FISCAL YEAR 1992 Cover Photo Photomicrograph of a rhopalium within a formalin-fixed cubomedusan jellyfish (Tripedalia cystophora). The rhopalium is attached to a stalk within a depression on the surface of the jellyfish. It contains a larger (top) and smaller (right) complex eye at right angles to one another as well as a statocyst (balancing organ). Each eye has a protruding cellular lens, retina, and pigmented epithelium. The picture was taken by Dr. Toichiro Kuwabara and was described in Piatigorsky, Horwitz, Kuwabara, and Cutress (1989) J. Comp. Physiol. A 164:577-587. NATIONAL EYE INSIIIUIE NATIONAL INSTITUTES OF HEALTH NIH LIBRARY MAR 2 I 1995 BLDGIO, 10 CENTER DR. BETHESDA, MD 20892-1150 Annual Report ^^c ^o^c-c,^ ckc^/,^;J Fiscal Year 1 992 U.S. Department of Health and Human Services Public Health Service National Institutes of Health "RE I .. 'S.i\ \ tl 1'" Table of Contents Statement of the Institute Director l Carl Kupfer. MD. Extramural and Collaborative Program 5 Report of the Associate Director 7 Jack A. McLaughlin, PhD. Retinal and Choroidal Diseases 7 Corneal Diseases 8 Cataract 9 Glaucoma 10 Strabismus, Amblyopia, and Visual Processing 10 Biometry and Epidemiology Program 13 Report of the Acting Associate Director 15 Roy Milton, PhD. Office of International Program Activities 17 Report of the Acting Assistant Director 19 Terrence Gillen, M.BA., MA. Office of Science Policy and Legislation 21 Report of the Acting Associate Director 23 Michael P. Davis Policy, Legislation, Planning, and Evaluation Section 23 Management Information Systems Section 23 Scientific Reporting Section 24 Office of the Scientific Director 27 Report of the Scientific Director 29 Robert B. Nussenblatt, MD. Office of the Scientific Director Francisco M. de Monasterio, MD., DSc. Physiological Studies of the Primate Visual System 33 Anatomical Studies of the Primate Visual System 35 Helen H. Hess, MD. Biochemistry of Retina and Pigmented Epithelium in Health and Disease 37 Laboratory of Immunology 41 Report of the Chief 43 Robert B. Nussenblatt, MD. Table of Contents NEI Annual Report-FY 1992 Section on Clinical Immunology Francois G. Roberge, MB. Study of Immunosuppressants for the Treatment of Uveitis in Animal Models 46 Benjamin I. Rubin, MD. Comparison of Surgical Treatment in Uveitis Patients With Glaucoma 49 Section on Clinical Immunoregulation Juan S. Lopez, MD. Magainin Therapy of Infectious Keratitis 50 Section on Experimental Immunology Charles E. Egwuagu, M.PH., PhD. Selective Accumulation of Vp8-Positive T Lymphocytes in Experimental Autoimmune Uveoretinitis 51 Ectopic Expression of Interferon-Gamma in the Eyes of Transgenic Mouse 54 Igal Gery, PhD. Immime Responses to Ocular Antigens 56 Section on Immimopathology Chi-Chao Chan, MD. Immunopathology in the Eyes With Experimental Uveitis 60 Immunopathology of Ocular Diseases in Humans 63 Cytokines and Ocular Antigens in the Eye 66 John J. Hooks, Ph.D. Interferon System in Cellular Function and Disease 68 Studies on the Bioregulatory Aspects of the Retinal Pigment EpitheUal CeU 71 Virus Infections in the Eye 74 Chandrasekharam N. Nagineni, PhD. Role of Retinal Pigment Epithelium in Retinal Disorders 78 Scott M. Whitcup, MD. The Diagnosis and Treatment of Human Uveitis 81 Ocular Toxicity of 2',3'-Dideoxyinosine (ddl) 84 CeU Adhesion Molecules in Ocular Inflammation 86 Section on Immimoregulation Rachel R. Caspi. PhD. Cellular and Immunogenetic Mechanisms in Uveitis 90 Experimental Autoimmune Uveitis in the Mouse 95 The Evaluation of the Antiflammins in the Anterior Uveitis 96 Marc D. de Smet, MD. Ocular Manifestations of the Acquired Immune Deficiency Syndrome 97 Charaaerization of Immime Responses to S-Antigen 100 Modulation of Immune Functions Using PE40 Derived Immimotoxins 103 Surgical Management of Uveitis 104 Robert B. Nussenblatt, MD. Immune Functions in Ocular Diseases of Obscure Etiology 107 Cyclosporine Therapy in Uveitis 108 Oral Administration of Antigen and the Ocular Immune Response Ill NEI A nnual Report-FY 1992 Table of Contents Laboratory of Mechanisms of Ocular Diseases 113 Report of the Acting Chief 115 /. Samuel Zigler. Jr., PhD. Section on Cataracts Deborah Carper, Ph.D. Structure and Expression of Polyol Pathway Enzymes 116 Donita L. Garland, PhD. Oxidation of Proteins in Cataractogenesis 118 James Fielding Hejtmancik, MD., Ph.D. Inherited Ocular Disease 121 Paul Russell, PhD. Characterization of the Lens 126 Cataract in the PhUly Mouse Strain 128 J. Samuel Zigler, Jr., PhD. Structure and Composition of Lens Crystallins with Respect to Cataractogenesis 130 Section on Pathophysiology Bruce A. Pfeffer, Ph.D. Ocular Cells Cultured Under Normal and Diabetic Conditions 133 W. Gerald Robison, Jr., Ph.D. Ultrastructure and Function of the Cells and Tissues of the Eye 134 Laboratory of Molecular and Developmental Biology 137 Report of the Chief 139 Joram Piatigorsky, PhD. Section on Cellular Differentiation Peggy S. Zelenka, Ph.D. Proto-Oncogene Expression During Lens Differentiation and Development 142 Section on Molecular Genetics Ana B. Chepelinsky. PhD. Genetically Engineering the Eye with the oA-Crystallin Promoter 146 Regulation of Expression of Lens Fiber Membrane Genes 149 R. Andrew Cuthbertson, MD., PhD. Physiological Expression of the Retinoblastoma- Associated Gene During Lens Cell Differentiation 152 Joram Piatigorsky, PhD. Crystallin Genes: Structure, Organization, Expression, and Evolution 153 Molecular Biology of the Cornea 159 Section on Molecular Structure and Function Robert Y. Kim, MD. Gene Expression in the Retinal Pigment Epithelium 161 Graeme J. Wistow, PhD. Molecular Biology and Functions of Lens Proteins 162 Table of Contents NEI Annual Report-FY 1992 Laboratory of Ocular Therapeutics 167 Report of the Chief 169 Peter F. Kador, Ph.D. Peter F. Kador, Ph.D. Pharmacology of Ocular Complications 170 Sanai Sato, M.D. Role of NADPH-Dependent Reductases in Ocular Complications 175 Laboratory of Retinal Cell and Molecular Biology 179 Report of the Chief 181 Gerald J. Chader, PhD. Section on Biochemistry Barbara Wiggert, Ph.D. Vitamin A and Ocular Tissues 184 Section on Gene Regulation Diane E. Borst, Ph.D., and Steven Bernstein, MD., Ph.D. Molecular Genetics of the Eye and Ocular Diseases 188 Gerald J. Chader, PhD. Metabolism of the Retina and Pigment Epithelium 191 Visual Control Mechanisms 194 T. Michael Redmond, PhD. Molecxilar Biology of Outer Retina-Specific Proteins 198 Section on Molecular Biology Toshimichi Shinohara, PhD. Molecular Biology of Phototransduction 201 Molecular Biology of Experimental Autoimmune Uveitis 204 Laboratory of Sensorimotor Research 207 Report of the Chief 209 Robert H. Wurtz, Ph.D. Section on Neural Modeling Lance M. Optican, PhD. Information Processing by Visual System Neurons 211 Section on Neuro-Ophthahnologic Mechanisms Michael E. Goldberg, MD. Cerebral Cortical Mechanisms for Eye Movements and Visual Attention 215 Section on Oculomotor Control Frederick A. Miles, PhD. Visual Motion and the Stabilization of Gaze 218 Section on Visual Behavior David Lee Robinson, PhD. Visuomotor Properties of Neurons in the Thalamus 221 Section on Visuomotor Integration Robert H. Wurtz, PhD. Visuomotor Processing in the Primate Brain 225 NEI Annual Re port-FY 1992 Table of Contents Ophthalmic Genetics and Clinical Services Branch 229 Report of the Chief 231 Muriel I. Kaiser-Kupfer, MJD. Section on Cataract and Corneal Diseases Manual B. Datiles, M.D. The Effects of Corneal Contact Lenses on the Cornea 234 Documentation and Monitoring of Opacities in the Human Lens 236 Use of Human Lens Material for Determining Possible Causes of Cataracts 239 Carl Kupfer, MD. Anterior Chamber Anomahes Associated with Glaucoma or Occular Hypertension 241 Section on Eye Services Rafael Caruso, MD. Clinical Psychophysics of the Visual System 243 Qinical Electrophysiology of the Visual System 245 Visual Function Diagnosis Service 248 Section on Ophthalmic Genetics Muriel I. Kaiser-Kupfer, MD. Pigment Dispersion With and Without Glaucoma 250 Visual Function and Ocular Pigmentation in Albinism 252 Gyrate Atrophy of the Choroid and Retina and Other Retinal Degenerations 254 NIH Interinstitute Genetics Program: The Genetics Clinic 257 A Double-Masked Controlled Randomized Qinical Trial of Topical Cysteamine 259 Index 261 Statement of the Institute Director statement of the Institute Director Carl Kupfer, M.D. Vision researchers supported by the National Eye Institute (NEI) once again have had a highly productive year. They have made outstanding progress in our effort to improve the prevention, diagnosis, and treatment of diseases and disorders that affect the eye and vision. Results from impor- tant clinical trials were reported this year, and several basic research studies have provided information that greatly improves our understanding of some of the pathological processes associated with blinding eye diseases. Clinical trial results were released recently from the NEI-supported Herpetic Eye Diseases Study (HEDS), a randomized, controlled clinical trial designed to evaluate whether oral acyclovir, given to patients in combination with steroid and antiviral eye drops, improves the management of active heipes simplex stromal keratitis. HEDS investigators reported that oral acyclovir is no better than a placebo in treating the condition. However, the role of acyclovir in preventing recurrences and progres- sion to a more severe form of the disease is still being examined by the HEDS research group. Results also were reported from the NEI-spon- sored Collaborative Corneal Transplantation Study (CCTS), which was designed to evaluate whether donor-recipient tissue typing helps to prevent trans- plant rejection. CCTS researchers found that people who received corneal transplants with well-matched antigens did not fare significantly better than those people receiving transplants without such a match. This wiU eliminate the need for costly tissue typing. Recently, researchers supported by the NEI demonstrated that a-crystallin belongs to a class of proteins called molecular chaperons. Laboratory tests have demonstrated that the biological function of a-crystallin may be to maintain the lens structural proteins in a transparent state. With this important discovery, it is now possible to investigate a number of long-standing questions in lens biology and cataractogenesis and to examine directly whether certain age-related lens changes can be prevented or reversed. The NEI-sponsored Optic Neuritis Treatment Trial (ONTT) compared oral corticosteroid (ie, predni- sone), intravenous corticosteroid methylprednisolone followed by oral prednisone, and placebo for the treatment of new cases of optic neuritis. ONTT investigators found that oral prednisone, the most conunon treatment for the disease, is ineffective when used alone in treating the disease and actually increases a person's risk for future attacks. The Age-Related Eye Disease Study has begun and wiU provide information on the development (incidence), course, and progression of age-related macular abnormalities and lens opacities. The study also will evaluate various possible risk factors for the development and/or progression of the two disorders, while another arm of the study will assess the effects of nutritional supplements and/or minerals on the development and progression of age-related macular degeneration and cataract. The first two National Eye Health Education Program public service campaigns were released in January. The glaucoma campaign — consisting of print, radio, and television public service announce- ments (PSAs) — and the diabetic eye disease cam- paign — ^using print PSAs — targeted potential audi- ences at risk for these diseases totaling more than 60 million people. The glaucoma television PSAs were aired more than 25,000 times in 47 States by 239 stations, representing more than $2.4 million in air time. The radio PSAs were aired more than 30,000 times in 49 States by 360 stations, representing $2.7 million worth of air time. Intramural investigators have developed the ability to produce large nimibers of retinal pigment epithe- hal (RPE) cells that have been used in ongoing transplantation experiments. They also have devel- oped a microinjection technique that allows place- ment of RPE cells into the proper space beneath the retina. Studies are currently under way to develop molecularly altered RPE cells that do not evoke an immune response and that, therefore, coixld be used as universal donors. Statement of the Institute Director NEI Annual Report-FY 1992 The role of various modifications of lens crystal- lins in the development of cataract is being investi- gated by NEI investigators. The primary focus is on metal-catalyzed oxidation, and gamma crystallin is being used as a model system. Using this system, these scientists have isolated a protein from bovine lenses that protects from this type of oxidation. Information from these types of studies may ulti- mately lead to the development of the means to prevent cataract formation in humans. Intramural scientists are using subtractive cloning to identify genes that are predominantly or exclusive- ly expressed in ocular tissues. Retina-specific genes located on the short arm of the X chromosome are being localized to determine whether they are disease linked. These are but a few of the highlights of the activities of NEI-supported extramural and intramural scientists during Fiscal Year 1992. The following reports provide more detail on these and other activities and accomplishments of each of the NEI's sections, branches, laboratories, offices, and programs during this past year. Carl Kupfer, M.D. Extramural and Collaborative Program Report of the Associate Director for the Extramural and Collaborative Program Jack A. McLaughlin, Ph.D. Extramural research activities supported by the Vision Research Program address the leading causes of blindness 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, as well as better diagnose, prevent, and treat, sight-threatening condi- tions; and to enhance the rehabilitation, training, and quality of life of individuals who are partially sighted or blind. In Fiscal Year 1992 the National Eye Institute (NEI) supported 1,241 research grants, 262 training awards, and 15 contracts for a total of $229,330,000. Individual expenditures in those areas were as follows: Research Grants Research Training Awards Research Contracts Total $216,957,000 7,294.000 5,079,000 $229,330,000 The NEI continues to emphasize investigator- initiated research with this group of mechanisms, accounting for 78% of the extramural budget. The following are highlights of the activities and accom- plishments of NEI-supported extramural investiga- tors. Retinal and Choroidal Diseases The retina, a delicate, light-sensitive tissue at the back of the eye, initiates the process of vision and transmits visual messages to the brain via the optic nerve. Collectively, diseases and disorders of the retina are the leading cause of blindness in the United States. Diabetic Retinopathy Approximately one-half of the estimated 14 million people in the United States w..o have diabetes experience at least early signs of diabetic retinopathy. Of this group, about 700,000 have serious retinal disease, and 8,000 people are blinded by this dis- order annually. Generally, the longer one has had diabetes, the greater is one's chances of developing diabetic retinopathy. Laser surgery is now being used successfully to treat two forms of diabetic retinopathy: (1) macular edema, in which a central portion of the retina swells and blurs vision, and (2) proliferative retinopathy, in which abnonnal, easily ruptured, new blood vessels grow onto the surface of the retina. The series of clinical trials that proved the value of laser treatment for diabetic retinopathy were supported by the NEI. The cost savings to the Federal Government associ- ated with improved detection and treatment of diabetic retinopathy and diabetic macular edema have been estimated to exceed $100 million annually. Opportunities for research include studies of newly discovered growth factors that control blood vessel growth, regulation of retinal blood flow, and new drugs and molecular genetic techniques to control retinal metabolism. Age-Related Macular Degeneration Age-related macular degeneration (AMD) is an important and growing public health problem; it is the leading cause of legal blindness for older Ameri- cans. By 1995 it is estimated that 1.7 million Americans will have decreased vision from AMD and 100,000 will be blind from the disease. The prevalence of decrease vision from AMD is expected to rise to 2.6 million by the year 2020, when an even greater percentage than now of our population will enter its retirement years. AMD destroys photo- receptor cells in the macula, the small specialized area of the central retina that is responsible for sharp, crisp vision. As the disease progresses, the acute vision that is needed for reading, driving a car, and Extramural and Collaborative Program NEI Annual Report-FY 1992 other daily activities is diminished, sometimes gradually, sometimes catastrophically. Unfortunately, there is no proven way to either prevent AMD or treat the vast majority of people who have the disease. An important new goal of the Age-Related Eye Disease Study is to evaluate the effect of high-dose antioxidant vitamins and zinc on the progression of AMD. Patients wiU be randomized to a high-dose dietary supplement or placebo and followed for a minimum of 7 years to assess AMD progression and cataract fonnation. NEI-funded basic scientists are searching for the cause of AMD. Molecular genetic approaches seem most likely to provide useful information about AMD, since there are no precise animal models for this disease. Retinitis Pigmentosa Retinitis pigmentosa (RP) is a diverse group of hereditary retinal diseases that causes a progressive degeneration of the rod and cone photoreceptors and loss of visual function. RP affects approximately 100,000 people in the United States. Currenfly, no effective treatments are available for the common types of RP. Molecular genetic techniques have enabled the identification of gene defects responsible for several forms of RP. NEI-supported scientists have found that some RP patients carry mutations in the gene encoding rhodopsin, the visual pigment critical for initiating the visual process. Other forms of RP can be investigated using these techniques. In parallel studies, transgenic mice have been produced that carry the human gene for one form of RP. Expres- sion of this gene in mice causes a retinal degenera- tion that is similar to that seen in humans. Further study of this animal model has enormous potential for delineatiaig the precise molecular and cellular mechanisms involved in the pathogenesis of RP. These types of studies have begim to provide the first real clues to understanding the mechanisms by which RP causes blindness. They are important first steps in the development of rational treatment strate- gies. Ocular Complications of AIDS Cytomegalovirus (CMV) retinitis is a potentially blinding disease of the retina that affects about 20% of people with AIDS (acquired immunodeficiency syndrome). The NEI supports a network of investi- gators with expertise in AIDS clinical research, retinal diseases, and clinical trial methodology to expedite the testing of new treatments for CMV retinitis and other ocular complications seen in AIDS patients. This network is called the Studies of the Ocular CompUcations of AIDS (SOCA). The CMV Retinitis Retreatinent Trial is the second multicenter clinical trial conducted by SOCA investigators. The study's primary objective is to compare the safety and efficacy of three therapeutic regimens for AIDS-related CMV retinitis in patients who were treated with foscamet or ganciclovir and whose retinitis progressed or recurred. The three regimens under investigation are (1) high-dose foscamet, (2) high-dose ganciclovir, and (3) combin- ation treatment with standard doses of foscamet and ganciclovir. Retinal Detachment The Sihcone Oil Study, a NEI-sponsored 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 after intraocular surgery for severe proliferative vitreoretinopathy (PVR). The study found that use of silicone oil is superior to use of long-acting gas in PVR treatment, resulting in a higher rate of successful retinal re- attachment. Corneal Diseases Diseases and injury to the cornea are the leading causes of visits to clinicians for eye care in the United States and rank among the most painful of eye conditions. NEI-supported research on comeal diseases includes studies of ocular infections and inflammation, problems with tears and the ocular surface, reflractive errors, comeal transplantation, and comeal wound healing. Ocular Herpetic Infection Second only to trauma as the leading cause of comeal blindness in the United States today, ocular herpes simplex virus keratitis accoimts for 500,000 reported cases yearly. Ocular herpes infections often begin as painful sores on the surface of the cornea NEI Annual Report-FY 1992 Extramural and Collaborative Program (ie, the transparent tissue at the front of the eye). Like heipes cold sores, these ocular lesions may recur periodically. Over time, the herpes virus may cause an inflammation deep inside the cornea. This deep infection is known as herpes simplex stromal keratitis, a condition which can lead to severe cor- neal scarring, inflammation of the interior of the eye, and even blindness. This disease affects otherwise healthy, productive individuals, leading to long periods of disabiUty and the need for chronic care. The costs associated with ocular herpetic infection are significant, totaling approximately $100 million per year in direct medical costs, not counting the costs associated with loss of work. The NEI-supported Herpetic Eye Diseases Study (HEDS) is a randomized, controlled clinical trial designed, in part, to evaluate whether oral acyclovir, given to patients in combination with steroid and antiviral eye drops, improves the management of active herpes simplex stromal keratitis. HEDS investigators recently reported that oral acyclovir is no more effective than a placebo in treating the condition. This indicates that the significant costs associated with the use of oral acyclovir can be avoided. The role of acyclovir in preventing recur- rences of herpetic eye diseases over the course of IVi years and the role of acyclovir in preventing progres- sion of superficial (ie, epitheUal) keratitis to the more severe stromal keratitis or iritis are currenfly being examined by the HEDS research group. Corneal Wound Healing The cornea's vulnerable position at the ftont of the eye places it at great risk for damage from infection, injury, and toxic agents. Such problems can lead to ulceration of the cornea and the formation of opaque scar tissue, with permanent visual impairment Therefore, an important program goal is to under- stand and enhance the processes that lead to normal healing of the ocular surface. Such knowledge may lead to better ways to promote normal healing of corneal woimds. The role of various growth factor cell-cell and cell-matrix interactions is an important area for research. Corneal Transplantation More than 40,000 corneal transplant operations are perfonned annually in the United States; however, about 1 in 10 patients receiving a corneal transplant is at high risk of rejecting the transplant. The NEI- sponsored Collaborative Corneal Transplantation Study (CCTS) was designed to evaluate whether donor-recipient tissue typing helps to prevent trans- plant rejection. Previous studies had suggested that closely matching the donor's HLA with those of the recipient increases the likelihood that the patient's immune system would accept, rather than reject, the donor tissue. CCTS researchers have found, how- ever, that people who received corneal transplants with well-matched antigens did not fare significanfly better than those people who received transplants without such a match. These findings indicate that tissue typing was not an important factor in trans- plant survival. Had donor-recipient tissue typing become standard practice in corneal transplantation, it would have significanfly increased the waiting period and the cost (by at least $1,000) for this operation. Cataract Cataract, a clouding of the eye lens, is the leading cause of blindness worldwide, disabling approx- imately 20 million people. In the United States cataract is responsible for more than 3.3 million cases of visual impairment and accounts for 1.5 million surgical procedures for extraction annually. Cataract surgery is the most frequent operation performed among older Americans. Reimbursement for cataract surgery (totaling more than $5 billion) currenfly accounts for approximately 12% of the entire Medicare Part B budget. The enormous burden of cataract will worsen in coming decades as the American population ages. Consequenfly, the three major goals of this program are as foUows: (1) to determine the causes and mechanisms of cataract formation, (2) to search for ways to slow or prevent the progression of cataract, and (3) to devel- op and evaluate new diagnostic and therapeutic techniques in cataract management Function of a-Crystallins The a-crystallins are major structural proteins of the lens which contribute to its structure and transparen- cy. Recenfly researchers supported by the NEI demonstrated that a-crystallin belongs to a class of proteins called molecular chaperons. Chaperons stabilize protein conformations, mediate the folding Extramural and Collaborative Program NEI Annual Report-FY 1992 and correct assembly of new proteins, catalyze the membrane translocation of secretory proteins, and prevent protein aggregation in the face of heat denaturation or other environmental stresses. Lab- oratory tests have demonstrated that the biological function of a-crystallin may be to maintain the lens structural proteins in a transparent state. With this important discovery, it is now possible to investigate a number of long-standing questions in lens biology and cataractogenesis and to examine directly whether certain age-related lens changes can be prevented or reversed. Glaucoma Glaucoma is a group of eye disorders in which a distinct type of optic nerve damage leads to blindness. As well as being a major pubUc health problem, glaucoma is a leading cause of blindness in the United States and the number one cause of blindness in African-Americans. Approximately 3 million Americans have glaucoma, but one in two is unaware of hisAier condition. As many as 120,000 are now blind from this disease. Blindness from glaucoma costs the United States Government more than an estimated $1.5 billion annually in health care expenditures, lost tax revenues, and Social Security benefits. The NEI's extramural program activities in glaucoma are directed toward imderstanding the mechanisms of the disease through basic research, identifying risk factors, and preventing blindness through the development of improved methods for early detection and treatment. Treatment for Ocular Hypertension Elevated intraocular pressure (lOP) is a key risk factor for the development of open-angle glaucoma. However, there is no consensus on whether eariy treatment of elevated TOP prevents or delays the onset of open-angle glaucoma. The Ocular Hyper- tension Treatment Study (OHTS) is an NEI-support- ed, randomized, multicenter clinical trial to determine whether medical reduction of lOP prevents or delays the onset of glaucomatous optic nerve and/or visual field damage in ocular hypertensive subjects. The OHTS win enroll 1,500 subjects who have elevated lOP in at least one eye with normal visual fields and optic discs in both eyes; these individuals are consid- ered to be at moderate risk for the development of open-angle glaucoma. Study participants will be assigned randomly to receive medical treatment to both eyes or no treatment to both eyes. The central OHTS units have been funded, and the protocol is nearing completion. Patient recruitment is projected to begin in early 1994. Treatment for Open-Angle Glaucoma The Collaborative Initial Glaucoma Treatment Trial, also in a stage of protocol refinement, wiU compare medications with a surgical filtration procedure as the initial treatment for newly diagnosed open-angle glaucoma. This study will not only compare the two modes of treatment but wiU carefiiUy evaluate the quality of life among patients receiving the different therapies. Juvenile Glaucoma Several NEI-supported research groups are exploring molecular genetic techniques in studies of childhood glaucomas. These diseases are the result of congeni- tal abnormalities that lead to moiphological defects in the anterior segment of the eye. One type is autosomal dominant juvenile glaucoma (ADJG). This disease lends itself to molecular biologic tech- niques for identifying the gene(s) responsible for this condition. As a first step in mapping the ADJG gene(s), laboratories have established pedigrees of families in which the disease occurs. A group of investigators at the University of Michigan has identified a kindred large enough to establish a linkage map, and work has begim to determine the chromosomal location of the gene(s). Strabismus, Amblyopia, and Visual Processing Developmental disorders such as strabismus (ie, misalignment of the eyes) and amblyopia (ie, commonly known as "lazy eye") affea 2-4% of the U.S. population. Correction of strabismus is one of the most frequently perfoimed of all surgical opera- tions (about 700,000 operations each year). Blind- ness from amblyopia is preventable if the condition is detected and treated early in life. In addition to research on strabismus and amblyopia, the program supports research on neuro-ophthalmological dis- 10 NEI Annual Report-FY 1992 Extramural and Collaborative Program orders, presbyopia, and refractive errors. This program also supports research on more effective means to improve the quality of life for people with visual impairments by helping them maximize their use of remaining vision or by devising aids to assist those without useful vision. Currently 3 miUion Americans have chronic visual conditions that are not correctable by eye glasses or contact lenses and that impair everyday function. About 900,000 Americans are legally blind, and approximately 100,000 are totally blind or have only the ability to perceive light but not formed images. Understanding visual pro- cessing and its disorders require new knowledge of the visual nervous system, including the molecular, genetic, chemical, cellular, and integrative processes that underlie perception and the control of eye movements. Optic Neuritis Optic neuritis is an acute debilitating inflammation of die optic nerve that affects more than 25,000 Ameri- cans annually, primarily women between the ages 18 and 45. People with the disease usually experience rapid vision loss. Left untreated, most patients regain normal vision after several months of gradual improvement, but some are left with at least some visual deficit Because a significant number of people who have an initial attack of optic neuritis later develop multiple sclerosis, many physicians consider optic neuritis as a precursor or manifestation of that disease. The NEI-sponsored Optic Neuritis Treatment Trial (ONTT) compared oral corticosteroid (ie, prednisone), intravenous corticosteroid methyl- prednisolone followed by oral prednisone, and placebo for the treatment of new cases of optic neuritis. ONTT investigators found that oral cortico- steroid, the most common treatment for the disease, is ineffective when used alone in treating the disease and actually increases a person's risk for future attacks. The investigators also found that 27% of people taking oral prednisone had at least one new attack of optic neuritis during the foUowup period. In contrast, patients in the intravenous methylpredni- solone group had a 13% rate of new attacks, and those given a placebo had a 15% rate of subsequent optic neuritis. The ONTT study points the way to potential annual savings of $26 million in the treatment of acute optic neuritis. About $4 mUlion wiU be saved due to decreases in the optic neuritis recurrence rate when prednisone is no longer used as the common treatment modality. Another $4 million in savings wiU result if intravenous and other less common, but expensive, treatment methods are discontinued. The remaining $18 miUion in potential annual savings wUl be realized if cosfly and urmecessary pretreat- ment magnetic resonance imaging and blood testing are not included as part of the pretreatment evalua- tion. Development and Regeneration of the Visual System Critical to our understanding of the regenerative process is knowledge of the factors that control the elongation and guidance of visual nerve axons. Research over the last decade shows that adult visual neurons do not synthesize all of the constituents that they did during their development. A class of molecules known as growth-associated proteins (ie, pretreatment), for example, is found in the develop- ing central nervous system but is "down-regulated" as development ends. Recent work by NEI grantees has demonstrated that experimental interruption of optic nerves can lead to a reexpression of some pretreatment. Investigators are now trying to eluci- date the factors that regulate the genetic expression of pretreatment and related molecules with the goal of enhancing regenerative capacity. Computational Neuroscience Stereopsis, or the process by which we see in three dimensions, remains a difficult phenomenon to understand. Most individuals see objects from two slightiy different perspectives due to the lateral separation of the two eyes. The brain has the ability to somehow combine these two different monocular views and provide depth perception. How does this happen? Developments in computational neurosci- ence have provided new theoretical insights into the nature of vision. With these concepts, neurophysiol- ogists now have a highly sophisticated framework by which to guide their experimental approach. Eye Movements and Neurological Disease One type of eye movement is the vestibular-ocular reflex (VOR). Normally, a head mm in one direc- tion causes the eyes to rotate in the opposite direc- tion so that the eyes continue to point at the same place in the stationary worid. This is referred to as VOR. Without VOR, vision would appear blurred 11 Extramural and Collaborative Program NEI Annual Report-FY 1992 during head turns because the eyes would move with research has led to many new tests for diagnosing respect to the surroundings. neurological disease in humans and localizing its site VOR is being studied by NEI-suppoited investiga- through various oculomotor symptoms. VOR studies tore in monkeys fitted with telescopic spectacles that ^PP^^r exceptionally promising in this regard, alter the visual scene. The investigators have found Researchere recently have found specific sites in the tiiat, after several days, VOR becomes modified to nervous system tiiat appear to be associated with the compensate for the altered visual input. The im- behavioral changes in animals. Thus, VOR research mediate goal of tiiis research is to better understand provides us with a real opportunity to study the the neural basis for this behavioral plasticity. The dynamics of how these plastic changes occur at the continued assimilation of advances ftom basic behavioral, synaptic, and even molecular levels. 