nussenblatt RB, Gery I, Caspi R: Predominant usage of 64 NEI Annual Report— FY 1993 Laboratory of Immunology Vp8.3 T cell receptor in a T cell line that induces uveoretinitis induced by two (Afferent retinal anti- experimental autoimmune uveoretinitis. J Clin gens. J Immunol 151:1627-1636, 1993. Immunol Immmopathol 65:152-160, 1992. Mahdi RM, Caspi RR, Nussenblatt RB, Gery I, Egwuagu CE, Caspi R, Mahdi R, Gery I, Nussenblatt Egwuagu CE: Selective accumulation of vps* T RB: Evidence for selective accumulation of lymphocytes in EAU. Invest Ophthalmol Vis Sci Vp8+ T lymphocytes in experimental autoimmune 34(4)(suppl): 1 144, 1993. 65 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00280-02 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Eaopic Expression of Inlerferon-Gamma in the Eyes of Transgenic Mice and Rats Induces Ocular Pathology and MHC Qass 11 Gene Expression PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atiiliation) PI: Charles E. Egwuagu Ph.D., M.P.H. Senior Scientist LI, NEI Others: Robert B. Nussenblan Chi-Chao Chan Ana B. Cbepelinsky Jorge Sztein Rashid Mahdi M.D. M.D. Ph.D. D.V.M., Ph.D. Scientific Director Head, Section on Immunopathology Head, Section on Regulation of Gene Expression Visiting Associate Biologist NEI LI, NEI LMDB, NEI LI, NEI LI, NEI COOPERATING UNITS (it any) LAB/BRANCH Laboratory of Immunology SECTION Section on Experimental Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.2 PROFESSIONAL: 1.2 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neitiier SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) To investigate the consequences of ectopic expression of interferon gamma (IFN-y) in the lens and how this lymphokine growth factor may influence the fate of cells committed to a particular differentiation state, we used the murine (xA-crystallin promoter (oACry) to direct the expression of IFN-y to the lens of transgenic mice. Two transgenic mouse lines were established using two distinct strains, Balb/c and P/B/N. In both the aACry-IFN-y/Balb/c and cxACry-IFN-y/FYB/N transgenic mice, the essential histopathological features observed were very similar at all developmental stages studied (i.e., 12- to 18-day embryos, newborns, adults), suggesting that maldevelopment of ocular tissues of these mice resulted from oAGry-IFN-Y expression. Recently we have generated transgenic rats using the oACry-IFN-y cDNA construct, making our transgenic rats the first transgenic rats to be generated at the National Institutes of Health. Constitutive expression of IFN-y in the lens induced ocular pathology and aberrant major histocompatibility complex (MHC) class-II protein synthesis in several ocular tissues. These transgenic mice and rats provide powerful models for understanding the molecular pathways governing synchronized programmed differentiation of ocular tissues and for studying the linkage between aberrant MHC expression and predisposition to autoimmune diseases. 66 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Objectives This project is designed to generate transgenic animals (rats and mice) that selectively secrete interferon-gamma (IFN-y) in their eye tissues for use in studying the bioregulatory actions of IFN-y in the eye. Because aberrant expression of major histocom- patibility complex (MHC) molecules is an early event in a number of human autoimmune diseases and IFN-y induces high levels of MHC class II protein biosynthesis, these transgenic mice and rats are ideally suited for studying (1) the effects of IFN- y on the physiology of the eye and (2) the role of elevated MHC class D in predisposition to auto- immune diseases such as diabetes and uveitis. Methods Transgenic animals were generated according to standard transgenic mouse techniques; however, for the transgenic rats, adjustments in the hormone treatment regimen were necessary to obtain embryos used for microinjection of the construct. Transgenic and wild-type (WT) phenotypes were characterized by histologic examination of representative tissue sections. We used conventional recombinant DNA techniques — such as Southern blot hybridization, polymerase chain reaction, cDNA construction, densitometry, and DNA sequencing — to characterize DNAs and RNAs derived from WT and transgenic mice. Major Findings The most notable effects of IFN-y in the transgenic mice include cataract, microphthalmia, blepharophim- osis, microphakia, impairment of lens fiber forma- tion, arrest of retinal differentiation, serous retinal detachment with the presence of macrophages in the subretinal space, persistent hyperplastic primary vitreous, and corneal vascularizatioa MHC class II mRNA levels were significantly increased in the transgenic eyes, and MHC class II proteins were expressed in the cornea, iris, ciliary body, choroid, lens, and retinal pigment epithelium. Significance to Biomedical Research and the Program of the Institute The generation of transgenic rats is a major technical accomplishment that now allows us to generate rats containing other DNA constructs. This is particularly important considering that several ocular diseases, such as uveitis and cataract, are better suited for study in the rat than in the mouse. Our data suggest that oACry-IFN-y transgenic mouse ocular cells express functional IFN-y receptors and that inter- action of IFN-y with its receptor induced biochemical and morphological changes in the transgenic eyes. These transgenic mice and rats provide a powerful model for understanding the molecular pathways that govern synchronized, programmed differentiation of ocular tissues and for studying the linkage between aberrant MHC expression and predisposition to autoimmune diseases. Proposed Course We intend to further dissect the molecular basis of IFN-y actions in the eye, placing particular emphasis on the rat model. A major focus will be the study of transcriptional factors that mediate oACry-IFN-y effects. NEI Research Program Retinal and Choroidal Diseases — ^Inflammatory Disorders Publications Egwuagu CE, Sztein J, Reid W, Chan C-C, Mahdi R, Nussenblatt RB, Chepelinsky AB: Ectopic expression of gamma interferon in the eyes of transgenic mice induces ocular pathology and MHC class n gene expression. Invest Ophthal- mol Vis Sci, in press. Egwuagu CE, Sztein J, Reid W, Chan C-C, Mahdi R, Nussenblatt RB, Chepelinsky AB: Ganmia interferon gene expression in the lens of trans- genic mice directed by the ctA-crystallin gene promoter. Invest Ophthalmol Vis Sci 34(4) (suppl):846, 1993. 67 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00069-16 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Immune Responses to Ocular Antigens PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Igal Gery Others: Alexander Kozhich Xiaowen Wu Nancy Miller-Rivero Barbara Vistica Norman Chanaud UI Robert B. Nussenblatt Ph.D. Head, Section on Experimental Inmiunology LI, NEI Ph.D. Visiting Fellow LLNEI M.D. Visiting Fellow LLNEI M.D. IRTA Fellow LLNEI B.A. Microbiologist LLNEI B.A. Special Volunteer LI, NEI M.D. Scientific Director NEI COOPERATING UNITS (it any) Biotechnology Unit, Laboratory of Cellular and Developmental Biology, National Institute of Diabetes and Digestive and Kidney Diseases (Joseph Shiloacfa, Ph.D.); Center for Neurologic Diseases, Brigbaro and Women's Hospital, Boston, MA (David Hafler, M.D.); Department of Medicine, Hadassah Hospital, Jerusalem, Israel (Shimon Slavin. M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Experimental Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 4.2 PROFESSIONAL: 3.8 OTHER: 0.4 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Targeted at learning about the pathogenesis of inflammatory eye diseases grouped under the term "uveitis," this project continued to focus mainly on learning about ocular antigens that induce experimental autoimmune uveoretinitis (EAU), an animal model for uveitis in man, and on procedures that modulate this disease. Three major achievements of this study are as follows: (1) We found tiiat the induction of EAU by bovine interphotoreceptor retinoid-binding protein (IRBP) in the Lewis rat involves a unique immunologic reaction in which lymphocytes sensitized against the immunodominant and uveitogenic determinant of this protein do not recognize the rat homolog of this determinant, but, rather, are stimulated by another peptide of the rat IRBP. This is the first description of a phenomenon in which a "siuTogate" peptide determinant of an organ- specific antigen is used to initiate an autoimmime pathogenic process. (2) To overcome the inaccessibility of large amounts of human S-antigen (S-Ag), we have used recombinant DNA technologies to produce this human antigen in Escherichia coli bacteria. The recombinant human S-Ag was found to aoss-react with native bovine S-Ag and to be similarly capable of inducing EAU in Lewis rats. In addition, the recombinant human S-Ag was used to identify the immunodominant peptide determinants of this antigen, i.e., the peptides recognized by lymphocytes sensitized against whole human S-Ag. Three regions of the molecule were found to harbor immunodominant determinants, with different peptides being selected as dominant in rats of different inbred strains. (3) A novel immunomodulator, linomide, wqs found to be effective in inhibiting EAU developing in rats and mice actively immunized with S-Ag or IRBP, respectively. This drug also inhibited the humoral and cellular immune responses in these animals. On the other hand, treatment with linomide had no effect on the development of EAU adoptively transferred by presensitized lymphocytes. The latter finding suggests that linomide cannot be useful for the treatment of uveitis in humans, in whom immunopathogenic processes are preactivated. 68 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Objectives Studies conducted during Fiscal Year (FY) 1993 were aimed at the following: (1) to fiirther analyze the immune responses to the immunodominant and highly uveitogenic peptide 1181-1191 of bovine interphotoreceptor retinoid-binding protein (IRBP); (2) to prepare recombinant human S-antigen (rHumS- Ag) and test its immunological properties — (a) its immunogenicity and uveitogenicity in rats, (b) its cross-reactivity with bovine S-antigen (S-Ag), and (c) identification of the peptide determinants in its sequence that are immunodominant in various rat strains; and (3) to investigate the effect of linomide, a novel immunomodulator, on the development of experimental autoimmune uveoretinitis (EAU) in rats and mice. Methods Peptides were synthesized on an Applied Biosystems synthesizer 430A. Bovine S-Ag was extracted from frozen retinas by Dr. Joseprti Shiloach (National Institute of Diabetes and Digestive and Kidney Diseases). rHumS-Ag was expressed in Escherichia coli according to the method described by H. F. Oettinger et al. (7 Neuroimmunol 44:157-162, 1993) using a cDNA coding for human S-Ag. A large batch of the recombinant antigen was prepared by Dr. Shiloach and was extracted by 8 M urea. Con- ventional procedures were used for immunization of animals and for testing their immune responses and development of EAU. The affinity of peptides toward major histocompatibility complex (MHC) molecules on antigen-presenting cells (APC) was assessed by the capacity of the tested peptide to inhibit the binding of biotinylated bovine IRBP peptide 1181-1191 to adherent cells from syngeneic spleens. Linomide was provided by Dr. S. Slavin (Hadassah Hospital, Jerusalem). The drug was administered to experimental animals via the drink- ing water at 1 mg/ml. Major Findings Studies concerning IRBP peptide 1181-1191. — ^As indicated in our report for FY 1992, the rat homolog of bovine IRBP peptide 1181-1191 is immuno- logically inactive in the Lewis rat and is not recog- nized by lymphocytes sensitized to the bovine sequence. Since the bovine peptide is immunodomi- nant and highly uveitogenic in Lewis rats (Sanui et al., J Exp Med 169:1947, 1989), lymphocytes sensi- tized against it must recognize a determinant of the rat ERBP molecule to initiate the pathogenic process of EAU. Our studies now show that this target determinant is likely to be the rat IRBP peptide 273- 283: (1) sequence 273-283 of IRBP, which is ex- tremely conserved, is identical in bovine and rat; (2) rat peptide 273-283 is recognized by lymphocytes sensitized against bovine peptide 1181-1191 and stimulates them to proliferate; and (3) moreover, rat 273-283 is superior to bovine 1181-1191 in its capacity to stimulate uveitogenicity in lymphocytes sensitized against the bovine peptide. The lack of immunogenicity of the rat IRBP peptide 1181-1191 in Lewis rats was found to be related to the poor afiinity of this peptide toward the MHC molecules on the APC of Lewis rats. (T lymphocytes recognize antigenic determinant only in the context of the MHC molecule.) The poor affinity of the rat peptide 1181-1191 was shown by its inability to competitively inhibit the binding of the bovine 1181-1191 to Lewis rat spleen cells. In line with the assumption that the lack of immunogenicity of rat peptide 1181-1191 in Lewis rats is due to its poor affinity to the Lewis MHC, we found that this rat peptide is immunogenic and uveitogenic in rats of the Buffalo inbred strain; the Buffalo MHC class n is RT1^ while the Lewis MHC molecule is RTl'. Moreover, bovine 1181-1191 was found to be nonuveitogenic and nonimmunogenic in the Buffalo rat. Studies with rHwnS-Ag. — ^Partially purified rHuraS-Ag, expressed in E. coli, was examined for its immunological activities in rats. Our findings were as follows: (1) rHumS-Ag resembled bovine S-Ag (extracted from retinas) in its capacity to induce EAU in Lewis rats; the minimal dose of both preparations was 2.5 pg per rat (when injected in complete Freund's adjuvant, along with Bordetella pertussis). (2) There were moderate levels of cross- reactivity between the two preparations when tested by either antibody or cellular immune responses. (3) Lymphocytes from rats immunized with rHumS- Ag were used to identify the immunodominant peptide determinants of human S-Ag, namely the determinants against which the immune response is targeted in animals immunized with the whole antigen. When tested against 40 overlapping syn- 69 Laboratory of Immunology NEI Annual Report— FY 1993 thetic peptides that extend the whole antigen se- quence, sensitized lymphocytes from Lewis rats immunized with rHumS-Ag responded against peptides derived from three sequence regions: 61-80, 171-190, and 281-310. The highest lymphocyte response was observed against peptide 281-300, which is also uveitogenic in Lewis rats. On the other hand, we observed low proliferative responses with peptides 341-360 and 351-370, which are highly uveitogenic in the Lewis rat. Testing the responses of rHumS-Ag-sensitized lymphocytes from rats of inbred strains other than Lewis revealed that the same three regions of human S-Ag provide the inamunodominant peptides for the response of all tested strains, but we saw remarkable differences among the strains with regard to their response to individual peptides. Thus, the highest response of Buffalo rats was aimed at peptides 61-80 and 71-90; ACI rats responded mainly to peptides 71-90 and 181-200, while BN rats responded at the highest levels against peptides 71-90 and 291-310. The effects of linomide in the EAU system. — Linomide (quinoline-3-carboxamide) is a unique ixnmunomodulator that both enhances natural killer cell activity and suppresses autoimmune processes. Treatment with linomide inhibited the development of actively induced EAU: In mice, 1 1 of 15 controls had EAU versus none of the 15 in the treated group; in rats, 11 of 12 controls developed EAU versus 5 of 12 in the treated group. The disease developed later and was less severe in treated rats than in control rats. Linomide treatment also significantly sup- pressed humoral and cellular immune responses in both mice and rats. In contrast, however, treatment with linomide had no effect on the development of adoptively transferred EAU or on the immune responses in the recipient animals. Significance to Biomedical Research and the Program of the Institute 1. Our findings with IRBP peptide 1181-1191 reveal for the first time a unique immunological system in which lymphocytes sensitized against an immunodominant peptide of a xenogeneic organ- specific protein do not recognize the autologous homolog but, instead, initiate the pathogenic auto- immune process by recognizing a "surrogate" peptide epitope of the autologous molecule. This unique phenomenon is made possible by the unusual four- fold structure of the IRBP molecule, allowing cross- reactivity between "repeats" on different regions of the molecule. In addition, our observations with this immunological system provide new evidence of the pivotal role of the affinity of a peptide toward the MHC in determining the immunogenicity of the peptide in animals using that MHC molecule. 2. The limited supply of human retinas has restricted in the past usage of the human S-Ag in the various immunological studies concerning the in- volvement of this retinal antigen in human diseases. Consequently the large majority of studies have been carried out with the bovine protein. The successful expression of immunologically active rHumS-Ag thus provides us with an essentially limitless supply of this antigen. It is expected that rHumS-Ag will become a useful tool for analyzing autoimmunity in uveitic patients. Moreover, rHumS-Ag may be the antigen of choice in clinical studies in which oral tolerance with retinal antigens will be used as a modality to modulate the pathogenic process of autoimmune-mediated uveitis. 3. Our study with linomide provides the first data concerning the effect of this immunomodulator on an autoimmune ocular disease. Moreover, oiu: data indicate that, unlike its effectiveness in inhibit- ing inunune responses of the afferent type, tinomide has no effect on the efferent limb of the immune re- sponse. This new observation underscores the uniqueness of the mode of action of this compound, but it casts doubt on the future usefulness of lino- mide in chnical conditions. Proposed Course Our future efforts will focus on the following issues: (1) the relationship between the affinity of IRBP peptides to MHC molecules and the immunogenicity and uveitogenicity of these peptides in rats of various inbred strains and (2) analysis of the system in which feeding with uveitogenic antigens protects animals against the development of EAU and immune re- sponse toward these antigens. In particular, we will examine the capacity of rHumS-Ag to induce oral tolerance. We will probe the possibility of using this antigen in the treatment of patients with uveitis. NEI Research Program Retina] and Choroidal Diseases — Inflammatory Disorders 70 NEI Annual Report— FY 1993 Laboratory of Immunology Publications Casper- Velu LE, Verougstraete C, Gery I, Nussen- blatt RB: Ultrastructural changes of retinal vascular endothelial cells at the onset of experi- mental autoimmune uveitis, in Dernouchamps JP, Verougstraete C, Caspers-Velu L, Tassignon MJ (eds): Recent Advances in Uveitis. Amsterdam, Kugler Publications, 1993, pp 103-110. Egwuagu CE, Bahmanyar S, Mahdi RM, Nussenblatt RB, Gery I, Caspi RR: Predominant usage of VP8.3 T cell receptor in a T cell line that induces experimental autoimmune uveoretinitis (EAU). Clin Immunol Immunopathol 65:152-160, 1992. Egwuagu CE, Mahdi RM, Nussenblatt RB, Gery I, Caspi RR: Evidence for selective accumulation of Vp8+ T lymphocytes in experimental auto- immune uveoretinitis induced with two different retinal antigens. J Immunol 151:1627-1636, 1993. Fujino Y, Li Q, Chung H, Hikita N, Nussenblatt RB, Gery I, Chan C-C: Immunopathology of experi- mental autoimmune uveoretinitis in primates. Autoimmunity 13:303-309, 1992. Gery I, Nussenblatt RB: Immunologic basis of uveitis, in Pepose JS, Holland GN, Wilhelmus KR (eds): Ocular Infection and Immunity. Philadelphia, Mosby-Year Book, Inc, in press. Kasner L, Chan C-C, Cordella-Miele E, Gery I: The effect of chlorpromazine on endotoxin-induced uveitis in the Lewis rat. Curr Eye Res 11:843- 848, 1992. Kasner L, Chan C-C, Whitcup SM, Gery I: The paradoxical effect of tumor necrosis factor alpha (TNF-a) in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 34:2911-2917, 1993. Oppenheim JJ, Gery I: From lymphodrek to inter- leukin 1 (IL-1). Immunol Today 14:232-234, 1993. Sasamoto Y, Kawano YI, Wiggert B, Chader GJ, Gery I: Induction of unresponsiveness in adult rats by immunodominant and nondominant pep- tides. Cell Immunol 152:286-292, 1993. Suh EDW, Vistica BP, Chan CC, Raber JM, Gery I, Nussenblatt RB: Splenectomy abrogates the induction of oral tolerance in experimental auto- immune uveoretinitis. Curr Eye Res 12:833-839, 1993. 71 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00288-01 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Gene Therapy for Ocular Genetic Disease PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Moncef Jendoubi Ph.D. Visiting Scientist LI, NEI Others: Noriko Esumi Daniel H. Lacorazza Luis J. Rivero Robert B. Nussenblatt M.D., Ph.D. Visiting Associate LI, NEI Ph.D. Visiting Fellow LI, NEI Ph.D. Visiting Fellow LLNEI M.D. Scientific Director NEI COOPERATING UNITS (it any) LAB/BRANCH Laboratory of Immunology SECTION Section on Genetics and Molecular Immunology INSTITUTE AND LOCATION NEI. NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 3.9 PROFESSIONAL: 3.9 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) n (a) Human subjects □ (a1) Minors n (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The goals of our main project are to get a better understanding of the physiological regulation of the ornithine 5-aminotransferase (OAT) and its regulation in vivo. In humans, the mutation of OAT leads to the degeneration of the choroid and retina, causing a gyrate atrophy disease. There is no treatment for this human genetic disease; the only feasible approach would be to submit the patient to gene therapy via modified somatic cell lines. To accomplish this task, we are focusing on (1) the regulation of OAT gene in vivo and (2) the genetic modification of somatic cell lines mediated by recombinant retroviruses. With the idea of applying gene therapy, Moloney murine leukemia virus-based recombinant retrovirus vectors have been constructed. The human OAT cDNA was placed under the control of the enhancer-promoter regulatory elements derived from the Moloney murine leukemia virus long-terminal repeat. The construct was transfected into a safe packaging cell line, GP-hE-86, to produce provirus particles. Supernatant from these ecotropic OAT producer cell lines was used to transduce mouse C57B1/6 embryonal fibroblasts and embryonic stem cells. We showed that the recombinant retrovirus transfers the OAT gene to the recipient cells, which produce an immunoreactive OAT. Northern blot analysis confirmed the presence of an OAT transcript in the transduced cell lines, even after a long period of time in vitro. 72 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Objectives Ornithine 5-aminotransferase (OAT, L-omithine:2- oxoacid aminotransferase, EC 2.6.1.13) is a nuclear- encoded mitochondrial matrix housekeeping gene that catalyzes the reversible transamination of orni- thine to glutamate semialdehyde. Biochemical analyses have shown that OAT is synthesized as 49- kD precursor monomer cleaved to a 45-kD protein on entry into the mitochondria, where assembly into the active homohexameric form of the enzyme occurs. This enzyme is expressed constitutively at low levels in the liver and kidney and at much higher levels in the retina, where OAT function seems to be critical for vision. Methods In humans, a genetic deficiency of OAT results in gyrate atrophy (GA) of the choroid and retina — a rare, blinding chorioretinal degeneration with associ- ated cataract, inherited as an autosomal recessive trait. Such OAT deficiency disrupts ornithine and arginine catabolism and results in a 10- to 15-fold accumulation of ornithine in all body fluids. In addition to their visual problems, some GA patients exhibit muscular dystrophy. Using a variety of techniques, including RNase A protection, single- strand conformational polymorphism analysis, and polymerase chain reaction, recent studies have shown that the unique OAT gene in GA patients contains firameshift, nonsense, and missense mutations that lead to the inactivation of the gene. A diet low in arginine has been shown to possibly delay the onset of this disease, but this approach is difficult to follow and may not be applicable to all patients. Therefore, since no therapy has been shown to be particularly effective, gene therapy in somatic cells (i.e., insertion of a functional OAT gene into patients' cells) could be a reasonable therapeutic alternative for patients suffering from GA. Vector construction. — ^The 1.4-kilobase (kb) EcoRI/HindHI fragment containing the entire human OAT cDNA was inserted into EcoRI/XhoI linearized retroviral vector LXSN to generate the recombinant vector pLXSN/OAT. All plasmids were grown in Escherichia coli strains HB 101 and DH 5 Alfa. Cell lines. — The murine retroviral packaging cell line GP+E-86 was grown in Dulbecco's modified Eagle's medium containing 10% (v/v) calf serum. We seeded ecotropic cells at 2 x 10^ cells per 10 cm dish and transfected with 10 ng of undigested pLXSN/OAT plasmid DNA, using the calcium- phosphate method. Twenty-four hours later cells were washed twice with phosphate-buffered saline (PBS) and grown in the presence of 1 mg/ml of geneticin (G418) for 1 week in 24- well plates. Supematants from different wells were harvested, filtered through 0.2 \xn\ filters, and tested for viral titer on 14-day-old C57BI/6 murine embryonal fibroblasts. After 1 day, the culture medium was replaced by G418 culture selection medium. Resis- tant clones were stained with methylene blue and counted. OAT immunodetection. — Subconfluent 10-cm dishes of transduced and nontransduced murine embryonal fibroblast cells were grown for 16 hours in the culture conditions described above, then harvested and lysed in 500 mM sodium chloride (NaCl), 50 mM Tris (pH 7.5), 1% NP40. Protein extracts from the same number of transduced and nontransduced cells were subjected to preparative sodium dodecyl sulfate/polyacrylamide gel electro- phoresis (SDS/PAGE) and transferred to nylon filters (Schleicher and Schuell). After saturation in PBS solution containing 5% nonfat dry milk, the strips were incubated with anti-OAT or with preimmune serum at the same dilution — 1/100 — ^for 1 hour at room temperature, then washed three times at 20°C for 20 minutes in 1% NP-40, 150 mM NaCl, and 50 mM Tris/HCl (pH 7.5). The strips were incubated with goal anti-IgG antibodies conjugated with peroxi- dase. Anti-antibodies were visualized by visiblof™ AP. Major Findings We have achieved the construction of Moloney murine leukemia virus (Mo-MuLV)-based recombi- nant retrovirus vectors expressing a human OAT cDNA under the control of a Mo-MuLV long-termi- nal repeat. Neomycin phosphotransferase was included as a selectable marker in these recombinant retroviruses. Murine embryonal primary fibroblasts, as well as embryonal stem cells, have been trans- duced with helper virus-free recombinant refrovirus particles generated from a GP-^E-86 packaging cell line previously ttansfected with the described con- struct. We have successfully transduced several cell lines, as shown by Northern hybridization analysis 73 Laboratory of Immunology NEI Annual Report— FY 1993 and immunodetection via rabbit polyclonal antibodies raised against human OAT. Significance to Biomedical Research and the Program of the Institute The improvement in visual function on reduction of ornithine accumulation suggests that the physio- pathology of GA may involve (1) a direct toxic effect of ornithine; (2) a deleterious effect of meta- bolic alterations, occurring as a result of hyperomi- thinemia; or (3) both. However, despite many efforts, no therapy has been totally successful in treating this genetic disease. It is possible to correct a genetic disease by directing the treatment to the site of the defect itself (i.e., "the mutated gene"), rather than against second- ary or pleiotropic effects due to the mutant gene. A retrovirus-based delivery vehicle is likely to be the best choice for introducing a functional gene into somatic cells, thus achieving gene therapy of heredi- tary genetic diseases. Since the first successful retrovirus-mediated transfer of the adenosine deamin- ase gene into lymphocytes of patients suffering from a lethal immune deficiency, gene ther^y has been viewed as a reasonable, feasible approach to treating human genetic diseases. Since then, work has focused on several other genes, such as those encod- ing low-density lipoprotein, factor IX, and the cystic fibrosis transmembrane conductance-regulator gene. Further research is under way, with the aim of clinical trial. As shown by several groups, retroviral vectors can stably introduce genes into a variety of cultured cells. Defective retroviruses have been proposed as carriers to transfer functional genes to patients suffering from human genetic diseases. To attempt gene transfer via somatic cell lines to patients suffering from genetic deficiency of OAT, we designed and made a Mo-MuLV-based retroviral vector carrying a functional human OAT gene. We analyzed its stable integration and expression in murine embryonal fibroblasts and embryonic stem cells. To test the production of OAT transcript from the recombinant retroviral virus present in the frans- duced cell lines, we performed Northern blot analy- sis, using a total RNA preparation from cell lines both transduced and nontransduced by OAT refro- virus. The results obtained show the production of significant amounts of mRNA transcript exclusively in the transduced cell lines that hybridize with an OAT cDNA probe. No reaction was detected in wild-type fibroblasts. The absence of cross-reaction with murine OAT may be due to the high stringency during Northern blot washes or to the fact that mouse embryonal fibroblasts do not produce OAT enzyme. Moreover, when Western blot analysis of proteins extracted from the various cell lines used (including transduced mouse embryonal fibroblasts) was performed with rabbit polyclonal antibodies raised against two peptides of human OAT, we observed a specific reaction in only those extracts prepared from die cell lines transduced by the OAT provirus. The same polyclonal antibodies tested in Western blot against protein exfracts from human retinal pigmented epithelium — fibroblasts and HeLa cell lines — showed a similar reaction on just one polypeptide of the same apparent molecular weight as that detected in the transduced fibroblasts. Taken together, these results reveal in transduced cells the expression of a single OAT transcript and a single OAT polypeptide. We show here the ability to produce a retrovirus vector carrying and expressing a functional human cDNA coding for OAT, opening up the possibility of considering replacement gene ther^y for OAT- deficient patients who suffer from GA disease. Proposed Course Our future efforts will focus on the following issues: (1) the enzymatic activity of OAT produced by genetically modified somatic cell lines, which we will further investigate via recombinant refrovirus; (2) assessment of the target cell types for gene product delivery; (3) investigation of the effect of overexpression of OAT in transgenic mice that express human OAT; and (4) characterization of the murine OAT genomic gene, prior to engineering the appropriate homologous recombination vector. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders 74 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00232-08 LI PERIOD COVERED October 1, 1992 to September 30. 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Interferon System in Cellular Function and Disease PRINCI PAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: Caroline Percopo M.S. Biologist LI, NEI Chandrasekharam Nagineni Ph.D. Visiting Scientist LI, NEI COOPERATING UNITS (if any) Department of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: PROFESSIONAL: OTHER: 0.5 0.2 0.3 CHECK APPROPRIATE BOX{ES) □ (a) Human subjects □ (b) Human tissues [x] (c) Neither □ (a1) Minors □ (a2) interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Cytokines, such as interferon-gamma (IFN-y) and interleukin 2 (IL-2), are a group of specialized hormone-like proteins which exert profound influences on cellular development and on a variety of cellular functions. This project has concentrated on studying the ways in which cytokines interact with cells of the immune system and with cells in the ocular miCToenvironment. We have shown that IFN-y and IL-2 are found within the inflamed eye in association with T-cell infiltration and major histocompatibility complex (MHC) class II antigen expression on infiltrating cells and on retinal pigment epithelium (RPE) cells. Furthermore, IFN-y- activated RPE cells can process and present antigeas to helper T lymphocytes. Experimentally we demonstrated that isolated human RPE cells can be induced to produce another lymphokine, IL-6, following incubation with IFN-y. IL-6 is a potent inflammatory cytokine capable of enhancing antibody production and cytotoxic T-cell activities. These studies indicate that cytokine-mediated activation of RPE cells may be a basic component of ocular immunity and an important aspect of RPE cell transplantation. Retinoblastoma cells are an important model for exploring human malignancy and differentiation. Using these cells we showed that EFN-y can regulate MHC cla.ss 1 genes at both transcriptional and posttranscriptional levels. In addition, this modulation is not associated with downregulation of N-myc oncogene expression. These observations indicate that IFN-y-induced MHC class 1 and class II antigen expression may serve as a local amplification system in autoimmune and inflammatory eye disease. A better understanding of the role of cytokines in the mechanisms involved in the development of autoimmunity and inflammation may be beneficial in developing treatments for these diseases. 75 PHS 6040 {Rev. 5/92) Laboratory of Immunology NEI Annual Report— FY 1993 Project Description Objectives This project is designed to determine the bioregula- tory actions of interferon (IFN) and other cytokines and to evaluate their regulatory actions in the patho- genesis of disease. Methods We assayed human IFN, using inhibition of vesicular stomatitis virus plaque formation in human amnion (WISH) cells. IFNs were characterized by neutral- ization of antiviral activity with monoclonal anti-IFN immunoglobulin. Interleukin 2 (IL-2) biological activity was assayed by induction of proliferation of CTL cells. Interleukin 6 (IL-6) activity was assayed by an enzyme-linked immunosorbent assay, immuno- blot assays, and Northern blots. Analytical flow cytometry was used to quantitate retinal proteins. Gene transcription techniques, such as Northern blot analysis and nuclear runoff transcription assays, are being used to evaluate IFN-y modulation of retinal proteins. Major Findings IFN activation of retinal pigment epithelial (RPE) cells. — Numerous studies indicate that a variety of autoinunune diseases are associated with the DFN-y- induced tissue-specific expression of major histocom- patibility complex (MHC) class 11 molecules. During the past 5 years, we have identified various steps that may be involved in ocular immunopathologic mecha- nisms. In these studies of retinal degenerations and autoimmune diseases, we showed that a critical regulatory cell in the retina, the RPE cell, is capable of expressing MHC class II determinants. We also can detect IFN-y in situ and in retinas from patients with inflammatory eye diseases, as well as in MHC class Il-positive RPE cells. In addition, freshly isolated hiunan RPE cells can express these determi- nants following treatment with IFN-y. In animal model systems, we found that inocula- tion of recombinant IFN-y induces la expression of ocular cells, and treatment with anti-la antibodies can eliminate or inhibit experimental autoimmune uveitis. Most recently we showed that the RPE cell may be playing an important role in ocular immunity, acting as a resident antigen-presenting cell in the retina. During the past year we have provided the most recent piece of experimental evidence implicating a role for cytokine-activated RPE cells in autoimmune phenomena by showing that the RPE cell is capable of producing the cytokine IL-6. RPE cell cultures were established from human donor eyes. These isolated RPE cells do not produce IL-6 alone; IFN-y induces these cells to produce IL-6 in a dose-depen- dent manner. Moreover, EFN-y can synergize with tumor necrosis factor (TNF) to produce IL-6 in human RPE cells. IL-6 is a potent cytokine which can act on B lymphocytes to induce growth and antibody production. It can also act on T lympho- cytes to induce IL-2 production, IL-2 receptor expression, and cell proliferation. These studies further substantiate the concept that cytokine-medi- ated activation of RPE cells may be a basic compo- nent of ocular immunity and may have major inmiu- nological consequences for RPE cell transplantation studies. Cytokine-induced modulation of cellular proteins in the retina and retinoblastoma. — Retinoblastoma cells are an important model for exploring human malignancy and differentiation. These multipotent embryonic cells are capable of differentiating into neuronal, glial-like, and RPE-like elements. We have shown that flow cytometric analysis can be used to measure the expression of both cytoplasmic and cell surface proteins in retinoblastoma cells. We used this technique to monitor changes in the expres- sion of selected cellular proteins after exposure to specific cytokines and found that MHC class I molecules were augmented by IFN-a and IFN-y but not by TNF. However, the MHC class 11 molecules were augmented by IFN-y but not by BFN-a or TNF. The neuronal markers IRBP and PR-6, the glial-like marker GFAP, and the RPE cell markers RPE-9 and RPE- 15 were not altered by any of the cytokines tested. The mechanism of induction of MHC class I and II antigens by IFN in retinoblastomas is not known. We therefore have initiated studies to compare IFN-a, BFN-P, and IFN-y in their ability to induce the expression of class I antigens and to investigate the role of transcriptional and posttranscriptional mechanisms in this induction. We found that IFN-y increased HLA class I antigen expression and in- duced a fivefold increase in its transcription rate. Posttranscriptionally IFN-P and IFN-y increased 76 NEI Annual Report— FY 1993 Laboratory of Immunology Steady-State mRNA for the HLA-B7 gene. These effects were not associated with down-regulation of N-myc oncogene nuclear transcription. Moreover, dexamethasone did not affect the IFN-y-induced expression of HLA class I molecules. These studies implicated both transcriptional and posttranscriptional mechanisms in the modulation of class I molecule expression by IFNs. Significance to Biomedical Research and the Program of the Institute These studies highlight the fact that the release of cytokines, such as IFN-7, within the ocular microen- vironment and the subsequent induction of cytokines and MHC class I and II antigen expression on resident and infiltrating cells may be critical elements in a cascade leading to ocular cell destruction. The retinal cell that may play a critical role in auto- immune uveitis is the RPE cell. IFN-y-induced activation of RPE cells may participate in auto- immune disease in the ocular microenvironment. Cytokines produced and localized in the eye may play critical roles in normal physiology, pathogenic mechanisms, and therapeutic approaches. Since the RPE cell is a pivotal regulatory cell in the retina, an understanding of how cytokines interact with this cell will shed light on avenues for therapeutic interven- tion in pathogenic states and in transplantation. Proposed Course We plan to continue our evaluation of the role of cytokines in autoinununity and inflammation. We are now developing systems in rat models to monitor directly the effects of altering cytokine production on inflammatory eye diseases. Moreover, we will continue to characterize the antigen-presenting ability of the RPE cell in a variety of antigens and viruses. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Barez S, Boumpas D, Percopo C, Anastassiou ED, Hooks JJ, Detrick B: Modulation of major histocompatibility complex (MHC) class I genes in human retinoblastoma cells by interferons: Evidence for both transcriptional and post-tran- scriptional regulation. Invest Ophthalmol Vis Set 34:2613-2621, 1993. Detrick B, Hooks JJ: Cytokines and effector mole- cules in human immunology, in Leffell MS, Bias WB, Donnenberg AD, Rose MR (eds): CRC Handbook of Human Immunology. Boca Raton, CRC Press, in press. 77 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00233-08 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (BO characters or less. Title must lit on or\e Ime between the borders.) Studies on the Bioregulatory Aspects of the Retinal Pigment Epithelial Cell PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: Chandrasekharam Nagineni Caroline Percopo T. Michael Redmond David Parks Robert B. Nussenblatt Ph.D. Visiting Scientist LLNEI M.S. Biologist LI, NEI Ph.D. Research Biologist LRCMB, NEI M.D. LI, NEI M.D. Scientific Director NEI COOPERATING UNITS (if any) Hopital St. Louis, Paris, France (Lawrence Boumsell, M.D.); University of Nice, France (Alain Bernard, M.D.); National Institute of Dental Research (Reuben Siraganian, M.D.); The Johns Hopkins University (Stanley A. Vinores, Ph.D.; Peter Campocbiaro, M.D.); Department of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS; 0.9 PROFESSIONAL: CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews 0.5 OTHER: 0.4 [x] (b) Human tissues □ (c) Neither SUMMARY OF VlfORK (Use standard unreduced type. Do not exceed the space provided.) The retinal pigment epithelial (RPE) cell plays a basic role in maintaining the structural and physiological integrity of the neural retina. We have isolated and propagated RPE cells in vitro and have developed monoclonal antibodies directed against human RPE cells. We are using these techniques and reagents to evaluate molecular, biochemical, and biological properties of the RPE cells. Since the monoclonal antibodies detect epitopes present solely on RPE cells, they provide a unique opportunity to evaluate a variety of aspects of RPE cell development and function. Studies on RPE cell development indicate that the epitopes appear only after the cells have begun terminal differentiation. Moreover, studies on RPE migration demonstrate the value of these antibodies in evaluating epiretinal membrane formation. The RPE epitope is a 67-kD protein that is closely associated with the microsomal membrane. A cDNA clone that has been isolated codes for a protein which does not match any other sequences in the data bases. Studies are in progress to propagate and transplant RPE cells in various animals. We have propagated human RPE cells in vitro and evaluated their ability to respond to cytokine activation. RPE cells respond to retinal aberrations by dying, proliferating, migrating, losing phagocytic function, expressing major histocompatibility complex (MHC) class II antigens, and presenting antigens to T lymphocytes. The techniques and reagents obtained in these studies allow us to evaluate the mechanisms involved in aberrant responses of RPE cells. Moreover, they provide a framework for evaluating RPE cell transplantation. 78 PHS 6040 {Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Objectives The aim of this project is to evaluate the molecular, biochemical, and various biologic properties of retinal pigment epithelial (RPE) cells in normal and disease states. Moreover, we are evaluating RPE cell transplantation. Methods RPE cells are isolated and propagated in vitro. Monoclonal antibodies are generated in mice by fusing mouse spleen cells with myeloma cells. Antibodies to the RPE cells are evaluated by immu- noperoxidase and Western blot assays. The effects of drugs and cytokines are evaluated by cell viabiUty and proliferation assays and by nuclear transcription runoff assays. Major Findings Evaluation of epitopes identified by monoclonal antibodies. — ^We have identified two mouse IgG3 monoclonal antibodies that react with RPE cells from a variety of species, ranging from man to frog. Since these antibodies detect epitopes present solely on RPE cells, they provide us with the unique opportunity to evaluate a variety of aspects of RPE cell development and function. Electron microscopic immunocytochemistry revealed labeling patterns for the two RPE antibodies that are very similar. In human eyes, staining was localized to surface and intracellular membranes and the cytoplasm. Staining occurred predominantly on the j^ical surface of the RPE cells. The RPE pro- tein, a 65-kD protein, was isolated by polyacrylamide gel electrophoresis and fransferred to nitrocellulose blot, and the sequence of its amino acid residues was determined. The amino acid sequence was used to design a synthetic cDNA probe. A bovine cDNA library was screened, and cDNA clones were isolated and characterized. The cDNA insert is 3,1 15 base pairs; the open reading frame encodes a protein of 533 amino acids. The RPE protein does not match any other sequence in the data bases. The protein expressed in Escherichia coli has a molecular weight similar to that of the native protein. In addition, we used Northern blotting with the cDNA to detect protein mRNA in RPE cells. In studies of the developmental expression of RPE and photoreceptor determinants in the rat retina, we had previously shown that the expression of these determinants in rats is absent the day of birth and detectable at postnatal Day 6. Recent studies show that RPE cells express their determinants shortly before the first outer segments are detected and that the posterior-anterior progression of outer segment formation matches a similar progression of the expression of the RPE determinants. These data indicate that the RPE resumes its maturation during the first postnatal week and that RPE maturation and outer segment growth can be correlated. RPE cell transplantation. — ^Recent studies indicate that RPE cell transplantation may be beneficial in restoration of the retinal architecture in selected retinal degenerations. It is essential to develop methods for large-scale preparations of RPE cell cultures for somatic cell genetic engineering manipu- lation. We are in the process of evaluating various parameters for human and rat RPE cell culture and transplantation. Preliminary smdies show that we can successfiiUy inoculate human RPE cells into the rat refina Evaluation of the immunologic parameters of this transplantation process is under way. Significance to Biomedical Research and the Program of the Institute The monoclonal antibodies developed in this smdy are the first directed solely at the RPE cell. These antibodies are potentially useful in identifying RPE cells in situ and in vitro. These antibodies, which can be used to monitor RPE cellular fiinctions, may provide a better understanding of the role of RPE cells in retinal degenerative disorders. Identification of the cDNA and proteins detected by the mono- clonal antibodies may provide the framework on which to evaluate specific RPE cell fiinctions. RPE cell transplantation to correct retinal degenerative processes is being actively investigated in a number of laboratories. The studies reported here provide the basis for evaluation of RPE cell transplantation. Proposed Course 1. We will continue to characterize these anti- bodies and their effects on cell function in vivo and in vitro. 2. We will isolate, propagate, and characterize RPE cells for transplantation studies in animals and 79 Laboratory of Immunology NEI Annual Report— FY 1993 humans. We will design effective ways to maintain the cell in culture and to measure and monitor cell function. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Hamel CP, Tsilou E, Harris E, Pfeffer BA, Hooks JJ, Detrick B, Redmond TM: A developmentally regulated microsomal protein specific for the pigment epithelium of the vertebrate retina. J Neurosci Res 34:414-425, 1993. Hamel CP, Tsilou E, Pfeffer BA, Hooks JJ, Detrick B, Redmond TM: Molecular cloning and expres- sion of RPE65, a novel retinal pigment epithe- lium-specific microsomal protein that is post- transcriptionally regulated in vitro. J Biol Chem 268:15751-15757, 1993. Vinores SA, Orman W, Hooks JJ, Detrick B, Camp- ochiaro PA: Ultrastructural localization of RPE- associated epitopes recognized by monoclonal antibodies in human RPE and their induction in human fibroblasts by vitreous. Graefe's Arch Clin Exp Ophthalmol 231:395-400, 1993. 80 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00240-07 LI PERIOD COVERED October 1, 1992 to September 30. 1993 TITLE OF PROJECT (BO characters or less. Title must lit on arte line between the borders.) Virus Infections in the Eye PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: Caroline Percopo Yun Wang Miguel Burnier Ingeborg Kirch Yusuke Komuraski M.S. M.D. M.D. M.D. M.D. Biologist Guest Worker Visiting Scientist Guest Worker Guest Worker LI, NfEI LI, NEI LI, NEI LLNEI LI, NEI COOPERATING UNITS (if any) DepartmeDi of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.); Department of Pathology, Uniformed Services University for Health Sciences (Katherine Holmes, Ph.D.); Department of Ophthalmology, Ruprecht-Karl's University, Heidelberg, Germany (Ellen Kraus-Mackiw, M.D.); Laboratory of Biology, NCI, NIH (Charles H. Evans, M.D., Ph.D.); Department of Medicine. The Johns Hopkins Medical School, Baltimore. MP (William Bums, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunology and Virology INSTITUTE AND LOCATION NEI. NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: PROFESSIONAL: OTHER: 1.0 0.8 0.2 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [xl (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Our studies of various virologic and immunopathologic processes that occur when viruses replicate in the ocular microenvironment comprise four areas: (1) coronavirus infection in ocular and optic nerve cells, (2) the possible roles of viruses in human diseases. (3) antiviral therapeutic actions of cytokines and drugs, and (4) molecular diagnosis and pathogenesis of cytomegalovirus (CMV) infections in man. We have estabhshed that murine coronavirus can induce ocular disease and may be used as a model system for studying retinal degenerative diseases. This model has many unique features. The virus is capable of inducing an acute infection in the presence of mild retinal vascular inflammation. Initial retinal damage is followed by clearance of the virus and progressive retinal degeneration, even months after the virus is gone. The virus replicates predominantly in MuUer cells and also can be detected in retinal pigment epithelium (RPE) and photoreceptor cells. Recent studies show that there are genetic and immunologic components to this disease. The retinal degenerative pathologic manifestations of the disease can be influenced by the genetics of the host, i.e.. some strains of mice are resistant to virus-induced retinal degenerative changes. The pathologic changes also are closely related to the development of antiretinal and anti-RPE antibodies. These findings suggest a role for autoimmunity in the pathogenesis. This disease may be considered a model for degenerative diseases of the pigment epithelium and photoreceptors in hiunans. The need for effective drug treatment and prevention of herpes virus and other viral diseases has assumed growing importance. We found that leukoregulin, a naturally occurring immunologic cytokine, not only increases the antiviral actions of the drug acyclovir but also directiy inhibits herpes simplex virus replication, demonstrating that combination immunotherqjy and chemotherapy can produce substantial inhibition of herpes virus replication and providing a rationale for applying this approach to treating virus infections. Studies initiated this past year indicate that CMV is capable of replicating within human RPE cells in vitro; however, replication is limited at the level of immediate early protein production. The low frequency of expression of immediate early viral proteins in RPE cells and the subsequent slow replication of CMV may be critical variables in terms of their relationship to viral persistence and activation within the retina. 81 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report— FY 1993 Project Description Objectives This project was designed to determine the various effects of virus infections on the ocular microenvi- ronment and to study modes of antiviral therapy. Methods This study involves the propagation and quantitation of viruses, such as herpes simplex virus type 1 (HSV-1), coronaviruses, and cytomegalovirus (CMV), both in vitro and in vivo. It also includes immunocytochemical analysis of infected cells and tissues. Techniques used in characterization of virus infection include flow cytometric analysis, Western blot analysis, Northern blot analysis of HSV thymi- dine kinase, in situ hybridization, and amplification of viral genes by polymerase chain reaction (PCR). Techniques used in characterization of antivirus antibodies include enzyme-linked immunosorbent assay and neutralization assays. Major Findings Coronavirus infection in the eye. — ^The murine coronavinis, mouse hepatitis virus (MHV), JHM strain, induces a retinal degenerative disease in adult Balb/c mice. Tlie disease consists of an acute phase lasting from 2 to 8 days, during which virus is detected within the retina and initial pathology is noted in the retinal pigment epithelium (RPE) and photoreceptor layers. This is followed by a late phase lasting from 1 to 14 weeks, during which virus is not detected but retinal degenerative changes continue, with reduction of the photoreceptor layer, loss of interphotoreceptor retinoid-binding protein, and retinal detachments. This model provides evidence that viruses can trigger retinal degenerative processes and may offer insight into pathogenic mechanisms in retinal degenerative diseases of humans. During the past year we have evaluated three aspects of this disease process: (1) immuno- logic aspects of the disease, (2) genetic predisposi- tion to the disease, and (3) use of electroretinography (ERG) to monitor the disease process. In the coronavirus-induced retinopathy, the late phase of the JHM-induced disease was associated with the lack of direct evidence for viral replication within the retina. This observation suggested that the continued degenerative process may be associated with alterations directly induced by virus replication during the first few days after infection, or it may be associated with additional factors. Because viruses are known to trigger an autoimmune phenomenon and some human retinopathies may be associated with autoantibody formation, we studied the possible production of antiretinal autoantibodies. We found that the retinal degenerative process is associated with the presence of antiretinal autoantibodies. In total, 22 of 23 sera samples collected from 10 to 70 days after JHM virus inoculation of Balb/c mice contained antiretinal autoantibodies. These autoanti- bodies were not found in sera from normal or mock- injected mice. Antibodies to retinal tissue were identified by two distinct patterns of immunoperoxidase staining on frozen sections of normal rat eyes — ^retinal autoanti- bodies and RPE autoantibodies. The antiretinal autoantibodies first appeared as IgM class antibodies which shifted to IgG class autoantibodies. The anti-RPE cell autoantibodies were predominantly of the IgG class. Sera positive for these autoantibodies did not stain with liver or kidney sections, but two of three did react with rat brain sections. We also evaluated a second mouse strain, CD-I, because these animals respond to JHM virus inocula- tion by developing only the early phase of this disease, i.e., vasculitis. On Day 10 postinoculation (pi) the retinal architecture had a normal ^pearance. In these mice, which are free of a retinal degenera- tion, antiretinal autoantibodies are not produced. However, as noted in the Balb/c mice, antivirus neutralizing antibodies were produced in the infected CD-I mice. These findings suggest a role for autoimmunity in the pathogenesis of murine corona- virus-induced retinal degeneration. Since the genetic composition of the host and the virus can determine the response to infection and the resulting pathology, we evaluated the effect of MHV infections on different strains of mice. The JHM and A59 strains of MHV were propagated in rat L2 cells. Balb/c, CD-I, and A/J mice were inoculated by the intravitreal route with 10"^ TCID50/O.5 pi of virus or with uninfected tissue culture preparations (mock injection). At various times after infection, the eyes were removed and evaluated histologically, and sera were assayed for the presence of virus- neutralizing antibody. Both JHM and A59 strains of MHV induced similar retinal diseases. 82 NEI Annual Report— FY 1993 Laboratory of Immunology We observed two distinct phases of coronavirus- induced retinopathy, retinal vasculitis (Days 3-6) and retinal degeneration (Days 10-20), in Balb/c mice. In contrast, we saw only the early stage of disease in CD-I mice. We observed typical retinal vasculitis at 3-6 days pi. However, by Day 10 pi the retinal architecture had returned to a normal appearance. No retinal degeneration was observed. The third strain, A/J mice, displayed a biphasic disease but with a mild degenerative component. All strains of mice responded to the retinopathy by developing antivirus-neutralizing antibody at similar levels. These studies demonstrate that the pathologic mani- festations of a virus infection in the retina can be influenced by the genetics of the host. The third phase of these studies incorporated ERG evaluation of the development of the virus-induced retinopathy. At various times after inoculation, animals were dark-adapted for 60 minutes and anesthetized, after which ERGs were recorded between a wick electrode touching the cornea and a needle electrode placed subcutaneously at the fore- head. Light stimuli were provided by a xenon arc light source and focused onto the eye via a fiberoptic bundle. We varied the light intensity via neutral - density filters. Five responses were averaged for each light intensity. On Day 3 pi all virus-infected mice had slightly depressed ERGs and retinal vasculitis was seen. In contrast, on Days 8 and 20 pi all these mice had subnormal or abolished responses, and retinal degen- erative changes were clearly apparent. On Day 10 pi 57% of the mice (4/7) had abolished responses and 43% (3/7) had subnormal responses. On Day 22 pi 50% (4/8) had abolished responses, while the remain- ing 50% had subnormal ERGs. Mice inoculated with virus-free tissue culture preparations had minor alterations in their ERG patterns. However, at Day 20 pi these mock-injected mice displayed ERG patterns similar to those of normal, uninfected mice. ERG studies in this murine model provide an indirect but objective means of measuring visual function, serving as the basis for future studies of treatment effects. In summary, this model is characterized by the replication of JHM virus in the retina, producing an acute necrotizing disease of the sensory retina that results in only a mild inflammatory response and a long-lasting disease (>14 weeks). In these studies we identified a progressive degenerative disease in the retina that may be initiated by an acute virus infection in the absence of major inflammatory response. During the past year these studies have clearly indicated that this retinal degenerative process has viral, immune, and genetic components. How the genetic and immunologic factors interact to influence the development of retinal degenerations is the intriguing aspect of this model. Possible role of viruses in human eye diseases. — We have initiated studies to evaluate the possible involvement of viruses in the pathogenic processes of a variety of human eye diseases. We are now collecting serum samples and ocular tissue in order to use seroepidemiologic approaches for the detection of virus and viral antigens via immunocytochemical staining, in situ hybridization, and PCR assays. Antiviral therapeutic actions of cytokines and drugs. — The need for effective treatment and preven- tion of herpesvirus and other viral diseases has assumed growing importance during the past 10 years. The development of targeted antiviral agents through combination ther^y is becoming an impor- tant strategy. One strategy consists of the develop- ment of cytokines or lymphokines in combination with chemother^y to treat malignancy and infec- tions. Using this approach, we recently showed that the cytokine leukoregulin could enhance the anti- HSV actions of acyclovir (ACV). Cytokines are a group of specialized hormone-like proteins that can exert profound influences on cellular development and a variety of cellular func- tions. As a lymphokine that performs unique regula- tory activities in transformed cells, the leukoregulin molecule, which is produced by a variety of lym- phoid cells, is a multifunctional cytokine. It can prevent chemical carcinogen transformation, inhibit neoplastic cell proliferation, and augment target cell sensitivity to natural killer cell cytotoxicity. Further- more, this cytokine has been shown to increase membrane permeability of tumor cells and to in- crease drug uptake in these cells. We recently showed that leukoregulin can selectively increase membrane permeability in HSV-1 -infected cells but not in normal (i.e., uninfected) cells. In addition, we have recently shown that leuko- regulin enhances the anti-HSV actions of ACV. The cells were exposed to the cytokine and/or ACV for only 3 hours early in the replication cycle. Because the continued presence of ACV greatly enhances antiviral activity, we evaluated the effect of the 83 Laboratory of Immunology NEI Annua] Report— FY 1993 continuous presence of leukoregulin on HSV replica- tion. Human amnion epithelial (WISH) cells were infected with HSV-1 (Wendy and F strains) and vesicular stomatitis virus (VSV). Following a 90- minute incubation period, we washed the cells and treated them with media, leukoregulin, ACV, or leukoregulin plus ACV. We evaluated virus replica- tion by plaque assays while testing virus and cellular protein expression by immunoblotting. The continu- ous presence of leukoregulin (0.1 unit) inhibited HSV-1 plaque formation by 50-80% in the Wendy and F strains, respectively. In contrast, leukoregulin did not affect VSV replication. Immunoblot analysis revealed that the expression of the 89-kD HSV-1 protein was inhibited by 50%, whereas the cellular protein, actin, was not affected by leukoregulin treatment Moreover, leukoregulin treatment did not alter the ability of the cells to incorporate tritiated thymidine. Initial evaluations of the effect of leukoregulin on HSV transcription indicated that the cytokine did not alter the level of expression of HSV tk mRNA. These studies show that leukoregulin not only enhances the antiviral actions of ACV but also can act to inhibit HSV-1 replication directly. These findings, which demonstrate that combination immu- notherapy (cytokines) and chemotherapy can substan- tially inhibit herpesvirus replication, provide rationale for the application of this approach to the interven- tion of virus infections. CMV replication within the retina. — CMV infec- tions are frequent complications in kidney and bone marrow transplant patients and HIV (human inmiu- nodeficiency virus) patients. Because the mecha- nisms by which CMV is activated and replicates within the retina are not known, we evaluated the ability of human CMV to initiate replication in human RPE cells and compared the results with finding in studies of human fibroblasts (HEL) and WISH cells. Human RPE cells obtained from donor eyes were propagated in vitro and infected at an input multiplicity of 1. CMV replication was evalu- ated in three ways: (1) detection of viral antigen by immunofluorescence and flow cytometry, (2) detec- tion of virus-induced cpe, and (3) assaying for infectious virus. We found no evidence of viral replication in the WISH cells. In contrast, CMV replication was detected in both RPE and HEL cells. In HEL cells, IE, E, and L proteins were detected at 5, 48, and 48 hours, respectively. In RPE cells these proteins were detected somewhat later — at 24, 48, and 72 hours. We noted a striking difference in the percentage of cells expressing IE protein: After 72 hours, 100% of the HEL cells expressed IE protein, whereas after 7 days, less than 1 % of the RPE cells expressed this protein. Analysis of the production of infectious virus revealed that viral infectivity and cpe were maximal at Day 5 in HEL cells and at Day 30 in RPE cells. This study demonstrates that, although all of the RPE cells were capable of becoming infected with CMV, less than 1% of the cells expressed IE viral proteins early in the infection cycle. The low fre- quency of expression of IE viral protein in RPE cells and the subsequent slow replication of CMV may be critical variables in terms of their relationship to viral persistence and activation within the retina. Significance to Biomedical Research and the Program of the Institute Elucidating the factors involved in viral spread and pathogenesis will yield a better understanding of diseases of viral etiology. We have established a new virus model for retinal degenerative processes in adult animals. This model has many unique features. It is capable of inducing acute infection in the presence of mild retinal vascular inflanmiation. The initial retinal damage is followed by clearance of the virus and progressive retinal destruction, even months after the virus is gone. Moreover, develop- ment of the retinal degenerative process is deter- mined by the genetics of the host; it involves the development of antiretinal autoantibodies. This model should assist us in understanding the patho- genesis of selected human diseases of unknown etiology. We have identified a cytokine, leukoregulin, that selectively increases membrane permeability in virus- infected cells. We also have shown that combined cytokine and drug therapy can produce substantial inhibition of HSV replication. Moreover, the contin- uous presence of the cytokine can directly inhibit virus replication. The data from these studies pro- vide rationale for the application of this approach to the interventive treatment of virus infections. We have shown that CMV replicates within RPE cells in a slow, limited manner. Evaluation of the molecular aspects of this defect may provide critical clues in terms of the virus' ability to establish 84 NEI Annual Report— FY 1993 Laboratory of Immunology persistent infections and the factors initiating viral activation within the retina. Proposed Course 1. We will continue to evaluate coronavirus infections of the eye. The role(s) of genetic factors and autoantibodies in the pathogenesis of retinal degenerations will be evaluated. The data obtained will be correlated with what is known about human retinal degenerative disorders. 2. We will initiate studies to determine whether certain viruses can replicate in retinal tissues and cells. Infected cells will be evaluated for the release or expression of uveitogenic proteins. 3. We will continue to collect samples and initiate studies to detect the involvement of viruses in human eye diseases. 4. We will continue to evaluate combinations of leukoregulin and chemotherapeutic agents for the management of virus infections. 5. We will evaluate the molecular diagnosis and pathogenesis of CMV infections in the eye. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Bumier M, Wang Y, Detrick B, Hooks JJ: Retinal manifestations of a murine coronavirus infection: A histopathological and ultrastructural study. Exp Pathol, in press. Hooks JJ: Ocular virology, in Tabbara K (ed): Infections of the Eye. Boston, Little, Brown & Co, in press. Hooks JJ, Percopo C, Wang Y, Detrick B: Retina and retinal pigment epithelial cell autoantibodies are produced during murine coronavirus retinop- athy. J Immunol 151:3381-3389, 1993. Wang Y, Detrick B, Hooks JJ: Coronavirus replica- tion within the retina: Analysis of cell tropism in mouse retinal cell cultures. Virology 193:124- 137, 1993. 85 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00287-01 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Toxoplasmosis Infections in the Eye PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: M. Cristina Martins Chandrasekharam Nagineni Miguel Burnier Robert B. Nussenblatt M.D. Guest Worker LI, NEI Ph.D. Visiting Scientist LLNEI M.D. Visiting Scientist LI, NEI M.D. Scientific Director NEI COOPERATING UNITS (if any) National Institute of Allergy and Infectious Diseases (R. Gazzinelli, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NTH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.5 PROFESSIONAL: 0.5 OTHER; 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (at) Minors □ (a2) Interviews □ (b) Human tissues [x| (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Toxoplasma gondii infections are a major source of visual loss and blindness. Ocular toxoplasmosis may occur as a result of congenital or acquired infections and as a manifestation of immunosuppression, particularly as a result of transplantation or AIDS (acquired immune deficiency syndrome). Due to the recent resurgence of acquired ocular toxoplasmosis in Brazil and the worldwide complications of toxoplasmosis in HIV (human immunodeficiency virus) infections, we initiated smdies to develop a model of acquired toxoplasmosis to evaluate the molecular mechanisms of pathogenesis and therapeutic strategies. We have developed an animal (murine) model of ocular toxoplasmosis that is characterized by retinal inflammation, chorioretinal scarring, retinal disorganization, and cyst formation. Retinal disease occurs in three different strains of mice following inoculation with toxoplasmosis by the subcutaneous or intraperitoneal routes. This model of acquired ocular toxoplasmosis is being used to evaluate the efficacy of new antiparasitic agents in controlling the development of retinal cyst formation and retinal inflammation. 86 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunoiogy Project Description Objectives This project was designed to develop an animal model of acquired ocular toxoplasmosis, which will be used to evaluate molecular mechanisms of ocular pathogenesis and to evaluate new antiparasitic drugs and cytokines. Methods This study involves the propagation and quantitation of Toxoplasmosis gondii strains in vitro and in vivo, as well as immunocytochemical analysis of infected cells and tissues. Techniques used in characteriza- tion of T. gondii infections include histopathology, immunocytochemistry, in situ hybridization, and Western blot analysis. Techniques used in character- ization of antitoxoplasmosis antibodies include enzyme-linked immimosorbent assay. Major Findings Adult Swiss Webster, C57BL6, and Balb/c mice were inoculated by the subcutaneous route or intra- peritoneal route with 10 T. gondii cysts (S2C9 or ME49 strains) in a 1 -ml volume. At various times after inoculation (i.e.. Days 7, 14, 21, 28, and 42), we sacrificed the mice and removed and fixed the eyes and brains in 10% buffered formalin. Fifteen hematoxylin and eosin-stained sections of brain and eye were evaluated for the presence of 7. gondii cysts. By Day 14, 100% of the mice had developed cysts in the brain. Retinal inflammation also was noted in 100% of the animals by Day 14, and chorioretinal scars were observed in mice inoculated with both strains of T. gondii. Retinal cysts were found in mice 28 and 42 days after inoculation with the MB49 strain and 14 and 42 days after inoculation with the S2C9 strain in Swiss Webster mice. T. gondii cysts in the retina were detected in C57BL6 mice at 14, 21, 28, and 42 days after inoculation with the S2C9 strain. This smdy has identified an animal model of ocular toxoplasmosis characterized by retinal inflammation, chorioretinal scarring, retinal disorganization, and cyst formation. Significance to Biomedical Research and the Program of the Institute This is the first model of acquired toxoplasmosis that consists of retinal inflammation, degeneration, and parasitic cyst formation. It will allow us to evaluate the efficacy of new antiparasitic drugs in controlling the development of retinal cyst formation and retinal inflanmiation and scarring. Proposed Course We will evaluate drugs and cytokines in the control of the ocular manifestations of acquired toxoplasmo- sis infections. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders 87 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00277-02 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Role of Retinal Pigment Epithelium in Retinal Disorders PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Chandrasekharam N. Nagineni Ph.D. Visiting Scientist LI, NEI Others: John J. Hooks Ph.D. Head, Section on Immunology LI, NEI and Virology COOPERATING UNITS (il any) Department of Pathology, George Washington University, Washington, DC (Barbara Detrick, Ph.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NTH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.0 PROFESSIONAL: 1.0 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The retinal pigment epithelium (RPE) plays a critical role in the regulation of retinal and choroidal function in normal and disease states. Due to limited availability of human tissues, an in vitro cell culture system is desired. Therefore we have developed and characterized the primary cell Unes of human RPE from donor eyes obtained from eye banks. Using human RPE cell cultures as a model, we conducted investigations to examine the various roles of RPE in the pathophysiology of retinal disorders. Human RPE cultures exposed to bacterial lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF-a), and interleukins 1 alpha and 1 beta (IL-loc, IL-lp) secreted large amounts of interleukin 6 (IL-6). Immunoblot and Northern blot analysis confirmed the presence of posttranslationally modified IL-6 protein and mRNA levels, respectively. Interferon-gamma (IFN-y) acted synergistically with other mediators to stimulate 3- to 5-fold increases in IL-6 secretion. We extended our studies to examine the expression and secretion of intercellular adhesion molecule- 1 (ICAM-1), a cell surface Ugand for lymphocyte function-associated antigen- 1 (LFA-1) expressed during inflammatory reactions, by human RPE. Wi^-y, TNF-oc, and IL-1 increased significantly both cell surface expression (detected by unmunofluorescence staining) and secretion into the medium (detected by ELISA). Secretion (shedding) of ICAM-1 by RPE cells in the presence of a mixture of EFN-y (100 u/ml), TNF-a (1 ng/ml), and IL-1 (I ng/ml) was cumulative, suggesting that combining these cytokines results in potent inflammatory reactions. The response of RPE cells to inflammatory mediators was rapid and sustained in the presence of stimulants but reversed to control levels quickly upon withdrawal, suggesting the reversibiUty of the responses of RPE to inflammatory signals. The effects of transforming growth factor beta (TGF-p) on RPE functions at cellular and molecular levels also are being studied. TGF-p increased the expression of heme oxygenase- 1, an enzyme reaction that generates the antioxidant bihrubin, which helps in cellular defense mechanisms against oxidative stfess. The results clearly show that human RPE cells respond to specific inflammatory signals or infections by increased cellular expression and secretion of IL-6 and lCAM-1, which may in tum perpetuate immune reactions in the pathogenesis and/or prevention of retinal and choroidal diseases. 