12 Biometry and Epidemiology Program Report of the Acting Associate Director for the Biometry and Epidemiology Program Roy Milton, Ph.D. The primary mission of the Biometry and Epi- demiology Program (BEP) is to plan, develop, and conduct human population studies concerned with the causes, prevention, and treatment of eye diseases and vision disorders. This includes studies of incidence and prevalence in defined populations, prospective and retrospective studies of risk factors, natural history studies, chiucal trials, genetic studies, and studies to evaluate diagnostic procedures. The BEP also conducts an education program in bio- metric and epidemiologic principles and methods for the vision research community, as well as provides biometric and epidemiologic assistance to the Nation- al Eye Institute's (NfEI's) scientific staff. The BEP began a prospective cohort study this fiscal year called the Age-Related Eye Disease Study (AREDS) that is designed to provide information on the development (ie, incidence), course, and progres- sion of age-related macular abnormalities and lens opacities. The study also wiU evaluate various possible risk factors for the development and/or progression of the two disorders. In addition, a multicenter, randomized clinical trial will assess the effects of nutritional supplements and/or minerals on the development and progression of age-related macular degeneration (AMD) and cataract. Eleven cUnical centers are now enrolling eligible partici- pants — with the goal of recruiting 4,600 persons — to be followed for at least 7 years. Randomization and followup begins in early 1993. The information resulting from AREDS should provide important stepping stones toward preventing AMD and cataract. A study of patients with a variety of retinal diseases continues to yield important information on the role of environmental and nutritional factors in the causation of eye disorders. In the first compo- nent of the study, patients with higher blood levels of nutrients with antioxidant characteristics, such as carotenoids, vitamin C, and vitamin E, were foimd to be at decreased risk of the most visually disabling form of macular degenerafion. This leading cause of blindness also was seen to share many risk factors with cardiovascular disease. A second component of the study now suggests that about 55% of nontrau- matic retinal detachments are attributable to myopia and that systemic factors play little role in the pathogenesis of retinal detachment. The study also is identifying risk factors for the following other retinal disorders that result in severe vision loss: branch retinal vein occlusion, central retinal vein occlusion, and macular holes. The NEI joined with the U.S. National Cancer Institute and the Cancer Institute of the Chinese Academy of Medical Sciences in conducting eye examinations in central China in 1991 at the end of a 5-year randomized trial of vitamin/mineral dietary supplements. Almost 6,000 adults were examined to see if that nutritional intervention might reduce the occurrence of age-related cataract and AMD. A manuscript describing the results has been prepared. In collaboration with Boston University, BEP is investigating familial relationships for age-related cataract and AMD using Framingham Study cohorts. Examination of 1,600 offspring of parents who were examined in the 1972-74 Framingham Eye Study was completed in late 1991. Analyses designed to identify genetic relationships have begun. 15 Office of International Program Activities Report of the Acting Assistant Director for International Program Activities Terrence Gillen, M.B.A., M.A. Over the past year, the National Eye Institute (NEI) has continued to support investigations of important blinding eye diseases with worldwide impact. These studies have been implemented through bilateral agreements with the U.S. Govern- ment, other types of country-to-country programs (such as those supported by the U.S. Agency for International Development), and through collabora- tive activities with the World Health Organization (WHO), the Pan-American Health Organization, and other foundations and private and voluntary organiza- tions (such as Lions Qubs International). The following are highlights of the ongoing activities and accomplishments of this office during the last year. 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 India, Italy, 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. Open-angle glaucoma is the leading cause of blindness in 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 develop- ment are largely imknowa Since 1988 the Barbados Eye Study has examined a cohort of more than 4,600 persons ages 40 to 86 as part of a population-based study to detennine the prevalence and risk factors for glaucoma and other eye disorders, such as age- related macular degeneration, cataraa, diabetic retinopathy, 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. Risk factor analysis also will be conducted for associations with development of glaucoma and to characterize those who have pro- gressive eye disease. Although not a major problem in the United States, the leading cause of blindness among pre- school-age children in Asia is vitamin A deficiency. The NEI supports a great deal of basic research on the interaction of nutrients, such as vitamins A, C, and E, on retinal and 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, which has shown an impressive reduction in childhood mortal- ity associated with improved vitamin A nutritional status, and other efforts to transfer this technology to alleviate world blindness are imder way. The NEI has continued its collaboration with the National Institute of Allergy and Infectious Diseases and three Brazilian scientific organizations in Sao Paulo to develop a research program on the basic mechanisms, epidemiology, and immimology of toxoplasmosis in Southern Brazil. The prevalence of ocular toxoplasmosis in Southern Brazil was found to be more that 30 times higher than previous esti- mates for the same condition elsewhere. In this population ocular toxoplasmosis appears to be a sequela of postnatal, rather than congenital, infection. The NEI and the Indian Coimcil of Medical Research (ICMR) have developed a collaborative blindness research program under the 1983 Indo-U.S. Science and Technology Initiative. This program includes projects to reduce blindness in India from cataract, Eales' disease, and vitamin A deficiency. Indian government funds for the work come through the ICMR, and U.S. Government funds are funneled through the National Science Foundation and the NEI. In addition, the NEI collaborates with Indian scientists under the U.S.-Indo Subcommission Program. 19 International Program Activities NEI Annual Report-FY 1992 NEI's Director, Deputy Director, and Special Advisor to the Director have participated as consul- tants to the World Bank in the development of a proposal by the government of India for a major new initiative in cataract blindness control. Technical meetings have been held in New Delhi and Madurai to provide the knowledge base upon which training and surgical guidelines can be developed for a twofold expansion of cataract surgery, with explicit attention to the quality and extent of vision restoration. An NEI-supported randomized clinical trial to compare intracapsular cataraa surgery plus aphakic spectacles versus extracapsular cataract extraction plus implantation of an intraocular lens is being conducted at the Aravind Eye Hospital in Madurai. The trial's primary comparison concerns operative and postoperative complications. Secondary evalua- tion endpoints include measurement of vision func- tion assessed by interview using a multi-item ques- tionnaire and appraisal of economic impact in terms of direct and indirect costs associated with blindness and cataract surgery. NEI staff continue to provide technical advice to Lions Qubs International in the development of its new $100 million SightFirst initiative, a global sight- conservation program aimed at substantially reducing the prevalence and incidence of preventable and curable vision loss. In Fiscal Year 1992 the NEI continued its activi- ties as a WHO Collaborating Center for the Preven- tion of Blindness. The NEI Director has continued to serve on the WHO's Special Advisory Panel in the Prevention of Blindness, and the Assistant Director for International Program Activities has been appointed to the Global Advisory Committee. Other NEI staff have, on request, provided consultation to the WHO program. 20 Office of Science Policy and Legislation Report of the Acting Associate Director for Science Policy and Legislation Michael P. Davis The Office of Science PoHcy and Legislation is responsible for all National Eye Institute (NEI) program planning, analysis, and evaluation activities, including the development and maintenance of a computerized management information system; legislative and other program coordination activities; and public information and scientific reporting, including health education, media relations, and scientific reporting services in support of the NEl's research programs. Policy, Legislation, Planning, and Evaluation Section One of the major activities of the Policy, Legisla- tion, Planning, and Evaluation Section (PLPES) this fiscal year has been the finalization and prepara- tion for publication of the latest in the series of NEI long-range plans. Vision Research — A National Plan: 1994-1998. Section staff were responsible for finalizing draft panel reports and sending them to recipients of NEI Merit awards for reviewing the science in their particular area of expertise. Com- ments also were soUcited from special reviewers from the optometric community. The PLPES staff worked with staff fi"om the NEI Extramural and Collaborative Program on incorporating comments and suggestions and in updating some sections of the reports. These revised reports were then sent to the panel chairs and each program planning panel member for final review and approval. Final com- ments from the panel members and chairs have been received and are being incorporated into the reports. Introductory narrative material and an executive summary are currently being drafted. Design work and internal layout for publication should be com- pleted by January 1993 for publication in February 1993. This PLPES also has served as the primary contact point for the NEI's involvement in the National Institutes of Health (NIH) strategic planning process. This involvement has included attendance by Section personnel at the National Task Force meeting and the NIH Director's Retreat on the NIH Strategic Plaa In addition, PLPES staff have been responsible for reviewing and providing comments on the various drafts of the NIH Strategic Plan. Section staff also have prepared material for the draft NEI plan detailing the development of the NIH plan and the initiatives proposed by the NEI for incorpor- ation and implementation. One of the primary activities of the PLPES staff is the assignment of scientific, disease-related, and other codes to awarded grants. Nearly 540 grants were coded during Fiscal Year (FY) 1992 using a state-of-the-art, computer-based coding system and a data base that allows quick and accurate storage, retrieval, and reporting of scientific, programmatic, and fiscal information concerning the Institute's research grants, contracts, and intramural projects. Because of this activity, the PLPES is the focal point within the Institute for responding to a variety of requests for specific program and expenditure infor- mation and for the extensive reporting of NEI program developments and accomphshments to the NEI, the U.S. Public Health Service, the U.S. Depart- ment of Health and Human Services, Congress, and nongovernmental organizations and individuals. The PLPES staff also are engaged in writing, editing, contributing to, and commenting on corre- spondence, reports, legislative analyses, public testimony, and other written materials from within and outside of the NEI. Management Information Systems Section Over the past fiscal year the Management Infor- mation Systems Section (MISS) staff imple- mented client-server data base systems to allow 23 Science Policy and Legislation NEI Annual Report-FY 1992 extramural staff to enter and retrieve grants informa- tion directly. The MISS staff also installed internet- working software, allowing high-speed communica- tions between NEI personal computers and other NIH staff, mainframe systems (such as the administrative data base. WYLBUR, and TSO), and other BITNET and INTERNET users worldwide. MISS personnel have enhanced the Institute's computer hardware configuration with the installation of an optical scanner, film recorder, automatic backup power supply, high-speed network printer interface cards, and some new computer worksta- tions. The MISS staff have smoothly implemented a major upgrade of its network operating system to a Microsoft local area network (LAN) manager and have added custom enhancements to facilitate log-on procedures. Full remote access capability for the network has been added, as well as enhanced daily backup procedures. MISS staff have upgraded software packages for spreadsheets, graphics, remote printing, calendar functions and scheduling, FAX interface, Windows applications, menus, and the NEI data base server. Custom spreadsheets for use in grants management have been developed, and MISS personnel have conducted special training seminars in the use of some of these software packages. All equipment has been ordered, and software configuration has begun to support a LAN at the new site for the Extramural and Collaborative Program. This system wUl be fully compatible with the NEI's building of 31 LANs. MISS staff also have been active on several trans- NIH computing committees and activities, including the following: the Office Technical Coordinators and its LAN subcommittee, the Extramural ADP Coordi- nating Committee and its steering committee, the Technical LAN Coordinators, the NIH Lead Users, the WordPerfect Woridng Group, the Procurement Decentralization Task Force, the IMPAC Database Conversion Advisory Group, and the Database Technology Group. The head of MISS has made presentations on client-server data base technology, and his technical recommendation for the new IMPAC data base system was incorporated as a policy recommendation by the NIH contractor for that effort. Scientific Reporting Section The Scientific Reporting Section (SRS) develops and disseminates information and education activities and programs for the public and health professionals. Responsibilities of the SRS include the following: the National Eye Health Education Program (NEHEP); development of publications and other information materials; preparation of responses to inquiries received from the press, public, and health professionals; the NEI exhibit program; preparation of reports to Congress; maintenance of a computerized data base of eye health-related educa- tional materials; and dissemination of the results of NEI-supported clinical trials. The SRS staff responded to more than 9,000 inquiries from the general public, patients and their families, students, health professionals, legislators, and the media in FY 1992, including 40 pieces of controlled correspondence representing congressional inquiries and Presidential greetings and proclama- tions. This is a significant increase over the 6,(XX) inquiries received last year. In addition, the SRS disseminated clinical trial results through clinical alerts and mass media activities for the Foscamet- Ganciclovir Cytomegalovirus Retinitis Trial of the Studies of the Ocular Complications of AIDS and the Optic Neuritis Treatment Trial. The SRS staff also developed the Eye Health Education (EY) Subfile on the Combined Health Information Database, which now includes more than 4(X) abstracted items. National Eye Health Education Program The NEHEP was foimaUy launched at a press conference in December 1991, reaching more than 65 million people with important health information through television, radio, newspapers, and magazines. The NEHEP Partnership now includes more than 40 public and private organizations that implement activities designed to reach individuals at high risk for glaucoma and diabetic eye disease. The fust two NEHEP public service campaigns were released in January 1992. The glaucoma campaign — consisting of print, radio, and television public service announcements (PSAs) — and the diabetic eye disease campaign — ^using print PSAs — 24 NEI Annual Report-FY 1992 Science Policy and Legislation targeted potential audiences at risk for these diseases totaling more than 60 million people. The glaucoma television PSAs were aired more than 25,000 times in 47 States by 239 stations, representing more than $2.4 million in air time. The radio PSAs were aired more than 30,000 times in 49 States by 360 stations, representing $2.7 million worth of air time. Three education kits were produced: the Educat- ing People With Diabetes Kit, the Glaucoma Com- munity Education Kit, and the Information Kit for Pharmacists, each providing a variety of materials designed to help health professionals and conununity leaders implement eye health education programs. Since becoming available in April, more than 16,000 kits have been distributed. To support local efforts of the NEHEP Partnership members, the NEHEP staff provided technical assistance by linking groups with additional resources in their area and assisting diem in designing programs for their specific target audiences. The NEHEP also provided funds to the Healthy People 2000 Project of the National Medical Association to evaluate eye health education strate- gies in two cities aimed at reaching African-Ameri- cans and providing them with important eye health information. Grants for three applied research projects on diabetic eye disease education were awarded this year. These projects — designed to evaluate cost- effective education strategies that increase awareness and knowledge of diabetic eye disease and encourage actions to prevent loss of vision — wiU be aimed at three different target audiences: (1) low income African- Americans, (2) Native Americans, and (3) all people with diabetes in a defined community. 25 Office of the Scientific Director Report of the Scientific Director Robert B. Nussenblatt, M.D. This past year has been one of continuing change and achievement in the Intramural Research Program of NEI. During this year woric has pro- gressed in both clinical and basic research spheres. Continued heavy emphasis in the area of AIDS (acquired immunodeficiency syndrome) has resulted in the initiation of a clinical study to evaluate the effectiveness of a sustained-release device for gan- cyclovir, placed in the eye for the treatment of cytomegalovirus (CMV) retinitis. A study to im- munomodulate uveitis and the diseases in uveitis patients without the use of medications has begun as well. In the area of basic research, there are exciting new concepts concerning gene regulation, and the initial steps of a gene therapy approach to eye disease have been taken. The following are a few highlights of research achievements by NEI intra- mural scientists in Fiscal Year 1992. Laboratory of Retinal Cell and Molecular Biology (LRCMB) The mission of the LRCMB is to uncover new aspects of functioning of the retinal pigment epithelium (RPE) complex in health and disease. The focus has been on the elucidation of new genes and biochemical mechanisms as well as learning the underlying causes of ocular disease. This work has been performed in both clinical and basic research. In collaboration with Dr. Muriel Kaiser-Kupfer (Ophthalmic Genetics and Clinical Services Branch), LRCMB researchers are investigating fatty acid uptake and metabolism in Bietti's crystallin retinoph athy and a tubulin acetylation defect in a form of Usher's syndrome in the hope of elucidating specific defects. Candidate genes for hereditary retinal diseases have been evaluated. The approach has included a method that uses subtractive cloning. Retina-specific genes and genes located on the short arm of the X chromosome are of special interest These genes are being chromosomaUy localized to see whether they are disease linked. Other work performed in the Laboratory includes the search for RPE-specific genes. The RPE is essential for the photoreceptive process. A major effort has been in the cloning of genes unique to the RPE and its function. A new, specific 65-kD protein of potential immunologic importance has been isolated and cloned ftom human RPE. Several transgenic studies have been initiated. A gene analysis system in transgenic mice and in transient transfections in cultured himian retinal blastoma cells have been established for interphotoreceptor retinoid- binding protein (IRBP). Another LRCMB area of interest has been the use of ribozymes as a mode of gene therapy. These ribozymes are specifically constructed RNA species that can control the expression of proteins within the cells. Linking these simplified gene forms to appro- priate promotors and utilizing a suitable transfer vector enables construction of new therapeutic modalities. It is hoped that this approach may be used in the future to treat autosomal dominant disorders that cannot now be managed. Laboratory of Ocular Therapeutics (LOT) The LOT'S focus is on the development, evalua- tion, and mechanism of action of new ophthal- mic drugs to treat eye diseases. The major emphasis has been in examining aldose reductase inhibitors and anticataract agents. Developing and pursuing the more effective and less toxic therapies, the LOT scientists have discovered an inhibitor unrelated to previous aldose reductase inhibitors. They are now conducting studies to characterize this inhibitor. In addition, LOT work has centered on elucida- tion of the specific mechanisms of aldose reductase- initiated diabetic complications. In these studies, the use of galactose-fed dogs has permitted the investiga- tors to follow the progression of retinal changes associated with diabetic retinopathy and to see the 29 Onice of the Scientific Director NEI Annual Report-FY 1992 dose-dependent arrest of these changes with aldose reductase inhibitors. Laboratory of Mechanisms of Ocular Diseases (LMOD) The LMOD's primary emphasis in the past year has been on cataract formation and the ocular complications of diabetes. The ' aboratory has had a longstanding interest in the evaluation of the vascular network of the retina during diabetes. Using elastase, LMOD researchers have developed a new procedure for the isolation and evaluation of the entire vascular network of the retina. Some in the group have continued to investigate the role of the polyol pathway in diabetic complications. They have determined the primary sequence of human sorbitol dehydrogenase. Others in the group have developed an in vitro system to assay the effectiveness of anticataract agents. The development of such an organ culture system wUl allow initial screening of compoimds before they are moved to animal models of cataract for the next stage of testing. More clinically oriented studies have also contin- ued. They include investigation of the modifications that lens crystallins undergo as they become cata- racts. The primary focus has been on metal-cata- lyzed oxidation using y-crystallin as a model system. Dr. Fielding Hejtmancik and his coinvestigators have successfully mapped to chromosome 11 two genes causing Usher's syndrome type I. Laboratory of Sensorimotor Research (LSR) This Laboratory of international standing focuses its research efforts on the brain mechanisms underlying visual sense and visually controlled movement. The group, which has concentrated on the processing of the visual input to the brain, has made several advances this past year. This woik has indicated that different visual areas in the brain may communicate via temporally modulated messages. Neurons in the visual areas of the cerebral cortex carry information about the form and color of the stimulus in the temporally modulated code. The results suggest that cortical neurons can convey information about many different features without confounding them. LSR scientists have also evaluated the effects of selective attention on visual processing, obtaining results that suggest the brain treats voluntary atten- tion differently from involuntary attention to salient and novel stimuli. In other work, they have studied the rapid or saccadic eye movements that shift the focus of the eye from one object of interest in the visual field to another. This year LSR investigators found that, wWle many neurons in the brain may give bursts of cell discharge before the onset of these saccades, other types of cells lying just below these bursts cells are related to the generation of saccades. This spread of activity may represent a type of neuroprocessing not previously recognized in the brain. Laboratory of Molecular and Developmental Biology (LMDB) The continuing focus of the LMDB is on under- standing the fundamentals of gene expression and cellular differentiation in the eye, particularly in the lens. The group has ventured into the complex area of protein-DNA interactions involved in the expression of crystaUin genes. Some of their experi- ments have shown that putative regulatory regions appear to be involved in the heat-shock response of these genes and in their expression in the lung and brain, both of which use a different promotor than those expressed in lens and other tissues. Others in the LMDB group have investigated the crystallins in invertebrates with ceUular lenses. These models have shown a convergent evolution that we hope wUl elucidate basic aspects of crystal- lins and their genes required for optical properties of the transparent lens. Of particular interest was their finding that the ectopic expression of interferon-y in the lens induced major histocompatibility complex class II gene expression in the eye disrupted the developmental program of the lens and retina. The transgenic mouse developed may become a useful model for the study of ocular autoimmune disease. It promises to provide insight into the tight control of gene expression and cell differentiation in the eye. Proto-oncogenes in the lens have been of ongoing interest to the LMDB. Recent work has revealed 30 NEI Annual Report-FY 1992 Office of the Scientific Director unexpected patterns that shed hght on the mecha- nisms of cell cycle rest during differentiation. Insights have also been gained on the cellular mecha- nisms that regulate proto-oncogene expression in the lens. Ophthalmic Genetics and Clinical Services Branch (OGCSB) The purpose of the Branch is to conduct clinical and laboratory research on gene expression and molecular interaction important to the eye and to apply chnicaUy relevant research findings to the prevention, diagnosis, and treatment of diseases affecting the eye and visual system. The OGCSB is also responsible for the essential psychophysical and electrophysiological diagnostic testing of visual function required by cUnical Intramural Research Programs of all of the Institutes. Work in the field of cataract has demonstrated that with aging there is acidic shift in proteins and an increased nimiber of polypeptide species in the molecular weight range of the crystaUins. These aging changes need to he differentiated fix)m changes occurring in cataract formation. Other OGCSB woric has centered on the disease gyrate atrophy. There appear to be many different single-point mutations in the ornithine aminotransfer- ase gene in gyrate atrophy patients. Dietary inter- vention studies utilizing an arginine-deficient diet have been quite promising, especially in delaying the onset of pathologic changes in young patients. Furthermore, foveal cone sensitivity was found to be abnormal in a group of patients with gyrate atrophy, suggesting that foveal cones have altered orientation and sensitivity before the encroachment on the foveal area by gyrate atrophic lesions. Other OGCSB researchers, looking at visually evoked potentials (VEP) in albinism, have noted two different patterns of VEP asymmetry in albinism that may be explained by differences in the reorganization of the geniculo-cortical pathway. Also this year, after confirming the usefulness of 0.5% cysteamine eyedrops in young patients with cystinosis, the Branch has expanded its study to include older patients, producing strikingly similar results. Laboratory of Immunology (LI) The Laboratory of Immunology is dedicated to the evaluation, diagnosis, and treatment of ocular inflammatory diseases. Work by this group has been both in clinical and basic research fields. In the chnical area, studies continue to center around the evaluation of the Molteno glaucoma implant and 5-fluorouracil, combined with trabulectomy. In addition, the LI group has made a major clinical investment in the study of AIDS. This study has dealt with better ways to diagnose the presence of CMV retinitis, including noninvasive mechanisms such as increased protein content in the anterior chamber, as weU as therapeutic studies on the use of such anti-CMV retinitis drugs as foscamet. Recently the laboratory began a randomized study to evaluate the usefulness of an intraocular slow-release device containing gancyclovir, over either a 4- or 8-month period to treat CMV retinitis. Other areas of interest to the group include the development of new aspects of immunosuppression. They have woiiced in developing rapamycin as a potential therapeutic aid in the treatment of uveitis. The Laboratory has also studied various virologic and immunopathologic processes that occur when viruses replicate in the ocular microenvironment. This project has used a coronavirus model for retinal degeneration. It is of interest that the virus is cap- able of inducing an acute infection in the presence of a mild inflammation, with the virus then being cleared and a subsequent retinal degeneration ensuing with no virus present The Laboratory has also developed a reproducible method for performing RPE cell transplants. The ultimate goal is to use molecularly altered RPE cells to develop universal donors, as well as to develop a reproducible animal model for the study of RPE interactions from both biochemical and immunologic points of view. Experimental uveitic models have been thorough- ly studied by the LI. Over the past year, researchers have developed an in vitro system to investigate mechanisms of specific unresponsiveness toward uveitogenic peptides. The group has also initiated a study to evaluate the effectiveness of oral administra- 31 Office of the Scientific Director NEI Annual Report-FY 1992 tion of various antigens on the ocular immune response. This approach, which has been tested in the animal model for severe intraocular inflammatory disease induced by uveitogenic antigens, is quite successful. A randomized masked trial to evaluate more fuUy the usefulness of S-antigen feeding in patients with intraocular inflammatory disease has now begun. Other areas of immunoregulation under investiga- tion include evaluation of adhesion molecules and their role in the induction of disease. Over the past year, one section has devoted itself to an NEI prior- ity, that of gene therapy. Work has centered on the development of a construct of ornithine aminotrans- ferase that can be used for gene therapy in patients who have gyrate atrophy. 32 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00065-15 OSD PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 charaaers or less. Title must tit 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 aflillation) PI: Francisco M. de Monasterio M.D., D.Sc. Medical Officer OSD, NEI COOPERATING UNITS (It any) LAB/BRANCH Office of the Scientific Director INSTITUTE AND LOCATION NEI, >fIH, Bethesda. MP 20892 TOTAL STAFF YEARS: 0.3 PROFESSIONAL: 0.3 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. The cells studied are flrom the lateral geniculate nucleus and the primary visual cortex of macaques. 33 PHS 6040 (Rev. 5/S2) Office of the Scientific Director NEI Annual Report-FY 1992 Project Description Objectives This project is designed to study the neural organiza- tion underlying the processing of visual information at different levels of the primate visual system. Methods The 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 researchers attempted to continue studying the responses from single ganglion cells and neurons of the primary visual cortex of macaque monkeys. The continuation of these studies, however, was impeded by underground construction work along the waU separating the laboratory rooms from the street as well as delays in obtaining needed additional equip- ment. This construction work, which has lasted for several months, shakes the ground to the extent of making microelectrode recordings impossible, and even resulted in damage to computer hard disks. Cell recordings have been suspended until the end of this construction. 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, so that an understanding 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) 34 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1, 1991 to September 30, 1992 PROJECT NUMBER ZOl EY 00122-12 OSD TITLE OF PROJECT (80 characters or lass. Title must lit on one line between the borders.) Anatomical Studies of the Primate Visual System PRINCIPAL INVESTIGATOR (List other prolesslonal personnel below the Principal Investigator.) (Name, title, laboratory, and Institute allUiaUon) PI: Francisco M. de Monasterio M.D., D.Sc. Medical Officer OSD, NEI COOPERATING UNITS (II any) LAB/BRANCH Office of the Scientific Director INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.7 PROFESSIONAL: 0.7 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects (b) Human tissues □ (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project focuses on the anatomical properties and organization of ceUs in the primate visual system with emphasis on the retina and the visual cortex. The studies include (1) the anatomical association of outer- retinal ceUs selectively stained with tissue-reactive dyes and (2) the distribution pattern of cones in the retina of human donors who have a diagnosis of diabetic retinopathy. Systematic light-microscopy of the anatomical association of macaque retina blue cones and horizontal and bipolar cells, wdth selective staining by several tissue-reactive dyes, has provided information on the probable circuitry of the blue-sensitive cone pathway of primate retina. Retinal cone studies of human eye donors with a history of diabetes continue. There is evidence that the cone population fix)m the retinas of these donors has a point pattern resembling that of cones selectively stained by tissue-reactive dyes. Although such dyes were not injected in the eyes from diabetic donors, in vitro staining wdth more conventional dyes shows a differential labeling of the receptors. Some cones are more densely stained than other cones, and the point pattern is similar to that of the blue-sensitive cones of the primate retina. 35 PHS 6040 (Rev. 5/92) Office of the Scientific Director NEI Annual Report-FY 1992 Project Description Objectives The puipose of this project is to study the anatomical properties and neural organization of the primate visual system. Methods Included in this research are retinal histological processing, intravitreal injection of dyes, computer modeling and spatial statistical analyses of point and area patterns, silver cell and myelin staining, histological processing of the cerebral cortex, deoxy- glucose labeling, autoradiography, and cytochrome oxidase labeling. Major Findings While both studies continue from Fiscal Year 1991, their continuation has been severely hampered by underground construction work along the wall separating the laboratory rooms from the street, as well as difficulties in obtaining new equipment This construction work, which has lasted for several months, shook the ground to the extent of making tissue sectioning and microphotography impossible. Evidence of a cone population with a point pattern distribution resembling that of the cones identified as blue-sensitive ones (ie, absence in the central-most region of the fovea, peak density in the parafovea, and regular though slightly disordered spacing in which stained cones are separated by two to three unstained cones) has been obtained in initial studies of the retinas of human donors with clinical history of diabetes and diabetic retinopathy. Due to the need for eyes fixed within 3 hours of death, additional material has been difficult to obtain. Results consistent with the above findings were obtained in two additional diabetic cases; however, the eyes were fixed several hours after death, and the retinal preservation was less than adequate. Because of the unpredictability of obtaining properly fixed tissue from donors with documented retinopathy, slow progress is expected in this study. Systematic examination of thin serial sections of macaque retinas stained with a tissue-reactive dye selective for blue-sensitive and some postreceptoral cells continues. The objective is to trace at the light microscopic level the anatomical relationship be- tween selectively stained blue cones, HI horizontal ceUs, and blue-cone (BC) bipolar cells of the area centralis of macaque retina. Data obtained so far provide evidence that, on the central macaque retina, each blue cone contacts one HI horizontal cell and an average of at least one BC bipolar cell. Because the latter neurons almost always show a significant lateral displacement from the contacting cones, it is difficult to trace their contacts in thin sections. Additional studies on whole mounted retinas, viewed en face, provide further evidence of a nearly unitary blue cone/Hl/BC-bipolar cell distribution. Observations of more peripheral retina, however, support the view that with increasing retinal eccen- tricity, each BC bipolar cell contacts more than one blue cone. Among our achievements is the use of Lucifer yellow VS dye to increase staining consis- tency for inner cells of the peripheral retina. Stained bipolar cells, which have anatomical properties similar to those of BC bipolar cells described in Golgi-stained retinas, appear to have weakly stained projections on two and sometimes three stained blue cones. Significance to Biomedical Research and the Program of the Institute Information on the anatomical properties of blue- sensitive cones is important not only to understand- ing the functional properties of these cones by investigators in different basic disciplines. These experimental results also aid clinical research and the diagnosis of acquired retinal disease. Preliminary 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 Oi:ganiza- tion, Neurotransmission, and Adaptation) 36 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00135-20 OSD PERIOD COVERED October 1. 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or less. Title must lit or\ one line between the borders.) Biochemistry of Retina and Pigmented Epithelium in Health and Disease PRINCIPAL INVESTIGATOR (Ust other prolasslonal personnel below the Principal Investigator.) (Name, title, laboratory, and Institute alliliatlon) PI: Helen H. Hess M.D. Medical Officer (Research) OSD. NEI COOPERATING UNITS (il any) LAB/BRANCH Office of the Scientific Direaor INSTITUTE AND LOCATION NEI, NIH. Bethesda. MD 20892 TOTAL STAFF YEARS: 1.0 PROFESSIONAL: 1.0 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.) The effects of nutrition, oxidation, and other environmental factors (light 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 accimiulates secondary to a phagocytic defect in retinal pigmented epithelium. Evidence was obtained that oxidative changes in polytmsaturated 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 10- to 40-lux intensity, and 27% of the rats developed mature cataracts by 7-12 months. Increased light intensity (cyclic or constant) increased the percentage of rats with mature cataracts, while dark-rearing from birth prevented PSO and matiue cataracts. Rhodopsin bleaching appears essential for retina light damage and PSO. In vitro, free retinaldehyde can act as a photosensitizer to generate singlet oxygen, an extremely energetic oxidant. Our results suggest a similar effect in vivo, with damage to both lipids and proteins. Antioxidants may slow or prevent cataracts in some diseases of the human retina. A purified diet (AIN-76A) fortified with antioxidants (0.4% p-carotene + 0.01% BHT) prevented PSO and mature cataracts in RCS rats. After a diet containing additional antioxidants (vitamin C, 1,000 mg/kg, and vitamin E, 150 mg/kg) was fed to dystrophic rats, histopathological examination showed retinal degeneration was retarded during the time the cataracts would have had their onset (23-53 postnatal days) if NIH-07 had been fed. Future studies are being direaed to exploring whether retinal degeneration can be delayed fiirther by increasing the vitamin E by a large (7-fold) factor. 37 PHS 6040 (Rev. 5/92) Office of the Scientific Director NEE Annual Report-FY 1992 Project Description Additional Personnel J. Samuel Zigler, Jr. Joseph J. Knapka Ph.D. Ph.D. E>ennis Bemaid M.S. Chief. LMOD, NEI Chief, Scientific Services Branch (SSB), Veterinary Research Program (VRP), National Center for Research Resources (NCRR) Nutritionist, SSB, VRP, NCRR Objectives This project is designed to study the biochemical relationships between lens, retinal photoreceptors, retina, retinal pigment epithelium (RPE), and biologi- cal fluids in health and disease. It also involves exploring the possibilities for slowing the rate of retinal degeneration and preventing the lens opacities and mature cataracts often associated with retinal degeneration in rats and humans. Diseases in which the RPE may be involved are of particular interest. Methods The Royal College of Surgeons (RCS) rat is being studied as an animal model of both hereditary retinal degeneration that results from a defect in the RPE and a type of cataract that is secondary to retinal degeneration. Nutritional biochemistry is being used as a tool to combat lipid peroxidation in the RCS rat retina and prevent water-soluble toxic aldehyde byproducts from reaching and damaging the lens. Defined diets are prepared and fed to congenic affected and unaffeaed 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 fi-om conception to date of observation. Clinical findings are recorded after indirect ophthalmoscopic and biomicroscopic slit-lamp examination. Post-mortem examinations of the eye include dissecting microsco- py and light microscopy of stained specimens. At appropriate times, photography is used to record data in vitro or in vivo. 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 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 protected. The most effective diet we have studied so far is a combination of AIN-76A purified diet containing twice the normal concentration of all minerals in the AIN mineral mix, plus 0.4% beta-carotene and 0.01% BHT, as weU as 1,000 mgA:g vitamin C and 150 mg/kg vitamin E. When the rats consumed this diet, histopathological examination showed retarded retinal degeneration during the time the cataracts would have had their onset (23-53 postnatal days) if NlH-07 diet had been fed. Future studies are being directed to exploring whether retinal degeneration can be delayed further by increasing the vitamin E sevenfold. Extreme sensitivity of the dystrophic retina to light damage and to peroxidation of its lipids may be as fundamental as the defect of the RPE; thus it may provide a clue to the underlying metaboUc disease. Significance to Biomedical Research and the Program of the Institute Retinal deteriorations are the major cause of imtreat- able bUndness in the United States and probably in the world. The retinal pigment epithelium is increas- ingly implicated as the primary site of many of these disease processes. Posterior subcapsular cataracts occur in humans with various types of hereditary retinal degenerations and in RCS rats, as well as in older persons and in some persons treated with steroids or exposed to various types of short-wave radiation. Nutritional and genetic faaors thought to play key roles in many hiunan diseases can often be studied in detail in animal models, giving an oppor- tunity to develop ways to slow, prevent, or cure the diseases. Environmental light intensity as a factor in retinal and lenticular diseases deserves more anen- tion. Information gained ftom these studies should contribute to our understanding of human disease and to initiating and conducting trials of possible thera- peutic measures. More than 1 million cataract 38 NEI Annual Report-FY 1992 Office of the Scientific Director operations are performed per year, and with an increase in the aging sector of the population, this number will increase. Any measures (eg, protective eye and headwear as well as dietary antioxidants) that may prevent or slow retinal deterioration and prevent or delay development of cataracts would increase the quality of daily life for millions of older people and decrease the costs of national medical care. Proposed Course The project will be continued. Further emphasis will be placed on controlled trials of nutritional regimens in RCS rats, toward the objective of preventing or delaying retinal degeneration and preventing cata- racts. Studies will continue on the effects of darkness and light and antioxidant factors in retinal degenera- tion and cataracts in RCS dystrophic rats as weU as RCS congenic control rats. Collaborations will con- tinue to investigate biochemical factors in cataracto- genesis and retinal degeneration. NEI Research Program Cataract — Pathogenetic Mechanisms Retina — Retinitis Pigmentosa and Other Inherited Disorders Publications Hess HH: Effect of nutritional factors on retinal degeneration and cataracts in pink-eyed tan- hooded Royal College of Surgeons (RCS) rats. Invest Ophthalmol Vis Set 33(4): 1186, 1992. 39 Laboratory of Immunology Report of the Chief, Laboratory of Immunology Robert B. Nussenblatt, M.D. In its 6th year, the Laboratory of Immunology has undergone some change. The section head posi- tion in the Section on Qinical Immunology, which was vacant a year ago, was flUed recently by Dr. Marc de Smet, the acting chief. The other sections are the Section on Immunology and Virology, headed by Dr. John J. Hooks; the Section on Experimental Immunology, headed by Dr. Igal Gery; and the Section on Immunoregulation, headed by the acting chief. Dr. Rachel Caspi. Dr. Gery continues as deputy chief of the Laboratory; his major role in the development of new areas of investigation for the Laboratory was recognized this past year when he received the Director's Award from Dr. Carl Kupfer. Two new sections recently have been formed. The first is the Section on Experimental Immunopathol- ogy, with Dr. Chi-Chao Chan as chief. The newly created Section on Molecular Biology, of which I am acting chief, includes Dr. Moncef Jendoubi and his group. The Fiscal Year (FY) 1992 work of these two sections will be mentioned within their previous section groups because they were created very recently, and the work was done prior to the new Sections' establishment. Section on Clinical Immunology The Section on Clinical Immunology has contin- ued to focus on major questions of clinical relevance. In one area, the treatment of secondary glaucoma due to uveitis, the studies are centered around evaluation of the use of the Molteno glau- coma implant and 5-fluorouracil (5FU) combined with trabeculectomy. Our observations continue to make us rather optimistic about the long-term effi- cacy of the Molteno implant; however, no definitive data are yet available. As this randomized study to evaluate both the efficacy and complications of these two modalities continues at the NEI, we are still recruiting patients with secondary glaucoma due to uveitis to reach numbers that will provide some idea as to the comparability of these two modalities of treatment The Section also has been studying patients with AIDS. Because of its time-consuming nature, this major endeavor has included clinicians from various Laboratory Sections. Since completing a Phase 2 randomized control study to evaluate the efficacy of foscamet in 24 patients with cytomegalovirus (CMV) retinitis, we have continued to evaluate the ability of foscamet as well as gancyclovir to prevent progres- sion of CMV retinitis. In collaboration with the National Institute of Allergy and Infectious Diseases, we have begun to evaluate the safety of administering anti-CMV hyperimmunoglobulin to patients at risk for CMV retinitis. To date, this treatment, given monthly to a small number of patients, has had no adverse effects. We also have continued to follow patients who have diagnosed CMV retinitis. Patients with advanced AIDS and a severe state of immunosuppression are often xmable to tolerate gancyclovir, even with the use of G-CSF; some also are unable to tolerate the renal toxicity of foscamet. Therefore, we have had to revert to the use of intravitreal injections. In the course of clinical therapy of two patients who had silicone oil placed in the eye because of retinal detachment (a complica- tion of CMV retinitis), we showed that intravitrial gancyclovir injections can be given safely as an effective means of arresting the progression of CMV retinitis. Other infections also can decrease vision in patients with AIDS. The second most common pathogen in most parts of the world is toxoplasmosis. We have evaluated and demonstrated the efficacy of a new agent, 566C80, in the treatment of ooilar toxoplasmosis. Recentiy a device that slowly releases gancyclovir directiy in the eye has been described. It may be an important alternative to systemic anti-CMV therapy for patients who cannot tolerate the intravenous infusions or who possibly do not have a specific 43 Laboratory of Immunology NEI Annual Report-FY 1992 indication for systemic therapy. We have developed a protocol that should allow us to evaluate the safety of the device. This protocol is now in effect, and the study will begin shortly. Other areas of interest to the Section concern the development of new aspects of immunosuppression. The group has worked through this year in develop- ing rapamycin as a potential therapeutic aid in the treatment of uveitis. Rapamycin, which has a mode of action different from that of cyclosporine and FK506, appears to be very effective in preventing the expression of experimentally induced uveitis. Section on Immunology and Virology Woric of the Section on Immunology and Virol- ogy has continued to emphasize several specif- ic areas. One area has been the study of various virologic and immunopathologic processes that occur when viruses replicate in the ocular microenviron- ment. This project's coronavirus model for retinal degeneration is capable of inducing an acute infec- tion in the presence of a mild inflammation but without the presence of a virus. The retinal destruc- tion occurred during the ensuing months in spite of the fact no virus was present The woric has shown that while the initial viral infection may induce some degree of degeneration, a potentially autoimmune response may be playing a role in this disorder as well. The group continues to be actively involved in the study of the retinal pigment epithehal (RPE) cell. The group has developed and characterized primary ceU lines of human RPE from donor eyes obtained from eye banks. Using these human RPE cell cul- tures as an in vitro model, the investigators have conducted a set of experiments to examine the various roles of the RPE in the pathophysiology of retinal disorders. Cultures of these cells exposed to bacterial lipopolysaccharide (LPS), tumor necrosis factor, and interleukin (IL-la and IL-lp) secreted large amounts of interleukin 6 (IL-6). This response, which is dose dependent, is sustained in the presence of stimulants. It is interesting that growth factors did not induce IL-6. In addition, the group has developed the ability to produce large nimibers of RPE cells that have been used in ongoing RPE transplantation experiments. Utilizing a microinjection technique, they have succeeded in injecting RPE cells into the proper space beneath the retina. The injection of RPE ceUs obtained from a non-major histocompatibility com- plex identical source induces a rejection phenome- non. This development has provided the whole laboratory with the model to study ways of altering the immune response to these important ceUs. Achieving priority status among the Section on Immunology and Virology has been the use of the molecular diagnosis and pathogenesis of CMV infections in man. In addition, the group has worked over the past year in the development of an animal ocular model for toxoplasmosis. Section on Experimental Immunology The Section on Experimental tomunology has maintained its long-term interest in the investi- gation of the pathogenesis of inflammatory eye diseases grouped under the term "uveitis." This group's work has centered around the evaluation of experimental autoimmune uveitis (EAU) with inter- photoreceptor retinoid-binding protein (IRBP). They found that the substitution of certain residues of the bovine IRB peptide 1181-1191 eliminated its high uveitogenicity. It is interesting that the corresponding sequence of the rat IRBP differs from the bovine sequence by two residues and is completely nonuve- itogenic. The group has developed an in vitro system to investigate mechanisms of specific unresponsive- ness toward uveitogenic peptides. In this system, lymphocytes sensitized toward peptide 1181-1191 were rendered unresponsive. The group also has examined several approaches aimed at inhibiting the immunopathogenic process of EAU. Injecting peptides that compete with the uveitogenic molecule, they have found that EAU induced by the highly uveitogenic IRB peptide 1181- 1191 is completely inhibited in rats coimmunized with peptide A 183, an analogue of the peptide derived firom a Mycobacterium tuberculosis protein. These investigators also have demonstrated that the procedure of oral tolerance, another approach to effectively inhibiting S-antigen (S-Ag)-induced EAU, is modulated by the small peptide 1181-1191. New immunosuppressive agents investigated by the group include mycophenolate mofetil, which is effective in 44 NEI Annual Report-FY 1992 Laboratory of Immunology preventing the expression of this animal model for a human disease. Section on Immunoregulation The Section on Immunoregulation's involvement in investigating many aspects of the immune response in the eye ranges from clinical to basic studies. The effect of Uie oral administration of various antigens on the ocular immune response has been tested in the animal model for severe intraocu- lar inflammatory disease (EAU) induced by both S-Ag and IRBP. Previous work showed that a CDA- positive putative suppressor ceU could be isolated from the spleen of such animals. Additional woik this year has shown that the role of the spleen is imperative in the induction of this toleragenic state. In a pilot study, two patients with evidence of in vitro proliferative responses to S-Ag began a S-Ag feeding protocol. A tolerant inmiune state appeared to be induced in both of these patients after feeding and a program to take them off their inflammatory disease medication was successful. We have now begun a randomized masked trial to evaluate more fully the usefulness of S-Ag feeding in patients with intraocular inflammatory disease. This Section has demonstrated the expression of endothelial leukocyte adhesion molecule 1 (ELAM-1) on the corneal endothelium in Lewis rats with induced uveitis. They have shown that treatment with the monoclonal antibody against Mac-1 inhibits the development of endotoxin-induced uveitis (EIU) in mice. Furthermore, they have demonstrated that monoclonal antibodies blocking intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function- associated antigen (LFA-1) inhibit the development of both EIU in rats and EAU in mice. These anti- bodies against ICAM-1 and LFA-1 also inhibited lymphocyte stimulation in vitro. The expression of cell adhesion molecules in animal models of uveitis such as ICAM-1 are expressed in the eye before the infiltration of inflammatory cells. Woric performed to evaluate human biopsy tissue from the eye has revealed the expression of ICAM-1, E-selectin, and vascular cell adhesion molecule (VCAM-1) in corneal graft tissue. Over the past year the Section has devoted itself to an NEI priority, gene therapy. Woik has centered on the development of a construct of ornithine aminotransferase that can be used for gene therapy in patients who have gyrate atrophy. The expression of this gene now can be effected in cells that have been transfected with the appropriate constnia. Also under way are plans to develop an animal model of gyrate atrophy, using homologous recombination techniques that originated in the Laboratory. These efforts already have yielded a model mouse deficient in the invariant chain of the major histocompatibility complex. 