88 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Objectives Human retinal pigment epithelial (RPE) cell cultures have been established from human donor eyes. Primary cell lines of RPE are used as an in vitro model to study the effects of growth factors, inflam- matory mediators, and toxic compounds on biochem- ical, cellular, and molecular aspects of RPE structure and function. The usefulness of RPE cultures also are being evaluated for transplantation to restore retinal function in hereditary and age-related disor- ders. Methods Primary cell cultures are prepared by initial seeding of either freshly isolated RPE cells or RPE-choroid explants. Cells are grown in minimum essential medium supplemented with 10% fetal calf serum, nonessential amino acids, and antibiotics. We are attempting to develop serum-free hormonally defined medium to render cultured cells more suitable for fransplantation therapy with minimal immune-related complications and consequent graft rejection. Techniques required for cell culture, immunofluo- rescence, cytokine, and ICAM-1 assays by enzyme- linked immunosorbent assay (ELISA), gel elecfro- phoresis, and Western and Northern blotting for pro- teins and RNA are developed and standardized in our laboratory to carry out these studies. Major Findings In the past, the age of the donor was considered very critical in preparing human RPE cultures because eyes from donors over 50 years did not yield fruitful cell lines, probably due to senescence-associated loss of viability. In these experiments, RPE cells were first dissociated from the eye cups by digestion with proteolytic enzymes, freatment that might have caused initial contamination with nonepitheUal cells, from which it is impossible to purify epithelial cells. Therefore, we modified this method, using RPE-cho- roid explants, native and without harsh enzyme freatment, to initiate cell growth. Then, by careful monitoring of clusters of cells growing around the explants, we were able (on the basis of morphology combined with experience) to select purely epithelial cells and discard nonepitheUal cells at the primary culture stage. Using this technique, we established primary cell lines of human RPE from eyes obtained from 81- and 87-year-old donors. The epitheUal nature of these cell lines was confirmed by inmiunochemical staining with monoclonal antibodies to cytokeratin. All of the cells expressed cytokeratin at different passages (3 to 10). Immunoblotting analysis of cellular proteins indicated cytokeratin 18 as the predominant cytokeratin in these cells. Because RPE is the only epithelial cell in the posterior segment (choroid-RPE-retina), these results establish without doubt that the cell lines developed are, in fact, RPE. The feasibility of using donor eyes from a population over 70 years of age for preparing RPE cultures is demonsfrated. Human RPE cultures secrete large quantities of inflammatory cytokine, interleukin 6 (IL-6), when exposed to inflammatory mediators — ^lipopolysacca- ride (LPS), tumor necrosis factor alpha (TNF-a), IL-la, and IL-ip. Western blot analysis revealed posttranscriptionally processed forms of IL-6 in the secreted proteins. Although interferon-y (IFN-y) induced the lowest levels of IL-6 by itself, it acted synergistically with other cytokines to stimulate threefold to fivefold increases in IL-6 secretion by RPE. Analysis of IL-6 secretion by ELISA and the expression of IL-6 mRNA by Northern blotting indicated rj^id, sustained RPE responses to inflam- matory mediators that can be reversed quickly upon withdrawal of the stimulus. Cell surface expression and secretion (shedding) of intercellular adhesion molecule 1 (ICAM-1), a cell surface glycoprotein ligand for lymphocytes, by RPE was significantiy stimulated by inflammatory cytokines — IFN-y, TNF-a, and IL-1. The presence of all these cyto- kines together appears to induce more potent secre- tion of ICAM-1. Regulation of the expression of ICAM-1 in RPE is under investigation through the use of monoclonal antibodies and cDNA probes. Our observations suggest that, in response to the presence of the inflammatory cytokines produced by macrophages and lymphocytes that, for example, infiltrate the eye during inflammation caused by infection or autoimmune disease, RPE can locally produce IL-6 and ICAM-1. In turn, IL-6 aids in the proliferation and differentiation of lymphoid cells to regulate immunological phenomena. Secreted or cell surface ICAM-1 expressed on RPE helps in homing and concentration of lymphocytes near the sites of inflammation for immunoregulation. 89 Laboratory of Immunology NEI Annual Report— FY 1993 Elevated levels of intravitreal IL-6, IL-1, TNF-a, and IFN-y were reported in proliferative and other noncomplicated retinal detachments. Moreover, intravitreal injection of IL-1, TNfF-a, or IL-6 induced uveitis in experimental animal models. These studies implicate local but not systemic increase in these cytokines as initiators of uveitis. Increases in the circulating ICAM-1 levels in the serum of uveitis patients and expression of ICAM-1 in epiretinal membranes in proliferative vitreoretinopathy, prolif- erative diabetic retinopathy, and macular pucker were reported, suggesting involvement of ICAM-1 in various diseases. Our studies indicate that RPE, possibly in association with other resident cells, reacts to inflammatory stimuli and participates in the immunopathologic mechanisms by secreting IL-6 and ICAM-1. Basic fibroblastic growth factor (bFGF) and transforming growth factor beta (TGF-|3) secreted by RPE are known to have both autocrine and paracrine actions on retina and choroid. bFGF and TGF-P are involved in various biological processes, such as cell proliferation, differentiation, wound healing, immu- nosuppression, and apoptosis. The roles of bFGF and TGF-P in RPE functions and the regulation of secretion of these growth factors by RPE are being investigated. We have studied the expression of heme oxygenase- 1 (HO-1), an enzyme known to respond to oxidative stress, heat shock, heavy metals, and inflammatory agents, that offer protection against oxidative damage. Among ocular tissues, RPE has the highest activity of HO-1. Using specific poly- clonal antibodies and PCR-generated probes, we have demonstrated that TGF-P increases HO-1 levels by fourfold to fivefold in human RPE cells within 4 hours. Toxic compounds — cadmium, lead, mercury, arsenite, and iodoacetate — are the most potent inducers of HO-1 in RPE. HO-1 catalyzes the oxidation of heme into biliverdin and carbon mon- oxide. Biliverdin is converted by nonlimiting enzy- matic reaction into bilirubin, an antioxidant that offers cells protection against heat and oxidative stress. Significance to Biomedical Research and the Program of the Institute Primary cell lines of human RPE are an ideal in vitro model for evaluation of several RPE functions and for further elucidation of the mechanisms of RPE involvement in the pathogenesis of retinal and choroidal diseases. These cells are potentially useful in cellular transplant therapy to correct hereditary and age-related macular degeneration defects in humans. Proposed Course Two of the major problems associated with human RPE cell cultures are (1) progressive loss of pigmen- tation upon serial passaging of cells and (2) lack of clear intercellular junctions and in vivo-like morpho- logical appearance. These changes may be due to cytoskeletal reorganization and partial dedifferentia- tion. Our immediate goal is to examine the mecha- nisms by which RPE cultures can be induced to resume in vivo characteristics. This will be achieved by selecting specific media composition, the addition of growth and differentiating factors, and/or culturing on suitable extracellular matrix. Development of a fully differentiated RPE cell line is crucial, not only for understanding cellular functions but also for cellular transplant therapy. Continuing to evaluate the effects of inflammatory cytokines and bacterial endotoxins on RPE cell cultures, we will address three areas: (1) influences of these factors on cellular cytoskeletal organization, intercellular junctions, and adhesion properties; (2) effects on cell functions (e.g., membrane perme- ability and solute transport and phagocytosis); and (3) characterization of proteins such as growth factors, cytokines, and proteolytic enzymes secreted by RPE in response to various stimuli. These studies are likely to shed light on the role of RPE in the pathophysiology of the retina and choroid, tissues that are in close proximity to and directly influenced by RPE. NEI Research Program Retinal Diseases — Inflammatory Diseases, Macular Degeneration, Photoreceptors, and Retinal Pigment Epithelium Publications Kutty RK, Nagineni CN, Kutty G, Hooks JJ, Chader GJ, Wiggert B: Transforming growth factor-p increases the expression of heme oxygenase 1 in human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 34(4)(suppl):1451, 1993. Nagineni CN, Detrick B, Hooks JJ: Interferon-y acts synergistically with inflammatory mediators to induce expression of interleukin 6 by human retinal pigment epithelial cells. Invest Ophthal- mol Vis Sci 34(4)(suppl):1020, 1993. 90 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00222-08 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Immunopathology in Eyes With Experimental and Clinical Ocular Diseases PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Chi-Chao Chan M.D. Head, Section on LI, NEI Immunopathology Others: Qian Li M.D. Visiting Associate LI, NEI Kourosh Dastgheib M.D. IRTA FeUow LI, NEI Deborah Luyo Technician LI, NEI Scon M. Whitcup M.D. Medical Officer LI, NEI Francois G. Roberge M.D. Visiting Scientist LI, NEI Rachel R. Caspi Ph.D. Visiting Scienust LI, NEI Igal Gery Ph.D. Deputy Chief LI, NEI Robert B. Nussenblan M.D. Scientific Director NEI COOPERATING UNITS (if any) Department of Ophthalmology, Kunime University, Kurume, Japan (Manabu Mochizuki, M.D.) LAB/BRANCH Laboratory of Inmiunology SECTION Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 4.4 PROFESSIONAL: 3.4 OTHER: 1.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (b) Human tissues [xj (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The identity and topographic localization of immunocompetent cells, the alteration of surface markers on ocular resident cells, and their cytokines in animals with experimental autoimmune uveoretinitis or endotoxin- induced uveitis (EAU, EIU) and in human ocular tissues with various diseases were analyzed by immunohistochemical smdies and in situ hybridization. T lymphocytes were the predominant infiltrating cells in EAU, yet both maaophages and polymorphonuclear neutrophils (PMNs) were the predominant infiltrating cells in EIU. Migration of the inflammatory cells from tlie vessels into the target site is directed by adhesion molecules, which can be expressed on vascular endothelium and other resident cells in the eye. Mast cells appear to participate in the immunopathogenesis of EAU and EIU. T-lymphocyte specificity is directed to small fragments of antigen bound to cell surface major histocompatibility complex (MHC) molecules, which are presented on the surface of specialized antigen-presenting cells. The expression of MHC class 11 antigens was observed on ocular resident cells such as retinal pigment epithelium (RPE), retinal endothelium, keratocytes, fibroblasts, and ciliary epithelium in rodents. Both the infiltrating cell subpopulation and the expression of class II antigens and adhesion molecules on ocular resident cells can be altered by various immunomodulating agents and cytokines. Specimens from human ocular tissues with various diseases — such as uveitis, retinal disease, conjunctival and corneal diseases, metabolic genetic diseases, and tumors — are studied using immunohistochemical and in situ hybridization techniques as well as light and electron microscopic evaluation. In uveitis, immunocompetent cells and lymphokines are valuable adjuncts to clinical diagnosis, and they are determinants of disease course and prognosis. In nonuveitic conditions, alteration of cellular membrane surface markers and intracytoskeleton of the ocular resident cells may imply damage and abnormalities in these diseases. Elucidating the immunopathological role of the relationships between infiltrating inflammatory or malignant cells and other resident cells in the clinical behavior of various diseases will increase our understanding of human ocular disorders. 91 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report— FY 1993 Project Description Objectives This program is designed to evaluate the clinical manifestation, histopathology, and immunopathology of the ocular tissue when experimental autoimmune uveoretinitis (EAU) and endotoxin-induced uveitis (EIU) are induced and/or modulated by various immunosuppressive agents in various animal species. Ocular tissues obtained from patients with various diseases, including inflammatory and noninflamma- tory disorders, also are studied. The infiltrating inflammatory cells, ocular resident cells and their products, and various lymphokines and cytokines are examined. The findings will help us understand ocular inflammation and the pathogenesis of each disease examined in humans. Methods Clinical examinations include flashlight and slit-lamp examinations as well as examination of the fundus of animals and patients under the dissecting microscope. Pathological examinations include routine histologic techniques for light and electron microscopy, immu- nofluorescence, avidin-biotin-peroxidase complex methods, and in situ hybridization techniques. Major Findings We continued to study the immunopathology of various inflammatory cells and ocular resident cells in different experimental models of uveitis. We have observed that, prior to the infiltration of inflamma- tory cells into the eye, the number of mast cells in the anterior uvea decreases and is consistent with mast cell degranulation in EAU and EIU. This observation suggests that anterior uveal mast cells participate in uveitis by releasing vasoactive amines to alter the integrity of the blood-ocular barrier and amplify the inflammatory process. Toward understanding the immunopathological process in various experimental uveitides, we have examined the efficacy of different anti-inflammatory mediators and immunomodulating agents in these animal models. For example, we have found that feeding animals rat chow mixed with CGS- 13080, a thromboxane synthetase inhibitor, suppresses the development of clinical and histopathological EAU. Inhibition of this enzyme results in a reduction of thromboxane B^ and an increase of prostaglandin E in the serum, thus altering the inflammatory mediator and suppressing EAU. Antibodies against adhesion molecules are able to abrogate EAU and EIU because expression of adhesion molecules precedes the flux of inflamma- tory cells into the eye. The cytokine cascade in the inflammatory process is complicated. Tumor necro- sis factor a has a paradoxical role in EIU. Immuno- suppressive agents — ^in particular the inhibitors of T- cell function, such as cyclosporine A, FK 506, and rapamycin, which interfere with the release of lymphokines — are potent and effective medications to treat EAU, a T-cell-mediated autoimmune uveo- retinitis. Using immimopathological techniques, we exam- ine ocular tissues obtained from patients with various ocular diseases to help visualize the pathology and the kinetics of the specific disease process. The findings provide useful information for understanding the pathological mechanisms of the disease, deter- mining the diagnosis, and guiding the subsequent management of patients. We found collagen dysgenesis in Reis-Buckler corneal dystrophy. The presence of immature collagen type III and poorly developed collagen type I may contribute to the pathogenesis of Reis-Buckler dystrophy. In Cogan-Reese syndrome, we found that the iris nevi are cells that originate in the neural crest and have numerous melanosomes, junctional com- plexes, and basement membrane. We demonstrated the presence of oB-crystallin, a major lens protein in retinoblastoma, suggesting that oB-crystallin is involved in tumor growth and/or is a marker for general oncogenic "stress" in retinoblastoma We also have shown the presence of tachyzoites and the role of T lymphocyte in congenital toxoplasmosis. Using in situ hybridization, we detected the RNAs of botii interleukins 2 and 4 in the conjunctiva of ocular onchocercal patients, suggesting that Th2 cells and their lymphokines are important for localized host responsiveness to ocular onchocerciasis. In practice, correct handling and processing of surgical specimens obtained from vitrectomy and/or chorioretinal biopsy can yield important information, in particular, the diagnosis of intraocular large B-cell lymphoma (central nervous system lymphoma) and progressive chorioretinal lesions of unknown etiol- ogy. Once the diagnosis is made, the appropriate treatment can be offered. 92 NEI Annual Report— FY 1993 Laboratory of Immunology We are investigating other experimental models, e.g., allergic conjunctivitis, melanin-protein-induced uveitis (EMIU), and acquired toxoplasmosis, and their resemblance to other ocular inflammatory diseases in humans. We hope to better understand the mechanisms of ocular inflammation and evaluate the effects of different therapeutic approaches in these different new models. Significance to Biomedical Research and the Program of the Institute Immunopathological findings on experimental uve- itides have provided information on various inflam- matory cells and ocular resident cells during the process of ocular inflammation. This information helps us to choose and evaluate novel pharmacologic agents and provide better therapeutic intervention of uveitis in humans. Studies of ocular tissues obtained from patients with various disorders have enabled us to gain information on the mechanism, diagnosis, and management of these ocular diseases. This informa- tion is useful in treating patients not only with uveitis but also with ocular tumors and congenital disorders. Proposed Course Various experimental models, including EAU, EIU, EMIU, allergic conjunctivitis, and acquired toxoplas- mosis, will be studied clinically, histopathologically, and immunopathologically in different species and strains. Various pharmacological agents and the role of cytokines, lymphokines, enzymes, and cellular surface markers will be evaluated in these models. Also, we propose continuation of analysis of human specimens in the study of their immunopathogenesis. NEI Research Program Retinal and Choroidal Disease — Inflammatory Disorders Publications Brezin AP, Kasner L, Thulliez P, Li Q, Daffos F, Nussenblatt RB, Chan C-C: Ocular toxoplasmo- sis in the fetus: Immunohistochemistry and DNA amplification. Invest Ophthalmol Vis Sci 34(4)(suppl):1001, 1993. Brezin AP, Kasner L, Thulliez P, Li Q, Daffos F, Nussenblatt RB, Chan C-C: Ocular toxoplasmo- sis in the fetus: Immunohistochemistry and DNA amplification. Retina, in press. Bucci FA Jr, Li Q, Luyo D, Tanner J, Chan C-C: Detection of T lymphocytes in patients with allergic conjunctivitis. Invest Ophthalmol Vis Sci 34(4)(suppl):853, 1993. Caspi RR, Chan C-C, Fujino Y, Najafian F, Grover S, Hansen CT, Wilder RL: Recruitment of antigen-nonspecific cells play a pivotal role in the pathogenesis of a T-cell-mediated organ-specific autoimmune disease, experimental autoimmune uveoretinitis. J Neuroimmunol 41:171-1^^, 1993. Caspi RR, Chan C-C, Fujino Y, Najafian F, Grover S, Hansen CT, Wilder RL: Recruitment of naive T cells plays a pivotal role in the pathogenesis of experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci 34(4)(suppl):902, 1993. Chan C-C, Cogan DG, Bucci FS, Barsky D, Li Q, Crawford MA: An anterior corneal dystrophy with dyscollagenosis (Reis-Buckers type?). Cornea 12:451-460, 1993. Chan C-C, Hikita N, Dastgheib K, Whitcup SM, Gery I, Nussenblatt RB: Immunopathology of experimental autoimmune anterior uveitis (EAAU). Invest Ophthalmol Vis Sci 34(4) (suppl):999, 1993. Chan C-C, Li Q, Brezin AP, Whitcup SM, Egwuagu C, Otteson EA, Nussenblatt RB: Immunopathol- ogy of ocular onchocerciasis. 3. Th-2 helper T cells in the conjunctiva. Ocular Immunol Inflam 1:71-77, 1993. Chepelinsky AB, Overbeek PA, Chan C-C, Jamieson S, Dickson C, Parker DM, Robinson W: Int2 ectopic expression induces differentiation of secretory epithelia in the eyes of transgenic mice. Invest Ophthalmol Vis Sci 34(4)(suppl):1222, 1993. Dastgheib K, Hikita N, Sredni B, Albeck M, Nussen- blatt RB, Chan C-C: Ocular inflammation stimu- lated by the immunomodulator ASIOI. Invest Ophthalmol Vis Sci 34(4)(suppl):1483, 1993. Egwuagu CE, Sztein J, Chan C-C, Reid W, Mahdi R, Nussenblatt RB, Chepelinsky AB: Gamma- interferon expression in the eyes of transgenic mice disrupts differentiation of the lens and retina. Invest Ophthalmol Vis Sci 34(4)(suppl): 1455, 1993. 93 Laboratory or Immunology NEI Annual Report— FY 1993 Fujino Y, Li Q, Chung H, Hikita N, Nussenblatt RB, Chan C-C: Immunopathology of experimental autoimmune uveoretinitis in primates. Autoimmu- nity 13:303-309, 1992. Kara Y, Caspi RR, Wiggert B, Chan C-C, Streilein JW: Use of ACAID to suppress interphotorecep- tor retinoid-binding protein-induced experimental autoimmune uveitis. Curr Eye Res 1 l(suppl):97- 100, 1992. Hikita N, Chan C-C, Mochizuki M, Maturi R, Nus- senblatt RB, Whitcup SM: Topical FK 506 inhibits endotoxin-induced uveitis (EIU). Invest Ophthalmol Vis Sci 34(4)(suppl):1480, 1993. Holland EJ, Chan C-C, Bergstrom L, Palestine AG, Nussenblatt RB: Kinetics of corneal transplant rejection in the rat penetrating keratoplasty model. Cornea, in press. Holland EJ, Hardten DR, Murali S, Lushine K, DeMartelaere S, Olevsky OM, Mindrup EA, Karlstad C, Chan C-C: Effect of topically admin- istered platelet-derived growth factor on corneal wound strength in penetrating keratoplasty. Invest Ophthalmol Vis Sci 34(4) (suppl):1375, 1993. Kasner L, Chan C-C, Whitcup SM, Gery I: The paradoxical effect of tumor necrosis factor alpha (TNF-a) in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 34:2911-2917, 1993. Kasner L, Chan C-C, Whitcup SM, Gery I: The paradoxical role of tumor necrosis factor-alpha in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 34(4)(suppl):1480, 1993. Kupfer C, Chan C-C, Bumier M Jr, Kaiser-Kupfer MI: Histopathologyofthe ICE syndrome. Trans Am Ophthalmol Soc 90:149-160, 1992. Lai JC, Chan C-C, Li Q, Whitcup SM: Treatment with corticosteroids and cyclosporine A inhibits the expression of cell adhesion molecules in experimental autoimmune uveitis (EAU). Invest Ophthalmol Vis Sci 34(4)(suppl):1206, 1993. Li Q, Fujino Y, Caspi RR, Najafian F, Nussenblatt RB, Chan C-C: Association between mast cells and the development of experimental autoimmune uveitis in different rat strains. Clin Immunol Immunopathol 65:294-299, 1992. Li Q, Hikita N, Whitcup SM, Nussenblatt RB, Chan C-C: Allergic conjunctivitis induced by com- pound 48/80 in C57BL/6NCR mice. Invest Ophthalmol Vis Sci 34(4)(suppl):857, 1993. Li Q, Lopez JS, Caspi RR, Roberge FG, Nussenblatt RB, Kador P, Chan C-C: Suppression of S- antigen-induced experimental autoimmune uveo- retinitis in Lewis rats by oral administration with CGS- 13080, a thromboxane synthetase inhibitor. Exp Eye Res, 57:601-608, 1993. Li Q, Whitcup SM, Fujino Y, Nussenblatt RB, Chan C-C: The role of mast cells in endotoxin induced uveitis. Invest Ophthalmol Vis Sci 34:256-259, 1993. Martin DF, DeBarge LR, Nussenblatt RB, Chan C-C, Roberge FG: Synergistic effect of rapamycin and cyclosporine A on the inhibition of experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci 34(4)(suppl):1476, 1993. Martin DF, Chan C-C, de Smet MD, Palestine AG, Davis JL, Whitcup SM, Burnier MN Jr, Nussen- blatt RB: The role of chorioretinal biopsy in the management of posterior uveitis. Ophthalmology 100:705-714, 1993. Parks DJ, Hikita N, Nagineni C, Hooks JJ, Chan C-C, Nussenblatt RB, de Smet MD: Immunohis- tochemistry of xenogeneic RPE transplants in the rat: A model for graft rejection. Invest Ophthal- mol Vis Sci 34(4)(suppl):1095, 1993. Pineda R U, Chan C-C, Ni M, Hayden BJ, Johnson MA, Chader GJ: Human retinoblastoma cells express oB-crystallin in vivo and in vitro. Curr Eye Res 12:239-245, 1993. Rizzo LV, Silver PB, Hakim F, Chan C-C, Wiggert B, Caspi RR: Establishment and characterization of an IRBP-specific T-cell line that induces EAU in BIO. A mice. Invest Ophthalmol Vis Sci 34(4) (suppl):1143, 1993. Roberge FG, Kozhich A, Chan C-C, Martin DF, Nussenblatt RB, de Smet MD: Inhibition of cellular transfer of experimental autoimmune uveoretinitis by rapamycin. Ocular Immunol Inflam 1:269-273, 1993. Roberge FG, Xu D, Chan C-C, de Smet MD, Nus- senblatt RB, Chen H: Treatment of autoimmune uveoretinitis in the rat with rapamycin, an inhibi- tor of lymphocyte growth factor signal transduc- tion. Curr Eye Res \2:\91-lQ'i,\99l. Shah DN, Piacentini MA, Bumier MN Jr, McLean IW, Nussenblatt RB, Chan C-C: Inflammatory 94 NEI Annual Report— FY 1993 Laboratory of Immunology cellular kinetics in sympathetic ophthalmia. A study of 29 traumatized (exciting) eyes. Ocular Immunol Inflam 1:255-262, 1993. Silver PB, Rizzo LV, Chan C-C, Donoso LA, Wig- gert B, Caspi RR: Identification of a putative epitope in the IRBP molecule that is uveitogenic for mice of the H-2b h^lotype. Invest Ophthal- mol Vis Sci 34(4)(suppl):1482, 1993. Vistica B, Gery I, Chan C-C, Nussenblatt RB, Whit- cup SM: Anti-ICAM-1 and anti-LFA-1 mono- clonal antibodies (mAbs) inhibit in vitro prolifera- tion of a uveitogenic cell line. Invest Ophthalmol Vis Sci 34(4)(suppl):1144, 1993. Westeren AC, Chan C-C, de Smet MD, Nussenblan RB, Roberge FR: Evaluation of the immunosup- pressant SK&F 106610 in the treatment of experi- mental autoimmune uveoretinitis. Invest Ophthal- mol Vis Sci 34(4)(suppl):1477, 1993. Whitcup SM, DeBarge LR, Caspi RR, Haming R, Nussenblatt RB, Chan C-C: Monoclonal anti- bodies against ICAM-1 (CD54) and LFA-1 (CD 11 a/CD 18) inhibit experimental autoimmune uveitis. Clin Immunol Immunopathol 67: 143-150, 1993. Whitcup SM, DeBarge LR, Rosen H, Nussenblatt RB, Chan C-C: Monoclonal antibody against CSl lb/CD 18 iniiibits endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 34:673-681, 1993. Whitcup SM, de Smet MD, Rubin BI, Palestine AG, Martin DF, Burnier M Jr, Chan C-C, Nussenblatt RB: Intraocular lymphoma: Clinical and histo- pathologic diagnosis. Ophthalmology 100:1399- 1406, 1993. Whitcup SM, Hikita N, Shirao M, Mochizuki M, Nussenblatt RB, Chan C-C: Effect of monoclonal antibodies against ICAM-1 (CD54) and LFA-1 alpha (CDl la) in the prevention and treatment of endotoxin-induced uveitis (EIU). Invest Ophthal- mol Vis Sci 34(4)(suppl):1143, 1993. Whitcup SM, Nussenblatt RB; Price FW Jr, Chan C-C: Expression of cell adhesion molecules in corneal graft failure. Cornea 12:475-480, 1993. 95 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00241-07 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) Immunopathology of Ocular Diseases in Humans PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aftiliatlon) PI: Chi-Cbao Chan Others: Robert B. Nussenblatt Qiao Li Marc D. de Smet Raymond DeBarge Scott M. Wfaitcup Juan Lopez Miguel Bumier Richard Fenton Dev Shah M.D. M.D. M.D. M.D. M.D. M.D. M.D. M.D. M.D. M.D. Chief, Section on Inununopathology Scientific Director Visiting Fellow Visiting Scientist Senior Staff Fellow Staff Medical Officer Visiting Associate Visiting Scientist Staff Fellow Visiting Associate LI, NEI NEI LI, NEI LI, NEI LLNEI LLNEI LI, NEI LI, NEI LLNEI LINE] COOPERATING UNITS (if any) Department of Ophthalmology, Armed Forces Institute of Pathology (Ian W. McLean, M.D.); University of Minnesota, Department of Ophthalmology (Edward J. Holland, M.D.); L'Hdpital de la Piti6, Paris, France (Phuc LeHoang, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.0 PROFESSIONAL: OTHER: 0.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project has been terminated and combined with Project No. ZOl EY 00222-08 LI. 96 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00264-04 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or lass. Title must tit on one line between the borders.) Cytokines and Ocular Antigens in the Eye PRINCIPAL INVESTIGATOR (List other profossional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Chi-Chao Chan M.D. Head, Section on LI, NEI Immunopathology Others: Robert B. Nussenblatt Igal Gery Qian Li Louis Kasner M.D. Ph.D. M.D. M.D. Scientific Director LI, NEI Head, Section on LI, NEI Experimental Immunology Visiting Fellow LI, NEI Fellow LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.0 PROFESSIONAL; 0.0 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) n (a) Human subjects □ (a1) Minors □ (a2) Interviews [xl (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use Standard unreduced type. Do not exceed the space provided.) This project has been terminated and combined with project number ZOl EY 00222-08 LI. 97 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00268-03 LI PERIOD COVERED October 1, 1992 to September 30. 1993 TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) The Diagnosis and Treatment of Human Uveitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, lataratory, and institute affiliation) PI: Scott M. Whitcup M.D. Staff Medical Officer LI, NEI Others: Robert B. Nussenblatt Marc D. de Smet Chi-Chao Chan M.D. M.D. M.D. Scientific Director Visiting Scientist Medical Officer NEI LI, NEI LI, NEI COOPERATING UNITS (if any) Department of Medicine, The Johns Hopkins University, Baltimore, MD (David R. MoUer, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Inmiunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews PROFESSIONAL; 1.0 OTHER: 0.0 □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The goal of this project is to develop improved methods for diagnosing and treating human uveitis. Three studies of diagnosis are as follows: (1) We examine biopsy and pathology specimens from patients with uveitis and AIDS to develop improved diagnostic tests and to understand better the pathophysiology of inflammatory eye disease. Ongoing studies of intraocular lymphoma show that multiple vitrectomies or lumbar punctures are required to diagnose about one- third of the patients. Appropriate, prompt handling of pathology specimens by an experienced cytopathologist remains critical to making the correct diagnosis. (2) To improve methods for diagnosing ocular sarcoidosis, we test lacrimal gland and conjunctival biopsies for the presence of interferon-gamma; Kveim antigen; interleukins 2, 3, 4, 6, and 8; and T-c^U receptors believed to be specific for this disease. This year we retrospectively reviewed 46 patients with biopsy-proven sarcoidosis and 21 with uveitis. In patients with ocular involvement, the most sensitive diagnostic test was the pulmonary diffusing capacity (DLCO), which diminished in 78% of patients tested. Corticotropin-releasing hormone tests are performed on patients with uveitis to determine whether a defective hypothalamic-pituitary-adrenal axis is associated with increased risk for autoimmune ocular inflammatory disease. (3) Our study of animals with endotoxin-induced uveitis (EIU) showed that tumor necrosis factor alpha causes a paradoxical exacerbation of ocular disease. In the area of treatment, we have three projects: (1) We are continuing a masked, randomized crossover study to compare acetazolamide with placebo for the treatment of uveitic cystoid macular edema; to date 31 patients have been recruited. (2) Topically applied FK 506 was used to treat EIU in the rat. Ocular inflammation was reduced significandy in animals treated with topical FK 506 (0.3% and 0.05%) when compared with control animals, a finding that may be useful in the treatment of acute ocular inflammation in humans. (3) The optimal therapy for intraocular lymphoma remains unclear; however, previous studies suggest that untreated patients die within 1 year of diagnosis. Retrospective review of 11 patients with intraocular lymphoma treated with radiation, chemotherapy, or both showed substantial treatment-related mortality. In a joint protocol with the National Cancer Institute, we now are investigating alternative treatment regimens for central nervous system lymphoma. 98 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Additional Personnel Emily Chew M.D. Visiting Scientist, BEP, NEI Frederick Ferris, III M.D. Chief, Clinical Trials Branch, BEP, NEI George P. Chrousos M.D. Diabetes Epidemiology Branch (DEB), National Institute of Child Health and Disease (NICHD) Daniel Martin M.D. Senior Staff Fellow, LI, NEI George Mastorakos M.D. Visiting Scientist, DEB, NICHD Igal Gery Ph.D. Deputy Chief, LI, NEI Clinical Protocol Numbers 90-EI-132 91-EI-30 91 -EI- 139 92-EI-0070 Objectives The goal of this study is to develop better methods for the diagnosis and treatment of human uveitis. We also are interested in defining the pathophysiol- ogy of inflammatory eye diseases by analyzing human tissue and animal models of uveitis. Methods Diagnosis of Uveitis 1. To improve the diagnostic yield of conjuncti- val and lacrimal gland biopsies for sarcoidosis, we are examining tissue specimens using immunohisto- chemical staining. Conjunctival and lacrimal gland biopsies will be performed on 10 patients with known sarcoidosis and snap frozen in O.C.T.® Immunohistochemical staining will be performed using an avidin-biotin-peroxidase complex. Primary monoclonal antibodies against T-cell markers, T-cell receptors, Kveim antigen, and various interieukins will be applied. The results will be compared with those of biopsies from patients with other uveitic conditions, such as Behcet's disease, to determine the specificity of these results. We also have reQ-ospec- tively reviewed the records of patients with biopsy- proven sarcoidosis to determine the sensitivity of current tests obtained to diagnose sarcoidosis. 2. Intraocular lymphoma often masquerades as an idiopathic uveitis, which delays the stan of appro- priate therapy. We continue to collect data on patients diagnosed with intraocular lymphoma. 3. We are performing corticotropin-releasing hormone tests to access the hypothalamic-pituitary- adrenal axis in patients with autoimmune uveitis. Subnormal Cortisol production in response to this hormone may predispose patients to the development of autoimmune disease. 4. The pathophysiology of endotoxin-induced uveitis (EIU) is being studied using immunohisto- chemistry, histology, and monoclonal antibodies against various cytokines. Treatment of Uveitis 1. The efficacy of acetazolamide for the treat- ment of uveitis-associated macular edema is being evaluated in a masked, crossover study comparing acetazolamide with placebo. Visual acuity and the height of the macular edema measured by fluorescein angiography are the primary endpoints. 2. We are testing the efficacy of topically applied FK 506, a new immunosuppressive agent, for the treatment of acute anterior uveitis, using the animal model of EIU in the rat Histologic evidence of intraocular inflammation and aqueous humor protein concentrations are compared between treated and control animals. 3. In an investigation of treatment for patients with intraocular lymphoma, we are reviewing both morbidity and mortality. In addition, we are partici- pating in a joint protocol with the National Cancer Institute to study chemotherapy on non-Hodgkin's lymphoma arising in the central nervous system (CNS) or the eye. 4. We are comparing frabeculectomy combined witii subconjunctival 5-fluorouracil to the Molteno implant for the treatment of glaucoma secondary to uveitis. Major Findings 1. We retrospectively reviewed 46 patients with biopsy-proven sarcoidosis, 21 with uveitis. In patients with ocular involvement, only 61% had abnormal chest x-rays; 36% had an elevated angio- tensin-converting enzyme. The most sensitive 99 Laboratory of Immunology NEI Annual Report— FY 1993 diagnostic test was the pulmonary diffusing capacity, which was diminished in 78% of the patients tested. There was no statistically significant difference between the test results of sarcoidosis patients with and without uveitis. Among 21 uveitis patients, 14 (67%) had visual acuity of 20/40 or worse in at least one eye. Poor visual acuity (20/200 or worse) was predominantly caused by secondary glaucoma. 2. Examination of the use of topical FK 506 for the treatment of EIU showed that the mean anterior chamber cell count per microliter and the median histologic grade of ocular inflammation (scale of to 4) were significantly decreased in rats treated with topical FK 506 0.05% and FK 506 0.3% when compared with those of placebo-treated rats. The blood levels of FK 506 in rats treated with topical 0.05% and 0.3% FK 506 were 1.2 and 2.9 mg/ml, respectively— more than tenfold lower than levels obtained with systemic therapy at a dose of 1 mg/kg. 3. Retrospective review of 11 patients with intraocular lymphoma treated with radiation, chemo- therapy, or both showed that 5 patients died a mean of 21 months after diagnosis while 6 have survived a mean of 33 months. We performed autopsies on three of the five patients who died. Interestingly, no residual lymphoma was found in any of the three, whose deaths were felt to result from treatment- related complications, predominantly severe leuko- encephalopathy. This substantial treatment-related mortality suggests that improved ther^)eutic regi- mens are needed. We are currently involved in a joint protocol with the National Cancer Institute, investigating alternative treatment regimens for CNS lymphoma. Significance to Biomedical Research and the Program of the Institute Uveitis accounts for about 10% of visual impairment in the United States. A major goal of the ^fEI is to improve the methods for diagnosing and treating uveitis in an attempt to preserve useful vision in patients with inflammatory eye disease. Proposed Course We will continue patient recruitment for the clinical trials of cystoid macular edema, corticotropin-releas- ing hormone, sarcoidosis, uveitic glaucoma, and intraocular lymphoma. We have completed our initial studies, showing the effectiveness of topically applied FK 506 for the treatment of EIU, and we are planning to investigate the use of Uposome-bound FK 506 to improve ocular penetration of topically applied compounds. In addition, studies on the effect of cytokines on ocular inflammatory disease will continue. NEI Research Program Retinal Diseases — Inflammatory Diseases Publications Chan C-C, Li Q, Brezin AP, Whitcup SM, Egwuagu C, Otteson EA, Nussenblatt RB: Immunopathol- ogy of ocular onchocerciasis. 3. Th-2 helper T cells in the conjunctiva Ocular Immunol Inflam \:1\-11, 1993. Fenton RM, Rubin BI, de Smet MD, Whitcup SM, Nussenblatt RB: A prospective study of 5-FU trabeculectomy vs. single plate Molteno implant in patients with panuveitis complicated by glauco- ma refractory to prior therapy. Invest Ophthalmol Vis Sci 34(4)(suppl):897, 1993. Hikita N, Chan C-C, Mochizuki M, Maturi R, Nus- senblatt RB, Whitcup SM: Topical FK 506 inhibits endotoxin-induced uveitis (EIU). Invest Ophthalmol Vis Sci 34(4)(suppl):1480, 1993. Kasner L, Chan C-C, Whitcup SM, Gery I: The paradoxical role of tumor necrosis factor-alpha in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 34(4)(suppl):1480, 1993. LI Q, Hikita N, Whitcup SM, Nussenblatt RB, Chan C-C: Allergic conjunctivitis induced by com- pound 48/80 in C57BL/6NCR mice. Invest Ophthalmol Vis Sci 34(4)(suppl):857, 1993. Li 0. Whitcup SM, Fujino Y, Nussenblatt RB, Chan C-C: The role of mast cells in endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 34:256-259, 1993. Martin DF, Chan C-C, de Smet MD, Palestine AG, Davis JL, Whitcup SM, Burnier M Jr, Nussenblatt RB: The role of chorioretinal biopsy in the management of posterior uveitis. Ophthalmology 100:705-714, 1993. Whitcup SM, de Smet MD, Rubin BI, Palestine AG, Martin DF, Burnier M Jr, Chan C-C, Nussenblatt RB: Intraocular lymphoma: Clinical and histo- pathologic diagnosis. Ophthalmology, 100:1399- 1406, 1993. Whitcup SM, Fenton RM, Pluda JM, de Smet MD, Nussenblatt RB, Chan C-C: Pneumocystis carinii and Mycobacterium avium-intracellulare infection of the choroid. Retina 12:331-335, 1992. 100 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1, 1992 to September 30, 1993 PROJECT NUMBER ZOl EY 00269-03 LI TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Ocular Toxicity of 2^3^-DideoxyinosiDe (ddl) PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Scott M. Whitcup M.D. Staff Medical Officer LI, NEI Others: Robert B. Nussenblatt Marc D. de Smet Rafael Caruso M.D. M.D. M.D. Scientific Director Visiting Scientist Visiting Scientist NEI LI, NEI OGCSB, NEI COOPERATING UNITS (If any) Pediatric Branch, National Cancer Institute (Philip A. Pizzo, M.D.); Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases (Clifford H. Lane, M.D.); Clinical Oncology Program, National Cancer Institute (Robert Yarchoan, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.4 PROFESSIONAL; 0.4 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 2',3'-Dideoxyinosine (ddl), a purine analog with antiretroviral activity currentiy used to treat patients with AIDS (acquired immune' deficiency syndrome), is being used to treat both adults and children in clinical protocols at the National Institutes of Health. The purpose of this study is to follow prospectively patients treated with ddl for the development of ocular complications secondary to drug toxicity. Ninety-five children with symptomatic (CDC class P-2) HIV (human immunodeficiency virus) infection were enrolled in a phase I/II study to assess the safety and antiretroviral activity of ddl. Five children developed peripheral atrophy of the retinal pigment epithelium during ddl therapy. The two children with the most severe retinal atrophy were enrolled in the smdy at the highest dose level studied (540 mg/m^/day). Electro-oculograms were abnormal in one of three patients with retinal toxicity who could be tested. A group of 75 adults treated with ddl are being followed with periodic fundus examinations and electro-oculograms. During the past year similar retinal lesions were found in one adult patient treated with ddl. 101 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report— FY 1993 Project Description Additional Personnel Daniel Martin Margaret Cheung David Parks John J. Hooks Caroline Percopo M.D. Senior Staff Fellow, LI, NEI M.D. Senior Staff Fellow M.D. Senior Staff Fellow Ph.D. Head, Section on Immunology and Virology, LI, NEI M.S. Biologist, LI, NEI Objectives The goal of this study is to monitor patients treated with 2',3'-dideoxyinosine (ddl) for the development of ocular complications. Methods Every 3-4 months patients treated with ddl are given complete eye examinations, including dilated oph- thalmoscopy and fundus photography of any abnor- mal retinal findings. Patients treated with the higher dosages of ddl also receive periodic electro-oculo- grams to assess the electrophysiologic function of the retinal pigment epithelium (RPE). Major Findings 1. Five children have now developed peripheral atrophy of the RPE during ddl therapy. The lesions are scalloped areas of RPE atrophy with hyperpig- mented borders. They occur predominantly in the midperiphery of the fundus in both eyes. These retinal lesions slowly progress if ddl therapy is continued, but central visual acuity has remained unaffected. During the past year no discrete retinal lesions have developed in any other children treated with ddl. 2. One adult patient treated with ddl developed progressive, well-circumscribed atrophic retinal lesions of the peripheral RPE, similar to those in the children. The lesions appeared after 32 months of ddl treatment; the total dosage received was 264 g (approximately 3.3 g/kg). Adjacent areas of RPE mottling also were seen. Visual acuity and electro- oculography were normal in this patient. Significance to Biomedical Research and the Program of the Institute ddl is a drug with in vitro and in vivo activity against HIV (human immunodeficiency virus) infec- tion. One mission of the NEI is to monitor patients for the development of ocular toxicity and to assess the effect such toxicity has on vision. Proposed Course We will continue to follow all patients treated with ddl at the NIH for signs of ocular manifestations of ddl toxicity or HIV infection. We are performing serial electro-oculograms in adults treated with ddl. NEI Research Program Retinal Diseases — ^Photoreceptors and Retinal Pig- ment Epithelium Publications Nguyen B-Y, Shay LE, Wyvill KM, Pluda JM, Brawley O, Cohen RB, Whitcup SM, Venzon DJ, Broder S, Yarchoan R: A pilot study of sequen- tial therapy with zidovudine (AZT) plus acyclo- vir,dideoxyinosine, dideoxcytidine in patients with severe human immunodeficiency virus infection. J Infect Dis 168:810-817, 1993. 102 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00270-03 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Cell Adhesion Molecules in Ocular Inflammation PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atfitiation) PI: Scott M. Whitcup M.D. Staff Medical Officer LI, NEI Others: Chi-Chao Chan Robert B. Nussenblatt M.D. M.D. Head, Section on Inmiunopathology Scientific Director LI, NEI NEI COOPERATING UNITS (if any) Biochemical and Molecular Pathology, Merck Sharp & Dohme Research Laboratories (Hugh Rosen, M.D.); Immunology Section, Roberts Pharmaceutical Corporation (Ron Haming, Ph.D.); Department of Ophthalmology, Kurume University School of Medicine, Fukuoka, Japan (Manabu Mochizuki, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.4 PROFESSIONAL: 0.4 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neitiier SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Cell adhesion molecules are surface proteins important for antigen sensitization and the migration of leukocytes to sites of inflammation. We are studying the expression of cell adhesion molecules in ocular inflammation, investigating the blocking of cell adhesion molecules as a treatment for uveitis and other ocular inflammatory diseases, and examining the effect of immunosuppressive agents on cell adhesion molecule expression in eyes with experimental autoimmune uveitis (EAU). We previously showed that intercellular adhesion molecule 1 (lCAM-1) is expressed in eyes with EAU before the infiltration of inflammatory cells. Further experiments showed that monoclonal antibodies against lCAM-1 and its counter-receptor lymphocyte function-associated antigen 1 (LFA-1) inhibit EAU development in mice. We demonstrated that cell adhesion molecules are important for both antigen sensitization and inflammatory cell infiltration into the eye, with additional sets of experiments showing the following: (1) that monoclonal antibodies against both lCAM-1 and LFA-1 will prevent inflammatory cell infilti-ation of the eye induced by endotoxin, and importanUy that these antibodies can inhibit ocular inflammation even when administered after signs of inflammation have been noted; and (2) that monoclonal antibodies against lCAM-1 and LFA-1 can inhibit in vitro proliferation of a uveitogenic cell line by interfering with the interaction between lymphocytes and antigen-presenting cells. These results suggest that cell adhesion molecules play an important role in the development of uveitis and that blockage of cell adhesion molecules may provide a new therapeutic approach for patients with inflammatory eye disease. Finally, we examined the effect of inununosuppressive agents on the expression of cell adhesion molecules in animals with EAU. Ocular expression of cell adhesion molecules was delayed and downregulated in animals treated with corticosteroids and cyclosporine following immunization with retinal S-antigen. Downregulation of cell adhesion molecule expression may be one of the mechanisms by which immunosuppressive agents inhibit ocular inflammation. 103 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEl Annual Report— FY 1993 Project Description Additional Personnel Rachel Caspi Ph.D. NEI Visiting Associate, LI, Igai Gery Ph.D. Deputy Chief, LI, NEI Qian Li M.D. NEI Visiting Fellow, LI, Objectives The goal of this project is to examine the role of cell adhesion molecules in ocular inflammation. We are studying the expression of cell adhesion molecules in eyes with uveitis and examining the effect of block- ing these adhesion molecules on the development of ocular inflammatory disease. We also are investigat- ing the role of ceU adhesion molecules in antigen sensitization. By blocking cell adhesion molecules or preventing the expression of these surface pro- teins, we hope to be better able to treat patients with ocular inflammatory disease. Methods Animal models of ocular inflammation. — ^Endotoxin- induced uveitis (EIU) is induced by injecting 100 ^g of Salmonella typhimurium endotoxin into one footpad of a Lewis rat or 200 |ig into one footpad of a C3H-hen mouse. Experimental autoimmune uveitis (EAU) in mice is induced by immunizing BIO.A mice with 50 \ig of interphotoreceptor retinoid- binding protein (IRBP) in complete Freund's adju- vant, with pertussis toxin injected intraperitoneally. Histology and immunohistochemistry of ocular inflammation. — ^Enucleated animal eyes and human ocular tissue are immediately snap frozen and em- bedded in O.C.T.® The expression of cell adhesion molecules and the presence of cytokines are then assessed by immunohistochemical staining with avidin-biotin-peroxidase complex (ABC) on frozen sections of ocular tissue. Eyes also are embedded in methyl methacrylate, and 4-|jm sections are examined for histologic evidence of inflammation. Treatment of ocular inflammation by blocking cell adhesion molecules. — In an attempt to inhibit the development of ocular inflammation, we treated animals with infraperitoneal injections of monoclonal antibodies against ICAM-1 or LFA-1 before the induction of either EIU and EAU. Effect of monoclonal antibodies against ICAM-1 or LFA-1 on cell proliferation of a uveitogenic cell line. — Mouse anti-rat ICAM-1 (CD54) monoclonal antibody (mAb), designated 1A29, and mouse anti-rat LFA-1 (CDl la) mAb, designated WT.l, were kindly provided by Dr. Miyasaka (Tokyo, Japan). mAbs were incubated with irradiated Lewis rat thymocytes (antigen-presenting cells [ APCs]) at concentrations of 10, 1, 0.1, and ^g/ml for 2 hours. These cells were then added to lymphocyte cultures comprised of CD4-(- T cells of a highly uveitogenic cell line, sensitized against IRBP-derived peptide R15 (se- quence 1181-1191), and stimulated with peptide R15 (1 or 0.01 )iM) or with concanavalin A (con A). The cultures, which consisted of 2 x 10^ lymphocytes and 1 X 10^ or 5 X 10^ APCs in 0.2 ml of medium, were processed as previously detailed (Cell Immunol 122:251, 1989). Effect of corticosteroids and cyclosporine A (CsA) on the expression of cell adhesion molecules in eyes with EAU. — ^EAU was induced in 36 female Lewis rats by injecting into one hind footpad 30 MS of retinal S-antigen in complete Freund's adjuvant containing 0.25 mg of Mycobacterium tuberculosis. Rats were then treated with daily intramuscular injections of 0.2 mg/kg methylprednisolone (MP), 3 mg/kg CsA, or olive oil as a confrol. Rats were sacrificed 0, 7, 10, 14, 21, and 28 days after immuni- zation. Each right eye was processed for routine histology, and each left eye was immediately sn£^ frozen for immunohistochemical staining, using an avidin-biotin-peroxidase technique and primary antibodies against ICAM-1 (CD54), LFA-1 alpha (CDl la), E-selectin, major histocompatibility com- plex (MHC) class n antigens (RTIB and RTID), CD4+ T cells (W3/25), and CD8+ T cells (0X8). Slides were then graded by two masked observers. Major Findings 1. When treatment was given at the time of endotoxin injection, the mean number of inflamma- tory cells infiltrating the eye on histologic sections was 469.2 ±51.9 (standard error of the mean [SEM]) for controls, 13.8 ± 2.6 for rats receiving anti-ICAM- 1 mAb (p < 0.001), and 195.8 ± 48.8 for rats receiv- ing anti-LFA-1 mAb (p < 0.001). When treated after the start of inflammatory disease, the mean number of infiltrating inflammatory cells ± SEM was 273.0 ± 30.7 for controls, 6.4 ±1.7 for rats receiving anti- 104 NEI Annual Report— FY 1993 Laboratory of Immunology ICAM-1 mAb (p < 0.001), and 54.2 ± 7.6 for rats receiving anti-LFA-1 mAb (p < 0.001). The mean numbers of cells per microliter of aqueous humor were 1867.6 ± 321.8 for controls, 21.7 ± 5.3 for rats receiving anti-ICAM-1 mAb (p < 0.001), and 295.1 ± 71.2 for rats receiving anti-LFA-1 mAb (p < 0.001). Treatment with mAbs against ICAM-1 and LFA-1 significantly inhibited the development of EIU and was effective in treating clinically evident ocular inflammatory disease. 2. In mice with EAU, ocular inflammation, graded clinically by examination of the fundus 14 and 21 days after immunization, was significantly decreased in animals treated with anti-ICAM-1 (p < 0.01 at days 14 and 21) and with anti-LFA-1 anti- body (p < 0.01 at days 14 and 21). 3. In cultures containing 5 x 10^ APCs, anti- LFA-1 mAb (10 ng/ml) inhibited the lymphocyte response to 1 and 0.01 \M of R15 by 58% and 74%, respectively. mAbs against ICAM-1 were less inhibitory, reducing the responses to R15 by 23% and 30% for doses of anti-LFA and R15 mAb, respectively. Decreasing the APC concentration had little effect on the antibody activity, while decreasing the mAb concentration to <1 jig/ml almost complete- ly eliminated their inhibitory cs^acity. mAbs against LFA-1 and ICAM-1 inhibited the interaction between APCs and lymphocytes of a uveitogenic cell line. We propose that this activity plays a major role in the inhibition of EAU in animals treated with mAbs against LFA-1 and ICAM-1. 4. By 14 days after inununization, ICAM-1, E- selecfin, and MHC class II antigens were strongly expressed on the vascular endothelium of the iris, ciliary body, choroid, and retina of control rats; infiltrating lymphocytes expressing LFA-1 also were noted in these eyes. In contrast, 28 days after immunization, rats treated with MP and CsA still had only mild expression of ICAM-1, E-selectin, and MHC class II antigens, and few infiltrating lympho- cytes were noted on histologic sections. Ocular expression of cell adhesion molecules was delayed and down-regulated in animals treated with MP and CsA following immunization with S-antigen. Ex- pression of class II antigens and infiltration with inflammatory cells also were diminished in eyes with decreased expression of cell adhesion molecules. Down-regulation of cell adhesion molecule expres- sion may be one of the mechanisms by which corticosteroids and CsA inhibit ocular inflammation. Significance to Biomedical Research and the Program of the Institute One major mission of the NEI is to understand the mechanisms of sight-threatening eye diseases so that new and effective therapies can be developed. The expression of cell adhesion molecules appears to be a fundamental mechanism in the development of intraocular inflammation. With this understanding, we hope to develop new anti-inflammatory therapy for ocular inflammation, which accounts for approxi- mately 10% of the visual impairment in the United States. Proposed Course We plan to continue our experiments on the expres- sion of cell adhesion molecules in eyes with ocular inflammatory diseases, including uveitis, corneal disease, and uveitic glaucoma. We are examining the roles of additional cell adhesion molecules such as VCAM-1 and VLA-4 in ocular inflammation. In addition, we plan to study the pharmacokinetics of antibodies against cell adhesion molecules, adminis- tered topically or intraocularly, to determine the feasibility of local therapy. NEI Research Program Retinal Diseases — Inflammatory Diseases Publications Lai JC, Chan C-C, LI Q, Whitcup SM: Treatinent with corticosteroids and cyclosporine A inhibits the expression of cell adhesion molecules in experimental autoimmune uveitis (EAU). Invest Ophthalmol Vis Sci 34(4)(suppl):1206, 1993. Vistica B, Gery I, Chan C-C, Nussenblatt RB, Whit- cup SM: Anti-ICAM-1 and anti-LFA-1 mono- clonal antibodies (mAbs) inhibit in vitro prolifera- tion of a uveitogenic cell line. Invest Ophthalmol Vis Sci 34(4)(suppl):1144, 1993. Whitcup SM, DeBarge LR, Caspi RR, Haming R, Nussenblatt RB, Chan C-C: Monoclonal anti- bodies against ICAM-1 (CD54) and LFA-1 (CDlla/CD18) inhibit experimental autoimmune uveitis. Clin Immunol Immunopathol 67: 143-150, 1993. Whitcup SM, DeBarge LR, Rosen H, Nussenblatt RB, Chan C-C: Monoclonal antibody against CDllb/CD18 inhibits endotoxin-induced uveitis. Invest Ophthalmol Vis Sci 34:673-681, 1993. 105 Laboratory of Immunology NEI Annual Report — FY 1993 Whitcup SM, Hikita N, Shirao M, Mochizuki M, Whitcup SM, Nussenblatt RB, Price FW Jr, Chan NussenblattRB, Chan C-C: Effect of monoclonal C-C: Expression of cell adhesion molecules in antibodies against ICAM-1 (CD54) and LFA-1 corneal graft failure. Cornea, 12:475-480, 1993. alpha (CDl la) in the prevention and treatment of endotoxin-induced uveitis (EIU). Invest Ophthal- mol Vis Sci 34(4)(suppl):1143, 1993. 106 DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00184-11 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (BO characters or less. Title must lit on one line between the borders.) Cellular and Immunogenetic Mechanisms in Uveitis PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Rachel R. Caspi Ph.D. Visiting Scientist LI, NEI Acting Head, Section on ' Immunoregulation Others: Phyllis Silver Luiz Rizzo Chj-Chao Chan B.S. Biologist LLNEI M.D. Visiting Associate LI, NEI M.D. Head, Section on Immunopathology LI, NEI COOPERATING UNITS (If any) Laboratory of Immunobiology, Rega Instituut, Katholjeke Universiteit, Leuwen, Belgium (A. Billiau, M.D.; H- Heremans, Ph.D.); Arthritis and Rheumatism Branch, National Institute of Arthritis and Musculoskeletal and Skin Diseases (Ronald L. Wilder, M.D., Ph.D.); Bone Marrow Transplantation Unit, National Cancer Institute (Frances Hakim, Ph.D.); Research and Development, WiUs Eye Hospital, Philadelphia, PA (Larry A. Donoso, M.D., Ph.D.) - LAB/BRANCH Laboratory of Immunology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 2.6 PROFESSIONAL: 2.6 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Cellular mechanisms in ocular immunologically mediated disease are being studied in animal models of experimental autoimmune uveoretinitis (EAU). Rats and mice are immunized with retinal -derived antigens, or synthetic peptides representing fragments of these antigens, to induce EAU. Susceptibility to disease is being evaluated in various strains of known genetic makeup in the hope of delineating the hereditary mechanisms that predispose to uveitis. EAU in rats and mice serves as a template for the evaluation of new drugs and compoimds as well as for the study and characterization of the participating cells and their factors. In vivo functional long-term T-cell lines and clones are developed from lymphoid organs of rats and mice immunized with uveitogenic ocular proteins. The functional properties and antigen receptors of these cells are studied to develop strategies for in vivo targeting of the autoimmune cells. The goal of these studies is to identify the immunogenetic factors predisposing to uveitic disease, learn about the pathogenic mechanisms involved, characterize the immunoreactive cells and their mediators, and finally to utilize this knowledge for designing rational approaches to immunotherapy. 107 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report— FY 1993 Project Description Additional Personnel Robert B. Nussenblatt M.D. Chief, LI, NEI Charles E. Egwuagu Ph.D. StaffFellow,LI,NEI Igal Gery Ph.D. Head, Section on ExperimentaJ Immunology, LI, NEI Objectives The development and study of animal models of experimental ocular autoimmune disease permits the study of cellular and genetic factors that may be involved in ocular autoimmunity in a general sense. Experimental autoimmune uveitis (EAU) in rats and mice serves as a template for the evaluation of new drugs and compounds as well as for the study and characterization of the participating cells and their factors. Long-term maintenance of T cells in vitro permits the investigators to separate and selectively grow various T-cell subsets. The goals are (1) to continue to establish and characterize the murine EAU model because the mouse offers some impor- tant advantages over other rodents as a model of EAU; (2) to use the EAU model in rodents for the study of cellular mechanisms in ocular autoimmuni- ty; this is done in large part by establishing and using retinal antigen-specific T-cell lines and clones, permitting us to identify and characterize cells capable of ocular immunomodulation, learn about migration and localization of autoimmune lympho- cytes, and study their interactions with other lym- phoid and nonlymphoid cells in eliciting effector mechanisms; (3) to use tiie EAU model as a template for the development of immunotherapeutic jqjproach- es designed to target autoimmune lymphocytes directly or to disrupt specific stages in the autoim- mune inflammatory cascade; and (4) to use the murine EAU model for the study of various genetic mechanisms controlling susceptibility to ocular autoimmune disease. The study and understanding of these parameters will help not only in the devel- opment of new therapies but possibly in the preven- tion of ocular disease. Methods Rats and mice of various strains are inmiunized with purified S-antigen (S-Ag) or interphotoreceptor retinoid-binding protein (IRBP) in complete Freund's adjuvant or with various pathogenic peptides derived from these proteins. After disease development, eyes are processed for histopathology and examined for disease, and lymphoid cells are taken from the blood, lymph nodes, or eyes. Cells thus obtained are placed in culture either with mitogen or with the retinal antigen with which the donor animal was immunized. Responses of the immune cells are studied. Cells also are expanded in culture and used in attempts to transfer EAU to nonimmune animals in order to find out tiie cell population responsible for disease induction. Long-term cell lines are devel- oped and in some cases are cloned by either the soft agar bilayer or tiie limiting dilution technique. These lines or clones are tiien tested for functional charac- teristics such as the ability to induce ocular disease, production of soluble mediators, expression of various cell surface molecules, response to therapeu- tic agents, and interactions with other cells in culture. Major Findings We previously had studied the importance of "non- specific" T-cell recriiitment in the immunopathogene- sis of uveitis by using congenitally athymic Lewis rats (LEW.mu/mu), which are deficient in functional endogenous T cells but are otherwise syngeneic with the euthymic Lewis rats that develop characteristical- ly severe EAU. The uveitogenic stimulus was delivered in the form of phenotypically and function- ally homogeneous pathogenic T-cell lines specific to the major pathogenic epitope of either the intracellu- lar photoreceptor protein, S-Ag, or the extracellular photoreceptor matiix protein, IRBP. Previous data indicated that, depending on the T-cell line used, EAU in athymic rats was either drastically reduced in severity or absent Susceptibility was restored when the athymic animals were reconstituted with immunocompetent T cells from syngeneic euthymic donors. We now have shown that the severity of inflam- mation and tissue damage are correlated with the proportion of lymphocytes in the intraocular infil- ti^ate: The infiltrate in euthymic rats was predomi- nantly lymphocytic, with smaller numbers of mono- cyte-macrophages and even fewer neuti-ophils, whereas the sparse infiltrate in athymics was largely monocytic and had a relatively high proportion of neutrophils and eosinophils. Reconstituted animals had an intermediate histological picture with respect 108 NEI Annual Report— FY 1993 Laboratory of Immunology to the infiltrating cell types and disease severity. Our results indicate that recruited nonspecific T cells play a major role in the pathogenesis of disease. Furthermore, the data suggest that the extent of dependence on recruitment may be influenced by the antigenic specificity of the T-cell line and could be connected to the "accessibility" of the target antigen in vivo. This is the first direct demonstration that recruitable "nonspecific" T lymphocytes are neces- sary for the expression of the disease itself. In collaboration with Dr. Charles Egwuagu, we are continuing to study the T-cell receptor (TCR) genes of these lines and clones on the molecular level. The data indicate that TCR variable-region gene usage in uveitis differs from that reported for some other autoimmune diseases and may be more heterogeneous. We currently are studying TCR expression in the inflamed eyes of Lewis rats immu- nized with various retinal antigens or adoptively transferred with T-cell lines of the appropriate specificity. Because our previous findings with athymic rats suggest that the majority of lymphocytes infiltrating the eye are likely to be recruited cells, we are look- ing at the earliest stages of disease induction as well as at cells that infiltrate the eye in athymic nude rats injected with pathogenic T cells. The results appear to confirm our previous findings: TCR Vp8.2 may be a pathogenic clonotype inS-Ag-EAU, whereas TCR vp8.3 may be one of the pathogenic clonotypes in IRBP-EAU. The results also indicate tiiat additional clonotypes such as Vpl4 may be involved. These findings could impact on the development of thera- peutic strategies designed to specifically target the pathogenic cells through their T-cell receptors. In the mouse model of EAU, we have developed a pathogenic T-cell line specific to the whole IRBP molecule in the BIO.A strain of mice (I-A"). The line was developed from draining lymph nodes of IRBP- immunized mice using a protocol similar to that used for the derivation of uveitogenic T-cell lines in the rat (alternating cycles of stimulation with antigen and expansion in interleukin 2). After the fourth weekly stimulation with IRBP, we tested the line for patho- genicity and found that it induced uveitis at 5 x 10* cells per mouse. The line elaborated an unrestricted lymphokine profile, suggesting that both Thl-type and Th2-type cells were present With continued stimulations in culture, the cell line became progressively more pathogenic. After 16 cycles the cell line was pathogenic at cell numbers as low as 10^ cells per mouse. The TCR profile of the line also changed wdth time in culture, with progres- sive enrichment in Vp8.2 and VP6 TCR -expressing cells (64% and 16%, respectively), suggesting that vp8.2 and Vp6 may represent pathogenic clonotypes in IRBP-EAU in the BIO.A mouse. The line current- ly is being cloned to test this hypothesis and to further characterize the pathogenic cells with respect to their Thl-like or Th2-like identity. Another IRBP-specific pathogenic T-cell line was developed, using a similar cultiu-e protocol, from eyes of uveitic BIO.A mice. The line was pathogenic at 5 x 10* cells per mouse. These results suggest that the pathogenic cells infiltrate and are physically present in the eye during uveitis. In all animal species, as well as in humans, the genetic makeup of an individual determines the regions of the uveitogenic protein molecule that evoke an autoimmune response. It is important to study the nature of these epitopes and their relation to different major histocompatibility complex types because the findings could potentially be extri^lated to the situation in humans. In collaboration with Dr. Larry Donoso (Wills Eye Hospital, Philadelphia), who synthesized overlapping peptides representing the entire sequence of the human IRBP molecule, we are engaged in an ongoing effort to identify epitopes that are pathogenic in the three previously identified susceptible mouse H-2 haplotypes, namely, H-2'', H-2', and H-2\ The peptides are being systematical- ly screened by immunizing animals from strains representing the three susceptible haplotypes. Pep- tides that cause EAU are being studied further with respect to the identity of the minimal pathogenic sequence, immunodominance, and the fine specificity of the response. Pathogenic T-cell lines are raised to these peptides, and their TCR usage is studied. Peptide LRHNPGGPSS AVPLLLSYFQ, represent- ing a highly conserved sequence in the IRBP mole- cule and sparming amino acids 461^80, was found to be pathogenic in C57BL/10, but not in the other mouse strains. The disease scores obtained with the peptide (50-250 |ig) were lower than those obtained with tiie whole IRBP molecule (50-100 ^ig). A lymphocyte line specific to the peptide was able to 109 Laboratory of Immunology NEI Annual Report— FY 1993 adoptively transfer low-grade uveitis to syngeneic recipients. Mice immunized with the peptide, or with whole IRBP, had positive DTH to the immuniz- ing, but not to the reciprocal, antigen. Lymphocytes of IRBP-immunized mice did not proliferate in vitro in response to the peptide; however, positive lympho- cyte responses to IRBP could sometimes be detected in peptide-immunized mice and the peptide-specific lymphocyte line proliferated in vitro to IRBP. Thus, peptide 461-480 appears to contain an epitope pathogenic to mice of the H-2'', but not H-2'' or H-2', haplotypes. The low pathogenicity of peptide 461- 480 in comparison to that of whole IRBP, as well as its lack of immunological recognition by IRBP- immunized mice, in vivo or in vitro, suggests that it may be a minor, immunologically nondominant epitope. This is the first uveitogenic epitope de- scribed for the mouse EAU model. Several addition- al sites, pathogenic for the other haplotypes, have been tentatively identified and are being studied. Significance to Biomedical Research and the Program of the Institute It has become increasingly clear that the cellular mechanisms and possibly the genetic mechanisms observed in animal models of uveitis reflect the mechanisms that operate in ocular immune-mediated disease in humans. The identificaUon and character- ization of the cells involved in ocular autoimmunity, and of their functions, will provide new understand- ing of inflammatory ocular diseases. Successful immunomodulation of EAU in animal models usually has served as a good predictor of the clinical success of a given therapeutic modality. Continued study of basic mechanisms involved in the immunopathogene- sis of uveitis in animal models will aid in the devel- opment of novel immunotherapeutic approaches for the control of uveitis in humans. Proposed Course This project will continue so that more information about the basic mechanisms in experimental uveitis may be obtained. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Caspi RR. Experimental autoimmune uveoretinitis in rats and mice, in Cohen I, Miller A (eds): Guide- hook to Animal Models for Autoimmune Diseases. Academic Press, in press. Caspi RR: Immunogenetic aspects of cUnical and experimental uveitis. Reg Immunol 4:321-330, 1992. Caspi RR, Chan C-C, Fujino Y, Najafian F, Grover S, Hansen CT, Wilder RL: Recruitment of naive T cells plays a pivotal role in the pathogenesis of experimental autoimmune uveoretinitis. Invest Ophthalmol Vis Sci 34(4)(suppl):902, 1993. Caspi RR, Chan C-C, Fujino Y, Najafian F, Grover S, Hansen CB, Wilder RL: Recruitment of antigen-nonspecific cells plays a pivotal role in the pathogenesis of a T-cell-mediated organ- specific autoimmune disease, experimental autoimmune uveoretinitis. J Neuroimmunol 47:177-188, 1993. Caspi R, Nussenblatt R: Natural and therapeutic control of ocular autoimmunity — rodent and man, in Kazatchkine M, Roitt I (eds): Autoimmunity. Wiley and Sons, Inc, in press. Egwoiagu CE, Mahdi RM, Gery I, Nussenblatt RB, Caspi RR: Evidence for selective accumulation of VP8+ T lymphocytes in experimental auto- immune uveoretinitis induced by two different retinal antigens. J Immunol 151:1627-1636, 1993. Mahdi RM, Caspi RR, Nussenblatt RB, Gery I, Egwuagu CE: Selective accumulation of Vp8-i- T lymphocytes in EAU. Invest Ophthalmol Vis Sci 34(4)(suppl):1144, 1993. Rizzo LV, Silver PB, Hakim F, Chan C-C, Wiggert B, Caspi RR: Establishment and characterization of an IRBP-specific T-cell line that induces EAU in BIO. A mice. Invest Ophthalmol Vis Sci 34(4) (suppl):1143, 1993. Silver PB, Rizzo LV, Chan C-C, Donoso LA, Wig- gert. B, Caspi RR: Identification of a putative epitope in the IRBP molecule that is uveitogenic for mice of the H-2'' haplotype. Invest Ophthal- mol Vis Sci 34(4)(suppl):1482, 1993. 110 NEI Annual Report-FY 1993 Laboratory of Immunology Whitcup SM, DeBarge LR, Caspi RR, Haming R, Nussenblatt RB, Chan C-C: Monoclonal anti- bodies against ICAM-1 (CD54) and LFA-1 (CD 11 a/CD 18) inhibit experimental autoimmune uveitis. Clin Immunol Immunopathol 67:143-150, 1993. Ill DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00218-08 LI PERIOD COVERED October 1. 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less Title must tit on one line between ttie borders.) Ocular Manifestations of the Acquired Immune Deficiency Syndrome PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator. J (Name, title, laboratory, and institute affiliation) PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI Others: Roben B. Nussenblan M.D. Scott Whitcup M.D. Margaret Cheung M.D. David Parks M.D. Dan Martin M.D. Francois Roberge M.D. Chi-Chao Chan M.D. Scientific Director NEI Senior Staff Fellow LI. NEI Senior Staff Fellow LI. NEI Senior Staff Fellow LI, NEI Senior Staff FeUow LI, NEI Visiting Scientist LI. NEI Head, Section on LI, NEI Immunopatbology COOPERATING UNITS (if any) Department of Critical Care Medicine, Clinical Center (Henry Masur, M.D.); Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases (H. Clifford Lane, M.D.); Pediatric Branch, National Cancer Institute (Phil A. Pizzo, M.D.) LAB/BRANCH ~~ Laboratory of Immunology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI. NIH, Bethesda, MD 20892 TOTAL STAFF YEARS; 2.