45 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00279-01 LI PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or lass. Title must lit on one line between the borders.) Study of Immunosuppressants for the Treatment of Uveitis in Animal Models PRINCIPAL INVESTIGATOR (List other prolasslonal personnel below the Principal Investigator) (Name, title, laboratory, and institute attillation) PI: Francois G. Roberge M.D. Visiting Scientist LI, NEI Others: Chi-Chao Chan M.D. Chief, Immunopathology LI, NEI Visiting Scientist LI, NEI Scientific Direaor NEI Senior Staff FeUow LI, NEI Senior Staff FeUow LI, NEI Visiting Fellow LI, NEI Chi-Chao Chan M.D. Marc D. de Smet M.D. Robert B. Nussenblatt M.D. Dan Martin M.D. Raymond DeBarge M.D. Alexander Kozhich Ph.D COOPERATING UNITS (If any) Laboratory of Experimental Surgery, Notre Dame Hospital, Montreal, Quebec, Canada (Dasheng Xu, M.D.; Huifang Chen, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Clinical Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: PROFESSIONAL: 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 pmvided.) The aim of the project is to develop alternative treatment for noninfectious intraocular inflammatory diseases (uveoretinitis). During this past year we concentrated on a group of noncytotoxic immimosuppressants comprising rapamycin (RAPA), cyclosporin A (CsA), and FK506. The work specifically focused on (1) RAPA treatment of experimental autoimmune uveoretinitis (EAU) and (2) combination therapy using RAPA with dexamethasone (Dex) or CsA. The noncytotoxic immimosuppressants inhibit the immune system function mainly by preventing the proliferation of activated lymphocytes. CsA and FK506 both prevent lymphokine gene expressioa RAPA exerts its action at a different level by interfering with the transduction of the proliferative signal of cellular growth factors. In our studies of the efficacy of RAPA in EAU in the Lewis rat, EAU was inhibited when RAPA treatment was instituted either on the day of immunization with S-antigen (S-Ag) or 7 days later (minimal effective dose, 0.1 mg/kg/day). The inhibition of EAU was correlated with reduced lymphocyte proliferation and antibody production to S-Ag. RAPA also was shown to prevent the adoptive transfer of EAU with a retinal antigen-specific T-ceU line, indicating that it could be effective in the treatment of ongoing disease. Drug combination is commonly used as a strategy to minimize the toxicity of each drug while achieving a fiill therapeutic effect. Our studies of the combination of RAPA with Dex or CsA in vitro have shown a potent synergistic action of these drugs. In combination, RAPA and CsA could be reduced by 10- and 3-fold, respectively, while Dex with RAPA allowed for a 4.5-fold reduction of both. 46 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Immunology Project Description Clinical Protocol Number 91-191 Objectives To determine the efficacy of rapamycin (RAP A) to inhibit experimental autoimmune uveitis (EAU), the approach was to test the effect of treatment during the afferent phase of the immune system acti.ation by starting treatment on the day of immunization. Afterward, testing during the efferent phase was enabled by instituting treatment 7 days after immuni- zation. Finally, the fuUy activated immune system was tested by inducing EAU by T-cell line transfer. In addition, we evaluated the biological correlates of the activity of RAPA by measuring the effect of various doses of treatment on antibody production as well as on the lymphocyte proliferative response to S-antigen (S-Ag). A major objective of the study of noncytotoxic immimosuppressants is to develop strategies of combination therapy that permit reducing the dose of the individual drugs to lower the toxic side effects while achieving a sufficient level of immunosuppres- sion. To determine the combined effect of RAPA with dexamethasone (Dex) or cyclosporine A (CsA), we planned in vitro inhibition studies of the prolifer- ation of S-Ag-primed lymphocytes with covaried concentrations of the drugs. The type of combination effect was to be determined by submitting the data to a calculation method that distinguishes between additive and synergistic effects. Methods EAU was induced either by immimization with S-Ag in Himter's adjuvant, or by adoptive transfer with an interphotoreceptor retinoid-binding protein (IRBP)- specific T-ceU line. Treatment was delivered over 14 days by continuous intravenous infusion by means of miniosmotic pimips implanted in the abdominal cavity and coimected to a lumbar vein. The treatment protocol was aimed at the afferent, efferent, and ftilly differentiated phase of the immune system activation. The combination effects of RAPA with Dex or CsA were evaluated in vitro by the proliferation of S-Ag- primed lymph node ceUs. The data were evaluated by the medium effect analysis method of Chou et al (in Synergism and Antagonism in Chemotherapy, Chou T-C and Rideout DC, eds, Academic Press, San Diego, 1991). Major Findings Actively induced autoimmune uveoretinitis could be inhibited with RAPA at a minimal effective dose of 0.1 mg/kg/day. The drug was effective not only when used during the stimulation phase of the immune system, but also when treatment was de- layed until the efferent phase, indicating that RAPA could be useful in the treatment of active disease. Because EAU in the rat has a very short evolution course once the eye is inflamed, the treatment cannot be reasonably tested during active disease. To ascer- tain the efficacy of RAPA in conditions mimicking disease conditions better, we treated animals in which EAU was induced by transfer of fuUy differ- entiated T cells. We found that the same 0.1 mg/kg/day dose was completely effective, indicating the RAPA would be appUcable in the treatment of ongoing disease. In vitro we foimd a strong synergistic effect on the inhibition of proliferation of lymphocytes to retinal S-Ag for the combination of RAPA with Dex or CsA. We calculated a dose-reduction factor for various dose combinations. For example, the combi- nation of Dex with RAPA allowed for a 4.5-fold reduction of both drugs, while the combination of RAPA with CsA yielded reduction factors of 10 for RAPA and 3 for CsA. Significance to Biomedical Research and the Program of the Institute The study has demonstrated the efficacy of RAPA in the treatment of a uveitis model, which indicates that, barring immanageable toxic side effects, the drug could be useful in the treatment of uveitis in humans. Peitiaps the most encouraging result is the strong synergistic effect of RAPA combinations with Dex and CsA, which would eliminate most of the toxicity of these drugs while maintaining good therapeutic control of the disease. Proposed Course Future woik on this project will include (1) evalua- tion of the toxic side effects of RAPA, (2) condua- ing in vivo combination treatment to evaluate the dose reduction factors found in the in vitro synergy, (3) evaluation of the effects of RAPA on the compli- 47 Laboratory of Immunology NEI Annua l Report-FY 1992 cation of uveitis, particularly subretinal membrane Roberge FG, Xu D, Chan C-C, de Smet MD, formation and neovascularization, and (3) evaluation Nussenblatt RB, Chen H: Treatment of experi- of a new noncytotoxic immunosuppressant (SK&F mental autoimmune uveoretinitis in the rat with 106610) in an aim to increase the combination's rapamycin, an inhibitor of lymphocyte growth potential to lower tiie side effects of the various factor signal Q-ansduction. Curr £ye /?«, in press, agents. Publications Roberge FG, Xu D, Chan C-C: A new effective and non-haraiful chemical adjuvant for the induction of experimental autoimmune uveoretinitis. Curr Eye Res 4:371-376, 1992. 48 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00263-03 LI PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or lass. Title must fit on orta Una betwaan the borders.) Comparison of Surgical Treatment in Uveitis Patient s With Glaucoma PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute affiliation) PI: Benjamin I. Rubin M.D. Special Volunteer LI, NEI Others: Robert B. Nussenblatt Marc de Smet Chi-Chao Chan M.D. M.D. M.D. Clinical Director Visiting Fellow Medical Officer >fEI LI, NEI LI, NEI COOPERATING UNITS (If any) LAB/BRANCH Laboratory of Immunology SECTION Section of Clinical Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ {a2) Interviews PROFESSIONAL: 0.0 OTHER: 0.0 13 (b) Human tissues Q (c) Neitiier SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project has been terminated. 49 PHS 6040 (Rov. SJ9Z) DEPARTMEm- OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1, 1991 to September 30, 1992 PROJECT NUMBER ZOl EY 00248-05 LI TITLE OF PROJECT (BO charaaers or lass. Title must til on one line between the borders.) Magainin Therapy of Infectious Keratitis PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aHUIation) PI: Juan S. Lopez M.D. Visiting Associate LI, NEI Others: Robert B. Nussenblatt M.D. Richard M. Fenton M.D. Clinical Director Senior Staff Fellow NEI LI, NEI COOPERATING UNITS (II any) Clinical Pathology Department, Qinical Center, NIH (Frida Stock, B.S.) LAB/BRANCH Laboratory of Immunology SECTION Section on Clinical Immunoregulati on INSTITUTE AND LOCATION NEI, NIH, Bethesda. MD 20892 TOTAL STAFF YEARS: 0.0 CHECK APPROPRIATE BOX(ES) n (a) Human subjects n (a1) Minors □ (a2) Interviews PROFESSIONAL: 0.0 OTHER: 0.0 n (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project has been terminated. 50 PHS 6040 (Rev. 5«2) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00262-03 LI PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (BO characters or lass. Title must tit on one line tetween the borders.) Selective Accumulation of VB8-Positive T Lymphocytes in Experimental Autoimmune Uveoret initis PRINCIPAL INVESTIGATOR (List other prolassional personnel below the Principal Investigator.) (Name, title, laboratory, and insUtute altUmtion) PI: Charles E. Egwuagu M.P.H., Ph.D. Scientist LI, NEI 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 LI, NEI Visiting Associate LI, NEI Biologist LI, NEI COOPERATING UNITS (11 any) Laboratory of Immimology SECTION Section on Experimental Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.8 PROFESSIONAL; 1.8 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interview/s □ (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-ceU-mediated autoimmune disease that serves as a model of human intraocular inflammatory disease (uveitis). It is initiated in susceptible animals by immunization with retinal antigens, such as interphotoreceptor retinoid-binding protein (IRBP) and S-antigen (SAg). Previous studies of T-cell receptor (TCR) usage by uveitogenic T cells suggested a possible connection between pathogenicity of T lymphocytes and usage of vps family genes. Here, we have analyzed the T-cell repertoire at the autoimmune site by examining Vp gene expression in the retinas of animals with SAg- or IRBP-induced uveitis. Our data show a remaricable bias for the use of vp8.2 TCR among Vp8-positive T cells in the retinas of animals with SAg-EAU, while in IRBP-EAU both Vp8.2-positive and Vp8.3-positive T cells were found. Similar results were obtained by DNA sequence analysis of 75 vps cDNA clones from uveitogenic T-ceU lines. Twenty clones isolated from SAg-specific T-cell lines were aU vp8.2 TCRs, whereas, among 55 vpS cDNAs from IRBP-specific lines, 22% were vp8.2; 33%, vp8.3; and 44%, Vp8.2 isoforms not previously reported, vps.l -positive T cells were absent from the VP8 repertoires of the retina or any of the T-cell lines. In addition, there was maiked heterogeneity of the CDR3 sequences utilized by the Vp8- positive uveitogenic T cells, which contrasts with the reported near homogeneity of encephalitogenic T cells involved in experimental allergic encephalomyelitis (EAE). Taken together, our data show that although there is a bias toward usage of vpS-positive cells, the T-cell response in EAU is not oligoclonal. Moreover, distinct Vp8 subfamilies were differentially activated by the autoantigens SAg and IRBP. In view of these findings, we believe that anti-VP therapy might have only limited usefulness in human uveitis because several autoantigens and an unknown number of immunopathogenic epitopes may be involved in the etiology of this diverse group of diseases. 51 PHS 6040 (Rev. 5fl2) Laboratory of Immunology NEI Annual Report-FY 1992 Project Description Objectives This project is aimed at gaining knowledge concern- ing the clonality of the T cells that mediate experi- mental autoimmune uveitis (EAU). It is noteworthy that similar studies in experimental allergic encepha- lomyelitis (EAE) foimd restricted usage of vps T- cell receptor (TCR) elements. This knowledge was exploited to protect animals against the disease by using antibodies specific to VP8.2 or by vaccination against portions of the receptor. Our effort during Fiscal Year 1991 focused on the analyses of the TCRs expressed at the autoimmune site to determine whether there is restricted TCR Vp usage in EAU. Methods Animals were inraiunized with either S-antigen (S-Ag) or interphotoreceptor retinoid-binding protein (IRBP), and at time points corresponding to the expected onset of EAU, as well as 1 day before and 1 day after expected onset, the animals were sacri- ficed and RNA was isolated from their retinas. cDNAs generated by reverse transcription were used to amplify all the known Lewis rat Vp TCR chains. The T-cell lines used in these studies were derived from the lymph nodes of Lewis rats immunized with either S-Ag or peptides derived from IRBP. Conven- tional recombinant DNA techniques such as Southern blot hybridization, polymerase chain reaction (PCR), cDNA construction, densitometry, and DNA sequenc- ing were used to analyze the resultant PCR products. Major Findings Analysis of TCR Vp5 repertoire induced by S-Ag and 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 vps genes in S-Ag-induced uveitis is restricted to vp8.2 TCR, while in IRBP-induced EAU multiple members of the Vp8 family (Vp8.2, vp8.3, and VP8.2 isoforms not previously reported) are expressed. Analysis of junctional sequences used by uveito- genic T cells. — In contrast to findings among the TCRs of lymphocytes specific against xenogenic antigens such as cytochrome c and sperm whale myoglobin, in which T cells that recognize the same minimal epitope express identical junctional se- quences, uveitogenic T ceUs with the same antigen fine specificity express different jimctional se- quences. We also found a subpopulation of T cells in IRBP- and S-Ag-specific T-ceU Unes that expressed similar jimctional sequences, suggesting that these TCRs may be specific to a common determinant, possibly a self-superantigen or coligand. Analysis of the fourth complimentarity-determin- ing region (CDR4). — ^Forty-five percent of vps family TCRs analyzed contain amino acid substitu- tions at positions corresponding to the CDR4 region of the Vp chain. Amino acid residues within this region (amino acids 67-75) have been shown to be involved in superantigen binding. Conservation of superantigen-binding sequences by a significant proportion of vpS* lymphocytes in these lines may not be coincidental and may suggest that lympho- cytes expressing these receptors could have functions other than binding conventional antigens plus major histocompatibility complex. Significance to Biomedical Research and the Program of the Institute Taken together, our data show that although there is a bias toward usage of vps* cells, the T-cell re- sponse in EAU is not oligoclonal, and distinct vps subfamilies were differentially activated by the autoantigens, S-Ag and IRBP. Tliese findings suggest that anti-Vpb therapy might have only limited usefulness in human uveitis since several autoanti- gens and an unknown number of immunopathogenic epitopes may be involved in the etiology of this diverse group of diseases. Proposed Course We intend to induce EAU in nude mice by injection of antigen-activated S-Ag- or IRBP-specific uveito- genic T-cell lines. Because these mice do not nor- mally produce T cells, we expect the intraretinal T-cell repertoire of these mice to be very limited. This finding would allow for a more concise analysis of the identity of autoaggressive T cells involved in uveitis. NEI Research Program Retinal and Choroidal EHseases — Inflammatory Disorders 52 NEI Annual Report-FY 1992 Laboratory of Immunology Publications Mahdi RM, Caspi RR, Tsai L, Nussenblatt RB, Gery Egwuagu CE. Bahmanyar S, Caspi R, Mahdi R, ^' Eg^uagu CE: T ceU receptor Vp gene usage in Nussenblatt R, Gery I: Predominant usage of experimental autoimmune uveoretinitis. Invest Vp8.3 T cell receptor in a T ceU line that induces Opthalmol Vis Sci 33(4): 1024, 1992. experimental autoimmune uveoretinitis. / Clin Immunol Immunopathol 65(2): 152-160, 1992. 53 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00280-01 LI PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) Ectopic Expression of Interferon-Gamma in the Eyes of Transgenic Mouse PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Charles E. Egwuagu M.P.H., Ph.D. Scientist LI, NEI Others: Robert B. Nussenblatt M.D. Chi-Chao Chan M.D. Ana Chepelinsky Ph.D. Jorge Sztein D.VM. Rashid Mahdi Ph.D. Scientific Director Chief, Section on Immunopathology Visiting Associate Biologist NEI LLNEI LMDB, NEI VRRS, NEI LLNEI COOPERATING UNITS (if any) Laboratory of Immunology Section on Experimental Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.4 PROFESSIONAL: 1.4 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.) Ectopic expression of interferon-gamma (IFN-y) in the lens provides a model for studying the clinical and biological effects of IFN-y in the normal development of the eye. In this project, two transgenic mouse lines expressing IFN-y under the direction of lens-specific oA-crystallin promoter were estabhshed using two distinct mouse strains, Balb/c and FVB/N. In both the oACry-IFN-Y/Balb/c and oACry-IFN-Y/FVB/N transgenic mice, the essential histopathological features observed were very similar at all developmental stages studied (12- to 18-day embryos, newborns, adults), suggesting that maldevelopment of ocular tissues of these mice resulted from oACry-IFN-y expression. The differentiation of tissues of surface ectodermal, neuroectodermal, and mesodermal origin was adversely affected. In the lens, normal differentiation of the anterior and posterior lens cells was perturbed, resulting in marked disorganization of the entire lens architecture and very little or no lens fiber formation. The retina was very convoluted with retinal detachment, retinal degeneration, partial loss of |diotoreceptors, and accumulation of intraocular macrophages in the subretinal space. Vitritis, iridocyclitis, and corneal and vitreous neovascularization were commonly observed. To investigate the molecular basis of IFN-y-mediated disruption of the normal developmental program of ocular tissues, we analyzed the abundance of mRNA transcripts of several lens genes: oA-crystallin, major intrinsic protein (MIP), oB-crystallin, macrophage inhibitory factor (MIF), transforming growth factor (TGPP), DNA-binding protein (dbpBA^-1). and major histocompatibihty complex (MHC) class n in oACry-IFN-Ymice and age-matched controls. Li the transgenic mice, upregulation of MHC class n gene was observed while MIP gene transcription was silenced. Transcription of the other lens genes was not affected. Although the oA-crystallin promoter is normally active only in lens tissue, its tissue specificity was abrogated in the oACry-II^-Y/Balb/c and oACry-IFN- Y/FVB/N transgenic mice; expression of oACry-IFN-Y also was found in the brain, kidney, Uver, ovary, spleen, testes, and thymus. Furthermore, upregulation of MHC class n also was found in the brain, kidney, liver, ovary, and testes. 54 PHS 6040 (Rev. S/92) NEI Annual Report-FY 1992 Laboratory of Immunology Project Description Objectives This project was designed to generate transgenic mice that selectively secrete interferon-gamma (IFN- Y) in their eye tissues for use in studying the bioregu- latory actions of IFN-y in nonlymphoid tissues. Because fiFN-y induces high levels of major histo- compatibility complex (MHC) class II proteins, the utility of these mice as a model for studying the role of elevated MHC class II in predisposition to auto- immune diseases such as diabetes and uveitis was also explored. Methods Transgenic animals were generated according to standard transgenic mouse techniques. The transgenic and wild-type (WT) phenotypes were characterized by histological examinations of representative tissue sections. Conventional recombinant DNA techniques such as Southern blot hybridization, polymerase chain reaction (PCR), cDNA construction, densitome- try, and DNA sequencing were used to characterize DNAs and RNAs derived firom WT and transgenic mice. Major Findings 1. Qinical symptoms manifested by the oACry- DFN-y mice included microphakia, blepharophimosis (decrease in the palpebral aperture), blepharosyn- echiae (adhesion of the eyelids to the eyeball), exudative retinal detachment, vitritis, iridocyclitis, partial loss of photoreceptors, and corneal neovascu- larization. 2. IFN-y gene expression affected normal eye development at the earliest stage studied, 18 days of embryonic development. The anterior chamber was not formed, and the retina was not differentiated into iimer and outer neuroblastic layers. In the lens, normal primary lens fibers were replaced by baUoon cells. 3. Expression of the oACry-IFN-y transgene abrogated the characteristic tissue-specificity of the ocA-crystallin promoter, we also foimd expression of the transgene in the brain, kidney, liver, ovary, spleen, testis, and thymus. 4. oA-crystallin and aA™-crystallin, previously thought to be expressed only in eye tissues, also were detected in normal mouse kidney, spleen, and thymus by PCR. 5. MHC class II gene expression was upregulated while major intrinsic protein (MIP) gene transcription was shut down in the transgenic mice, suggesting that IFN-y can function as a transcriptional regulator in nonlymphoid cells. 6. Immunoperoxidase antibody staining of y-crys- tallin and MIP, both markers of lens fibers, was decreased, while MHC class n staining was increased in the transgenic mice compared with that in normal age-matched controls, indicating that the synthesis of these proteins also was affected by eaopic expres- sion of IFN-y. Significance to Biomedical Research and the Program of the Institute We have generated a transgenic mouse system that can serve as a model for human autoimmune dis- eases. This mouse is also a useful system for study- ing the molecular events relating to the normal developmental program of ocular tissues. Because these transgenic mice are MlP-deficient mutants, they afford a useful model to begin characterizing the role of this gap junction protein in the physiology of the eye. Proposed Course We will further dissect the molecular basis of IFN-y actions in the eye, placing particular emphasis on the transcriptional factors that mediate (xACry-IFN-y effects. We also wiU mate the otACry-IFN-y mice with transgenic mice with ocular tumors to determine whether constitutive expression of IFN-y in the eye would lead to tumor regression in the progeny. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Egwuagu CE, Sztein J, Reid W, Chan C-C, Mahdi R, Nussenblatt RB, Chepelinsky C: Gamma interferon gene expression in the lens of trans- genic mice directed by the ccA-crystallin gene promoter. Invest Opthalmol Vis Sci 33(4):846, 1992. 55 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00069-15 LI PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 charaaers or less. Title must lit on one line between the borders.) Immune Restx)nses to Ocular Antigens PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator) (Name, title, laboratory, and Institute BllUiation) PI: Igal Gery Others: Yoichi Kawano Yoichi Sasamoto AJexander Kozhich Barbara Vistica Belinda Stiirkey Robert B. NussenblaB Gerald J. Chader Barbara Wiggert Ph-D. Head, Section on Experimental Imimmology LI. NEI M.D. Visiting Fellow U. NEI M.D. Visiting Fellow LI. NEI Ph-D. Visiting Fellow LI, NEI B.A. Microbiologist LI, NEI B.A. HHMI-NIH Scholar LI. NEI M.D. Scientiric Director NEI Ph.D. Chief LRCMB. NEI Ph.D. Head, Section on Biochemistry LRCMB.NH COOPERATING UNITS (il any) Metabolism Branch, Division of Cancer Biology, Diagnosis, and Centers, National Cancer Institute, NIH (Jay A. Berzofsky, M.D.); Synlex Research, Palo Alto, CA (Anthony C. Allison, M.D.) LAB/BRANCH Laboratory of Immunology Section on Experimental Immunology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.2 PROFESSIONAL: 2.8 0.4 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.) Targeted at learning about the pathogenesis of inflammatory eye diseases grouped under the term "uveitis," this project in FY 1992 focused mainly on learning about ocular antigens that induce experimental autoimmime uveoretinitis (EAU), an animal model for uveitis in man, and on procedures that modulate this disease. Major achievements are three: (1) We found that the substitution of certain residues of the bovine IRBP peptide 1181-1191 eliminated its high uveitogenicity. The corresponding sequence of the rat IRBP differs from the bovine sequence by two residues and was found to be completely nonuveitogenic. (2) To investigate mechanisms of specific unresponsiveness toward uveitogenic peptides, we developed an in vitro system in which lymphocytes sensitized toward peptide 1181-1191 are rendered imresponsive. We have defined the process to be highly specific and to involve active metabolic events. (3) We have examined three procedures aimed at inhibiting the immunopathogenic process of EAU: (a) EUA induced in rats by IRBP peptide 1181- 1 191 was completely inhibited when this peptide was coinjected with a competing peptide, A183, an analog of a peptide derived from a Mycobacterium tuberculosis protein. On the other hand, nonuveitogenic analogs of peptide 1181-1191 were inactive in this system, (b) The procedure of oral tolerance, shown in another project to inhibit S-antigen-induced EAU effectively, also was found to modulate EAU induced by the smaU peptide 1181-1191. Furthermore, using analogs of peptide 1181-1191 we have shown that oral tolerance is highly specific: Only cross-reactive analogs were effective, (c) We have examined a new immunosuppressive drug, mycophenolate mofetil (MM), for inhibition of EAU. MM completely inhibited both actively induced and adoptively transferred EAU when given at doses that had no apparent side effects. 56 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Immunology Project Description Objectives This project is designed (1) to collect additional information on the immunodominant and highly uveitogenic peptide detenninant 1181-1191 of interphotoreceptor retinoid-binding protein (IRBP), with regard to (a) the residues pivotal for the inunu- nological activities of this molecule and (b) the capacities of the rat sequence corresponding to the bovine IRBP 1181-1191; (2) to analyze further the procedure in which lymphocytes sensitized against p)eptide 1 181-1 191 are rendered unresponsive ("aner- gic") to this uveitogenic molecule; and (3) to investi- gate procedures aimed at suppressing the immuno- pathogenic processes of experimental autoimmune uveoretinitis (EAU). The procedures included (a) in- jection of peptides with the capacity to compete with the uveitogenic peptide, (b) induction of specific tolerance by oral administration of the uveitogenic peptide, and (c) treatment with a new immunosup- pressive drug, mycophenolate mofetU (MM). Methods Synthetic peptides were provided by A.B.I. (Foster City, CA) or synthesized on an Applied Biosystems synthesizer 430A. MM, supplied by Syntex Research (Palo Alto, CA), was administered by gavage. EAU induction and measurements of immune responses were performed as described in our pubhcations. The procedures for induction of anergy in vitro are described in previous NEI annual reports. Major Findings Studies concerning IRBP peptide 1181-1191. — In a previous study (Fiscal Year [FY] 1990) we showed that certain residues of peptide 1 1 8 1 - 1 1 9 1 are pivotal for its immunodominance and high uveitogenicity in Lewis rats; substitution of these residues with alanine markedly reduced or even completely abolished the activities of this peptide. We extended this approach by testing analogs in which other amino acids were used for substitution. The data further underscored the pivotal role of residues 1 1 88, 1 1 89, and 1 190 and established the role of residue 1183. The recently published sequence of rat IRBP shows that within detenninant 1181-1191 the rat sequence differs from that of the bovine by two residues: Position 1188 is threonine in the rat and valine in the bovine sequence, while position 1 190 is asparagine in the rat and aspartic acid in the bovine. Both residues are pivotal for the bovine molecule's activities, and no cross-reactivity was detected between the bovine and rat peptides. Moreover, the rat peptide was nonuveitogenic and even nonimmu- nogenic in the Lewis rat: Neither EAU nor immune responses were detected in rats immunized with this molecule. We found that a peptide in which only the valine at 1188 was substituted with threonine was weakly uveitogenic and immunogenic; it showed a low-level cross-reactivity with bovine peptide 1181- 1191. Induction of anergy in vitro. — We had previously developed an in vitro system in which specific anergy (ie, uiuiesponsiveness) is induced toward uveitogenic peptides (FY 1991). We further analyzed the system, producing the following findings: (1) Testing additional peptide analogs of bovine IRBP peptide 1181-1191 showed that the system is highly specific: Only analogs with cross-reactivity toward the native peptide were capable of inducing anergy in lymphocytes specific toward this peptide. Accordingly, the rat homologue of peptide 1181- 1191 (see above) failed to show any effect in this system. (2) Induction of anergy was accompanied by an increase in inositol phosphate synthesis in the test lymphocytes. The relationship between anergy induction and increased inositol phosphate synthesis was shown by the complete correlation between the two processes when different analogs of peptide 1181-1191 were tested. (3) Antibodies against rat major histocompatibility complex class II antigens had no effect on induction of anergy. On the other hand, the antibodies tested here, OX-3 and OX-6, effectively inhibited the proliferative response of the lymphocytes toward peptide 1181-1191 in the con- ventional system in which antigen-presenting cells are present. Modulation of EAU. — We have assessed three procedures for their capacity to modulate the immu- nopathogenic processes of EAU. Because published studies have shown that experimental autoimmime diseases can be inhibited by injecting "competing" peptides along with the immimopathogenic antigens (eg, Proc Natl Acad Sci 88.9633, 1991), we tested two types of peptides for their capacity to inhibit EAU induced by IRBP determinant 1181-1191: (1) four nonuveitogenic analogs of 1181-1191 with substitution of pivotal 57 Laboratory of Immunology NEI A-^nual Report-FY 1992 residues (see above), which we found to be inactive in this system, and (2) an unrelated peptide with known competitive capacity. This peptide, designated "A183," an analog of a nonamer determinant of a heat-shock protein of Mycobacterium tuberculosis, was found to inhibit the development of adjuvant arthritis and experimental allergic encephalomyelitis in Lewis rats. Peptide A 183 completely inhibited the development of EAU induced by peptide 1181-1191 when injected at the molar ratio of 1 :500 and caused partial inhibitior. 