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects [x] (a1) Minors □ (a2) Interviews PROFESSIONAL: 2.0 OTHER: 0.0 [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Patients suffering from AIDS (acquired immune deficiency syndrome) are at risk of developing significant ocular problems, either as a result of HIV (human immunodeficiency virus) itself or as a result of opportunistic infection. Some of these problems can lead to blindness if left untreated. Among the many pathogens that can lead to blindness, cytomegalovirus (CMV) is by far the most common. In FY 1993 the two major emphases have been (1) CMV retinitis detection and therapy and (2) pediatric AIDS. All currently available drugs are virostatic. Early detection is the most effective means of ensuring that pafients will preserve long- term vision. We have continued to evaluate the useftilness of a laser photometric device to help screen patients for the presence of ocular inflammation and/or CMV retinitis. We also have looked at the use of alternative methods of followup using tangent saeens and Amsler grids to help determine early recurrences. We have initiated a study using an implantable slow-release device for ganciclovir. This study is still in its early stages. Due to the sustained nature of the release, it is possible that this approach will lead to prolonged remissions, as compared to standard therapy. In FY 1993 we continued to evaluate the incidence of ocular infection in about 220 children with AIDS. The incidence of complications is rare, which has prompted us to reduce the frequency of foUowups to every 6 months instead of every 4 months. However, it is still important that parents or guardians monitor their AIDS-affected children for symptoms and signs of visual loss. 112 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Additional Personnel Susan Mellow R.N. Clinical Protocol Number 90-EI-208 Objectives This project's primary objective is to develop meth- ods of identifying and treating known ocular compli- cations of HIV (human immunodeficiency virus) infection in such a way as to prevent significant loss of vision. The second, equally important objective is the identification of new manifestations of ocular involvement from the AIDS (acquired immune deficiency syndrome) virus itself and related oppor- tunistic ocular infections. The third objective is to recognize complications related to the therapeutic agents used or the mode of administration. Methods This project entails the clinical evaluation, diagnosis, and treatment of retinitis in AIDS patients. It also involves the development of novel methods of ther^y for the various forms of retinitis observed. Study of pathologic tissue also is used to better understand the nature of the infectious processes. Major Findings In the past year our major effort has centered on the evaluation and treatment of cytomegalovirus (CMV) retinitis. CMV is a major vision-threatening infec- tion found in patients with AIDS. Preservation of useful vision for a prolonged period of time requires early detection. In the present context, this can only be done by careful funduscopic examination by a trained eye care professional. In asymptomatic individuals, such exams are usually normal. Exami- nations in these patients are both time consuming and costiy, with very few having any ocular lesions. The development of a screening device that could help to detect, within the eye, conditions predispos- ing to CMV retinitis and other disorders is highly desirable. A device capable of detecting even minute fluctu- ations in the anterior chamber flare has been evalu- ated over the past 2 years. It consists of a low- intensity laser beam focused on the anterior chamber of the eye. Under normal circumstances, none of the incident light is reflected, as there is neither protein nor cells in the anterior chamber. In the presence of inflammation, part of the laser beam is reflected back through the cornea to a detector that measures the intensity of the reflected light in photons per milli- second. The intensity of this reflected light corre- lates directiy with the intensity of inflammation in an affected eye. On average the test itself takes only 15 minutes for both eyes, and it is simple enough that it can be performed in a nonophthalmic cUnic. We recentiy have evaluated data from 80 patients with and without CMV retinitis who were subjected to this test We found that the technique was 100% sensitive in detecting patients with CMV retinitis when the cutoff count was 8.0 photons per milli- second; the corresponding specificity was 77%. Early detection can lead to preservation of vision, but it also commits the patient to life-long intra- venous (IV) therapy with anti-CMV drugs. A device recentiy has been developed that slowly releases ganciclovir directiy into the eye. This alternative to systemic therj^jy may be particularly useful in patients who cannot tolerate IV infusions of antiviral drugs. We have developed a protocol to evaluate the safety and efficacy of the device in patients who have not been treated previously with an anti-CMV agent. The protocol is designed to compare the rates of progression between patients in whom ther^y is delayed with the rates in patients implanted with a device that releases the drug over 8 months. Only patients with peripheral non-sight-threatening disease are ehgible for the study. One important concern has been the lack of systemic coverage and its possible effects on patient survival. While the study is not designed to answer this question, patients will be followed carefully to determine whether this form of therapy has any adverse effect on their survival. Lack of systemic side effects from anti-CMV therapy and the patient's ability to stay on anti-HIV agents may in fact be of greater benefit to survival. So far, a dozen patients have been recruited out of a total of 35. We have continued to follow about 200 children who developed AIDS by various means. We have been particularly struck by the lower incidence of CMV in this population, where the overall incidence is about 1.6%. However, ia children who have low total T-cell counts (below 100/mm^), the risk in- 113 Laboratory of Immunology NEI Annual Report— FY 1993 creases to 16%. By following children every 6 months, we have been able to detect all cases of ocular involvement, provided that the children's parents or guardians are periodically screened for the presence of vision loss. Significance to Biomedical Research and the Program of the Institute The AIDS epidemic is a major public health concern. CMV retinitis remains the number one cause of blindness among patients infected with the AIDS virus. Early diagnosis is important because all drugs currently available are only virostatic and not viro- cidal; thus, some progression of the lesion is seen in more than 50% of patients, despite anti-CMV ther- apy. Inasmuch as most patients present late with well-established disease, a device able to screen and identify patients with early lesions is highly desir- able. New therapeutic modalities that are cost- effective and reduce the incidence of progression or the development of resistant strains are necessary. Reports of strains resistant to gancyclovir are increas- ing in number. The number of children infected with AIDS is on the rise. A good understanding of the epidemiology of AIDS in terms of ocular disease is highly desir- able. We are therefore continuing to follow these children prospectively to identify the frequency and type(s) of ocular complications they are likely to develop. Proposed Course In the coming fiscal year we plan to evaluate further the laser photometer to determine possible ways of increasing the specificity of the device in detecting CMV retinitis. We will continue to evaluate the slow-release device in patients with newly diagnosed CMV retinitis. We also are plarming to use new therapeutic agents for the treatment of CMV retinitis. NEI Research Program Retina] and Choroidal Diseases — Inflanmiatory Disorders Publications de Smet MD: Ocular consequences of human inmiu- nodeficiency virus infection. Ophthalmol Clin North Am 6:117-126, 1993. de Smet MD, Butler KM, Rubm BI, Whitcup SM, DeBarge LR, Martin DF, Pizzo PA, Nussenblatt RB: The ocular complications of HIV in the pediatric population, in Demouchamps JP, Verougstraete C, Capsers-Velu L, Tassignon MJ (eds): Recent Advances in Uveitis, Proceedings of the Third International Symposium on Uveitis. New York, Kugler Publications, 1993, pp 315-319. Muccioli M, Belfort R, Podgor M, Sampaio P, Hayashi S, Neves R, Lottemberg C, Kim MK, de Smet M, Nussenblatt RB: The diagnosis of intraocular inflammation and CMV retinitis in HIV infected patients by laser flare photometry. Invest Ophthalmol Vis Sci 34(4)(suppl):1110, 1993. Polls MA, de Smet MD, Baird BF, Mellow S, Falloon J, Davey RT, Kovacs JA, Palestine AG, Nussenblatt RB, Masur H, Lane HC: Increased survival of a cohort of patients with acquired immimodeficiency syndrome and cytomegalovirus retinitis who received sodium phosphonoformate (foscamet). Am J Med 94:115-1^0, 1993. Whitcup SM, Fenton RM, Pluda JM, de Smet MD, Nussenblatt RB, Chan C-C: Pneumocystis carinii and Mycobacterium avium-intracellulare infection of the choroid. Retina 12:331-335, 1992. 114 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00266-04 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must til on one line between the borders.) Characterization of Immune Responses to S- Antigen PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI Others: Igal Gery Robert B. Nussenblatt Margaret Cheung Fran90is Roberge Ph.D. M.D. M.D. M.D. Head, Section on LI, NEI Experimental Immunology Scientific Director NEI Senior Staff Fellow LI, NEI Visiting Scientist LI, NEI COOPERATING UNITS (it any) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI, NTH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.6 PROFESSIONAL: 0.6 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) One of the characteristics of S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP), which are retinal-specific antigens, is the ability to induce an intense autoimmune inflammation in the eyes of experimental animals when injected in the presence of an adjuvant. This disease, called experimental autoimmune uveitis (EAU), is critically dependent on T cells and antigen processing by appropriate antigen- presenting cells (APCs). Antigen processing, which occurs within the endocytic vesicles of the ATCs, results in the production of small polypeptide subunits. These small polypeptides must then be protected from further degra£lation and transported to the cell surface where the interaction with the T cell takes place. In FY 1993 we identified an intracellular protein that is capable of binding a major epitope of IRBP. It was identified first in rat B cells, which are good antigen-presenting cells. This binding protein qjpears to belong to the heat shock family of proteins, and its production appears to be upregulated under conditions of cellular stress. These stresses can be exogenous, such as heat, or they can result from more physiologic stresses, such as stimulation by lectins and bacterial cell wall products. This protein appears to be present not only in animal cells but also can be detected in human B cells. 115 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report— FY 1993 Project Description Additional Personnel Sumeet Mainigi Kalpana Rengerajan Ph.D. Gerald J. Chader Ph.D. Barbara Wiggert Ph.D. Biologist, LI, NEI Biologist, LRCMB. NEI Chief, LRCMB, NEI Head, Section on Biochemistry, LRCMB, NEI Clinical Protocol Numbers 84-EI-214 79-EI-49 Objectives In Fiscal Year (FY) 1993 the study has concentrated mainly on identification and isolation of the intracel- lular binding proteins involved in preventing degra- dation of key immunopathogenic epitopes of inter- photoreceptor retinoid-binding protein (IRBP) and S- antigen (S-Ag) within antigen-presenting cells of Lewis rats and humans. Methods Using B-cell lysate, we isolated the intracellular binding protein for R-15 (1169-1191), a major immunopathogenic epitope of IRBP, on a cyanogen bromide Sepharose 4b column. Because this particu- lar protein is produced in very small amounts within cells, large numbers of B cells were needed. In rats, these were first obtained by panning, but to increase the yield and the purity of the cell population, we used a magnetic separator with paramagnetic beads. The efficiency of the separation was greatly in- creased by this approach, and it required much less time. This approach also ensured a highly viable population of cells, which appeared to be much more responsive to physiologic stressors than those ob- tained by paiming. R-15 also has been shown to cause an immune resptonse in peripheral blood lymphocytes of some patients with uveitis; thus, an attempt was made to identify and isolate a similar intracellular binding protein in human antigen-presenting cells — namely, the B cell. To produce large quantities of B cells, we used the Epstein-Barr virus (EBV) to transform peripheral blood lymphocytes from patients and normal controls. EBV causes B cells to proliferate indefinitely. Although these cells are infected with a virus and are perpetually in a blastic phase, their antigen-presenting capabilities are not affected. Once transformed, these cells can be grown in very high numbers in a variety of cell growth systems. A combination of stirrer flasks, tissue culture flasks, and cell factories was found to be the most efficient way of growing these cells in sufficient numbers. Once the appropriate cell number was obtained, we subjected the cells to specific physiologic stressors to increase the production of the cellular binding protein. Major Findings Our previous studies in the Lewis rat have shown that several fi-agments of S-Ag are able to induce a strong immune response when tested in vitro. These fragments are normally produced by endocytic enzyme degradation of a parent protein such as S-Ag or IRBP. Partially degraded fi-agments must then be protected firom further degradation and transported to the cell surface, where they can associate with class II antigens. Recently it has been suggested that proteins belonging to the heat shock family of proteins might play a role in preventing enzymatic degradation and in carrying antigens to the cell surface. In FY 1993 we showed that antigen-pre- senting cells contain a protein that is able to bind to the immunodominant determinant of IRBP (sequence 1169-1191). This pepfide-bihding protein has a molecular weight similar to that of other heat shock proteins (HSP). By Western blot, it stains positively to monoclonal antibodies directed against the consti- tutive and inducible forms of HSP70. Binding does not appear to occur to all peptide firagments of IRBP, as shown in some preliminary experiments using Sepharose columns activated with different epitopes of IRBP. We also have isolated a similar protein from transformed human B cells. This protein has the same molecular weight and staining characteris- tics as the protein isolated from the rat cells. In both cell types, the protein is produced in larger amounts when the cell is activated by exogenous stress (heat) or by agents such as lipopolysaccharide. In addition to the 70-kD protein, there appears to be a secondary peak at 40-kD that is nearly always present. Its exact nature and role remain to be determined. 116 NEI Annual Report— FY 1993 Laboratory of Immunology Significance to Biomedical Research and the Program of the Institute Intracellular processing is a crucial step in the generation of an immune response. These studies suggest that certain intracellular proteins may play a determining role in the selection of the peptidic determinants that are ultimately presented at the cell surface. In addition, exogenous and endogenous factors appear to regulate the synthesis of these proteins. Identification of these intracellular proteins and the mechanisms that regulate their synthesis may give us further insights on the mechanisms of antigen presentation and possibly a means of regulating aberrant antigen presentation. Proposed Course In the coming year the main emphasis will be on further characterization of the intracellular binding protein. We will attempt to determine the binding characteristics of the protein and to determine the factors that can enhance its synthesis. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications de Kozak Y, Mirshahi M, Stiemer R, de Smet M, Frank R, Faure JP: Modulation of S-antigen induced EAU by neonatal injection of peptides from S-Ag or TNF-a or by anti-idiotypic anti- body. Exp Eye Res 55(suppl 1):S84, 1992. de Kozak Y, Stiemer RH, Mirshahi M, Frank RW, de Smet M, Faure JP: Humoral immune response against S-antigen/TNF-alpha common epitope in rat EAU suppressed by the monoclonal antibody S2D2. CurrEyeRes ll(suppl):119-127, 1992. de Smet MD, Mainigi S, Nussenblatt RB: Immuno- genicity and immunopathogenicity of peptide determinants of human S-Ag in various rat strains. Invest Ophthalmol Vis Sci 34(4)(suppl): 1143, 1993. Rengarajan K, de Smet MD, Chader GJ, Wiggert B: B cells in Behcet patients contain a heat shock protein that binds to a fragment of IRBP. Clini- cal Immunology Society, Denver, CO, 1993, p72A. Rengarajan K, de Smet MD, Chader GJ, Wiggert B: Identification of a heat shock protein that binds to a peptide causing autoimmune uveitis. Keystone Meeting on Molecular Chaperones: Function in Protein Folding and Cellular Metabolism. Key- stone, CO, October 1992. Rengarajan K, de Smet MD, Chader GJ, Wiggert B: Identification of a heat shock protein that binds to peptide 1169-1191 of IRBP causing autoimmune uveitis. Invest Ophthalmol Vis Sci 34(4)(suppl): 1482, 1993. 117 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00276-02 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the boraers.) Surgical Management of Uveitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute afliliationj PI: Others: Marc D. de Smet Francois Roberge Margaret Cheung Scott Whitcup David Parks Dan Martin David Callanan Naofurm Hikita Ray DeBarge Richard Feoton M.D. Visiting Scientist LI, ^fEI M.D. Visiting Scientist LI, NEI M.D. Senior Staff Fellow LI, NEI M.D. Senior Staff Fellow LI, NEI M.D. Senior Staff Fellow LI, NEI M.D. Senior Staff Fellow LLNEI M.D. Senior Staff Fellow LI, NEI M.D. Visiting Associate LLNEI M.D. Senior Staff Fellow LI. NEI M.D. Senior Staff Fellow LLNEI COOPERATING UNITS (It any) Clinical Oncology Program, Medicine Branch, National Cancer Institute (Robert Wittes, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Inamunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.75 PROFESSIONAL: 1.75 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Patients with uveitis often develop ocular complications that require surgery to prevent permanent loss of vision. Surgery in these patients has been particularly challenging because the surgery itself can induce severe inflammation. The exact timing of the surgery and the choice of postoperative immunosuppressive therapy given to the patient often determine the outcome. Proper handling of the specimen is essential, particularly in cases of intraocular lymphoma in which the lymphoma cells are particularly fragile. In patients with recurrent disease, doing an air-fluid exchange can provide the necessary cells to make a diagnosis. Glaucoma remains an important complication in patients with uveitis. In all cases, standard trabeculectomies stop functioning after a few months. We are continuing to compare the use of 5-FU and. Molteno implants in patients with uveitis and glaucoma who require surgery. We have enrolled 12 patients in the smdy. The use of intraocular lenses following cataract extraction in patients with uveitis is being addressed in a randomized double-masked study to compare modified intraocular lenses with standard lenses in patients with uveitis that has been under control for at least 3 months. So far three patients have been enrolled in the study, and no significant complications have been seen. In experimental models, we evaluated the effect of different methods of inmiunomodulation on graft rejection. In a corneal graft rejection model in rats, we evaluated the kinetics of inflammatory cell infiltration into the graft with and without FK 506 treatement. We also began to evaluate the effect of feeding class I and class II antigens on the rejection rate of corneal grafts. We also have initiated a study of retinal pigment epithelial cell transplantation in the Lewis rat. The immunohistochemical characteristics of the graft were studied for several weeks. Results thus far indicate that rejection occurs at the same rate as in any other tissue. 118 PHS 6040 (Rev, 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Additional Personnel Igal Gery Chi-Chao Chan Susan Mellow Susan Whitcher Ph.D. Head, Section on Experimental Immunology, LI, NEI M.D. Head, Section on Immunopathology, LI, NEI R.N. Nurse, LI, NEI Psychologist, LI, NEI Clinical Protocol Numbers 79-EI-49 87-EI-104 92-EI-157 Objectives The objectives of this project are as follows: (1) to develop rational surgical approaches for patients with intraocular inflammation, because appropriate surgi- cal modalities are needed to properly manage the complications that arise with chronic intraocular inflammation; (2) to devise rational methods for sampling intraocular tissues and to develop the methodology needed to obtain clinically useful information from limited tissue samples; (3) to test new methods of suppressing graft rejection in animal models; and (4) to determine the immunology of graft rejection in the subretinal space. Methods Patients who have developed ocular complications as a result of ocular inflammation and who require surgery and patients for whom an appropriate diag- nosis can only be made with surgery are eligible for one of the patient protocols described above. Pa- tients with vitritis and retinitis of unknown etiology in whom a nonspecific trial of immunosuppression is contraindicated may, according to the protocol, undergo vitrectomy and chorioretinal or endoretinal biopsy to obtain a diagnosis. The tissue specimen is partitioned for microbiology, electron microscopy, immunohistochemistry, and polymerase chain reac- tion. Patients with a suspected intraocular lymphoma undergo an intraocular lymphoma workup with appropriate computed tomography or magnetic resonance imaging scans and lumbar punctures. If these are negative, a vitrectomy is performed and the cells are studied by immunohistochemistry. Patients who have intraocular lymphoma are entered in a central nervous system (CNS) lymphoma protocol and followed prospectively. Patients with glaucoma and uveitis are entered in a double-masked trial of either trabeculectomy with 5-fluorouracil or Molteno implant. They are then followed prospectively to determine the degree of postoperative inflammation and to determine how effective the procedure is over time. For patients with cataracts and uveitis under good control and minimal intraocular inflammation, the protocol calls for a cataract extraction and random- ization to a standard intraocular lens or a modified lens with a heparin coating. Patients are then moni- tored postoperatively for the appearance of inflamma- tion via laser cell flare meter. They also are moni- tored for the appearance of cellular deposits on the lens surface. In animals, we test the efficacy of oral feeding of class I and class II antigens in preventing corneal graft rejection. We also evaluate the kinetics of inflammatory cell infiltration in corneal grafts that are treated with a placebo or FK 506. This model uses heterotopic grafts taken from Fisher rats and sewn into Lewis rats. To evaluate the rejection characteristics of retinal pigment epithelial (RPE) cells under conditions that would favor rejection, human RPE cells are implanted into the subretinal space of Lewis rats using a fransscleral approach. Animals are serially sacrificed at preset times to determine the severity of rejection and the type of cell infiltration occurring within the grafted tissue. Standard immunohistochemistry for avidin-biotin- peroxidase reactions is used in these experiments. Finally, using the endotoxin-induced uveitis model, we are testing a method of measuring, in a noninvas- ive way, the inflammation present in the anterior chamber of rats. The device used is a laser photom- eter that already is being used in patients to monitor anterior chamber inflammation. Measurements of anterior chamber inflammation made with the device are correlated with measurements of the protein in the anterior chamber. Major Findings In Fiscal Year 1993 we performed several diagnostic vitrectomies for intraocular lymphoma. This particu- lar lymphoma, a subtype of CNS lymphomas, is on 119 Laboratory of Immunology NEI Annual Report— FY 1993 the rise. There are three times more CNS lym- phomas being diagnosed today than 10 years ago. Prior to performing a vitrectomy, we perform a complete workup, including an MRI brain scan and lumbar puncture on each patient Several patients were referred to us after unsuccessful attempts to diagnose the tumors elsewhere. Invariably, after reviewing the charts, we found that the specimens had been poorly processed or had been allowed to sit for too long. We have found that it is imperative to bring a specimen to the pathology laboratory while the vitrectomy is under way to ensure adequate cell viability. In a patient who has had a previous vitrectomy and in whom there are still cells floating in the vitreal cavity, a simple air/fluid exchange may be all that is necessary to make the diagnosis. We have found that pretreatment with pulse methylprednisolone is an effective way of decreasing preoperative inflammation. This has been particular- ly useful in cases in which it has been necessary to perform a vitrectomy while there was still evidence of inflammation. In patients undergoing the Molteno implant interim analysis seems to suggest that Molteno implants maintain a lower pressure for a longer period of time. Most trabeculectomies fail by about 1 8 months, while Moltenos are still functioning after 24 months. We also have found that in all postoperative cases, the use of topical nonsteroidal agents such as diclofinac significantly reduce the amount of inflammation. This is particularly visible in glaucoma patients. No data analysis is yet avail- able on the intraocular lens trial, which has just begun. In the animal studies, we were able to demon- strate that topical drops of FK 506 are an effective means of stopping corneal graft rejection. FK 506 has a predominant effect on T cells, inhibiting both the activation of these cells and their recruitment into the transplanted tissue. It appears to down-regulate the expression of both class I and class II antigens as well as adhesion molecules in the transplanted cornea Using these drops, we are now looking at the influx of cells into the graft tissue to determine the early events involved in graft rejection. Prelimi- nary studies of feeding lymphocytes of donor ani- mals to recipients prior to corneal grafting are showing promising results. There appears to be a delay in corneal graft rejection, but the effect is not yet statistically significant Studies on the kinetics of RPE rejection in the subretinal space of Lewis rats reveal that, when human RPE cells are used, graft rejection occurs within 14 days. This is the same rate of rejection observed for other tissue grafts; The infiltrating cell population is mixed, containing both T and B cells. This is the first good demonstration of graft rejection in RPE transplantation. Prior to these experiments, several claims had been made that graft rejection did not occur. Studies using the laser photometer have shown that it is possible to take measurements of anterior chamber flare from rat eyes. The major advantages of this technique are that it can be performed on live, anesthetized animals; measure- ments can be repeated over time; and the measure- ments are given as numerical values, making data analysis much easier. Significance to Biomedical Research and the Program of the Institute Uveitis is the cause of 10% of visual impairment in the United States. Ocular complications that require surgery for correction are common in these patients, despite adequate inmiunosuppression. Developing appropriate surgical modalities of treatment is thus an important endeavor. Similarly, conditions exist in which appropriate therapy can only be given once the proper diagnosis has been made from intraocular tissue. Developing the means of obtaining a minimal amount of tissue and properly processing it is thus of major significance. Proposed Course This study will continue to investigate methods of surgically managing patients with uveitis. Patient enrollment continues. In the animal models, we will continue to study new methods of modulating graft rejection. We also will proceed with our study of the immunology of RPE transplantation and factors that may influence rejection, such as the method of graft insertion. A transvitreal approach may prove to be less inflammatory. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications DeBarge LR, Fenton R, Nussenblatt RB, de Smet MD: Quantitative determination of the flare in 120 NEI Annual Report— FY 1993 Laboratory of Immunology the anterior chamber of the rat by a noninvasive method. Invest Ophthalmol Vis Sci 34(4)(suppl): 1481, 1993. Fenton RM, Rubin BI, de Smet MD, Whitcup SW, Nussenblatt RB: A prospective study of 5-FU trabeculectomy vs. single plate Molteno implant in patients with panuveitis complicated by glauco- ma refractory to prior therapy. Invest Ophthalmol Vis Sci 34{4)(suppl):897, 1993. Hikita N, Lopez JS, Chan C-C, Mochizuki M, Nussenblatt RB, de Smet MD: Effect of topical FK 506 on the rejection of corneal allograft in the Lewis rat. 2nd International Symposium on Ocular Inflammation, Jerusalem, Israel, Aug 30- Sep 3, 1992. Martin DP, Chan C-C, de Smet MD, Palestine AG, Davis JL, Whitcup SW, Bumier MN, Nussenblatt RB: The role of chorioretinal biopsy in the management of posterior uveitis. Ophthalmology 100:705-714, 1993. Parks DJ, Hikita N, Nagineni C, Hooks JJ, Chan C-C, Nussenblatt RB, de Smet MD: Immuno- histochemistry of xenogeneic RPE transplants in the rat: A model for graft rejection. Invest Oph- thalmol Vis Sci 34(4)(suppl):1095, 1993. 121 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00115-15 LI PERIOD COVERED October 1, 1992 to September 30. 1993 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Cyclosporine Therapy in Uveitis PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atliliation) PI: Robert B. Nussenblatt M.D. Scientific Director NEI Others: Marc D. de Smet Scott Whitcup Chi-Chao Chan Richard Fenton Dan Martin M.D. M.D. M.D. M.D. M.D. Senior Staff Fellow Senior Staff Fellow Head, Section on Immunopathology Special Volunteer Senior Staff Fellow LI, NEI LI, NEI LI, NEI LI, NEI LI, NEI COOPERATING UNITS (il any) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI. NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.75 PROFESSIONAL: 0.75 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) To test its efficacy in the treatment of uveitis, cyclosporine — an endecapeptide fungal product with specific anti-T-cell charaaeristics — ^is being administered to patients with sight-threatening ocular inflammatory disease of noninfectious origin who have failed on either corticosteroid or cytotoxic agent therapy. Within the context of these ongoing studies, the combined use of cyclosporine A and ketaconazole has been tested in a randomized masked study of a small group of patients whose uveitis was well controlled with cyclosporine. The combination allowed a significant reduction in the dose of cyclosporine needed to control the disease. In some instances the dose could be reduced by as much as 90%. No significant increase in side effects was noted. A phase I/II randomized trial using cyclosporine A and cyclosporine G has ended. There is a definite trend showing that combined use of a cyclosporine and low-to-moderate steroid doses are efficacious in preventing the progression of uveitis. An effective dose of cyclosporine appears to be around 5 mg/kg. At this dosage, toxicity has been reduced for up to 12 months of foUowup. Cyclosporine G was more effective than cyclosporine A in treating cystoid macular edema. 122 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Immunology Project Description Additional Personnel Bany Grubbs Clinical Protocol Number 81-EI-33 Biologist, LI, NEI Objectives Cyclosporine, an endecapeptide obtained from fungi, has been shown to have specific anti-T-cell activity (Transplant Proc 12:234, 1980). We have reponed cyclosporine's exceptional effectiveness in preventing the induction of S-antigen (S-Ag) autoimmune uveitis in rats, as well as in inhibiting the disease once immunization has occurred (J Clin Invest 67:1228, 1981). The goal of this study is to test cyclosporine A (CsA) versus cyclosporine G (CsG) to test their efficacy in treating patients with bilateral sight-threatening posterior uveitis of an autoimmune nature. Methods Patients 18 years or older, of either sex (females not pregnant) who have not done well on more conven- tional medical ther^y have been admitted to this study. All patients must have bilateral sight-threaten- ing uveitis of noninfectious etiology that was not satisfactorily controlled by either corticosteroid or cytotoxic agent therapy. Lymphocyte cultures are prepared, and the immune cells are tested against various crude ocular extracts, as well as purified human S-Ag, to assess evidence of cellular immune memory, which is considered to be the in vitro equivalent of the ananmestic response in vivo. Patients chosen are treated with CsA or a new analog called CsG in a phase I/II trial to evaluate the safety and activity of CsG versus CsA. During this period, the patients' clinical and immunologic courses are closely monitored. Specific attention is given to renal function change, a frequent side effect. Pa- tients who need to continue CsA for over 1 year because of their ocular disease may be asked to undergo renal biopsy for evaluation of the reversible and irreversible components to CsA renal toxicity. Some patients entered on previous CsA studies still followed in tiie eye clinic will continue to be moni- tored for their renal function to determine how and when cyclosporine dosage can safely be tapered. Major Findings CsA has been effective in the treatment of some cases of posterior uveitis. Decreased inflammatory activity and improved visual acuity was seen in most patients Created to date. The particular responsive- ness to this agent by patients with the ocular mani- festations of Behget's disease has been corroborated by a masked randomized trial performed in Japan. The improvement in the chnical condition was supported by a concomitant improvement in elecfro- physiologic test results, particularly in contrast sensitivity. Patients freated with CsA had no abnormalities of natural killer cell activity before the initiation of tiierapy, nor was any noted afterward. CsA signifi- cantiy decreased skin test responsiveness but did not alter lymphocyte proliferation or antibody production in patients. Renal toxicity has been noted in some patients on long-term therapy, necessitating the addition of systemic corticosteroids and a decrease in CsA dosage. At 3 months approximately 78% of the patients entering this open study were considered therapeutic successes, while 62% were considered successes at 1 year. Seventeen patients treated long term with CsA underwent renal biopsy. These biopsy specimens were read in a masked fashion by a group of renal disease specialists who compared these biopsies to those from age-matched conttols. An irreversible component of CsA toxicity could be identified: in the main, renal tubular atrophy accompanied by intersti- tial fibrosis. The majority of the individuals' biop- sies had normal serum creatinine values, but a correlation could be made between the alterations noted and previous serum creatinine elevations for some period of time. The cyclosporine A/G trial has shown that the two cyclosporines have overall equal value in treating uveitis. However, CsG was more effective than CsA in reducing cystoid macular edema, particularly at lower dosages. Significance to Biomedical Research and the Program of the Institute Uveitis is one of the most frustrating problems in all of ophthalmology. Present modes of therapy for patients with severe ocular inflammatory disease are inadequate and nonspecific. CsA appears effective in treating posterior uveitis of noninfectious etiology. This is tiie first new agent in decades to be found 123 Laboratory of Immunology NEI Annual Report— FY 1993 useful in treating the severe form of this condition; therefore, it is important that the optimum therapeutic schedule be developed. Newer therapeutic strategies have already begun. Proposed Course Newer studies to look at various cyclosporine combi- nations will continue. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Clinical use of Int Ophthalmol Publications de Smet MD, Nussenblatt RB: cyclosporine in ocular disease. Clin 33(4):31-45, 1993. Nussenblatt RB, de Smet MD, Rubin B, Freidlin V, Whitcup SM, Davis J, et al: A masked random- ized, dose-response study between cyclosporine A and G in the treatment of sight-threatening uveitis of noninfectious origin. Am J Ophthalmol 115:583-591, 1993. 124 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00278-02 LI PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Oral Admimstration of Antigen and the Ocular Immune Response PRINCIPAL INVESTIGATOR (List other protessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Robert B. Nussenblatt M.D. Scientific Director LI, NEI Others: Igal Gery Susan Whitcher Marc D. de Smet Ph.D. M.S. M.D. Head, Section on LI, NEI Experimental Immunology Clinical Protocol Assistant LI, NEI Visiting Scientist LI, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI, NTH, Bethesda, MP 20892 TOTAL STAFF YEARS 0.8 PROFESSIONAL: OTHER: 0.3 0.5 CHECK APPROPRIATE BOX{ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The effect of oral administration of various antigens on the ocular immune response has been tested in the animal model for a severe intraocular inflammatory disease, experimental autoimmune uveioretinitis, which is induced by both retinal S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP). Oral tolerance could be induced by repeatedly feeding rats S-Ag. A putative suppressor cell that was CDS positive could be isolated from the spleen of such animals and transferred to other animals to induce a similar toleragenic effect. In addition, the role of the spleen was confirmed in ongoing animal experiments. A randomized, masked trial to evaluate the usefulness of S-Ag feeding in patients with intraocular inflammatory diseases has been put together. A pilot study performed in two patients showed the induction of such tolerance. 125 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report— FY 1993 Project Description Objectives Exploring means of immunomodulation has been a major role in this laboratory. While extensive experimentation has used various immunosuppressive agents, there also has been a major thrust in attempts to use other modes of immunosuppression. The goal of this series of experiments, both in animals and in humans, is to test the efficacy of oral tolerance with uveitogenic antigens in the treatment of animals induced with experimental autoimmune uveitis and in patients with bilateral sight-threatening posterior and intermediate uveitis of an autoimmune nature. Methods Six- to 10- week-old Lewis rats of either sex are used for these experiments. Animals are fed various antigens both before and after the induction of experimental uveioretinitis. The antigens include whole molecules such as the retinal S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein (IRBP), as well as their fragments. In a subset of experiments, some animals also undergo splenectomy before the initiation of the exjjeriments; other ani- mals receive sham procedures. We are attempting to evaluate the clinical course of the disease and corrob- orate the clinical observations with histopathology at various points after initiation of the experiments. The goal is to evaluate the role of the spleen, as well as the role of various fragments, in the ability to induce this toleragenic state. In the studies performed with patients, individuals who have bilateral uveitis of a noninfectious cause and are 18 years or older (either sex) are considered for the study. In addition, their lymphocytes must demonstrate an in vitro proliferative response to the retinal S-Ag. The patients also need to be on sys- temic immunosuppressive ther^y, whether it be corticosteroids, cytotoxic agents, or cyclosporine. The goal of this study is to determine whetlier the addition of oral feeding of retinal antigeas will induce a toleragenic state in individuals who need high amounts of immunosuppressive therapy to control their disease. This study is performed in a randomized, double- masked fashion in which some patients receive S-Ag, other patients receive a retinal mixture containing several antigens, and still other patients receive placebo. The intent is to reduce the amount of immunosuppressive therapy that the patients are taking. We hope that a toleragenic state can be induced by feeding these antigens. Major Findings In the animal study, the spleen appears to play an important role in the induction of oral tolerance of S-Ag. In addition, the spleen is essential for adop- tive transfer of tolerance by splenocytes from donors fed S-Ag. Thus, it would be logical to assume that the spleen acts as a site for induction and/or amplifi- cation of cells with suppressive activity. The pilot study demonstrated that, at least in two patients, a toleragenic state can be induced by feeding antigen at the dosages planned for this study. One patient with par planitis and one with Behcet's disease have been able either to come off medication completely or to be reduced to exceptionally low dosages. Significance to Biomedical Research and the Program of the Institute Uveitis is one of the most frustrating problems in all of ophthalmology. The present modes of ther^y for patients with severe ocular inflammatory disease all have limitations, in particular because of their sec- ondary effects. By identifying patients with an immune response to the retinal S-Ag, we will be able to induce an immunosuppressive state without the use of pharmacologic agents. Furthermore, the induced tolerance would be antigen specific. Proposed Course The randomized study will begin shortly. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Nussenblatt RB, de Smet MD, Weiner HL, Gery I: The treatment of the ocular complicafions of Behcet's disease with oral tolerizafion, in Wechs- ler B, Godeau P (eds): Sixth International Con- ference on Behcet's Disease. New York: Ex- cerpta Medica, 1993. 126 NEI Annual Report— FY 1993 Laboratory of Immunology Weiner HL, Miller A, Khoury SJ, Zhang ZJ, AL- Sabbagh A, Brod SA, Lider O, Higgins P, Sobel R, Matsui M, Sayegh M, Carpenter C, Eisenbarth G, Nussenblatt RB, Hafler DA: Suppression of organ-specific autoimmune diseases by oral administration of autoantigens. Progress in Immunology VII. Eight International Congress of Immunology, Budapest, 1992. 127 Laboratory of Mechanisms of Ocular Diseases Report of the Acting Chief, Laboratory of Mechanisms of Ocular Diseases J. Samuel Zigler, Jr., Ph.D. Investigators in the Laboratory of Mechanisms of Ocular Diseases (LMOD) have continued to conduct studies on a broad range of topics relating to the biology of various tissues in the normal eye and the molecular mechanisms responsible for certain ocular diseases. The major emphasis has been on cataract and the various ocular compUcations of diabetes. Section on Cataract Dr. Deborah Carper and her colleagues have concentrated their efforts on the role of aldose reductase, which produces polyols, in causing diabet- ic complications and on the possible effect of sorbi- tol dehydrogenase, which metabolizes polyols, in protecting against such pathologies. Specifically, site-directed mutagenesis studies have demonstrated that the histidine at position 110 of aldose reductase is critical for catalytic activity. Also, a study has been instituted on a family with congenital cataracts, whose members have a probable genetic defect in the sorbitol dehydrogenase gene. Dr. Donita Garland's group has made major advances in its collaborative study on the protein composition of normal human lens and cataracts. The addition of a scanner capable of quantifying the complex images obtained by two-dimensional elec- trophoresis and software with which to compare and analyze the data provides the tools necessary to address the important questions raised by this investi- gation. In addition, this group is investigating the effects of metals, including copper, iron, and zinc, on the lens crystallins and has found that both oxidation and aggregation are induced by such exposure in vitro. Dr. Fielding Hejtmancik and his group are study- ing structure/function relationships of P-crystallins and doing gene-mapping studies on a variety of genetic diseases with ocular findings. One such disease is Usher's syndrome type I, for which two independent genes have been m^ped on chromo- some 2. One of these genes, which causes Usher's syndrome in the Acadian population, has been localized within a 6cM portion of the chromosome. Dr. Paul Russell's group has concentrated its efforts on the biology of the lens epithelium and on development of lens organ culture techniques. Smdies on lens epithelium have included analysis of protective mechanisms induced by various types of stress and analysis of the process whereby lens epithelial cells differentiate into fibers. These studies include tissue culture approaches as well as analyses of the epithelial layer from intact lenses. A novel method has been developed to assess the integrity of lenses in organ culture. This technique provides quality control information which allows the re- searcher to reject imperfect lenses before committing them into experiments. Dr. Samuel Zigler' s group also has been working with the lens organ culture system, using it as a means of screening potential anticataract drugs. This group also is investigating the functions of lens crystallins, in particular the role of a-crystallin as a molecular chaperone. Definitive proof of noncova- lent complex formation between a-crystallin and the early non-nafive forms of denaturing proteins has been obtained, as has evidence for marked differ- ences in the protection of apo- and holo- forms of some enzymes. Section on Pathophysiology Dr. W. Gerald Robison, Jr., has continued to refine and better characterize the rat model for diabetic retinopathy. Multiple angiopathies were present in the retinas of these rats following 24 months of galactose feeding. In contrast, galactose- fed animals given an aldose reductase inhibitor did not develop such pathologies. The data support the hypothesis that aldose reductase is the primary player in the formation of retinopathy in this model. 131 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00201-09 LMOD PERIOD COVERED October 1. 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Structure and Expression of Polyol Pathway Enzymes PRINCIPAL INVESTIGATOR (List oltier prolassional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Deborah Carper Others: Susan Old Takeshi Iwata Ph.D. Ph.D. Ph.D. Biologist Staff Fellow Visiting Associate LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.0 PROFESSIONAL: 3.0 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors [~| (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) In diabetes, the accumulation of sorbitol is believed to be a Icey factor in initiating cataract, retinopathy, and neuropathy. We are interested in controlling the accumulation of sorbitol by regulating the action of the two enzymes of the sorbitol pathway: (1) aldose reductase (AR), which reduces glucose to sorbitol, and (2) sorbitol dehydrogenase (SDH), which oxidizes sorbitol to fructose. Our aim is to design innovative methods to inhibit the action of AR or increase SDH, with the purpose of reducing sorbitol accumulation in diabetic tissues. Site-directed mutagenesis of AR has been a major priority of our laboratory. We have made amino acid substitutions in the rat and human AR and determined that some of these changes affect the kinetics of the protein with its substrate. For example, when histidine at position 1 10 was changed to glutamine, the activity of AR was reduced dramatically. TTie HI lOQ mutant protein showed very little activity with glyceraldehyde (1% of normal) and no activity with ;7-nitrobenzaldehyde. Other histidine substitutions did not acutely alter the kinetics of AR, supporting the finding that HI 10 plays an essential role in catalysis. These structure/ function studies should help define the active site and locate the target areas of the current AR inhibitors. Another strategy to control sorbitol accumulation is to regulate SDH. We have determined the primary sequence of human SDH and characterized part of the SDH gene. Molecular genetic studies also are under way to test for an SDH genetic defect in a family presenting with congenital cataracts and lowered SDH enzyme activity. By evaluating the expression of SDH at the gene level, we may be able to evaluate its role in sorbitol accumulation in diabetes and other genetic diseases. 132 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The objective of this project is to study regulation of the enzymes of the polyol pathway. Methods The methods employed include molecular biology, protein chemistry, and cell biology techniques. Major Findings Structure/function studies. — Mutant forms of the aldose reductase (AR) protein were synthesized using the polymerase chain reaction. Sequencing verified the amino acid substitution. The mutant proteins were expressed in bacteria, purified on three columns using different biochemical characteristics of the protein, then tested for enzyme activity. Of the six histidines we mutated, histidine at position 110 was discovered to play an essential role in enzyme catalysis. When histidine 110 was changed to glutamine, AR activity was reduced to only 1% of the normal. The K^ for glyceraldehyde changed from 0.12 mM for the normal to 12.5 mM for the HI 10 mutant. No reductase activity was observed for HI lOQ using p-nitrobenzaldehyde as a substrate. Ultraviolet circular dichoism and NADPH fluores- cence quenching indicated that HllOQ was not substantially altered in structure or in its ability to bind NADPH. Because of its location in the active site pocket, histidine 110 has been proposed to be a hydrogen donor in the catalytic mechanism of AR. From these findings, using site-directed mutagenesis, we conclude that HI 10 plays a critical role in the catalytic mechanism of AR, although further studies will be needed to determine the exact nature of its action. Expression of human AR in transgenic mice. — A cDNA that encodes human AR was ligated with a ^- crystaUin promoter. The construct was injected into mice. Several mice were found to carry the trans- gene and were bred to produce separate Fj genera- tions. Evaluation of the presence of the enzyme and its polyol product are now under way. Expression of human AR in transgenic mice will facilitate in vivo drug design studies. Characterization of sorbitol dehydrogenase (SDH) in a family with congenital cataracts. — We have obtained and sequenced over 50% of the gene for human SDH. Previously we determined the complete coding sequence of the protein. With this information we have begun studies to determine a possible genetic defect in a family reported to have reduced levels of SDH and congenital cataracts. Our preliminary nucleotide-sequencing data have indi- cated a difference between this family and normal controls. Significance to Biomedical Research and the Program of the Institute AR has been implicated in diabetic cataracts, retinop- athy, and neuropathy. Side effects and lack of efficacy of AR inhibitors in diabetic clinical trials have emphasized the need for innovative approaches to AR inhibition. Our research is a rational approach to designing new types of inhibitors by characteriz- ing the structure/function aspects of the protein and evaluating the signals that regulate this enzyme. In addition, we feel that by understanding the regulation of SDH — ^the other enzyme of the polyol pathway — we may be able to modulate more fully the accumu- lation of sorbitol in diabetes. Proposed Course The project will continue via site-directed mutagene- sis of AR protein to localize the critical amino acid residues in the active and inhibitor binding sites. We will complete the structure of SDH and analyze the gene in a family with congenital cataracts. NEI Research Program Cataract — Molecular Genetics Publications Bateman JB, Kojis T, Diep A, Klisak I, Heinzmaim BS, Carper D, Nishimura C, Mohandas T, Sparkes RS: Mapping of aldose reductase gene sequences to human chromosomes 1, 3, 7, 9, 11, 14 and 18. Genomics, in press. Lin L-R, Carper D, Yokoyama T, Reddy V: Effect of hypertonicity on aldose reductase, alphaB- crystallin and organic osmolytes in retinal pig- ment epithelium. Invest Ophthalmol Vis Sci 34:2352-2359, 1993. 133 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT HHUJbUI INUMOtM ZOl EY 00189-10 LMOD PERIOD COVERED October 1. 1992 to September 30. 1993 TITLE OF PROJECT (80 characters or less Title must lit on one line between the borders.) Oxidation of Proteins in Cataractogenesis PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Donita L. Garland Ph.D. Research Chemist LMOD, NEI Others: Jose Jimenez Lorenzo Merola Kenichi Matsuno Ph.D. M.S. Ph.D. Visiting Fellow Chemist IRTA LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (it any) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI. NTH, Bethesda, MD 20892 TOTAL STAFF YEARS: 4.0 PROFESSIONAL: OTHER: 4.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The proteins of the normal human lens and cataracts of various etiologies are being characterized. These studies include identifying the major protein species and the modified forms of these proteins, mapping the protein composition tiiroughout the lens, and quantitadng changes in the levels of these proteins in cataracts. An enzyme that protects proteins against thiol-dependent oxidative inactivation has been identified in bovine, rat, and primate lens. The enzyme has been purified and identified by sequence analysis. Seventy percent of the amino acid sequence has been obtained. The interaction of copper, iron, and zinc with a number of proteins, including lens crystallins, has been studied. All three metals alter the solubility of the lens crystallins and many of the other proteins studied. Copper and iron are metals generally thought to be involved in the metal-catalyzed oxidation of proteins. In addition, these studies show them capable of inducing aggregate formation. 134 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The immediate objectives of tiiis project are (1) to. identify and characterize the types of protein modifi- cations found in cataracts of various etiologies, (2) to investigate the role of oxidation in the formation of these modifications, (3) to study the interaction between metals and crystallins and the effects of these interactions on crystallin solubility and aggre- gate formation, and (4) to characterize one of the enzymes that protect lens proteins against thiol- dependent metal-catalyzed oxidation. Methods Bovine, rat, and human tissues were used for these studies. After human lens material was obtained from donors' eyes via cataract surgery, we employed classical methods to purify bovine and rat lens proteins. Other methods used were standard proce- dures for studying proteins, including two-dimension- al gel electrophoresis, high-pressure liquid chroma- tography, ultraviolet/visible spectroscopy, fluores- cence, circular dichroism, electron spin resonance, amino acid analysis, and inmiunotechniques. Major Findings 1. Copper, zinc, and iron, but not calcium, induced aggregate formation in bovine lens extracts and solutions of the crystallins. Aggregation, mea- sured by light scattering, was time dependent, occur- ring at metal-to-protein ratios greater than 1.0 and varying, depending on the metal and protein. Zinc induced the aggregation of P- and a- but not y- crystallin. Copper and zinc induced aggregation of a number of other proteins, but they had no effect on lysozyme and papain. One explanation for the lack of effect on these two proteins is that they are basic proteins. However, copper induced aggregation of y- crystallin is also a basic protein. The affinity of copper and zinc for these proteins is relatively low (K^ max is about 10"^ M). The addition of EDTA, DETAPAC, L-histidine, or L- cysteine prevented zinc- and copper-induced protein aggregation and caused complete disaggregation. These studies suggest that histidine is the amino acid involved in the aggregation induced by zinc and possibly copper. The treatment of a- and ^-crystal- lin and trypsin inhibitor with diethylpyrocarbonate prevented aggregation. Increasing the pH to 8.0 substantially increased the zinc-induced aggregation of a-crystallin but not that of P-crystallin. No pH- induced change in conformation of either protein was observed by fluorescence studies, suggesting the effect may have been on the pK of an amino acid. The presence of salt decreased the metal-induced aggregation of a- and p-crystallin. No metal-induced changes in secondary and tertiary structures of these proteins were observed by fluorescence and circular dichroism spectroscopy. The mechanism of metal- induced aggregation is not clear, but it is likely that it primarily involves cross-linking rather than confor- mation-induced protein-protein interactioa The presence of small amounts of zinc in the buffer reduced the thermal stability of a-crystallin and hemoglobin. Zinc concentrations greater than 20 ^iM induced cell membrane damage to rat lenses in culture, as measured by choline and rubidium uptake. Atomic absorption analysis of bovine crystallins indicate the presence of zinc associated with a- and P-crystallin. These studies clearly demonstrate that metals are known to be involved in oxidative damage and protection against oxidative damage bind crystallins. This interaction induces aggregate formation, a phenomenon that has been linked to cataractogenesis. 2. A protein that appears to function in detoxifi- cation of the products of thiol-dependent oxidation has been demonstrated in cow, monkey, and rat lens and human trabecular meshwork cells. The protein has been purified to ^parent homogeneity, and about 70% of the amino acid sequence has been obtained. The enzyme has been identified from the protein sequence as one of the detoxificafion enzymes found in most cells. The absence of secondary sequences and the copurification of the antioxidant activity and the detoxification enzyme during two separate schemes su-ongly indicate that the antioxidant activity is associated with this detoxification enzyme, not with a contaminant in the preparation. There are no previous reports of the antioxidant activity of tiiis enzyme. 3. Analysis of the proteins in human cataract specimens by two-dimensional gel electrophoresis has continued and the techniques have been opti- mized. Preparation procedures to facilitate analysis of aspirated lens material (primarily outer cortex 135 Laboratory of Mechanisms of Ocular Diseases NEI Annual Report— FY 1993 obtained during extracapsular cataract surgery) have been established. These procedures allow us to determine the relative amount of the major proteins present and the oxidation state of these proteins in the cataract. We have identified alterations in protein patterns and are doing quantitative analyses of the changes. Correlations between the altered patterns and cataract etiologies are being sought 4. We have developed capillary gel chromatogra- phy procedures that allow the separation and accurate quantitation of sorbitol and galactitol. Significance to Biomedical Research and the Program of the Institute Oxidative processes have long been considered a major contributing factor in senile cataracts. Metal- catalyzed oxidation of the crystallins leads to protein modifications that mimic those seen in aging and senile cataracts and in brunescent lenses. These studies continue to demonstrate the potential for metal involvement in cataract formation. Not onJy do these metals facilitate oxidative modification of the proteins, they also can induce protein aggrega- tion. Understanding the lens' mechanisms of protecting itself against oxidative damage is important for developing interventions. These studies demonstrate the presence in the lens of an enzyme that protects against thiol-dependent oxidation. This activity is associated with a detoxification enzyme present in most cells and is induced imder oxidative stress. The importance of characterizing the proteins in the human lens — normal and cataractous — ^is obvious. It will give us a wealth of information on aging processes, mechanisms involved in cataractogenesis, and metabolic processes in this unique tissue. Proposed Course We will focus our studies for Fiscal Year 1994 on the following: (1) continuing the investigation of metal-catalyzed oxidation of lens proteins, (2) de- tailed characterization of the interaction of metals witii crystalUns and the effects on solubility, (3) anal- ysis of human lens proteins in cataracts and the normal lens, and (4) molecular biology and immuno- logical characterization of the enzyme that protects against thiol-dependent oxidation reactions. NEI Research Program Cataract — ^Lens Development and Aging Publications Bettelheim FA, Reid MB, Garland D: Hydration of gamma crystallins. Exp Eye Res, in press. Giannessi M, Del Corso A, Cappiello M, Vatarelli M, Marini I, Barsacchi D, Garland D, Camici M, Mura U: Thiol-dependent metal catalyzed oxidation of bovine lens aldose reductase: I. Studies on the modification process. Arch Biochem Biophys 300:423^29, 1992. 136 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00272-03 LMOD PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on arte lirte between trie borders.} Inherited Ocular Diseases _^_^ PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: James Fielding Hejtmancik M.D., Ph.D. Medical Officer LMOD, NEI Others: John Hope Radha Ayyagari Ling Lee Anthony Lloyd T. Padma Ph.D. Ph.D. M.S. M.D. Ph.D. Senior Fellow Special Volunteer Chemist IRTA Fellow Visiting Scientist LMOD, NfEI LMOD, NEI LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (if any) Baylor College of Medicine (J. Towbin, B. Periyman, T. Ashizawa, P. Overbeek); Univ. of Iowa (R. Smith); Univ. of Texas-Houston (S. Daiger); Ocular Genetics and Clinical Services Branch, NEI, NIH (M. Kaiser- Kupfer); Washington Univ. at St. Louis (M. Petrash, R. Hayes); Massachusetts Institute of Technology (G. Benedek, J. Pande); Osmania Univ., Hyderabad, India (J.S. Murty) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: PROFESSIONAL; 5.15 5.15 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) [xj (a) Human subjects Ix] (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The study of inherited visual diseases provides a means by which both normal and aberrant visual processes might be understood. In addition to directly elucidating the pathophysiology of the inherited disease under study, these studies can provide insights into the structure-function relationships of the molecular components of the visual system and their normal physiology. This laboratory is using a number of approaches to study inherited visual diseases affecting the lens and retina. Lens crystallins comprise over 90% of the soluble protein of the lens and are heavily modified in most cataracts. The effects which specific modifications of P- and y-crystallin structure produce on crystallin functions, such as stability and formation of macromolecular aggregates, are being studied in tissue culture cells transformed with normal and modified pA3/Al-crystalHn genes. Regions of the P-crystallin molecule of special interest include the amino terminal arm and tlie Greek key motifs of the core domains. The effects which these modifications have on lens transparency also are being studied in a transgenic mouse system in which a modified PA3/A1 gene is driven by an aA-crystalUn promoter. A second approach to understanding inherited visual diseases uses principles of positional cloning to identify genes important in human inherited diseases. Human diseases currently undergoing linkage analysis, gene isolation, or characterization of mutations include Usher syndrome, myotonic dystrophy, Duchenne muscular dystrophy. Long QT syndrome, cataracts, and a variety of X-linked syndromes. We currently are collecting families with autosomal recessive retinitis pigmentosa in preparation for study of this important group of diseases. 137 PHS 6040 (Rev. 5/92) Laboratory of Mechanisms of Ocular Diseases NEI Annual Report— FY 1993 Project Description Objectives The long-range objectives of this project include increasing the understanding of inherited visual diseases, with the eventual aims of increasing the diagnostic ability for these diseases and providing a foundation for developing rational therapies based on a thorough knowledge of their molecular pathophysi- ology. These long-range objectives will be approached by pursuing the specific aims of identify- ing genes involved in inherited visual diseases and elucidating the mechanisms by which mutations in these genes cause disease. Methods Conventional cloning technology is utilized in preparing sequences for gene expression studies. These include ligation with T4 DNA ligase, screen- ing by NaOH miniprep methodology, and ^^P-labeled DNA probes, as well as allele-specific oligonucleo- tide hybridization to screen for specific single-base settings. Sequence changes are introduced by site- specific mutagenesis via standard methodology. Gene expression in Chinese hamster ovary cells (RJK 88) and in insect cells (SF9) is enabled by the baculovirus expression system. Protein expression is monitored by standard two-dimensional gel electro- phoresis followed by immunoblotting. Association behavior is assessed by elation volume on sieve FPLC. Crystallin and other cDNAs and genomic frag- ments are isolated by library screening with cloned genes or oligonucleotides using routine metiiods. Sequencing is performed by cycling or using auto- mated fluorescent technology (ABI). Until recentiy, linkage analysis has involved conventional Southern blotting. Cell lines from NIH Eye Chnic patients and their family members are immortalized by Epstein Barr virus ti-ansformation. DNA is isolated by standard methodology and digested by restriction endonucleases. After agarose gel electrophoresis and Southern transfer, the result- ing blot is probed with isolated DNA fi-agmenLs labeled with ^^P by oligonucleotide labeling. Recent- ly, short tandem repeat (microsatellite) markers have been analyzed by polymerase chain reaction per- formed in the presence of labeled oligonucleotides and analyzed on sequencing gels. Linkage data recorded on computerized spreadsheets are subject to both two-point and multipoint analysis with the LINKAGE program package. Major Findings 1. The P-crystallins, their structure, and the mechanisms by which heterogeneity arises among this family of proteins are being investigated. The |3A3-crystallin is identical to PAl except for an additional 17-amino-acid N-terminal extension. The same gene is believed to encode and express both polypeptides. The PA3/A1 coding sequences were ligated behind the RSV promoter, and RJK 88 fibroblast cells were stably transfected with this construction. In addition, the PA3/A1 coding se- quences were inserted into the Bluebac expression vector (Stratagene) and expressed in SF9 cells. A single 26-kD protein, the predicted size of pAl- crystallin, was detected on Western blots of soluble extracts of stable clones using antibodies raised to crystallin peptides. However, the RJK 88 ceUs, transformed with the same cDNA except with codons gln7 and leulO mutated in vitro to stop codons, express only a 24-kD protein, the predicted size of the |3A1 -crystallin. Thus, it appears that the up- su-eam (PA3) start codon is preferentially used in cell lines, although the downstream (pAl) start codon can be used. In SF9 cells, a protein with the same amino terminal sequence as the PAl -crystallin is produced when the baculovirus-infected cells are grown past their prime. This is temporally correlated with the disappearance of the pA3-crystallin band, suggesting that the smaller band is created by processing or degradation of the larger in this system. In addition, clones for the mouse PA2-, PB1-, pB2-, and PB3- crystallin have been isolated and sequenced in preparation for characterization of their roles in P- crystallin aggregation. 2. We have constructed an additional crystallin in wliich the amino-terminal arm was deleted and replaced by a glycine residue, an extension identical to that found in 72-crystalIin. This new crystallin has been expressed in RJK 88 and SF9 cells (Bluebac vector) and has an appropriate migration on Laemmli gels, CD-spectrum, and amino acid sequence. The activity of this P-crystallin in association with the typical 200- to 250-kD aggregates has been tested by FPLC on superdex 75 and superose columns. The nonnal pA3 polypeptide readily associates into 138 NEI Annual Report— FY 1993 Laboratory of Mechanisms of Ocular Diseases homodimers, whereas the truncated |3A3 associates minimally if at all. SF9 cells expressing the recom- binant crystallins were grown in ^^S-containing medium, then purified and re-associated with an excess of lens extract containing normal crystallins (unlabeled) using limited urea denaturation followed by dialysis. Aggregation to form P-crystallin was assessed by FPLC on sizing columns. The recombi- nant full-length p-crystallin peptide aggregates into both dimers and tetramers, with the dimer peak migrating slightly before the p-light peak; however, the truncated pA3-crystallin migrates slightly behind the P-light peak and does not form obvious tetra- mers. These data strongly suggest that the amino- terminal arm of P-crystallins assists in the incorpora- tion of p-crystallins into higher order aggregates. 3. We have constructed a pA3-crystallin in which the entire connecting peptide from the first to the second domain has been replaced with the corre- sponding sequence from 72-crystalfin. This construc- tion should test the hypothesis that the connecting peptide, which crystallographic data show is extend- ed in the P-crystallins and curved back on itself in the y-crystallins, is responsible in this fashion for the P-crystallins' tendency to dimerize. The P-crystallin with the modified connecting peptide was subjected to the same tests of aggregation described in Section 2 above; it behaved essentially as the normal (un- modified) PA3-crystallin. The secondary structure of the modified P-crystallin currentiy is being confirmed with CD analysis. 4. Studies of phase transition properties of the y- crystallin gene family have begun in collaboration with Drs. Mark Petrash (Washington University, St. Louis) and George Benedek (MIT, Boston). The bovine yB-crystallin has been modified at two of the four residues proposed to be critical for phase fransition behavior. Phase transition analysis of the expressed unmodified yZ-crystallin has begun at MIT. 5. We also studied human genetic diseases that involve the eye. In addition to elucidating the pathogenesis of visual symptoms in inherited diseases, our efforts have provided reagents and information applicable to genomic analysis in general. Genetic markers in the myotonic dystrophy region have been used to confirm the diagnostic usefulness of bilateral lens opacities in the diagnosis of myotonic dystrophy; the data were confirmed by examining the trinucleotide repeat shown to be expanded in persons affected by myotonic dystrophy. The phenomenon of anticipation, long controversial in myotonic dystrophy, was shown to occur with statistical significance in the families enrolled in our study. In addition, earlier age of onset through anticipation was correlated with expansion of the trinucleotide repeat, although the correlation was not perfect We have isolated a cDNA clone correspond- ing to the dystrophin gene product from a mouse lens library and are characterizing it. 6. Ophthalmologic diseases in humans have been studied by linkage analysis of RFLP markers. Diseases we have mapped within the past year include Long QT syndrome, X-linked agammaglobu- linemia, and Usher's syndrome type I. In addition, clinical and genetic heterogeneity of Usher's syn- drome within the Acadian population of Louisiana has been explored in detail. Genetic analysis con- firms the clinical impression that both type I and n of Usher's syndrome are found in the Acadian population, even within a single extended pedigree. The heterogeneity analysis described above implies this is due to segregation of two different, unlinked genes within this population. Two genes causing Usher's syndrome type I have been m^ped. In Acadians, the genetic locus is on chromosome 1 Ip, while in the British families in our study, the gene is on chromosome llq. When subjected to the most stringent heterogeneity analyses (both the H0M0G2 program and M test), these findings are significant at p < 0.01. This surprising finding implies that multiple genes can cause the rather specific clinical findings in Usher's syndrome. In detailed study of the Usher's syndrome gene on chromosome lip, we have used fine linkage map- ping and haplotype analysis to localize it to a 6-cM interval between tiie markers Dl 1S861 and Dl 1S928. Several large families with autosomal dominant and recessive cataracts have been ascertained, and samples have been collected. Genotyping of micro- satellite markers has begun for four of these families and will initially be concentrated in regions around candidate genes. Significance to Biomedical Research and the Program of the Institute Elucidation of the genetic defects causing visual disability will have implications far beyond the patient population suffering from the specific syn- dromes under study. Inherited diseases provide a means by which the molecular pathophysiology of 139 Laboratory of Mechanisms of Ocular Diseases NEI Annual Report— FY 1993 the visual system may be understood. This knowl- edge can then be applied to a broad spectrum of diseases. This rationale also applies to the study of inherited diseases of which visual defects are only a small part. Thus, while our studies of myotonic dystrophy already have resulted in improved diagnos- tic abilities, the mechanism by which cataracts occur in this disease will provide insight into cataracto- genesis in other hereditary syndromes as well as in age-related and nonspecific cataracts. Proposed Course 1. We will continue studies on the structure- function relationships of lens crystallins, concentrat- ing on how modifications of the terminal arms and possibly the interconnecting peptide between the two domains affect aggregation of P-crystallins. We also will continue to explore the effects that modificatioas of the Greek key motifs have on crystallin stability and, when applicable, lens transparency. In addition, we will explore the effects of modifications of y- crystallin sequences on the protein phase transitions and its relationship to cold cataract. 2. Sample collection and linkage analysis of a variety of human diseases will continue. The main emphasis will be on inherited visual diseases, espe- cially Usher's syndrome type II. We are initiating a linkage study of autosomal dominant cataracts in families ascertained in collaboration with Dr. Muriel Kaiser (Ophthalmic Genetics and Clinical Services Branch) and of autosomal recessive cataracts ascer- tained in collaboration with Dr. J.S. Murty (Osmania University, India). This study will be coordinated with a new project to categorize and map expressed sequences of the human lens and the ongoing mecha- nistic studies on lens crystallins described above. Together these projects should provide a coordinated effort to elucidate the mechanisms of cataractogene- sis in the human lens. NEI Research Program Cataract — Molecular Genetics Publications Ashizawa T, Dubel JR, Dunne PW, Dunne CJ, Fu Y-H, Pizzuti A, Caskey CT, Boerwinkle E, Perryman MB, Epstein HF, Hejtmancik JF: Anticipation in myotonic dystrophy: Complex- relationships between clinical findings and struc- ture of the GCT repeat. Neurology 42: 1877-1893, 1992. Ashizawa T, Dunne CJ, Dubel JR, Perryman MB, Epstein HF, Boerwinkle E, Hejtmancik JF: Anticipation in myotonic dystrophy: Statistical verification based on clinical and haplotype findings. Newro/o^ 42:1871-1877, 1992. Ashizawa T, Hejtmancik JF, Liu J, Perryman MB, Epstein HF, Koch DD: Diagnostic value of ophthalmologic findings in myotonic dystrophy: Comparison with risks calculated by haplotype analysis of closely linked restriction length poly- morphisms. Am J Med Genet 42:55-60, 1992. Ayyagari R, Smith RJH, Lee EC, Kimberling WJ, Jay M, Bird A, Hejtmancik JF: Heterogeneity of Usher syndrome type I, in Anderson RE, Holly- field JG, Lavail MM (eds): Degenerative Retinal Disorders: Clinical and Laboratory Investiga- tions. New York, Alan R. Liss Inc., 1992. Hejtmancik JF: Neurology of the visual system, in Conn PM (ed): Neurology, 1992. Hejtmancik JF, Black S, Harris S, Ward PA, Calla- way C, Ledbetter D, Morris J, Leech SH, Pollack MS: Congenital 21 -hydroxylase deficiency as a new deletion mutation. Detection in a proband during subsequent prenatal diagnosis by HLA typing and DNA analysis. Hum Immunol 35:246- 252, 1992. Hejtmancik JF, Kaiser-Kupfer MI, Piatigorsky J: Inherited disorders of the eye lens, in: The Metabolic Basis of Inherited Disease. New York, McGraw Hill, 1992. Hejtmancik JF, Piadgorsky J: Molecular biology of the eye lens, in Raviola E, Dowling J (eds): Principles and Practice of Ophthalmology. Philadelphia, WB Saunders, 1992. Hejtmancik JF, Roberts R: Molecular genetics and the application of linkage analysis, in Roberts R (ed): Molecular Basis of Cardiology. London, Blackwell Scientific Publications, 1993, pp 355- 381.' Keats BJ, Todorov AA, Pelias MZ, Hejtmancik JF, Kimberling WJ, Leppert M, Lewis RA, Smith RJ: Linkage studies of Usher syndrome type I: Exclusion results from the Usher syndrome consortium. Genomics 14:707-714, 1992. 140 NEI Annual Report— FY 1993 Laboratory of Mechanisms of Ocular Diseases Muller B, Dechant C, Meng G, Liechti-Gallati S, Doherty RA, Hejtmancik JF, Bakker E, Read AP, Jeanpierre M, Fischbeck KH: Estimation of the male and female mutation rates in Duchenne muscular dystrophy (DMD). Hum Genet 89:204- 206, 1992. Nickerson JM, Hejtmancik JF: Molecular biology and genetics of the retina, in Tasman W, Jaeger E (eds): Biomedical Foundations of Clinical Ophthalmology. Philadelphia: Lippincott, 1992. Parolini O, Hejtmancik JF, Allen RC, Belmont JW, Lassiter GL, Henry MJ, Barker DF, Conley ME: Linkage analysis and physical mapping near the gene for X-linked agammaglobulinemia at Xq22. Genomics 15:342-349, 1993. Smith RJH, Berlin C, Hejtmancik JF, Laties A, Lewis RA, Keats B, Kimberling WJ, Moller CG, Pelias MA, Tranebjaerg L: Clinical diagnosis of the Usher syndromes. Am J Med Genet, 1992. Smith RJH, Lee EC, Kimberling WJ, Daiger SP, Pelias MZ, Keats BJB, Jay M, Bird A, Reardon W, Guest M, Ayyagari R, Hejtmancik JF: Local- ization of two genes for Usher syndrome type I to chromosome 11. Genomics 14:995-1002, 1992. Smith RJH, Pelias MZ, Daiger SP, Keats B, Kimber- ling W, Hejtmancik JF: Clinical variability and genetic heterogeneity within the Acadian Usher population. Am J Med Genet 43:964-969, 1992. Towbin J A, Hejtmancik JF, Brink P, Gelb B, Zhu XM, Chamberlain JS, McCabe ER, Swift M: X- linked dilated cardiomyopathy. Molecular genetic evidence of linkage to the Duchenne muscular dystrophy (dystrophin) gene at the Xp21 locus. Circulation 87:1854-1865, 1993. 141 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1. 1992 to September 30. 