5f the disease at the ratios of 1 :40 and 1:100. A183 also inhibited the cellular immune response to 1181-1191 but, unexpectedly, A 183 did not competitively inhibit the proliferative response of lymphocytes toward 1181-1191. Another project G^I protocol 90-EI-116) had shown that feeding rats uveitogenic proteins or peptides derived from their sequence effectively inhibits the development of EAU induced by these molecules. We tested the fine specificity of this procedure by testing analogs of peptide 1181-1191 for their capacity, when fed, to induce a state of tolerance toward the native peptide. The data showed good correlation between the analogs in inducing such tolerance and in their cross-reactivity toward peptide 1181-1191, which is evident in their recogni- tion by lymphocytes sensitized against the native peptide. MM, an inhibitor of purine synthesis (Eugui et al, Scmd J Immunol 33:161, 1991), had been found to efficiently inhibit S-antigen (S-Ag)-induced EAU when given daily at 30 mg/kg/day, on days 0-13 postimmunization. At a higher dose, 60 mg/kg/day, we foimd MM inhibited EAU, even when adminis- tered on days 7-20. In addition, MM at 30 mg/kg/day inhibited the development of EAU adoptively trans- ferred by lymphocytes sensitized against S-Ag. These data thus show that MM can inhibit both the afferent and efferent limbs of the immunopathogenic process of EAU. Significance to Biomedical Research and the Program of the Institute 1. Peptide 1181-1191 is the immunodominant determinant of bovine IRBP in Lewis rats. It is highly uveitogenic and immunogenic in these ani- mals. Our studies of this molecule provide new infonnation concerning residues that are pivotal for its inmiimologic activities. Of particular interest are the findings with the rat homologue of peptide 1181- 1 191. The complete lack of uveitogenicity, immuno- genicity, and cross-reactivity with the bovine peptide was unexpected. This finding suggests that a unique mechanism should be used by lymphocytes sensitized against bovine peptide 1 181-1 191 for the recognition of the target antigen in the rat eye when initiating the immunopathogenic process of EAU. 2. The system of anergy induction in vitro has provided useful new information concerning the mechanisms whereby lymphocytes can be rendered unresponsive toward uveitogenic peptide. Moreover, this information should be applicable for developing novel procedures specifically for suppressing re- sponses toward immunopathogenic antigens. 3. Our EAU modulation studies have yielded several findings of interest: (a) Using peptides to inhibit uveitogenic processes is feasible, but mecha- nisms other than competitive inhibition also are involved, (b) Oral tolerance can be induced by peptides as small as 1181-1191, and the effect is highly specific, (c) MM inhibits the development of EAU by affecting both the afferent and efferent immunopathogenic processes of disease at doses that have no apparent adverse side effects. Thus, this compound should be considered as a potential new drug for treatment of uveitis. Proposed Course Our future efforts will focus on several issues. (1) We will further investigate the enigmatic finding concerning the rat homologue of peptide 1181-1191 (see above) by testing several potential explanations for this observation. (2) We will further analyze the induction of anergy in vitro with regard to the features of the phenomenon and the biochemical processes involved. (3) In particular, we will focus on procedures and agents that modulate the immuno- pathogenic processes of EAU. More studies will be conducted to understand better the induction of anergy, both in vitro and in vivo. We will test MM and additional immunosuppressive agents, and we will examine the possible synergy between com- pounds to achieve effective combinations of drugs at significantly lower doses. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders 58 NEI Annual Report-FY 1992 Laboratory of Immunology Publications Beraud E, Kotake S, Caspi RR, Oddo SM, Chan C-C, Gery I, Nussenblatt RB: Control of experi- mental autoimmune uveoretinitis by low dose T- cell vaccination. Cell Immunol 140:112-122, 1992. Egwuagu CE, Bahmanyar S, Mahdi RM, Nussenblatt RB, Gery I, Caspi RR: Predominant usage of V|38.3 T cell receptor in a T cell line that induces experimental autoimmune uveoretinitis (EAU). Clin Immunol Immunopathol, in press. Egwuagu CE, Chow C, Beraud E, Caspi RR, Mahdi RM, Brezin AP, Nussenblatt RB, Gery I: T cell receptor P-chain usage in experimental auto- immune uveoretinitis. J Autoimmun 4:315-324, 1991. Fujino Y, Chan C-C, de Smet MD, Hikita N, Gery I, Mochizuki M, Nussenblatt RB: FK506 treattnent of experimental autoimmune uveoretinitis in primates. Transplant Proc 23:3335-3338, 1991. Fujino Y, Mochizuki M, Chan C-C, Raber J, Kotake S, Gery I, Nussenblatt RB: FK506 treattnent of S-antigen induced uveitis in primates. Curr Eye Res 10:679-690, 1991. 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, in press. Kawano Y-I, Sasamoto Y, Kotake S, Thurau SR, Wiggert B, Gery I: Trials of vaccination against experimental autoimmune uveoretinitis with a T-cell receptor peptide. Curr Eye Res 10:789-795, 1991. Kotake S, Redmond TM, Wiggert B, Vistica B, Sanui H, Chader GJ, Gery I: Unusual immuno- logic properties of the uveitogenic interphoto- receptor retinoid-binding protein-derived peptide R23. Invest Ophthalmol Vis Sci 32:2058-2064, 1991. Kotake S, Sasamoto Y, Kawano Y-I, Sanui H, Wiggert B, Chader GJ, Gery I: The existence of two completely distinct antigenic sites within a decapeptide. Cell Immunol 140:123-129, 1992. Lipham WJ, Redmond TM, Takahashi H, Berzofsky JA, Wiggert B, Chader GJ, Gery I: Recognition of peptides that are immunopathogenic but cryp- tic. Mechanisms that allow lymphocytes sensitized against cryptic peptides to initiate pathogenic autoimmune processes. J Immunol 146:3757- 3762, 1991. Sasamoto Y, Kawano Y-I, Bouligny R, Wiggert B, Chader GJ, Gery I: Immimomodulation of experi- mental autoimmune uveoretinitis by intravenous injection of uveitogenic peptides. Invest Ophthalmol Vis Sci 33:2641-2649, 1992. Wiggert B, Kutty G, Long KO, Inouye L. Gery I, Chader GJ, Aguirre GD: Interphotoreceptor retinoid-binding protein (IRBP) in progressive rod-cone degeneration (PRCD) — Biochemical, immimocytochemical and immunologic studies. Exp Eye Res 53:389-398, 1991. Yamamoto JH, Okajima O, Mochizuki M, Shinohara T, Wiggert B, Chader GJ, Gery I, Nussenblatt RB: Cellular iimnune responses to retinal antigens in retinitis pigmentosa. Graefe's Arch Clin Exp Ophthalmol 230:119-123, 1992. 59 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00222-07 LI PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or loss. Title must li on one line between the borders.) Immunopathology in the Eyes With Experimental Uveitis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute alliliation) PI: Chi-ChaoChan M.D. Head, Section on Imnumopathology Rachel R. Caspi Ph.D. Visiting Scientist Igal Gery Ph.D. Deputy Chief QianLi M.D. Visiting Associate Robert B. NussenblaD M.D. Scientific Director Scott M. Whitcup M.D. Medical Officer Fran9ois G. Robeige M.D. Visiting Scientist Deborah Luyo Technician Louis Kasner M.D. IRTA U, NEI LI, NEI LI. NEI LI. NEI NEI LI. NEI LI. NEI LI, NEI LI, NEI COOPERATING UNITS (11 any) Department of Ophthalmology, Kurume University, Kurume, Japan (Manabu Mochizuki, M.D.); Service Universtaire d'Ophtalmologie, H6pital Ophtalmique Jules Gonin, Lausanne, Switzerland (Carl P. Herbort, M.D.) LAB/BRANCH Laboratory of Immunology Section on Immunopathology INSTrrUTE AND LOCATION NEI, MH, Bethesda, MP 20892 TOTAL STAFF YEARS: 2.50 PROFESSIONAL: 1.25 1.25 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 protrided.) The identity and topographic localization of immunocompetent cells and the alteration of surface markers on ocular resident ceUs in mice and rodents with experimental autoimmune uveoretinitis (EAU) were analyzed by immunohistochemical studies and in situ hybridization. T lymphocytes were the predominantly infiltrating cells in rat EAU, yet both macrophages and T helpersAnducers were the predominantly infiltrating cells in mouse EAU. The migration of inflammatory cells from vessels into target sites is directed by adhesion molecules that can be expressed on vascular endothelium and other resident cells in the eye. 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 II antigens was observed on ocular resident cells such as retinal pigment epithelium (RPE), retinal vascular endothelium, keratocytes, fibroblasts, and ciliary epithelium in rodents. The expression of MHC class n antigens is confined to ocular resident cells immediately at the inflammatory sites in mouse EAU, which is characterized by focal lesions and chronicity. Both the infiltrating cell subpopulation and the expression of class U antigens on ocular resident cells can be altered by various immunomodulating agents. Experimental endotoxin-induced uveitis (EIU) is a model for anterior uveitis in humans. The mechanism of this inflammation is still imclear, although activation of phospholipase A2 (PLA2) may play a role in the initiation and propagation of this disease. The partioilar vascular structure of the anterior uvea and the role of adhesion molecules (such as intercellular adhesion molecule and endothelial leukocyte adhesion molecule) and their ligand (lymphocyte function-associated antigen) also were examined in EIU and EAU models. Inhibition of PLA2 (chloipromazine, antiflammin, etc.) and adhesion molecules can abrogate EIU. This project will be combined with projea numbers ZOl EY 00241-06 LI and ZOl EY 00264-03 LI, entitled "Immunopathology of Experimental and Clinical Ocular Diseases." 60 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Immunology Project Description Additional Personnel Louis Kasner M.D. LI, NEI 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. The infiltrating inflammatory cells, ocular resident cells and their products, various lymphokines, and cytokines are examined. The findings wiU help us understand ocular inflammation. Methods Clinical examinations include flashlight, slit-lamp, and fundus examination under the dissecting micro- scope. Pathological examinations include routine histologic techniques for light and electron microsco- py, immunofluorescence, avidin-biotin-peroxidase complex methods, and in situ hybridization tech- niques. Major Findings The kinetics of infiltrating cells inside the eyes with EAU in the rat showed predominantly CD4 cells in the early stage and predominantly CDS cells in the late stage. In the mouse, both CD4 and CDllc/18 ceUs predominated in the eye. In the monkey, an influx of CD 19 cells among the CD3 cells and subretinal fibrosis coexisted in the late stage. Prior to the infiltration of inflammatory cells, we identified mast cell degranulation, expression of adhesion molecules, and major histocompatibility complex (MHC) class II antigens on ocular resident cells. Inmiunosuppressive medications could alter the kinetics of the infiltrating cells and the markers of the resident cells. Mast-ceU degranulation, the expression of adhe- sion molecules, and MHC class II antigens on ocular resident cells also were observed prior to the ocular infiltration of inflammatory cells in EIU. Significance to Biomedical Research and the Program of the Institute Different imraunopathological findings of EAU can be presented in various species using die same antigen. The patiiology of uveitis in human beings may capture a single moment of the changing kinet- ics and can be influenced by the treatment Proposed Course EAU and EIU wiU be studied clinically and histo- pathologically in rodent, murine, and primate models. Various immunosuppressive agents will be used to modulate the disease. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Beraud E, Kotake S, Caspi RR, Oddo SM, Chan C-C, Gery I, Nussenblatt RB: Control of experi- mental autoimmune uveoretinitis by low dose T cell vaccination. Cell Immunol 140:112-122, 1992. Caspi RR, Chan C-C, Fujino Y, Oddo S, Najafian F, Bahmanyar S, Heremans H, Wilder RL, Wiggert B: Genetic factors in susceptibility and resistance to experimental autoimmune uveoretinitis. Curr Eye Res ll(suppl):81-86, 1992. Caspi RR, Grubbs BG, Chan C-C, Chader GJ, Wiggert B: Genetic control of susceptibility to experimental autoimmune uveoretinitis in the mouse model: Concomitant regulation by MHC and non-MHC genes. J Immunol 184:2384-2389, 1992. Fujino Y, Li Q, Chung H, Hikita N, Gery I, Nussen- blatt RB, Chan C-C: Immunopatiiology of experi- mental autoimmune uveoretinitis in primates. Autoimmunity, in press. Fujino Y, Mochizuki M, Chan C-C, Raber J, Kotake S, Gery I, Nussenblatt RB: FK506 treatinent of S-antigen induced uveitis in primates. Curr Eye Res 10:679-690, 1991. Hara Y, Caspi RR, Wiggert B, Chan C-C, WUbanks GA, Streibin JW: Suppression of experimental 61 Laboratory of Immunology NEI Annual Report-FY 1992 autoimmune uveitis in mice by induction of anterior chamber associated immune deviation with interphotoreceptor retinoid binding protein. J Immunol 148:1685-1692, 1992. Holland EJ, Chan C-C, Wetzig R, Palestine AG, Nussenblatt RB: Qinical and immunohistologic studies of corneal rejection in the rat penetrating keratoplasty model. Cornea 10:347-380, 1991. 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 1 1 :843- 848, 1992. 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, Whitcup SM, Fujino Y, Nussenblatt RB, Chan C-C: The role of mast cells in endotoxin induced uveitis. Invest Ophthalmol Vis Sci, in press. Roberge FG, Caspi RR, Chan C-C, Nussenblatt RB: Inhibition of T lymphocyte proliferation by retinal glial Muller cells: Reversal of inhibition by glucocorticoids. /AMroJ/wnM/wO' 4:307-314, 1991. Roberge FG, Xu D, Chan C-C: A new effective and non-harmful chemical adjuvant for the induction of experimental autoimmune uveoretinitis. Curr Eye Res 11:371-376, 1992. Thuran SR, Caspi RR, Chan C-C, Weiner HL, Nussenblatt RB: Immunologic suppression of experimental autoimmune uveitis. Fortschr Oph- thalmol 88:404^07, 1991. Thuran SR, Chan C-C, Suh E, Nussenblatt RB: Induction of oral tolerance to S-antigen induced experimental autoimmune uveitis by a uveitogenic 20mer peptide. J Autoimmunity 4:507-516, 1991. Whitcup SM, Wakefield D, Li Q, Nussenblatt RB, Chan C-C: Endothehal leukocyte adhesion mole- cule- 1 in endotoxin induced uveitis. Invest Oph- thalmol Vis Sci 33:2626-2630, 1992. 62 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00241-06 LI PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (BO characters or lass. Title must fit on one line between the borders.) Immunopathologv of Ocular Diseases in Humans PRINCIPAL INVESTIGATOR (Usl other professional personnel below the Princtpal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Chi-Chao Chan M.D. Chief. Section on LI, NEI ImmunopaUiology Oiben: Robert B. Nussenblaa M.D. Scientific Director NEI Qian Li M.D. Visiting Fellow LI. NEI Marc D. de Smel M.D. Visiting Scientist LI, NEI Raymond DeBarge M.D. Senior Staff FeUow U. NEI Scott M. Whilcup MX). Staff Medical Officer U. NEI Juan Lopez M.D. Visiting Associate LI, NEI Miguel Bumier M.D. ''isiting Scientist LI, NEI Richard Fenton MJ). oiaff Fellow LI, NEI Dev Shah M.D. Visiting Associate LI, NEI COOPERATING UNITS (it any) Department of Ophthalmology, Armed Forces Institute of Pathology (Lan W. McLean, M.D.); University of Minnesota, Department of Ophthalmology (Edward J. Holland, M.D.); L'H6pital de la Piti6, Paris, France (Phuc LeHoang, M.D.) LAB/BRANCH Laboratory of Immunology Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda. MD 20892 TOTAL STAFF YEARS: 2.5 PROFESSIONAL: 2.5 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [3 (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) 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 weU as light and electron microscopic evaluation. In uveitis, immunocompetent ceUs and lymphokines are valuable adjimcts to clinical diagnosis, as well as determinants of disease course and prognosis. In nonuveitic conditions, alteration of cellular membrane surface maricers and intracytoskeleton of the ocular resident cells may imply damage and abnormalities in these diseases. Elucidating the role of the relationships between infiltrating inflammatory or malignant ceUs and other resident cells in the clinical behavior of various diseases will increase our understanding of human ocular disorders. This project will be terminated and combined with Project No. ZOl EY 00222-07 LI next year. 63 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report-FY 1992 Project Description Additional Personnel Debrah Luyo Technician, LI, NEI Objectives The program is designed to evaluate the pathology of infiltrating cells and ocular resident cells in various ocular diseases, including inflammatory and non- inflammatory disorders. The study emphasizes the cell surface maiicers and cell products and brings information on the pathogenesis of each disease. Methods Immunohistochemistry and in situ hybridization, in addition to routine light and electron microscopic techniques, are applied. Major Findings Infiltrating immunocompetent cells and their prod- ucts, lymphokines, are vital in the development of ocular inflammation. However, these ceUs also play a role in noninflammatory diseases, such as intraocu- lar lymphoma (large B-cell lymphoma) and orbital lymphoma. Expression of some cell maricers such as adhesion molecules and major histocompatibility complex class II antigens are important for homing the infiltrating cells and immune responses. The handling and processing of himian specimens are critical for evaluation and diagnosis, in particular, diagnostic vitrectomy and chorioretinal biopsy. Significance to Biomedical Research and the Program of the Institute T lymphocyte-mediated immune response is usually involved in noninfectious inflammation in the eye. The routine use of immunohistochemical techniques and the proper handling of specimens — in particular the biopsies from the vitreous, uvea, and retina — will increase the diagnostic yield and help the therapeutic choice. In addition, they will help us understand the mechanism of the disease. Proposed Course The continued evaluation of specimens obtained from patients with different ocular disorders is proposed in the study of their immunopathogenesis. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Bumier MN Jr, Piacentini MA, Shah DN, Chan C-C. Nussenblatt RB, McLean IW: Differentiation between benign and malignant orbital lymphocyt- ic lesions: The use of proliferating cell nuclear antigen. Invest Ophthalmol Vis Sci 33(4):1245, 1992. Chan C-C, Li Q, Brezin AP. Whitcup SM, Egwuagu C, Otterson EA, Nussenblatt RB: Detection of IL-2 and IL-4 mRNA in the conjunctiva of patients with onchocerciasis by in situ hybridiza- tion. Invest Ophthalmol Vis Sci 33(4): 1320. 1992. Chan C-C. Li Q, Kikuchi T. Shinohara T. Nussen- blatt RB: Enhancement of S-antigen and its mRNA in the irises from uveitic patients. J Autoimmunity, in press. Chan C-C, Palestine AG, Davis LD. de Smet MD, McLean IW. Bumier M. Drouihet MD, Nussen- blatt RB: The role of chorioretinal biopsy in inflammatory eye disease. Ophthalmology 98:1281-1286, 1991. Davis JL, Chan C-C, Nussenblatt RB: Diagnostic vitrectomy in intermediate uveitis. Dev Ophthal- mol 23:120-132, \992. Davis JL, Chan C-C. Nussenblatt RB: Immimology of intermediate uveitis. Dev Ophthalmol 23:7 \-&5, 1992. Davis JL, Solomon D, Nussenblatt RB. Palestine AG, Chan C-C: Immunocytochemical staining of vitreous cells: Indication, techniques and results. Ophthalmology 99:250-256. 1992. Fenton RM. Soylu M, deSmet MD, Chan C-C, Nussenblatt RB: The use of indirect immunofluo- rescence testing and guinea pig lip mucosa for the evaluation of Behcet's and non-Behcet's uveitis. Invest Ophthalmol Vis Sci 33(4):942. 1992. Lopez JS. Chan C-C. Bumier M, Rubin B, Nussen- blatt RB: Immunohistochemistry findings in primary intra-ocular lymphoma. Am J Ophthalmol 112:472-474, 1991. Nussenblatt RB, Palestine AG, Chan C-C. Stevens G Jr. Mellow S. Green SB: Randomized double- 64 NEI Annual Report-FY 1992 Laboratory of Immunology masked study of cyclosporine compared to pred- nisolone in the treatment of endogenous uveitis. Am J Ophthalmol 112:138-146, 1991. Piacentini MA, Shah DN, Chan C-C. Nussenblatt RB, Bumier MN Jn The orbital pseudotumor spectrum: An immunopathological study of 40 cases. Invest Ophthalmol Vis Set 33(4): 1197, 1992. Shah DN, Piacentini MA, Bumier MN Jr, McLean IW, Nussenblatt RB, Chan C-C: Inflammatory cellular subsets in sympathetic ophthalmia: Corre lation with the disease kinetics. Invest Ophthalmol Vis Sci 33(4):929, 1992. Whitcup SM, Chan C-C, Li Q, Nussenblatt RB: Expression of cell adhesion molecules in posterior uveitis. Arch Ophthalmol 110:662-666, 1992. Whitcup SM, Fenton RM, Pluda JM, de Smet MD, Nussenblatt RB, Chan C-C: Pneumocystis carinii and mycobacterium avium — intracellulare infec- tion of the choroid. P^.tina 12:331-335, 1992. 65 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00264-03 LI PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 charaaers or less. Title must fit on one line between the borders.) Cytokines and Ocular Antigens in the Eye PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Chi-Chao Qian M.D. Head, Section on LI, NEI Inununopathology Others: Robert B. Nussenblatt Igal Gery Qian Li Louis Kasner M.D. Scientific Director LI, NEI Ph.D. Head, Section on Experimental Immunology LI, NEI M.D. Visiting Fellow LLNEl M.D. Fellow LLNEI COOPERATING UNITS (if any) LAB/BRANCH Laboratory of Immimology Section on Immunopathology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: PROFESSIONAL: 1.0 1.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x] (b) Human tissues Q (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Cytokines are communication signals between leukocytes and organ resident cells. Interleukin 1 (IL-1), a pleiotropic cytokine produced by many ceU types — most notably macrophages, can stimulate a general inflammatory reaction by causing the activation of a variety of cells which then, among their other functions, release a cascade of other cytokines. One of these cytokines is interleukin 8 (IL-8), which takes the more direct action of activating neutrophils and T lymphocytes. CD4 ceUs can be subtyped to T helper 1 and 2 (Th-1 and Th-2) cells on the basis of the cytokines they release. Th-1 cells produce IL-2, interferon-gamma (IFN-7), and tumor necrosis factor (TNF), while Th-2 cells produce IL-4, IL-5, IL-6, and IL-10. Th-1 ceUs are involved in the cell-mediated immime response, while Th-2 ceUs are involved in allergy and extracellular parasitic infection. Cytokines, lymphokines, and adhesion molecules also are located in ocular inflammatory sites of patients with uveitis and animals with experimental uveitis. Ocular antigens play an important role in autoimmime diseases. S-antigen (S-Ag), a retinal soluble antigen, can induce experimental autoimmune uveoretinitis (EAU). The expression of S-Ag mRNA of nonretinal tissues in fetuses may suggest its involvement in certain ocular diseases. The expression of S-Ag mRNA was foimd in the iris of some patients with uveitis after prolongation of corticosteroid therapy. This finding was confirmed by treating EAU rats with long-term steroids. This project will be terminated and combined with project number ZOl EY 00222-07 LI next year. 66 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Immunology Project Description Additional Personnel Scott M. Whitcup M.D. Toshimichi Shinohara Ph.D. Staff Medical Officer, LI, NEI Head, Section on Molecular Biology, LRCMB, NEI Objectives The program is designed to evaluate the presence and localization of various cytokines and their mRNAs as well as ocular antigens and enzymes in normal and diseased eyes from animals and/or humans. Methods Immunohistochemistry and in situ hybridization techniques are applied to evaluate the ocular tissues on slides. The amounts of cytokines in the ocular fluids (aqueous humor and vitreous) also are ana- lyzed. Intraocular injection and/or systemic injection of cytokines is used to study their direct effects on the eye. Major Findings Interleukin 4 (IL-4) mRNA is found in the conjuncti- va of patients with ocular onchocerciasis. This fmding suggests that Th-2 helper T cells play an immunopathological role in this parasitic disease. S-antigen (S-Ag) and its mRNA are found not only in the retina but also in the ocular tissues arising from neurocrest in the fetus. S-Ag mRNA is detected in the iris of some uveitic patients who have undergone prolonged corticosteroid treamient. The observation is confirmed via animal studies. Significance to Biomedical Research and the Program of the Institute Cytokine functions are complex, but the analysis of a relatively simple regulatory network suggests that activities determined in vitro are highly relevant to physiological and immunological functions as well as the pathogenesis in vivo. In the future, pharmacolog- ically switching different cytokines, antigens, surface markers (eg, adhesion molecules), and enzymes on and/or off may be useful in the therapeutic interven- tion of ocular disorders. The side effects of corticosteroids include cataract formation and glaucoma. These adverse effects may be induced by the aberrant release of signals, such as S-Ag on the trabecular meshworic and lens. Proposed Course Ocular tissue and cultured cells wiU be evaluated. NEI Research Program Retinal and Choroidal Diseases — Inflanunatory Disorders Publications Chan C-C, Li Q, Kikuchi T, Shinohara T, Nussen- blatt RB: Enhancement of S-antigen and its mRNA in the irises from uveitic patients. J Autoimmunity 5:719-732, 1992. Fujino Y, Li Q, Chung H, Hikita N, Gery I, Nussen- blatt RB, Chan C-C: Immunopathology of experi- mental autoimmune uveoretinitis in primates. Autoimmunity, in press. Grossman S, Lopez JS, Shah DN, Rubin B, Altman D, Reznik R, Testa D, Feldman J, Chan C-C, Nussenblatt RB: Inhibition of endotoxin-induced uveitis (EIU) in Lewis rats by AA-313. Invest Ophthalmol Vis Sci 33(4):933, 1992. Kasner L, Chan C-C, CordeUa-Miele E, Gery I: The effect of chlorpromazine on endotoxin-induced uveitis in the Lewis rat. Curr Eye Res, in press. Lopez JS, Li Q, Caspi RR, Nussenblatt RB, Kador P, Chan C-C: Use of oral CGS- 13080 to suppress the development of experimental autoimmune uveitis in the Lewis rat. Invest Ophthalmol Vis Sci 33(4):933, 1992. Ni M, Yamaki K, Kikuchi T, Ferrick M, Shinohara T, Nussenblatt RB, Chan C-C: Development expression of S-antigen in fetal human and rat eye. Curr Eye Res 11:219-229, 1992. Whitcup SM, Chan C-C, Li Q, Nussenblatt RB: Expression of ceU adhesion molecules in posterior uveitis. Arch Ophthalmol 110:662-666,1992. Whitcup SM, Wakefield D, Li Q, Nussenblatt RB, Chan C-C: Endothelial leukocyte adhesion mole- cule- 1 in endotoxin induced uveitis. Invest Oph- thalmol Vis Sci 33:2626-2630, 1992. 67 DEPARTMEtfT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00232-07 LI PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (SO characters or less. Title must fit on one line between the borders.) Interferon System in Cellular Function and Disease PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute altiliatlon) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: Caroline Percopo Chandrasekharam Nagineni M. Cristina Martins M.S. Biologist LI, NEI Ph.D. Visiting Scientist LI, NEI M.D. Guest Researcher 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: 0.65 PROFESSIONAL: 0.40 0.25 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.) Cytokines, such as interferon-gamma (IFN-y) and interleukin 2 (IL-2), are a group of specialized hormone-like proteins that 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 microenvironment. We have shown that IFN-y and IL-2 are found within the inflamed eye in association with T-cell infiltration and major histocompatibUity 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 antigens 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 ceU transplantation. Retinoblastoma cells arc an important model for exploring human malignancy and differentiation. Using these cells, we have shown that IFN-y can regulate MHC class I 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 I and n antigen expression may serve as a local amplification system in autoimmune and inflammatory eye disease. A better imderstandkig 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. 68 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Immunology 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 fonnation in human amnion (WISH) cells. IFNs were characterized by neutraliza- tion 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 blot assays. We used analytical flow cytometry to quantitate retinal pro- teins. We are using gene transcription techniques, such as Northern blot analysis and nuclear run-off transcription assays, to evaluate EFN-y modulation of retinal proteins. Major Findings IFN activation of retinal pigment epithelial (RPE) cells. — ^Numerous studies indicate that a variety of autoimmune diseases are associated with the IFN-y- induced tissue-specific expression of major histocom- patibility complex (MHC) class II molecules. During the past 5 years we 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 ceU in the retina, the RPE cell, is capable of expressing MHC class II detenninants. We can detect IFN-Y in situ as well as MHC class Il-positive RPE cells in retinas from patients with inflammatory eye diseases. In addition, freshly isolated human RPE cells can express these detenninants following treatment with IFN-y. In animal model systems, we found that inoculation of recombinant DFN-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 play an important role in ocular immimity, acting as a resident antigen-presenting cell in the retina. During the past year we provided the most recent piece of experimental evidence indicating a role for cytokine-activated RPE cells in autoimmune phenom- ena by showing that the RPE ceU is capable of producing the cytokine IL-6. The RPE cell cultures were established from human donor eyes. The isolated RPE ceUs alone did not produce IL-6. However, IFN-y induced these ceUs to produce IL-6 in a dose-dependent manner. Moreover, IFN-y can synergize with tumor necrosis factor (TNF) to produce IL-6 in human RPE cells. IL-6, a potent cytokine, can act on B lymphocytes to induce growth and antibody production. It also can act on T lym- phocytes 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. It may have major immuno- logical consequences for RPE cell transplantation studies. Cytoldne-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 expression 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. The MHC class II molecules were augmented by IFN-y, but by neither IFN-a nor TNF. The neu- ronal maricers interphotoreceptor retinoid-binding protein (IRBP) and photoreceptor protein (PR-6), the glial-like maricer 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 uitknown. We have therefore initiated studies to compare IFN- a, IFN-p, and IFN-y in their abUity to induce the expression of class I antigens and to investigate the role of transcriptional and posttranscriptional mecha- nisms in this induction. We found that IFN-y in- creases HLA-class I antigen expression and induces a 5-fold increase in its transcription rate. Posttrans- 69 Laboratory of Immunology NEI Annual Report-FY 1992 criptionally, IFN-P and IFN-y increased steady-state mRNA for the HLA-B7 gene. These effects were not associated with downregulation of N-myc oncogene nuclear transcription. Moreover, dexamethasone did not affect the IFN-y-induced expression of HLA-class I molecules. These studies demonstrate that both transcriptional and posttranscriptional mechanisms are implicated in the modulation of class 1 molecule expression by IFNs. Significance to Biomedical Research and the Program of tlie Institute These studies highlight the fact that the release of cytokines, such as IFN-y, within the ocular micro- environment and the subsequent induction of cjlo- kines and MHC class I and class II antigen expres- sion on resident and infiltrating cells may be critical elements in a cascading effect leading to ocular ceU destruction. Within the retina, the ceU that may play a critical role in autoimmune uveitis is the RPE ceU. in^-y-induced RPE ceU activation may participate in autoimmune disease in the ocular microenvironment. Cytokines produced and localized in the eye may play a critical role in normal physiology, pathogenic mechanisms, and therapeutic approaches. Because the RPE ceU is a pivotal regulatory cell in the retina, an imderstanding 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 autoimmunity and inflammatioa 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 to 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 histo- compatibility complex (MHC) class I genes in human retinoblastoma cells by interferons: Evi- dence for both transcriptional and post-transcrip- tional regulation. Invest Ophthalmol Vis Sci, in press. Detrick B, Hooks JJ: Autoimmune aspects of ocular disease, in Rose N, MacKay I (eds): The Auto- immune Diseases - II. New Yoric, Academic Press, 1992, pp 345-361. Hooks JJ, Detrick B: Evaluation of the interferon system, in Rose N, DeMacario EC, Fahey J, Friedman H, Penn G (eds): Manual of Clinical Laboratory Immunology, ed 4. Washington, DC, ASM PubUcations, 1992, pp 240-244. 70 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00233-07 LI PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Studies on the Bioregulatory Aspects of the Retinal Pigment Epithelial Cell PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute attiliationj PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: Chandrasekharam Nagineni Caroline Percopo Christian Hamel T. Michael Redmond Ph.D. Visiting Scientist LLNEI M.S. Biologist LI, NEI M.D. LLNEI Ph.D. LI, NEI COOPERATING UNITS (il any) Hopital St. Louis, Paris, France (Lawrence Boumsell, M.D.); University of Nice, France (Alain Bernard, M.D.); National Institute of Dental Research, NIH (Reuben Siraganian, M.D.); The Johns Hopkins University (Stanley A. Vinores, Ph.D.; Peter Campochiaro, M.D.); Depaitment of Pathology, The George Washington University Medical Center (Barbara Detrick, Ph.D.) LAB/BRANCH Laboratory of Immimology SECTION Section on Immunology and Virology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.25 PROFESSIONAL: 1.05 0.20 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 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 ceUs. Because the monoclonal antibodies detect epitopes present solely on RPE cells, they provide us with the unique opportunity to evaluate a variety of aspects of RPE ceU development and function. Studies on RPE cell development indicate that the epitopes appear only after the ceUs have begun terminal differentiation. Moreover, studies on RPE migration also 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. We have isolated a cDNA clone that codes for a protein which matches no other sec^uence in the data bases. Studies are also 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 ceUs 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 RPE ceU responses. Moreover, they provide the framework to evaluate RPE cell transplantation. 71 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report-FY 1992 Project Description Objectives The aim of this project is to evaluate the molecular, biochemical, and varied biologic properties of retinal pigment epithelium (RPE) cells in normal and disease states. Moreover, we are evaluating RPE cell transplantation. Methods RPE ceUs were isolated and propagated in vitro. We generated monoclonal antibodies in mice by fusing mouse spleen ceUs with myeloma cells. Antibodies to RPE cells were evaluated by immunoperoxidase assays and by Western blot assays. The effect of drugs and cytokines are evaluated by cell viability 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 human to frog. Whereas these antibodies detect epitopes present solely on RPE ceUs, they provide us the unique opportunity to evaluate a variety of aspects of RPE cell development and function. Electron microscopic inununocytochemistry revealed very similar labeling patterns for the two RPE antibodies. In human eyes, staining was local- ized to surface and intracellular membranes and the cytoplasm. Staining occurred predominantly on the apical surface of the RPE cells. The RPE protein is a 65-kD protein. We isolated this protein by poly- acrylamide gel electrophoresis, transferred it to nitrocellulose blot, and determined the sequence of amino acid residues. 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 protein does not match any other sequence in the data bases. The protein ex- pressed in Escherichia coli has a molecular weight similar to that of the native protein. Northern blotting with the cDNA detected protein mRNA in RPE cells. We have studied the developmental expression of RPE and photoreceptor determinants in the rat retina. We had previously shown that the expression of this detenninant in rats is absent the day of birth but detectable at postnatal day 6. Recent studies show that RPE cells express their determinants shortly before the first outer segment formation matches a similar progression of the expression of the RPE detemiinants. 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 retinal architecture in selected retinal degenerations. It is essential to develop methods for large-scale preparations of RPE ceU cultures for somatic cell genetic engineering manipulations. We are evaluating various parameters for human and rat RPE cell culture and transplantation. Preliminary studies show that we can successfully inoculate human RPE ceUs into the rat retina. Studies are in progress to evaluate the immunologic parameters of this transplantation process. Significance to Biomedical Research and the Program of the Institute The monoclonal antibodies developed in this study are the first directed solely at the RPE ceU. These antibodies are potentially useftil in the identification of RPE cells in situ and in vitro. These antibodies, which can be used to monitor RPE cellular functions, may be used in providing a better understanding of the role of RPE cells in retinal degenerative dis- orders. RPE cell transplantation to correct retinal degenerative processes is being actively investigated in a number of laboratories. The studies reported here provide the fi^mework to evaluate RPE cell transplantation. Proposed Course We will continue to characterize these antibodies as well as the effect of these antibodies on cell function in vivo and in vitro. We will isolate, propagate, and characterize RPE cells for transplantation studies in animals and human. We will design effective ways to maintain the cell in culture and design ways to measure and monitor cell function. 72 NEI Annual Report-FY 1992 Laboratory of Immunology NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Hamel CP, Tsilou E, Harris E, Pfeffer BA, Hooks JJ, Detrick B, Redmond TM: Characterization of orpenin, a developmentally regulated protein specific for pigment epithelium of the vertebrate retina. / Neurosci, in press. Vinores SA, Orman W, Hooks JJ, Detrick B, Campochiaro PA: Ultrastructural locaUzation of RPE associated epitopes recognized by mono- clonal antibodies in human RPE and their induc- tion in human fibroblasts by vitreous. Graefes Arch Klin Ophthalmol, in press. 73 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00240-06 LI PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Virus Infections in the Eye PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute afliliation) PI: John J. Hooks Ph.D. Head, Section on LI, NEI Immunology and Virology Others: Caroline Percopo M.S. Biologist LI, NEI Yun Wang M.D. Guest Worker LI, NEI Miguel Bumier M.D. Visiting Scientist LI, NEI Ingeborg Kirch M.D. Guest Worker LI, NEI COOPERATING UNITS (If any) Department of Pathology, The George Washingtoti University Medical Center (Barbara Detrick, Ph.D.); Department of Pathology, Unifonned Services University for Health Sciences (Katherine Hobnes, Ph.D.); Department of Ophthalmology, Ruprecht-Karl's University, Heidelberg, Gennany (Ellen Kraus- Mackiw, M.D.); Laboratory of Biology. NCI, NIH (Charles H. Evans, M.D.. Ph-D.) LAB/BRANCH Laboratory of Immunology Section of Immunology and Virology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 0.8 PROFESSIONAL: 0.5 0.3 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects [3 (b) Human tissues □ (c) Neither □ (a1) Minors □ (a2) Interviews 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 three areas: (1) coronavirus infection in ocular and optic nerve cells; (2) the possible roles of viruses in human diseases; and (3) antiviral therapeutic actions of cytokines and drugs. A fourth area of study initiated during the past year is the evaluation of molecular diagnosis and pathogenesis of cytomegalovirus infections in man. We have established 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 inflanmiation. Initial retinal damage is followed by clearance of the virus and progressive retinal degeneration, even months after the virus is gone. In vitro studies and electron microscopic evaluation of in situ infection indicate that the virus replicates predominantly in MiiUer cells and can also be detected in retinal pigment epithelium (RPE) and photoreceptor cells. These results demonstrate that the virus can induce biochemical and morphological changes in the retina that persist and progress long after the virus is detectable. This disease may be considered a model for degenerative diseases of the pigment epithelium and photoreceptors in humans. The need for effective drug treatment and prevention of herpes virus and other viral diseases has assimied growing importance. We found that leukoregulin, a natur^y occurring immunologic cytokine, not only increases the antiviral actions of the drug acyclovir but also directly inhibits herpes simplex virus replication. These findings, which demonstrate that combination immunotherapy and chemotherapy can produce substantial inhibition of herpes virus replication, provide a rationale for the application of this approach to the treatment of virus infections. 74 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Immunology Project Description Objectives This project was designed to detemiine 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 in vitro and in vivo, as weU as immunocytochemical analysis of infected cells and tissues. Techniques used in characterizing virus infection include Western blot analysis, Northern blot analysis of HSV thymidine kinase, and in situ hybridization. Techniques used in the characterization of antivirus antibodies include enzyme-linked immunosorbent assay and neutraliza- tion assays. Major Findings Coronavirus infection in the eye. — The murine coronavirus, mouse hepatitis virus (MHV), JHM strain, induces a retinal degenerative disease in adult Balb/c mice. The disease consists of an acute phase lasting 2-8 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 photoreceptor layer, loss of interphotoreceptor retinoid-binding protein, and retinal detachments. This model provides evidence that viruses can indeed trigger retinal degenerative processes and may provide insight into pathogenic mechanisms in retinal degenerative diseases of maa Coronaviruses in general and the murine MHV, JHM strain in partictilar, induce a number of intrigu- ing pathologic processes. Central nervous system involvement following JHM virus iirfections can consist of acute infections or chronic demyelinating disease. The acute infection is associated with viral replication in selected cells while chronic disease is associated with both virus replication and immune reactivity. Many of the steps involved in the patho- genic processes of these infections have been eluci- dated by in vitro studies. Some of the studies have shown that virus pathogenesis is associated with infection of specific cell typ)es, such as glial cells, neurons, and oligodendrocytes. However, viral pathogenesis has also been associated with ceU-to- ceU interactions, release of cytokines, and the state of differentiation of the cells. The in vivo interactions occurring in the retina are complex and difficult to assess. During the past year we evaluated the cellular basis of JHM virus retinal tropism. We prepared retinal cultures and RPE-retinal mixed cell cultures from eyes obtained from Balb/c mice. We evaluated the ability of JHM virus to infect and repUcate in these retinal cultures by light micros- copy, immunofluorescent staining, electron micros- copy, and virus isolation. Retinal cultures alone failed to support JHM vims replication. No cyto- pathology was observed and no virus could be detected in supernatant fluid, suggesting that cell-to- cell interactions may be critical because virus parti- cles and virus antigens could be seen in vivo within the neural retina and the RPE. In contrast to the retinal cultures, RPE-retinal mixed cultures were supportive of JHM virus replica- tion. Syncytial cytopathology was observed for the first 4 days, and virus was isolated from supernatant fluids. By electron microscopy, we found intracellu- lar virus within vacuoles and extracellular virus at the plasma membrane. After day 4, a persistent virus infection was established in which cells produced virus for 5 weeks without cytopathic effects or cell death. Double-labeling immunofluorescent studies of RPE-retinal mixed cultures showed the virus coex- pressed with a MiiUer cell marker, glutamine synthe- tase. This cell is the most prominent glial element in the retina. These studies demonstrate that JHM virus is capable of establishing a persistent virus infection in RPE-retinal mixed (MiiUer) cell cultures. More- over, these data suggest that cell-to-ceU interactions influence the establishment of coronavirus infections in the retina. Preliminary studies indicate that the genetic background of the host can actually determine susceptibility to coronavirus-induced retinal degener- ative disease. Moreover, preliminary studies also indicate that the pathogenesis of retinal degeneration may be associated with the development of antireti- nal autoantibodies. Thus, both genetic and immuno- logic factors may contribute to the pathogenic process. In summary, this model is characterized by the replication of JHM virus in the retina, producing an 75 Laboratory of Immunology NEI Annual Report-FY 1992 acute necrotizing disease of the sensory retina and resulting in only a mild inflammatory response but a longlasting disease (over 14 weeks). These studies indicate that a progressive degenerative disease in the retina may be initiated by an acute virus infection in the absence of major inflammatory response. Possible role of viruses in human eye disease. — 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 collect- ing serum samples and ocular tissue in order to use seroepidemiologic approaches to detect virus and viral antigens via immunocytochemical staining, in situ hybridization, and polymerase chain reaction assays. Antiviral therapeutic actions of cytokines and drugs. — ^The need for effective treatment and preven- tion of heipes virus and other viral diseases has grown in importance during the last 10 years. The development of targeted antiviral agents through combination therapy is becoming an important strategy. One of these strategies consists of the development of cytokines or lymphokines combined with chemotherapy 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 on a variety of cell func- tions. Leukoregulin is a lymphokine that performs unique regulatory functions in transformed ceUs. This molecule, produced by a variety of lymphoid cells, has been shown to be a multifunctional cytokine. It can prevent chemical carcinogen transformation, inhibit neoplastic cell proliferation, and augment target cell sensitivity to natural killer cell cytotox- icity. Furthennore, this cytokine has been shown to increase membrane permeability of tumor cells and to increase drug uptake in these ceUs. Recently we showed that leukoregulin can selectively increase membrane permeability in HSV-1 -infected cells but not in normal (ie, uninfected) cells. The increase in membrane permeability attribut- able to leukoregulin is associated with an increase in the antiviral actions of ACV. In these studies, the virus was added to the cell cultures for a 2-hour incubation after which the cjtokine and/or antiviral drug was added for 3 hours. The drugs and cytokine were removed, and the production of virus at 24 and 72 hours was evaluated. Under these conditions, the infected ceUs were exposed to the cytokine or drug for only 3 hours early in the replication cycle. Although short-terai exposure of the cells to leuko- regulin alone did not alter HSV-1 replication, it dramatically augmented the antiviral actions of ACV. During the past year we characterized the anti- HSV-1 actions of leukoregulin. The continuous presence of leukoregulin during the replication of HSV-1 significantly decreases the production of new virus and the translation of HSV-1 -specific proteins. Human amnion 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. Virus rephcation was evaluated by plaque assays while virus and cellular protein expression was tested by immunoblot assays. The continuous presence of leukoregulin (0.1 unitAnL) inhibited HSV-1 plaque formation by 50% and 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 cell protein actin was not affected by leukoregulin treatment. Moreover, leukoregulin treatment did not alter the ability of the cells to incorporate tritiated thymidine. 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 demonstrate that combination immunotherapy (cytokines) and chemo- therapy can produce substantial inhibition of herpes virus replication, thus providing rationale for the application of this approach to the interventive treatment of virus infections. Significance to Biomedical Research and the Program of the Institute Elucidating the factors involved in viral spread and pathogenesis wiU aid better imderstanding of diseases of viral etiology. We have established a new virus model for retinal degenerative processes in adult animals. This model, which has many unique fea- tures, is capable of inducing an acute infection in the presence of mild retinal vascular inflammation. The initial retinal damage is followed by clearance of the virus and progressive retinal destruction, even months after the virus is gone. This model should 76 NEI Annual Report-FY 1992 Laboratory of Immunology assist us in understanding the pathogenesis of select- ed human diseases of unknown etiology. We have identified leukoregulin, a cytokine that selectively increases membrane permeability in virus- infected cells. We also have shown that combined cytokine and drug therapy can substantially inhibit herpes simplex virus replication. Moreover, the continuous presence of the cytokine can directly inhibit virus replication. The data from these studies provides rationale for the application of this approach to the interventive treatment of virus infections. Proposed Course This project wiU continue several lines of investiga- tion. 1. We will evaluate coronavirus infections of the eye. Ocular tissue wiU be evaluated by electron microscopy. We will study the ability of the virus infection to potentiate autoimmune phenomena and correlate the data obtained 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 wiU 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 Hooks JJ, Detrick B, Evans CH: Leukoregulin, a novel cytokine, enhances the anti -herpesvirus activity of Acyclovir. Clin Immunol Immuno- pathol 60:244-253, 1991. Robbins SG, Wiggert B, Kutty GA, Chader GJ, Detrick B, Hooks JJ: Redistribution and reduction of IRBP during ocular coronavirus infection. Invest Ophthalmol Vis Sci 33:60-67, 1992. Wang Y, Detrick B, Hooks JJ: Coronavirus replica- tion within the retina: Analysis of cell tropism in muse retinal ceU cultures. Virology, in press. 77 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00277-01 LI PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or lass. Title must tit or> one line between the borders.) Role of Retinal Pigment Epithelium in Retinal Disorders PRINCIPAL INVESTIGATOR (List other prolessional 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 'ing the T-cell receptor (TCR) genes of these lines and clones on tlie molecular level. The data indicate that TCR variable region gene usage in uveitis differs from that reported for other autoim- mune diseases and may be more heterogeneous. On the basis of in vitro and in vivo results, TCR Vp8.2 appears to be a pathogenic clonot>pe in S-Ag-EAU, whereas TCR \'p8.3 may be one of the pathogenic clonot>pes in IRBP-EAU. These findings impact on the development of therapeutic strategies that would specifically target the pathogenic cells via their TCR. We studied the importance of "nonspecific" T-C€ll recruitment in the immunopathogenesis of uveitis, using congenitally athN-mic Lewis rats (LEW.mu/mu) deficient in functional endogenous T cells, but otherwise SNTigeneic with the euthj-mic Lewis rats that develop characteristically severe EAU. The uveitogenic stimulus was delivered in the form of phenot>pically and functionally homogeneous patho- genic T-cell lines, specific to the major pathogenic epitope of either the intracellular photoreceptor protein, S-Ag, or the extracellular photoreceptor matrix protein, IRBP. Depending on the T-cell line used, EAU in ath>Tnic rats was eitl^r drastically reduced in se\'erity or absent. Susceptibilit>' was restored when the athNinic animals were reconstituted with immunocompetent T cells from s>Tigeneic euthNinic donors. Our results indic;ite that recruited nonspecific T cells play a major role in the pathogenesis of disease and suggest that the extent of dependence on this phenomenon may be influenced by die antigenic specificity- of the T-cell line. This is the first direct demonstration that recruitable nonspecific T h-mpho- CNtes are necessan- for the expression of the disease itself. This finding impacts upon the development of immunotherapeutic strategies, which must take into accoimt the magnitude of the recruitment phenome- non. In collaboration with Drs. Chi-Chao Chan and Scott WTiitcup, we ha\'e shown that adhesion mole- cules are expressed on the ocular vasculamre before the onset and during the active phase of EAU in the mouse model. Moreover, these molecules have a functional significance, because anti-adhesion mole- cule therapy with monoclonal antibodies to ICAM-1 and LFA-1 is able to suppress induction of EAU. Anti-adhesion molecule therapy is one of the means by which recruitment of nonspecific cells from the circulation can be controlled. This finding has potential clinical significance, because the same adhesion molecules are expressed in the human disease ae well. Studies of genetic control of susceptibility and resistance to uveitis, using various mouse strains, were continued and extended. In experiments using congenic and intra-H-2 recombinant strains, we showed that expression of EAU requires both a genetically susceptible major histocompatibility complex class n region, and a "permissive" genetic background. One of the genetic factors determining the "permissiveness" or "nonpermissiveness" of the genetic background was shown to be connected to the regulation of the lymphokine IFN-y. Neutraliza- tion of endogenously produced IFN-y abrogated EAU resistance in one low-responder strain of mice. Additional resistance mechanisms, such as the influence of the genetically determined T-cell reper- toire, which affects recognition of pathogenic epi- topes, are now being studied. In collaboration with Dr. Wayne Streilein (Uni- versity of Miami, PL), we are exploiting the mouse EAU model for the study of immunologically in- duced suppression of ocular autoimmunity by a phenomenon known as anterior chamber-mediated immune dcNiation (ACAID). We induced protection against EAU by pretreating the animals with the uveitogen IRBP, injecting it in the anterior chamber of the eye before the uveitogenic challenge. The protecti>'e effect was found to reside in a splenic population of T cells that could be isolated and could transfer the protection to unmanipulated hosts. Adoptive transfer of these ACAID-induced suppres- sor cells aborted disease in a previously immunized host, even when pathological signs were already present We also showed that ACAID-like protection from EAU can be induced not only by injection of IRBP into the anterior chamber, but also by infusion of monocne/macrophages of the F4/80 phenotype that had been pretreated in Nitro with IRBP in the presence of supematants from immune-privileged sites (, amniotic fluid, aqueous humor). These studies. 92 NEI Annual Report-FY 1992 Laboratory of Immunology which contribute to the understanding of the natural mechanisms that protect from ocular autoimmunity, might be exploited in the future for the development of clinically relevant strategies for immunomodula- tion. 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 identification and character- ization of the cells involved in ocular autoimmunity, and of their functions, will provide new understand- ing of inflammatory ocular diseases. Successful immimomodulation of EAU in animal models has thus far usually served as a good predictor of the clinical success of a given therapeutic modality. The continued study of basic mechanisms involved in the immimopathogenesis of uveitis in animal models will aid in the development of novel immunotherapeutic approaches for the control of uveitis in humans. Proposed Course This project wiU 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 Beraud E, Kotake S, Caspi RR, Oddo S, Chan C-C, Gery I, Nussenblatt RB: Control of experimental autoimmune uveitis (EAU) by low dose T cell vaccinatioa Cell Immunol 140:112-122, 1992. Caspi RR: Eye, autoimmune disease, in Roitt IM, Delves PJ (eds): Encyclopaedia of Immunology. San Diego, Academic Press, 1992, vol 2, pp 535- 537. Caspi RR: Immunogenetic aspects of clinical and experimental uveitis. Reg Immunol 1992, in press. Caspi RR, Chan C-C, Fujino Y, Oddo S, Najafian F, Bahmanyar S, Wilder RL, Wiggert B: Genetic factors in susceptibility and resistance to experi- mental autoimmune uveoretinitis. Curr Eye Res ll(suppl):81-86, 1992. Caspi RR, Chan C-C, Grubbs BG, Chader GJ, Wiggert B: Genetic control of susceptibility to experimental autoimmune uveoretinitis in the mouse model: Concomitant regulation by MHC and non-MHC genes. J Immunol 148:2384-2389, 1992. Caspi RR, Nussenblatt RB: Natural and therapeutic control of ocular autoimmunity — rodent and man, in Roitt I, Kazatchkine M (eds): Autoimmunity. New Yoric, Wiley and Sons, Inc., in press. de Smet MD, Dayan M, Caspi R, Hafler DA, Gery I, Nussenblatt RB: Determination of the precursor frequency of response to S-Ag in uveitis patients and controls. Invest Ophthalmol Vis Sci 33(suppl):930, 1992. DeBarge LR, Chan C-C, Caspi RR, Haming R, Nussenblatt RB, Whitcup SM: Expression of cell adhesion molecules in mice with experimental autoiimnune uveitis. Invest Ophthalmol Vis Sci 33(suppl):796, 1992. Egwuagu CE, Bahmanyar S, Mahdi RM, Brezin AP, Nussenblatt RB, Gery I, Caspi RR: Predominant usage of Vp8.3 gene in a T cell receptor in a T cell line that induces experimental autoimmune uveoretinitis. Clin Immunol Immunopathol 65:152-160, 1992. Egwuagu CE, Beraud E, Chow C, Caspi R, Mahdi R, Brezin AP, Nussenblatt RB, Gery I: T ceU receptor p-chain usage in experimental auto- immune uveoretinitis. J Autoimmunity 4:315-324, 1991. Hara Y, Caspi R, Streilein JW: Analysis of an in vitro created ACAID-inducing signal that prevents IRBP-specific experimental autoimmune uveitis. Invest Ophthalmol Vis Sci 33(suppl):1025, 1992. Hara Y, Caspi RR, Wiggen B, Chan C-C, WHbanks GA, StreUein JW: Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber associated immune deviation with interphotoreceptor retinoid binding protein. J Immunol 148:1685-1692, 1992. Hara Y, Caspi RR, Wiggen B, Dorf M, Streilein JW: Analysis of the ACAID-inducing signal that induces systemic immune deviation similar to that elicited by antigen injected in the anterior cham- ber of the eye. J Immunol 149:1524-1530, 1992. 93 Laboratory of Immunology NEI Annual Report-FY 1992 Helbig H, Thurau SR, Nussenblatt RB, Caspi RR: [Active immunoregulation by cells of the ciliary body]. Fortschr Ophthalmol 88:299-303, 1991. Li Q, Fujino Y, Caspi RR, Najafian F, Nussenblatt RB, Chan C-C: Association between mast cells and development of experimental autoimmune uveitis in different rat strains. Clin Immunol Immunopathol 65:294-299, 1992. Lopez JS, Li Q, Caspi RR, Nussenblatt RB, Kador P, Chan C-C: Use of oral CGS- 13080 to suppress the development of experimental autoimmune uveitis in the Lewis rat. Invest Ophthalmol Vis Sci 33(suppl):933, 1992. Mahdi RM, Caspi RR, Tsai L, Nussenblatt RB, Gery I, Egwuagu CE: T cell receptor Vp gene usage in experimental autoimmune uveoretinitis (EAU). Invest Ophthalmol Vis Sci 33(suppl):1024, 1992. Mastorakos G, Silver PB, Bouzas EA, Caspi RR, Chan C-C, Chrousos GP: Immunoreactive cortico- tropin-releasing hormone in experimental uveitis. Invest Ophthalmol Vis Sci 33(suppl):933, 1992. Roberge FG, Caspi RR, Chan C-C, Nussenblatt RB: Inhibition of T lymphocyte proliferation by retinal glial MiiUer ceUs: Reversal of inhibition by gluco- corticoid. J Autoimmunity 4:307-314, 1991. Rubin BI, Najafian F, Caspi RR, Sehgal SN, Nussen- blatt RB: Synergistic interactions of cyclosporine and rapamycin to inhibit EAU in the Lewis rat. Invest Ophthalmol Vis Sc' 33(suppl):934, 1992. Savion S, Grover S, Kawano Y, Caspi RR: Uveito- genicity of T cell lines in the rat does not corre- late with their production of TNF, IFN-y or IL-3. Invest Ophthalmol Vis Sci 33(suppl):932, 1992. Thurau SR, Caspi RR, Chan C-C, Weiner HL, Nussenblatt RB: [Immunologic suppression of experimental autoimmune uveitis]. Fortschr Ophthalmol 88:404^07, 1991. 