1993 PROJECT NUMBER ZOl EY 00237-08 LMOD TITLE OF PROJECT (80 characters or loss. Title must In on one line between the borders.) Characterization of the Lens PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Nante. title, laboratory, and institute affiliation) PI: Paul Russell Ph.D. Research Chemist LMOD, NEI Others; Carolyn Chambers Geoffrey Kidd Santa Tumminia Ph.D. Ph.D. Ph.D. Senior Staff Fellow Senior Staff Fellow Senior Staff Fellow LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (il any) LAB/BRANCH Laboratory of Mechanisms of Ocu lar Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH. Bethesda, MP 20892 TOTAL STAFF YEARS: 4.0 CHECK APPROPRIATE BOX(ES) D (a) Human subjects n (a1) Minors □ (a2) Interviews PROFESSIONAL: 4.0 OTHER: 0.0 B (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.) ' ' We are conunuing to develop an in vitro model to check anticataract agents. The organ culture system utilizes lenses from rats and inonkeys. We have developed a method to screen the lenses to detennine which might have been damaged m dissecuon. This is the first method to adequately predict the integrity of the lens at a very early stage in culmrerfens always been correct. Many lenses can mamtain clarity but are not able to transport ions and amino acids normally By Ifr? "^''^''^l^^^^' ^^ ^so l^ve been able to show that the main protein in the organ culmre medium is albumin most probably as a result of residual protein sticking to the lens during dissection. The albumin can be taken chilC.nf.H^fhTH""'^ ""'"^ ^ 'i^'"' '""'' ""^ '"^' P''^'"^ ^"^ ^^^ «"' «f ^^ l«"s- Cunously, when the lens is ■of hvfh r 'ah7'" Pf™'''' "" ' '""'"^ '°^ ^'^'^^^^ ^^^^^' ^^ P«-2 ^'^^ i^ o°e of the principal proteins ixnreLTii'l .1 ''h " t"^' organ-culnired lenses have begun analysis of the types of messenger RNA Zsag^ " ' ^' "'""^ """"^'^ '^"''' '° '*'''™^' '^^ sequences of these stress-related lTi,r°" '^h ^T ^^'"^fr ^"^ """^"^ "^ ''"'^y '^^ 'P"'^^'" ""^^"O"^- ^^ '^on'^en's the protective mechanisms present ui the lens epithelium to prevent damage from oxidative sQ-ess. Work with the lens epitheUum cell lines has shown that the major oxidoreductase activated in an oxidative sffess system appears to be D-T diaphorase. C-crystallin also an oxidoreductase. is responsive to the oxidative stress and increases in the lens cells. The increa^in the ^-crystalhn cannot by itself account for the large increase in oxidoreductase found in the stressed cells The second quesuon concerns cellular differentiation into fiber cells. The region of the lens where this process occurs tends to be wni^r^'' f^ ^^^^ ""?' '''"' conditions. We have separated certain steps in the differentiation process and will be better able to explore the stages at which cataracts might develop in the equatorial region of the lens. Work continues on the human PB-2 crystallin, which now has been successfully cloned and sequenced. The deduced 2TZZ T J rr '""^"""'^ ^^ '°°'*''' '^^- ^'""^ ^'^ P^*'^" '^ developmentally regulated, investigation into the promoter activity of this gene is continuing. ^ & . eauu.. muj 142 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The purposes of this project are (1) to understand the basic biological processes of the human lens and how they are altered in cataract formation, (2) to develop model systems with which to mimic these processes, and (3) to use these model systems to develop methods to test anticataract agents. Methods Among numerous biochemical and molecular biolog- ical methods used in this research are Northern, Southern, and Western blotting of mRNA, DNA, and proteins. In addition, various methods for quantita- tion of these components, such as slot-blotting, are done. The polymerase chain reaction is used, as is nucleic acid sequencing. Major Findings 1. An organ culture model system to test anticat- aract agents has been standardized. Part of the model involves screening the proteins in the culture medium to determine lens integrity. 2. With lenses from young animals, glutathione is rapidly lost in the organ culture system; however, this might not be the case with an older animal. 3. One of the major proteins that leak from the lens under conditions of stress is PB2-crystallin. 4. The PB2-crysta]lin has been cloned and sequenced from human lens. It has about 907c similarity with the mouse pB2-crystaUin that we had sequenced previously. Our deduced sequence for the human crystallin has been confirmed by another group. 5. ^-crystallin, an oxidoreductase found in guinea pigs and camels, has been found in the mouse lens. Lens cells from transgenic animals also have i^- crystallin. 6. Lens cells under oxidative stress react to the stress by increasing oxidoreductase activity. The activity that appears to be most responsible for the amelioration of the effects of oxidative radicals is D- T diaphorase, although i^-crystallin also is activated under oxidative stress conditions. 7. In tissue cultures, lens cells form so called "lentoid bodies." We have shown that lentoid body formation is an early step in the maturation process of the lens cell. The lentoid body can form without the activation of certain lens-specific proteins. Lentoid body formation is similar to the elonga- tion process that occurs in the equator of the lens. In this equatorial area, many cataracts originate. Thus, understanding the steps in the differentiation proce- dure is necessary to understand cataract formation. 8. Some of the proteins present in the aqueous humor of the eye have been identified. The aqueous humor is the fluid that nourishes the lens in vivo. The eight proteins that have been confirmed to be in the aqueous of the monkey are albumin, transferrin, ceruloplasmin, plasminogen, fibrinogen, a-1 antitryp- sin, HDL, and cystatin. Significance to Biomedical Research and the Program of the Institute The development of systems to study the lens is vital to understanding the mechanisms involved in cataract formation. The new methods and protocols that we have developed for organ-cultured lenses have enabled us to standardize this useful technique for the study of cataract development. Working under definable, reproducible conditions is an advantage in formulating model systems to study conditions that lead to loss of cell function. These studies will enable us to devise systems to study anticataract agents. Publications Chambers C, Russell P: Sequence of the human lens betaB2-crystallin encoding cDNA. Gene 133:295-296, 1993. Du X-Y, Russell P, Zigler JS Jr: Potentiation of protein oxidation in cultured lenses by 2-deoxy- glucose and BCNU. Curr Eye Res 11:475^78, 1992. Kidd GL, Reddan Jr, Russell P: Differentiation and angiogenic potential in two mammalian lens epithelial cell lines. Differentiation, in press. Qin C, Rao PJ, Tumminia SJ, Zigler JS Jr, Russell P: Loss of glutathione in the organ cultured rat lens. Invest Ophthalmol Vw5d34(4)(suppl):758, 1993. Russell P, Epstein DL: Protein analysis of monkey aqueous humor. Curr Eye Res 11:1239-1243, 1992. 143 Laboratory of Mechanisms of Ocular Diseases NEI Annual Report— FY 1993 Russell P, Epstein DL: Protein analysis of the rhesus monkey aqueous humor. Invest Ophthal- mol Vis Sci 34(4)(suppl):1280, 1993. Russell P, Koretz J, Epstein DL: Is primary open- angle glaucoma caused by small proteins? Med Hypothesis, in press. Russell P, Zigler JS Jr: Analysis of the effects of eye bank storage conditions on primate lens epithelium. Exp Eye Res 54:153-155, 1992. Tumminia SJ, Qin C, Zigler JS Jr, Russell P: Asses- sibility of the viability and integrity of mammali- an lenses in organ culture. Invest Ophthalmol Vis Sci 34(4)(suppl):756, 1993. Tumminia SJ, Rao PV, Zigler JS Jr, Russell P: Xenobiotic induction of quinone oxidoreductase activity in lens epithelial cells. Biochim Biophys Acta 8:251-259, 1993. 144 DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00252-05 LMOD PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Cataract in the Philly Mouse Strain PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Paul Russell Ph.D. Research Chemist LMOD, NEI Others: Carolyn Chambers Ph.D. Senior Staff Fellow LMOD, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews PROFESSIONAL: 0.0 OTHER: 0.0 □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project has been terminated. 145 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00105-14 LMOD PERIOD COVERED October 1. 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less T1II9 must lit on one line between the borders.) Structure and Composition of Lens Crystallins with Respect to Cataractogenesis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atliliation) PI: J. Samuel Zigler, Jr. Ph.D. Research Biologist LMOD, NEI Others: Vasantha Rao Pedro Gonzalez Chuan Qin Ph.D. Ph.D. M.D. Visiting Associate Visiting Fellow Visiting Fellow LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (il any) Jules Stein Eye Institute, UCLA (J. Horowitz, B. Bateman); Laboratory of Molecular and Developmental Biology, NEI (G. Wistow, D. Lee); National Cancer Institute (M. Krishna); Centre for Cellular and Molecular Biology, Hyderabad, India (D. Balasubramanian; M. Rao) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 4.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews PROFESSIONAL: 4.0 OTHER: 0.0 [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project, directed toward elucidation of the molecular mechanisms responsible for cataractogenesis, places special emphasis on the role of the structure and function of the lens crystallins. Until recently, the crystallins were thought to be simply structural elements of the lens protein matrix, without any specific quantifiable biological function. Two recent discoveries have provided new insights and approaches to the physiological roles of the crystallin: (1) the crystallins either are functionally active enzymes or are at least related to proteins with specific biological activities and (2) a-crystallin is a molecular chaperone that can prevent the aggregation of denaturing proteins. Our group is studying the chaperone function of a-cTystallin, with the goal of establishing its significance in the intact lens. Using the isolated crystallin, we have shown that it forms stable complexes with target proteins, specifically interacting with denaturing proteins at the earliest stage of denaturation. The specificity of this interaction has been demonstrated by the fact that in some instances it is strongly dependent on the availability of obligate cofactors of the target proteins. Studies on "enzyme/crystallins" focus on i;-crystallin, a major protein in the lenses of certain mammals (e.g., guinea pigs, camelids). In guinea pigs a mutation in the ^-crystallin gene causes hereditary nuclear cataracts, and our goal is to understand how the mutation affects the lenticular function(s) of the protein, leading to cataract. The ^-crystallin system also is being used to investigate the mechanisms of lens-specific expression of crystallin genes. Lens organ culture is being used as both a means of testing potential anticataract agents and a system for analyzing the responses of intact lenses to various cataractogenic stresses. The changes in gene expression induced in primate lenses by stress are being studied to identify specific proteins and processes important in combating stress. This information will facilitate more rational design of strategies to accomplish our ultimate goal: the prevention or delay of cataractogenesis. 146 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The primary objectives of this project are (1) to elucidate at the molecular level processes responsible for cataract development, (2) to investigate the structures and functions of the lens crystallins, and (3) to develop and use model systems for screening potential anticataract agents. Methods Conventional protein chemical techniques employed are chromatography, electrophoresis, and isoelectro- focusing. Immunological studies of lens proteins use specific antisera. Physicochemical analyses on the proteins are performed using high-pressure liquid chromatogrj^hy, fluorescence, and circular dichroism techniques. In lens organ culture experiments involving rat or monkey lenses, we use active trans- port and membrane permeability parameters to monitor the effects of various stresses on the cultured lenses. Techniques used in analysis of nucleic acids include RNA and DNA isolation, cDNA and gene cloning, DNA sequencing, various electrophoretic methods, and the polymerase chain reaction. Major Findings 1. a-crystallin acts as a molecular ch^erone, forming stable complexes with various other proteins undergoing denaturation and preventing their aggre- gation. Once fully denatured, the target proteins do not associate with a-crystallin. Thus, like other chaperone proteins, a-crystallin specifically recog- nizes and binds proteins only in the very early stage of denaturation. 2. Further evidence for the specificity of this reaction is provided by the finding that, with certain proteins, protection from aggregation by a-crystallin is dependent on the presence of cofactors. For example, ^-crystallin/quinone reductase, an NADPH- requiring enzyme, is efficientiy protected only in the presence of NADPH. 3. The ^-crystallin cDNA from the lens of the llama has been sequenced and found to be highly similar to that of other mammals analyzed. The llama is a camelid and therefore of particular interest because, like guinea pigs, these animals have very high levels of lenticular ^-crystallia 4. Our analysis of the llama ^-crystallin gene has revealed two promoters, one of which regulates normal low level expression in many tissues. This promoter exists in all ^-crystallin genes examined. A second lens-specific promoter is found only in species in which the protein is also a major lens protein (e.g., guinea pig and llama). Interestingly, the promoter in the llama gene is unrelated to the lens-specific promoter previously characterized in the guinea pig, suggesting that ^-crystallin/quinone reductase was recruited as a lens protein at least two different times during evolution. 5. The human ^-crystaUin gene has been local- ized to chromosome lp22-p3P, and six restriction fragment-length polymorphisms (RFLPs) have been identified within the gene. 6. Analysis of the sequences of all known lens crystallins reveals that, in general, they are not designed for high intracellular (metabolic) stability. Therefore, the extremely long half-lives of crystallins must result largely from the environment within the lens rather than from intrinsic properties of the proteins themselves. 7. The organ-cultured rat lens loses 40% of its glutathione (GSH) during the first 24 hours of culture and more than 60% by 72 hours. This loss occurs even in the absence of Oj and, thus, is not the result of oxidative stress in the culture system. Interestingly, monkey lenses cultured for up to 48 hours showed no decrease in GSH. 8. Huorescence spectra of intact human lenses over a wide age range demonstrate different amounts and numbers of fluorophors in lenses from the United States relative to lenses collected and ana- lyzed in India. It is hoped that these analyses will give clues to the molecular mechanisms underlying the increased pigmentation and earlier onset of cataract in India. Significance to Biomedical Research and the Program of the Institute Cataract is a major public health problem worldwide. Better understanding of the biochemistry of the normal lens and of the molecular changes that occur during aging and cataract development are essential if this disease is to be controlled. Our smdies are aimed primarily at elucidating the role of the lens crystallins, the primary structural elements of the normally transparent lens matrix, in the processes 147 Laboratory of Mechanisms of Ocular Diseases NEI Annual Report — ^FY 1993 leading to opacification. Such knowledge should contribute to the development of means of interven- tion that can prevent or delay the process of cataract development. Proposed Course We will continue to (1) work to establish viable model systems for testing anticataract agents and use these systems to assess the efficacy of various types of compounds, including antioxidants, (2) complete analysis of the molecular basis underiying the high lens-specific expression of an enzyme/crystallin C^- crystallin), (3) further investigate the chaperone-like function of a-crystallin and determine its physiologi- cal significance in the normal lens and in cataract, and (4) evaluate gene expression in lenses under stress to seek proteins critical in the response to stress. NEI Research Program Cataract — Pathogenetic Mechanisms Publications Gonzalez P, Rao PV, Zigler JS Jr: Molecular clon- ing and sequencing of zeta-crystallin/quinone reductase cDNA from human liver. Biochem Biophys Res Commim 191:902-907, 1993. Jomvall H, Persson B, Du Bois GC, Lavers GC, Chen JH, Gonzalez P, Rao PV, Zigler JSJr: Zeta-crystallin versus other members of the alcohol dehydrogenase superfamily: Variability as a functional characteristic. FEBS Lett 322:240- 244, 1993. Lee DC, Gonzalez P, Rao V, Zigler JS Jr, Wislow GJ: Carbonyl-metabolizing enzymes and their relatives recruited as structural proteins in the eye lens. Adv Exp Med Biol '\:\59-\()^, 1993. Rao CM, Zigler JS Jr: Are crystallins designed for high intracellular stability? Exp Eye Res 56:615- 619, 1993. Rao PV, Horwitz J, Zigler JS Jr: Alpha-crystallin, a molecular chaperone, forms a stable complex with carbonic anhydrase upon heat denaturation. Biochem Biophys Res Commun 190:786-793, 1993. Rao PV, Zigler JS Jr: Mutant zeta-crystallin from guinea pig hereditary cataracts has altered struc- tural and enzymatic properties. Exp Eye Res 54:627-630, 1992. Tumminia SJ, Rao Pv, Zigler JS Jr, Russell P: Xenobiotic induction of quinone oxidoreductase activity in lens epithelial cells. Biochim Biophys Acta, 1203:251-253, 1993. Zigler JS Jr: Lens proteins, in Albert DM, Jakobiec F (eds): Principles and Practice of Ophthalmol- ogy. Basic Sciences Philadelphia, JB Saunders Co, 1994, pp 97-113. I 148 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00149-20 LMOD PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Ultrastructure and Function of the Cells and Tissues of the Eye PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: W. Gerald Robison, Jr. Ph.D. Head, Section on LMOD, NEI Pathophysiology Others: Nora Laver Anne Groome Joe Hackett Evita By nam Joel Glover M.D. Special Volunteer LMOD, NEI B.S. Histology Technician LMOD, NEI B.S. Biologist LMOD, NEI B.S. Microbiologist LMOD, NEI B.S. Biologist LMOD, NEI COOPERATING UNITS (if any) Alcon Laboratories, Inc. (Billie M. York, Jr., Ph.D.) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Pathophysiology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 4.75 PROFESSIONAL: 1.0 OTHER: 3.75 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects O (a1) Minors □ (a2) interviews [xl (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Using the galactose-fed rat model for diabetic retinopathy, which was first developed in this laboratory, we designed intervention studies to test the possibility of delaying, halting, or reversing retinopathy soon after the earliest capillary lesions could be documented. Weanling male SD rats were divided into five groups, three of which received either normal lab chow or a 50% galactose diet with or without an aldose reductase inhibitor (ARI:ca. 11 mg/kg/day AL-3152) and two of which received 50% galactose for 6 months and then intervention, by either addition of inhibitor or removal of galactose. From each rat killed at 6, 18, and 24 months, one retina was prepared for obtaining electron miaographs of capillary transections and the other was used for whole mounts of isolated retinal vessels. We captured images of whole and transected capillaries and analyzed them using computer hardware and programs specially designed for 1,024 x 1,024 x 8-bit resolution. Based on several quantitative assessments, including basement membrane thickness, PAS stain intensity, acellularity, dilation, tortuosity, length, and microaneurysms, the retinopathy was graded on a scale of 1 to 10. At 6 months, when intervention began, untreated galactose-fed rats exhibited a 30%, statistically significant (p < 0.01) increase in capillary basement membrane thickness and grade- 1 retinopathy overall. By 18 months, the same group had grade-7 retinopathy whereas rats receiving intervention with either AL-3152-enriched or galactose -firee diets exhibited only grade-2 retinopathy, and rats fed control diet or galactose plus AL-3152 throughout 18 months showed none. At 24 months, untreated rats had grade- 10 retinopathy, and both intervention groups had grade-8 retinopathy. Thus, intervention at 6 months delays but does not halt or reverse the progression of galactose-induced retinopathy. We plan to attempt, by dietary manipulation, to produce rat models that develop the diabetic-like retinal angiopathies sooner. Also, using cell culture, we will investigate possible mechanisms of endothelial cell proliferation and subsequent pathologies. 149 PHS 6040 (Rev. 5/92) Laboratory of Mechanisms of Ocular Diseases NEI Annual Report — FY 1993 Project Description Objectives This project is designed to use special diets in vivo and controlled media in cell cultures of ocular tissues to mimic the diabetic state in order to determine whether diabetic-like tissue changes can be prevented by aldose reductase inhibitors (ARIs). Methods Weanling male Sprague-Dawley rats were divided into five groups, three of which received either normal lab chow or a 50% galactose diet, with or without an ARJ (ca 11 mg/kg/day AL-3152), and two groups which received 50% galactose for 6 months and then intervention by either the addition of an inhibitor or the removal of galactose Rats were killed at 6, 18, and 24 months. A new enzyme digestion procedure (elastase method) developed in this lab was used on the retina of one eye of each rat to remove all retinal tissues except the vessels. This provided a whole mount of the retinal vasculature and permitted the recognition of degenerated peri- cytes ("ghosts") and all of the more advanced angi- opathies by light microscopy. The retina of the other eye of each pair was sectioned and examined by electron microscopy. Images of whole and tran- sected capillaries were captured and analyzed by using computer hardware and programs specially designed for 1024 x 1024 x 8-bit resolution. Based on several quantitative assessments— including basement membrane thickness, PAS stain intensity acellularity, dilation, tortuosity, length, and microaneurysms— the retinopathy was graded on a scale of 1 to 10. Tissue cultures of human, bovine and canine retinal capillary pericytes and leas' epithehal cells were used to investigate the mechamsm(s) underlying the diabetic angiopathies. Major Findings Vascular whole mounts of capillaries of rats fed galactose for 24 months, prepared by our new enzyme digestion procedure, exhibited multiple reunal angiopathies identical to those typical of human background diabetic retinopathy. These angiopathies did not occur in the retinas of rats fed a galactose diet with an ARI. The presence of aldose reductase was demonstrated in cultured retinal pericytes (1) by immunohistochemistry, shown by the antibody against human placental aldose reductase; (2) by its activity, shown by measurements of xylitol production in cells grown in a medium supplemented with xylose; and (3) by the detection of messenger RNA for aldose reductase. There was a compro- mi.sed proliferation rate in pericytes, compared with endothelial cells incubated in high (30 mM) sugar concentrations, suggesting toxicity of polyol at the cellular level. Aldose reductase appears to be involved in all the retinal complications of diabetes, from pericyte degeneration to microaneurysms. Significance to Biomedical Research and the Program of the Institute Diabetic retinopathy is mainly a disease of retinal capillaries. Recently potentially beneficial treatments and animal models have become available. How- ever, demonstration of the earliest vessel lesions has relied on the 30-year-old trypsin digestion method for the isolation of retinal vessels. Until now, basic experimental studies and drug testing on diabetic reanopathy have been limited by the lack of reliable and convenient animal models. Now, besides the alloxan diabetic dog and the galactosemic dog, there IS a galactosemic rat model. All this has been possible because aldose reductase is involved in diabetic retinopathy. Aldose reductase, which has been implicated in sugar cataracts, certain corneal healing defects, and peripheral neuropathy of diabetic and galactosemic animals, now appears to be in- volved in all lesions of background diabetic retinopa- thy. While the normal physiological role of this enzyme in most tissues remains unknown, under the conditions of high plasma sugar concentrations encountered in diabetes and galactosemia, aldose reductase converts these sugars to their respective sugar alcohols (polyols). Tliese polyols are not readily metabolized, nor do they penetrate cell membranes easily. Thus, once formed at significant rates, they may accumulate to very high levels in cells, leading to hypenonicity, alteration of ion permeabiUty, and eventual cell death, with conse- quent tissue changes such as cataract formation. Treatment of diabetic or galactosemic rats with potent ARIs, such as sorbinil or tolrestat, decreases tlie accumulation of polyols, which in turn appears to prevent the formation of cataracts in lenses, defective healing in scraped corneas, thickening of basement membranes in retinal capillaries, and decreased conduction velocity in nerves. 150 NEI Annual Report— FY 1993 Laboratory of Mechanisms of Ocular Diseases By using a novel vessel preparation method, we have shown for the first time that the rat can be a good model for human diabetic retinopathy and that demonstration of early lesions can be improved. Pericyte loss, endothehal cell proliferation, micro- aneurysms, shunts, occlusions, dilations, and all the other microangiopathies that we found in the galac- tose-fed rat are identical to the histopathologies that characterize human background diabetic retinopathy. Until now, the only other experimental animal model has been the diabetic or galactosemic dog. We have shown for the first time that diabetic-like retinopathy in galactosemic rats can be prevented with an ARI. Proposed Course The following studies are proposed for Fiscal Year 1993. We will extend the intervention studies to determine how late one can interrupt the disease process and still obtain beneficial results by treatment with various ARIs. We also will examine the early formation of intracellular vacuoles, cell transport systems, the mechanism of basement membrane synthesis, and the relationships of these changes to aldose reductase in isolated retinal cells grown under diabetic conditions. We will manipulate the rat diets to shorten the time when diabetic-like retinal angiop- athies appear, thus improving the rat as a model for diabetic retinopathy. NEI Research Program Retinal Diseases — Diabetic Retinopathy Publications Laver NM, Robison WG Jr: Proliferative retinopa- thy stage in long-term galactose fed rats. Invest Ophthalmol Vis Sci 34(4)(suppl):713, 1993. Laver N, Robison WG Jr, Calvin HI, Fu S-CJ: Early epithelial lesions in cataracts of GSH-depleted mouse pups. Exp Eye Res 57:493-498, 1993. Laver NM, Robison WG Jr, Hansen BC: Demon- stration of retinal histopathologies in spontaneous- ly diabetic monkeys. Am J Clin Pathol 99(3): 349, 1993. Laver NM, Robison WG Jr, Pfeffer BA: Novel procedures for isolating intact retinal vascular beds from diabetic humans and animal models. Invest Ophthalmol Vis Sci 34:2097-2104, 1993. Matthews GP, Laver N, Robison WG Jr: Electro- physiological and histological evaluation of inner retina in galactosemic rats. Invest Ophthalmol Vis Sci 34(4)(suppl):720, 1993. Robison WG Jr, Laver N: Ocular lesions in animal models of human diabetes, in Shafirir E (ed): Frontiers in Diabetes Research, Lessons from Animal Diabetes IV. London, Smith-Gordon and Company Limited, 1993, pp 145-163. Robison WG Jr, Laver NM, York BM, Chandler ML, Lou MF: ARI intervention studies of galac- tose induced retinopathy by computer analysis of retinal vessel images. Invest Ophthalmol Vis Sci 34(4)(suppl):718, 1993. 151 Laboratory of Molecular and Developmental Biology Report of the Chief, Laboratory of Molecular and Developmental Biology Joram Piatigorsky, Ph.D. In its 12th year, the Laboratory of Molecular and Developmental Biology (LMDB) has been ex- panded by two sections — the Section on Regulation of Gene Expression, headed by Dr. Ana B. Chepelin- sky, and the Section on Transgenic Animals and Genomic Manipulation, headed by Dr. Eric Wawrou- sek. Dr. Chepelinsky, a valued member of the LMDB since its beginning, has augmented her research area, the crystallins, to include membrane proteins and their genes, as well as the effects of growth factors on eye development. Dr. Wawrousek, a former postdoctoral fellow at the LMDB, has returned to the National Eye Institute (NEI) after a brief sojourn in industry. His Section wears two hats. The first provides a service for the NEI — ^the creation of transgenic mice; the second performs research involving site-specific gene recombination. The addition of these two sections has increased the expertise of the LMDB and extended our usefulness to the NEI. The other sections of the LMDB include the Section on Molecular Genetics, headed by Dr. Joram Piatigorsky; the Section on Cellular Differenti- ation, headed by Dr. Peggy S. Zelenka; and the Section on Molecular Structure and Function, headed by Dr. Graeme J. Wistow. Not all developments are happy ones. Sadly, Ms. Dawn Chicchirichi, the LMDB secretary since the Laboratory's beginning, retired due to illness. Ms. Chicchirichi gave 11 years of devoted and excellent assistance to the LMDB and will be greatly missed. She has been replaced by Ms. Linda Willett. Ms. Willett already has become an invaluable mem- ber of the LMDB, and we are extremely lucky to have her with us. I also take this opportunity to thank the many NEI staff members who gave us great support and help during the difficult months of Ms. Chicchirichi 's illness so that we could keep functioning during the transition period. The LMDB's primary goal is to perform basic research on the molecular biology of the eye. Although particular attention is directed to the lens, the cornea and retina have not escaped our efforts; research projects also have focused on the role of growth factors on eye development. In addition, because lens crystalUns are multifunctional proteins that are expressed outside the lens and eye, the scope of our research has increased during the last few years to include new areas of metabolism and gene expression in various tissues. Moreover, many of the transcription factors involved in the expression of crystallin genes are present in many tissues and are used to control numerous biological processes. Consequently, our studies on eye genes have implica- tions for many areas of molecular, cellular, and evolutionary biology. This is reflected in the pleth- ora of general journals in which we publish our scientific discoveries and the fact that we often attend meetings that focus on broad issues of genet- ics, development, evolution, and molecular biology. Thus, the original twin purposes of using the visual system as a model for the structure, expression, and evolution of genes and incorporating general princi- ples of molecular biology to understanding the visual system continue as the core of our thinking. There have been many individual research accom- plishments by LMDB staff this year. These accom- plishments are detailed in the specific annual reports. In general, much attention has been given to the identification of regulatory elements required for expression of genes in the eye and other tissues. Many of these regulatory elements are commonly found in genes; however, each has its own special properties. The diversity of elements used for expression of eye genes is impressive, ensuring that we will be busy for many years sorting them all out. To complicate things even more, we have shown that the regulatory elements may be functionally redun- dant, i.e., removing one does not necessarily eliminate the expression of the gene. There are also many different nuclear proteins that bind to the DNA regulatory elements, and this year we have cloned a number of them. One of our biggest challenges is to determine which binding proteins are actually in- volved in regulating the genes in the animal. 155 Laboratory of Molecular and Developmental Biolog> NEI Annual Report — FY 1993 Our studies on the expression of proto-oncogenes and cyclins have linked the normal process of cellular differentiation in the lens with the cell cycle and growth control, providing another example of the broad relevance of our research. In addition, the use of crystalhn promoters for directing various growth factors to the lens have extended cellular studies to consideration of growth of the entire eye. These genetic engineering experiments have opened the opportunity to develop animal models for auto- immune diseases of the eye, fostering communication between the LMDB and the NEI Laboratory of Immunology. The addition of the transgenic facility has had a major impact in increasing the dialog between the LMDB and other NEI laboratories. We look forward to additional cross-fertilization of ideas in the years to come. 156 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00238-08 LMDB PERIOD COVERED October 1. 1992 to September 30. 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Proto-Oncogene Expression During Lens Differentiation and Development PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Peggy S. Zelenka Ph.D. Head, Section on CeUular LMDB, NEI Others: Jo Ann Rinaudo Chun Yun Gao Emmanuel Vacchiano Anoradba Rampalli Jaspreet Arora Graeme Wistow Differentiation Ph.D. Staff Fellow M.D., Ph.D. Staff Fellow Ph.D. Staff Fellow Ph.D. Visiting Fellow Ph.D. Visiting Fellow Ph.D. Head, Section on Molecular Structure and Function LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI COOPERATING UNITS (if any) Department of Surgery, New Jersey Medical and Dental College (Thomas Lysz, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Cellular Differentiation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 5.2 PROFESSIONAL 5.2 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues jx] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project to investigate the expression of proto-oncogenes and other cell cycle regulatory genes in the embryonic chicken lens seeks to determine their relationship to cell growth, quiescence, and differentiation. The normal developmental profiles of five nuclear proto-oncogene mRNAs (c-myc, N-myc, c-fos, c-jun, and p53) and the cell cycle regulatory protein, cyclin B, have been completed, and the profile of retinoblastoma (Rb) expression is in progress. The finding that cyclin B is present in lens-fiber cells suggests that lens-fiber cell differentiation may represent an aberrant form of the cell cycle. In addition to cyclin B, a number of other proteins normally associated with proliferating cells are expressed in postmitotic, differentiating lens cells. These include cyclin A, c-myc, c-fos, c-jun, and p53. Moreover, preliminary studies using explanted embryonic chicken lens epithelia indicate that the order of expression of these genes during differentiation is the same as during proliferation, further strengthening the link between differentiation and the cell cycle. The functional role of each of these genes during leas differentiation now is being explored through the use of retroviral vectors, transfection of exogenous DNA, and the production of transgenic mice. In addition, regulatory mechanisms governing the changes in proto-oncogene expression that accompany differentiation are being explored. 157 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biology ^fEI Annual Report— FY 1993 Project Description Additional Personnel Milton Berger B.A. Tania Tolstoshev Graeme Wistow Ph.D. Special Volunteer, LMDB, NfEI H.S. Student, Special Volunteer, LMDB, NEI Head, Section on Molecular Structure and Function, LMDB, NEI Objectives In this project we seek to determine whether the expression of specific proto-oncogenes is altered during lens cell differentiation and, if so, to deter- mine the mechanism of gene regulation and the function of the corresponding proto-oncogene prod- ucts in the developing lens. The objective is to develop a greater understanding of the mechanisms underlying lens cell growth and differentiation. Methods Techniques of molecular biology are used in con- junction with traditional cell biology techniques. Conventional methods are employed for protein and nucleic acid analysis, including polyacrylamide gel electrophoresis, RNA and DNA isolation, polymerase chain reaction (PCR), reverse transcription PCR (RT/ PCR), nucleic acid hybridization, in vitro transfec- tion, in situ hybridization, immunocytochemistry, and immunoblotting. DNA/protein interactions are studied using DNAse I footprinting, electrophoretic mobility shift assays, and ultraviolet (UV) cross- linking. Studies employ lens epithelia and lens fibers of embryonic chickens, explants of embryonic chicken lens epithelia, primary cultures of embryonic chicken lens epithelial cells, and other avian and manmialian cell lines. In addition, transgenic mice are produced to test the function of proto-oncogenes and cell cycle regulatory proteins in the lens in vivo. Major Findings In the past year major progress has been made on studies of the cell cycle regulatory protein, cyclin B. Expression of this protein is known to be cell-cycle dependent in proliferating cells, appearing in the S and G2 phases of the cell cycle. Work done by Dr. Chun Gao has demonstrated that cyclin B is ex- pressed in differentiating lens fiber cells. The presence of cyclin B mRNA was shown by RT/PCR, followed by sequencing of the PCR product. Immunoblotting with an antibody specific for cyclin B following two-dimensional gel electrophoresis confirmed that the protein is also present in lens fiber cells. In situ hybridization of sections of 14-day embry- onic lenses with riboprobes for cyclin B mRNA showed that the mRNA is abundant in the differenti- ating cells at the lens equator and in the nucleated fiber cells, but it cannot be detected in the enucleated fiber cells. Cyclin B from lens fiber cells was affinity-purified by chromatography on pi 3™" Sepharose. Because the pl3 protein binds to p34'=*^ kinase, purificadon of cyclin B by this process provides evidence that it is complexed with the p34""^ protein. The kinase activity of this complex was demonstrated by using histone HI as a substrate. Kinase activity could be increased about twofold by phosphatase treatment. These results indicate that cyclin B, a protein normally expressed in the G2 phase of the cell cycle, is present in differentiating lens fiber cells. Since the cyclin B/p34"''=^ complex is known to be responsible for chromosomal condensation and nuclear envelope breakdown in mitotic cells, finding this complex in lens fibers and in the enzymatically active, dephos- phorylated form provides evidence that this same biochemical mechanism may be responsible for chromosomal condensation and nuclear envelope breakdown during fiber cell differentiation. Because cyclin B normally is expressed only in the G2 phase of the cell cycle, its presence in differ- entiating lens fiber cells suggested that the differenti- ation process itself may be an aberrant form of the cell cycle. To test this possibility, Dr. Anuradha Rampalli has initiated experiments to examine the order of expression of a number of cell-cycle mark- ers in differentiating explants of 6-day-old embryonic chick lens epithelia. Preliminary results show a strong, early induction of c-fos, c-jun, c-myc, and N- myc, followed after a lag of 5-7 hours by induction of p53 and after a lag of 18-24 hours by induction of the heat shock protein HSP70. Since c-fos, c-jun, and c-myc are normally expressed in early Gl, p53 in late Gl, and HSP70 during the S and G2 phases in proliferating cells, the order of induction seen 158 NEI Annual Report— FY 1993 Laboratory of Molecular and Developmental Biology during differentiation parallels that of the nonnal cell cycle. Additional S-phase markers under investiga- tion include PCNA (the 5 subunit of DNA polymer- ase) and thymidine kinase. Cyclins A and B will be examined as markers for the G2 phase. The induc- tion of N-myc is not typical of proliferating cells and, thus, marks a significant difference between the two processes. The tumor supressor genes Rb and p53 seem to play key roles in preventing Gl cells from entering the S phase, making their role in lens differentiation particularly interesting. Studies by other investiga- tors have shown that inactivation of these gene products by SV40 T antigen prevents terminal differentiation of lens fiber cells. Dr. Rampalli has completed a developmental study of p53 expression in the embryonic chick lens, and a companion study of Rb expression is in progress. Her results clearly show that p53 mRNA and protein are expressed in differentiating cells at the lens equator and in the newly formed fiber cells, consistent with the data from differentiating explants and with the idea that differentiation and cell-cycle progression share important features. A major focus of this project continues to be the biological function of the proto-oncogenes expressed in the lens. Preliminary evidence, reported last year, that c-myc regulates expression of the x-crystallin/a- enolase gene has now been extended to demonstrate that the c-myc protein itself is involved in binding to the T-crystallin promoter. Interestingly, a x-crystal- lin/chloramphenicol acetyltransferase (CAT) coastruct with a mutation in the potential c-myc binding site was shown to be expressed at somewhat higher levels in cultured lens cells than was the wild-type construction, although cotransfection of a c-myc expression vector was no longer required to stimulate this expression. This observation raises the possibil- ity that c-myc and a negative regulatory protein may compete for binding to the same or overlapping sites. The possibility that N-myc may be such a negative factor is under investigation. The function of c-jun in the embryonic chicken lens has been explored using wild-type and mutated cDNAs for chicken c-jun cloned into the avian retroviral vector RCAS. Use of this vector permits fransfer of the c-jun constructs to cultured cells with efficiencies approaching 100%, making it possible to test for the effects of c-jun on DNA synthesis, differentiation, and expression of endogenous genes. Results obtained by Drs. Jo Aim Rinaudo and Emmanuel Vacchiano indicate that a negative domi- nant mutation of c-jun increases the levels of endoge- nous oA-crystallin mRNA twofold above the already high levels present in the control cells. The levels of PA3/A1 mRNA were not affected in the same cells, suggesting that multiple, independent pathways may operate in differentiating lens cells, only some of which are affected by c-jun. Overexpression of wild-type c-jun in chicken lens epithelial cells by means of the RCAS vector greatiy enhances cell proliferation and may immortalize the cells. Cells infected with a retrovirus bearing the c- jun cDNA have now undergone nine passages, with no apparent decrease in proliferative capacity. Furthermore, the cells seem to have retained their ability to differentiate to lens fibers; lentoid bodies form in the cultures when they are permitted to become confluent. If these cells are immortalized, they will be extremely useful for future studies of gene expression and differentiation. Noting the similarities between lens fiber cell differentiation and a^ptosis. Dr. Vacchiano has employed the c-jun rettoviral vector to investigate the role of c-jun in proliferation, differentiation, and apoptosis in chicken embryo lens epithelial cells. His results indicate that c-jun overexpression stimu- lates proliferation in the presence of serum, but it does not prevent differentiation once confluence is attained. In the absence of adequate levels of serum or growth factors, however, overexpression of c-jun increases the rate at which the cells enter apoptosis. Dr. Vacchiano also is investigating the possible role of bcl-2 expression in regulating lens cell growth and differentiation. This proto-oncogene has been shown in other cell types to block apoptosis induced by overexpression of e-myc or p53. Using RT/PCR, he has demonstrated that bcl-2 is expressed in the embryonic chicken lens. He is now preparing constructs that will permit overexpression of this gene in both cultured chicken lens epithelial cells and transgenic mice to determine its effect on apoptosis and differentiation of lens cells. Inhibition of differ- entiation by bcl-2 would indicate an important biochemical link between fiber cell formation and apoptosis. Our ongoing collaboration with Dr. Thomas Lysz (University of Medicine and Dentistry of New Jersey) has now demonstrated that endogenous production of 12-hydroxyeicosatetraenoic acid (12- 159 Laboratory of Molecular and Developmental Biology NEI Annual Report — F\' 1993 HETE), a lipoxygenase pathway metabolite of arachidonic acid, is a required step in DNA synthesis stimulated by epithelial growth factor in the neonatal rat lens. Dr. Jaspreet Arora has used RT/PCR to demonstrate that 12-HETE synthesis is required for expression of two proto-oncogenes, c-fos and c-myc, whereas expression of c-jun seems to be independent of this pathway. Because inhibition of either c-fos or c-myc is sufficient to cause cell-cycle arrest, these findings indicate that 12-HETE production is a key control point in the lens epithelial cell cycle. Significance to Biomedical Research and the Program of the Institute The proto-oncogenes are normal cellular homologs of retroviral oncogenes. Since retroviral transformation disrupts cell growth and differentiation, it is likely that the proto-oncogenes are involved in the normal regulation of these processes. A study of proto- oncogene expression during lens cell differentiation may elucidate basic regulatory processes underlying lens cell growth and differentiation. Many types of cataract are associated with abnormal lens epithelial cell growth and inhibition of lens fiber cell differen- tiation. In addition, a number of other eye diseases involve loss of normal controls on cell proliferation. An understanding of the basic controls of cell growth and differentiation would further our understanding of these disease states. Proposed Course The following studies are in progress or are proposed for Fiscal Year 1994: 1. We will test the hypothesis that the cyclin B/ p34'=^'=^ kinase is responsible for nuclear loss in differentiating lens cells by producing transgenic mice that express the Weel* kinase in lens fiber cells. This kinase inactivates the cyclin B/p34"''=^ kinase and would be expected to delay or prevent nuclear loss if cyclin B/p34"''^ is required. 2. Due to the unexpected finding of the cyclin B/ p34«)c2 jynase in differentiating lens fibers and the noted similarities between lens differentiation and apoptosis, we will examine whether cyclin B is expressed in apoptotic cells. We will test a variety of apoptotic cells of divergent origins for the pres- ence of cyclin B and cyclin B/p34"''^ kinase activity. 3. We will continue study of the time course of the expression of cell-cycle-dependent genes in differentiating explants of lens epithelia, with the addition of S and G2 phase markers to determine the extent to which differentiation resembles cell-cycle progression. 4. We will examine ftirther the effect of c-myc on transcription of the x-crystallin/a-enolase gene by transfection studies in cells that do not express N- myc. We also will examine the effect on the endog- enous duck gene by transfection into duck fibroblasts and lens epithelial cells. 5. We will examine the effect of the N-myc proto-oncogene on transcription directed by the x- crystallin/a-enolase gene using the techniques previ- ously used to smdy the role of c-myc. 6. We will extend the collaborative effort with Dr. Lysz to other growth factors, as well as examine the mechanism by which 12-HETE affects expression of c-fos and c-myc. We will experiment to deter- mine whether human lenses possess the 12-lipoxy- genase pathway. 7. We will explore the possible role of post- translational modifications of Rb and p53 proteins in lens cell differentiation. NEI Research Program Cataract — The Normal Lens Publications Dash A, Chung S, Zelenka PS: Expression of HSP70 mRNA in the embryonic chicken lens: Association with differentiation. Exp Eye Res, in press. Piatigorsky J, Zelenka PS: Transcriptional regulation of crystallin genes: cis elements, trans-factors, and signal transduction systems in die lens. Adv Develop Biochem 1:211-256, 1992. Wistow GJ, Shaughnessy MP, Lee DC, Hodin J, Zelenka PS: A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens. Proc Natl Acad Sci USA 90:1272- 1275, 1993. 160 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00126-12 LMDB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must tit on one Ime between the borders.) Crystallin Genes: Structure, Organization, Expression, and Evolution PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Joram Piatigorsky Others: James B. Brady Sambath Chung Ales Cvekl Melioda Duncaii Peter Frederikse Rashmi Gopal-Srivastava John Haynes John G. Dagao Ph.D. Chief LMDB, NEI Ph.D. IRTA LMDB, NEI B.A. Technician LMDB, NEI Ph.D. Visiting Fellow LMDB, NEI Ph.D. IRTA LMDB, NEI Ph.D. Senior Staff Fellow LMDB, NEI Ph.D. Staff FeUow LMDB, NEI Ph.D. IRTA LMDB, NEI Howard Hughes Medical Institute/ LMDB, NEI NIH FeUow (Additional personnel listed under P rogram Description.) COOPERATING UNITS (if any) Jules Stein Eye Instimte, UCLA (J. Horwitz, Ph.D.); National Institute of Child Health and Human Development, NIH (K. Becker, Ph.D.; K. Ozato, Ph.D.); University of Southern California (V.M. Weis. Ph.D.; M. McFall-Ngai, Ph.D.); Medical College of Virginia (D.M. Stover, Ph.D.; Z.E. Zehner, Ph.D.); N.K. Koltzov Developmental Biology Institute, Russian Academy of Sciences, Moscow (R.D. Zinovieva, Ph.D.); Uniformed Services University of the Health Sciences (S. Bassnet, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Molecular Genetics INSTITUTE AND LOCATION NEI, NIH. Bethesda. MP 20892 TOTAL STAFF YEARS: 14.0 PROFESSIONAL 10.8 OTHER: 3.2 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The structure, expression, and evolution of crystallin genes of vertebrates and invertebrates are being studied. The four functional promoter elements described in the aA-crystallin gene of the mouse (DEI, oA-CRYBPl, PEl, and PE2) and five in that of the chicken (DE3, DE2A, DE2B, DEIA, and DEIB) are surprisingly different, considering the gene is orthologous in these species with the same high-level expression in the lens. We have cloned putative trans-acting factors which bind to the mouse oA-CRYBPl and chicken DE2A sites. The cxA-CRYBPl gene has been cloned and characterized; cDNA analyses indicate that it produces alternatively spliced mRNAs. The oA-CRYBPl protein also spears to be cleaved in a tissue-specific fashion. The mouse DEI site appears to bind a member of the CREB/ATF family. Four functional elements (oBE-l, aBE-2, (xBE-3, and MRF) have been identified in the mouse oB-crystallin enhancer; oBE-l, aBE-2, and txBE-3 are used in muscle and lens, while MRF binds myoD and myogenin and is muscle specific. We have identified in the chicken PA3/A1 -crystallin gene an enhancer containing an AP-1 consensus-binding sequence which increases lens transcription but is not necessary for lens specificity. Transfection and gel mobility shift experiments indicate that the PL-1 and PL-2 functional elements of the chicken pBl -crystallin promoter and the AP-l/ARE sequence of two squid crystallin promoters are necessary for activity in transfected chicken lens cells and bind similar nuclear proteins of the chicken lens. Six chicken nuclear proteins that bind to the PL-1 sequence have been cloned. Transgenic mouse experiments indicate that the 52-crystallin enhancer works efficiently in the lens and also has modest activity in the cornea, brain, and retina. Squid glutathione S-transferase (GST) and two squid S-crystallin cDNAs have been expressed; GST is very active while the S-crystallins show little if any activity. Cephalopod cDNAs for Q-crystallin/ALDH, intermediate filament protein, and a- and P-tubuIin have been cloned; the genes for all but a-tubulin were lens specific. Cloned cubomedusan jellyfish J3-crystallin has been shown to be a novel protein. 161 PHS 6040 (Rev. 5/92) Laboraton of Molecular and Developmental Biolog> NEI Annual Report— FY 1993 Project Description Additional Personnel Cynthia Jaworski Ph.D. Marc Kantorow Ph.D. Xuan Li Ph.D. Joan B. McDermon M.S. Barbara Norman Christina M. Sax Ph.D. Stanisiav 1. Tomarev Ph.D. Staff Fellow, LMDB. NEI IRTA. LMDB, NEI Visiting Associate, LMDB. NEI Biologist, LMDB, NEI Chemist, LMDB, NEI Senior Staff Fellow, LMDB. NEI Visiting Scientist, LMDb"^ NEI Objectives The objective of this project is to understand the structure, organization, expression, and evolution of the gene families encoding the lens crystallins in the animal kingdom. Particular attention is given to the regulation of crystallin gene expression in the devel- oping lens and. in the case of multifunctional crystal- lins and enzyme-crystallins, in nonlens tissues. Methods Conventional methods used for analysis of proteias and nucleic acids include polyacrylamide and agarose gel electrophoresis, RNA and DNA isolation, molec- ular hybridization (Southern and Northern blots), cDNA and gene cloning, DNA sequencing, recombi- nant DNA construction and mutagenesis, in situ hybridization, expression of recombinant DNAs in transfected cells and transgenic mice, polymerase chain reactions, primer extension and SI protection experiments, in vitro and in vivo footprinting, gel mobility shift analysis, chromatographic purification of proteins, and Western immunoblotting. Major Findings a-crystallin. — There are two a-crystallin genes, aA and otB. Although both are expressed principally in the lens, the oB gene is expressed constitutive! y in many other tissues and is inducible by stress. By contrast, there is very little expression of aA in other tissues (although there is some), and it is not induc- ible by stress. We have been continuing our studies on the molecular basis for expression of the mouse and chicken otA and the mouse oB-crystallin genes. At least four separate control sequences have been identified for the mouse ocA-crystallin gene: DEI (-111 to -97), oA-CRYBPl region (-75 to -48), TATA/PEl (-35 to -19), and PE2 (-h24 to -^43). Our current evidence suggests that the DEI site is a cyclic AMP-responsive element (CRE) that binds a member of the CREB/ATF family of transcription factors. PE2 contains both an API and a glucocorti- coid-responsive element. The oA-CRYBPl site binds a ubiquitous protein that we have cloned and called oA-CRYBPl. However, this site also con- tains a consensus sequence for the transcription factor, NF-kB. Immunoblotting experiments indicate that the oA-CRYBPl site binds various tissue-specific forms of the ocA-CRYBPl protein. Although there is no evidence indicating that NF-kB is used as a tran- scription factor for the expression of the mouse otA-crystallin gene, this cannot be ruled out at the present time. The otA-CRYBPl gene has been cloned and shown to consist of at least seven exons. Its entire cDNA sequence is almost completed, except for a small stretch in the middle. The en- coded protein contains at least four zinc fingers and is approximately 3(X),000 Daltons (D). It is homolo- gous to the human PRDII-BFl/MBP transcription factor. Since oA-CRYBPl is smaller than 300,000 D on Western blots, it appears as if the protein has been cleaved before use. Sequencing multiple cDNAs has indicated that the primary transcript of oA-CRYBPl is alternafively spliced, providing another possible basis for hetero- geneity of this putative transcription factor. A series of mutated constructs inserted into transgenic mice have shown that the DEI and oA-CRYBPl sites are functionally redundant. It appears necessary for at least one of these control sites to interact with PEl and/or PE2 for lens-specific expression to occur. Work over the past 5 years has shown, surprising- ly, that different control elements are used in expres- sion of the orthologous mouse and chicken oA-crys- talUn genes. We have identified the following control sequences for the chicken gene: DE3 (-153 to -140), DE2A (-144 to -134; this overiaps with DE3), DE2B (-128 to -118), DEI A (-114 to -104), and DEIB (-100 to -93). The (xA-CRYBPl se- quence in the chicken gene differs from that in the mouse gene by one nucleotide, and although it footprints with nuclear proteins from the chicken lens in DNAse I protection experiments, it does not 162 NEI Annual Report— FY 1993 Laboratory of Molecular and Developmental Biology appear to be functional, as judged by mutagenesis and transfection tests. Three cDNAs encoding proteins that bind to the DE2A sequence have been cloned from an embryonic heart and are currently under investigation. In contrast to the DEI region of the mouse oA-crystallin gene, the DEI A and DEIB sequences of the chicken aA gene do not appear to bind a member of the CREB/ATF family of tran- scription factors. In 1991 we reported the presence of an enhancer at positions -426 to -259 of the oB-crystallin gene of the mouse. This sequence behaves as a strong enhancer for expression in cultured muscle cells and as a weak enhancer in cultured lens cells. We have now established by mutagenesis and footprinting experiments the presence of at least four functional elements witliin this enhancer: oBE-l (-420 to -397), aBE-2 (-360 to -327), aBE-3 (-319 to -303), and the muscle regulatory factor (MRF) binding site (-300 to -280). The MRF contains an E box and is activated by the binding of either MyoD or myogenin. Last year we indicated by DNAse I footprinting experiments that the -148/-118 sequence was necessary for the specific expression of the oB gene in the lens. This year transgenic mouse experiments have shown that this sequence is necessary for lens-specific expression but the enhancer is not. The -426/-259 enhancer sequence is necessary for expression of the ccB gene in skeletal muscle and heart, and it quantitatively boosts expression in the lens. Thus, cxBE-1, otBE-2, and aBE-3 are used for expression of the oB-crystallin gene in both lens and muscle cells, but the MRF element is used only in the muscle cells. This is the first example of a muscle-specific control element in a crystallin gene. ^-crystallin. — ^We have been investigating the |3B1 and PA3/A1 -crystallin genes in the chicken for several years. This year we have added the PBl- crystallin gene of the mouse to our studies. Previous experiments have established the importance of the PL-1 and PL-2 control elements for activity of the chicken PBl promoter in transfection experiments. The PL-1 and PL-2 sequences are present between position -122 and the TATA box; they resemble AP-1 binding consensus sequences and compete with AP-1 sites for binding nuclear proteins. This year we have selected six different cDNAs from an embryonic chicken heart library on the basis of binding to multimerized PL-1 sequences. They are currently under investigation. The -434/+30 sequence of the chicken PBl -crystallin gene has been shown to contain the information for lens specificity in transgenic mouse experiments. Deletion experi- ments are in progress to determine whether the PL-1 and PL-2 elements are necessary for lens specificity. Finally, we have cloned and are characterizing an approximately 18-kbp fragment that contains the mouse PBl -crystallin gene. We are continuing our studies on the chicken pA3/Al -crystallin gene. The -287/-254 sequence has been shown to possess enhancer activity for expression in transfected embryonic chicken lens epithelial cells. It contains an AP-1 consensus sequence and binds multiple nuclear proteins in gel mobility shift experiments. A T-rich tract located between positions -218 and -168 appears to sup- press activity of the -287/-254 enhancer in transfec- tion experiments. A series of transgenic mice have been produced using the reporter gene chlorampheni- col acetyltransferase (CAT). Fusion genes containing the -382/+22 or -143/+22 fragments of the pA3/Al gene directed lens-specific expression, indicating that neither the -2877-254 enhancer nor the -218/-168 T tract is necessary for lens specificity. d-crystallin. — There are two linked 5-crystallin genes in the chicken. The 5' gene is specialized for lens expression, while the 3' gene encodes arginino- succinate lyase (ASL), making this an enzyme-crys- talhn. Although only the 82 gene encodes a protein with ASL activity, both genes are expressed in a developmentally regulated fashion in the lens, cor- nea, retina, heart, and brain of the chicken embryo. This year we generated transgenic mice carrying fusion genes comprising various combinations of the 5-crystalUn promoters and enhancers (located in the third intron of the natural genes) attached to the bacteria] CAT reporter gene. The enhancers of both 6-crystallin genes caused extremely high expression of the CAT gene in the lens of the transgenic mice, suggesting that a silencer for lens expression of the 52-crystallin gene is operative in the chicken. Evidence also was obtained indicating that the 5-crystallin enhancers influence expression of the transgenes in the transgenic mouse cornea, retina, and brain in a way that is consistent with the expres- sion of the natural genes in these tissues in the embryonic chicken. The 6-crystallin locus has been cloned from the duck genome to compare the regulatory sequences 163 Laboratory of Molecular and Developmental BiologA' NEI Annual Report— FY 1993 for these two genes in the duck and the chicken. We have undertaken this project because, in contrast to the chicken 52 gene, the duck 82-crystallin gene is very active in the lens. It should be able to help us understand the mechanism of suppression of the 52 enhancer in the chicken lens. So far, we have established that the duck 5-crystallin genes are linked as in the chicken; further characterization is in progress. S-crysiallin. — Although much research had been performed on the lens crystallins of venebrates, very little information was available concerning the major lens proteins of the complex eyes of invertebrates. Thus, several years ago we began studying inverte- brate crystallins. Particular attention has been given to the crystallins of cephalopods (squids and octopi) because these species have the prototypical cellular invenebrate lenses that have evolved independent of those of vertebrates. We showed earlier that S-crys- tallins are encoded by a family of at least 10 genes that are expressed specifically in the lens and are related to the glutathione S-transferase (GST) genes of vertebrates. Last year we cloned the squid gene encoding GST. In contrast to the S-crystallin genes, the GST gene is expressed principally in the digestive gland (analogous to the venebrate liver) and very little in the lens or other tissues of the squid. This year we have expressed the cDNAs for the GST gene and for a minor (SLl 1) and major (SL20-1) S-crystallin gene of the squid in a bacterial extract and assayed for GST activity. The results showed that squid GST has more enzymatic activity than any other inverte- brate or venebrate GST ever reported. The major SL20-1 crystallin had essentially no GST activity, and the minor SLll crystallin had slight GST activ- ity. Thus, the S-crystallins generally lost GST activity as they specialized for expression in the lens. Interestingly, the loss of GST activity of SL20-1 is associated with the insertion of a novel peptide in the center of the protein. There is no insertion in the SLl 1 protein, which has some GST activity. Muta- genesis experiments are in progress to identify the active sites for substrate binding and for enzymatic function. Transfection experiments using the CAT reponer gene and embryonic chicken lens epithelial cells have been conducted in order to identify putative control elements of the S-crystallin genes. An overlapping AP-1/antioxidant responsive element (ARE), present just upstream of the TATA box of the SL20-1 and SLll crystallin genes of the squid, is required for promoter activity in the transfected chicken lens cells. A similar sequence is present in the PL-1 and PL-2 functional elements of the chicken PBl -crystal- lin gene. Gel mobility shift and competition experi- ments have provided evidence that the chicken PL-1 and PL-2 and squid AP-l/ARE regulatory sequences bind similar nuclear proteins of the chicken lens. These data raise the possibility that entirely different, nonhomologous crystallin genes of the chicken and squid have convergently evolved a similar c«-acting regulatory element for high expression in the lens. It is especially interesting that this element is a stress-responsive gene regulatory element, providing a further link between crystallins and stress proteins. ^.-crystallin. — In addition to the major S-crystal- lins of cephalopods, a minor crystallin, called r2-crystallin, is related to aldehyde dehydrogenase (ALDH). This is the only known invertebrate crystallin that has a vertebrate counterpan (i.e., r| -crystallin/ ALDH found in the elephant shrew). Last year we cloned i2-crystallin cDNA. This year we finished characterizing the cDNA and the expres- sion pattern of the Q-crystallin gene. Sequence comparisons have suggested that vertebrate ALDHl/ ALDH2 gene duplication occurred after the diver- gence of cephalopods from the line giving rise to vertebrates but before the separation of squid and octopus. Southern blots are consistent with the presence of few, possibly only one, gene for ii-crystallin in octopus and squid, and Northern blots indicate that this gene is expressed specifically in the lens. However, it is of particular interest that Q-crystallin is the dominant protein in the muscle-derived, cellular lens of the ventral light organ in one squid (i.e., Euprymna scolopes). This indicates that the same gene has been recruited to be a crystallin in two entirely different lenses developing from differ- ent tissues. No ALDH activity has been found for i2-crystallin. These results are consistent with the idea that, like the S-crystallins, fl-crystallin evolved by duplication of an ancestral gene encoding ALDH and subsequently specialized for refraction in the transparent lens while losing enzymatic activity and expression in other tissues. J-crystallin. — In addition to the cephalopod crystallins, we have been studying the crystallins of cubomedusan jellyfish, which also have complex 164 NEI Annual Report— FY 1993 Laboratory of Molecular and Developmental Biology eyes with cellular lenses. We discovered several years ago that these lenses contain three apparently unrelated crystallins (Jl, J2, and J3). Last year we cloned three genes encoding Jl-crystallin polypep- tides and showed that they lack introns and encode novel proteins. This year we have initiated studies on the J2- and J3-crystallins. Sequences of tryptic peptides have indicated that the J2 and J3 polypep- tides are different proteins, despite their similarity in molecular mass (20 and 19 kD, respectively). A J3-crystallin cDNA has been cloned and partially sequenced. Like Jl, J3-crystallin appears to be a novel protein; no homolog is reported in the data base. Interestingly, although the 19-kD J3 polypep- tide is considerably smaller than the 35-kD Jl polypeptides. Northern blots indicate that the J3 mRNA is approximately 1.4 kb in length rather than the 1 kb size of the J 1 mRNAs. We are now cloning the J3 gene(s). Cytoskeletal proteins. — ^This year we completed analysis, initiated last year, of the intermediate filament (IF) protein and tubulin cDNAs of cephalo- pod lenses. Northern blots show that a-tubulin mRNA is present in all tissues examined, while the P-tubulin and EF mRNAs are lens specific. The proteins encoded in the tubulin cDNA sequences are very similar (87-93% identical) to the corresponding tubulins of insects and vertebrates, as expected for the high degree of conservation among these cyto- skeletal proteins. In the IF protein, the central rod region is more highly conserved than the head and tail regions, yet even the rod region shows at most 39% identity with any other known rod region of an IF protein, namely with that of the squid neuronal IF protein. The rod regions of the squid lens IF protein contained the six heptads characteristic of nuclear lamins, consistent with an evolutionary relationship between IF proteins and lamins. We had previously investigated the regulatory elements of the chicken vimentin gene because it is expressed relatively highly in the lens. Earlier transfection experiments revealed the presence of both positive- and negative-acting sequence elements within the first 767 nucleotides of the 5'-flanking region of the gene. This year we identified a silen- cer between positions -1360 and -1156 and an activator between positions -1612 and -1360 of the chicken vimentin gene. These regions, which con- tain numerous consensus sequences for the binding of transcription factors implicated in the expression of different crystallin genes, deserve further smdy. Significance to Biomedical Research and the Program of the Institute The crystallins comprise a diverse family of differ- entially expressed proteins that are required for the optical properties of the transparent lens. Under- standing the structure, function, and evolution of these protein families and their genes contributes to our knowledge of embryonic development, eukary- otic gene expression, cell differentiation, molecular evolution, the visual system, and cataract. That crystallins are multifunctional proteins expressed in lens and nonlens tissues adds another dimension of interest and has implications for metabolism, cell biology, and drug and gene therapy. Proposed Course The following studies are proposed for Fiscal Year 1994: 1. We will continue identifying ds-acting ele- ments in crystallin genes by mutagenesis and expres- sion studies. 2. We will investigate the interaction of cis ele- ments of the crystallin genes by footprinting and function studies. 3. We will continue cloning and characterizing putative transcription factors for crystallin genes by binding studies. 4. We will complete the sequences of the cxA- CRYBPl cDNA and gene in the mouse. 5. We will continue mutagenesis studies of the squid GST and S-crystallin cDNA to relate structur- ally the enzymatic and refractive functions of these proteins. 6. We will continue cloning and characterizing the jellyfish crystallin genes. 7. We will investigate the natiu-e of the noncrys- tallin functions of the a-crystallin polypeptides. 8. We will continue investigations on the similar- ities and differences of the IF proteins of squid and vertebrates by conducting structural and function studies. NEI Research Program Lens and Cataract — Molecular Biology 165 Laboraton of Molecular and Developmental Biolog> NEI Annual Report— FY 1993 Publications Brady JP, Piatigorsky J: Cloning and characteriza- tion of a novel zinc-finger protein-encoding cDNA from the mouse eye lens. Gene 124:207-214. 1993. Hejtmancik JF, Kaiser MI, Piatigorsky J: Molecular biology of inherited disorders of the eye lens, in Scriver CR, Beaudet AL, Sly WS, Valle D (eds): The Metabolic Basis of Inherited Disease, ed 7. New York, McGraw-Hill P*ublishing Co, in press. Hejtmancik JF, Piatigorsky J: Molecular biology of the eye lens, in Alben DM, Jakobiec FA (eds): Principles and Practice of Ophthalmology: Vie Harvard System. Philadelphia, WB Saunders Co, 1993. pp 168-181. Kantorow M, Becker K, Sax CM, Ozato K, Piatigor- sky J: Binding of tissue-specific forms of oA- CRYBPl to their regulatory sequence in the mouse OLA-crystallin-encoding gene: Double-label immunoblotting of UV-crossIinked complexes. Gene 131:159-165, 1993. Kantorow M, Cvekl A, Sax CM, Piatigorsky J: Protein-DNA interactions of the mouse aA-crys- tallin control regions. Differences between expressing and non-expressing cells. J Mol Biol 230:425-435, 1993. Klement JF, Cvekl A, Piatigorsky J: Functional elements DE2A, DE2B, and DEI A and the TATA box are required for activity of the chicken oA-crystallin gene in transfected lens epithelial cells. J Biol Chem It'i-.fnil-bliA, 1993. Li X, Zelenka PS, Piatigorsky J: Differential expres- sion of the two 5-crystallin genes in lens and non-lens tissues: Shift favoring 62 expres,sion from embryonic to adult chickens. Dev Dynamics 196:114-123, 1993. Piatigorsky J: Gene sharing in the visual system. Trans Am Ophthalmol Soc, in press. Piatigorsky J: Puzzle of crystallin diversity in eye lenses. Dev Dynamics 196:267-272, 1993. Piatigorsky J, Horwitz J, Norman BL: J 1 -crystal lins of the cubomedusan jellyfish lens constitute a novel family encoded in at least three intronless genes. J Biol Chem 268:11894-11901, 1993. Sax CM, Klement JF, Piatigorsky J: Role of the otA-CRYBPl site in lens-specific expression of the aA-crystallin gene, in Loveh PS, Mongkolsuk S, Trempy JS (eds): Biotechnology and Environ- mental Science. New York, Plenum Press, 1992, pp 27-33. Sax CM, Ilagan JG, Piatigorsky J: Functional redundancy of the DE-1 and oA-CRYBPl regula- tory sites of the mouse ocA-crystallin promoter. Nucleic Acid Res 21:2633-2640, 1993. Sax CM, Piatigorsky J: Expression of the a-crystal- lin/small heat shock protein/molecular chaperone genes in the lens and other tissues. Adv Enzymol, in press. Sax CM, Stover DM, Hagan JG, Zehner ZE, Piati- gorsky J: Functional analysis of chicken vimentin distal promoter regions in cultured lens cells. Gene 130:266-281, 1993. Tomarev SI, Zinovieva RD, Piatigorsky J: Primary structure and lens-specific expression of genes for an intermediate filament protein and a P-tubulin in cephalopods. Biochim Biophys Acta, in press. Tomarev SI, Zinovieva RD, Weis VM, Chepelinsky AB, Piatigorsky J, McFall-Ngai MJ: Abundant mRNAs in the squid light organ encode proteins with a high similarity to mammalian peroxidases. Gene 132:219-226, 1993. Zinovieva RD, Tomarev SI, Piatigorsky J: Aldehyde dehydrogenase-derived fl-crystallins of squid and octopus. Specialization for lens expression. J Biol Chem 268:11449-11455, 1993. 166 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00259-04 LMDB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the tiorders.) Molecular Biology of the Cornea PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Joram Piatigorsky Ph.D. Chief LMDB, NEI Others: W. Todd Kayes Ph.D. IRTA LMDB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Molecular Genetics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.6 PROFESSIONAL: 0.6 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) In the past 2 years we have cloned a number of abundant cDNAs from the corneal epithelial cells of the mouse. Some of these encode metabolic enzymes, suggesting that enzymes may act as crystallins in the transparent cornea as they do in the lens. Indeed we have found that different species accumulate different enzymes in their corneal epithelial cells, reminiscent of the taxon specificity observed for enzyme-crystallins of the lens. Aldehyde dehydrogenase (ALDH) class III is the major protein in the corneal epithelial cells of mammals. We have employed the RACE technique to clone the 5' end of the ALDH class 3 corneal mRNA, identified the sequences that should constitute the 5' exon of the gene, and cloned the ALDH class 3 gene in an 18 kbp genomic fragment This firagment is undergoing analysis. 