94 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00258-04 LI PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 charaaars or lass. Title must tit on one line between the borders.) Experimental Autoimmune Uveitis in the Mouse PRINCIPAL INVESTIGATOR (Usi other prolesslonal personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Rachel R. Caspi Ph.D. Visiiing Scienlist LI, NEI Sua Bahmanyar Chi-Chao Chan Shlomo Grossman Sujata Grover Bairy Grubbs Martha Kirfoy Reza Mozayeni Benjamin Rubin Phyllis Silver M.D. Staff Fellow M.D. Medical Officer Ph-D. Special Volunteer M.S. Biologist B.S. Biologist M.S. Biologist B.S. Biologist M.D. Staff Fellow B.S. Biologist LI, NEI LI, NEI LI, NEI LI. NEI LI, NEI LI. NEI LI. NEI LI. NEI LI. NEI COOPERATING UNITS (If any) Laboratory of Immunobiology, Rega Instituut, Katholieke Universiteit Leuwen, Belgium (A. Billiau, M.D.); Department of Microbiology and Immunology, University of Miami School of Medicine (Wayne Streilein, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI. NIH. Bethesda, MP 20892 TOTAL .STAFF YEARS: 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews PROFESSIONAL: 0.0 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 as a separate entity. Further work in the mouse EAU model has been folded into project number ZOl EY 00184-10 LI. 95 PHS 6040 (Rev. 5«2) OEPARTMErn* OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00271-02 LI PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or /ass. Title must til on or\e line between the borders.) The Evaluation of the Antiflammins in the Anterior Uveitis PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Chi-Chao Chan M.D. Medical Officer LI, NEI Others: Robert B. Nussenblatt M.D. Qinical Director Juan S. Lopez M.D. Visiting Fellow Susan Mellow RJ^. Nurse Specialist NEI LI. NEI LI, NEI COOPERATING UNITS (If any) LAB/BRANCH Laboratory of Immimology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI. NIH. Bethesda, MP 20892 TOTAL STAFF YEARS: 0.0 PROFESSIONAL: 0.0 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.) This project has been terminated. 96 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00218-07 LI PERIOD COVERED October 1. 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Ocular Manifestations of the Acquired Immune Deficiency Syndrome PRINCIPAL INVESTIGATOR (List other prolesslonal personnel below the Principal Investigator.) (Name, title, laboratory, and institute altillallon) PI- Marc D. de Smet M.D. Visiting Scienlisl UNEl Omers: Robert B. Nussenblatt M.D. Clm«:al Director ^ Juan Lopez M.D. Visiting Fellow U. ^ ScouXtcup M.D. Senior Staff gjlow U NH Margaret ChetBtg M.D. Senior Staff Feow U. NH David Paries M.D. Senior Staff Feow L . NH Richard ftnton M.D. Senior Staff Feow U, NH Dan Martin M.D. Senior Staff Fellow L . NE Raymond DeBarge M.D. Senior Suff Fellow U. NE Miguel Bumier M.D. Visitrng SaenUst L . NE ■r^ •* _._T^-L 11* n Viciiinp Scientist LI, Nbl Francois Roberge Nia Visiting Sciennst COOPERATING UNITS ^/A any; » . t^ x r i. . e Department of Critical Care Medicine. Qinical Center, NIH (Henry Masur, M.D.); Laboratory of Immunoregulation, National Institute of Allergy and Infectious Diseases, NIH (H. CUfford Lane, M.D.); Pediatric Branch, National Cancer Institute. NIH (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 PROFESSIONAL: 2.0 OTHER: 0.0 CHECK APPROPRIATE BOX(ES) (xl (a) Human subjects E (b) Human tissues D (c) Neither 13 (a1) Minors □ (a2) Interview's SUMMARY OF W/ORK (Use standard unreduced type. Do not exceed the space provided.) Patients suffering from AIDS are at risk of developing significant ocular problems either as a result of the human immunodeficiency virus (HIV) itself or as a result of opportiinistic infections. Some of these problems can lead to blindness if left untreated. Among tiie many pathogens that can lead to blindness, cytomegalovims (CMV) is by far the most common. In FY 1992 several approaches have been taken to investigate this disease. Some CMV retinitis studies focused on prevention. Early initiation of therapy is important for the preservation of useful vision. However, all the drugs available are virostatic, not virocidal; patients are required to take medication for life if they want to preserve their sight. If one could prevent the development of CMV retinitis, it would be considered a major advance. In tiiis regard, we have participated in a phase I trial of immune globulin in patients at risk for development of CMV retinitis. Other CMV retinitis studies focused on tiierapy. We have continued to evaluate the ability of foscamet and ganciclovir to prevent progression of CMV retinitis. Two particular questions evaluated were (1) the possible options in the treaonent of patients with end-stage CMV retinitis who have silicone oU in tiieir eyes foUowing retinal detachment and (2) die possibility of using a drug delivery system for the eye, which would obviate the need for systemic therapy. In stiidies of pediatiic MDS during FY 1992, we continued to evaluate the incidence of ocular infection in about 220 children with AIDS. The incidence of compUcations is lower in children tiian in adults. However, CMV retinitis in children is more difficult to treat. In a subpopulation of pediatric patients treated with 2',3'-dideoxyinosine (ddl), peripheral retinal changes suggesting drug toxicity have been observed and reported as our monitoring of these children continues. Otiier FY 1992 studies on AIDS include evaluation of a new antiviral dnig for the treatment of toxoplasmosis in AIDS patients' eyes. 97 PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report-FY 1992 Project Description Additional Personnel Susan Mellow R.N. Clinical Protocol Number 90-EI-208 Objectives The foremost objective of this project is to evaluate means of identifying and treating known ocular compUcations of AIDS, using novel approaches. A second, equally important objective is the identifica- tion of new manifestations of ocular involvement from the AIDS virus itself and related opportunistic ocular infections. The third objective is to recognize complications related to the therapeutic agents used. Methods This project entails the clinical evaluation, diagnosis, and treatment of retinitis in AIDS patients. It also involves the development of novel methods of therapy for the various forms of retinitis observed. Study of pathologic tissue is used to enable better understanding of the nature of infectious processes. Major Findings In the past year, our major effort was still centered on the evaluation and treatment of cytomegalovirus (CMV) retinitis. CMV is a major vision-threatening infection found in patients with AIDS. While early detection can lead to preservation of vision, it also commits the patient to lifelong intravenous therapy with anti-CMV drugs. Prevention of CMV retinitis in patients at risk would be a significant advance. Most AIDS patients develop CMV end-organ disease when their CMV immunoglobulin has dropped to a very low level. Administering exoge- nous anti-CMV hyperimmunoglobulin thus has the potential to prevent end-organ disease fit)m appear- ing. In collaboration with the National Institute of AUergy and Infectious Diseases, we have begun to evaluate the safety of administering anti-CMV hyperimmune globulin to patients at risk for CMV retinitis. To date, the treatment, given on a monthly basis, has been administered to eight patients. No adverse effects of this therapy have been noted. Recently a device that slowly releases gancyclovir directly into the eye has been described. This might be an interesting alternative to systemic therapy for patients who cannot tolerate intravenous infusions. We have developed a protocol that should allow us to evaluate the safety of the device. The protocol will test two dosage regimens: in one, the drug will be released over a 4-month period; in the other, it wiU be released over 8 months. The protocol, which has demanded considerable effort by several investiga- tors, is now ready for patient enrollment. We have continued to follow patients with CMV retinitis. Patients with advanced AIDS and a severe state of immunosuppression are often imable to tolerate gancyclovir, even with the use of G-CSF. In some of these patients who are unable to tolerate the renal toxicity of foscamet, we have had to revert to using intravitreal injections. One particular problem arises in patients with intraocular silicone oil follow- ing retinal detachment surgery, which occurs in up to 30% of patients with CMV retinitis. Silicone oil has been considered a contraindication to intravitreal injection. We have shown in two patients with silicone oil that intravitreal gancyclovir injections can be safely given, thus they are an effective means of arresting the progression of CMV retinitis. Other infections can cause decreased vision in patients with AIDS. The second most common pathogen in many parts of the world is toxoplas- mosis, a particularly difficult infection to treat, inasmuch as the most common drug, sulfacetamide, can cause a Stevens-Johnson-like syndrome. We have evaluated and demonstrated the efficacy of a new agent, 566C80, in the treatment of ocular toxoplas- mosis. It holds promise as an alternative to the medications now available. Finally, we have continued to follow about 220 children who developed AIDS by various means. Whereas the overall incidence of CMV is about 1.6%, we have been particularly struck by the lower incidence of CMV in this population. However, in children who have low total T-ceU counts (below 100 per cubic millimeter), the risk increases to 16%. Also, children who have acquired HIV in utero or shortly after birth are at increased risk of having strabismus. 98 NEI Annua! Report-FY 1992 Laboratory of Immunology 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. Prophylaxis would offer the best defense against costly and cumbersome lifelong therapy. In addition, an approach that could deliver a drug directly to the eye would be a valuable adjunct in those patients who are unable to tolerate systemic therapy. The number of infected children 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 thus continuing to follow these children prospectively to identify the frequency and type of ocular complications which they are likely to devel- op. Proposed Course Further studies on the ocular manifestations of AIDS are planned. We will continue to monitor pediatric patients. We will, in the course of Fiscal Year 1993, evaluate the intraocular device and study further the use of hyperimmune globulin as a means of prophy- laxis against CMV retinitis. We also wiU evaluate new anti-CMV drugs as they become available for clinical trials. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Butler KM, de Smet MD, Husson RN, MueUer B, Manjunath K, Montnella K, Lovato G, JaroUnski P, Nussenblatt RB, Pizzo PA: Treatment of aggressive CMV retinitis with ganciclovir in combination with foscamet in a child with human immunodeficiency virus infection. J Pediatr 120:483-486, 1992. de Smet MD: Differential diagnosis of retinitis and choroiditis in patients with acquired immunodefi- ciency syndrome. Am J Med 92(Suppl 24):S17-S21, 1992. de Smet MD, Butler KM, Rubin BI, Whitcup SM, Lopez J, DeBarge R, Martin D, Pizzo PA. Nussenblatt RB : The ocular complications of HIV in the pediatric population. Proceedings of the 3rd International Symposium on Uveitis. Brussels, Kluwer, 1992, in press. de Smet MD, Nussenblatt RB: Ocular manifestations of AIDS. JAMA 266:3019-3022. 1992. Lopez JS, de Smet MD, Masur H, MueUer BU, Pizzo PA, Nussenblatt RB: Orally administered 566C80 for treatment of ocular toxoplasmosis in a patient with the acquired immunodeficiency syndrome. Am J Ophthalmol 113:331-333, 1992. Whitcup SM, Butler KM, Caruso R, de Smet MD, Rubin B, Husson RN, Lopez JS, Belfort R Jr, Pizzo PA, Nussenblatt RB: Retinal toxicity in HIV-infected children treated with 2',3'-dideoxy- inosine (ddl). Am J Ophthalmol 113:1-7, 1992. 99 DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00266-03 LI PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must lit 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 alllliation) PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI Others: Igal Gery Robert B. Nussenblatt Margaret Cheimg Ph.D. Head, Section on LI, NEI Experimental Immunology M.D. Clinical Director NEI M.D. Senior Staff Fellow LI, NEI COOPERATING UNITS (it any) Department of Ophthalmology, Kurume University, Kurume, Japan (M. Mochizuki, M.D.); INSERM-86, Laboratory of Ocular Inununopathology, Paris, France (J.-P. Faure, Ph.D.); L'H6pital de la Piti6, Paris, France (P. LeHoang, M.D.); Hadassah Hospital, Jerusalem, Israel (D. Ben Ezra, M.D.). LAB/BRANCH Laboratory of Immunology SECTION Section on Immimoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.6 PROFESSIONAL: 0.6 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects n (a1) Minors □ (a2) Interviews [^ (b) Human tissues Q (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) One of the characteristics of soluble retinal antigen (S-Ag) is its 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 (APQ. In FY 1992, we further characterized the immunogenic and pathogenic sites of human S-Ag in the Lewis rat, using overlapping fragments of S-Ag, thus determining that there are several immunogenic sites in the S-Ag molecule as well as at least three immunopathogenic sites. Of these immunopathogenic sites, only one is immunodominant — a fragment located at sequence 341-360. The immunogenic sites for the Lewis rat do not correspond to the sites identified in the human. While most of the sites recognized by the rat immune system are clustered close to the C-terminal end of the molecule, in the human most of the activity is located in the N-terminal region. We also have tested the response of the immunodominant sequence, as well as the nondominant immunopathogenic sequences in several other rat strains in an attempt to determine whether factors within the major histocompatibility complex (MHQ class II as well as outside MHC class II may play a role in the establishment of an immunogenic and an immimopathogenic response. In human subjects, we have tested new means of establishing ceU lines by seeding multiple wells with S-Ag primed peripheral blood cells and maintaining the ceUs in culture for 14 days in medium emiched in T-cell growth factors. This approach yields a low number of cell lines which can then be further expanded by additional rounds of stimulation. In addition, this approach allows us to determine the precursor frequency of cells reactive to S-Ag in the peripheral blood of patients and controls. This precursor frequency, which is of the order of 1 in 10 million circulating cells in patients, is lower in controls. The ceU lines established using this technique wiU now be further characterized in terms of their response to fragments of human S-Ag as weU as their T-cell receptor usage. 100 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Immunology Project Description Additional Personnel Sumeet Mainigi Biologist, LI, NEI Molly Dayan B.Sc. Biologist, LI, NEI Gerald J. Chader Ph.D. Chief, LRCMB, NEI Barbara Wiggert Ph.D. Head, Section on Biochemistry, LRCMB, NEI Clinical Protocol Numbers 84-EI-214 79-EW9 Objectives In Fiscal Year 1992 the study has been aimed at mainly two areas: (1) a determination of the immu- nogenicity of the fragments of human S-antigen (S-Ag) in a defined experimental animal, the Lewis rat, and in other inbred rat strains that either use the same major histocompatibility complex (MHC) class II antigen or a different antigen, and (2) characteriza- tion of the immune response profile to S-Ag in patients with well-defined uveitic conditions, using standard proliferation assays as well as multiple short-term T-cell lines. Methods Overlapping synthetic peptides of human S-Ag, each consisting of 20 amino acids, were provided by Applied Biosystems (Foster Qty, CA). In the animal study, we immunized groups of animals with the various peptides, using conventional methods. The animals were examined daily for the appearance of uveitis. Between days 12 and 14, the cell-mediated immune response was determined in a standard in vitro proliferation assay. Several different rat strains were used for these studies. The strains were chosen on the basis of their class n usage. Certain strains shared the same class II antigen as the Lewis rat while others differed completely in the class n antigens they expressed. Lymphocytes obtained from the peripheral blood of patients with uveitis in various stages of activity were tested for their response to synthetic peptides in a standard microculture assay. Their response to bovine S-Ag was determined in the same assay. These results were correlated with responses found in normal volunteers provided by the NEI Eye Clinic. Short-term cell lines were established in a number of patients to determine the precursor frequency of reactive cells and to establish responsive cell lines. Cells were first pulsed with bovine S-Ag, then cultured in standard 96-weU microliter plates. The mediimi was changed frequently and enriched in T- ceU growth factors. In a few patients, this assay was repeated several times to determine the profile of response during and after inflammatory episodes. Major Findings Our studies in the Lewis rat have shown that, even in the experimental model, it is possible to generate an immunogenic response to multiple epitopes of the S-Ag sequence. Several of these epitopes are also pathogenic, with the most active located close to the C-terminal end of the molecule and corresponding to sequence 340-360 of human S-Ag. We then tested this fragment as well as some nondominant but pathogenic fragments in different rat strains to determine the degree of restriction to specific MHC class II antigens. The immunodominant epitope was pathogenic in all the strains tested although the level of pathogenicity differed. These strain differences were due not only to differences in class II antigen but also in non-MHC factors; pathogenicity in the Fisher strain, which shares the same MHC class II, was maricedly depressed. For further characterization of the human immune response, it is necessary to establish cell lines and clones. These should help us to determine the exact peptide sequences responsible for the inmiune reaction. To obtain these cell lines, we have estab- lished multiple short-term cell lines. The frequency of positive responses in the controls was around 0-7% for a precursor frequency on the order of 1 in 10 million circulating lymphocytes. In patients, two patterns of response were found. There was either a very high response rate, on the order of 30-40% of wells, or a very low response rate, similar to that of controls. Such a high response rate corresponds to a precursor frequency of about 1 in 1 million circulat- ing mononuclear cells. The highest response rates were observed shortly after attacks of intraocular inflammation, and they developed once the inflam- matory episodes had resolved. This suggests that the T cells marginate during periods of intraocular inflammation and that their numbers expand during inflammatory events. We also have noted that the standard proliferation assay becomes very weak after 101 Laboratory of Immunology NEI Annual Report-FY 1992 an inflammatory episode, suggesting that suppressive factors are present in the peripheral blood following episodes of inflammation. Significance to Biomedical Research and the Program of the Institute These studies wiU help to define the parallels be- tween the animal models and human autoimmune ocular diseases. Findings in the rat suggest that an immunodominant epitope may be recognized by more than one MHC class II antigen. This suggests that if one is able to identify such an epitope in man, it may be possible to use it to induce tolerance and prevent rejection from occurring. The establishment of specific human cell lines, which stiU remains a priority, win help us to characterize the interactions between the human T cells and their antigen-present- ing cells. Proposed Course In the coming year, the main focus of attention will be on determining the HLA class II restriction of the immune response to fragments of S-Ag. Concentrat- ing mainly on human material, we will try to char- acterize the T-ceU receptor usage as well as the MHC class n being used in various weU-defined uveitic entities. NEI Research Program Retinal and Choroidal Diseases — ^Inflammatory Disorders Publications de Smet MD, Dayan M, Caspi R, Hafler DA, Gery I, Nussenblatt RB: Determination of the precursor frequency to S-Ag in uveitis patients and controls. Invest Ophthalmol Vis Sci 33(4):930, 1992. Dayan M, de Smet MD, Caspi R, Roberge FG, Gery I, Nussenblatt RB: Determination of the response to S-Ag over time in patients with Behfet's disease. Second International Symposium on Ocular Inflammation. Jerusalem, Israel, p 230, 1992. 102 DEPARTMEMT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00267-03 LI PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Modulation of Immune Functions Using PE40 Derived Immunotoxins PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator) (Name, title, laboratory, and institute emalion) PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI Others: Juan Lopez M.D. Visiting Fellow Robert B. Nussenblatt M.D. Clinical Director Stephan S. Thurau M.D. Special Volunteer LI, NEI NEI LLNEI COOPERATING UNITS (It any) Laboratory of Molecular Biology, National Cancer Institute (Ira Pastan, M.D.) Laboratory of Immunology Section on Immunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.0 PROFESSIONAL: 0.0 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.) This project has been tenninated. 103 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00276-01 LI PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 charactBrs or laes. Title must III on one line between the borders.) Surgical Management of Uveitis PRINCIPAL INVESTIGATOR /Usl other proleeslonal personnel below the Principal Investigator.) (Name, title, laboratory, and Institute atlillatlon) PI: Marc D. de Smet M.D. Visiting Scientist LI, NEI Othen: Fran90is Roberge M.D. Visiting Scientist LI, NEI Margaret Cheung M.D. Senior Staff FeUow LI, NEI Scott Whitn^) M.D. Senior Staff Fellow LI, NEI David Parks M.D. Senior Staff Fellow LI. NEI Juan Lopez M.D. Visiting Fellow LI, NEI Ray DeBarge M.D. Senior Staff Fellow LI. NEI Richard Fenion M.D. Senior Staff Fellow LI, NEI Dan Martin M.D. Senior Staff Fellow LI, NEI David Callanan M.D. Senior Staff Fellow LI, NEI Naofumi Hilciu M.D. Visiting Associate LI, NEI COOPERATING UNITS (II any) Clinical Oncology Program, Medicine Branch, National Cancer Institute, NIH (Robert Wittes, M.D.) LAB/BRANCH Laboratory of Immimology Section on Immunoregulation INSTfTUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.75 PROFESSIONAL: 1.75 0.0 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects |3 (b) Human tissues □ (c) Neither □ (a1) Minors □ (a2) Interviews 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 exaa timing of the surgery and the postoperative immunosuppressive therapy given to the patient often determine the outcome. Several conditions also exist where there is a need to obtain a tissue diagnosis before proceeding with treatment We have used vitreaomy and chorioretinal biopsy to help us make an appropriate diagnosis in such cases. Proper handling of the specimen is essential, particularly in cases of intraocular lymphoma in which the lymphoma cells are particularly fragile. Glaucoma remains an important complication in patients with uveitis. Standard trabeculectomies always stop functioning after a few months. We are continuing to compare the uses of 5-FU and Molteno implants in patients with uveitis and glaucoma who require surgery. We now have 10 patients enrolled in the study. The use of intraocular lenses following cataract extraction in patients with uveitis wUl be addressed in a randomized double-masked study to compare modified intraocular lenses with standard lenses in patients with uveitis that is quiet In a rat model, we tested the use of topical FK506 as an immunosuppressant When heterotopic corneal grafts were performed in Lewis rats to determine tiie ability of these drops to prevent corneal graft rejection, a 0.3% solution of FK506 was found effective. 104 PHS 6040 (Rev. S/92) NEI Annual Report-FY 1992 Laboratory of Immunology Project Description Additional Personnel Igal Gery Ph.D. Chi-Chao Chan M.D. Susan Mellow R.N. Susan Whitcher Head, Section on Experimental Immunology, NEI, LI Head, Section on Immunopathology, NEI, LI Nurse, NEI, LI Psychologist, NEI, LI Clinical Protocol Numbers 79-EI-49 87-EI-104 92-EI-157 Objectives One objective of this study is to develop rational surgical approaches for patients with intraocular inflammation. The development of appropriate surgical modalities for patients with uveitis wiU enable proper management of the complications that arise with chronic intraocular inflammation. A second objective is to devise rational methods for sampling intraocular tissues and to develop the methodology to obtain clinically useful information from limited tissue samples. A third objective is to test new therapeutic modalities for postoperative immunosuppression in animal models. Methods Patients with ocular inflammation who have devel- oped ocular complications that require surgery or patients in whom an appropriate diagnosis can be made only with surgery are eligible for one of the protocols. Patients with vitritis and retinitis of un- known etiology in whom a nonspecific trial of immunosuppression is contraindicated may, accord- ing to the protocol, undergo vitrectomy and chorio- retinal or endoretinal biopsy to obtain a diagnosis. The tissue specimen is partitioned for microbiology, electron microscopy, immunohistochemistry, and polymerase chain reaction. Patients with a suspected intraocular lymphoma undergo an intraocular lymphoma workup with appropriate computer tomography or magnetic resonance imaging (MRI) scans and lumbar punc- tures. When these are negative, a vitrectomy is perfomied and the cells are studied by immunohisto- chemistry. Patients who have intraocular lymphomas 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-FU or Molteno implant. They then are followed prospectively to determine the degree of postopera- tive inflammation and how effective the procedure was over time. For patients with cataracts and uveitis under good control and with minimal intraocular inflammation, the protocol calls for a cataract extraction and randomization to a standard intraocular lens or a modified lens with a heparin coating. We then monitor patients postoperatively for the appearance of inflammation, using a laser cell flare meter. Patients also are monitored for the appearance of cellular deposits on the lens surface. In animals, we have tested topical immunosup- pression in a corneal transplant model. In this model, rejection of the graft occurs in 100% of animals within 1 week of stopping immimosuppression. Donor corneas are taken from Fisher rats, while die recipients are Lewis rats. Major Findings In Fiscal Year 1992, we performed several diagnostic vitrectomies for intraocular lymphoma. This particu- lar lymphoma, a subtype of O^S lymphomas, is on the rise; three times more CNS lymphomas are diagnosed today than 10 years ago. In each case of intraocular lymphoma, the diagnosis was made after a complete woricup with MRI brain scans and limibar pimcture. We have found that adding culture medium to the vitrectomy container improves the survival of the lymphoma cells and makes the cytologic diag- nosis easier. Enough culture medium is added to give a 10-20% concentration in a full vitrectomy speci- men container. One patient who recentiy died after a recurrence had no signs of systemic disease. There was evidence only of diffuse brain dissemination. We have found that pretreatment with pulsed methylprednisolone is an effective way of decreasing preoperative inflammation. This pretreatment has been particularly useful in cases in which it has been necessary to perfonn a vitrectomy while there was still evidence of inflammation. Postoperatively these patients did not develop any significant inflamma- lOS Laboratory of Immunology NEI Annual Report-FY 1992 tion. Data analyses are not yet available on the glaucoma trial or the intraocular lens trial; both trials are still recruiting patients. In the animal studies, we demonstrated that topical drops of FK506 are an effective means of stopping corneal graft rejection. FK506 predominant- ly affects T cells, inhibiting both their activation and their recruitment into the transplanted tissue, as demonstrated by immunohistochemistry. It appears to downregulate the expression of both class I and class II antigens in the transplanted cornea. This eiiect is probably mediated by inhibiting T-ceU cytokine secretion. Thus topical FK506 can prevent the onset of inflammation in several pathways and may be effective in the treatment of several immunologically based surface disorders. 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 immunosuppression. Developing appropriate surgical modalities of treatment is an important endeavor. Similarly, conditions exist in which the appropriate therapy can be given only when the proper diagnosis has been made from intraocular tissue. Developing means of obtaining a minimal amount of tissue and properly processing it is thus of major significance. Proposed Course This project will continue with study of methods of surgically managing patients with uveitis. Patient enrollment continues. We also will study in animal models new methods to sample intraocular tissue and to test new immunomodulatory agents. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Chan C-C, Palestine AG, Davis JL, de Smet MD, McLean IW, Bumier M, Drouilhet JH, Nussen- blatt RB: Role of chorioretinal biopsy in inflam- matory eye disease. Ophthalmology 98:1281-1286, 1991. Hikita N, Lopez JS, de Smet MD, Chan C-C, Mochi- zuki M, Nussenblatt RB: Use of topical FK-506 in the corneal graft rejection model in lewis rats. Invest Ophthalmol Vis Set 33(4):832, 1992. Whitcup SM, Belfort R Jr, de Smet MD, Palestine AG, Nussenblatt RB, Chan C-C: Immunohisto- chemistry of the inflammatory response in Propi- onibacterium acnes endophthalmitis. Arch Oph- thalmol 109:978-979, 1991. 106 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00075-14 LI PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 charaaers or less. TillB must tit on one line between the borders.) Immune Functions in Ocular Diseases of Obsoire Etiology PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aftiliaUon) PI: Robert B. Nussenblatt M.D. Clinical Director NEI Others: Marc de Smet Rashid Mahdi Scott M. Whitcup M.D. Visiting Scientist LI, NEI Biologist LI, NEI M.D. Staff Medical Officer LI, NEI COOPERATING UNITS (II any) Department of Ophthalmology, University of Kurume, Kurume, Japan (Manabu Mochizuki, M.D.) LAB/BRANCH Laboratory of Immunology SECTION Section on Immunoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.0 PROFESSIONAL: 0.0 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.) This project has been terminated. 