167 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biology NEI Annual Report — FY 1993 Project Description Objectives The project's objectives are to identify and character- ize the genes that are preferentially expressed in the epithelium and endothelium of the cornea and to study the molecular basis for their expression in this transparent structure. Methods Conventional molecular biology methods of cloning, sequencing, recombinant DNA construction, transfec- tion, and transgenic mouse production are used. Major Findings Aldehyde dehydrogenase (ALDH) class 3 comprises approximately 40% of the soluble protein of the corneal epithelial cells of the mouse and other mammals. Such abundance for a metabolic enzyme suggests that this protein is acting like an enzyme- crystallin and has a refractive function in the cornea as crystallins do in the lens. Last year we reported the cloning of the mouse ALDH gene. We thought at that time that we had approximately 2 kbp of the 5'-flanking region as well as the structural gene. Thus, we constructed a P-galactosidase fusion gene with what we believed was 1.1 kbp of the ALDH 5'- flanking sequence and used this construct as a transgene in transgenic mice. Analysis of the progeny of these mice during this fiscal year showed that the transgene had been incorporated but that it was not expressed in any tissue, not even the cornea. We thus extracted more corneal RNA from mice and employed the RACE technique to clone the 5' end of the ALDH cDNA. The sequence of the resulting clones showed that there is an additional exon 5' beyond what we had cloned, implying that our P-galactosidase fusion gene lacked the ALDH promoter and other regulatory sequences for expression. Consequently the ALDH gene was cloned from a mouse genomic library. The 18-kbp, cloned DNA is now undergoing analysis. Significance to Biomedical Research and the Program of the Institute The molecular biology of corneal epithelium and endothelium has not advanced to the same extent as that of the collagenous stroma; consequently, it should be investigated. The cornea is a transparent, ectodermaily derived tissue like the lens; thus com- parative studies between it and the lens are of special interest with respect to transparency. Moreover, because of our finding that corneal epithelial cells show taxon-specific gene sharing of metabolic en- zymes as does the lens, our major tissue of research, comparative studies on the cornea and lens, is of obvious importance from developmental and evolu- tionary viewpoints. Finally, the cornea is particularly accessible for gene therapy on account of its expo- sure to the siuface and its association with numerous hereditary diseases. Proposed Course In Fiscal Year 1994 we will (1) analyze and se- quence the mouse ALDH class 3 gene expressed in the cornea, (2) identify the promoter and functional regulatory elements for the ALDH class 3 gene by transfection experiments, and (3) create and analyze transgenic mice containing various truncated 5'- flanking sequences of the ALDH class 3 gene to identify the sequences responsible for high expres- sion in the corneal epithelial cells. NEI Research Program Lens and Cataract — Molecular Biology 168 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00255-05 LMDB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Molecular Biology and Functions of Lens Proteins PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Graenje J. Wistow Ph.D. Chief, Section on Molecular LMDB, NEI Stnicture and Function Others: Vishwas Paralkar Caroline Graham Lorenzo Segovia Jill Richardson Cynthia Jaworski Peggy Zelenka Ph.D. B.S. Ph.D. Ph.D. Pb.D. Ph.D. Visiting Associate Biologist Visiting Fellow Visiting Fellow Chemist, Section on Molecular Genetics Chief, Section on Cellular Differentiation LMDB. NEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI COOPERATING UNITS (if any) DNX, Inc., Princeton, NJ LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Molecular Structure and Function INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ {a2) Interviews PROFESSIONAL; 4.8 OTHER: 1.0 □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Crystallins are stress-related proteins or enzymes that often maintain dual roles without gene duplication. We have shown that the putative lens promoter of NADPH: quinone oxidoreductase/^-crystallin is highly lens specific and by itself is responsible for the recruitment of this gene as a crystallin. An important functional element (ZPE) that binds different complexes in lens and nonlens-cell extracts has been identified. The full sequence of Ti-crystallin, another mammalian enzyme crystallin, has been obtained and shows identity with the enzyme ALDHl, another example of gene recruitmenL n-Crystallin, originally discovered in marsupial lenses, is a novel enzyme with NADPH-binding activity. Immunohistochemistry suggests it is preferentially expressed in vertebrate photoreceptors. The gene for ^ has been cloned. Not all important lens proteins are crystallins. We have shown that MIF, a lymphokine, is expressed in differentiating lens cells. The gene for human MIF has been cloned, and expression studies are under way. Lens P2 protein, another potential marker for the differentiation process and a possible intermediary messenger has been cloned and shown to belong to a lipid/retinoid-binding family. Members of the C/EBP family, transcription factors with important roles in differentiation and candidates for promoter binding in some crystallin genes, have been detected in the lens, where tlieir pattern of expression is developmentally regulated. 169 PHS 6040 (Rev. 5/92) Laboratorj' of Molecular and Developmental Biology NEI Annual Report— FY 1993 Project Description Objectives We are investigating the molecular basis for normal lens structure and function, including the character- ization of crystallins and the mechanisms of their normal expression. This also has led to the identifi- cation of molecules, such as transcription and growth factors, involved in lens cell differentiation. The interplay of such factors is an essential pan of normal lens development and function. Methods A wide range of molecular biology techniques are used, including RNA analysis, gene and cDNA cloning and sequencing, functional gene promoter analysis in cultured cells and in transgenic mice, and polymerase chain reaction (PCR). We perform some protein analysis and make use of commercial facil- ities for protein sequencing. Major Findings Gene recruitment: Enzyme crystallins. — A novel enzyme with quinone reductase activity has under- gone gene recruitment in certain mammals, acquiring a second function as the lens structural protein ^- crystallin. In work by Drs. Douglas Lee and Jill Richardson, we have shown that recruitment of this taxon-specific crystallin can be explained by the leas specificity of an alternative promoter. By itself this promoter confers strongly lens-preferred expression in both cultured cells and transgenic niice. While proximal regions of the promoter have some activity in the transgenic brain, this is abol- ished by the addition of more distal regions. The minimal active promoter contains a region, ZPE C^- protected element), including a consensus C/EBP binding site, which is strongly and identically foot- printed by nuclear extracts from both mouse and rabbit cultured lens cells in which the promoter is active. In fibroblast nuclear extract, the ZPE's more restricted footprint is flanked by other protected regions that are absent or only weakly footprinted in lens cell extracts. In gel shift assays, the ZPE sequence forms specific complexes with lens-cell extracts but not with fibroblast extracts. Additional transgenic mice have been generated and show that sequences between positions -385 and -533 are required for suppression of promoter expjression in the brain. )j-Crystallin is the major component of the eye lens in several Australian marsupials. It also is a novel enzyme in other manunals, including humans, where it has nonlens expression in neural tissue (including retina), muscle, and kidney. It has strik- ing sequence similarity with bacterial ornithine cyclodeaminases, suggesting an unusual role in ornithine metabolism. Dr. Lorenzo Segovia has shown that p-crystallin preferentially binds NADPH, consistent with its role as a reductase. Immunohisto- chemistry of the developing rat shows a remarkable, intense reaction with retinal photoreceptors; in fact, our anti-p antiserum detects the earliest marker known for photoreceptor cells. Since the retina is susceptible to ornithine toxicity in gyrate atrophy, the expression of a novel ornithine-metaboUzing enzyme in photoreceptors could be significant. The kangaroo gene for this enzyme crystallin has been cloned and is being analyzed. Another major enzyme crystallin (up to 25% of total protein) in mammals is Ti-crystallin found in elephant shrews. Ms. Caroline Graham has cloned the complete cDNA sequence for this protein from two different species. As predicted, sequence data confirm that Ti-crystallin is ALDHl and that it is the product of a single recruited gene. cDNA clones will be expressed in baaerial hosts to characterize enzyme activity. Dr. Cynthia Jaworski has completed the cDNA sequence of human oA-crystallin and has thereby corrected sequence errors in older protein data ocA- crystallin is the single major component of the human lens and is related to small heat shock pro- teins (shsp). The quaternary structure of a-crystal- lins and shsps is both controversial and of great interest. New model structures were predicted on the basis of existing biochemical data. Briefly, these correspond to cubic and dodecahedral structures with coaserved intermolecular contacts. These predictions are stimulating biophysical investigations in other laboratories. oB-crystallin is multifunctional, serving as both a major structural protein in the lens and as an shsp in other tissues in mammals. By cloning and North- em analysis. Dr. Lee showed similarly that aB- crystallin mRNA is present in all the mamre tissues examined in a bird {Anas platyrhynchos), although 170 NEI Annual Report— FY 1993 Laboratory of Molecular and Developmental Biology there are some differences in the pattern of tran- scripts seen. Interestingly, sequence analysis not only shows that duck otB-crystallin is a member of the shsp family, as expected, but that this family shares more distant similarity with another heat shock protein (hsp) family, the highly conserved HSP70s of both eukaryotes and prokaryotes. This raises the interesting possibility that large and small hsps may share structural and perhaps functional features. Molecular markers of differentiation. — Macro- phage migration inhibitory factor (MIF) was original- ly identified as a lymphokine. However, recent work strongly suggests a role for MEF beyond the immune system. Expressed specifically in the differentiating cells of the immunologically privileged eye lens and brain, it is a delayed early response gene in fibro- blasts but is expressed in many tissues. In contrast to previous reports, we have found evidence for a single gene for MEF in the human genome. Dr. Vishwas Paralkar has shown that this small gene has three exons separated by introns of only 189 and 95 bp and covers less than 1 kb. The cloned se- quence also includes 1 kb of 5'-flanking region covering the putative promoter. Primer extension and 5' RACE PCR of human brain RNA both indicate the presence of a single transcription start site in a TATA-less promoter. Northern blot analysis shows a single-size MIF mRNA in all human tissues examined. MIF mRNA is particularly abundant in the kidney and is ex- pressed at high levels in many other tissues but is at low levels in muscle and the pancreas. The relative- ly abundant expression of MIF in lens may have clinical significance, with the possibility of involve- ment in lens-induced endophthalmitis and uveitis. Another potential differentiation marker also was discovered by protein microsequencing of a 14-kD band in calf lens extract. Dr. Jaworski has now obtained the complete cDNA sequence for this protein by PCR methods. The protein is a member of the lipid/retinoic acid-binding family of P2 pro- teins. Since retinoic acid has now been implicated in ■y-crystallin gene expression, this protein could have a direct role in mediating lens gene expression. We are interested in connections between differ- entiation in the well-studied adipocyte system and in the eye lens. In both systems there is a switch in c- myc expression during differentiation: Stem cells are marked by expression of a-enolase while P2-like proteins also may be associated with differentiation. Furthermore, C/EBP-like proteins are candidates for binding to an essential, tissue-specific region of the ^-crystallin gene lens promoter. Dr. Richardson is examining whether C/EBP-like proteins are expressed in the lens. She has results from immunohistochem- istry associating expression of C/EBP p and 5 with rat lens epithelia, the relatively undifferentiated "stem cell" population. C/EBP 5 is most abundant in the central, quiescent cells, while (3 comes on down- stream in regions where cells are migrating and dividing. Both are undetectable in the terminally differentiated lens fiber cells. The human Marner cataract maps to chromosome 16 near the locus of several cadherins, which are important cell adhesion molecules. The cataract manifests as opacities at the Y-sutures, suggesting that a cell-cell contact or adhesion process could be defective. We examined whether a cadherin could be involved in this cataract. Immunochemical methods suggest that N-cadherin is expressed in the eye lens, but having no sequence data, this could be a cross-reaction with a related, perhaps lens-specific, variant. Ms. Graham used PCR to amplify cadherin- related sequences in human fetal lens and sequenced multiple clones. All were identical to known human N-cadherin. We then checked the chromosomal location of N-cadherin by PCR, using chromosome- specific template DNA, confirming its published position on chromosome 18. It is the only major cadherin not on chromosome 16. These results suggest that the principal cadherin expressed in the eye lens is identical to N-cadherin and that there is no lens-specific variant of this family. The only remaining possibilities of a con- nection between cadherins and the Marner cataract are that another cadherin is expressed at low levels in the lens or with a particular development pattern, or that the mutation causes inappropriate expression in the lens of another cadherin. Significance to Biomedical Research and the Program of the Institute The discovery of fundamental mechanisms in the differentiation and evolution of complex tissues has had important results in our understanding of impor- tant processes in evolution and in tissue-specific expression. In the process, we have discovered a novel enzyme that has possible significance in the 171 Laboratory of Molecular and Developmental Biology NEI Annual Report — FY 1993 human retina. We also have discovered important markers for cellular differentiation, including proteias with inflammation-related lymphokine activity. Proposed Course 1. We will continue examining the molecular mechanisms for lens-preferred expression and for gene recruitment in ^, Ti, n, and x-crystallins. 2. We will characterize the function and nonlens role of |i-crystallin. 3. We will explore the molecular biology and fimction of MIF expressed in the lens and its pos- sible role in eye inflammation. 4. We will determine the function and pattern of expression of the lens P2 protein, a possible second messenger in signal transduction. NEI Research Program Cataract — Molecular Genetics Publications Chen H, Phillips HA, Callen DF, Kim RY, Wistow GJ, Antonarakis SE: Localization of the human gene for mu-crystallin to chromosome 16p. Genomics 14:1115-1116, 1992. de Jong WW, Leunissen JAM, Wistow GJ: Eye lens crystallins and the phylogeny of placental orders: Evidence for a macroscelid-paenungulate clade? in Szalay FS, Novacek MJ, McKenna MC (eds): Mammal Phylogeny: Placentals. New York, Springer Verlag, 1992, pp 5-12. Hodin J, Wistow G: 5'-RACE PCR of mRNA for three taxon-specific crystallins: For each gene one promoter controls both lens and non-lens expression. Biochem Biophys Res Commun 190:391-396, 1993. Kim RY, Gasser R, Wistow GJ: Mu-crystallin is a mammalian homologue of Agrobacterium orni- thine cyclodeaminase and is expressed in human retina. Proc Natl Acad Sci USA 899292-9296 1992. Kim RY, Wistow GJ: The cDNA RPEl and mono- clonal antibody HMB-50 define gene products preferentially expressed in retinal pigment epithe- lium. Exp Eye Res 55:657-662, 1992. Kim RY, Wistow GJ: Expression of the duck a-enolase/T-crystallin gene in transgenic mice. FASEB J 7:464-469, 1993. Lee DC, Gonzalez P, Rao PV, Zigler JS Jr, Wistow GJ: Carbonyl -metabolizing enzymes and their relatives recruited as structural proteins in the eye lens, in Weiner H (ed): Advances in Experimen- tal Medicine and Biology, 284: Enzymology and Molecular Biology of Carbonyl Metabolism. New York, Plenum Press, 1993, vol 4, p 159-168. Lee DC, Kim RY, Wistow GJ: An avian oB-crystal- lin. Non-lens expression and sequence similar- ities with both small (HSP27) and large (HSP70) heat shock proteins. J Mol Biol 232:1221-1226, 1993. Wistow G: Lens crystallins: A model system for gene recruitment, in Zimmer EA, White TJ, Cann RL, Wilson AC (eds): Methods in Enzymology: Molecular Evolution Producing the Biochemical Data. San Diego, Academic Press, 1993, vol 224, pp 563-575. Wistow G: Lens crystallins: Gene recruitment and evolutionary dynamism. Trends Biochem Sci 18:301-306, 1993. Wistow G: Possible tetramer-based quaternary structures for a-crystallins and small heat-shock proteins. Exp Eye Res 56:729-732, 1993. Wistow G: I^otein structure and introns. Nature 346:107-108, 1993. Wistow GJ, Shaughnessy MP, Lee DC, Hodin J, Zelenka PS: A macrophage migration inhibitory factor is expressed in the differentiating cells of the eye lens. Proc Natl Acad Sci USA 90:1272-1275, 1993. 172 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00251-06 LMDB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (BO characters or less. Title must tit on one line between ti)e borders.) Genetically Engineering the Eye with the ccA-Crystallin Promoter PRINCIPAL INVESTIGATOR (List other protessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Ana B. Chepelinsky Ph.D. Head, Section on LMDB, NEI Regulation of Gene * Expression Others: Devonne M. Parker B.S. Biologist LMDB, NEI COOPERATING UNITS (if any) Department of Cell Biology, Baylor College of Medicine. Howard Hughes Medical Institute (Paul Overbeek, Ph.D.; Michael Robinson, Ph.D.); Imperial Cancer Research Fund, London, England (Clive Dickson, Ph.D.); Gerontological Research Unit, National Institute of Health and Medical Research, Paris, France (Yves Courtois, Ph.D.: Maryvonne Laurent, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Regulation of Gene Expression INSTITUTE AND LOCATION NEI. NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.9 PROFESSIONAL: 0.4 OTHER: 0.5 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Gamma interferon (IFN-y) expression was directed to the eyes of transgenic mice to investigate the possible role of this lymphokine in ocular pathogenesis. The most notable effects of IFN-y in these transgenic mice include microphthalmia, blepharophimosis, microphakia, impairment of lens fiber formation, arrest of retinal differentiation, retinal detachment, and persistent hyperplastic primary vitreous. Major histocompatibility complex (MHC) class n mRNA levels were significantly increased in tiie transgenic eyes, and MHC class II proteins were expressed in the cornea, iris, ciliary body, choroid, lens, and retinal pigment epithelium. Int-2/fibroblast growth factor (FGF)-3 expression was directed to the eye to investigate how the aberrant expression of this growth factor would affect the developmental program of the eye. The ectopic expression of int-2 during the embryonic development of the lens affected the differentiation of the entire eye, highlighted by the appearance of intraocular secretory glandular epithelium, similar to dermoid-like pathology. 173 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biolo^ NEI Annual Report— FY 1993 Project Description Additional Personnel Charles Egwuagu Ph.D. Scientist, Public Health Service, LI, NEI Chi-Chao Chan M.D. Chief, Section on Immunopathology, LI, NEI Robert B. Nussenblatt M.D. Clinical Director, LI, NEI Jorge Sztein D.V.M. Visiting Associate, Veterinary Research Resources Service, NEI Objectives The objective of this project is to understand how aberrant genetic expression of interferon gamma (EFN-y), int-2, or acidic fibroblast growth factor (aFGF), under the control of the oA-crystallin promoter, perturbs normal eye development in transgenic mice. Methods Recombinant DNA techniques used in this study include plasmid construction, oligonucleotide se- quencing, Southern and Northern hybridizations, DNA sequencing, primer extension, polymerase chain reaction (PCR), reversed transcription PCR, immuno- histochemistry, and the production and analysis of transgenic mice. Major Findings IFN-y. — This project is conducted in collaboration with Drs. Charles Egwuagu, Jorge Sztein, Chi-Chao Chan, and Roben B. Nussenblatt from the NEI Laboratory of Immunology. The aberrant expression of IFN-y in the lens of transgenic mice allowed us to study the effect of IFN-y on the normal development of the eye and the regulation of major histocompati- bility complex (MHC) class II gene expression by IFN-y in a nonlymphoid tissue such as the lens. We generated transgenic mice containing as a transgene the murine aA-crystallin promoter (-366/ +46) fused to the murine IFN-y coding sequence. The ectopic expression of IFN-y in the lens of the transgenic mouse affected the growth of the whole eye, resulting in microphthalmia and microphakia. The lens fiber cells were replaced by balloon cells, differentiation of the neuroretina into iimer and outer neuroblastic layers was arrested at the embryonic stage, and retinal detachment and the presence of macrophages in the subretinal space were observed J in the adult mouse eye. 1 Constitutive expression of EFN-y in the lens induced aberrant MHC class n protein synthesis in several ocular tissues. This transgenic mouse serves as an animal model to (1) facilitate understanding of the molecular pathways governing synchronized programmed differentiation of ocular tissues and (2) enable study of the linkage between aberrant MHC class II expression and predisposition to autoimmune diseases. int-2. — This project is conducted in collaboration with Drs. Paul Overbeek and Michael Robinson (Baylor College of Medicine) and Dr. Clive Dickson (Imperial Cancer Research Fund). To assess whether ectopic expression of the proto-pncogene int-2/FGF-3 would perturb normal eye development, we directed its expression to the eyes of transgenic mice with the miuine oA-crystallin promoter. We obtained three lines of transgenic mice expressing the oA-crystal- lin/int-2 transgene. These adult transgenic mice presented microphthalmia characterized by intraocu- lar hyperplastic glandular structures replacing the normal iris, ciliary body, and lens; the retinas showed various degrees of dysplasia. The intraocular glandular structures of these mice stained positively for int-2 and Muc-1, a marker for secretory epithelia. We also observed a marked increase in Muc-1 mRNA levels and a drastic de- crease in MIP (major intrinsic protein) mRNA levels, a marker of lens fibers, in the eyes of the adult trans- genic mice. Proptosis of the transgenic eye, ob- served as early as day 15 of embryonic development, was followed by expulsion of the lens through the cornea and detachment of the imdifferentiated retina. The newborn mice presented a "scabby eye" pheno- type. Ectopic expression of the growth factor int-2 during the embryonic development of the lens affected the differentiation of the entire eye, high- lighted by the appearance of intraocular secretory glandular epithelium, similar to dermoid-like patholo- gy, in the adult eye. aFGF. — In collaboration with Drs. Overbeek and Robinson , as well as Dr. Yves Courtois (Institute for Gerontological Research, INSERM), we injected into mouse embryos a recombinant DNA containing the aA-crystallin promoter (-366/+46) fused to the bovine aFGF cDNA. Three lines of transgenic mice 174 NEI Annual Report— FY 1993 Laboratory of Molecular and Developmental Biology were obtained; no particular phenotype was observed. We currently are analyzing these mice for expression of the transgene. Significance to Biomedical Research and the Program of the Institute The aberrant expression of IFN-y or int-2 will allow us to elucidate the mechanisms underlying eye development. At the same time, it opens new avenues in the development of animal models for studies of eye pathologies and of gene regulation in the eye. Proposed Course The following studies will continue during Fiscal Year 1994: 1. We will characterize further the effect of BFN- Y on the regulation of gene expression in the eyes of the transgenic mice. 2. We will continue to study the effect of int-2 on gene expression in the eyes of the transgenic mice and try to understand its role in eye growth and differentiatioa NEI Research Program Cataract — Molecular Genetics Publications Chepelinsky AB, Overbeek PA, Chan C-C, Jamieson S, Dickson C, Parker DM, Robinson M: Int-2 ectopic expression induces differentiation of secretory epithelia in the eyes of transgenic mice. Invest Ophthalmol Vis Sci 34(4)(suppl):1222, 1993. Egwuagu CF, Sztein J, Chan C-C, Reid W, Mahdi R, Nussenblatt RB, Chepelinsky AB: Ectopic expression of gamma interferon in the eyes of transgenic mice induces ocular pathology and MHC class n gene expression. Invest Ophthal- mol Vis Sci, in press. Egwuagu CF, Sztein J, Chan C-C, Reid W, Mahdi R, Nussenblatt RB, Chepelinsky AB: Gamma interferon expression in the eyes of transgenic mice disrupts differentiation of the lens and retina. Invest Ophthalmol Vis Sci 34(4)(suppl): 1455, 1993. 175 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00253-05 LMDB PERIOD COVERED October 1. 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less Title must lit on one line between the borders.) Regulation of Expression of Lens Fiber Membrane Genes PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute afliliation) PI: Ana B. Chepelinsky Ph.D. Head, Section on LMDB, NEI Regulation of Gene Expression Others: Chiaki Ohtaka-Maruyama Xiaoyan Wang LaShawn R. Drew Devonne M. Parker Ph.D. Visiting Fellow LMDB, NfEl M.D. IRTA Fellow LMDB. NEI B.S. Chemist LMDB, NEI B.S. Biologist LMDB, NEI COOPERATING UNITS (il any) Harvard University (David Paul, Ph.D.); Columbia University (Jorge Fischbarg, M.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Regulation of Gene Expression INSTITUTE AN^ LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.2 PROFESSIONAL: 2.0 OTHER: 1.2 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project studies the regulation of expression of genes encoding lens fiber membrane chaimel proteins. We are focusing on the regulation of expression of the gene encoding MIP, the major intrinsic protein of the lens fiber membrane, which is specifically expressed in the ocular lens fibers and belongs to an ancient superfamily of transmembrane channel proteins. We characterized 2,840 bp of 5' flanking sequence of the human MIP gene to study the cis regulatory elements responsible for the tissue specificity and developmental regulation of the MIP gene. We found that a DNA fragment containing 253 bp of 5' flanking sequence and 42 bp of exon 1 of the human MIP gene fused to the reporter chloramphenicol acetyltransferase (CAT) gene is able to express the CAT gene in cultured lens cells. We are studying the interaction of transcription factors with the cis regulatory elements of the MIP gene and its effect on the in vitro transcription of the MIP gene in Drosophila nuclear extracts. Purified human Spl and AP2 interact with cis regulatory elements of the MIP promoter and activate the in vitro transcription of the MIP promoter, suggesting their involvement in the regulation of MIP gene transcription. These studies will further our understanding of the role of general u-anscription factors on the tissue-specific expression of the MIP gene. 176 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Molecular and Developmental Biology Project Description Objectives The objective of this project is to elucidate the mechanisms involved in the regulation of expression of fiber membrane genes involved in cell-cell com- munication in the lens. The identification of the cis regulatory elements of these genes and their inter- action with trans-acting factors is essential for understanding the regulation of gene expression in the lens. Methods Recombinant DNA techniques used in this study include screening genomic libraries, subcloning, plasmid construction, oligonucleotide synthesis. Southern and Northern hybridizations, DNA sequenc- ing, primer extension, polymerase chain reaction (PCR), reversed transcription PCR, gel mobility shift assays, DNA footprinting, methylation interference, subcellular fractionation to obtain nuclear extracts, in vitro transcription, tissue culture techniques (includ- ing transfection of primary lens explants and cell lines), and analysis of transgenic mice. Major Findings Cis regulatory elements of the human major intrinsic protein (MIP) gene promoter. — We characterized 2,840 bp of 5'-flanking sequence of the human NOP gene to identify the cis regulatory elements responsi- ble for the tissue specificity and developmental regulation of the MIP gene. We found that a DNA fragment containing 253 bp of 5'-flanking sequence and 42 bp of exon 1 of the human MIP gene fused to the reporter gene chloramphenicol acetyltrans- ferase (CAT) directs CAT gene expression to leas cells in transient assays. Therefore, the -253/-i42 sequence of the human MIP gene contains informa- tion for lens cell expression, suggesting that cis regulatory elements responsible for the lens-specific expression of the MIP gene are localized within this domain. Several motifs known to bind transcription factors in other genes are present in the 5'-flanking sequence of the MIP gene. To elucidate whether those motifs are involved in the regulation of MIP gene expres- sion, we are studying their interaction with several transcripton factors. Analysis by DNA footprinting and gel mobility shift assays show that purified human Spl and AK interact with several domains of the MIP promoter. They also activate the in vitro transcription of the MIP promoter in Drosophila nuclear extracts. We found that the initiation site of transcription of the human MIP gene was the same in vivo and in vitro. These results suggest that SPl and AP2 are involved in the regulation of transcrip- tion of the MIP gene. We have generated several lines of transgenic mice containing 253 bp of the human MIP gene 5'- flanking sequence with 42 bp of exon 1 fused to the CAT gene as a transgene. In one transgenic line, the CAT gene expressed specifically in the ocular lens. Expression of the CAT gene was observed in the ovaries of the females of four additional transgenic lines, although no expression was observed in any tissue of the male progeny. We currentiy are map- ping other regulatory elements localized between -2,8(X) bp and the initiation site of transcription of the MIP gene that may be required for proper tissue specificity. We also are cloning the murine MIP gene in order to study the mouse MIP promoter in its homologous in vivo environment 3' untranslated sequence of the MIP gene. — We are sequencing the 3'-flanking region of the human MIP gene to characterize the polyadenylation sites and the processing of the two MIP transcripts ob- served in vivo. Cloning of the connexin 46 gene. — ^We are cloning the coimexin 46 gene, which encodes one of the lens fiber g^ junction proteins, to be able to study how its expression is regulated in the lens. CHIP28 expression in cornea endothelial cells. — In collaboration with Dr. Jorge Fischbarg (Columbia University), we are characterizing the member of the MIP family responsible for the CHIP28-like water channels observed in primary cultures of bovine cornea endothelial cells. Sequencing data indicate that it is CHIP28. Significance to Biomedical Research and the Program of the Institute Since the differentiation of lens epithelial cells into fiber cells results in a dramatic increase of new plasma membrane synthesis by elongating cells, proper membrane biosynthesis and physiology are of utmost importance in maintaining the transparent state of the lens. Membrane protein synthesis is regulated in a temporal and spatial manner in the lens. Alterations in lens membranes, particularly involving MIP, have been observed during cataracto- genesis and aging. Therefore, studies on MIP gene 177 Laboratory of Molecular and Developmental Biologj' NEI Annual Report— FY 1993 expression should further our understanding, not only of the mechanisms involved in the regulation of gene expression in the normal lens, but also of its disrup- tion during disease. Proposed Course The following studies will continue during Fiscal Year 1994: 1 . Charaaerization of the cis regulatory elements of the human MIP promoter. 2. Isolation of the murine MIP gene promoter and its comparison with its human homolog. 3. Study of the interaction of the MIP gene cis regulatory elements with transcription factors. 4. Characterization of the 3'-flanking region of the human MIP gene. NEI Research Program Cataract — Molecular Genetics Publications Chepelinsky AB: The MIP transmembrane chaimel family, in Peracchia C (ed): Handbook of Mem- brane Channels: Molecular and Cellular Physiol- ogy. Academic Press, in press. Ohtaka-Maruyama C, Drew LR, Pisano MM, Chepe- linsky AB: Regulatory elements of the MIP gene promoter. Invest Ophthalmol Vis Sci 34(4) (suppl):1342, 1993. Ohtaka-Maruyama C, Drew LR, Pisano MM, Chepe- linsky AB: Transcriptional regulation of the human MIP gene. J Cellular Biochem 17A (suppl):167, 1993. Tomarev SI, Zinovieva RD, Weis VM, Chepelinsky AB, Piatigorsky J, McFall-Ngai MJ: Abundant mRNAs in the squid light organ encode proteins with a high similarity to mammalian peroxidases. Gene 132:219-226, 1993. 178 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00285-01 LMDB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) NEI Central Transgenic Animal Production Facility PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Eric Wawrousek Ph.D. Research Biologist LMDB, NEI Others: Susan DiCamillo R. Steven Lee Mariana Gonzalez-Baez B.S. Chemist LMDB, NEI B.S. Biologist LMDB, NEI Biological Science LMDB, NEI Lab Aide, Stay-in- School Program LMDB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Transgenic Animal and Genome Manipulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 2.7 PROFESSIONAL: 0.5 OTHER: 2.2 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The NEI Central Transgenic Animal Production Facility is a research support facility for all NEI intramural researchers requiring the use of transgenic mice in their research programs. We are providing transgenic animal support to 18 researchers from four laboratories in the NEI (Laboratory of Immunology, Laboratory of Mechanisms of Ocular Diseases, Laboratory of Molecular and Developmental Biology, and Laboratory of Retinal Cell and Molecular Biology); in our program, there are 52 DNA constructs at various stages of completion. NEI researchers using molecular biology techniques to study the eye submit DNA constructs to our section for production of transgenic mice. We create transgenic mice by standard procedures, then biopsy and perform DNA analysis on the mice bom from tliese procedures to identify positive mice. At researchers' request, we mate positive transgenic mice, wean litters, biopsy and analyze DNA from successive generations of transgenic mice, and provide the transgenic animals to researchers for use in their experiments. Over the year we have generated 129 transgenic mice from 26 DNA constructs; set up 231 matings of transgenic mice; weaned, tagged, and tail-biopsied 3,033 mice; and isolated DNA and performed DNA analysis on 2,313 biopsy samples. We are working toward creating an embryo cryopreservation and banking program to provide long- term storage of important transgenic lines to eliminate the need to maintain live mice. In addition to service functions, we collaborate with NEI researchers on transgenic animal projects. This year we collaborated with Dr. Igal Gery (Laboratory of Immunology, NEI) in creating DNA constructs, and subsequently transgenic mice, to examine the immunologic consequences of expressing foreign antigens in the encapsulated ocular lens. 179 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biology NEI Annual Report— FY 1993 Project Description Objectives This project has been established to (1) produce transgenic animals for use in NEI's eye research, (2) supply ancillary services related to maintenance of transgenic animals, (3) provide advice and exper- tise in matters of transgenic animal projects to all NEI intramural researchers using this technology in their research, and (4) act as a central facility for all transgenic animal work conducted in the NEI Intra- mural Research Program in order to coordinate and conserve resources and utihze severely limited animal housing space with maximum efQciency. We provide a comprehensive program for short- and long-term storage of transgenic animal lines, both as live animals and as frozen embryos. Methods Standard methods are used for microinjecting DNA into the pronucleus of one-celled mouse embryos and surgically reimplanting the injected embryos into foster mothers for development. Conventional molecular biology techniques are used to isolate and analyze DNA from biopsy samples of transgenic mice. Data on all transgenic mice are maintained in a computerized relational data base accessed by programs written within our group. Major Findings Production of new transgenic mouse lines. — We have generated 129 new transgenic founder mice from 26 constructs submitted by researchers in 4 NEI intra- mural laboratories. These constructs are quite diverse in nature, reflecting the diversity of research being performed in the NEI. Some of the general categories of constructs are (1) promoter/reporter constructs in which the promoter of an eye gene is fused to a reporter gene to assess transcriptional activity in the transgenic mouse, (2) eye-specific or ubiquitous promoters driving expression of genes believed to be involved in eye pathologies to as.sess their roles in pathological conditions in a transgenic mouse model, and (3) other constructs for probing normal eye function and pathological conditions in the mouse. Maintenance of transgenic mouse lines. — Transgenic mouse lines are derived by mating of the original transgenic founder mice and derivation of successive generations of progeny, which are then used in biomedical research. To generate lines of transgenic mice from our transgenic founder mice, we have set up 231 mouse matings and weaned, tagged, and biopsied 3,033 mice resulting from matings and microinjection procedures. DNA analyses. — Approximately 10-25% of mice bom from microinjected embryos are transgenic; similarly, approximately 50% of mice resulting from a transgenic mouse mating are transgenic. A r^id, efficient, and rehable method of identifying transgene positive and negative mice is in place in our group. We have processed 2,313 biopsy samples to obtain DNA and have performed analyses on these samples to determine whether the mice were transgene positive. Embryo cryopreservation and banking. — ^We have been experimenting with freezing mouse embryos for banking of important lines of ttansgenic mice. Slow freezing of embryos in vials and in sfraws has been attempted. The sfraw method has the advantages of being simpler and more time efficient and will be pursued further. Reconstitution of mice has been accomplished by transferring thawed embryos into the oviduct or into the uterus of foster mothers. We have had the best results with oviductal transfers and will pursue this methodology. Our reproducibility in freezing and thawing embryos and in reconstitut- ing mouse lines from thawed embryos is not yet sufficient to begin large-scale banking of embryos with confidence that we can regenerate the banked lines. Significance to Biomedical Research and the Program of the Institute Transgenic mice are currently the only readily attainable system for studying gene expression in the context of an entire, intact animal. While tissue culture can yield a great deal of information in many studies, true understanding of how a particular gene affects an organ (such as the eye) or an entire organ- ism can be obtained only by studying that gene in tlie intact organism. We play a pivotal role in many NEI inframural research projects by providing the technology and expertise to insert into the mouse genes related to normal eye development and patho- logical eye conditions. Proposed Course In Fiscal Year 1994 we will continue our research as follows: 180 NEI Annual Report— FY 1993 Laboratory of Molecular and Developmental Biology 1. We will continue producing new transgenic mice for NEI researchers as required for their re- search projects. 2. We will continue breeding and maintaining the existing transgenic mouse lines needed for ongoing NEI research. 3. We will continue our efforts to reproducibly freeze and thaw mouse embryos and reconstitute mouse lines from the thawed embryos. Once we are certain that we can regenerate transgenic mouse lines from frozen embryos, we will begin cryopreservation and banking of some existing transgenic mouse lines, which are important to keep but which are not currently in use. This will free some of our limited animal housing space and ensure that important lines of mice will not be lost due to aging and loss of fertility. NEI Research Program Cataract — Molecular Genetics 181 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED PROJECT NUMBER ZOl EY 00286-01 LMDB October 1. 1992 to September 30. 1993 TITLE OF PROJECT (80 characters or less Title must tit on one line between the borders.) g-Crystallin Gene Disruption in the Mouse PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute altiliation) PI: Eric Wawrousek Ph.D. Research Biologist LMDB, NEI COOPERATING UNITS (il any) University of Maryland Medical School (Nicholas Ambulos, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Transgenic Animal and Genome Manipulation INSTITUTE AND LOCATION NEI, NIH. Bethesda, MD 20892 TOTAL STAFF YEARS: 0.5 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects n (a1) Minors □ (a2) Interviews PROFESSIONAL: 0.5 OTHER: 0.0 n (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The a-crystallins, which comprise a large fraction of the soluble protein in the vertebrate lens, where they are beUeved to function solely as structural proteins, are the first crystallins to be expressed in the developing mouse lens and are a relatively small family of crystaUins encoded by only two genes, the oA- and oB- crystalhn genes. The a-crystallins exhibit molecular chaperone activity and, at least in the case of oB- crystallin, have been shown to be expressed in a variety of nonlenUcular tissues, where their function is unknown. Toward understanding the role of the a-crystallins in lens and nonlens tissues, we are attempting functional deletion of a-crystallins by disrupting the a-crystallin genes in mice. We are employing the technique of homologous recombination in pluripotent mouse embryonic stem cells, followed by the generation of chimeric mice containing the altered stem cells. We have isolated and mapped 15-kb clones containing the OA- and oB-crystallin gene loci from a mouse 129SV library (the same strain as most of the embryonic stem cell Lnes currently in use). Construction of the aA-crystallin "knockout vector" is near completion In collaborauon with Dr. Nicholas Ambulos (University of Maryland Medical School), we have nearly completed double-stranded sequencing of the mouse aA-crystallin gene (5 kb) and single-stranded sequencing of an addiuonal 4 kb of the 5 -flanking sequence. We currently are generating constructs to look for enhancer regions m the aA-crystallin introns and in the 5'- and 3'-flanking regions. 182 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Molecular and Developmental Biology Project Description Additional Personnel Ellen Liberman Ph.D. Division of Basic Vision Research, NEI Objectives This project was designed to disrupt the a-crystallin genes (otA and oB) in the mouse to study their effect on normal lens and eye development. Disruption of the genes essentially will delete these proteins from the mouse, enabling us to analyze the effects these proteins have on expression of other lens proteins, developmental regulation and morphology of the lens and other eye structures, and the role of these pro- teins in nonlenticular tissues. Methods Standard molecular biology techniques are used to clone the a-crystallin genes and construct "gene knockout" vectors. Disruption of the genes will be accomplished by the newly developed technology of homologous recombination in pluripotent mouse embryonic stem cells, followed by insertion of the genetically altered cells into blastocyst mouse embry- os to generate chimeric mice with the gene disrup- tion. Chimeric "knockout" mice will be bred to generate mice with heterozygous and homozygous knockouts. Major Findings Cloning of mouse a-crystallin genes. — To maximize the success of homologous recombination in the planned experiments, we have isolated clones for clA- and oB-crystallin from a mouse 129 SV genom- ic library. This is the mouse strain from which most currently used embryonic stem cells are derived. Use of isogeneic DNA has been shown to improve greatly the yield of correctly targeted gene disrup- tions. Two overlapping aA clones and five overlap- ping (xB clones have been isolated and mapped. A 15-kb oA clone with 9 kb of 5'- and 2 kb of 3'- flanking sequence and a 16-kb ctB clone with 7 kb of 5'- and 6 kb of 3'-flanking sequence will be used in construction of the knockout veaor. Construction of knockout vectors. — Construction of the aA-crystallin gene knockout vector is nearly complete. The vector contains 9 kb of aA 5'-flank- ing sequence, a selectable neomycin resistance gene cassette disrupting the aA gene, 1.3 kb of oA sequence, and a negative selectable marker (HSV tk). Sequencing of mouse aA-crystallin gene. — ^We are now sequencing the mouse aA-crystallin gene locus in collaboration with Dr. Nicholas Ambulos. A double-stranded sequence of the gene is nearly complete, and a single-stranded sequence of 4 kb of 5'-flanking sequence has been done. Having the sequence of the locus will enable easy interpretation of DNA analysis of stem cells and mouse biopsies carrying the gene knockout. It also will facilitate identification of binding sites for regulatory protein in portions of the gene not yet studied. Transcriptional regulation of mouse aA-crystallin gene. — We are constructing vectors containing portions of the aA-crystallin gene locus to search for transcriptional enhancer elements. A base vector containing the oA-crystalUn promoter (-366 to +46) fused to the bacterial CAT reporter gene is under construction. Large pieces of the aA locus will be inserted into the base vector and tested for enhancer activity in transient transfection assays in cultured cells. Significance to Biomedical Research and the Program of the Institute Deletion of the a-crystallin proteins, individually or together, will provide a fundamental understanding of how these proteins function during normal lens development and how they may influence the struc- ture and function of the lens and the entire eye. In addition, it would give us insight into the function of these proteins in nonlenticular tissues, which in turn could help us imderstand some of their more subtle roles in the eye. Proposed Course In Fiscal Year 1994 we will continue our investiga- tion as follows: 1 . We will continue construction of aA and aB knockout vectors so that mice deficient in either of these genes can be created. We also will perform the gene knockouts by introducing the targeting vector into mouse embryonic stem cells, selecting appropriately altered cells, and then creating chimeric mice from these cells. Deletion of a single allele of either aA or aB can be studied to assess the gene dosage effect (50% reduction of protein). Breeding to homozygosity (deletion of both alleles) will allow us to study the consequences of complete absence of 183 Laboraton of Molecular and Developmental Biolog> NEI Annual Report — FY 1993 the individua] protein. Eventually we will mate aA and oB icnockout mice to produce mice totally devoid of a-crystallin. 2. We will complete sequencing of the aA- crystallin gene locus. Although much is known about regulation of the mouse ocA-crystallin gene, the complete sequence of the gene has not yet been detennined. Knowing the complete sequence of the gene and flanking regions will be beneficial in the location of possible regulatory sites not in the imme- diate 5'-flanking region of the gene. This knowledge will be invaluable in analysis of gene knockouts in stem cells and mice. 3. We will continue construction of vectors for use in transient transfection assays to locate addition- al regulatory elements in and around the oA-crystal- lin gene. This, along with the sequence of the locus, will help us to identify potential sites influencing the levels of gene expression. NEI Research Program Cataract — Lens Development and Aging I 1 184 Laboratory of Ocular Therapeutics Report of the Chief, Laboratory of Ocular Therapeutics Peter F. Kador, Ph.D. The Laboratory of Ocular Therapeutics (LOT) focuses on the development, evaluation, and mechanism of action of new ophthalmic drugs to treat eye diseases. The LOT research team is exam- ining aldose reductase inhibitors (ARIs) and anticata- ract agents. In pursuing the development of more effective and less toxic ARIs, the efforts are pro- gressing toward the development of an inhibitor unrelated to previous ARIs. A patent application has been made, and efforts are concentrated on further characterizing this inhibitor using biochemical, pharmacological, and computer molecular design techniques. Studies designed to elucidate the specific mechanism(s) by which aldose reductase initiates diabetic complications also are being conducted. In studies utihzing galactose-fed dogs, LOT investigators have established that retinal changes associated with diabetic retinopathy progress to the proliferative stage. The dog represents the first animal model that demonstrates clinical and histolog- ical changes found in all stages of retinopathy. Studies are now focused on the development of proliferative retinopathy in long-term galactose-fed dogs. In addition, investigators are analyzing the specific role of aldose reductase in neuropathy, thyroid changes, and immune system responses. Models for the evaluations of lens changes by magnetic resonance imaging also are being developed. 187 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00003-20 LOT PERIOD COVERED October 1, 1992 to September 30. 1993 TITLE OF PROJECT (80 characlars or less. Title must tit on one line between the borders.) Pharmacology of Ocular Complications PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, PI: Peter F. Kador Others: WiUjam Greentree JuD Inoue YoDg Lee Martii] Lizak Amta Bartoszko- Malik Kazuhiko Mori Heike Neuenschwander Libaniel Rodriguez Maneo Scbaffbauser Ph.D. Chief D.V.M. IRTA FeUow M.S., Phann. Special Volunteer Ph.D. Staff Fellow Ph.D. Staff Fellow Ph.D. Visiting Fellow M.D., Ph.D. Visiting Fellow M.D. Special Volunteer Ph.D. Staff Fellow Ph.D. Visiting Fellow and institute affiliation) LOT, NEI LOT, NEI LOT. NEI LOT, NEI LOT, NEI LOT, NEI LOT, NEI LOT. NEI LOT. NEI LOT. NEI COOPERATING UNITS (it any) LAB/BRANCH Laboratory of Ocular Therapeutics SECTION INSTITUTE AND LOCATION NEI, NIH. Bethesda, MP 20892 TOTAL STAFF YEARS: 6.75 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews PROFESSIONAL: 6.75 OTHER: 0.0 [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.) Events leading to the onset of various ocular complications are being investigated. Specific studies include the role of the enzymes aldose reductase and aldehyde reductase in the onset and progression of retinopathy, cataract, keratopathy, pupil function changes, and iris and ciliary process structure changes associated with diabetes and galactosemia. In addition, methods for either delaying or preventing the onset and progression of these complications through the pharmacological control of these enzymes are being developed. Also being studied are events leading to the formation of several types of cataracts, as well as methods for controlling the onset of these cataracts through pharmacological intervention. 188 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Ocular Therapeutics Project Description Additional Personnel Robert Balaban Ph.D. Duane Miller Ph.D. National Heart, Lung and Blood Institute Ohio State University College of Pharmacy, Columbus, OH Objectives This project is designed to (1) gain insight into the mechanisms by which polyol-induced ocular diabetic complications and cataracts are formed and (2) devel- op methods for their regulation. Methods Diabetes can be induced experimentally in animals through the injection of streptozotocin. Diabetes- related complications linked to the sorbitol pathway also can be induced in animals such as rats and dogs by feeding them a galactose-enriched diet. Cataract formation and clinical retinal changes in experimental animals can be monitored through fundus photogra- phy. Biochemical smdies used for the purification of enzymes include column chromatography, polyacryl- amide gel electrophoresis (PAGE), isoelectric focus- ing, chromatofocusing, and high-pressure liquid chromatography (HPLC). Polyol levels were deter- mined by gas-liquid chromatography (GLC). Immu- nological analyses include the use of enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), Western blots, and immunohistochemical techniques employing the coupled antibody DAB- PAP technique. Computational methods for enzyme analysis, inhibitor structure-activity studies, and pupil-function changes require the use of the NIH PROPHET computer system and Charm and Quanta computer systems from Molecular Design. Major Findings Biochemical studies. — Studies on defining the inhibi- tor site of aldose reductase and aldehyde reductase are continuing with the evaluation of a number of Michael addition affinity-labeled aldose reductase inhibitors (ARIs). Analogs of the ARIs AL1576 and alrestatin possessing sterically diverse substituents, Michael addition adducts, and haloalkyl groups have been synthesized and evaluated in vitro for their ability to inhibit and irreversibly bind rat lens aldose reductase and rat kidney aldehyde reductase. Inhibitory potency in general was reduced in a series of alrestatin analogs in which the carboxyl acid was condensed with sulfonamide groups; however, inhibition increased with 5-substitution with either nitro or Michael addition adducts. In the spirofluor- enehydantoin series, substitution in the 2 position with similar open- or constrained-ring Michael addition adducts did not result in increased inhibi- tion. The selective introduction of Michael addition adducts can result in increased inhibitory activity and irreversible binding of aldose reductase but not aldehyde reductase. Specific nucleophilic residues on the enzyme also are being identified by subjecting the alkylated enzyme to trypsin digestion and subse- quent chemical ionization mass spectroscopy. The pharmacophore requirements of the inhibitor site and the location of reactive nucleophilic and electrophilic sites are being refined through the use of molecular modeling. The theoretical tools utilized for this study include the AMI quantum chemical method and the fitting methods of Quanta 3.3 con- ducted on an SGI Crimson computer. These results were then correlated to observed inhibitions of rat lens aldose reductase. Geometry optimization and energetics calculations have been conducted for a series of carboxyl acid-containing analogs of 9- spirofluorenehydantoin. These calculations were conducted on the charged (-1) rather than neutral species because they are assumed to be charged at physiological pH. Super- position of these geometry-optimized compounds on AL1576 were conducted by utilizing both torsional flexible and rigid body fits, and the spatial regions at which reversible nucleophihc siibstimtion takes place were then compared. The spirohydantoin AL1576 was chosen as a template in this study because (1) it contains a rigid hydantoin ring having two probable pharmacophores in which the relative position in space is fixed and (2) it has been shown to have high affinity for aldose reductase. These studies indicate that a negative charge center resides in the vicinity of the 2-oxygen atom of the hydantoin ring while the 4- carbonyl represents a region where a nucleophilic substitution likely takes place. Understanding the pharmacophore requirements will lead to the rational development of new ARIs. New, potent ARIs have been uncovered, and patent application has been made. 189 Laborator) of Ocular Therapeutics NEI Annual Report— FY 1993 Recent studies have demonstrated that the dog is an excellent animal model for investigating ocular diabetic complications. Cataract development in the dog is similar to cataract development in humans with diabetes. The cataracts are characterized by formation of anterior and posterior superficial cortical opacities with the posterior polar region being more advanced. Furthermore, the dog develops advanced retinal changes similar to those clinically observed in human diabetics. Polyol formation initiated by the NADPH-dependent enzyme aldose reductase has been demonstrated to initiate these lens and retinal changes. Magnetization transfer contrast (MTC) is a method in magnetic resonance imaging (MRI) that generates high-contrast images based on characteris- tic tissue differences resulting from the interaction of water and macromolecules. We are applying the MTC to document cataract formation and other structural changes of the anterior segment associated with diabetes eye complications in galactose-fed dogs. Dogs, sedated with acepromazine (i.m. injec- tion), intubated, and then ventilated with 1.0-1.5% halothane are administered succinylcholine chloride to prevent eye movements and placed in a General Electric 2-T Omega MRI system. M^ and M„ images are acquired using gradient- recalled echo sequences, with and without the satura- tion pulses, respectively, consisting of rf-irradiation 10 kHz off-resonance from the free-water proton signal. The Ti^^, image data are obtained using one- short T,-imaging, employing an inversion pulse followed by a series of small tip-angle pulses that sample the relaxation curve. These images are then compared with photographs obtained with photo-slit- lamp and retroillumination photography. The results indicate that MTC not only generates excellent images of the lens but also aids in the visualization of fine structures in the anterior segment, including the cornea, iris, ciliary bodies, choroid membrane, and Schlemm's canal. "F-NMR spectroscopy also is being used to measure in vivo aldose reductase activity in the dog lens by measuring the conversion of 3-deoxy-3- fluoroglucose to 3-deoxy-3-fluorosorbitol. This work is an extension of the in vivo evaluation of aldose reductase activity in rabbit lenses. Initial spatial coordinates for lenses are calculated from 'H-images determined on a 2.0 Tesla GE Omega-CSI spectrom- eter. The SLOOP (spectral localization with optimal pointspread function) technique is then used with a proton decoupler to measure the accumulation of sorbitol in the rabbit lens. A double spin-echo sequence is utilized with selective excitation and refocusing pulses and with optimized phase-encoding gradient pulses using 1-sec repetition times and 25- msec echo times. SLOOP experiments indicate that 3-deoxy-3-fluorosorbitol can be observed in spectra of the anterior portion of the lens when adequate amounts of 3-deoxy-3-fluoroglucose are adminis- tered. Retinal studies. — Vascular changes associated with diabetic retinopathy can be produced experi- mentally in beagles fed a 30% galactose diet In studies designed to clarify the initiating lesions and progression of diabetic retinopathy, we have docu- mented the progression of retinal lesions from background through the proliferative stage in the dog with ophthalmoscopic, fluorescein angiographic, and histopathologic findings. Initial retinal changes include aldose reductase-linked formation of pericyte ghosts and subsequent development of acellular capillaries, microaneurysms, and intraretinal hemor- rhages. This early retinopathy progresses to include the appearance of occluded vessels, areas of nonper- fusion, and intraretinal microvascular abnormalities (IRMA). Finally proliferative retinopathy develops, including the formation of fibrovascular membranes seen histologically on both the retinal surface and the posterior hyaloid membrane. Pericyte ghost formation and the subsequent appearance of microaneurysms, intraretinal hemor- rhages, and acellular capillaries associated with background retinopathy have been arrested in a dose- dependent maimer in 36- to 38-month prevention studies utilizing 0.5, 5, 10, and 16 mg/kg/day of the ARI M79175. The dog represents the first animal model to demonstrate all the clinical and histological retinal vessel changes observed in human diabetics. Significance to Biomedical Research and the Program of the Institute Loss of vision from cataract and diabetic retinopathy are significant; therefore, methods for the pharmaco- logical control of these ocular complications are required. We have developed an animal model that demonstrates advanced retinal vessel changes that are virtually identical both clinically and histologically to those observed in advanced diabetic retinopathy. Our present studies in dogs demonstrate for the first 190 NEI Annual Report— FY 1993 Laboratory of Ocular Therapeutics time that loss of retinal pericytes, associated with aldose reductase, initiates retinal changes associated with both background and advanced diabetic retinop- athy and that adniinistration of ARIs in prevention studies can ameliorate the loss of pericytes and subsequent microaneurysms and retinal hemorrhages in a dose-dependent manner. The successful devel- opment of noninvasive methods for monitoring aldose reductase activity by nuclear magnetic reso- nance (NMR) procedures may have a direct impact on ongoing and plaimed clinical trials in which this procedure could serve as a quantitative indicator of drug efficacy. Cataract is one of the major causes of bhndness in the developing world. In addition, loss of vision due to cataract is one of the major health problems of both people with diabetes and the aging population in the United States. Proposed Course These studies will be continued. Currently discov- ered ARIs will be evaluated and developed pharma- cologically. The inhibitor site will be probed further through the use of affinity labels so that more potent and specific inhibitors may be developed. Studies will be continued on the mechanisms through which aldose reductase induces diabetic complications in various tissues. NEI Research Program Retina] Disease — Diabetic Retinopathy, Sickle Cell Retinopathy, and Other Vascular Abnormalities Publications Bartoszko-Malik A, Schaffhauser M, Ghany-Abdel Y, Miller DD, Kador PF: Evaluation of novel aldose reductase inhibitor site probes. Invest Ophthalmol Vis Set 34(suppl):280, 1993. Fukase S, Mori K, Sato S, Kador PF: Comparison of NADPH-dependent reductases in dog lens and leukocytes. Invest Ophthalmol Vis Sci 34(suppl): 757, 1993. Kador PF: Intermediary metabolism of the lens, in Raviola E, Dowling J (eds): Principles and Practice of Ophthalmology. New York, Wiley Press, in press. Kador PF, Takahashi Y, Schaffhauser M: Vorbeug- ung diabetischer Komplikationen im Auge mit Aldosereduktase-Hemmem. Diabetes und Stojf- wechsel, in press. Kador PF, Takahashi Y, Sato S, Wyman M: Retinal vessel changes in galactose-fed dogs treated with the aldose reductase inhibitors M79175 and FK366. Invest Ophthalmol Vis Sci 34(suppl):64, 1993. Kador PF, Takahashi Y, Wyman M, Ferris F III: Arch Ophthalmol 111:585, 1993. Lee YS, Peralstein R, Kador PF: Molecular model- ing of aldose reductase inhibitors. J Med Chem, in press. LI Q, Lopez JS, Caspi RR, Roberge FG, Nussenblatt RB, Kador PF, Chan C-C: Suppression of S- antigen induced experimental autoimmune uveoretinitis in Lewis rats by oral administration with COS- 13080, a thromboxane synthetase inhibitor. Exp Eye Res 57:601-608, 1993. Mori K, Ceckler TL, Kador PF, Balaban RS: Mag- netic resonance imaging of the galactosemic dog eye using magnetization transfer contrast. Invest Ophthalmol Vis Sci 34(suppl):1061, 1993. Ogawa K, Yamawaki I, Matsusita Y, Nomura N, Kador PF, Kinoshita JH: Synthesis of substituted 2,4-dioxo-thienopyriniidine-l -acetic acids and their evaluation as aldose reductase inhibitors. Eur J Med Chem, in press. Okamoto S, Terubayashi H, Tsutsumi M, Ikebe H, Nishimuna C, Kador P, Akagi Y: Localization of aldose reductase mRNA in the rat lens. Nippon Ganka Gakkai Zasshi 96:1373-1378, 1992. Reddy VN, Lin L-R, Giblin FJ, Lou M, Kador PF, Kinoshita JH: The efficacy of aldose reductase inhibitors on polyol accimiulation in human lens and retinal pigment epithelium in tissue culture. J Ocular Pharmacol 8:43-52, 1992. Smar MW, Ares J, Nakayama T, Itabe H, Kador PF, Miller DD: Selective irreversible inhibitors of aldose reductase. J Med Chem 35:1117-1120, 1992. Takahashi Y, Augustin W, Wyman M, Kador PF: Quantitation of retinal vessel changes associated with diabetic retinopathy in galactose-fed dogs. J Ocular Pharmacol, 9:257-269, 1993. Waldbillig RJ, Jones BE, Schoen TJ, Heidersbach S, Bitar MS, Van Kuijk FJGM, de Juan E, Kador PF, Chader GJ: Vitreal insulin-like growth factor binding proteins (IGFBPs) are increased in human and animal diabetics: Implications for under- 191 Laboratory of Ocular Therapeutics NEI Annual Report — ^FY 1993 standing diabetic retinopathy. J Cliri Invest, in press. Woel V, Kuszak JR. Takahashi Y, Wyman M, Kador PF: An ultrastructural analysis of posterior migrating lens epithelial cells in cataracts of galactose-fed dogs. Invest Ophthalmol Vis Sci 34(suppl):916, 1993. 192 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00275-02 LOT PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (BO characters or less. Title must tit or one line betweer) tt\e borders.) Role of NADPH-Dependent Reductases in Ocular Complications PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Sanai Sato M.D., Ph.D. Visiting Scientist LOT, NEI Others: Peter F. Kador Ph.D. Chief LOT, NEI Shigeru Fukase M.D. Visiting Associate LOT, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Ocular Therapeutics SECTION INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.35 PROFESSIONAL: 1.35 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects [x| (b) Human tissues □ (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The increased influx of glucose into the sorbitol pathway in diabetes results in the accumulation of sugar alcohol sorbitol, which is linked to the pathogenesis of various diabetic complications such as retinopathy, neuropathy, and nephropathy. The role of aldose reductase and related enzymes in polyol formation and the subsequent onset of these complicadons are being investigated. 193 PHS 6040 (Rev. 5/92) Laboratory' of Ocular Therapeutics NEI Annual Report — FY 1993 Project Description Objectives In diabetes, excess glucose results in an increased influx of glucose into the polyol pathway. The accumulated sugar alcohol, sorbitol, has been linked to the onset of various diabetic complications such as cataract formation, retinopathy, neuropathy, and nephropathy. The development of potent aldose reductase inhibitors (ARIs) represents a new pharma- ceutical approach to the treatment of diabetic compli- cations. Understanding NADPH-dependent enzymes in target tissues is a required first step in the design of ARIs. This project is designed to investigate aldose reductase and its related enzymes in various tissues where diabetic changes occur. Methods Biochemical techniques include gel filtration, affinity chromatography, electrophoresis, immunoblotting, and isoelectric focusing on high-pressure liquid chromatography. Gas chromatography is used to identify and quantitate sugars. In vitro culture techniques include the culture of retinal capillary pericytes and endothelial cells, leukocytes, and fibroblasts. The results, including enzyme kinetic evaluations, are calculated using the NIH PROPHET computer system. Major Findings Human kidney. — Like that of the rat and dog, human kidney cortex contains predominantly aldehyde reductase rather than aldose reductase, whereas the medulla contains aldose reductase. The kinetic properties of aldose and aldehyde reductases purified from human kidney are essentially identical to those of the rat and dog kidney enzymes. Moreover, as demonstrated with rat kidney enzymes, aldehyde reductase — in addition to aldose reductase — ^generates polyols from both glucose and galactose in an in vitro incubation system. As in rat kidney, aldehyde reductase contributes to polyol formation in human kidney cortex, where diabetic changes occur. The use of animal models is based on the premise that similar pathological changes occur in both experi- mental animals and humans. Evidence that both human kidney aldose and aldehyde reductases are similar to the rat and dog enzymes in kinetic proper- ties and inhibition by aldose reductase inhibitors gives enzymatic rationale for this approach. Rat lens. — Rat lens displays dehydrogenase activity with naphthalene dihydrodiol as substrate. This dehydrogenase activity corresponds to aldose reductase throughout all purification procedures, which include gel filtration, affinity chromatography, and chromatofocusing. The dehydrogenase activity is observed with the highly purified aldose reductase and also with recombinant enzymes from the rat lens aldose reductase clone. The evidence indicates that, in rat lens, dehydrogenase activity in the presence of naphthalene dihydrodiol comes from aldose reduc- tase. Significance to Biomedical Research and the Program of the Institute Despite the establishment of insulin therapy, the risk of loss of vision due to diabetic complications is still significantly high. Based on evidence that excess amounts of polyols are linked to the onset of diabetic complications, worldwide efforts have been made to develop ARIs. The fiill understanding of NADPH- dependent reductases and polyol formation wall provide essential information on their clinical poten- cy and contribute to further development of more potent inhibitors. Evidence that aldose reductase responds to naphthalene dihydrodiol with dehydroge- nase activity may expose new facets of the role(s) of aldose reductase in certain types of toxic cataract Proposed Course We will continue our evaluation of the location and enzymatic properties of aldose and aldehyde reduc- tases in various tissues in which diabetic complica- tions occur. To investigate retinal pericytes and endothelial cells as keys in diabetic retinopathy, we will utilize cell culture techniques. Leukocytes and various leukemia cells also will be investigated. NEI Research Program Diabetic Complications Cataract Research Publications Fukase S, Mori K, Sato S, Kador PF: Comparison of NADPH-dependent reductases in dog lens and 194 NEI Annual Report — ^FY 1993 Laboratory of Ocular Therapeutics leukocytes. Invest Ophthalmol Vis Sci 34(4) Sato S, Kador PF: Human kidney aldose and alde- (suppl):757, 1993. hyde reductases. J Diab Compl 7:179-187, 1993. Sato S: Aldose reductase is the major protein associ- Sato S, Lin L-R, Reddy V, Kador PF: Aldose ated with naphthalene dihydrodiol dehydrogenase reductase in human retinal pigment epithelium, activity in rat lens. Invest Ophthalmol Vis Sci Exp Eye Res 57:235-241, 1993. 34:3172-3178, 1993. 195 Laboratory of Retinal Cell and Molecular Biology Report of the Chief, Laboratory of Retinal Cell and Molecular Biology Gerald J. Chader, Ph.D. The research focus of members of the Laboratory of Retinal Cell and Molecular Biology (LRCMB) is on elucidating new genes and biochemi- cal mechanisms and learning the underlying causes of ocular diseases. In this way, we hope to intervene in the disease process before substantial damage to vision has been done or to ^ply rational methods of gene therapy before the terminal stages of the disease have been reached. Most of the approaches taken are molecular biological, molecular genetic, and/or candidate gene approaches. The work of the Laboratory members is within the following National Institutes of Health strategic initiatives and National Eye Institute priorities: (1) molecular medicine, (2) gene research and gene therapy, and/or (3) research of high clinical relevance. Within the LRCMB, the following three areas are emphasized: 1. Molecular biology and molecular genetics, 2. Gene therapy, and 3. Immunopathology. Molecular Biology and Molecular Genetics Specific advances made in the area of molecular biology and molecular genetics are discussed below. Retina-specific genes. — Several genes that are pre- dominantly or exclusively expressed in ocular tissues have been identified by subtractive cloning. Retina- specific genes and genes located on the short arm of the X-chromosome are pinpointed. These genes are being localized chromosomally to determine whether they are linked to eye diseases. Concurrently, genomic cloning and sequencing are being done to generate appropriate markers and polymorphisms. Cell lines are being immortahzed in tissue culture such that subsequent laboratory experiments can be conducted. Genes specific to retinal pigment epithelium (RPE). — ^Little is known about the specific comple- ment of genes in the RPE or how these could be involved in diseases of the visual system. Thus, cloning of genes unique to RPE and its functioning is of importance. A new 65-kD protein of potential immunologic importance has been isolated from the human RPE. The gene has been cloned, allowing for smdy of tissue-specific expression. This gene is the first RPE-specific gene to be reported and character- ized. Photoreceptor-specific genes. — ^An effort has been made to identify and characterize genes for proteins and enzymes that are critical in functioning of the photoreceptor outer segment. For example, two proteins of the phototransduction cascade, S-antigen (S-Ag) and phosducin, have been well characterized as to their expression control. Both proteins are specific to the rod neuron and interact with visual cycle components (e.g., opsin). cDNAs and genomics for S-Ag and phosducin have been cloned and thoroughly analyzed, allowing for current advan- ces in our understanding of expression, function, and pathology of the gene products. Interphotoreceptor retinoid-binding protein (IRBP). — ^IRBP also is a critical link in the chain of enzymes and proteins that make up the visual cycle. Because of the huge size of the gene, however, it is very difficult to sequence in potential disease cases. We are thus cloning the homolog of the human IRBP gene from Drosophila megalogaster (i.e., fhiitfly). Interestingly, it maps to an area of the Drosophila genome that is rich in mutants of ocular disease. Knowing the characteristics (e.g., erg) of the dif- ferent diseases in the fly, we hope to pinpoint a specific human population with similar characteristics and examine the gene for defects in specific human famihes. Pigment epithelium-derived factor (PEDF). — We have identified a novel neurotrophic protein, PEDF, synthesized by fetal human RPE cells that may be critical in the development of retinal photoreceptors. At very low concentration, PEDF causes the exten- 199 Laboratory of Retinal Cell and Molecular Biologj NEI Annual Report— FY 1993 sion of elaborate neuronal processes from cultured retinoblastoma cells. Since these cells are thought to be derived from photoreceptor cone cells, we hope that PEDF can be as effective on cone neuron development in vivo. An important possibility is that, in the Royal College of Surgeons rat, a defect in the PEDF gene could cause retinal degeneration. Also, we are continuing evaluation of the clinical use of PEDF in retinal transplantation in collaboration with Dr. M. del Cerro. The molecular biology of this potentially very important neurofrophic agent is now being studied for application to retinal dysfunc- tions. Fatty acid and tubulin defects in retinal degenera- tion. — In collaboration with Dr. Muriel Kaiser- Kupfer (Ophthalmic Genetics and Clinical Services Branch), we are investigating fatty acid uptake and metabolism in Bietti's crystalline retinopathy and a tubulin acetylation defect in a form of atypical retinitis pigmentosa (RP) for which we hope to elucidate the specific defects. Significant progress has been made in pinpointing the metabolic problems expressed in both these hereditary conditions. Gene Therapy of Retinal Diseases Points of focus and advances made witliin the area of gene ther^y are discussed below. Transgenic studies. — ^The IRBP and S-Ag genes are the best studied retinal genes other than rhodop- sin. Ci5-acting elements controlling the IRBP and S-Ag promoters, and thus their protein expression, have been identified in transfected human cells and in ttansgenic mice. Transgenic studies, in particular, have helped to uncover factors controlling gene activation in the embryonic period, specifically in the photoreceptor cell. Gene analysis systems in transgenic mice and in fransient fransfections in cultured himian retinoblasto- ma cells have been established for IRBP. Much of the 5'-flanking region of IRBP has been thoroughly examined to date. Similarly, a good deal of progress has been made with the S-Ag promoter. Enhancer elements necessary for expression are being defined through target mutagenesis studies. Tissue- and stage-specific elements, including TATA and CAAT boxes, are being defined as to retinal expression. This work is important in that specific molecules can be "gene-targeted" to the retina with precision. Gene therapy. — Ribozymes are specifically constructed RNA species that can control expression of proteins within cells. By linking these simplified gene forms to appropriate promoters and utilizing a suitable transfer vector, we can construct new thera- peutic modalities. Gene therapy can then be planned to freat autosomal dominant disorders that are cur- rently uimianageable. Ribozyme constructs for IRBP have been de- signed and are being studied in a ttansfected human retinoblastoma cell system. Concurrently, fransgenic mice have been reared to determine if an RP-like condition is produced through down-regulating IRBP synthesis. Once perfected, ribozymes should be useful, not only with IRBP-related retinopathies but in conditions such as diabetic retinopathy and reti- nopathy of prematurity, disorders which probably involve overexpression of normal proteins such as growth factors. Immunopathology Most of this work is in collaboration with inves- tigators in the Laboratory of Immunology. The focus is on the induction of experimental auto- immune uveitis. Immunopathology. — Collaborative work with Dr. Igal Gery continues on the study of uveitis. The immunopathological site(s) of IRBP are being dissected in the Lewis rat and in the human with the final goal of controlling or preventing the disease process in man. Immunogenetics. — Collaborative work with Dr. Rachel Caspi has established the IRBP-mouse model for experimental autoimmune uveoretinitis as very useful for studying the genetics of the disease and its relapsing characteristics. Antigen presentation. — Collaborative work with Drs. Gery, Marc de Smet, and Robert Nussenblatt has demonsfrated the presence of a 70-kD cell surface protein of B cells that specifically binds the major immunopathological determinant of IRBP. It is thought that this protein may function as a molec- ular chaperone in antigen presentation. It is a likely candidate for gene therapy in uveoretinitis. 200 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00070-16 LRCMB PERIOD COVERED October 1, 1992 to September 30. 1993 TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) Vitamin A and Ocular Tissues PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Barbara Wiggert Ph.D. Head, Section on LRCMB, NEI Biochemistry Others: Kalpana Rengarajan R. Krishnan Kutty Todd Duncan Geetha Kutty Ph.D. Ph.D. M.S. M.S. Visiting Fellow Senior Staff Fellow Biologist Visiting Associate LRCMB, NEI LRCMB, NEI LRCMB, NEI LRCMB, NEI COOPERATING UNITS frf any; ^ „ ,^ ^ U. Lund, Sweden (T. van Veen, Ph.D.); U. Illinois Coll. of Med., Chicago (D. Pepperberg, Ph.D., T.-I. Okajima, Ph.D., H. Ripps, Ph.D.); Med. U.S.C. (R. Crouch, Ph.D., S. Hazard, Ph.D.): SLU Inst F. Kir. Sweden (K. Narfstrom, D.V.M., Ph.D.); U. Hosp., Utrecht, The Netherlands (B. Zonnenberg, M.D., Ph.D.); U. Maryland Med. Sch. (M. Rodrigues, M.D., Ph.D.); Medical College of Georgia (S. Smith, Ph.D.); Emory Eye Center (J. Nickerson, Ph.D.) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology SECTION Section on Biochemistry INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 5.0 PROFESSIONAL: 3.0 OTHER: 2.0 CHECK APPROPRIATE BOX{ES) □ (a) Human subjects □ (a1) Minors n (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Studies on the physicochemical characteristics of a fatty acid-binding site on the interphotoreceptor retinoid- binding protein (IRBP) with fluorescent fatty acid analogs demonstrated that fatty acids were bound in a hydrophobic environment, that there was a single, specific fatty acid-binding site for each molecule of IRBP, and that there was nonradiative energy traasfer from tryptophan residues to bound ligand. Probing the microenvironment of bound fluorophore with a quencher indicated a liighly structured binding site. Studies of the formation and release of ll-m retinal by the retinal pigment epithelium at a physiological concentration of IRBP demonstrated that a sequential (i.e., unbranched) pathway mediates the processing of all-rran^ retinol to ll-cis retinal and its transfer to IRBP. In the mi'"'mi"' mutant mouse model of retinal degeneration, retinyl palmitate was elevated fourfold and IRBP was elevated twofold in the eyes of affected mice, as compared with that in controls at 6-8 weeks of postnatal development. At the same time, IRBP mRNA was not elevated. The elevation in retinyl palmitate may be a significant factor in the retinal degeneration in this mutant, and IRBP turnover may be affected by an aberration in retinoid metabolism. A 72-kDa heat shock protein (hsp) which bound specifically to peptide 1169-1191, a potent uveitogenic determinant of IRBP, has been identified in Lewis rat B cells and Epstein Barr virus-transformed B cells from normal human donors and uveitis patients. This hsp has a potential role in antigen processing and presentation by antigen-presenting cells. 201 PHS 6040 (Rev. 5/92) Laboratory' of Retinal Cell and Molecular Bioloe>' NEI Annual Report— FY 1993 Project Description Additional Personnel Igal Gery Ph.D. Rachel Caspi Ph.D. Tatiana Putilina Ph.D. Mark de Smet M.D. Head, Section on Experimental Immunology, LI, NEI Visiting Associate, LI, NEI Visiting Associate, LRCMB, NEI Visiting Scientist, LI, NEI Objectives The purpose of this research project is to investigate the role of specific retinoid-binding proteins, such as interphotoreceptor retinoid-binding protein (IRBP), in mediating the action of retinoids in both normal and diseased ocular tissues. Methods Affinity chromatography, fluorescence spectroscopy, high-performance liquid chromatography, SDS- polyacrylamide gel electrophoresis. Western blotting. Northern blotting, slot-blotting, and the enzyme- linked immunosorbent assay were used to study retinoid-binding proteins. Major Findings The physicochemical characteristics of a fatty acid- binding site on IRBP were examined using a set of fluorescent fatty acid analogs with an anthracene moiety attached at different positions along the hydrocarbon chain. The results demonstrated that fatty acids were bound in a hydrophobic environ- ment, as indicated by a blue shift in fluorescence maxima and by an increase in quantum yield of the bound ligand. A single, specific fatty acid-binding site existed for each molecule of IRBP with an apparent K^3.6xlO'^M. There was nonradiative energy transfer from tryptophan residues to bound ligand, as well as fluorescence energy transfer to all- trans retinol when this ligand was bound to IRBP. The interactions of IRBP and bound fatty acids were sensitive to denaturation by increasing concen- trations of urea as judged by changes in nonradiative energy transfer efficiency and the quantum yield of the bound probe. Quantum yields of bound fatty acid analogs varied with position of the fluorophore along the hydrocarbon chain and had the lowest values for the fluorophore located at the midpoint. Probing the microenvirorunent of bound fluorophore with a quencher indicated a highly structured binding site. In studies of the formation and release of 11 -cis retinal by the retinal pigment epithelium (RPE) in the toad eyecup preparation, the time course of the specific activity of radio-labeled l\-cis retinal at a fixed, near-physiological IRBP concentration demon- strated that a sequential (i.e., unbranched) pathway mediates the processing of zl\-trans retinol to ll-cis retinal and its transfer to IRBP. In the mutant mouse model of retinal degenera- tion, levels of retinyl palmitate in the eyes were elevated fourfold greater than controls by 8 weeks, and the levels remained elevated through 42 weeks. Levels of ll-cis retinal, dll-trans retinol, and all- trans retinal were similar to those of controls, as were plasma retinol levels. Levels of IRBP at 2-4 weeks were similar in affected and control animals, but by 6 weeks IRBP levels were twofold greater in the affected mice than in controls and remained high for several weeks until degeneration of photoreceptor cells took place. At the same time, IRBP mRNA was not increased in the affected mice, indicating that IRBP turnover probably decreased in the disease. The elevation of retinyl palmitate may be a signifi- cant factor in the retinal degeneration seen in the mouse mutant, and IRBP turnover may be affected by an aberration in retinoid metabolism. Peptide 1169-1191 is a potent uveitopathogenic determinant of IRBP. Recently a new class of proteins known as chaperones, which are part of the heat shock protein (hsp) family, have been implicated in antigen presentation and appear to prevent further degradation of antigen by lysosomes. A 72-kD protein that bound specifically to peptide 1169-1191 was found in Lewis rat B cells and EBV-transformed B cells from normal human donors and uveitis patients. This protein reacted with antibodies specific for both constituitively expressed and induc- ible 72/73-kD HSP 70 proteins and could have a potential role in antigen processing and presentation by antigen-presenting cells. Large-scale purification of IRBP was continued for studies on the production of experimental auto- immune uveitis (EAU) in rats and mice and possible modes of suppression of the disease. 202 NEI Annual Report— FY 1993 Laboratory of Retinal Cell and Molecular Biology Significance to Biomedical Research and the Program of the Institute Because of its importance in normal photoreceptor cell physiology (i.e., in facilitating the transport of retinoids during the visual cycle as well as the transport of fatty acids that are essential to normal function), abnormalities in IRBP function resulting from changes in concentration, distribution, or affinity for retinoids or fatty acids could be important either directly or indirectly in visual cell pathogen- esis. Proposed Course Studies on the fatty acid- and retinoid-binding sites on IRBP will be continued to elucidate the relation- ship between the two ligands and the possible effect of fatty acid binding on the binding of retinoids and the function of IRBP in the interphotoreceptor matrix. We also will continue to smdy the physio- logical role of ERBP in the visual cycle, particularly the effect of removing IRBP during retinal detach- ment on regeneration of visual pigment. Continued studies on the mi^'mi"" mouse model of retinal degeneration will include studies of retinoid metabo- lism in the RPE to determine the mechanism causing large elevations in retinyl palmitate in mutant mice. Further work also will be carried out to characterize the 72-kD hsp, which binds the uveitogenic peptide 1169-1191 of IRBP, and its possible role in human disease. We will continue to conduct large-scale purification of IRBP protein for studies of EAU. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications Hara Y, Caspi RR, Wiggert B, Chan CC, Streilin JW: Use of ACAID to suppress interphotore- ceptor retinoid binding protein-induced experi- mental autoinunune uveitis. Curr Eye Res 1 1:97- 100, 1992. Kutty RK, Kutty G, Duncan T, Nickerson J, Chader GJ, Wiggert B: Radioanalytic estimation of amplification products generated by RT-PCR using (alpha-"P) deoxynucleotide triphosphate. Biotechniques, 15:808, 811-812, 1993. Pepperberg DR, Okajima TL, Wiggert B, Ripps H, Crouch RK, Chader GJ: Interphotoreceptor retinoid-binding protein (IRBP), in Molecular Neurobiology. Clifton, NJ, Humana Press Inc, 1993, in press. Putilina T, Sittenfeld D, Chader GJ, Wiggert B: Smdy of a fatty acid binding site of interphotore- ceptor retinoid-binding protein using fluorescent fatty acids. Biochemistry 32:3797-3803, 1993. Rajagopalan S, Rodrigues MM, Wiggert B, Advani SH, Nair CN, Nickerson JM: Retinoblastoma: Interphotoreceptor retinoid-binding protein mRNA analysis by polymerase chain reaction. Ophthal- mic Paediatr Genet, in press. Sasamoto Y, Kawano YI, Bouligny R, Wiggert B, Chader GJ, Gery I: Immunomodulation of exper- imental autoimmune uveoretinitis by intravenous injection of uveitogenic peptides. Invest Ophthal- mol Vis Sci 33:2641-2649, 1992. 203 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00196-10 LRCMB PERIOD COVERED October 1. 1992 to September 30. 1993 TITLE OF PROJECT (SO crmraclars or lass Tilla must In on one line between the borders j Molecular Genetics of the Eve and Ocular Diseases PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, latmralory. and institute alliliation) Ph.D. Senior Staff Fellow LRCMB, NEI PI Others: Diane E. Borst Steven Bernstein Ph.D.. M.D. Senior Staff Fellow LRCMB, NEI COOPERATING UNITS (if any) Emor> Universit>. Atlanta, GA (J.M. Nickerson, Ph.D., J-S. Si, M.D.); University of Michigan, Ann Arbor (E. Farr, M.D.); University of Texas. Dallas (R. Hammer. Ph.D.) LAB/BRANCH Laboratory of Retinal Cell a nd Molecular Biology SECTION Section on Gene Regulation INSTITUTE AND LOCATION NEI. NIH. Bethesda. MP 20892 I TOTAL STAFF YEARS: PROFESSIONAL: 2.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects n (a1) Minors □ (a2) Interviews 2.0 OTHER: 0.0 [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.) Interphotoreceptor retinoid-binding protein (IRBP) is an abundant glycolipoprotein that is expressed in the reuna and pineal gland. IRBP mRNA is synthesized by the photoreceptor cells of the retina. We are charactenzing the cw-elements regulating IRBP expression, using a transient transfection assay and transgenic I^o^t; '^"^ ^^ ^° conserved areas of sequence in the 5'-flanking regions of the bovine, human, and mouse IRBP genes, one from -1 to -350 and another at -1200 to -1410. Tlie 5'-flanking region is necessary for expression of IRBP in transient transfection assays in Y79 retinoblastoma cell cultures. In transgenic mice the same region also shows promoter activity in the retina and pineal, demonstrating that tissue specificity is engendered within the tested 5'-flanking regions of the gene 204 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Retinal Cell and Molecular Biology Project Description Additional Personnel Eric Wawrousek Ph.D. Research Biologist, OSD, NEI Objectives This research is designed to (1) define the cw-acting elements and trans-acting factors that regulate inter- photoreceptor retinoid-binding protein (IRBP) gene expression in a tissue and/or developmentally specific manner, (2) down-regulate eye-specific genes by exogenously derived genetic elements introduced either as antisense/catalytic RNA or antisense DNA oligonucleotides, and (3) use antisense technology in determining the pathophysiological role of aldose reductase in diabetic pathology. In addition to the obvious gene therapeutic possibilities, these ap- proaches also can be used when total deletion of a target gene would be deleterious to the siu^'ival of the organism. Methods These studies use conventional techniques for clon- ing and analysis of nucleic acids. Transgenic mice have been made using standard techniques. Trans- genic rats containing an antisense construct for aldose reductase are being produced in collaboration with Dr. Robert Hammer. Chloramphenicol acetyl transferase (CAT) activity is measiu^ed by an en- zyme-linked immunosorbent assay (ELISA) or the biphase assay. Catalytic RNA/antisense constructs (ribozymes) have been used for permanent transfection of cell lines that actively transcribe the messenger for IRBP. In addition, the transgenic animals produced express high levels of ribozymes targeted against endogenous IRBP mRNA. IRBP mRNA levels are measured quantitatively by techniques based on polymerase chain reaction (PCR), and by Northern analysis. Major Findings Gene expression. — Constructions containing pre- sumptive elements of the IRBP promoter joined to an indicator gene (CAT) were made with both bovine and mouse IRBP promoters for study of the expres- sion of the IRBP gene. Two sites in the 5'-flanking (promoter) region show significant homologies across species, and we have made constructions containing (1) both conserved blocks, (2) only one of the two blocks, or (3) neither blocks. These constructions were tested in several systems, including retinoblas- toma cells (Y79 and WERI), frog oocytes, mixed pinealocyte primary cultures, transformed pinealo- cytes, and normal mouse fibroblasts. There is promoter activity in Y79 cells transfected with the IRBP promoter-CAT constructs containing both coaserved blocks of sequence. This is the first report of transfection of any retinoblastoma cell line yielding successful transient expression. In each block there is gel-shift experi- mental evidence for the binding of rron^-acting factors confirmed in the proximal upstream area by DNAse footprinting experiments (collaboration with Drs. John Nickerson and Jing-Sheng Si). Southwest- em blot analysis reveals a 120,(KK)-MW protein that binds to the -300 region of the promoter. This binding activity is not unique to the retina, being present also in the heart, kidney, and lung; however, it is not ubiquitous. DNA methylation. — DNA methylation is known to play a role in the regulation of gene expression. Experiments were done to determine the methylation state of the IRBP promoter in various tissues. DNA isolated from different tissues was digested with either Msp I or Hpa II and size-fractionated on agarose gels. Msp I and Hpa II are isoschizomers, but Msp I will digest the sequence when the 3' cytosine is methylated, whereas Hpa II will not. Southern blots of mouse tail, liver, and retina DNA were probed with a labeled 1.6-kb piece of the mouse IRBP promoter. The autoradiographs show that the IRBP promoter is hypomethylated in the retina but not in the liver or the tail. This indicates that DNA methylation may somehow be involved in the tissue-specific regulation of IRBP gene expres- sion. Downregulation of gene expression. — The effect of site specificity and varying complement length on ribozyme activity in vitro has been studied using in vitro partial duplex transcription, cloned ribozyme templates, and substrate fragments. Ribozyme activ- ity can be "tuned" in vitro by varying complement length. This tuning is unique and target site specific. We have developed ribozymes, targeted against different sites in the IRBP mRNA, which have high in vitro activity. These ribosymes have been used to generate transgenic mice that express these ribo- zymes in ocular and other tissues. Preliminary data 205 Laboraton ot Retinal Cell and Molecular Biology NEI Annual Report— FY 1993 show that there are apparently significant differences in the embryonic survival and tissue-specific expres- sion of transgene and IRBP mRNA in these different constructs. We are also using mouse retinoblastoma cell lines derived from mice transfected with SV-40 large T antigen. We have characterized these lines in terms of photoreceptor-specific gene expression; they also express high levels of IRBP mRNA. Sequence analysis of the mouse IRBP genome. — Sequencing the genomic clones encoding the mouse IRBP gene has shown that the mouse IRBP gene is similar in the coding regions to both human and bovine genes. They differ, however, in that the mouse fourth exon contains a 3'-untranslated region that is intermediate in length (1.0 kb) between bovine (2.4 kb) and human (0.7 kb) orthologs. We are examining the sequence to determine alternative splice sites that may explain the imique appearance of two IRBP mRNA-size classes, as well as the difference between the forms of uveitis in rat and mouse species. Significance to Biomedical Research and the Program of the Institute Elucidation of the gene sequences of IRBP is funda- mental to understanding normal retina development and function. Fmdings from the transgenic mice carrying the IRBP ribozyme construct will yield much useful information on the role of IRBP in development, as well as the function of the retina during relative IRBP deficiency. Proposed Course We have finished the major structural studies on the IRBP gene. With this foundation of information and battery of cloned genes, we have begun to study the regulation of IRBP gene expression. Related ques- tions about the consequences of abnormal or absent IRBP function can be investigated in transgenic mice and in vitro systems. Gene expression.— A deletion series of IRBP- promoter plasmids has been made, and preliminary experiments indicate that both the distal and proxi- mal conserved sequences are important for expres- sion of the IRBP gene in Y79 cells. However, fewer than 205 bases of the 5'-flanking region are needed for basal promoter activity in the Y79 cells. Some of these constructions have been injected into fertil- ized mouse eggs, and offspring are being examined for the expression of the constructions. We will compare the expression of these constructions in transgenic animals and Y79 cells under different culture conditions. Preliminary studies show that the transgene is active in development as early as embry- onic Day 9, but high levels of expression coincide with the beginning of outer segment elongation. Steady state adult levels are not reached until about postnatal Day 30. In future studies, we will examine in other trans- genic mouse lines gene expression in both the retina and several other tissues during development. We will characterize the cw-acting DNA sequences that bind proteins in the promoter region by making alterations to these sequences. We plan to isolate the proteins that bind to these elements by screening retina cDNA expression libraries by the established Southwestern blotting procedure. Preliminary screenings have yielded two potential clones. Downregulation of gene expression. — Mouse lines containing the IRBP ribozyme constructs are being analyzed concurrently with ocular histology and electrophysiological studies to assess the role of IRBP deficiency in ocular pathology. Transgenic rats expressing antisense for aldose reductase are currently being analyzed. A downregulation of aldose reductase in these animals should yield delayed galactose-induced cataractogenesis and resistance to diabetes-induced histopathology, con- firming the importance of AR in these pathologic states. NEI Research Program Retinal Diseases — Photoreceptors and Retinal Pig- ment Epithelium Publications Humayun M, Bernstein SL, Gould HB, Chavis RM: Orbital childhood acute lymphoblastic leukemia as the initial presentation. J Pediatr Ophthalmol Strabismus 29:252-255, 1992. 206 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00124-13 LRCMB PERIOD COVERED October 1. 1992 to September 30. 1993 TITLE OF PROJECT (BO characters or less. Title must fit art arte Ime between the borders.) Metabolism of the Retina and Pigment Epithelium PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Gerald J. Chader Ph.D. Chief LRCMB, NEI Others: Robert Waldbillig Bruce Pfeffer Joyce Tombran-Tink Stephen Gaudet S. Patricia Becerra Timothy Schoen Ph.D. Expert LRCMB, NEI Ph.D. Senior Staff Fellow LRCMB, NEI Ph.D. Staff Fellow LRCMB, NEI Ph.D. Staff Fellow LRCMB, NEI Ph.D. Visiting Scientist LRCMB, NEI B.S. Biologist LRCMB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology SECTION Section on Gene Regulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 6.5 PROFESSIONAL: OTHER: 5.5 1.0 CHECK APPROPRIATE BOX(ES) r~| (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Studies are focused on an understanding of the molecular biology and molecular genetics of the retina and hereditary retinal degenerations. The retina and pigment epithelium are neuroepithelial tissues that work in close cooperation. Specific growth and differentiating factors found in the eye guide development and interactions of individual ocular tissues to form a functional visual system. For example, ocular tissues synthesize a number of growth factors. There now appear to be several systems that could self-regulate growth and metabolic activity in the retinal pigment epithelium and that could be involved in eye diseases. In this regard, we have cloned and characterized a unique differentiating protein secreted from fetal human pigment epithelial cells, called pigment-epithelium-derived factor, that is neurotrophic to cultured human retinoblastoma cells and may affect neural retinal development in vivo. This protein maps to chromosome 17p, where there is a cluster of cancer-related genes. It is a prime candidate in the hereditary retinal dystrophy observed in the Royal College of Surgeons rat 207 PHS 6040 (Rev. 5/92) Laborator> of Retinal Cell and Molecular Biolo}!:^ NEI Annual Report — FY 1993 Project Description Objectives Our objective is to obtain a better understanding of the molecular biology and molecular genetics of ocuJar tissues in health and disease. Study of growth and differentiation factors, be they protein (e.g., pigment epithelium-derived factor fPEDn) or poly- p)eptide (e.g., insulin-like growth factor [IGF]-!), is critical in obtaining a view of the events that control the early development of the eye and in maintaining normal function in the adult. Methods Molecular biological, genetic, and immunocytochem- ical techniques are used. Tissue culture is used to grow cells. In particular, the human retinoblastoma cell line, Y79, is used as a test system for differenti- ating agents. Major Findings Hereditary diseases often occur in the presence of genes important in cell division and differentiation. PEDF seems to be such a gene product. It is se- creted by cultured fetal human pigment epitiielial cells and appears to be present in the normal adult interphotoreceptor matrix. The protein migrates at approximately 54 kD on SDS-polyacrylamide gels. PEDF causes marked differentiation of human Y79 retinoblastoma cells in cultiire. This differentiation is charaaerized by an extensive elongation of neu- rite-like processes and a gatiiering of cells into "rosette-like" aggregates. Immunocytochemisti7 shows that the expression of specific neuronal markers also is enhanced. Thus, PEDF is a unique protein, synthesized and secreted by retinal pigment epithelial cells, tiiat could direct early development, even early in embryogenesis. It may be that PEDF also is present after the important developmental period and may help to maintain retinal cell viability in the adult retina. We have cloned tiie cDNA for tiie PEDF gene, and have determined tiiat the protein is a member of tiie SERPIN (serine protease inhibitor) superfamily of genes. Some members of tiiis family are known to promote cellular differentiation, making it more probable that PEDF has a major, similar role in tiie retina. Using fluorescent in situ hybridization, polymerase chain reaction, and Soutiiern blottin", we have localized the PEDF gene to the short arm of human chromosome 17. Through analysis of somatic cell hybrids containing only specific regions of 17p and 17q, we have further pinpointed PEDF to 17pl3- .1. It is important that PEDF colocalizes to the chromosomal area that contains the Li-Fraumeni cancer gene and a number of yet unknown cancer genes. Thus, PEDF may be part of an important cluster of genes involved in cellular proliferation and cancer as well as a prime candidate gene in the retinal dystrophy in the Royal College of Surgeons rat. The recombinant protein, which has now been expressed in Escherichia coli cells, has been shown to be an active neurotrophic agent. The availability of relatively large amounts of recombinant PEDF should allow for more direct studies on its r61e(s) in ocular development and disease. In parallel work, we have evidence implicating IGF-1 in visual development. In the eye, IGF-1 seems to participate in the attainment of overall size of the eye and in the function of individual ocular tissues and cell types. Specific IGF-binding proteins (IGFBPs) are known to control the bioavailability of the IGFs and thus are important regulators of IGF activity in health and disease. The vitreous and several ocular tissues contain very high levels of IGFBPs that are not derived from extraocular sources. The ciliary body is the probable source of synthesis of at least one of the vitreal binding pro- teins (BPs), specifically IGFBP2. We believe that the ciliary body probably secretes the BP into the vitreous, where it could be a major factor in regulat- ing developmental programs in the eye. Interesting- ly, the cornea exhibits exceptionally high amounts of IGFBP activity. Its role in corneal metabolism is yet unknown but, because of their growth-regulating potential, BPs could be involved in important pro- cesses such as wound healing and corneal complica- tions of diabetes. Significance to Biomedical Research and the Program of the Institute Determining the genes tiiat conti-ol normal ocular growth, differentiation, and function and studying tiiem on molecular biological and molecular genetic levels will aid us in understanding eye diseases, especially tiiose of a hereditary, early developmental nature. With such knowledge, we can apply rational metiiods of gene tiierapy to ocular diseases. 208 IVEI Annual Report— FY 1993 Laboratory of Retinal Cell and Molecular Biology Proposed Course The molecular biology and molecular genetics of ocular development will be further examined. We will investigate the factors that affect normal and abnormal growth. Examination and analysis of the full PEDF gene will help to elucidate its presumptive role(s) in retinal development The recombinant PEDF protein will be used to elucidate the role of the novel new protein in retinal disease processes. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications Becerra SP, Palmer I, Kumar A, Steele F, Shiloach J, Notario V, Chader GJ: Overexpression of fetal human pigment epithelium-derived factor in Escherichia coli: A functionally active neuro- trophic factor. J Biol Chem, 268:23148-23156, 1993. Boje KM, Skolnick P, Raber J, Fletcher RT, Chader GJ: Strychrine-insensitive glycine receptors in embryonic chick retin: characteristics and modulation of NMDA neurotoxicity. Neurochem Int 20:473-486, 1992. Gaudet SJ, Chader GJ: Partial purification and characterization of arylamine-N-acetyltransferase in bovine retina. Curr Eye Res 11:1185-1192, 1992. Gaudet SJ, Hayden BJ, Chader GJ, Namboodiri MA: Differentia] regulation of arylamine and arylalkyl- amine N-acetyltransferases in human retinoblas- toma (Y-79) cells. Neurochem Int 22:271-275, 1993. Schoen TJ, Beebe DC, Clemmons DR, Chader GJ, Waldbillig RJ: Local synthesis and develop- mental regulation of avian vitreal insulin-like growth factor-binding proteins: A model for independent regulation in extravascular and vascular compartments. Endocrinology 131:2846- 2854, 1992. Steele FR, Chader GJ, Johnson LV, Tombran-Tink J: Pigment epithelium-derived factor (PEDF): Neurotrophic activity and identification as a unique member of the serine protease inhibitor (SERPIN) gene family. Proc Natl Acad Sci USA 90:1526-1530, 1993. Tombran-Tink J, Li A, Johnson MA, Johnson LV, Chader GJ: Neurotrophic activity of interphotore- ceptor matrix on human Y79 retinoblastoma cells. J Comp Neurol 317:175-186, 1992. 209 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1. 1992 to September 30. 1993 PROJECT NUMBER ZOl EY 00148-20 LRCMB TITLE OF PROJECT (80 characters or lass Title must In on one line between the boraers.) Visual Control Mechanisms PRINCIPAL INVESTIGATOR (Lisl oiner protessional personnel below me Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Gerald J. Chader Ph.D. Chief LRCMB, NEI Others: Paul Wong Tatiana Putilina Ignacio Rodriguez Jun Li Susan Gentleman R. Theodore Fletcher Ph.D. Ph.D. Ph.D. M.D. Ph.D. M.S. Visiting Fellow Visiting Associate Staff Fellow Visiting Associate Biologist Chemist LRCMB, NEI LRCMB, NEI LRCMB, NEI LRCMB, NEI LRCMB, NEI LRCMB, NEI COOPERATING UNITS (il any) School of Vetennao' Medjcme, University of Pennsylvania (G. Aguuie. D.V.M., Ph.D.); Department of Anaiomy. Erasmus Univeisity, Roaerdani, The Netherkmds (S. Sanyal. Ph.D.); Department of Zoology. University of Lund, Lund, Sweden (T- van Veen, Ph.D.); Instituto Nazionale per la Ricera sul Cancro, Genova, Italy (A. Albmi. Ph.D., D. Noonan, Ph.D.) LAB/BRANCH ' Laboratory of Retinal Cell and Molecular Biolopy SECTION '^^- Section on Gene Regulation INSTITUTE AND LOCATION NEI. NIH, Bethesda, MP 20892 I TOTAL STAFF YEARS: 6.5 CHECK APPROPRIATE BOX(ES) n (a) Human subjects n (al) Minors □ (a2) Interviews PROFESSIONAL: 5.5 OTHER: 1.0 [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) ^ ' hJTw'th.T.If"'"^^- °^.'^' '''^°' '^'P'"'^ °" knowledge of normal complements of tissue-specific genes and how they change m disease, we are studying the expression of specific gene products rdated to several redS^f^o '?'• ''.r'^f "^'^'^^'^ '^'' '^"'^^ diseases of the retina'such^ etTbl^ma^r retmitis pigmentosa will result We have developed new techniques to clone and sequence retinH^dfic S : S' ^^^7, ^' ^" '"^ 'r' "^^ '^'''^' "^ ™P^^^ '^^^''^^ matrix p"et"^5 S^fi'c^lv f V S H r"'"''^!' ''"^ retinoblastoma cell growth and promote differentiation. 210 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Retinal Cell and Molecular Biology Project Description Objectives Normal expression of genes in the retinal photorecep- tor neuron is crucial to visual function in the adult. Thus, the factors that code for normal gene control and expression in himian retina and in animal models of retinal degeneration are of primary interest. We also have mounted a major effort to develop new molecular biological techniques such that unique retinal and retinal pigment epithelial genes can be identified, cloned, and sequenced for ultimate use in screening human populations with inherited diseases of the visual system. Methods Standard molecular biological, biochemical, and neurochemical techniques are employed. Histochem- ical techniques are used when necessary. Major Findings 1. Laminin is a ubiquitous extracellular matrix protein that has profound effects on a variety of cell types. For example, both gene and protein expres- sion in culnired human Y79 retinoblastoma cells are switched from a photoreceptor to a conventional neuronal pathway by addition of this basement membrane glycoprotein in culture. Unlike other cell systems where laminin influences differentiation, Y79 cells cannot attach to or chemotactically respond to laminin. Cyclic AMP (cAMP) is also an intracel- lular messenger that can influence differentiation in several cell types. Using cultured human retinoblas- toma cells as a model system, we have found both laminin and cAMP to have major positive influences on photoreceptor differentiation. 2. We are interested in developing new molecular biological techniques that will allow for more effi- cient identification of highly expressed genes of the retina-pigment epithelium complex. Each tissue of the body expresses a unique complement of genes that are transcribed and translated at a high level. In the retina and pigment epithelium, several very specific proteins are highly expressed, such that photoreception and the visual process can take place. Similarly, it is often a genetic defect in these tissue- specific genes that results in a hereditary degenera- tion such as retinitis pigmentosa We have devel- oped and are using new methods for rapid polymer- ase chain reaction-based construction of specifically enriched libraries from very small retinal samples. This is especially important because tissue samples are limited for studying early development and rare pathology samples. An important methodological advance involves subtractive cloning on an immobi- lizing base. We are now applying these techniques to the study of apoptosis (i.e., programmed cell death) in the retina and to the elucidation of fatty acid-binding proteins in normal and degenerating retinas. In apoptosis, in particular, it now appears that programmed cell death may be a common mechanism by which many hereditary defects initiate photoreceptor cell death. Significance of Biomedical Research and the Program of the Institute To control a hereditary disease process in a tissue and to reverse it through gene therapy, one must identify the normal complement of unique genes expressed in that tissue. This is especially true in an early degenerative process (e.g., retinitis pigmentosa) and in other hereditary diseases, such as retinoblas- toma, in which abnormal changes are subtie and can be masked by normal developmental switches in gene expression. Thus, studying apoptosis and similar processes in the retina will lead to better methods for gene ther^y in the neural retina Proposed Course Molecular biological and developmental control mechanisms in the retina and pigment epithelium will continue. In particular, we will investigate gene expression in normal retinas and in retinas affected with specific genetic diseases. Apoptosis will continue to be a focus, since futiu-e gene therapy in retinal degenerations may depend on understanding how to prevent death of the photoreceptor neuron. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications Albini A, Melchiori A, Garofalo A, Noonan DM, Basolo F, Taraboletti G, Chader GJ, Gavazzi R: Matrigel promotes retinoblastoma cell growth in vitro and in vivo. Int J Cancer 52:234-240, 1992. 211 Laboraton of Retinal Cell and Molecular Biology NEI Annual Report— FY 1993 Hooks JJ, Robbins S, Wiggen B, Chader G, Detrick B: Can viruses trigger retinal degenerative processes? in Hollyfield JG, Anderson RE, LaVail MM (eds): Progress in Clinical Biological Research, Degenerative Retinal Disorders: Clini- cal and Laboratory' Investigations, in press. Kutty G, Duncan T, Nickerson J, Si JS, van Veen T, Chader GJ, Wiggen B: Light deprivation pro- foundly affects gene expression of interphotore- ceptor retinoid-binding protein in the developing mouse eye. Exp Eye Res, in press. Pepperberg DR, Okajima TI, Wiggert B, Ripps H, Crouch RK, Chader GJ: Interphotoreceptor reti- noid-binding protein (IRBP) — oiolecular-biology and physiological role in the visual cycle of rhodopsin. Mol Neurobiol 7:61-85, 1993. Putilina T, Smith S, Gentleman S, Chader GJ: Rapid PCR-based construction of specifically enriched libraries firom small retina samples. Exp Eye Res 54:825-826, 1992. Wong P, Putilina T, Chader GJ, Tenniswood M: The human gene encoding TRPM-2 exists as a single gene locus on the short arm of chromo- some 8. Am J Human Genet, in press. 212 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00260-04 LRCMB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. We must fit on one line between the borders.) Molecular Biology of Outer Retina-Specific Proteins PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, lalx>ratory, and institute affiliation) PI: T. Michael Redmond Ph.D. Research Biologist LRCMB, hfEI Others: Suyan Liu M.D., Ph.D. Visiting Fellow LRCMB, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology SECTION Section on Gene Regulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 2.0 PROFESSIONAL: 2.0 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Retinal pigment epithelium (RPE) cells and photoreceptor cells are functionally and developmentally closely integrated. Derangements of the RPE are involved in certain retinal diseases. However, the RPE is poorly understood at the molecular level. We are characterizing RPE65, a developmentally regulated, conserved 65- kD RPE-specific microsomal membrane-associated protein. We have cloned the cDNA for RPE65 and found that it encodes a novel protein. This protein does not have any predicted transmembrane segments, yet it has a strong affinity for phospholipids, which may be related to its function. The cDNA sequence is being used to overexpress RPE65 protein for functional studies. The potential role of the protein in inducing uveitis also will be studied using recombinant protein. The lack of translation of RPE65 mRNA in cultured RPE cells is being investigated as a possible mechanism of |X)Sttranscriptional regulation that may have a bearing on the RPE-retina relationship as well as on RPE transplantation studies. We have isolated a full-lengtJi genomic clone for RPE65. It is at least 22 kb in length. We have used the cDNA and genomic sequences to localize the human gene for RPE65 to chromosome lp31 and the mouse homolog to distal chromosome 3. These do not correspond to any ocular disease gene localized so far. Nonetheless, RPE65 remains a candidate gene for RPE-invoIved disease. 213 PHS 6040 (Rev. 5/92) Laboratory of Retinal Cell and Molecular Biology NEI Annual Report— FY 1993 Project Description Objectives The retinal pigment epithelium (RPE) and the photo- receptor cell layer of the neural retina form a func- tionally and developmentally interdependent com- plex. Dysfunction of the RPE, accordingly, is deleterious to the photoreceptors and, hence, to vision itself. Despite these important considerations, little is known about the RPE at the molecular level. In this laboratory, we are cloning proteins specific- ally or preferentially expressed in the RPE with the aim of understanding mechanisms important to the RPE. Our major emphasis is on a 65-kD protein that we have named RPE65. We also are studying other RPE-expressed proteins. Methods Molecular cloning and biochemical and protein chemistry techniques are employed in this study. In addition, we are performing automated fluorescent DNA sequencing and gene mapping. Major Findings 1. RPE65 is a developmentally regulated, mem- brane-associated, nonglycosylated 65-kD protein restricted to and conserved in vertebrate RPE. It is the major protein of the RPE microsomal fraction. This protein displays a calcium-independent affinity for phospholipids. 2. We have cloned a composite 3,1 15-bp cDNA for this protein and have shown it to encode a novel protein of 533 aa that matches exactly the authentic protein sequences from peptide fragments of RPE65. Recombinant protein expressed in Escherichia coli has the same molecular weight as native RPE65 and is recognized by the RPE9 monoclonal antibody. 3. mRNA for the protein, which is restricted to RPE, is abundant in primary cultures of RPE. However, these cultures do not express the protein, suggesting that the message is posttranscriptionally regulated in vitro. 4. We have isolated a human genomic clone for RPE65. It is at least 22 kb in length. We are now mapping and sequencing it 5. We have localized the gene for the RPE65 to human chromosome lp31 and to the far distal end of mouse chromosome 3. Neither of these loci matches tliat of a known ocular disease or phenotype. Significance to Biomedical Research and the Program of the Institute TTie RPE is poorly characterized at the molecular level, despite its pivotal role in the maintenance of photoreceptor function and, hence, in vision itself. We have identified RPE65 as a conserved, RPE- specific molecule that is developmentally expressed. cDNA sequencing demonstrates that it is a novel protein. The function of this protein, while not yet clear, may be related to its affinity for phospholipids. Elucidation of the basis for its posttranscriptional regulation in vitro may have significant bearing on the culture of RPE cells. This has some clinical significance because RPE cell transplantation is receiving much attention as a possible mode of intervention in treating some retinal diseases. In addition, because of its RPE specificity, the RPE65 gene can be considered a potential candidate gene for retinal disease. At present, however, neither its human nor its mouse chromosomal locations match those of any mapped disease loci. As more disease loci are matched, this may change. Again, in view of its RPE-specific expression, elucidation of its gene structure may uncover RPE-specific regulatory elements. Finally, in view of the involvement of the RPE in uveitis, it is possible that RPE65 is uveito- genic. Now that we have cloned the cDNA, it will be possible to overexpress the protein to test this hypothesis. Proposed Course 1 . The basis for the posttranscriptional regulation of RPE65 will be investigated. 2. The structure of the human RPE65 gene will be studied. The gene will be sequenced, and its regulatory regions will be analyzed. The mouse gene RPE65 will be compared with that of the human. 3. RPE65 will be tested as a possible RPE auto- antigen. RPE65 protein will be overexpressed for this purpose. 4. Elucidation of the structure and function of RPE65 will continue. This will involve use of a variety of approaches. 5. Other RPE proteins will be cloned. 214 NEI Annual Report — ^FY 1993 Laboratory of Retinal Cell and Molecular Biology NEI Research Program Retina] Diseases — Photoreceptors and Retinal Pig- ment Epitlielium Publications Hamel CP, Tsilou E, Harris E, Pfeffer BA, Hooks JJ, Detrick B, Redmond TM: A developmentally regulated microsomal protein specific for the pigment epithelium of the vertebrate retina. J Neurosci Res 34:414-425, 1993. Hamel CP, Tsilou E, Pfeffer BA, Hooks JJ, Detrick B, Redmond TM: Molecular cloning and expres- sion of RPE65, a novel retinal pigment epitheli- um-specific microsomal protein that is post- transcriptionally regulated in vitro. J Biol Chem 268:15751-15757, 1993. Redmond TM, Tsilou E, Pfeffer BA, Detrick B, Hooks JJ, Hamel CP: Cloning and expression of a novel retinal pigment epithelium-specific 65 kDa microsomal protein. Invest Ophthalmol Vis Sci 34(suppl):982, 1993. 215 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1. 1992 to September 30. 1993 PROJECT NUMBER ZOl EY 00132-12 LRCMB TITLE OF PROJECT (80 characlers or less Title must lit on one line between the borders.) Molecular Biology of Phototransduction PRINCIPAL INVESTIGATOR (List otner prolessionai personnel below tne Principal Investigator.) (Name, title, laboratory, and institute atlilialion) PI: Toshimichi Shinohara Ph.D. Head, Section on LRCMB, NEI Molecular Biology Others: Takanobu Kikuchi Ph.D. Visiting Associate LRCMB, NEI COOPERATING UNITS (il any) ~~ ' ' Mount Sinai Hospital, Toronto, Canada (Martin Breitman, Ph.D.); Department of Anatomy, Nagoya University School of Medicine, Tsurumai, Showa-Ku, Nagoya, Japan (J. Usukura, M.D.) LAB/BRANCH Laboratory of Retinal Cel l and Molecular Biology SECTION Section on Molecular Biology INSTITUTE AND LOCATION NEI, NTH. Bethesda, MD 20892 TOTAL STAFF YEARS: 1.5 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects n (a1) Minors □ (a2) Interviews PROFESSIONAL: 1.5 OTHER: 0.0 n (b) Human tissues jx] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) ' ' ~ We have characterized the S-antigen genes from human and mouse and the 33-K protein genes from mouse and human. ITie S-antigen genes were approximately 50 kbp in length, contained 16 exons and 15 introns and comprised 97% mtron and 3% exon. The 5'-flanking regions of the genes, approximately 1.5 kbp bn^ had no known regulatory elements for transcription, such as TATA, GC, or CCAAT boxes. Regulatory sequences and nuclear factors governing ti.ssue-restricted expression of the mouse airestin gene direct low evels of retina-specific gene expression, sequences extending upstream to position -209 suorxm Sn the f ^Tr^^'" " "^^ "'"^' " "^" " '^^^^^^'^'^ expression in'the lens, pS^eTZl^TS h^ ?mer TGACCT thTr^'f''" '''l^' -'''' ' "^^°" "^'^^ ^'^^^ ^' ^-'^ repeats of Te a^d Bd3 thm^oh n r P™"""'"' ^'°'^' ^'' "PP^^^^'y ^^tina-specific nuclear factors-Bpl, Bp2, auid Bp3-through overiapping sequences centered between positions -25 and -15 Bpl and Bd3 also P^cffrjnef m' "''^'' T"" '°""' " "^^ ^"™°^" ^^^^°°^ °f --^^ «*- vertebrrt pltoretptor- specific genes. Moreover, the consensus binding site for Bpl, designated PCEl is identical to RCSl an element known to play a criUcal role in eliciting photoreceptor-s^ific gene express^nln dS.^ 2'J^ogaster. The results suggest that PCEl and RCSl are function^ly, as well as s^^crally S2 that, despite marked differences in the fly and venebrate visual system, th transcriplnTiSL^Tnv^I^d in photoreceptor-specific gene expression has been strongly conserved evolutioT^^y ™"'^"''^ '"'"'^^^ 216 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Retinal Cell and Molecular Biology Project Description Objectives The objectives of this project are (1) to understand the basic mechanism of phototransduction in the retina and (2) to understand the structure, function, and evolution of the proteins present in photoreceptor rod cells and pinealocytes. Methods Conventional methods for analysis of proteins and nucleic acids being used include protein purification and RNA and DNA isolation, characterization, and sequence determination. Various recombinant DNA techniques also being used include a Baculovirus expression vector system, synthesis of point mutation clones, characterization of promoters, and transgenic animals. We also have synthesized and used purified oligopeptides and oligonucleotides. Major Findings 1 . The gene sequences of S-antigen (S- Ag) from human and mouse were determined. It is 50 kbp in length and has 15 introns and 16 exons. The small- est exon encodes for three amino acids. 2. The intron-exon map sequence of the moase S-Ag gene has been well conserved. Approximately 97% of the S-Ag gene is intron and 3% is exon. 3. The human and mouse S-Ag cDNAs have been subcloned into two expression vectors and have been expressed. The products of S-Ag cDNA were purified by column chromatography and prepared for crystallization. 4. The 5'-flanking sequence of the human and mouse S-Ag genes were determined. Promoter activity was demonstrated in the in vivo and in vitro transcriptional assays. 5. Although the S-Ag promoter sequences are highly conserved between human and mouse, pro- moter activity was found at different locations of the 5'-flanking region in the human and mouse genes. This result suggests that the promoter activity is highly specific to tissues and species. 6. The mouse S-Ag promoter, 1,300 bp in length, was fused with the chloroamphenicol acetyl- transferase (CAT) gene, and that gene was intro- duced into transgenic mice. The transgenic animals expressed CAT activity only in the retina and pineal gland. This result indicates that the promoters have a tissue-specific enhancer and promoter activity. 7. The opsin promoter was fused with a diph- theria toxin gene, and that fusion gene was intro- duced into transgenic mice, which subsequentiy lost only the photoreceptor rod cell layer. 8. Several cDNAs of Shuzin, a retinal photore- ceptor protein, were isolated from human and cow retinal cDNA libraries (k-gtll), and the entire DNA sequences were determined. The deduced protein has sequence similarity with TFIID. Its gene also was isolated from a genomic library and its DNA sequence was determined. It is composed of two introns and three exons. 9. Two genes of 33-kD ROS-specific proteins have been isolated from the retinal libraries of human and mouse, and the entire DNA sequence of these genes have been determined. They have four exons and three introns. 10. The proximal promoter sequence positions -38 to -1-304 are sufficient to direct low levels of retina-specific gene expressioiL 11. The proximal promoter binds three retinal specific nuclear factors (Bpl, Bp2, and Bp3) through overlapping sequences centered between positions -25 and -15. 12. The distal promoter sequence positions -205 to -185, a region which contains two direct repeats of the hexamer, TGACCT. 13. We found a consensus retinal photoreceptor- specific site (PCEl). 14. The transcriptional machinery involved in photoreceptor-specific gene expression has been strongly evolutionarily conserved. Significance to Biomedical Research and the Program of the Institute Eyes have remarkable properties in functioning efficientiy over a wide range of illuminations. Rod cells, having photosensitive rhodopsin, are more sensitive to dim light; they adapt in the dark to increase their sensitivity. However, rod cells cease tiieir sensitive phototransduction in bright light. In contrast, cone cells do not operate in dim light but are operative in bright light Rhodopsin, transducin, phosphodiesterase, rhodopsin kinase, and S-Ag have been known to be associated with the phototransduc- tion cascade. Rhodopsin kinase and S-Ag are 217 Laboratory of Retinal Cell and Molecular Biologj' NEI Annual Report — FY 1993 considered to be the imponant proteins for light- dependent modulation of phototransduction. To understand this light-dependent modulatory mecha- nism in rod outer segments, we have characterized S-Ag, Shuzin, and 33K protein as well as their genes. Interestingly, other signal transduction systems have cascades similar to that of phototrans- duction (one of the best characterized receptor-medi- ated signaJ transduction processes). In the photo- transduction cascade, the shutoff mechanism appears to be modulated by the phosphorylation and dephos- phorylation of rhodopsin. Studying this modulation mechanism is important for understanding photo- transduction as well as for understanding signal transduction in general. In addition, we think that the night blindness of vision may in part be associ- ated with light adaptation. Proposed Course The following studies are in progress or have been proposed for Fiscal Year 1993: 1. Identification of the S-Ag promoter using transgenic animals. 2. Identification of m-acting factors of the S-Ag and 33K protein promoter. 3. The knockout of genes of S-Ag and phos- ducin. Investigation of a functional role for S-Ag and 33K protein, the homologous recombination between a mutant gene and a normal gene will be induced in ES cell culture. The recombinant ES cells will be introduced into a transgenic animal system in order to produce a mutant mouse. NEI Research Program Retinal Diseases — Photoreceptors and Retinal Pig- ment Epithelium Publications Abe T, Kikuchi T, Chang T, Shinohara T: The sequence of the mouse phosducin gene and its 5'- flanking region. Gene, 133:179-186, 1993. Danciger M, Kozak CA, Abe T, Shinohara T, Farber DB: The gene for retinal rod 33-kDa protein is on mouse chromosome 2, near lamb2. Cytogenet Cell Genet, 56:202-205, 1991. Kikuchi T, Raju K, Breitman ML, Shinohara T: The proximal promoter of the mouse arrestin gene directs gene expression in photoreceptor cells and contains an evolutionarily conserved retinal factor-binding site. Mol Cell Biol 13:4400-4408, 1993. Shinohara T, Kikuchi T, Tsuda M, Yamaki K: A family of retinal S-antigen (arrestin) and their genes: Comparative analyses of human, mouse, rat, bovine, and Drosophila. Camp Biochem Physiol, 103:505-509, 1992. Usukura J, Khoo W, Abe T, Shinohara T, Breitman ML: Abnormal development of cone cells in transgenic mice ablated of cone rod photoreceptor cells. Ann N Y Acad Sci, in press. Usukura J, Khoo W, Abe T, Shinohara T, Breitman M: Cone cells fail to develop normally in trans- genic mice ablated of rod photoreceptor cells. Tissue Cell, in press. 218 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00250-06 LRCMB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on or\e Ime between the borders.) Molecular Biology of Experimental Autoimmune Uveitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Toshimichi Shinohara Ph.D. Head, Section on LRCMB, NEI Molecular Biology Others: Dhirendra Singh Siradanahalli Guru Shirley Yu Ph.D. Ph.D. B.S. Visiting Associate Visiting Fellow Biologist LRCMB, NfEI LRCMB, NEI LRCMB, NEI COOPERATING UNITS (if any) Department of Ophthalmology, Miami University, Miami, FL (D. Hamasaki, Ph.D.); Department of Anatomy, Nagoya University School of Medicine, Tsuramai, Showa-ku, Nagoya, Japan (Jiro Usukura, M.D.) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology SECTION Section on Molecular Biology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 3.5 PROFESSIONAL: 2.5 OTHER: 1.0 CHECK APPROPRIATE BOX(ES) [xj (a) Human subjects [x] (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) We had previously determined amino acid sequences of liuman, mouse, rat, and bovine retinal S-antigen (S-Ag) and rat pineal gland S-Ag. Immunogenic sites and four uveitopathogenic sites of S-Ag also were determined; two immunogenic sequences were highly conserved among the species. Many proteins in the National BiomediC£il Research Foundation data base have a sequence similar to that of a uveitopathogenic site. We chemically synthesized many peptides, some of which induced experimental autoimmune uveitis (EAU) and experimental autoimmune pinealitis (EAP) in Lewis rats. In addition, we found native yeast histone H3 enable of inducing EAU. To understand the role in autoimmunity of infectious microorganisms which have cross-reactive antigens, we injected Lewis rats with peptide M, together with one of six different killed bacteria, with or without incomplete Freund's adjuvant (IF A). The rats injected with IFA developed EAU. To assess the impact of infection by live microorganisms, we injected low doses of live Escherichia coli expressing S-Ag and baker's yeast with a cross-reactive antigen into the rats several times. The rats injected with either Uve E. coli or live yeast developed EAU. We conclude that infection by microorganisms which have cross-reactive antigens can break immune tolerance to self-antigens and induce inflammatory autoimmune diseases. As an extension of our previous EAU research, we speculated that some types of cataracts may be induced by autoimmune insults. To investigate this issue, we conducted similar experiments: Three groups of four rats were injected three times with lens homogenate, P-crystallins, or a P-crystallin (p-Al) emulsified with complete Freund's adjuvant (CFA). All the animals developed severe damage in lens epithelial cells 5 weeks from the date of the first injection. The rats injected with a synthetic peptide derived from Salmonella typhimurium protein, which has five amino acid residues identical to rat P-crystallin (P-B2), also induced similar damage. Infection by microbes having antigens homologous to the lens antigens can induce high levels of auto;intibodies that provoke lens epithelial cell damage. Thus, autoimmune insult in lens epitheUal cells may be an etiology of an initial stage of cataractogenesis. Our future research will focus more on autoimmunity in lens cataractogenesis. 219 PHS 6040 (Rev. 5/92) Laboraton of Retinal Cell and Molecular Bioloj^ NEI Annual Report — FY 1993 Project Description Objectives The objectives of this project are to understand the basic etiology of autoimmune inflammation including uveitis and to find possible treatments for human uveitis. Methods The conventional methods for analysis of proteins and nucleic acids used include the following: protein purification; RNA and DNA isolation, characteriza- tion, and sequencing; molecular cloning; screening of clones; in situ hybridization; immunocytochemistry; and chromosome mapping. We also have synthe- sized and used oligopeptides and oligonucleotides. Bovine, murine, primate, and human materials are used. Animal experiments are carried out with Lewis rats and monkeys. T-cell response and adop- tive transfer are done with lymph node or spleen cells of rat. Major Findings 1. Local sequence homology was found between peptide M and several other foreign proteins, includ- ing potato proteinase inhibitor Da, Escherichia coli hypothetical protein, hepatitis B virus probable DNA polymerase, Moloney murine sarcoma virus gag- polyprotein, Moloney murine leukemia virus gag-pol polyprotein, Baboon endogenous virus gag-pol polyprotein, and Baker's yeast histone H3. 2. The synthetic peptides of the above-mentioned proteins induced experimental autoimmune uveitis (EAU) in Lewds rats; its pathology was similar to that of EAU induced by pepUde M or native S-anti- gen (S-Ag). 3. For the first time we proposed and showed the evidence that molecular mimicry plays a role in the process of pathogenesis of EAU and perhaps in autoimmune diseases in general. 4. Oral administration of histone H3 peptide suppressed EAU in the Lewis rats. 5. The suppression of EAU by histone H3 also was found in the EAU induced by the S-Ag. Thus, the tolerance also cross-reacted with the ''peptide, which has molecular mimicry. 6. The T-lymphocytes obtained from rats immu- nized with peptide M or yeast histone H3 transferred disease (i.e., EAU) in the naive rats (adoptive transfer) when stimulated either with peptide M or histone H3. In addition, oral tolerance was adop>- tively transferred from rats fed peptide M or histone H3 to the naive rats. 7. Infection by microorganisms which have cross-reactive antigens can break immune tolerance to a self-antigen and induce inflammatory auto- immune diseases. Significance to Biomedical Research and the Program of the Institute Uveitis is a leading cause of visual handicap in the United States and throughout the worid. For many decades, physicians have suspected some types of uveitis to be induced by bacterial and viral infec- tions; however, there is no clear link between infec- tion and disease. Autoimmune processes are thought to play a significant role in the pathogenesis of disease. Molecular mimicry— a process by which an immune response, directed against a nonself protein, ctoss- reacts with a normal host protein — may play a role in autoimmunity. Here we have proposed the idea of molecular mimicry and showed evidence that molec- ular mimicry may play a role in the pathogenesis of EAU. In addition, we have provided evidence that infection is a possible cause of autoimmune inflam- mation. These fmdings provide an important clue for understanding the etiology of autoimmune inflamma- tory diseases in humaa Proposed Course The following studies are in progress or proposed for Fiscal Year 1993: 1 . We will conduct further evaluation of foreign proteins similar to S-Ag that induce EAU. 2. We will characterize peptide M with respect to the minimum number of amino acids required for induction of EAU. 3. We will study the induction of EAU in trans- genic mice that express foreign proteins in photo- receptor cells. 4. We will further characterize molecular mimic- ry and its role in EAU and human uveitis. NEI Research Program Retina] Diseases— Inflammatory Diseases 220 NEI Annual Report— FY 1993 Laboratory of Retinal Cell and Molecular Biology Publications Chan CC, Li Q, KiJkuchi T, Shinohara T, Nussenblatt RB: Enhancement of S-antigen and its mRNA in the irides of uveitic patients. J Autoimmun 5:719- 732, 1992. Eto K, Suzuki S, Singh VK, Shinohara T: Immuni- zation with recombinant Escherichia coli express- ing retinal S-antigen induced experimental auto- immune uveitis (EAU) in Lewis rats. Cell Immu- nol 147:203-214, 1993. Hamasaki DI, Sato H, Santhanakrishnan S, Shinohara T: Correlation between the physiological and morphological changes in the experimental auto- immune uveitis induced by peptide G of S-anti- gen. Exp Eye Res, in press. Nityanad S, Singh VK, Shinohara T, Paul AK, Singh VK, Agarwal PK, Agarwal SS: Cellular immune response of patients with uveitis to peptide M, a retinal S-antigen fragment. J Clin Immunol, in press. Sunil S, Eto K, Singh VK, Shinohara T: Oligopep- tides of three to five residues derived fi^om uve- itopathogenic sites of retinal S-antigen induce experimental autoimmune uveitis (EAU) in Lewis rats. Cell Immunol 148:198-207, 1993. 221 Laboratory of Sensorimotor Research Report of the Chief, Laboratory of Sensorimotor Research Robert H. Wurtz, Ph.D. One of the most admired human abilities is that of skilled motor control — be it hitting a baseball in Baltimore or returning a teimis serve on Long Island. These abilities are highly sophisticated sensory motor tasks; they depend heavily on vision. The Laboratory of Sensorimotor Research concen- trates on such sensory motor tasks, particularly in relation to the visual control of eye movements. Our goal is to understand the systems within the brain that process visual information and produce these eye movements and to understand what happens when disease or trauma leads these to fail. While our main interests are the systems in humans, we are fortunate to have a superb animal model, the Rhesus monkey, which allows us to explore not only the exact behav- ioral mechanisms related to visual motor behavior but also the underlying brain mechanisms controlling such behavior. Our investigations are best illustrated by a selection from the work of each of the five sections within the Laboratory. It previously has been shown that humans who wear spectacle lenses are able to generate saccades that differ in amplitude between the two eyes exactly as required by the different magnifications of the lenses, and usually it has been assumed that this ability results from some neural adaptive mechanism that adjusts for this over time. I>r. Miles' group has found that such an ad^tation period is not necessary. These investigators found that humans immediately adjusted the amplitude of the eye movement in ways appropriate for the size of the stimulus. They hypothesize that it is not adaptation that is control- ling binocular alignment of the eyes in this case but rather the use of the horizontal disparity in the image detected by the visual system. They were able to show exactly the same phenomena in the monkey, opening the way for extensive quantitative analysis of the parameters confrolling these saccadic eye movements and the possibility of determining the brain mechanisms imderlying this control. Section on Oculomotor Control Section on Yisuomotor Integration One of the most frequent uses of vision for the control of eye movement is in the generation of rapid or saccadic eye movements — those eye move- ments that move the eyes from one part of the field to another. Such shifts allow us to look from one area of the field of interest to another. Both eyes move together to maintain binocular alignment, which is critical for good depth vision. Dr. Fred Miles and his collaborators are able to measure these eye movements with great accuracy in both humans and monkeys to determine how we solve a problem in generating these saccades (i.e., what happens when humans or monkeys are first confronted with images that differ in size for the two eyes). Such a differ- ence in image size results when spectacle lenses cause the two eyes to see images that differ in size by several percent for each diopter of difference in correction between the eyes. Another case illusfrating the strong visual control of movement is from the work that my collab- orators and I have done on the control of our move- ments through the environment and the stabilization of our posture. It has been shown in humans that motion through the visual field produces a specific pattern of large field visual motion, referred to as "optic flow." The nature of this large field visual motion is thought to provide information about our direction of movement through the envirormient. It also provides information to control our posture; humans sway back and forth substantially more with their eyes closed than with their eyes open. In the past year we have tested whether monkeys use such visual information to control posture by training the monkey to stand oh a small platform that measures how much the monkey sways and in what direction. By projecting onto a screen a pattern of 225 Laboratorv of Sensorimotor Research NEI Annual Report— FY 1993 motion simulating the motion that would occur as the monkey leaned forward or back or side to side, we have been able to measure the monkey's postural changes. We have shown that the monkey responds to this visual stimulation and that the response is, in most respects, similar to that reported for humans. This now provides us with the ability to investigate further the regions of the brain that we know process this type of visual motion and to see whether alter- ations of these regions alter the monkey's use of the visual stimulation for the control of posture. Section on Neural Modeling The way neurons convey visual information has been studied by Dr. Lance Optican and his collaborators over the past several years. While in most studies of neurons within the brain the neuronal activity has been taken as the total number of action potentials or spike discharges emitted in response to a visual stimulus. Dr. Optican has shown that more information is conveyed if one looks at the temporal patterning of the action potentials as well as their total number. An understanding of this extra visual information may lead to a better understanding of visual perception. Dr. Optican and his collaborators previously have shown that neurons in a number of visual areas (i.e., VI, V2, V3, V4, and the inferior temporal cortex) both encode and transmit informa- tion about patterns that vary in form, color, bright- ness, and duration by a temporal code that represents these stimulus-dependent messages. In their present experiments. Dr. Optican's group trained monkeys to choose a particular stimulus according to whether it matched a previously given cue stimulus. They found that the temporal pattern of cell discharge varied with not only the stimulus falling on the receptive field of the cell but also with the nature of the cue stimulus. Furthermore, they found that each stimulus could be represented as the product of two wave forms that were specific for the features paired in each stimulus— for example, color and pattern. Thus, unique codes for every possible combination of visual features are not required. These experiments address the major issue in understanding information processing within the brain— the code by which this information is transmitted. This work clearly shows that neurons convey information about visual features by using a temporal code. This work may provide a key to understanding how the brain processes infor- mation to form a visual perception. Section on Visual Behavior Determining which visual stimulus is of impor- tance for visual processing is one of the critical functions of the visual system. We look at only one part of the visual field at a time; the selection of the region of the visual field to look at next is the function referred to as "selective visual attention." Dr. David Lee Robinson has continued to explore the neurons in the brain that give evidence of participat- ing in this attention process. He and his colleagues have conducted experiments on the superior collicu- lus to understand its role in visual attention. They have discovered that neurons there discharge at the appearance of certain visual stimuli and that these signals help indicate a change in the direction of attention. Other cells located in parts of the collicu- lus that are connected to the fovea also respond to visual stimuli, and here their activity starts the engagement of attention by images on the fovea These are the first data to demonstrate a visual function of the superior colliculus that is not related to eye movements. Section on Neuro-Ophthalmologic Mechanisms One of our most effective methods of studying the systems within the brain is the alteration of that system by electiical stimulation or chemical injection (as in Dr. Robinson's experiments). Such modification allows us to test hypotiieses about the contribution of a given set of cells within the system to the brain's function in controlling behavior. Dr. Michael Goldberg and his collaborators recentiy have been able to use such a technique, not only in the monkey model of the control of eye movements but also in humans. Dr. Goldberg previously had shown the characteristics of cells in a part of die frontal cortex of tiie monkey referred to as tiie firontal eye fields. Recent work using PET scanning identi- fied the approximate region in the human where such frontal eye fields are located. By using the technique of focal magnetic stimulation, which changes the 226 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00256-05 LSR PERIOD COVERED October 1. 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Information Processing by Visual System Neurons PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: Lance M. Optican Others: John W. McCIurkin Arthur V. Hays Brad J. Zoltick Jennifer A. Zarbock Merk Na Chee-Orts Marc H. Cohen Ph.D. Ph.D. B.A. M.A. B.A. Ph.D. M.S£. Chief, Neural Modeling Section Staff Fellow Electronics Engineer Computer Programmer Electronics Engineer Visiting Associate Visiting Associate LSR, NEI LSR, NEI LSR, NEI LSR, NEI LSR, NEI LSR, NEI LSR, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Sensorimotor Research SECTION Neural Modeling Section INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 5.6 PROFESSIONAL: 4.0 OTHER: 1.6 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors |~| (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Our studies indicate that different visual areas in the brain may communicate via temporally modulated messages. We showed previously that neuroas in different areas of the brain encode and transmit information about stationary, two-dimensional pictures that vary in form, brightness, and duration. We also showed that information about remembered visual features was carried by a temporal code. Now we have extended those studies to show that neurons in the visual cortex (areas VI, V2, V3, and V4) carry information about the form, color, luminance, and size of a stimulus in a temporally modulated code. Our results suggest that cortical neurons are able to convey information about many different features without confounding them. The mechanism for encoding these multiple messages uses temporal modulation to multiplex the different messages on the neuron's response in a separable way. 228 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Sensorimotor Research electrical activity of the brain in a small region below the skull, they have been able to show that stimulation of the frontal eye field of humans alters saccadic eye movements. Furthermore, they have been able to show that the type of alteration depends on the time at which the stimulation is given before the onset of the saccade. Thus, identification of a region in the brain of the monlcey has led to the localization, and now the modification, of a similar area in human subjects. A part of the activity of the Laboratory this year involved the move of those laboratories devoted primarily to research on monkeys into the new Silvio O. Conte Building (Building 49). This move was completed by June 22. The new building provides superb facilities for nonhuman primates, fine labora- tories for investigation, and a few small rooms for offices. 227 NEI Annual Report— FY 1993 Laboratory of Sensorimotor Research Project Description Objectives Perception and recognition of complex visual pic- tures depend on the normal function of interconnect- ed brain regions extending from the retina through the inferior temporal cortex. The properties of these regions are derived from the function of the single neurons within them. Thus, to understand how visual perception occurs, we must learn how infor- mation is encoded by the neurons in each stage of processing. If we could understand this neuronal code, it might be possible to distinguish between information related to the physical properties of a stimulus (e.g., form, luminance, color, and size) and information related to its behavioral significance (e.g., leading to a reward). Individual neurons in all the visual areas smdied thus far (retinal ganglion cell fibers; lateral geniculate nucleus neurons; pulvinar neurons; cortical neurons in visual areas VI, V2, V3, and V4; and inferior temporal cortical neurons) encode and transmit information about stationary, two-dimensional pictures that vary in form, color, brightness, and duration. The neurons use a multidimensional temporal code to represent and transmit their stimu- lus-dependent messages. We now have shown that visual neurons convey complex messages about (1) a stimulus' physical parameters and (2) its behavioral significance. Using information theory, we can begin to explore how physical and behavioral com- ponents of a neuron's response contribute to higher visual cognitive functions such as perception, atten- tion, and memory. Major Findings We have developed a new approach to studying single neurons in which they are treated as communi- cation channels that transmit information about visual pictures in their responses. This has allowed us to apply methods from signal processing, statistics, systems analysis, and information theory to under- stand single neurons. According to a commonly held view of neuronal function, the strength of a neuron's response repre- sents how closely the stimulus matches the receptive field's characteristics (e.g., orientation or color). Thus, if response strength were the only parameter a neuron could use to encode information, different stimulus features would be confounded by individual neurons. Using informational analysis, we have shown that information about different stimulus parameters is not confounded but is carried across tlie different parts of the multidimensional neuronal code. In recent experiments, we recorded responses of neurons in four visual cortical areas — VI, V2, V3, and V4 — of a monkey trained to choose one of three parafoveal stimuli on the basis of whether their color or pattern matched that of a cue stimulus. These responses were modulated by the pattern and color of the stimulus on the receptive field and by the pattern or color of the preceding cue. In other experiments, stimuli consisted of either colored bars that were isoluminant with the background or black or white bars that varied in size. Information about stimulus features developed continuously, but not uniformly, throughout the time-coiu"se of the neuronal responses. Most of the information was encoded in the initial 50-60 msec of the response. Some neurons also encoded a large amount of information in a second 50-msec interval, beginning 20-30 msec after the first These results show that neiirons in VI -V4 carry information about the color, pattern, contrast, and size of stimuli. Rnally, the development of informa- tion over time in different areas suggests that temp- orally modulated waves of activity may form a code for visual information. In fact, the response to each stimulus could be represented as the product of two waveforms that were specific for the features paired in each stimulus (e.g., color and pattern, color and orientation, contrast and orientation, or size and orientation). Feature-specific waveforms for each color, pattern, contrast, orientation, and size were isolated from the neuronal responses by a neural net. The product of these feature waveforms predicted the neuronal responses to stimuli with feature combina- tions not used to train the neural net (e.g., novel- colored patterns). Feature waveforms were often similar for all neurons within a cortical area. To compare these waveforms across cortical areas, we pooled all the responses from neurons within each area. Waveforms encoding pattern were strikingly similar across all areas, irrespective of the behavioral task. Waveforms encoding color in the color/pattern task differed between cortical areas, but there was a striking similarity for waveforms encoding color in the 229 Laborator\' of Sensorimotor Research NEI Annual Report— FY 1993 isoluminant-color/orientation paradigm. These resulLs suggest that neurons convey information about compound visual features by multiplexing feature- specific messages together. The invariance of the code waveforms suggests that information about a stimulus feature is represented similarly in all visual areas. Significance to Biomedical Research and the Program of the Institute This project studies how visual information is en- coded and transmitted by neurons. Knowledge of these fundamental processes is important for under- standing deficits of visual processing, such as those occurring in amblyopia, and for developing visual prosthetic devices to compensate for field defects or blindness. Proposed Course Discovering that the responses of visual system neurons are multidimensional led to the discovery that information about multiple stimulus features may not be confounded by single neurons, a result with important, even revolutionary, consequences. We now know that a substantial part of the temporal modulation arises after visual information has left the retina. Our latest results show that the neural code arises due to the influence of feedback. Ever since we found evidence of a neural code and saw a possible structure for it, we have been trying to delineate it The properties of the code should give clues about the functions performed by the neurons. Now that we have shown that some of the temporal codes are invariant across cells, and even across areas, a new theory of visual information processing is required. This theory will treat the visual system more as a concurrent processing system than as a hierarchical cascade of independent areas. Both these issues are being pursued. In addition, our findings have suggested previous- ly unconsidered principles as the basis for interac- tions among neurons. To investigate these princi- ples, we need to collect and analyze data from several simultaneously recorded neurons. Thus, we have been developing the apparatus needed to make multiple, simultaneous single-neuronal recordings. The apparatus should be completed sometime during the next year. The simultaneously recorded respons- es will be related to each other through use of recent extensions to methods of signal identification, which should allow us to develop models that will describe, relatively rapidly, the roles of single neurons as components of larger networks. These studies should yield a better understanding of the information transmission mechanisms, such as pattern perception and recognition, used for cognitive functions. Our findings suggest a completely new concep- tional framework in which to investigate neuronal function. One presumed reason for the huge number of single neurons has been the necessity to uncon- found stimulus features. However, we propose that the simultaneous messages about different features can be used as tags, so that the messages which arise in different processing regions of the visual system can be reunited into a unified percept This would provide the mechanism to build a whole perception across many processing regions. With the use of new computational equipment, we are exploring this hypothesis both experimentally and theoretically. NEI Research Program Strabismus, Amblyopia, and Visual Processing — Visual Processing and Functional Organization (Structure and Function of Central Visual Pathways) Publications Chee-Orts MN, Optican LM: Cluster method for analysis of transnutted information in multivariate neuronal data. Biol Cybem 69:29-35, 1993. Eskandar EN, Optican LM, Richmond BJ: Role of inferior temporal neurons in visual memory: II. Comparing tempwral waveforms arising fi-om vision and memory. J Neurophysiol 68: 1296-1306, 1992. Eskandar EN, Richmond BJ, Optican LM: Role of inferior temporal neurons in visual memory: I. Temporal encoding of information about visual images, recalled images, and behavioral context. J Neurophysiol 68:1277-1295, 1992. Kapoula Z, Robinson DA, Optican LM: Visually induced cross-axis postsaccadic eye drift. J Neurophysiol 69: 1 03 1 - 1 043 , 1 993. McClurkin JW, Zarbock JA, Optican LM: Temporal codes in monkey sQ-iate cortex for colors, patterns and memories, in Peters AA, Rockland KS (eds): Primary Visual Cortex of Primates, Cerebral Cortex. New York, Plenum, 1993, vol 10, in press. 230 NEI Annual Report— FY 1993 Laboratory of Siensorimotor Research Zee DS, FitzGibbon EJ, Optican LM: Saccade- vergence interactions in humans. J Neurophysiol 68:1624-1641, 1992. 231 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00049-15 LSR PERIOD COVERED October 1. 1992 to September 30. 1993 TITLE OF PROJECT (80 charaaars or less Tula must lit on one line between the borders.) Cerebral Cortical Mechanisms for Eye Movements and Visual Attention PRINCIPAL INVESTIGATOR (List other protessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute afliliation) PI: Michael E. Goldberg M.D. Chief, Neuro-Ophthalmologic LSR, NEI Mechanisms Section Others: Edmond J. FitzGibbon Carol L. Colby Suzanne Y. Musil M.D. Ph.D. Ph.D. Medical Officer Senior Staff Fellow Staff Fellow LSR, NEI LSR, NEI LSR, NEI COOPERATING UNITS (il any) Medical Neurology Branch, National Institute of Neurological Disorders and Stroke LAB/BRANCH Laboratory of Sensorimotor Research SECTION Neuro-Ophthalmologic Mechanisms Section INSTITUTE AND LOCATION NEI. NIH. Bethesda, MD 20892 TOTAL STAFF YEARS: 5.3 PROFESSIONAL: 4.0 OTHER: 1.3 n (b) Human tissues [x] (c) Neither CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.) Two Unes of inquiry were followed to determine how the cerebral cortex and its efferent regions control eye movements and visuospatial attention. In one, focal transcranial magnetic stimulation of the human fi-ontal eye field was used to determine the effect that frontal eye field activity can have on the generation of saccadic eye movements. Depending on the relationship of the exogenous stimulation to the ongoing processes of saccade initiation, such stimulation can facilitate or interfere with saccade generation. In the other, single neuron recording was used to probe the mechanisms whereby the parietal cortex of the monkey achieves spatial accuracy. Neuronal behavior in a double-step task is entirely predicted by presaccadic and predictive-shift activity in a single-step task. Tlie activity of parietal neurons is most consistent with their specifying a shift of visual attention of particular amplitude and direction. 232 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Sensorimotor Research Project Description Objectives This section has concentrated on two aspects of the physiology and phenomenology of higher visual and oculomotor processing in the monkey and man, especially as these functions relate to the parietal and frontal regions of the cerebral cortex, their afferent regions, and their efferent targets. Previous work in this laboratory has shown that neurons in the parietal cortex have neiu"ons that discharge in response to visual stimuli and before saccadic eye movements. There is a predictive aspect of these neurons' visual responses: Neurons respond to stimuli in which spatial location will be brought into their visual receptive fields by impending saccadic eye move- ments. The double-step task of Hallett and Lightstone has been considered the paradigmatic example of accur- ate spatial behavior. Humans and monkeys perform this task accurately, making accurate eye movements to successive, briefly flashed targets, despite a dissonance between the retinal location of the target and the amplitude and direction of the saccade necessary to fixate it Work in the laboratory during this year has concentrated on comparing the activity of neurons in the double-step task to see whether that activity could be explained by their activity in more simple tasks. We have demonstrated a neuronal mechanism for the initiation and targeting of saccadic eye move- ments in the monkey frontal eye field, where neurons discharge predictively before purposeful saccades and intracortical electrical miaostimulation elicit sac- cades. In this period, we used the technique of transcranial magnetic stimulation in human subjects to stimulate the frontal eye field selectively at various times in the generation of visually guided saccades in order to see whether electrical stimula- tion of this region affected saccades in a manner consonant with the single-neuron data from the monkey. Methods Monkeys were implanted with magnetic search coils for the measurement of eye position, along with devices for temporary restraint and electrophysiologi- cal recording and stimulation. The monkeys were trained to perform a number of visuomotor tasks. including fixation, saccades, and smooth pursuit. Microelectrodes placed in the lateral intraparietal area single neurons enabled smdy while the monkey performed various visuomotor tasks. Normal volunteers were instructed to fixate a central target and make a saccade as quickly as possible in response to the appearance of a peripheral light Eye movements were measured by an infrared eye tracker. In randomly interleaved trials, focal transcranial magnetic stimulation was delivered through a figure-eight-shaped coil over the presumed site of the frontal eye field, which was located with reference to the hand motor representation. Major Findings Transcranial magnetic stimulation j^jplied long before the onset of the expected saccade produced shorter saccadic reaction times and increased saccade acceleration. Conversely, transcranial magnetic stimulation ^plied shortiy before the onset of the expected saccade yielded longer saccadic reaction times and decreased saccade acceleration. Similar effects were observed when subjects were instructed to perform antisaccades. Independent of the effect on saccadic reaction times, transcranial magnetic stimulation produced transient divergence of the eyes immediately preceding saccade onset. When trans- cranial magnetic stimulation occurred during an ongoing saccade, it transientiy arrested or slowed the eye movement. In summary, transcranial magnetic stimulation can facilitate or retard saccadic reaction time and can affect the metrics of saccadic eye movements. When it occurs at a time at which it might interfere with ongoing frontal eye field pro- cessing, it slows saccades. When it appears at a time at which it could reasonably be expected to substimte for normal processing, it speeds up saccadic reaction times. Neurons in the lateral intraparietal have visual and sometimes presaccadic responses. The visual re- sponses are sometimes predictive, occurring before a saccade that will bring the spatial location of a visual target into the neuron's receptive field. Neurons that discharge in the double step tasks have presaccadic responses, predictive responses, or both. The activity in the double-step task approximates the sum of the presaccadic and predictive response. These data illustrate that the response in the double-step task emerges as a consequence of the neuron's response in relation to single eye movements and that it is 233 Laborator>' of Sensorimotor Research NEI Annual Report— FY 1993 unnecessary to postulate a special mechanism to account for activity in the double-step task. Significance to Biomedical Research and the Program of the Institute Understanding the way in which the cerebral cortex and its afferent regions guide eye movement; and modulate visual attention and learning is useful as a model for the neural control of other, more compli- cated behaviors. It is also a key to understanding and developing treatments for disorders of the neural control of vision, eye movements, and attention. Proposed Course TTie frontal eye fields will be examined to see whether they have predictive responses. The activity of neurons in both the frontal eye fields and the parietal cortex will be examined under the more natural condition of visual search. NEI Research Program Strabismus, Amblyopia, and Visual Processing — Visual Processing and Functional Organization (Structure and Function of Central Visual Pathways) Publications Colby CL, Duhamel JR, Goldberg ME: The analysis of visual space by the lateral intraparietal area of the monkey: The role of extraretinal signals. Prog Brain Res 95:307-316, 1993. Colby CL, Duhamel JR, Goldberg ME: Ventral intraparietal area of the macaque: Anatomic location and visual response properties. J Neuro- physiol 69:902-914, 1993. Duhamel JR, Goldberg ME, FitzGibbon EJ, Sirigu A, Grafman J: Saccadic dysmetria in a patient with a right frontoparietal lesion: The importance of corollary discharge for accurate spatial behav- ior. Brain 115:1387-1402, 1992. Goldberg ME, Musil SY, FitzGibbon EJ, Smith MK, Olson CR: The role of the cerebellum in the control of saccadic eye movements, in Mano N (ed): Cerebellum and Basal Ganglia in the Control of Movement. Amsterdam, Elsevier, 1993, in press. Olson CR, Musil SY, Goldberg, ME: Superior cingulate cortex and visuospatial cognition: Properties of single neurons in the behaving monkey, in Vogt BA, Gabriel M (eds): The Neurobiology of Cingulate Cortex and Limbic Thalamus. Boston, Birkhauser, 1993, in press. Segraves MA, Park K: The relationship of monkey frontal eye field activity to saccade dynamics. J Neurophysiol 69:lSS0-nS9, 1993. Stanton GB, Bruce CJ, Goldberg ME: Topogr^hy of projections to the fi-ontal lobe fi-om macaque frontal eye fields. J Comp Neurol 330:286-301, 1993. 234 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00153-11 LSR PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or lass. Title must fit on arte line between the borders.) Visual Motion and the Stabilization of Gaze PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Frederick A. Miles D.Phil. Senior Research LSR, NEI Physiologist Others: Urs Schwarz Claudio Busettini M.D. Ph.D. Visiting Associate Visiting Fellow LSR, NEI LSR, NFEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Sensorimotor Research SECTION Oculomotor Control Section INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 2.5 PROFESSIONAL 1.3 OTHER: 1.2 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ {a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) It has been known for some time that corrected anisometropes who wear spectacle lenses of different power in front of the two eyes are able to generate saccades that differ in amplitude in the two eyes exactly as required by the differing magnifications of the spectacle lenses. A common assumption is that this ability results from the operation of a neiu^al adaptive mechanism, which, over time, gradually adjusts the relative amplitudes of the saccades produced by the two eyes. We now report that when the scenes viewed by the two eyes suddenly differ in size, the two eyes produce saccades that inmiediately differ in amplitude without any prior period of adaptation. Two slide projectors and an arrangement of orthogonal polarizing filters were used to present overlapping stationary random dot patterns simultaneously yet separately to the two eyes. The right eye always saw the same pattern while the left eye saw a pattern that was initially identical (pretest) and later replaced by one that was 8% smaller (test). The positions of both eyes were recorded with the electromagnetic search coil, and horizontal saccades were elicited by target spots projected onto the pattern through polarizing filters so as to be visible only to the right eye. Subjects were three humans and three rhesus monkeys. Immediately upon viewing the smaller pattern, the left eye produced horizontal saccades that were significantly smaller than those produced by the right eye. This indicates that the saccadic system has some ability to cope immediately with aniseikonia, the compensation being almost complete in some subjects. We suggest that the important cue in these experiments is horizontal disparity and that the saccadic system uses this to scale the relative amplitudes of the saccades produced by the two eyes. 235 PHS 6040 (Rev. 5/92) Laboraton of Sensorimotor Research NEI Annual Report— FY 1993 Project Description Objectives In recent years there has been considerable interest in the binocular alignment of the two eyes because it is critical for good stereoscopic depth vision. The advent of new techniques for recording eye position with high precision in both monkeys and humans has, for the first time, permitted detailed studies of the relative alignment of the two eyes on objects at varying distances in a three-dimensional world. We have used the high-resolution electromagnetic search coil to examine the saccadic eye movements of both monkeys and humans when they are first confronted with images that differ in size for the two eyes. Correction of anisometropia with spectacle lenses causes the two eyes to see images that differ in size by 2-3% for each diopter of difference. It has been previously shown by others that human subjects who wear such spectacles are able to generate saccades that differ in amplitude between the two eyes exactly as required by the differing magnifications of the spectacle lenses. It has been commonly assumed that this ability results from the operation of a neural adaptive mechanism that over time gradually adjusts the relative amplitudes of the saccades produced by the two eyes. However, previous investigators had not recorded eye movements immediately after the subjects put on the spectacles; hence, they did not know whether this behavior was immediate or required a period of adaptation. Therefore, we looked at the saccadic eye movements produced by both human and monkey subjects when first con- fronted with the challenge of aniseikonia (i.e., un- equally sized images seen by the two eyes). Methods The subjects (i.e., three humans and three rhesus monkeys) faced a tangent screen onto which were projected two superimposed images, each of which was visible to only one of the two eyes. This we achieved using a special screen and polarizing filters so that one of the images was polarized in a plane orthogonal to the other. When viewing the screen through goggles with cross-polarizing filters, each eye could see only one of the images, which were computer-generated random dot patterns. We record- ed the positions of both eyes using the electromag- netic search coil method while the subjects made saccadic eye movements between target spots pro- jected onto the screen through polarizing filters so as to be visible only to the right eye. The targets were located 10 degrees to the right and left of straight ahead. The right eye always saw the same random dot pattern, whereas the image seen by the left eye could be either the same or 8% smaller, resulting in a gradient of binocular disparities across the scene (i.e., slight differences in the locations of the images on the two retinas). Fifty leftward and fifty right- ward saccades were recorded when the patterns seen by the two eyes were idenfical (pretest) and another 50 each when the two patterns differed in size (test). Major Findings When the patterns were identical in size, the two eyes made saccades that were essentially identical in amplitude, though not velocity. Immediately on viewing the smaller pattern, the left eye produced horizontal saccades significantly smaller than those produced by the right eye. Similar observations were made using scenes that differed only in their horizon- tal dimensions, when the pattern of disparity was like that experienced by an observer who views a vertical surface slanting away from him or her. A striking feature of such stimuli was that, at best, the human observers had only a very weak perception of such a slanting surface. However, the horizontal saccades of all subjects immediately showed considerable compensation for the aniseikonia; in some subjects it was almost complete. For example, when the left eye saw a pattern that was 8% smaller horizontally, the saccadic amplitude ratios (left eye/right eye) of the three humans during the test period were on average smaller than those during the pretest by 7.3%, 4.0%, and 7.3% for rightward saccades and by 5.5%, 3.8%, and 7.4% for leftward saccades. Similar saccadic data were obtained from the three rhesus monkeys. Significance to Biomedical Research and the Program of the Institute These data indicate that, when suddenly confi-onted with aniseikonic images, the saccadic systems of both monkeys and humans are able to make saccades that differ appropriately in size. Furthermore, in the experiments, the only cue provided was (horizontal) disparity, indicating that the saccadic system can directly utilize such cues to adjust the relative amplitudes of the saccades produced by the two eyes. 236 NEI Annual Report— FY 1993 Laboratory of Sensorimotor Research It also is interesting that the motor system responded to the disparity cues, even though human subjects failed to perceive them. Proposed Course Future experiments will ftuther examine the saccadic eye movements associated with aniseikonia in both human subjects and monkeys. The research will involve parametric studies of these asymmetric saccades to establish the limits of the system and their impact on saccadic dynamics. NEI Research Program Strabismus, Amblyopia, and Visual Processing — Image Formation and Stabilization (Ocular Motility) Publications Kimmig HG, Miles FA, Schwarz U: Effects of stationary textured backgrounds on the initiation of pursuit eye movements in monkeys. J Neuro- physiol 68:2147-2164, 1992. Miles FA: The sensing of rotational and transla- tional optic flow by the primate optokinetic system. Rev Oculomot Res 5:393-403, 1993. Miles FA, Busettini C, Schwarz U: Ocular responses to linear motion, in Shimazu H, Shinoda Y (eds): Vestibular and Brain Stem Control of Eye, Head and Body Movements. Tokyo, Japanese Scientific Societies Press/Karger 1992, pp 379-395. Miles FA, Wallman J: Prologue. Rev Oculomot Res 5:v-viii, 1993. 237 KHUJtO I rNUMDtn DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT ZOl EY 00045-15 LSR PERIOD COVERED October 1. 1992 to September 30. 1993 TITLE OF PROJECT (80 characters or loss. Title must In on one line berween the borders.) Visuomotor Properties of Neurons in the Thalamus PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atliliation) PI: David Lee Robinson Ph.D. Section Chief LSR, NEI Others: Alexander A. Kustov Ph.D. Fogarty Fellow NEI COOPERATING UNITS (il any) Department of Anatomy, Howard University (Robert J. Cowie, Ph.D.) LAB/BRANCH Laboratory of Sensorimotor Research SECTION Visual Behavior Section INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 2.4 PROFESSIONAL: 1.0 OTHER: 1.4 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type Do not exceed the space provided.) Visual stimuli excite the retina and often produce a shift of attention. To understand some of the brain mechanisms involved in shifts of attention, we recorded the activity of neurons in the superior colliculus while monkeys performed various tasks. We discovered that neurons in the representations of the peripheral visual fields respond uniformly to targets, regardless of the direction of the animal's attention. This occurs whether attention is shifted by visual cues or by more cognitive processes. In contrast, neurons within parietal cortex are modulated by the direction of the animal's attention. Neurons within the foveal representation of the colliculus respond differentially to fixation targets, depending on the behavioral task. While the animal is simply fixating, these neurons are only weakly responsive; during more attention-demanding tasks, there are more brisk responses of these same cells. We injected the colliculus with muscimol, a GABA-agonist, and discovered that it produced a slowing of responses to all targets within the injected visual field. The neurons recorded at the injection site had increased spontaneous activity and were also more responsive to visual stimuli. These data are some of the first to demonstrate an attentional contribution of the colliculus, independent of eye movements. The results suggest that the superior colliculus provides a visual trigger signal for shifts of attention. 238 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Laboratory of Sensorimotor Research Project Description Objectives Visual stimuli continually excite the eye, and some of these elicit a shift of attention. The present studies were conducted to understand some of the mechanisms the brain uses to mediate attentional shifts. We sought to discover the physiological mechanisms used within the superior coUiculus to produce visual activity that initiates shifts of atten- tion. In addition, we sought to learn the functional contributions this collicular activity might make to the whole organism. Because we previously had studied the contributions of pareital cortex to atten- tion, this smdy was intended to compare these two areas. Methods To study the activity of the superior colliculus during attentional behavior, we trained monkeys to enter a primate chair, sit quietly,. fixate spots of light, and make eye movements to them. After preliminary training, the monkeys were implanted with several recording devices during sterile surgery. Scleral search coils were implanted in the monkeys for recording eye movements and eye position. The monkeys learned to contact a bar at the beginning of each trial. They then fixated on a spot of light projected onto a screen and released the bar when- ever target lights were flashed onto the screen. The monkeys also learned to make eye movements to spots of light flashed onto the screen, as well as make fine discriminations of selected fixation targets. At the sites of interesting cellular activity, small marking lesions were made for later localization on histological sections. Major Findings As we previously had demonstrated, monkeys re- spond faster to visual targets that are preceded by visual cues at the same location than to visual targets that follow cues at other points. It is hypothesized that the cue shifts attention to that point and thereby improves visual performance. We studied neurons within the superficial layers of the superior colliculus while monkeys performed this and related tasks. Collicular neurons compose a uniform population of cells with consistent latencies and response magnitudes. Data from our previous studies of parietal cortex show that cells in that region make up at least three subgroups; most of these neurons could be driven by our collicular afferents. All collicular neurons responded to both the onset and offset of visual cues that controlled the monkeys' attention. In addition, the responses to these cues were uniform across the temporal intervals used. When these cues excited collicular neurons, they produced a very stong period of refractoriness that was much more intense than the one measured for parietal cells. When collicular neurons were tested with the attentional cue outside the visual receptive field but positioned to produce attentional effects, collicular cells responded equivalently to all targets, regardless of where attention was directed. Cells in parietal cortex tested under these same conditions were differentially modulated, depending on the direction of the animals' attention. When collicular neurons were tested via tasks that cognitively controlled the direction of the animals' attention, there also was consistent response independent of the attentional direction. We also studied neurons within the foveal repre- sentation of the colliculus. When these cells were analyzed at the time that attention was shifted to the cue, there were no changes in the activity levels of the cells. These data suggest that the foveal parts of the colliculus do not participate in the shifting of attention. However, the cells had different activity patterns, depending on the behavioral task. When the animals simply had to fixate a spot of light to obtain a reward, there was only weak responding to the onset of the fixation point. These cells were excitable by other lights, so there was no overall suppression of their excitability. When the animal fixated the identical tight during the performance of the attentional cuing task, there was a much stronger response from collicular neurons. The cells respond- ed as if the act of attending had facilitated their visual responsiveness. Comparably strong responses were obtained when the animal actively attended to the same fixation point during a special foveal attention task. These data suggest that the foveal region of the colliculus is under attentional control, enhancing visual excitability, whereas the peripheral collicular representation is not attentionally modu- lated. To evaluate the contribution of these collicular signals to the monkeys' behavior, we altered collicu- lar activity by microinjections of muscimol, a 239 Laboratory' of Sensorimotor Research NEI Annual Report — FY 1993 GABA-agonist. After an injection of muscimol, the moniceys were slow to respond to all visual targets that appeared within the affected region of visual space. However, collicular neurons at the site of the injection were more responsive after the injections, contrary to observations obtained from microionto- phoresis. Now there was an increase in spontaneous activity of the collicular neurons. In addition, these cells discharged more intensely to the cues and targets. No changes in cellular activity were ob- served with injections of saline. These data suggest that the effects of limited injections of muscimol are not always inhibitory and do not decrease transmission through a structure. They also suggest that the discharge of collicular neurons provides a visual trigger signal that can lead to a shift of attention. When this signal is modified, in this case by the production of increasing and distracting discharges, there is a generalized break- down in performance. Significance to Biomedical Research and the Program of the Institute Various disorders of the brain produce abnormal attention and eye movements. Some of these, such as progressive supranuclear palsy, involve dysfunc- tion of the superior colliculus. The data obtained this year help to define the contribution of the colliculus to these symptoms. Understanding these processes might facilitate early detection of such disease processes. For rehabilitation of people with damage to parts of the brain, it is important to know the normal capacities of certain neural centers and the ways in which one brain area can take over the functions of damaged areas. By gaining a clearer understanding of the contribution of the colliculus to visual behavior, we can gain insights into its capacity to acquire other functions. Proposed Course One of the major interests in this Section is visual attention. We recently have discovered that saccadic eye movements can be initiated at very shon laten- cies. Under certain conditions, including manipula- tion of the state and direction of attention, saccadic eye movements can begin rapidly. These movements are termed "express saccades." Whereas our previ- ous studies have demonstrated that certain regions of tlie parietal cortex and pulvinar are related to visuo- spatial attention, our future studies will attempt to determine the conoibutions of the pulvinar and parietal cortex to express saccades. Rhesus monkeys will be trained on a variety of eye movement tasks that will reliably evoke express saccades. Subse- quently, we will electrically excite or chemically inactivate these portions of the brain to determine how their attentional mechanisms contribute to the initiation of saccadic eye movements. NEI Research Program Strabismus, Amblyopia, and Visual Processing — Visual Processing and Functional Organization (Structure and Function of Central Visual Pathways) Publications Bowman EM, Brown VJ, Kertzman C, Schwarz U, Robinson DL: Covert orienting of attention in macaques. I. Effects of behavioral context J Neurophysiol 70:431-443, 1993. Brown VJ, Schwarz U, Bowman EM, Fuhr P, Robinson DL, Hallett M: Dopamine dependent reaction time deficits in patients with Parkinson's disease are task specific. Neuropsychologia 31:459-469, 1993. Robinson DL: Functional contributions of the primate pulvinar. Prog Brain Res 95:371-380, 1993. Robinson DL, Cowie RJ: Attentional engagement and the pulvinar. Behav Brain Sci, in press. 240 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00109-13 LSR PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Visuomotor Processing in the Primate Brain PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Robert H. Wurtz Pli.D. Chief LSR, NEI Others: Charles J. Duffy Hiroshi Aizawa Gregg H. Recanzone M.D., Ph.D. Staff Fellow LSR, NEI Ph.D. Visiting Fellow LSR, NEI Ph.D. Guest Researcher LSR, NEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Sensorimotor Research SECTION Visuomotor Integration Section INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 3.5 PROFESSIONAL: 2.0 OTHER: 1.5 CHECK APPROPRIATE BOX(ES) [x| (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Saccadic or rapid eye movements shift the direction of gaze from one part of the visual field to another, but most of normal vision occurs during the period of visual fixation between these saccades. This year we tested the hypothesis that fixation cells in the rostral superior colliculus (SC) suppress the activity of saccade-related cells in the posterior colliculus and thus also suppress saccades. Electrical stimulation of fixation cells interrupted saccades and the burst of activity in the posterior colliculus that precedes saccades. Stimulation of the rostral pole also lengthened the interval between a series of saccades that result from stimulation of the posterior SC. Both results are consistent with the hypothesis that the fixation cells in the rostral SC regulate the generation of saccades. As we move through the environment, we generate a full-field visual motion — a pattern of optic flow. We have previously studied cells in the cerebral cortex of monkey that appear to be selective for such flow. This year we began experiments to test the extent to which monkeys respond to this visual flow stimuli. When the monkey stood on a platform that allowed postural changes, we could detect in the monkey a sway related to the optic flow. Vibration of the platform increased reliance on flow. These experiments demonstrate the sensitivity of the monkey to flow stimuli and open tlie way to testing the effect of removal of the flow sensitive cortical neurons on the use of optic flow stimuli. 241 PHS 6040 (Rev. 5/92) Laboratory of Sensorimotor Research NEI Annual Report— FY 1993 Project Description Objectives Saccadic eye movement shifts the eyes rapidly from one item of interest in the visuaJ field to another. The neuronal organization underlying this system has been studied intensively in the monJcey during the past 20 years, particularly in this Laboratory. Our work this year centered on a brainstem structure that is related to the generation of these saccadic eye movements — the superior coUiculus. As we move through the environment, we use the resulting motion of the visual field as a whole, referred to as "optic flow," to provide stabilization of posture and possible guidance of movement. We had previously studied the visual processing related to such control in an area of the cerebral cortex, the medial superior temporal region of the superior temporal sulcus. Our experiments this year concentrated on the demonstra- tion of the use of these stimuli by the monkey. Methods The Rhesus monkey, Macaca mulatta, is an incom- parable model of the visual system of humans. Its saccadic eye movements are nearly identical to those of humans. The response of cells to optic flow field stimulation suggests that the monkey also uses such full-field stimulation in its behavior, although this is less well understood. In the study of saccadic eye movements, the monkey is awake and trained to perform a visual motor task that involves making saccadic eye movements from one target to another. Because the cooperation of the monkey is required to obtain a high level of visual performance, the mon- key is rewarded for making these eye movements. The entire experiment is managed by an online computer that turns on the stimuli, rewards the monkey, collects the neuronal and behavioral data, and stores that data on magnetic disks for later computer analysis. In the experiment on the effects of full-field visual stimulation, the monkey is free to move within a cage; his posture is measured by strain gauges attached to a flat platform on which the monkey is trained to stand. Full-field visual stimula- tion is projected on one side of the cage, which the monkey faces. Major Findings We had determined previously that damage to the superior colliculus produces immediate deficits in the generation of saccadic eye movements. We infer, therefore, that cells within the colliculus are related to the generation of saccades. This year we com- pleted a detailed study of the types of cells within the superior colliculus. We had detailed the two types of cells related to the generation of saccades — (1) burst cells and (2) preparatory or buildup cells— in last year's annual report This year we completed experiments and analysis on the remaining major cell type within the superior colliculus — fixation cells. These cells do not discharge with a saccadic eye movement. Rather, they increase their discharge rate when the monkey actively fixates on an object and decrease their discharge during saccadic eye move- ments. The period of this decrease is roughly equivalent to the duradon of the saccadic eye move- ment, suggesting that the pause of these cells is necessary for the generation of the saccadic eye movement. Two studies demonstrated that this interaction between fixation cells and saccade-related cells does occur. In the first, we electrically stimulated the fixation cells while recording both the eye movement and discharge of the saccade-related cells within the colliculus. We found that the stimulation of the fixation zone interrupted the saccadic eye movement and reduced the discharge of the saccade-related cells. The reduction in activity, a presumed indica- tion of inhibition, was greater for the burst cells than for the buildup cells. This action on the saccade cells is consistent witii our hypothesis that the fixation cells within the colliculus act to inhibit tiie generation of saccadic eye movements while the monkey is actively fixating. The second set of experiments on the action of tile fixation cells was to test tiie effect of tiieir stimulation on tiie generation of a "staircase" of saccades. Ttiis staircase occurs when the saccade- related cells of tiie superior colliculus are stimulated over a period of several hundreds of milliseconds. The result of tiiis stimulation is a series of individual saccades witii a pause between each saccade. 242 NEI Annual Report— FY 1993 Laboratory of Sensorimotor Research Why there would be such a series of identical saccades rather than one saccade alone has been a puzzle for more than 20 years. One explanation might be that, when the saccade-related cells are stimulated, they are active in the absence of a pause in the fixation cells such that the eye is not held in its new position after the saccade by the activity of the fixation cells. To test this notion, we electrically stimulated the fixation zone to see whether it altered the time between saccades; it did. As we stimulated the fixation cells, we increased the period between successive saccades in the staircase, indicating that it was the activity in the fixation zone that determined the spacing between the saccades. This finding is also consistent with the hypothesis that the fixation cells, when active, inhibit the generation of saccadic eye movements. Our second set of investigafions on the effect of large-field visual stimulation has concentrated not so much on the activity of cells within this area but on the consequence of the visual stimulation to the monkey's behavior. One of the goals of our research is not only to determine the relationship of cell activity to visual stimulation and behavior but to determine whether removal of these cells leads to a change in visual motor behavior. Thus, in the current set of experiments, we have begun to test the effect of the large-field stimulation on the monkey's behavior in order to eventually test whether removal of these cells affects that behavior. Our strategy has been to use the well-known effect of full-field visual stimulation on human posture. Whether such stimulation is used by mon- keys has never been determined. We measured the monkey's posture while full-field stimulation was given by using strain gauges attached to a platform on which the monkey was trained to stand. We oscillated the full-field motion over a period of several seconds and observed a swaying motion of the monkey synchronous with the moving visual field. We were struck, however, that the monkey was able to compensate, using other mechanisms, so that repeated presentations of the visual stimulation no longer produced the sway. One mechanism that could contribute to this compensation is proprioception, the sense that indicates the position of muscles and joints. To reduce this sensation in the monkey, we attached a low-frequency vibrator to the platform, thereby reducing the amount of information proprioception and increasing the monkey's sensitivity to visual stimulation. This increased sensitivity persisted over time. These experiments seem to show that (1) mon- keys are sensitive to ftill-field stimulation, as are humans, and (2) the use of the visual stimuli is coupled with the use of proprioception, and probably vestibular sensation, to control posture. Significance to Biomedical Research and the Program of the Institute The saccadic eye movement system usually has been taken in isolation as a system that moves the eye from one point of the visual field to another. Our studies on the fixation cells within the superior colliculus and their effect on the generation of saccadic eye movements demonstrate that a system for visual fixation is as important as the generation of saccades. Thus, the saccade moves the eye from one point to another, and the fixation system holds the eye in position. All of our vision results from the period of fixation, so that understanding this fixation system is essential to understanding normal vision. Just as the study of this visual fixation is not generally studied in primates, it is not frequentiy smdied in the clinic. We hope that clarification of the interaction of the saccadic and fixation systems in the monkey will stimulate analysis of fixation in the human as well. The effect of full-field or optic flow stimulation smdied in the monkey on postural responses attempts to establish the monkey as a model of the effect of vision on postural control. These experiments move the study of the monkey from one of just eye move- ment control to one of control of movement within tiie experiment, and it is increasingly recognized that one very important aspect of the use of visual stimulation is the control of movement in the envi- ronment, including posture. Our experiment estab- lished the monkey as a model for the study of these systems. Again in humans, damage to the parietal cortex is known to produce certain types of disorien- tation, and our studies in the monkey open the possibility of dissecting the types of disorientation, particularly those related to full-field visual stimula- tion. Proposed Course In the analysis of the fixation and saccadic systems, a major next step will be the development of a precise model of the saccadic-fixation control system. 243 Laboratory of Sensorimotor Research NEI Annual Report— FY 1993 This research, to be conducted in this Laboratory in collaboration with Dr. Lance Optican, of LSR, will serve a heuristic function — summarizing the explo- sive growth of knowledge of the superior colliculus in the past several years. It also will produce a computer-based simulation that will allow tests not only on the visual saccadic-fixation system of the monkey but on that of the human. In experiments on full-field visual stimulation, we will attempt to determine the effect of damage to the cortical areas most likely to be related to the monkey's use of optic flow. These experiments will indicate whether we can identify a specific system that relates optic flow to the behavioral use of this optic flow. NEI Research Program Strabismus, Ambylopia, and Visual Processing — Visual Processing and Functional Organization (Structure and Function of Central Visual Pathways) Publications Duffy CJ, Wurtz RH: An illusory transformation of optic flow fields. Vis Res 33:1481-1490, 1993. Munoz DP, Wurtz RH: Role of the rostral superior colliculus in active visual fixation and execution of express saccades. J Neurophysiol 67:1000- 1002, 1992. Munoz DP, Wurtz RH: Fixation cells in the monkey superior colliculus. I. Characteristics of fixation cells. J Neurophysiol, 70:559-575, 1993. Munoz DP, Wurtz RH: Fixation cells in the monkey superior colliculus. II. Reversible activation and deactivation. J Neurophysiol, 70:576-589, 1993. Wurtz RH, Munoz DP: Role of monkey superior colliculus in control of saccades and fixation. Cog Neurosci, in press. 244 Ophthalmic Genetics and Clinical Services Branch Report of the Acting Chief, Ophthalmic Genetics and CUnical Services Branch Muriel I. Kaiser-Kupfer, M.D. The Ophthalmic Genetics and Clinical Services Branch within the National Eye Institute (NEI) Intramural Research Program has been operational since February 1989. The Branch is organized into four sections: Ophthalmic Genetics, Acting Chief Muriel I. Kaiser-Kupfer, M.D.; Cataract and Corneal Diseases, Chief Manuel B. Datiles, M.D.; Ophthal- mic Pathology, Acting Chief W. Gerald Robison, Jr., M.D., Ph.D.; and Clinical Services, Acting Chief Rafael C. Caruso, M.D. The purpose of the Branch is to conduct clinical and laboratory research on gene expression and molecular interactions important to the eye and to apply clinically relevant research findings to the prevention, diagnosis, and treatment of diseases affecting the eye and visual system. Such disorders include corneal and retinal diseases, cataract, and visual pathway abnormalities. The Branch is responsible for the essential psychophysical and electrophysiological diagnostic tests of visual function required by clinical intra- mural research programs of all the Institutes. In addition, it processes ocular clinical biopsy and autopsy materials. The Branch differs from other NEI laboratories engaged in molecular investigations because its emphasis is the translation of appropriate research findings directly to the clinical setting. This Branch is also a point of focus for the trans-National Institutes of Health (NIH) emphasis on research in genetics, more effectively aligning its organizational structure within the Institute's Intramural Research Program. Since beginning its operation, the Branch has shown considerable growth and productivity. Section on Cataract and Corneal Diseases The Section on Cataract and Corneal Diseases continued to pursue research on the anterior segment, especially the short- and long-term effects of contact lens wear on the cornea. Analysis of the data may be helpful in understanding the dynamics of contact lens-cornea interaction, the risk to corneal tissues, and how systemic or local ocular disorders may increase the risk of wearing contact lenses. Corneal endothelial morphology is being studied by specular microscopy to compare the endothelial status in patients wearing different types of lenses. The development of automated computer analysis is under way to facilitate data analysis, which currently is performed by hand and is therefore time consum- ing and laborious. This Section has been particularly productive in studies using different systems to develop objective and subjective methods of monitoring and document- ing opacities in the human lens. Reproducibility studies on objective systems include the use of the Scheimpflug cameras (Zeiss and Oxford) and the retroillumination cameras (Neitz and Oxford). Sub- jective systems or methods — such as the LOCS n grading system and the effects of cataracts on visual perception, contrast sensitivity, and glare — may be useful in identifying additional parameters. These systems are being used to study the natural history of various cataracts, such as presenile, senile or age- related, steroid-induced, radiation, diabetic, retinitis pigmentosa, gyrate atrophy (GA), and neurofibroma- tosis 2 (NF2). Genetic linkage studies are under way to pursue the gene(s) of congenital cataracts. Moni- toring and documenting human cataract development is a crucial step toward the ultimate testing of several medications that might be helpful in preventing or reversing human cataracts. Research in cataractogenesis has been hampered by the extreme scarcity of tissue and an abrupt shift in surgical technique, from intracapsular (intact lens) to extracapsular (fragmented lens). Through the collaborative efforts of cataract surgeons and basic researchers, efforts are under way to develop and modify techniques to study materials that become available at surgery and can be well documented clinically. We are carefully documenting the cataract 247 Ophthalmic Genetics and Clinical Services Branch NEI Annual Report— FY 1993 preoperative! y, using clinical and photographic LOGS II grading, as well as Zeiss Scheimpflug and Oxford retroilluniination videophotography and image analysis. Cataracts are extracted extracapsularly, followed immediately by implantation of intraocular lenses. Specimens obtained are examined histologi- cally via light and electron microscopy and biochem- ically via two-dimensional gel electrophoresis (PHAST and LSB systems). Cataractous specimens are compared with normal tissues obtained from eye bank eyes. Abnormal proteins are identified by immunoblotting techniques, as well as by protein sequencing. It has been demonstrated that with aging there is an acidic shift of proteins and an increased number of polypeptide species in the molecular weight range of the crystallins. These aging changes need to be differentiated from changes occurring in cataract formation. Investigators in this Section have been in the forefiront of recognizing the role of the neural crest in normal and abnormal development of the anterior segment. Studies continue on anterior chamber abnormalities and iridocorneal endothelial syndrome patients. Section on Ophthalmic Genetics Studies by the Ophthalmic Genetics Section have emphasized retinal degeneration and ophthalmic involvement in systemic genetic diseases. This Section has been a leader in studying GA of the choroid and retina The accumulation of natural history data and the work on definition of the genetic abnormalities have been unique. Evidence for bio- chemical, clinical, and molecular heterogeneity continues to be confirmed. There appear to be many different single-point mutations in the ornithine aminotransferase gene in GA patients. Dietary intervention studies utilizing an arginine deficient diet have been promising, especially in young patients, in whom a delay in the onset of pathologic changes has been demonstrated. Foveal cone sensitivity (assessed by measurement of increment thresholds) and orientation (esfimated by measurement of the Stiles-Crawford effect) were found to be abnormal in a group of patients with GA. These results suggest that foveal cones are altered in their orientation and sensitivity before the encroachment on the foveal area by the atrophic lesions of GA. Albinism has been associated in animals with an anatomic anomaly of the visual pathways character- ized by excessive crossing of the retinogeniculate fibers, with two different modes of geniculocortical projection. In humans, indirect evidence of the same anomaly is demonstrated by asymmetry in visually evoked potentials (VEPs) elicited by pattern-reversal stimulation. Recent studies using appearing-disap- pearing patterns claimed VEP asymmetry to be diagnostic and proposed a uniform type of asym- metry. We used the same recording conditions to determine the diagnostic value of VEP in albinism and to attempt to correlate the VEP results with clinical features. This study shows that there are two different patterns of VEP asymmetry in albinism, which may be explained by differences in reorganization of the geniculocortical pathway. VEP asymmetry occurs frequently but may not be constant in this condition. However, its value is decreased in some cases in which the low amplitude of the responses makes interpretation difficult. Furthermore, there is no correlation of the type of asymmetry with any other feature of albinism. Collaboration with the Interinstitute Genetics Program has continued, with active participation by the Genetics Clinic. During the past year, we have seen approximately 200 individuals representing approximately 60 different disease categories. Be- cause of the high frequency of ocular involvement in these cases, almost all of these patients were evalu- ated by the Ophthalmic Genetics staff. NF2, otherwise known as bilateral acoustic neuroma, is inherited as an autosomal dominant disorder. Multiple members of several large pedi- grees, as well as a large number of unrelated fami- lies, have been studied in collaboration with Dr. Dilys Parry (National Cancer Institute). An important original observation was the striking frequency of posterior capsular cataract in patients with NF2 (BO- SS %). In addition, 30% of the patients have shown associated cortical cataracts. These findings are helpful in establishing a diagnosis of NF2 in at-risk patients. The etiology of die cataract is unclear; however, it is interesting that the gene locus for bilateral acoustic neuromas is on chromosome 22, as is the gene for pB -crystalline. Combined pigment 248 NEI Annual Report— FY 1993 Ophthalmic Genetics and Clinical Services Branch epithelial and retinal hamartomas appear to be another ocular marker for some patients with severe NF2. GA is a condition amenable to gene therapy; preliminary laboratory studies are under way toward that goal. Usher's syndrome, congenital deafness, and retinitis pigmentosa patients are being studied; molecular techniques are used to map the gene and to identify the responsible mutation. Finally, the results from the continuing double- masked, controlled clinical trial of topical cysteamine patients with nephropathic cystinosis are exciting. Since confirming the usefulness of 0.5% cysteamine eye drops in the young patients, we expanded our study to include older patients, with similarly striking results. Particularly important is the fact that these patients have shown dramatic relief from their ocular symptoms, with a decrease in crystals in the treated eye and a significant improvement in their quality of Ufe. The results of VEP studies showed that two waveforms, which frequently show the same surface- positive polarity but are generated by stimulation of each hemifield, combine to generate peaks of the full-field VEP. Our results indicate that the sum of the asymmet- rical contributions of both eyes (either hemisphere of each) is responsible for the symmetrical VEP elicited by binocular stimulation with a full-field stimulus. An asynmietrical, full-field VEP may occur in normal subjects and does not imply an abnormality in the visual pathways. Studies of dark adaption (DA) in patients with retinal dystrophies indicated that a complete evalua- tion of DA should include, in addition to measure- ment of DA, the time constant of adaptation, which provides information about the rate at which this final threshold is reached. The time constant serves as a clinically relevant parameter in both the diagno- sis of retinopathies and the followup of individual patients over time. Section on Clinical Services The Clinical Services Section has been active in characterizing psychophysical and electrophysio- logical findings in patients with diseases that affect the eye and the visual system. Continued documenta- tion by noninvasive techniques has shown that more and more refined and accurate classification of diseases is possible. Psychophysical and electrophysi- ological information is particularly helpful in under- standing the pathogenesis of disease, as well as being available for use as a marker in various treatment modalities. Section on Ophthalmic Pathology The Section on Ophthalmic Pathology has pro- vided technical support services to investigators involved in clinical and basic research as well as to those performing routine pathology. Careftil monitor- ing of the volume of material handled shows a steady increase in processing by the Laboratory, with excellent results. Considerable savings to the Institute have resulted from the elimination of costly contract services. 249 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1, 1992 to September 30. 1993 PROJECT NUMBER ZOl EY 00187-10 OGCSB TITLE OF PROJECT (80 cheracters or less Tillg must lit on one line between the borders ) The Effects of Corneal Contact Lenses on the Cornea PRINCIPAL INVESTIGATOR (List other prolessional personnel below tne Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Manuel B. Datiles M.D. Medical Officer OGCSB, NEI Others: Gregory P. Kracher O.D. Expert OGCSB, NEI Lessie McCain R.N. Nurse Specialist OGCSB, NEI Louella Lopez M.D. Visiting Associate OGCSB, NEI Doretha Leftwood B.A. Computer Specialist OGCSB, NEI Anup Mahurkar B.S. Visiting Associate OGCSB, NEI COOPERATING UNITS (if any) Image Processing and Analysis Laboratory, Division of Computer Research and Technology NIH (Mark Vivino, B.S.) LAB/BRANCH I Ophthalmic Genetics and Clinical Services Branch SECTION Section on Cataract and Corneal Diseases INSTITUTE AND LOCATION NEI. NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.575 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews PROFESSIONAL; 0.450 OTHER: 0.125 n (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This investigation of the short-term as well as the long-term effects of contact lens wear on the cornea includes specular microscopy smdies of changes in corneal curvature, corneal epitheUal morphology, and corneal endothehal cell morphology. Analysis of the data obtained will help us understand the dynamics involved in Uie mteraction between a contact lens and the cornea, the risk to corneal tissues, and how a systemic or local disorder may increase these risks. In addition, we are studying the differences in corneal endothelial status or wearers of soft compared with hard contact lenses. Animal models showing corneal endothelial abnormalities similar to those in long-term contact lens wearers also are bemg explored in diabetic and galactosemic animal models. Treatment with aldose reductase inhibitors helps prevent these corneal abnormalities. 250 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Ophthalmic Genetics and Clinical Services Branch Project Description Clinical Protocol Number 84 EI-133 Objectives The objective of this project is to investigate the effects of contact lens wear on corneal tissues, including the study of factors that increase or de- crease the potential risk of injury to corneal tissues by contact lens wear. Methods We used each patient's complete history, ophthalmo- logic examination, photography, keratotometry, pachymetry, and specular microscopy of the corneal endothelium. Major Findings We have found that diabetes may increase the risk of complications from contact lenses in the first 6 months of wear. In addition, we have found changes in the corneal endothelium after long-term wear of contact lenses. These changes include polymegathism and pleomorphism. Furthermore, 2 years after some of our patients discontinued wearing contact lenses, we found a trend toward recovery but no statistically significant change. In addition, we found that diabetic and galactos- emic animals have these endothelial abnormalities and that treatment with aldose reductase inhibitors prevented these abnormalities. Significance to Biomedical Research and the Program of the Institute Contact lenses are commonly used for correction of errors of refraction as well as for ther^y. However, our knowledge of the interaction of contact lenses with the cornea and tears is limited. In addition, risks associated with wearing contact lenses are poorly understood. Understanding the interaction between contact lenses and corneal tissues will allow us to determine why some patients cannot wear contact lenses and provide methods to avoid some of the complications associated with contact lens wear. Proposed Course The following studies are in progress or proposed for next year: (1) continued examination of patients who have worn hard contact lenses and have now shifted to gas-permeable or soft contact lenses, (2) recruit- ment of patients who plan to discontinue contact lens wear or to shift from one type of contact lens to another; and (3) development of automated computer analysis of all types to facilitate the analysis of data. NEI Research Program Corneal Diseases — Corneal Edema, Endothelial Dysfunction, Dystrophies and Inherited Disease 251 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00188-10 OGCSB PERIOD COVERED October 1. 1992 to September 30. 1993 TITLE OF PROJECT (80 cnaraclers or less. Title must tit on one line between the borders.) Documentation and Monitoring of Opacities in the Human Lens PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attlliation) PI: Manuel B. Datiles M.D. Medical Officer OGCSB, NEI Others: Benjamin Magno M.D. Maria Susan Lasa M.D. Louella Lopez M.D. Anup Mahurkar B.S. Doretha Leftwood B.A. Joan Lee Visiting Associate Visiting Associate Visiting Associate Visiting Associate Computer Specialist Clinic Coordinator OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (il any) Image Processing and Analysis Laboratory, Division of Computer Research and Technology (DCRT), NIH (Benes Trus, Ph.D.; Mark Vivino, B.S.); Biomedical, Engineering and Instrumentation Branch. DCRT, NIH (Michael Unser, Ph.D.); Epidemiology Branch, NEI NIH (Michael Unser, Ph.D.); Clinical and Diagnostic Trials Section, National Cancer Institute, NIH (Sylvan Green, M.D.); Nuclear Medicine, CImical Center. NIH (Joseph Frank, M.D.) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Cataract and Corneal Diseases INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.725 PROFESSIONAL: 2.30 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews OTHER: 1.425 n (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project uses different systems to develop objective and subjective methods to monitor and document opacities in the human lens. We are actively recruiting patients with and without cataracts for reproducibility studies on the objective systems— the Scheimpflug (Zeiss and Oxford) and retroillumination (Neitz and Oxford) cameras. Our study of subjective systems or methods, such as the LOGS H grading system, and the effects of cataracts on visual perception, contrast sensitivity, and glare may be useful in identifying additional parameters for monitoring cataract presence, progression, or regression. We are now using these systems to study the natural history of various cataracts, such as presenile, senile, or age-related, steroid-induced, radiation, diabetic, retiniUs pigmentosa, gyrate atrophy, and neurofibromatosis U cataracts. This study will prepare the way for eventual clinical trials of anticataract drugs. Genetic linkage studies under way are pursuing tlie gene(s) of congenital cataract 252 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Ophthalmic Genetics and Clinical Services Branch Project Description Additional Personnel Malina A. Drews- M.D. Visiting Fellow, Bankiewicz OGCSB, NEI Yvonne Duglas-Tabor B.S. Biologist, OGCSB, NEI Marvin Podgor M.D. Epidemiologist, NEI Robert Sperduto M.D. Chief, Epidemiology Section, NEI Laura Wozencraft B.S. Genetic Counselor, OGCSB, NEI Mark H. Scott M.D. Senior Staff Fellow, OGCSB, NEI J. Fielding M.D., Staff Scientist Hejtmancik Ph.D. LMOD, NEI Clinical Protocol Number 84-EI-132 Objectives The objective of this project is to formulate means of documenting cataract formation and progression. This is an important step prior to undertaking clinical trials of drugs purported to prevent cataract and cataract progression. Family studies are involved in looking for the gene for congenital cataract via linkage studies. Methods Complete ophthalmologic examination, including contrast sensitivity, glare testing, and potential acuity testing, are performed for each person in the study. Techniques used to measure and evaluate cataracts include Scheimpflug photography, retroillumination photogr^hy, specular microscopy, and laser light- scanning spectroscopy. Major Findings We have found that clinical grading of cataracts using the LOCS II system is a means of quantitative- ly detecting the progression of age-related cataracts within 1 year. In addition, we found that in various types of cataracts, glare and contrast sensitivity testing show abnormal results only in the severe or more advanced grades. The only exception was in posterior subcapsular cataracts, which showed an ab- normality in contrast and glare sensitivity in the early stages, based on LOCS II grading. In a study of pure nuclear cataracts, we found a significant correlation between lens nuclear density (assessed by either LOCS II grading or Scheimpflug photography) and contrast sensitivity loss of intermediate and high spatial frequencies. In our continued development of objective, semiautomated methods of detecting and following cataracts, we now are able to perform quickly densi- tometry of Scheimpflug nuclear cataract images and compare the results with previous images to detect significant changes, which are expressed in optical density units. For posterior subcapsular and cortical cataract, we also have developed a semiautomated method of quantitating the cataracts in square milli- meters using retroillumination photography. Significance to Biomedical Research and the Program of the Institute The monitoring and documenting of human cataract progression is a crucial step toward the ultimate testing of several medications believed capable of preventing or reversing human cataracts. This step is also important in categorizing types of cataracts in various parts of the world and correlating them with physical and genetic factors iii specific geographic regions. Subjective methods of determining visual function are also important to determine the degree of handi- cap that cataract patients have in coping with daily activities. Since in our studies none of the subjective methods could demonstrate subjective experiences in association with early cataracts, research is needed to develop more sensitive techniques. Proposed Course We will continue the study and development of subjective and objective methods of documenting and monitoring human cataracts. We will pursue the improvement and automation of the present system of lens photography (e.g., Scheimpflug, retroillu- mination, and laser-light spectroscopy), as well as exploration of possible applications of new techno- logical advances. Appropriate population groups for study will be identified. A^£/ Research Program Cataracts — Epidemiology of Cataract 253 Ophthalmic Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Publications Datiles M: Clinical evaluation of cataracts, in Tasman W, Jaeger E (eds): Duane's Clinical Ophthalmology. Philadelphia, Lippincon Co., 1992, Vol. I 73B. pp 1-16. Datiles M, Magno B, Leftwood D, Friedlin V, Vivino M: Longitudinal study of age related nuclear cataracts using the NEI Scheimpflug Imaging System. Investig Ophthalmol Vis Sci 34:4a, 943, 1993. Fox PC, Datiles M. Atkinson JC, Scott J, Fletcher D, Valdez IH, et al: Prednisone and piroxicam for treatment of primary Sjogren's syndrome. Clin Exp Rheumatol 11:149-156, 1993. Genhart M, Kelly K, Coursey R, Datiles M, Rosen- thal N: Effects of bright light on mood in normal elderly women. Psychiatry Res 41 -.SI -91, 1993. Kashima K, Trus BL, Unser M, Datiles MB, Ed- wards PA, Sibug M: Aging studies on normal volunteer lenses using the Scheimpflug slit lamp camera. Invest Ophthalmol Vis Sci 34:263-269, 1993. Kashima K, Unser M, Datiles MB, Trus BL, Ed- wards PA: Minimum views required to charac- terize cataracts when using the Scheimpflug camera. Graef Arch Ophthalmol, 231: 687-691, 1993. Lasa S, Datiles MB: Longitudinal study of cortical cataracts using retroillumination photographs. Investig Ophthalmol Vis Sci 34:4a, 943, 1993. Lasa S, Podgor M, Datiles M, Magno B: Glare sensi- tivity in early cataracts. Br J Ophthalmol 77:489- 491, 1993. Lopez ML, Datiles M: Longitudinal study of age related posterior subcapsular opacities using ret- roillumination photographs. Investig Opthalmol Vis Sci 34:4a, 943, 1993. Magno B, Datiles M, Friedlin V: Reproducibility of the NEI Scheimpflug Cataract Imaging System. Investig Ophthalmol Vis Sci 34:4a, 943, 1993. Magno BL, Datiles MB, Lasa SM: Senile cataract progression studies using the Lens Opacity Clas- sification System. Invest Ophthalmol Vis Sci 34:2138-2141, 1993. 254 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00212-08 OGCSB PERIOD COVERED October 1, 1992 to September 30. 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Use of Human Lens Material for Determining Possible Causes of Cataracts PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Manuel B. Datiles M.D. Medical Officer OGCSB, NEI Others: Maria Susan Lasa M.D. Benjamin Magno M.D. Yvonne Tabor B.S. Louella Lopez M.D. I*ushpa Sran M.D. Visiting Associate Visiting Associate Biological Technician Visiting Associate Medical Officer OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) Laboratory of Mechanisms of Ocular Diseases, NEI CDonita Garland, Ph.D.); Laboratory of Immunology, NEI (Miguel Bumier, Jr., M.D.) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Cataract and Corneal Diseases INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 3.05 PROFESSIONAL: 1.75 OTHER: 1.30 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) There is an extreme scarcity of properly documented and classified human cataract material because of an abrupt shift of cataract surgical technique from intracapsular (intact lens) to extracapsular (fragmented lens) with the advent of the use of intraocular lenses. We are exploring ways by which fragmented lens materials can be maximally used in cataract basic research through close collaboration with cataract surgeons and basic researchers and through modification of techniques by both groups. We are now carefully documenting the cataracts preoperatively, using clinical and photographic LOCS II grading and Scheimpflug (Zeiss and Oxford) retroillumination (Oxford) video photography and image analysis. Cataracts are extracted extracapsularly with implantation of an intraocular lens. Specimens obtained are examined histologically, using light and electron microscopy, and biochemically, using two-dimensional gel elecfrophoresis (PHAST and LSB systems). Cataractous specimens are compared with normal tissues obtained from eye bank eyes. Abnormal proteins are identified using immunoblotting techniques as well as protein sequencing. 255 PHS 6040 (Rev. 5/92) Ophtbalmjc Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Additional Personnel Samuel Zigler Ph.D. Head, Section on Cataracts, LMOD, NEI Clinical Protocol Number 84-EI-194 Objectives In light of the present and future scarcity of intact human cataracts for basic research, this study was designed to develop methods in which fragmented lens materials can be maximally used for biochemical and genetic research. Methods We examined patients who have different forms of cataracts and documented their cataracts, as described in project ZOl EY 00188-10, Documentation and Monitoring of Opacities in the Human Lens. After extracapsular cataract extraction, fragmented lens materials — which include the anterior lens capsule, lens nucleus, and aspirated lens cortical material — undergo protein and biochemical analyses. Some of the specimens are frozen for futtire genetic studies. We also have undertaken studies to determine the visual results of current techniques of cataract surgery and how to modify and improve them further. Major Findings We have found that a successful collaboration requires a close professional relationship between the clinician-surgeon and the basic researcher. Although the two professionals have different immediate priorities (one being patient care; the other, adequacy of tissue samples for laboratory studies), the ultimate goal of alleviating human suffering is the same. The two-dimensional gel electrophoresis technique may be extremely useful in determining lens protein changes using very small amounts of tissue (300 meg). Aging results in acidic shifting of proteins, an increased number of polypeptide species in the molecular weight range of the crystallins, and cova- lent cross-linking of crystallins — changes that need to be differentiated from changes occurring in cataract formation. We also have found that lens material aspirated through the irrigator-aspirator or phako- emulsifier lose some crystallins; optimum samples are those we obtain separately, thus avoiding oxidation. Significance to Biomedical Research and the Program of the Institute A severe setback is being dealt many cataract proj- ects because of the lack of human cataract material available for basic research studies. While the current technique, which involves fragmenting the cataract during extraction, is extremely successful and effec- tive, there is no foreseeable change back to utiliza- tion of the intracapsular method (i.e., removal of the lens in toto). Hence, it is imperative that we modify our basic research methodology to adapt to the use of fragmented lens materials in order to continue basic lens research projects that deal with human materials. Proposed Course We will continue to pursue the development of the use of firagmented lens material in basic research experiments. Using two-dimensional gel electro- phoresis, we will study ways in which surgeons can modify their surgical techniques without compromis- ing patient care while providing scientists with critical lens tissue for basic research. In addition, we will investigate ways in which scientists can work with surgeons in handling lens materials to maximize the quality of specimens for basic research. NEI Research Program Cataract — ^Treatment of Cataract and Correction of Aphakia Publications Datiles M, Kinoshita J: Pathogenesis of cataracts, in Tasman W, Jaeger E (eds): Duane's Clinical Ophthalmology. Philadelphia, Lippincott Co., 1991, 72B, pp 1-14. Garland D, Tabor Y, Zigler JS, Datiles MB: Protein analysis of lens cortical aspirates obtained at surgery. Investig Ophthalmol Vis Sci 34:4a, 1335, 1993. 256 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00281-01 OGCSB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (BO characters or less. Title must lit or) one \ir\e between the borders.) Addendum to Use of Human Lens Material for Determining Possible Causes of Cataracts PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alliliation) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEl Others: J. Fielding Hejtmancik Mark H. Scott M.D., Ph.D. M.D. Senior Staff Fellow LMOD, NET OGCSB, NEl COOPERATING UNITS (it any) Marshall Parks, M.D. (Private Practice), Washington, DC LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Cataract and Corneal Diseases INSTITUTE AND LOCATION NEl, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.325 PROFESSIONAL: 1.325 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects jx] (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Although the etiologies of some secondary cataracts are becoming better understood and certain animal models show promise for elucidating the relationships between lens crystalline and hereditary cataract, littie is known about the causes of congenital cataracts in humans. To date, the classification of different congenital cataracts has been cumbersome and imperfect. A better understanding of cataractogenesis will come through an understanding of the molecular components of the lens of the eye and the ways in which lesions of these components are manifested, structurally and functionally, as opacities of the lens. Animal studies have suggested that alterations in lens crystalline can cause hereditary cataracts, making them reasonable candidate genes for causing hereditary cataracts in humans. In addition, it is ^parent that hereditary lesions that mimic or contribute additively to envirormiental stress known to cause cataracts might be candidate genes for causing hereditary cataracts. The work in this project is designed to concentrate specifically on congenital and hereditary cataracts and to take full advantage of molecular technology developed for linkage analysis. Studies performed on informative families will include collecting blood specimens from available family members and, when possible, analyzing lens material from patients who undergo cataract surgery. 257 PHS 6040 (Rev. 5/92) Ophtbalmjc Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Clinical Protocol Number 84 EI -194 A Objectives The objective of this study is to do linkage analysis using molecular genetic techniques in families with congenital hereditary cataracts. Methods Linkage analysis (i.e., gene mapping) will be carried out by following the inheritance of genetic markers in families with hereditary cataracts. In informative families, blood specimens will be obtained and analyzed for gene marker linkage analysis. Major Findings Under way at present is the enrollment of families with congenital cataracts. A large family with known autosomal dominant congenital cataract has been analyzed. This analysis is the first to provide evidence of intraocular phenotypic heterogeneity in a family with autosomal dominant congenital cata- ract. Studies for markers for gene analysis are under way. Significance to Biomedical Research and the Program of the Institute By studying patients with congenital inherited cataract, it may be possible to find the specific gene responsible for the development of congenital cataracts. Proposed Course More famiUes who have congenital cataract will be recruited, and linkage analysis will be performed on these families. NEI Research Program Cataract — Treatment of Cataract and Correction of Aphakia Publications Hejtmancik JF, Kaiser-Kupfer MI, Piatigorsky J: Molecular biology and inherited disorders of the eye lens, in Scriver CR, Beaudet AL, Sly WS, Valle D (eds): Metabolics Basics of Inherited Disease. McGraw-Hill Inc., submitted. Scott MH, Hejtmancik JF, Wozencraft LA, Reuter LM, Parks MM, Kaiser-Kupfer MI: Autosomal dominant congenital cataract: Interocular pheno- typic heterogeneity. Ophthalmology, submitted. 258 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00084-15 OGCSB PERIOD COVERED October 1. 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or /ess. Title must fit on one tine between the borders.) Anterior Chamber Anomalies Associated With Glaucoma or Ocular Hypertension PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Carl Kupfer M.D. Director NEI Others: Muriel I. Kaiser-Kupfer Lessie McCain Manuel B. Datiles Maria Susan Lasa Benjamin V. Magno Louella Lopez M.D. Chief OGCSB, NEI R.N. Nurse Specialist OGCSB, NEI M.D. Medical Officer OGCSB, NEI M.D. Visiting Associate OGCSB, NEI M.D. Visiting Associate OGCSB, NEI M.D. Visiting Associate OGCSB, NEI COOPERATING UNITS (if any) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Cataract and Corneal Diseases INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.0 PROFESSIONAL: 0.7 OTHER: 0.3 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Recent embryological research has indicated the role of the neural crest in contributing to all coimective tissues anterior to the lens epithelium. Therefore, the group of developmental anomalies of the anterior chamber with glaucoma or ocular hypertension is being reviewed. 259 PHS 6040 (Rev. 5/92) Ophtbalmjc Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Clinical Protocol Number 77-EI-119 Objectives The object of this study is to determine whether congenita] or developmental anomalies of the anteri- or chamber are related to faulty migration or terminal differentiation of neural crest tissue. Methods Patients of all ages with congenital or developmental anomalies of the anterior chamber are being exam- ined to determine involvement of cornea, trabecular meshwork, iris stroma, lens, and ciliary body. When intractable glaucoma cannot be controlled with medication, surgery is performed and the specimens are examined histologically. When the lenses become cataractous, cataract extractions are performed and the lens epithelium is grown in tissue culture. When the cornea is opaque and corneal transplantation is indicated, that procedure is performed and the corneal specimen is examined histologically. Major Findings It appears that, in this group of anomalies of anterior chamber development, there are pathological changes in one or several tissues derived from neural crest. These changes include corneal stroma, corneal endothelium, anterior iris stroma, Descemet's mem- brane, and trabecular meshwork endothelium. We recently performed trabeculectomies on patients with the irido-comeal-endothelial syndrome. Histopathologically, we found the presence of a membrane covering the trabecular meshwork. That membrane may have caused the peripheral anterior synechias and glaucoma. Significance to Biomedical Research and the Program of the Institute A better understanding of the pathogenesis of this glaucoma may help by improving diagnosis and treatment The presence of this membrane may explain the glaucoma's progressive nature and suggest possible surgical or laser treatments as a way to control or prevent the progression of the disease. Proposed Course Patients with other anomalies Of the anterior cham- ber, including congenital cataracts, will be examined for abnormalities in tissue derived from neural crests. We will continue to study cases of congenital corneal disorders to uncover the cause and to determine treatment choices for these cases. NEI Research Program Glaucoma— Other Glaucoma (Developmental, Con- genital, and Infantile Glaucoma) Publications Kupfer C, Chan C-C, Bumier M Jr, Kaiser-Kupfer MI: Histopathology of the ICE syndrome. Trans Am Ophthalmol Sac 90:149-160, 1992. 260 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00123-13 OGCSB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (BO characters or less. Title must lit on one line between the borders.) Clinical Psychophysics of the Visual System PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Rafael Caruso M.D. Visiting Scientist OGCSB, NEI Others: Muriel I. Kaiser-Kupfer M.D. Malina Drews-Bankiewicz M.D. Doris J. Collie A.A. Patricia A. Mercer M.P.A. Leanne M. Reuter B.S. Chief Visiting Associate Ophthalmic Technician Ophthalmic Technician Ophthalmic Technician OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Eye Services Section INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.49 PROFESSIONAL: 0.41 OTHER: 1.08 CHECK APPROPRIATE BOX{ES) |x| (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The visual function of patients with ocular diseases or lesions in the visual pathways and of normal subjects is measured using psychophysical techniques. The data are correlated with those obtained with electrophysiological tests of visual fiinction. The results contribute to the diagnosis of ocular and neural disorders that affect vision and are needed to characterize their nature and evolution. They are also valuable in the assessment of how different forms of treatment affect the outcome of these diseases. 261 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Clinical Protocol Number 93-EI-0131 Objectives The aims of this project are (1) to apply and develop psychophysical techniques for the study of vision in the clinical setting, (2) to characterize the human visual system's normal function, and (3) to analyze the patterns of its alteration in ocular diseases and lesions of the visual pathways. Methods Several psychophysical techniques are employed as discussed below: Perimetry. — Visual fields are explored with kinetic quantitative perimetry and static quantitative perimetry. Color vision. — Central color vision is estimated using HRR pseudoisochromatic plates, Ishihara pseudoisochromatic plates, Famsworth's Tritan plate, Famsworth-Munsell D-15 panel, Lanthony's desatur- ated D-15 panel, Famsworth-Munsell 100 hue test, and the Nagel anomaloscope. Adaptometry. — Dark-adapted rod and cone thresh- olds are measured with a modified Goldmann-Week- ers adaptometer. Spatial vision. — ^The spatial contrast sensitivity function is determined using sinusoidal luminance gratings. A two-alternative temporal forced-choice technique is used for a criterion-free judgment of threshold visibility. Luminance and chromatic increment thresholds. — These are measured with a two-channel Maxwellian view instrument. This instrument also is used to assess retinal receptor orientation by measuring the Stiles-Crawford effect (SCE of the first kind). Major Findings The diagnostic value of the time constant of the dark adaptation (DA) function as an estimator for the evaluation of visual function was studied in patients with retinal degenerations. Seven groups (70 sub- jects) were included in this study: pa'ients with retinitis pigmeniosa TIP) (12), gyrate atrophy (GA) (15), cone dystrophy (11), juvenile macular dystro- phy (9), fundus flavimaculatus (FF) (33), other retinal degenerations (6), and normal subjects (14). The results obtained on one eye chosen at random in each subject were analyzed. Measurements were made with a modified Goldmann-Weekers adaptome- ter. The subjects were initially light adapted with a 2,550 cd/m^ background for 5 minutes. DA func- tions were obtained by measuring the change in absolute threshold with time (using von Bekesy tracking) for 30 minutes. The following model (Pugh, 1975) was used to fit each of the two limbs of the DA function: time Threshold = a + b • e " In this model, "a" is the final DA threshold (in dB), "b" is the magnitude of DA (in dB), and "c" is the time constant of DA (in minutes). Analysis of variance was used to examine differences between the means of each parameter among the study groups. Statistically significant differences in final threshold ("a") and time constant ("c") were seen in both the cone- and rod-mediated limbs of the DA function. Abnormally high time constants in the cone-mediated limb were observed in RP and GA patients. FF patients who had a normal final thresh- old had abnormal elevation in the time constant of rod-mediated limbs. The usual DA estimator is the final absolute threshold. These findings suggest that a complete evaluation of DA should include, in addition to a measurement of the final threshold, the time constant of adaptation, which provides informa- tion about the rate at which this final threshold is reached. The time constant serves as a clinically relevant parameter in both the diagnosis of retinopa- thies and the followup of individual patients over time. Significance to Biomedical Research and the Program of the Institute Psychophysical techniques are noninvasive methods useful in the diagnosis and management of ocular diseases and visual pathway lesions. In addition to the application of validated tests, the development of new techniques contributes to the elucidation of the pathophysiological mechanisms of visual disorders. Proposed Course Clinical psychophysical studies of visual fiincfion in diseases of the eye and visual pathways will be 262 NEI Annual Report— FY 1993 Ophthalmic Gen etics and Clinical Services Branch continued. We will introduce modifications that are Publications expected to enhance the diagnostic value of the r^ d i- • »** ^ t>^ r^ • t^ ^ techniques described. Drews-Bankiewicz MA, Caruso RC, Kaiser-Kupfer MI: The clinical relevance of the time course of NEI Research Program '^^*' adaptation in reUnal degenerative diseases. * Invest Ophthalmol Vis Sci 34(4)(suppl):1077, Retinal and Choroidal Diseases — Noninvasive 1993. Techniques in the Study of Retinal Disorders Strabismus Amblyopia, and Visual Processing — Visual Processing and Amblyopia 263 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00144-12 OGCSB PERIOD COVERED October 1. 1992 to September 30. 1993 TITLE OF PROJECT (80 characters or less Title must lit on arte line between the borders.) Clinical Electrophysiology of the Visual System PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, ana institute affiliation) PI: Rafael Caruso M.D. Visiting Scientist OGCSB, NEI Others: Muriel I. Kaiser-Kupfer M.D. Malina Drews-Bankiewicz M.D. Patricia A. Mercer M.P.A. Doris J. Collie A.A. Leanne M. Reuter B.S. Chief Visiting Associate Ophthalmic Technician Ophthalmic Technician Ophthalmic Technician OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Eye Services Section INSTITUTE AND LOCATION , NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.41 PROFESSIONAL: 0.41 CHECK APPROPRIATE BOX{ES) [x] (a) Human subjects [x] (a1) Minors □ (a2) Interviews OTHER: 1.0 □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The visual function of patients with ocular diseases or lesions in the visual pathways and of normal subjects is measured objectively using electrophysiological techniques. The data are correlated with those obtained with psychophysical tests of visual function. The results contribute to the diagnosis of ocular and neural disorders that affect vision and are needed to characterize their nature and evolution. They are also valuable in the assessment of the effects of different forms of treatment on the outcome of these diseases. 264 PHS 6040 (Rev. 5/92) NEI Annual Report— FY 1993 Ophthalmic Genetics and Clinical Services Branch Project Description Clinical Protocol Numbers 91-EI-26 82-EI-55 Objectives The aims of this project are (1) to apply and develop electrophysiological techniques for the study of visual function in the clinical setting, (2) to charac- terize the normal electrical activity of the human visual system, and (3) to analyze the patterns of its alteration in ocular diseases and lesions of the visual pathways. Methods The electrophysiological techniques employed involve recording potentials generated by the retinal pigment epithelium (electrooculogram), the neural retina (electroretinogram), and the central visual pathways (visually evoked potentials [VEPs]). These potentials are elicited by unstructured stimuli (Ganz- feld full-field or focal stimulation) and spatially structured stimuli (sinusoidal gratings or checker- board patterns). Major Findings The transient pattern reversal VEP elicited by stimu- lation of the central visual field is characterized by a typical negative-positive-negative (NPN) waveform. Previous reports using hemifield stimulation suggest that the peaks similar to those elicited by full-field stimulation arise primarily from the ipsilateral electrode sites. In this study, we investigated the contributions of both ipsilateral and contralateral electrode sites to the origin of the full-field VEP peaks. Fifty normal human subjects were studied. VEPs elicited by the contrast reversal of a 4 cycles/degree sinusoidal grating were recorded over five electrode sites 5 cm above the inion (midline) and 5 cm (occipital) and 10 cm (temporal) to the left and right of the midline. VEPs were elicited by binocular and monocular stimulation of the full (26° X 20°) field and of the left and right (13° x 20°) hemifields. VEPs elicited by stimulation of a hemifield have been described as consisting of NPN waveforms over the ipsilateral electrodes and variable low-amplitude responses of opposite polarity (i.e., surface-negative) over the contralateral electrodes. However, in our study, hemifield stimuli elicited asymmetrical sur- face-positive waveforms on both sides of the midline in most cases (92%). The amplitude of these wave- forms was larger over the electrodes ipsilateral to the stimulated hemifield ("paradoxical" lateralization). Their main positive peak occurred earlier over the electrodes contralateral to the stimulated hemifield. The peaks of the fiill field VEP at each electrode site were generated by the algebraic sum of the peaks of the hemifield VEPs. This sum of two hemifield responses closely matched the full-field VEP, with negative peaks being frequently generated by a single hemisphere. A summation of two waveforms, which very frequently show the same surface-positive polarity and are generated by stimulation of each hemifield, generates the peaks of the full-field VEP. The symmetry of pattern-reversal VEPs recorded to the left and right of the midline has been proposed as a valuable estimator of the functional integrity of the visual pathway. We explored the variability of VEP symmetry and analyzed the contribution of both eyes and both hemispheres to this symmetrical response. Fifty normal human subjects underwent an ophthalmologic examination, including a 30° visual field test (static perimetry). Transient VEPs elicited by the contrast reversal of a 4 cycles/degree sinu- soidal grating were recorded over five electrode sites 5 cm above the inion. VEPs were elicited by binoc- ular and monocular stimulation of the fijll (26° x 20°) field and of the left and right (13° x 20°) hemifields. Full field stimulus. — After binocular stimulation, mean VEP amplitudes were symmetrica] about the midline. However, asymmetries in the amplitude of VEPs were seen in individual subjects. Therefore, 95% tolerance intervals about the mean amplitude differences (left lead minus right lead) are large (-11.42 to 9.55 nV for the occipital sites and -4.80 to 5.18 |iV for the temporal sites). Monocular stimulation of either OD or OS generated larger mean amplitudes over the ipsilateral electrodes. This voltage difference was not large but significant (p < 0.001). Hemifield stimulus. — VEPs elicited by stimulation of a hemifield were asymmetrical. They consistently showed larger amplitudes over the ipsilateral elec- trodes (paradoxical lateralization). The sum of the two asymmetrical hemifield responses was synmietri- 265 Ophthalmjc Genetics and Ciinical Services Branch ^fEI Annual Report — FY 1993 ca] and closely matched the full-field VEP. Our results indicate that the sum of the asymmetrical contribution of either hemisphere and either eye are responsible for the symmetrical VEP elicited by binocular stimulation with a full-field sfimulus. An asymmetrical full-field VEP may be seen in normal subjects and does not imply an abnormality in the visual pathways. Significance to Biomedical Research and the Program of the Institute Electrophysiological techniques are noninvasive methods useful in the diagnosis and management of ocular diseases and visual pathway lesions. In addi- tion to validating tests, the new techniques developed contribute to the elucidation of the pathophysiologi- cal mechanisms of visual disorders. Proposed Course We will continue clinical electrophysiological studies of visual function in diseases of the eye and visual pathways, introducing modifications expected to enhance the diagnostic value of the techniques described. NEI Research Program Rednal and Choroidal Diseases — Noninvasive Techniques in the Study of Retinal Disorders Strabismus, Amblyopia, and Visual Processing — Visual Processing and Amblyopia Publications Caruso RC, Reuter LM, Muller SF, Drews- Bankiewicz MA, Kaiser-Kupfer Ml: Origin of the peaks of the human transient pattern reversal visual evoked potential. Invest Ophthalmol Vis 5a34(4)(suppl):1276, 1993. Reuter LM, Caruso RC, Muller SF, Drews- Bankiewicz MA, Bouzas EA, Kaiser-Kupfer MI: The pattern reversal visual evoked potendals: Symmetrical or asymmetrical. Invest Ophthalmol Vis Sci 34(4)(suppl):1276, 1993. 266 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00257-05 OGCSB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Visual Function Diagnosis Service PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Rafael Caruso M.D. Visiting Scientist OGCSB, NEI Others: Miuiel I. Kaiser-Kupfer M.D. Tracy T. Nolan M.A. Dessie Koutsandreas C.O.A. Anne Randall C.O.M.T. Linda Goodman C.O.T. Chief Health Technician Ophthalmic Technician Ophthalmic Technician Ophthalmic Technician OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Eye Services Section INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 6.81 PROFESSIONAL: 0.15 OTHER: 6.66 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This general service project provides diagnostic support for all research protocols conducted by the Clinical Sections of the NEI and other Institutes that require an assessment of visual function. Psychophysical and electrophysiological techniques are used to detect and quantify visual loss due to disorders of the ocular media, uvea, retina, optic nerve, and central visual pathways. 267 PHS 6040 (Rev. 5/92) OpfathalmJc Genetics and Clinical Services Branch NEI Annual Report — FY 1993 Project Description Additional Personnel R. Patrick McDaniel Ophthalmic Technician, OGCSB, NEI Antoinette LaReau Ophthalmic Technician, OGCSB, NEI Sueli Muller Special Volunteer, OGCSB, NEI Objectives The aim of this project is to provide accurate mea- surements of visual function for the differential diagnosis of visual loss. The first step in this process is detection of a visual deficit (i.e., determining whether visual loss has occurred). The second step is the quantification of a detected deficit The third is an analysis of the characteristics of the visual deficit to determine the site of the lesion responsible for this symptom (topographic diagnosis). The final step is correlation with other clinical findings to ascribe the visual deficit to a given pathological process. Methods The psychophysical techniques employed include commercially available and laboratory-developed techniques for the measurement of visual acuity, visual fields, color vision, dark adaptation, spatial contrast sensitivity, and glare disability. The electrophysiological techniques used include recording potentials generated by the retinal pigment epithelium (electrooculogram), the neural retina (electroretinogram), and the central visual patiiways (visually evoked potentials). Major Findings During the period October 1, 1992, through Septem- ber 30, 1993, we performed tiie following tests: Kinetic perimetry 312 Static perimetry 413 Screening perimetry 102 Manifest refraction 1,313 Color vision tests 193 Adaptometry 45 Contrast sensitivity tests 18 Glare disability tests 5 Ganzfeld electroretinography I81 Focal electroretinography 50 Electrooculography 102 Visually evoked potentials 117 This represents an increase of 57% over the tests performed diuing the same period in Fiscal Year 1992. Significance to Biomedical Research and the Program of the Institute This project provides all tests of visual function for patients who visit the NEI Eye Clinic. In the major- ity of ophthalmologic diseases, visual loss is the most meaningful finding. In most clinical research protocols involving diseases of the eye and visual pathways, visual deficit is used as an indicator of die progress of a disease or the effect of a treatment. Therefore, sensitive and accuriate measurements of visual function are essential for tiiese clinical re- search projects. Proposed Course The provision of clinical electrophysiological and psychophysical tests of visual function for patients with diseases of the eye and visual pathways will be continued. We will introduce modifications that are expected to enhance the diagnostic value of the techniques described. NEI Research Program Retinal and Choroidal Diseases — Noninvasive Techniques in the Study of Retinal Disorders Strabismus, Amblyopia and Visual Processing Visual Processing and Amblyopia 268 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00011-19 OGCSB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (BO characters or less. Title must fit on one line between the borders.) Pigment Dispersion With and Without Glaucoma PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEI Others: Lessie McCain Mark H. Scott R.N. M.D. Nurse Specialist Senior Staff Fellow OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Ophthalmic Genetics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.25 PROFESSIONAL: 0.15 OTHER: 0.1 CHECK APPROPRIATE BOX(ES) [x| (a) Human subjects \x\ (a1) Minors [x] (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The purpose of this project is to determine the risks of patients with pigment dispersion syndrome for glaucoma. Comparisons of patients with and without glaucoma are made on the basis of diagnostic tests, genetic screening, and aqueous humor dynamics. The data acquired may enable determination of pigment dispersion syndrome patients' risk of developing glaucoma, as well as adding to the understanding of the pathology of the disease. 269 PHS 6040 (Rev, 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Additional Personnel Marvin Podgor M.S. Statistician, Biometry and Epidemiology Program, NfEI Clinical Protocol Number 76-EI-189 Objectives This project was designed (1) to compare patients with and without glaucoma who have pigment dispersion by documenting and following the clinical features and courses of disease and by evaluating performance on a variety of diagnostic tests; (2) to determine the presence of abnormal aqueous humor dynamics in glaucoma and nonglaucoma patients with pigmentary dispersion; and (3) to compare the association of pigment dispersion, with and without glaucoma, with possible genetic markers. Methods A complete evaluation includes the following: complete family history with detailed pedigree; best- corrected visual acuity with manifest refraction; slit- lamp biomicroscopy; visual field examination (Goldmann I-,e and l4e); q)planation Goldmann tension; photography of iris color, iris transillumina- tion, and Krukenberg spindle; A-scan, anterior chamber depth, and anterior chamber volume mea- surements; goniophotography; static perimetry; baseline tonography and water-drinking tonography 1 hour later, when indicated; fasting blood sugar, when indicated; dilated ophthalmoscopic examination (using 2.5% phenylephrine and 1% cyclogel); and stereophotographs of the optic nervehead. Major Findings One hundred sixty-four patients were classified into three groups: (1) pigment dispersion syndrome (PDS) without abnormal ocular pressure, (2) PDS witli ocular hypertension, and (3) PDS with glaucoma (PDS-(-GL). Analysis of baseline characteristics with respect to anatomical and physiological parameters has yielded the following conclusions: 1 . It appears that the majority of patients recruited have PDS with a benign course: They do not develop ocular hypertension or glaucoma. 2. Consequently, family members of PDS patients should be alerted and appropriately screened. PDS may be familial and can show a dominant inheritance pattern. 3. Analyses of graded iris transillumination, the amount of pigment deposited on the trabecular meshwork, and the anterior chamber depth have demonstrated no significant differences among the three categories of PDS. Thus, pigment deposited in the angle may be only a secondary factor adversely affecting an already compromised outflow facility that is primarily a result of open-angle glaucoma. 4. It also appears that those patients who develop ocular hypertension and demonstrate early field changes can be managed medically by control of intraocular pressure and reversal of early field loss. Patients who develop glaucoma do not appear to be more difficult to treat than patients with open-angle glaucoma. 5. The phenomenon of unilateral or asymmetric PDS, in which there is little difference between the measurements of the two eyes, is being investigated in followup studies. 6. In our series, refinal detachment does not appear to occur with any greater frequency than with high myopia. A history of asymptomatic and nonpro- gressive peripheral retinal holes was noted in two patients. 7. The data from this study have been computer- ized, and an indepth analysis is under way. Significance to Biomedical Research and the Program of the Institute These results may facilitate determination of the risk of the development of glaucoma for patients with pigment dispersion. Specifically, it may be possible to identify which features have predictive value in forecasting which PDS patients will develop visual field defects. In addition, the data will aid investiga- tion of the relationship of "pigmentary" glaucoma to tile known characteristics of open-angle glaucoma. Proposed Course This project will be continued for 3 more years to obtain additional data on the patients enrolled in the study and to recruit more patients to add to the knowledge about PDS. NEI Research Program Glaucoma — Other Glaucomas (Developmental, Congenital, and Infantile Glaucomas) 270 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00060-15 OGCSB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) Visual Function and Ocular Pigmentation in Albinism PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEI Others: Lessie McCain R.N. Rafael Caruso M.D. Evrydiki Bouzas M.D. Malina Drews-Bankiewicz M.D. Nurse Specialist Visiting Scientist Visiting Scientist Visiting Associate OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Ophthalmic Genetics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.31 PROFESSIONAL: 0.26 OTHER: 0.05 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects \x\ (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Patients with hypomelanotic disorders, such as ocular albinism, oculocutaneous albinism, Chediak-Higashi disease, Hermansky-Pudlak syndrome, and iris traasillumination defects, are being recruited to determine visual fiinction with these conditions and to evaluate the changes in visual function over time. Family members are evaluated in an attempt to determine factors which may identify the heterozygous state. 271 PHS 6040 (Rev. 5/92) Opfathalmjc Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Clinical Protocol Number 76-EI-207 Objectives The objectives of this study are (1) to relate the level of visual function to the amount of ocular pigmenta- tion, especially iris and retinal pigmentation; (2) to correlate the amount of nystagmus with visual acuity and iris pigmentation; (3) to determine whether ocular pigmentation, visual acuity, and nystagmus change with age; (4) to identify the heterozygous state of family members; and (5) to determine whether abnormalities of crossing of the optic nerve fibers can be correlated with the lack of pigmentation and whether previous reports of crossing abnormali- ties can be confirmed. Methods For each patient, a complete family history with detailed pedigree is compiled and the following procedures are performed: best-corrected visual acuity near and at a distance with refraction; slit- lamp examination; psychophysical testing, including D-15 and Munsell 100 hue as well as rod and cone thresholds; dilated ophthalmoscopic examination; photography to document hair color, eye color, iris transillumination, and the status of the disc and macula; visually evoked response testing; and, in selected patients, contrast sensitivity measurements. Information on family members is collected by examination of best-corrected visual acuity, slit-lamp examination of iris, photography of iris transillumina- tion, and fundus examination when vision is not corrected to 20/20. Major Findings Major findings were as follows: 1. Examination of 82 patients and family mem- bers indicated that transillumination of the iris may occur in the absence of recognized albinism. The pattern, which appears to be punctate, may be present in a diffuse manner or limited to the 6 o'clock sector. The finding is not associated with nystagmus. 2. These patients presented with marked iris transillumination, reduced pigmentation of the fundus, and no nystagmus, but they had decreased visual acuity, which has improved in conjunction with an increase in the pigmentation of the fundae. 3. Visually evoked responses were normal in some patients, but in a subset of albinos, there was evidence of nonceniform pattern of asynmietry due to the miswiring of the visual pathways. The low amplitude of the visually evoked potentials recorded in a consecutive series of patients shows the difQ- culties of studying the phenomenon in a clinical setting. Significance to Biomedical Research and the Program of the Institute These data may allow identification of the carrier state of albinism, which would be important in genetic counseling. Determinafion of whether the development of the fovea is abnormal in albinism, whether this abnormal foveal development is the cause of the decreased visual acuity in albinism, or, alternatively, whether decreased visual acuity is secondary to hypopigmentation and the resultant light scatter and glare may be possible. Collection of these data also will facilitate ascertainment of whether visual acuity improves with age and whether this correlates with changes in pigmentation. In addition, studies are being conducted to verily the reported findings of abnormalities of the aossing fibers, as measiu-ed by visually evoked responses, contrast sensitivity, degree of nystagmus, and amount of pigmentation. Proposed Course This project will be continued for 5 more years to obtain additional data. NEI Research Program Retinal and Choroidal Diseases — Developmental and Hereditary Disorders Publications Bouzas EA, Caruso RC, Drews-Bankiewicz MA, Kaiser-Kupfer MI: Evoked potential analysis of visual pathways in human albinism. Ophthalmol- ogy, submitted. 272 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1. 1992 to September 30. 1993 PROJECT NUMBER ZOl EY 00083-16 OGCSB TITLE OF PROJECT (80 characters or less. Title must fit on one line betweerj the borders.) Gyrate Atrophy of the C horoid and Retina and Other Retinal Degenerations PRINCIPAL INVESTIGATOR (List other professional personr,el below the Prmapal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEI Others: Evrydiki Bouzas Lessie McCain Rafael Caruso Pushpa K. Sran Doris Collie M.D. R.N. M.D. M.D. A.A. Visiting Scientist Nurse Specialist Visiting Scientist Medical Officer Ophthalmic Technician OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) ' " " ' — Howard Hughes Medical Institute Laboratory and Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, MD (David L. Valle, M.D.) LAB/BRANCH ~~ ' Ophthalmic Genetics and Clinica l Services Branch SECTION Section on Ophthalmic Genetics INSTITUTE AND LOCATION "^ NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.15 CHECK APPROPRIATE BOX(ES) HJ (a) Human subjects [x] (a1) Minors Q (a2) Interviews PROFESSIONAL: 0.75 OTHER: 0.40 n (t>) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Patients with gyrate atrophy of the choroid and retina are examined systematically to confirm the diagnosis. Skin fibroblasts from affected patients and family members are grown in tissue culture and assayed for ornithine aminotransferase activity. The results are evaluated for correlation with the presence of homozygosity or heterozygosity for the disease trait. Each patient is given a trial of pyridoxine to see whether serum concentration of ornithine can be reduced; if so, the patient is classified as a "responder," and treatment with pyridoxine is continued. Nonresponder and responder patients are then placed on a low-arginine, low- protein diet with supplemental amino acids and observed for arrest or improvement of die disease. If patients are not considered eligible for the diet, or if they ^pear unable to comply with the dietary regimen, we follow them to record the natural progression of the condition. Patients with other forms of retinal degeneration, such as retinitis pigmentosa, fundus flavimaculatus, juvenile retinoschisis, and Usher's syndrome, also are examined. The courses of their diseases are compared with those of gyrate ati-ophy patients. 273 PHS 6040 (Rev. 5/92) OpbtbalmJc Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Additional Personnel Laura Wozencraft J. Fielding Hejtmancik Susan Gentleman M.S. Genetic Counselor, OGCSB, NEI Ph.D. Medical Officer, LMOD, NEI Ph.D. Biologist, LRCMB, NEI Clinical Protocol Number 78 EI-01 Objectives This project is being conducted (1) to determine the biochemical processes responsible for the elevated plasma ornithine and the chorioretinal lesions that occur in gyrate atrophy (GA) of the choroid and retina; (2) to determine which patients respond to pyridoxine treatment with a decrease in plasma ornithine concentration; (3) to determine whether treating "responders" with pyridoxine and nonrespon- ders with an arginine-deficient diet will arrest the progress of chorioretinal atrophy; (4) to study the natural history of this condition when intervention is not undertaken and to determine the degree of heterogeneity; (5) to define the molecular mutations and compare the molecular defect with the clinical features of the disease; and (6) to characterize and follow the progression of lens opacities, obtaining lens specimens at the time of cataract extraction for protein analysis. Methods Patients suspected of having GA of the choroid and retina are examined according to a standard set of procedures to confirm the diagnosis. Plasma ornithine concentration is measured periodically. Punch biop- sies of the skin are grown in tissue culture, their ornithine aminotransferase activity is measured, and patient molecular defect is characterized. Complete evaluation of ocular function in these patients in- cludes best-corrected visual acuity, Goldmann visual fields, color vision, cone thresholds, dark adaptation, electroretinogram (ERG), foveal electroretinogram (FERG), electrooculogram (EOG), contrast sensitiv- ity, and Stiles-Crawford effect. Major Findings Gyrate atrophy (GA), a rare autosomal recessive disorder, is associated with hyperornithinemia, overflow ornithinuria, and a deficiency of activity of the mitochondrial enzyme ornithine-6- aminotransferase (OAT). Although rare, the condition has been described worldwide in all races. Thirty-six patients have been recruited and evaluated in this study. The patients' ethnic origins vary and include African-American, Asian Indian, English, Fiimish, German, Israeli, Lebanese, Portuguese, Scottish, Turkish, and Welsh. In this study, among 44 patients, 22 females and 22 males range in age from 2.5 to 65 years, with 10 children less than age 12 at the time of recruitment. Observations of these patients have enabled docu- mentation of both clinical evidence and laboratory heterogeneity. Analysis of the mutation that causes GA of the choroid and retina has been undertaken by Drs. David Valle and Grant Mitchell and colleagues of The Johns Hopkins University. They have ana- lyzed probands fi-om 72 GA pedigrees. No gross structural alterations of the OAT gene have been de- tected; 85% of the probands express nearly normal amounts of normal-sized OAT roRNA. The remain- der express little or no OAT mRNA (n = 5) or an mRNA with an altered size (n = 2). Western blot studies showed the OAT antigen to be absent in 67% of the mRNA+ mutants and all of the mRNA- mutants. A total of 14 mutations have been deline- ated at the molecular level: 10 missense mutations (Mil, R180T, L402P, C93F, Y55H, R154L, A270P, R271KL, G375V, and P417L/L437F); a single nucleotide deletion at cDNA position +159 (H53fs); an interesting in-fi-ame three-nucleotide deletion of A la- 184 (A185F0), and a nonsense mutation at a CpG dinucleotide (R396ter). The functional consequences of several mutations have been examined by substituting the mutations into otherwise wild-type OAT cDNA in the expres- sion vector P9 1023b and transfecting the recombi- nant constructs into CHO-Kl cells that lack endoge- nous OAT mRNA or protein. Three (R180T, L402P, A184D0) have been shown to encode a CRM+, enzymatically inactive protein, while Mil — as ex- pected for an initiation codon alteration — has a CRM- phenotype. Studies are under way to correlate mutational heterogeneity with clinical and biochemi- cal heterogeneity. 274 NEI Annual Report — ^FY 1993 Ophthalmic Genetics and Clinical Services Branch The earliest clinical and electrophysiologic fea- tures were documented in the two youngest patients (ages 2.5 and 3 years). The minimal evidence of clinical retinal changes when significant reduction of rod and cone function is seen by electroretinographic studies is noteworthy. Clinical and biochemical evidence of genetic heterogeneity is present in these patients. Fewer than 10% of patients have been reported to have a 30-50% decrease in plasma ornithine following treatment with vitamin Bg. Only one of our patients showed an in vivo response to this treatment. Com- parisons of sibships reveal that there is a greater degree of interfamilial variability than intrafamilial variability. Whereas arginine is the precursor of ornithine in the metabolic pathway of ornithine metabolism, we have undertaken a dietary intervention study limiting arginine. Of 25 patients placed on a low-protein (i.e., low arginine) diet, all sustained significant reduction of ornithine during hospitalization; however, the diet was discontinued in 4 Finnish patients following their discharge because of poor compliance and in 7 other patients because of a variety of factors. Of 15 patients remaining on the diet, 4 have excellent control; 4, fair control; and 4, erratic control. One young child was followed for too short a period of time to assess control. Ophthalmologic evaluations are performed on all patients every 6 to 12 months, travel permitting. In the two patients with the best biochemical control for the longest time (11 and 12 years old, respectively), there was evidence of improved visual function. One patient, after being on the diet for 14 months, showed improved dark adaptation and aver- age ERG and color vision. This improvement was sustained for 30 months, then the ERG amplitude showed a small but definite reduction. The second patient who had lowered plasma ornithine levels and who had been on the diet for 11 years showed progressive improvement in visual field and color vision and has since remained stable. A third patient, despite fair control, was stable for 36 months but has deteriorated for the past 18 months. It should be noted that she was the oldest patient and had the most advanced disease at the outset. Other patients followed for various periods of time currently appear stable. Of particular interest are the children who were ages 2.5 to 9 years old at the outset of diet. The results indicate that as a result of dietary intervention the course of the disease in the younger of each sibship has been improved, compared with that of the older sibling. All but one patient over age 1 1 have had progres- sive cataracts in the posterior capsule. They present a uniform histologic picture and can be identified by their characteristic pattern in image analysis. Significance to Biomedical Research and the Program of the Institute GA of the choroid and retina is the first isolated of the genetically determined severe retinal degenera- tions for which a specific biochemical marker and concomitant enzyme defect have been demonstrated. Designed to test the efficacy of treatment for this bUnding eye disease, this smdy will serve as a model for the investigation of other genetically determined retinal degenerations. Smdy of the two young patients is the best opportunity for the evaluation of diet control. This disease is a likely candidate for future smdies to begin gene therapy. Proposed Course This project will be continued for 3 more years to assess further the knowledge concerning the reduc- tion of ornithine to halt chorioretinal degeneration. NEI Research Program Retinal and Choroidal Disease — Development and Hereditary Disorders Publications Brody LC, Mitchell GA, Obie C, Michaud J, Steel G, Fontaine G, Robert M-F, Sipila I, Kaiser- Kupfer M, Valle D: Ornithine o-aminotransferase mutations in gyrate atrophy. J Biol Chem 267:3302-3307, 1992. 275 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00163-11 OGCSB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must In on one line between the borders.) NIH Interinstitute Genetics Program: The Genetics Clinic PRINCIPAL INVESTIGATOR (List other protessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Muriel 1. Kaiser-Kupfer M.D. Chief OGCSB, NEI Others: Evrydiki Bouzas M.D. Mark Scott M.D. Lessie McCain R.N. Anren Li M.D. Laura Wozencraft M.S. Visiting Scientist Senior Staff Fellow Nurse Specialist Visiting Associate Genetic Counselor OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) Interinstitute Medical Genetics Program, NIH LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Ophthalmic Genetics INSTITUTE AND LOCATION NEI, NIH. Bethesda, MP 20892 TOTAL STAFF YEARS: 0.8 PROFESSIONAL: 0.3 OTHER: 0.5 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects [x] (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The Interinstitute Genetics Program and the Genetics Clinic supported by the Clinical Center offer a multidisciplinary approach to patients with genetic disease (ZOl CP 05139-06 CEB). Involved in the program are researchers from all Institutes. Patients evaluated in the clinic represent a broad spectrum of genetic diseases. During the past year, approximately 200 persons seen represented about 60 distinct disease categories. Due to the high frequency of ocular involvement in many of the cases, almost all the patients were evaluated by Clinical Branch staff or were discussed in consultation. The clinic serves as a source of interesting case material concerning patients with inherited or developmental abnormalities of the visual system. 276 PHS 6040 (Rev. 5/92) NEI Annual Report — FY 1993 Ophthalmic Genetics and Clinical Services Branch Project Description Clinical Protocol Number Interinstitute Medical Genetics Program Objectives The objectives of this Program are (1) to evaluate patients with ocular abnormalities associated with genetic disease in the context of a multidisciplinary approach to the patient; (2) to provide genetic counseling to patients at risk for inherited ocular disease; (3) to recommend and advise appropriate evaluation for the ocular problem; and (4) to provide training in the diagnosis, counseling, and treatment of individuals with or at risk for genetic disease, as well as in the research approach to genetic disease. Methods Referred patients are examined, and the appropriate diagnostic ophthalmologic workup is recommended. Major Findings 1. Iris nodules were seen commonly in the classic cases of neurofibromatosis (NFl) and less frequently seen in patients with less-well-defined disease. They were seen rarely in patients with bilateral acoustic neuroma (BAN or NF2). Interestingly, in a series of 14 consecutive patients with Cushing's disease, two patients (14%) had typical, unilateral lisch nodules. To our knowledge, the association of NFl on lisch nodules with Cushing's disease has not been de- scribed. The association of Cushing's disease and lisch nodules may represent a mild form of multiple endocrine neoplasia of the mixed type. It is possible that a common underiying mechanism leads to the overgrowth of melanocytes in the iris and cortico- trophs in the pituitary. Patients with NF2 showed increased frequency of posterior capsular cataracts, which serve as an excellent marker, being present in 80% of individuals with NF2. A new finding is the association of peripheral cortical cataracts in 37.8% of NF2 patients. In a group of severely affected ^fF2 patients, it appears that combined pigment epithelial and retinal hamartomas are also an ocular marker for NF2. In fact, there may be a predilection for the macula in some cases. 2. Serious ocular complications were observed in 13 long-term postrenal transplantation nephropathic cystinosis patients. These complications included decreased visual acuity and visual function, as measured by psychophysical and electrodiagnostic tests, band keratopathy, and posterior synechia. Corneal transplantation may be necessary in cases with debilitating symptoms from recurrent erosion after all other treatment modalities have failed. In two such patients, the corneal grafts have remained clear for as long as 6 years. 3. Ophthalmic studies performed in a population of patients with endogenous Cushing's syndrome revealed that posterior subcapsular cataracts were an infrequent phenomenon compared with exogenous Cushing's syndrome. Although an uncommon find- ing, central serous chorioretinopathy was seen in 3 of 60 patients (5%), suggesting that glucocorticoids may play a role in the development of the disease. Significance to Biomedical Research and the Program of the Institute Genetic and developmental anomalies of the eye are a major cause of blindness and visual disability; they are responsible for about 35% of the cases of blind- ness in developed nations. Involvement with the Interinstitute Genetics Program affords a systematic approach to studying these and other conditions associated with genetic diseases. Proposed Course The project is in a growth phase and will be expand- ing in future years. NEI Research Program Retina] and Choroidal Disease — Development and Hereditary Disorders Publications Bouzas EA, Kransewich D, Koufroumanidis M, Papadimitriou A, Marini JC, Kaiser-Kupfer MI: Ophthalmological examination in the diagnosis of Proteus syndrome. Ophthalmology 100:334-338, 1993. Bouzas EA, Freidlin V, Parry DM, Eldridge R, Kaiser-Kupfer MI: Lens opacities in neurofibro- matosis 2: Further significant correlation. Br J Ophthalmol 77:354-357, 1993. Bouzas EA, Mastorakos G, Chrousos GP, Kaiser- Kupfer MI: Lisch nodules in Gushing disease. Arch Ophthalmol 111:439, 1993. 277 Opbtbaimjc Genetics and Clinical Services Branch ^fEI Annual Report— FY 1993 Bouzas EA, Mastorakos GM, Chrousos GP, Kaiser- Kupfer MI: Posterior subcapsular cataract is infrequent in endogenous Gushing syndrome. Invest Ophthalmol Vis Sci 34(4)(suppl):1064, 1993. Bouzas EA, Mastorakos G, Friedmann T, Scott MI, Chrousos GP, Kaiser-Kupfer MI: Posterior subcapsular cataracts in endogenous Cusing Syndrome: An uncommon manifestation. Invest Ophthalmol Vis Sci 34:3497-3500, 1993. Bouzas EA, Parry DM, Eldridge R, Kaiser-Kupfer MI: Visual impairment in patients with neuro- fibromatosis 2. Neurology 43:22-623, 1993. Bouzas EA, Scott MH, Mastorakos GP, Chrousos GP, Kaiser-Kupfer MI: Central serous chorioreti- nopathy in endogenous hypercortisolism. Arch Ophthalmol, 111: 1229-1233, 1993. Kaiser-Kupfer MI, Bouzas EA: Ocular manifestations of metabolic disorders. Curr Opin Ophthalmol 3:221-227, 1992. Mastorakos G, Bouzas EA, Burnier MN, Chrousos GP, Chrousos GA: Presence of immunoreactive corticotropin releasing hormone in the optic nerve but not the inflammatory infiltrate of allergic optic neuritis/encephalomyelitis. Invest Ophthal- mol Vis Sci 34(4)(suppl):1000, 1993. 278 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00211-08 OGCSB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 charactars or less. Title must tit on one line between the borders.) A Double-Masked Controlled Randomized Clinical Trial of Topical Cysteamine PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, latmratory, and institute affiliation) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEI Others: Lessie McCain Manuel Datiles Evrydiki Bouzas Mark Scott Anren Li R.N. M.D. M.D. M.D. M.D. Nurse Specialist Medical Officer Visiting Scientist Senior Staff Fellow Visiting Associate OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) Human Genetics Branch, National Institute of Child Health and Human Development, NIH, Bethesda, MD (William Gahl, M.D., Ph.D.) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Ophthalmic Genetics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.70 PROFESSIONAL: 0.45 OTHER: 0.25 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects [x] (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Nephropathic cystinosis is an autosomal, recessively inherited storage disease in which noiy)rotein cystine accumulates within cellular lysosomes due to a defect in lysosomal cystine transport. Ocular manifestations include photophobia; crystal deposits in the cornea, conjunctiva, and iris; and depigmentation of the retina. Ten years ago, cysteamine, a free thiol that depletes cystine from cells, was introduced in the therapy of cystinotic patients. Although patients had improved growth and stabilized renal function, there was no noticeable effect on the accumulation of corneal crystals. Recent studies showed that corneal cells in tissue culture are readily depleted of cystine by the introduction of cysteamine, making feasible the use of topical ophthalmic cysteamine to circumvent the humoral route. After appropriate animal smdies to test for complications revealed none, we began a double-masked chnical trial to test the efficacy of topical cysteamine (0.1% and 0.5%) in humans. To date, in 14 of 29 young patients the code was successfully broken; of the 15 remaining, 2 died, 1 discontinued medication, and 12 are still in the trial with poor compliance and have not been seen for followup. Because of the success in the younger patients, this study was expanded to include older patients, 3 to 31 years of age. The findings have been most exciting: Twenty-three patients have shown a significant decrease in crystals in treated eyes as well as improvements in comfort, i.e., relief of pain and photophobia. This study has resulted in significantly improved quality of life for the successfully treated patients. Because of the success of this clinical trial, and evidence from the cysteamine-benzalkonium trial (Protocol Number 93 EI-0230), the Food and Drug Administration has requested that all patients in this protocol be switched to cysteamine plus benzalkonium and receive medication in both eyes. Each patient then will be judged by a comparison with his or her own natural history. 279 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Additional Personnel Ernest M. Kuehl Chief, Photography Section, OGCSB, NEI Clinical Protocol Number 86-EI-62 Objectives The purpose of this project is to test the efficacy of topical cysteamine in patients with nephropathic cystinosis. Methods Slit-lamp examination and photography of the cornea are performed by a masked observer to determine whether there is a difference in the quantity of crystals seen in the cornea. Major Findings Topical cysteamine eyedrops (0.5%) are well toler- ated. The crystal accumulation is reversible in very young patients, who do not have crystals packing the cornea, as well as in older patients in which the crystals pack the cornea. Significance to Biomedical Research and the Program of the Institute The continued accumulation of crystals in the cornea appears to lead to increasing discomfort in cystinosis patients, who develop severe photophobia with recurrent corneal erosions. Topical cysteamine treatment, which has been found to halt the process, has led to an improvement in the quality of life of these patients. Proposed Course This study will be replaced by a study in which the crystal accumulation will be compared with the natural history of the condition. NEI Research Program Corneal Diseases — Ocular Surface Problems GDrug Delivery and Toxicity) 280 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00282-01 OGCSB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must lit on one line between t/ie borders.) Usher's Syndrome — Clinical and Molecular Studies PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEl Others: J. Fielding Hejtmancik Mark H. Scott Rafael C. Caruso Laura A. Wozencraft Anita Pikus M.D., Ph.D. M.D. M.D. M.S. M.A. Medical Officer Senior Staff Fellow Visiting Scientist Genetic Counselor Chief, Audiology Unit LMOD, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI HS/NOB, NIH COOPERATING UNITS (if any) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Ophthalmic Genetics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.6 PROFESSIONAL: OTHER: 0.6 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects |x] (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The purpose of this project is to document the clinical features of Usher's syndrome, to refine the localization, and eventually to isolate the genes causing this disease. 281 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Clinical Protocol Number 93 EI-0161 Objectives The objectives of this study are (1) to relate the level of visual fimction to the amount of ocular pigmenta- tion, especially iris and retinal pigmentation; (2) to correlate the amoimt of nystagmus with visual acuity and iris pigmentation; (3) to determine whether ocular pigmentation, visual acuity, and nystagmus change with age; (4) to identify the heterozygous state of family members; and (5) to determine whether abnormalities of crossing of the optic nerve fibers can be correlated with the lack of pigmentation and whether previous reports in abnormalities of crossing can be confirmed. Methods Included in the evaluation will be audiometric, vestibular, ophthalmologic, and electrophysiologic and electrodiagnostic testing. These clinical findings will help classify the features of the different types of Usher's syndrome, as well as correlate the phenotypic features with the genetic mutation. To identify the genetic mutation, we will study informa- tive families, collecting blood specimens from all available family members for studies that will utilize molecular technology developed for linkage analysis. In cases in which there are no other affected family members, blood specimens will be obtained to study specific gene mutations when the specific gene or genes for Usher's syndrome are identifiable. Major Findings The recruitment for this project has begun: 40 patients have been recruited. Patients are being evaluated, and their blood specimens are being collected and maintained in the laboratory. Linkage analysis on these families has not yet begun. Significance to Biomedical Research and the Program of the Institute By molecular studies of patients with Usher's syn- drome, mutations may be correlated with clinical findings and genes responsible for Usher's syndrome may be defined, leading to the possibility of genetic therapy at some point. Proposed Course Patient recruitment into the study will be continued. NEI Research Program Retinal and Choroidal Disease — Development and Hereditary Disorders 282 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00283-01 OGCSB PERIOD COVERED July 13, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must fit on one line between the borders.) A Double-Masked Controlled Randomized Clinical Trial of Topical Cysteamine PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEl COOPERATING UNITS (if any) Human Genetics Branch, National Institute of Child Health and Human Development, NIH (William A. Gahl, M.D., Ph.D.) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Ophthalmic Genetics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.2 PROFESSIONAL: 0.1 OTHER: 0.1 CHECK APPROPRIATE BOX(ES) Fxl (a) Human subjects [x] (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF W/ORK (Use standard unreduced type. Do not exceed the space provided.) Cysteamine ophthalmic drops prepared for commercial availability must pose no risk for contamination and subsequent infection. This study is designed to demonstrate conclusively that benzalkonium chloride plus cysteamine is a safe preparation that is effective when administered every waking hour to patients who have nephropathic cystinosis in corneal crystals. 283 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Clinical Protocol Number 93 EI-0230 Objectives The purposes of this study are to determine whether the addition of benzalkonium ctiloride to cysteamine eyedrops is a safe preparation and whether this preparation is effective in removing crystals from patients with cystinosis. Methods Thirty patients were to be entered into this study. These were nephropathic cystinosis patients for whom the code had been successfully broken in conjunction with protocol 86 EI-62. Each patient was randomized, with one eye always serving as a comparison to the fellow eye. One eye was treated with cysteamine alone; the second, with cysteamine 0.5% plus benzalkonium 0.101%. The primary outcome parameter was the safety of the additive benzalkonium. There were periodic checks of retinas for irritation attributable to benzalkonium. Efficacy for cysteamine was evaluated over a 6-month period. Major Findings In the 20 patients who have been em-olled in this protocol so far, there is no evidence of toxicity from the addition of benzalkonium to the cysteamine eye drops. Furthermore, if used as required, the cyste- amine plus benzalkonium appears to be as effective as the cysteamine without benzalkonium. Significance to Biomedical Research and the Program of the Institute Ensuring that benzalkonium added to cysteamine eyedrops is not toxic but still effective will move this drug one step closer to new drug approval by the FDA. Proposed Course Since the toxicity has been proven to be nil and the drug is still effective, this protocol will be termi- nated. NEI Research Program Corneal Diseases— Ocular Surface Problems (Drug Delivery and Toxicity) 284 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PHOJECT NUMBER ZOl EY 00284-01 OGCSB PERIOD COVERED October 1, 1992 to September 30, 1993 TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) Characteristics of Macular Scotomas in Patients With Primary Monofixation Syndrome PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute afliliation) PI: Mark H.Scott M.D. Senior Staff Fellow OGCSB, NEI Others: Rafael Caruso M.D. Muriel I. Kaiser-Kupfer M.D. Visiting Scientist Chief OGCSB, NEI OGCSB, NEI COOPERATING UNITS (if any) Marshall M. Parks, M.D. (Private Practice), Washington, DC LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Ophthalmic Genetics INSTITUTE AND LOCATION NEI, NTH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.175 PROFESSIONAL: 0.175 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects [x] (a1) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The monofixation syndrome (MPS) is a defective form of binocular vision characterized by preservation of extramacular function with absence of macular fusion. Fusion is defined as the ability to perceive simultaneously similar images projected onto corresponding areas of each retina. The fusing of images is a binocular phenomenon that occurs in the higher-order parastriate and peristriate areas of the prestriate visuomotor cortex (Brodmann areas 18 and 19, respectively). Patients in this protocol have been examined by Goldmann perimetry and the Lancaster red-green test to map the facultative macular scotoma in the nonfixating eyes in patients with primary MFS, surgically corrected congenital esotropia, and anisometropic amblyopia. The characteristics of the scotomas in each population of patients will be compared. The results of this study will contribute to the understanding of primary MFS by testing the hypothesis that primary MFS is a mild expression of a gene or series of genes that causes congenital esotropia and that these genes exert their variable expression on the binocular neurons of the central macular fusion area. 285 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report— FY 1993 Project Description Clinical Protocol Number 93 EI -0067 Objectives The objectives of this study are to plot the character- istics of macular scotomas in patients with primary monofixation syndrome (MPS) and to gain data to test the hypothesis that such MPS scotomas may result from expression of a particular gene or series of genes that cause congenital esotropia. Methods Patients entering the study undergo a complete ophthalmic examination by Goldmann perimetry. By use of the Lancaster red-green test, the facultative macular scotoma is mapped out in the nonfixating eyes of patients with primary MPS, surgically cor- rected congenital esotropia and anisometropic amblyopia. The kinetic mode of the perimeter is used to plot the size and shape of the scotoma; the static mode of the perimeter is used to determine the depth of suppression within the scotomatous region. Lancaster red-green tests both standard and auto- mated versions also are used to plot the size of the scotomas. The characteristics of the scotomas in each population of patients can then be compared with each other. Major Findings Although the study is in its preliminary phases, it has been possible to plot the scotomas as planned. Analysis of the data awaits further recruitment. Significance to Biomedical Research and the Program of the Institute A better understanding of mechanisms of develop- ment of scotomata will help in elucidating the etiologies of certain potential blinding conditions such as amblyopia Proposed Course Work will continue with the completion of the analysis of data from patients who have been re- cruited into the study. NEI Research Program Developmental and Strabismus 286 Index Index Acetazolamide 99 Acidic fibroblast growth factor 174 Action potentials 226 Acyclovir 83 Adaptometry 262 Adhesion molecules 59 Aggregation 139 AIDS (acquired immune deficiency syndrome) 43, 45, 57 Albinism 248, 272 Aldehyde dehydrogenase 168 reductase 189 Aldose reductase 43, 131, 133, 189, 194 inhibitor(s) 44, 131, 149, 187, 189, 194, 251 Animal model 126 autoimmune disease 44 diabetic 251 dog 44 beagle 190 diabetic 194 galactose-fed 187, 190 galactosemic 251 monkey 220, 239, 242 macaque 48 retinas 50 Rhesus 43, 225 mouse Balb/c 82 C3H-hen, EAU 104 CD-I 82 chromosome 214 chromosomal locations 214 EAU model 58, 108 mutant 202 ocular toxoplasmosis 59 transgenic 67, 168, 174, 205 rat diabetic 194 retinopathy 43, 131 EAU 108 galactose-fed 150 galactosemic model 149 lens 194 Lewis 116, 126,220 B cells 202 EAU 58, 61, 104, 119 Royal College of Surgeons (RCS) 52 transgenic 44, 67, 155, 180 uveitis 99 Anterior chamber anomalies 260 Anticataract agents 44, 143, 146, 187 drugs 131 Antigen(s) cross-reactive 220 S- 115, 116 Antisense ribozymes 205 Antiviral therapy 82 Apoptosis 211 Aqueous humor dynamics 270 Arginine-deficient diet 274 Autoimmune diseases predisposition 67 Autoimmune inflammation etiology 220 B Benzalkonium chloride 284 Bilateral acoustic neuroma 248, 277 Binocular alignment 225 Biochemical 52 Bionutrition 52 Brain behavior control 226 mechanisms 43, 225 c Cadherins 171 Cataract(s) 43,45, 131, 135, 139, 189, 247, 256 (see Congenital cataracts) clinical grading 253 development 146 formation 143, 253 Marner 171 mature proteins 52, 136 sugar 149 Cataractogenesis 247 Cell adhesion 59 molecules 104, 171 cycle regulatory protein, cyclin B 158 flare meter 45 Cell-cell communication in the lens 177 Cellular differentiation 44, 155 289 Index NEI Annual Report— FY 1993 Cerebra] cortex 233 Chaperones 202 Chromosomal locations human 214 mouse (see Animal model, mouse) Clinical immunology 57 services 249 Collagen dysgenesis 92 Color vision 262 Combination therapy 61 Complications from contact lenses 251 Cone neuron development 45 Cones blue-sensitive 50 green-sensitive 50 red-sensitive 50 Congenita] cataracts 133 esotropia 286 hereditary cataracts 258 Contrast sensitivity 45 Cornea 168 cyrstals in 280 disease 247 endothelium 168 epithelium 168 healing defects 149 Coronaviruses 82 Corticosteroids 126 Cortisol 99 Crossing of the optic nerve fibers 272, 282 Cryopreservation 181 Crystallin 45 (see Human A-crystallin) a-crystallin 131, 146, 162, 183 oB-crystallin 59 p-crystalUn(s) 43, 131, 138, 163 PB2 crystallin 143 6-crystallin gene 163 ^-crystallin 146 Q-crystalUn 164 aggregation 135 enzyme- 168 J- 164 S- 164 Cushing's syndrome 277 Cyclins 44 Cyclosporine 123, 126 A 61 Cysteamine 249 eyedrops 284 topical 280 Cytokines 59, 76. 104 Cytomegalovirus (CMV) 82 retinitis 43,45, 57, 113 Cytoskeletal proteins 165 Cytotoxic agents 126 D Depth vision 225 Dexamethasone 61 Diabetes 67, 133, 189 Diabetic complications 131, 187, 194 retinopathy 44, 149, 190 Diagnosis 99 noninvasive methods 262 Dideoxyinosine(ddl) 2', 3'- 102 Diet low-arginine 275 Dietary intervention 45, 248 Distal promoter (see Promoter, distal) E EBV-transformed B cells 202 Electrical activity of the human visual system 265 stimulation 242 Electrodiagnostic information 249 Electrophysiological techniques 265, 268 Embryonic stem cells 183 Embryos 180 Endothelial abnormalities 251 Endotoxin-induced uveitis (EIU) 61, 92, 99 Enzyme digestion procedure elastase 149 Experimental autoimmune uveitis (EAU) 45, 58, 64, 69, 92, 104, 108, 126, 202, 220 Extracapsular cataract extraction 256 Eye development 175 physiology 67 Eye movement(s) 239 control 44 rapid 43 saccadic 43 F Fatty acid-binding proteins 211 site on IRBP 202 FK 506 99, 120 Fluorescence energy transfer 202 Fluorescent fatty acid analogs 202 Fluorouracil 99, 119 Frontal eye field 227 290 NEI Annual Report— FY 1993 Index GABA-agonist 240 Galactose 44 Galactosemia 189 Ganciclovir 43, 113 slow-release implant 45, 57 Gene(s) Y-crystallin 171 expression 44, 59, 205, 247 knockout 183 lens 177 regulation 45, 155, 162 mechanism of 158 retina-pigment epithelium complex 21 1 retina-specific genes 45, 199 therapy 43, 45, 46, 199, 208 (see Gyrate atrophy) Genetic(s) counseling 272, 277 disease 277 disorders 43 engineering 44 ophthalmic 247 Genomic manipulation 155 Genotyping 139 Glare testing 45 Glaucoma 260, 270 Glutathione 143, 146 Graft rejection immunology of 119 suppression of 1 19 Growth factors 89 Gyrate atrophy 45, 46, 247, 274 gene therapy 58 H Heat shock protein 202 HSP70 116,202 Hereditary diseases 211 Herpes simplex virus type 1 (HSV-1) 82 Histidine 43, 133 HIV (human inmiunodeficiency virus) infection 102 Homologous recombination 183 Horizontal disparity in the images 43 Human otA-crystallin 170 chromosome 214 frontal eye field 233 kidney cortex 194 retinal pigment epithelial (RPE) cell 89 S-antigen 69, 123 Immune responses 69 Immunology experimental 58 Immunomodulation 126 Immunopathology experimental 59 Immunoregulation 58 Immunosuppressive agents 92 treatment 61 Immunotherapy 108 Implantable slow-release device 113 (see ganciclovir slow-release implant) Inflammatory mediators 89 lasulin-like growth factor [IGF]-1 208 Intercellular adhesion molecule 1 (ICAM-1) 59 Interferon 76 gamma (IFN-y) 59, 67 Interinstitute Genetics Program 248 Interleukin 1 (IL-1) 59 2(E.-2) 76 receptor 45 6(IL-6) 59,76 Interphotoreceptor matrix 208 retinoid-binding protein (IRBP) 69, 116, 199, 205 Intraocular inflammation 1 19 lymphoma 99, 119 promoter 205 turnover 202 Irido-comeal-endothelial (ICE) syndrome 260 K J J-crystallin (see Crystallin, J-) L Laser examination 113 Lens 44, 52, 143 biology 131 cell differentiation 158, 170 crystallins 44, 131, 146, 162 development 162 materials 256 opacities 247, 274 organ culture 131 structure and function 170 Lewis rat (see Animal model, rat) Leukoregulin 83 291 Index ^fEI Annual Report— FY 1993 Linkage analysis 258, 282 Linomide 58, 70 Lipid peroxidation 52 Lipopolysaccharide G-PS) . -,116 LOCS-2 grading system 45 Long QT syndrome 139 Luminance and chromatic increment thresholds 262 Lymphocyte function-associated antigen 1 (LFA-1) 59 Lymphokines 92 M Macaque retinas (see Monkey, Macaque) Macrophage migration inhibitory factor 171 Macular scotomas 286 Magnetic resonance imaging 187 Magnetization transfer contrast 190 Marner cataract (see Cataract(s)) Metals 131 Methylprednisolone 120 Microinjecting DNA 180 Miniosmotic pump 61 Molecular biology 57, 168, 170, 208 function 155 genetic techniques 258 genetics 155, 208, 209 interactions 247 markers of differentiation 171 structure 155 Molteno glaucoma implant 57, 1 19 Monoclonal antibodies 104 antibody therapy 45 Monofixation syndrome 286 Mouse chromosome (see Animal model, mouse) Movement visual control 43 Myotonic dystrophy 139 N NADPH 147 -dependent enzymes 194 Nephropathic cystinosis 249, 277, 280, 284 Neural crest 260 Neurofibromatosis 247, 277 Neuronal discharge 226 Nitric oxide 61 Nonradiative energy transfer 202 Nystagmus 272 O Objective systems 45 Ocular autoimmune diseases 64 complications of diabetes 43 development 209 diseases 199 inherited 277 hypertension 270 immunomodulation 58 inflammation 104 inflammatory diseases 45 pigmentation 282 Opacities in the human lens 45 Ophthalmic drugs 187 Oral tolerization 57 Ornithine aminotransferase gene 248 Ornithine-6-aminotransferase (OAT) 274 gene 57 Oxidation 131 thiol-dependent metal-catalyzed 135 Oxidative stress 143, 146 P Parietal cortex 233, 239 Pathology ophthalmic 249 Peptide 1169-1191 202 Perimetry 262 Peripheral vision fields 239 PET scaiming 226 Phospholipids 214 Photophobia 280 Photoreceptor cell 214 -specific genes 199 site 217 Pigment dispersion syndrome (PDS) 270 epithelium 211 derived factor (PEDF) 45, 199, 208 Pigmentation iris 272 retinal 272 ocular 272 Plasma ornithine 274 Polyol 133 formation 190 pathway 194 Post-transcriptional regulation 214 Primate visual system 50 292 NEI Annua] Report — ^FY 1993 Index Proliferative retinopattiy 187 Promoter oA-crystallin 174 distal 217 proximal 217 Protein 72-kD 202, 203 Proto-oncogene(s) 44, 158 int-2 174 Psychophysical diagnostic information 249 techniques 262, 268 Pyridoxine treatment 274 Q R Rapamycin 45, 61 Regulatory cell in the retina 76 element 44 Retina 52,211 -specific genes (see Genes) Retinal 11 -CIS 202 antigen-specific T-cell lines 108 degeneration 52,202,211 degenerative disorders 79 disease 247 fatty acid defea 200 genetic 248 tubulin defect 200 vasculature 149 Retinal pigment epithelium(al) (RPE) 45 52 59, 102, 202, 214 cell(s) 76,79, 119 transplantation 79 -specific expression 214 gene(s) 45, 199 transplants 45 Retinitis 119 CMV (see Cytomegalovirus (CMV), retinitis) pigmentosa 247 Retinoblastoma 59 Retinoic acid 171 Retinoid metabolism 202 Retinyl palmitate 202 Rostral superior colliculus 242 RPE (see Retinal pigment epithelium[al]) S-Ag 115, 116 promoter sequences 217 -induced uveitis (see uveitis, S-Ag-induced) Saccadic eye movements 225, 233, 242 Sampling intraocular tissue 1 19 Sarcoidosis 99 Selective visual attention 226 Shift of attention 239 Single neuron recording 233 Site-directed mutagenesis 43 Sorbitol dehydrogenase 43, 131, 133 Spatial vision 262 Spectral property transformations 48 Stabilization of posture 44, 225 Stimulus-dependent messages 226 Sugar cataracts (see Cataract(s)) Superior colliculus 239 T T lymphocyte(s) role of 64, 92 T-cell receptor (TCR) genes 58 therapies 64 Targeting veaor 183 Temporal code 44 patterning 226 Temporally modulated code 229 Tolerance 220 Toxic compounds 89 Toxoplasmosis 45 ocular 59 model 45 Trabeculectomy 99 Tumor necrosis factor (TNF-a) 59, 76 U Usher's syndrome 139, 282 type I 43, 131, 139 Uveitis 67, 92, 99, 123, 126, 214, 220 IRBP-induced 58 S-Ag-induced 58 Uveitogenic antigens 126 Uveitopathogenic determinant 202 Uveoretinitis 61 V Virology 59 Virus infections 82 293 Index NEI Annual Report— FY 1993 Visual cortex (areas VI, V2. V3, and V4) 229 deficit 268 loss differential diagnosis 268 motion 226 motor behavior 225 pathway 48 abnormalities 247 perception 44, 226 sense 43 stimuli 242 Visually evoked potentials (VEP) 248, 265 Visuospatial attention 233 Vitritis 119 w X X-linked agammaglobulinemia 139 Y 294 Hoft '?^^"'er Drive 3 1