107 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOIEY 00115-14 LI PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 charaaers or less. Title must lit on one line between the borders.) Cyclosporine Therapy in Uveitis PRINCIPAL INVESTIGATOR (List other prolassional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aUlliatton) PI: Robert B. Nussenblatt M.D. Others: Marc de Smet M.D. Scott Whitcup M.D. Chi-Chao Chan M.D. Richard Fenton M.D. Raymond DeBarge M.D. Dan Martin M.D. Clinical Director Senior Staff Fellow Senior Staff Fellow Head, Section on Immimopathology Special Volunteer Senio: Staff FeUow Senior Staff Fellow NEI LI, NEI LI, NEI LI, NEI LI, NEI LI, NEI LLNEI COOPERATING UNITS (11 any) LAB/BHANCH Laboratory of Immimology SECTION Section on Immunoregulation INSTFTUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.5 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews PROFESSIONAL: 0.5 0.0 □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Cyclosporine, an endecapeptide fungal produa with specific anti-T-cell characteristics, is being administered to patients with sight-threatening ocular inflammatory disease of noninfectious origin who have failed on either corticosteroid or cytotoxic agent therapy to test its efficacy in the treatment of uveitis. 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 l/U 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 is 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 followup. Cyclosporine G was more effective than cyclosporine A in treating cystoid macular edema. 108 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Immunology Project Description Additional Personnel Barry Grubbs Biologist, LI, NEI Clinical Protocol Number 81-EI-33 Objectives Cyclosporine, an endecapeptide obtained from fungi, has been shown to have specific anti-T-cell activity (Transplant Proc 12:234, 1980). We have reported cyclosporine 's exceptional effectiveness in preventing the induction of S-antigen autoimmune uveitis in rats, as well as in inhibiting the disease once immu- nization 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 of age or older, of either sex (females not pregnant), who have not done well on more conventional medical therapy were admitted to this study. All patients must have bilateral sight- threatening uveitis of noninfectious etiology that has not been satisfactorily controlled by either corticoste- roid or cytotoxic agent therapy. Lymphocyte cultures are prepared, and the inunune ceUs are tested against various crude ocular extracts, as well as purified human S-antigen, to assess evidence of cellular immune memory, which is considered to be the in vitro equivalent of the anamnestic response in vivo. Patients chosen are treated with CsA or a new analog CsG in a phase I/II trial to evaluate the safety and activity of CsG versus CsA. During this period, the patients' clinical and inmiunologic course is closely monitored. Specific attention is given to renal func- tion changes, a frequent side effect. Patients who need to continue CsA because of their ocular disease for over 1 year may be asked to undergo renal biopsy to evaluate the reversible and irreversible components to CsA renal toxicity. Some patients who have been entered on previous CsA studies and have continued to be followed in the eye clinic will continue to be monitored for their renal function to determine how and when cyclosporine can safely be tapered. Major Findings CsA has been effective in the treatment of some cases of posterior uveitis. An improvement in the inflammatory activity and visual acuity was seen in most patients treated to date. The particular respon- siveness of patients with the ocular manifestations of Behcet's disease to this agent has been corroborated by a masked randomized trial performed in Japan. The improvement in the clinical condition was supported by a concomitant improvement in electro- physiologic testing, particularly contrast sensitivity. Patients treated with CsA had no abnormalities of natural kiUer ceU activity before the initiation of therapy, nor was any noted afterward. CsA signifi- cantly 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-tenn 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 controls. An irreversible component of CsA toxicity could be identified, in the main being renal tubular atrophy accompanied by interstitial fibrosis. The majority of these individuals* biopsies had normal serum creatinine values, but a correlation could be made between the alterations noted and previous sertmi 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 in reducing cystoid macular edema than was CsA, 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 109 Laboratory of Immunology NEI Annual Report-FY 1992 inadequate and nonspecific. CsA appears effective in Proposed Course treating posterior uveitis of noninfectious etiology. Newer studies to look at various cyclosporine combi- This is the first new agent in decades to be found ^^^^^ ^ ^^^^^^ ^ useful in treating the severe foim of this condition; *riT'.^ h'""?" Ti!''''^'^P'^'™'*''''P'"'^' NEI Research Program schedule be developed. Newer therapeutic strategies * have already begun. Retinal and Choroidal Diseases — Inflammatory Disorders 110 DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00278-01 LI PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 charactars or less. Title must lit on one line between the borders.) Oral Administration of Antigen and the Ocular Immune Response PRINCIPAL INVESTIGATOR (Ust other proteeslonal personnel below the Principal Investigator.) (Name, title, laboratory, and InsUtule affiliation) PI: Robert B. Nussenblatt M.D. Scientific Director LI, NEI Others: Igal Gery Ph.D. Susan Whitcher M.S. Marc D. de Smet M.D. Molly Dayan M.S. Irwin Suh B.A. Head, Section on LI, NEI Experimental Immunology Clinical Protocol Assistant LI, NEI Visiting Scientist LI, NEI Special Volunteer LI, lilil IRTA FeUow LI. NEI COOPERATING UNITS (If any) LAB/BRANCH Laboratory of Immunology SECTION Section on Immimoregulation INSTITUTE AND LOCATION NEI, NIH, Bethesda. MP 20892 TOTAL STAFF YEARS: 1.75 PROFESSIONAL: 0.50 1.25 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 the oral administration of various antigens on the ocular immime response has been tested in the animal model for severe intraocular inflammatory disease experimental autoimmune uveioretinitis induced by both retinal S-antigen and interphotoreceptor retinoid-binding protein (IRBP). Oral tolerance could be induced by repeatedly feeding rats S-antigen. A putative suppressor cell that was CD8 positive could be isolated fi-om the spleen of such animals and transferred to other animals to induce a similar toleragenic effea. In addition, the role of the spleen was confirmed in ongoing animal experiments. A randomized masked trial to evaluate the usefulness of S-antigen feeding in patients with intraocular inflammatory diseases has been put together. A pilot smdy perfomied in two patients gave evidence of the induction of such tolerance. Ill PHS 6040 (Rev. 5/92) Laboratory of Immunology NEI Annual Report-FY 1992 Project Description Objectives Exploring means of immunomodulation has been a major role of this laboratory. Although the Labora- tory has extensive experience using various immuno- suppressive agents, there also has been a major thrust in attempts to use other modes of immunosuppres- sion. The goal of this series of experiments, in animals as well as 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 The 6- to 10- week-old Lewis rats (either sex) used for these experiments will be fed before and after the induction of experimental uveioretinitis with various antigens. The antigens include whole molecules such as the retinal S-antigen (S-Ag) and interphotoreceptor retinoid-binding protein, as well as their fragments. In a subset of experiments, some animals also wUl imdergo splenectomy before initiation of the experi- ments; others will be subjected to sham procedures. We will attempt to evaluate the clinical course of their disease and corroborate the clinical observations with histopathology at various points after the initiation of the experiments. The goal is to evaluate the role of the spleen as well as the roles of various fragments in the ability to induce this tolerogenic state. In these studies, the patients will be individuals, 18 years or older, either sex, who have bilateral uveitis of a noninfectious cause. In addition, their lymphocytes must demonstrate an in vitro prolifera- tive response to the retinal S-Ag. The patients also need to be on systemic immimosuppressive therapy, be it corticosteroids, cytotoxic agents, or cyclospor- ine. The goal of this study is, in individuals who need high amoimts of immunosuppressive therapy to control their disease, to determine whether the addition of oral feeding of retinal antigens will induce a toleragenic state. This study will be per- fonned in a randomized double-masked fashion. Some patients will receive S-Ag; some others, a retinal mixture containing several antigens; still others, placebo. The intent is to reduce the amount of immunosuppressive therapy the patients are taking. It is hoped that a toleragenic state can be induced by feeding these antigens. Major Findings In animal work, the spleen appears to play an impor- tant role in the induction of oral tolerance of S-Ag. In addition, the spleen is essential for adoptive transfer of tolerance by splenocytes from S-Ag-fed donors. Thus, it would be logical to assume that the spleen acts as a sight for induction and/or amplifica- tion of cells with suppressive activity. The pilot study has demonstrated that — at least in two patients — a toleragenic state can be induced with the feeding of antigen at the dosages in this study. One patient with par planitis and one with Behcet's disease have been able to either come off their medication completely or 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 aU of ophthalmology. All the present modes of therapy for patients with severe ocular inflammatory disease have limitations, particularly because of their second- ary side effects. In patients identified as having an immune response to the retinal S-Ag, we will be able to induce an immunosuppressive state without the use of pharmacologic agents. Furthenmore, the induction of this tolerance wiU be antigen specific. Proposed Course The randomized study will begin shortly. NEI Research Program Retinal and Choroidal Diseases — Inflammatory Disorders Publications Thurau SR, Chan C-C, Suh E. Nussenblatt RB: Suppression of S-antigen induced experimental autoimmune uveitis by a 20mer peptide. / Auto- immunity 4:507-516, 1991. Weiner HL, Zhang ZH, Khoury SJ, Miller A, Al-Sabbagh A, Brod SA, Lider O, Higgins P, Sobel R, Nussenblatt RB, Haffler DA: Antigen- driven peripheral inmiune tolerance: Suppression of organ-specific autoimmune diseases by oral administration of autoantigens. Ann NY Acad Sci 636:227-232, 1991. 112 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 made significant progress during the past year in our mission to determine the molecular mechanisms underlying diseases affecting the eye. As in the past, the primary emphases have been on cataract and the ocular complications of diabetes. Section on Cataract Dr. Deborah Carper has continued to investigate the role of the polyol pathway in diabetic com- plications. Site-directed mutagenesis of specific residues of aldose reductase has identified certain residues important to the enzyme's catalytic function. The primary sequence of human sorbitol dehydroge- nase also has been determined, and studies are under way to elucidate how its gene is regulated. The groups headed by Drs. Paul Russell and Sam Zigler have been involved in the development of an in vitro system for assaying the effectiveness of anti- cataract agents. The successful development of such an organ culture system wiU allow initial screening of compoimds before moving to animal models of cataraa for the next phase of testing. Dr. Donita Garland is investigating various modifications of lens crystallins and their potential role in cataractogenesis. The primary focus has been on metal-catalyzed oxidation; y-crystallin is used as a model system. A protein that protects against such oxidation in the eyes of many species has been isolated from bovine lens. Dr. Fielding Hejtmancik and his colleagues have been successful in mapping to human chromosome 1 1 two genes causing Usher's syndrome type I. The fact that this disease can be caused by more than one gene is a surprising and potentially very important step toward understanding the paUiogenesis of this retinal degenerative disease. Section on Pathophysiology Dr. W.G. Robison, Jr., head of the Section on Pathophysiology has made significant advances in his studies on diabetic retinopathy. Using the enzyme elastase, he has developed a new procedure for isolation and evaluation of the entire vascular net- work of the retina. Coupled with the rat model system previously developed, this technique has proven that background diabetic retinopathy can be experimentally produced in rats, thereby facilitating both analysis of the pathogenesis of this disease and testing of potential therapeutic agents. lis DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1, 1991 to September 30. 1992 PROJECT NUMBER ZOl EY 00201-08 LMOD 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 (Ust other prolessional personnel below the Principal Investigator) (Name, title, laboratory, and institute alliliaUon) PI: Deborah Caiper Ph.D. Biologist LMOD, NEI Others: Susan Old Takeshi Iwata Ph.D. Ph.D. Staff FeUow Visiting Associate LMOD, NEI LMOD. NEI COOPERATING UNITS (il 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 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects n (a1) Minors n (a2) Interviews PROFESSIONAL: 3.0 OTHER: 0.0 S (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) In diabetes, the accumulation of soititol is believed to be a key factor in initiating cataract, retinopathy, and neuropathy. We are interested in controlling the accumulation of sorbitol by regulating the action of two enzymes of the sorbitol pathway: Aldose reductase (AR) reduces glucose to sorbitol, and sorbitol dehydrogenase (SDH) oxidizes soititol 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 tiie rat and human AR and determined tiiat some of these changes affect tiie kinetics of the protein witii its substrate. For example, when cysteine at position 298 is changed to a serine, tiie kinetics of glyceraldehyde increases 2-fold, whUe xylose decreases to 15% of normal. These structiire/function smdies should help define the active site and locate the target areas of die current AR inhibitors. Anotiier sti-ategy to control sorbitol accumulation is to regulate SDH. We have initiated studies to understand tiie structiire of tiiis protein and its action in vitro. The sequence of human SDH has been determined using cDNA analysis. Nortiiem blots indicate tiiat levels of SDH mRNA are higher in kidney tiian in otiier tissues, such as lung, liver, brain, and heart. A major portion of tiie SDH gene also has been obtained. These tools will allow us to study tiie regulation of SDH at tiie gene level. 116 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The objective of the project is to study the regulation of the enzymes of the polyol pathway. Methods The methods employed include molecular biology, protein chemistry, and cell biology techniques. Major Findings 1. Structure/function studies. — Several mutant forms of the aldose reductase (AR) protein were synthesized using the polymerase chain reaction (PCR). Sequencing verified the amino acid substitu- tion. The mutant proteins were expressed in bacteria, purified on three columns using different biochemical characteristics of the protein, then tested for enzyme and inhibitor activity. The change from histidine to glutamine in rat AR at amino acid positions 41 and 187 affected neither the substrate binding rate rior protein inhibition by sorbinil. A change ftom cys- teine to serine at position 298 altered the properties of AR. Glyceraldehyde was a better substrate for the mutant protein than the wild type. In contrast, xylose was a poor substrate for the mutant enzyme. Whereas cysteine 298 is located in the active site, this finding suggests an important role for this amino acid. 2. Cellular regulation of AR and sodiumJpotas- sium ATPase. — Sodium/potassium ATPase activity changes as sorbitol accumulates. In our studies on the induction of AR and its product, sorbitol, we found that sodium/potassium ATPase activity increased in the early stages of hypertonic stress. We then extended this finding by measuring the mRNA levels of the enzyme under various stress conditions. Sodiimi/potassium ATPase was synthesized by the reverse transcriptase PCR method. The DNA pro- ducts were subcloned and verified by sequencing. The DNA products were then used as probes on Northern blots of dog lens epithelial cells grown in hyperglycemic and hypertonic conditions. We found that by 24 hours, medium containing 150 mM NaQ increased the mRNA levels of sodiimi^tassiimi ATPase approximately eightfold. In medium contain- ing 300 mM glucose, the mRNA levels also in- creased, but at a slower rate. These studies show a correlation in the response of AR and sodium^tas- sium ATPase to stress. 3. Primary structure of sorbitol dehydrogenase (SDH). — In determining the complete sequence of human SDH, we employed two different methods: (1) cloning from a human cDNA library, which gave approximately 80% of the structure, and (2) use of the rapid amplification of cDNA ends (RACE) system, which completed the sequence. Using the SDH DNA as a probe, we obtained part of the gene for SDH, which we now are sequencing. 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 diabetes clinical trials have emphasized the need for innovative approaches to AR inJiibition. 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 wUl continue via site-directed mutagene- sis of AR protein to localize the critical amino acids residues in the active and inhibitor binding sites. We will complete the structure of SDH and use these DNA tools for studying the regulation of both AR and SDH in culture. NEI Research Program Cataract — ^Molecular Genetics Publications Dasgupta S, Hohman T, Carper D: Hypertonic stress induces oB-crystaUin expression. Exp Eye Res 54:461-470, 1992. Graham C, Szpirer C, Levan G, Carper D: Character- ization of the aldose reductase-encoding gene famUy in rat Gene 107:159-267, 1991. Lin L-R, Carper D, Yokoyama T, Reddy V: Effect of hypertonicity on aldose reductase, ocB-crystal- lin and organic osmolytes in retinal pigment epithelium. Invest Ophthalmol Vis Sci, in press. 117 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00189-09 LMOD PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or lass. Title must lit on one line between the borders.) Oxidation of PnJteins in Cataractogenesis PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aHlllallon) PI: Donita L. Garland Ph.D. Research Chemist LMOD, NEI Others: Jose Jimenez Lorenzo Merola Mark Reid Ph.D. M.S. B.A. Visiting Fellow Chemist Technician LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (il any) LAB/BRANCH Laboratory of Mechanisms of Ocular E>iseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 3.6 PROFESSIONAL: 3.0 0.6 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.) Oxidative changes of lens proteins are thought to occur with aging and to contribute to the development of cataracts. The goals of this project are to determine (1) the extent of oxidative modification of crystallins and metabolic enzymes in both normal and cataractous lenses, (2) the nature of the modifications and mechanisms leading to the changes, and (3) the modifications on the structure and fimction of lens proteins. Bovine and rat lenses are used. The approach is to study the modifications of lens proteins after treatment in vitro by metal-catalyzed oxidation systems. The focus of these studies has been on the modification of bovine y-crystaUins as a model system. There are distinct differences in the interaction of metals with each of the purified y-crystallins. The structural modifications induced by treatment of the crystallins with metal-catalyzed oxidation have been studied. A protein that presumably protects against metal-catalyzed oxidation in cells has been identified in the eye of rats, bovine, primates, and humans; the bovine lens protein has been purified. We have studied human lens proteins by two-dimensional polyacrylamide gel electrophoresis. These studies have included identification of the human lens proteins, protein distribution within the lens, age-related changes in composition, and identification of the age-related modifications that the proteins undergo. 118 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Mechanisms of Ocular Diseases Project Description Objectives Oxidative modifications of lens proteins are thought to occur with aging and to contribute to the develop- ment of cataracts. The objectives of these studies are (1) to detennine the extent to which oxidation contributes to the modification of lens crystallins and enzymes in normal lenses and cataracts, (2) to detennine the nature and *he mechanisms of the modifications, (3) to determine the effect of the modifications on the structure and function of the proteins, and (4) to develop methodology to assess levels of oxidation in tissues and cells in culture. Methods Bovine, rat, and human tissues were used for these studies. We employed classical methods to purify bovine and rat lens proteins. Other methods used were standard procedures for studying proteins, including amino acid analysis, high-pressure liquid chromatography (HPLC), electrophoresis, isoelectric focusing, circular dichroism, and two-dimensional gel electrophoresis. For metal analyses we used a multi- element atomic absorption spectrometer. Major Findings 1. A protein that protects enzymes against inacti- vation by thiol-dependent metal-catalyzed oxidative systems but not other metal-catalyzed oxidation systems is present in rat and bovine lens and in human trabecular meshwork cells. The protein has been purified and now is being sequenced. 2. When lyophilized, y-crystallins undergo struc- tural changes that we have demonstrated by changes in isoelectric focusing, hydration, and denaturation properties. 3. Continuing studies on the metal-catalyzed oxidation of y-crystallins, we have focused on demonstrating differences in copper and iron binding to the proteins and on differences in amino acid modification by the copper-ascorbate oxidation system. 4. A great deal of effort has gone into a project done in collaboration with the Ophthalmic Genetics and Clinical Services Branch. We are analyzing proteins of lens samples obtained from normal donors and from extracted cataracts by two-dimen- sional polyacrylamide electrophoresis. The initial focus has been on setting up equipment, optimizing the technique for processing these samples, and sample preparation. Analyses have primarily been done on samples obtained by aspiration during cataract surgery. Many proteins have been identified by immuno- blotting techniques and protein sequencing. In the analyses of nonnal donor lenses of various ages, we have identified many proteins and determined the extent of modification of these proteins using immu- noblotting techniques. Significance to Biomedical Research and the Program of the Institute Oxidative processes have long been considered a major contributing factor in senile cataracts. Treat- ment of lens proteins in vitro by mixed-function oxidation systems leads to protein modifications that mimic those seen in aging and senile cataracts and in brunescent lenses. The use of these oxidation systems to study the modification in vitro may facilitate our understanding of what role oxidation plays in aging and cataractogenesis. Also, it may be possible to use such an in vitro system to screen for agents that can prevent or protect against oxidative damage in the lens. Proposed Course The following studies are proposed for Fiscal Year 1993: 1. The major focus will be continuing the studies on the oxidative modification of lens proteins by metal-catalyzed oxidation systems. The work will include purifying the peptides containing the modi- fied amino acids, identifying the modified amino acid, and studying effects of the modification on structure. These studies will be done on y-crystallins. 2. The studies on the interaction of copper with j- crystallins will be continued. 3. The analysis of human lens proteins by two- dimensional electroi^oresis will continue. Proteins in various types of cataracts will be compared. NEI Research Program Cataract — Lens Development and Aging 119 Laboratory of Mechanisms of Ocular Diseases NEI Annual Report-FY 1992 Publications Bettelheim FA, Reid MB, McPhie P, Garland D: On the stability of bovine gamma n crystallin. Biochem Biophys Res Commun 187:39-44, 1992. Datiles MB, Schumer DJ, Zigler JS Jr, Russell P, Anderson L, Garland D: Two-dimensional gel electrophoresis analysis of human lens proteins. Curr Eye Res 7:669-677. 1992. Garland DL: Ascorbic acid and the eye. Am J Clin AfM/r54:1198S-1202S, 1991. Giannessi M, Del Corso A, Cappiello 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, in press. Russell P, Yamada T, Su G-T, Garland D, Zigler JS Jn Effects of naphthalene metabolites on cultured cells from eye lens. Free Rod Biol Med 10:255- 261, 1991. 120 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00272-02 LMOD PERIOD COVERED October 1. 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or lass. TiUa must tit or) orta Una between the borders.) Inherited Ocular Disease PRINCIPAL INVESTIGATOR (List other prolasslonal personnel below the Principal Investigator.) (Name, title, laboratory, end Institute altUiatlon) PI: James Fielding Hejtmancik M.D., Ph.D. Medical Officer LMOD, NEI Others: John Hope Radha Ayyagari Ling Lee Anthony Lloyd Ph.D. Ph.D. M.S. M.D. Senior Fellow Special Volunteer Chemist IRTA FeUow LMOD, NEI LMOD, NEI LMOD, NEI LMOD, NEI COOPERATING UNITS (II any) Baylor College of Medicine (J. Towbin, B. Peiryman, T. Ashizawa, P. Oveibeek); Univ. of Iowa (R. Smilh); Univ. of Texas-Houston (S. Daiger); Ocular Genetics and Clinical Services Branch, NEI, NIH (M. Kaiser); Washington Univ. at Sl 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. MD 20892 TOTAL STAFF YEARS: 5.0 PROFESSIONAL; 4.0 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.) 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 investigation, 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 ciystaUins, which comprise over 90% of the soluble protein of the lens, are heavily modified in most cataracts. The effects of specific modifications of P- and y-crystaUin structure on crystaUin functions, such as stability and formation of macromolecular aggregates, are studied in cultured cells transformed with normal and modified p A3/Al-crystallin genes. Regions of the P-crystallin molecule of special interest include the amino terminal arm and the Greek key motifs of the core domains. The effects of these modifications on lens transparency also are being studied in a transgenic mouse system in which a modified P-A3/A1 gene is driven by an oA-crystallin promoter. A second approach to understanding inherited visual diseases uses principles of positional cloning to identify genes important in human inherited diseases. Human diseases undergoing linkage analysis, gene isolation, or characterization of mutations include Usher's syndrome, myotonic dystrophy, Duchenne muscular dystrophy, long qt syndrome, and a variety of X-linked syndromes. We are now recruiting families with inherited cataracts in preparation for study of this important group of diseases. 121 PHS 6040 (Rev. 5/92) Laboratory of Mechanisms of Ocular Diseases NEI Annual Report-FY 1992 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 pathophys- iology. These long-range objectives will be approached by pursuing two specific aims: identify- ing genes involved in inherited visual diseases and elucidating the mechanisms by which mutations in these genes cause disease. Methods Conventional cloning technology utilized in prepar- ing sequences for gene expression studies includes ligation with T4 DNA ligase, screening by NaOH miniprep methodology and ^^P-labeled DNA probes, as weU as allele-specific oligonucleotide hybridiza- tion to screen for specific single-base settings. We introduced sequence changes by site-specific muta- genesis using standard methodology. Gene expression in either Chinese hamster ovary cells (RJK 88) or insect ceUs (Sf9) uses the baculovirus expression system. Protein expression is monitored by standard two-dimensional gel electrophoresis followed by immunoblotting. Association behavior is assessed by elution volume on seive FPLC. Isolation of crystallin and other cDNAs and genomic fragments by library screening with cloned genes or oligonucleotides involves routine methods. Sequencing is conducted by cycling or automated fluorescent technology (ABI). Until recentiy, linkage analysis has been per- formed by conventional Soutiiem blotting. Cell lines from patients and other family members are immor- talized by Epstein-Barr virus transformation. DNA is isolated by standard methodology and digested by restriction endonucleases. After agarose gel electro- phoresis. Southern transfer is performed, and the resulting blot is probed with isolated DNA Augments labeled witii ^ by oligonucleotide labeling. Recent- ly, we analyzed short tandem repeat (STR, micro- satellite) markers by polymerase chain reaction (PCR) performed in the presence of labeled oligo- nucleotides on sequencing gels. Linkage data are recorded on a computerized spreadsheet; for both two-point and multipoint analysis, we use the LINK- AGE program package. Major Findings 1. We modified the Greek key motifs of the PA3/A1 -crystallin and then expressed them in transgenic mice. The highly conserved glycine at position 23 assumes torsional angles impossible for any other amino acid; it is required to maintain the sef-ondary structure of the Greek key motif. We changed this glycine to a proline residue and created additional transgenic mice, using a PA3/A1 peptide with a stop codon inserted just before the fourth Greek key motif, essentially creating p-crysiallin with only three motifs. Although we have obtained animals containing the genomic insertion by South- em blot and PCR analysis, they do not have cata- racts. The levels of expression of the transgenes cumentiy are being characterized. 2. The P-crystallins, their structure, and the mechanisms by which heterogeneity arises among this family of proteins are under investigation. The |3A3-crystaUin is identical to PAI except for an additional 17-amino-acid N-teiroinal extension. The same gene is believed to encode and express both polypeptides. We ligated the PA3/A1 coding sequences behind the RSV promoter, and RJK 88 fibroblast cells were transfected stably with this construction. In addition, the PA3/A1 coding sequences were inserted into the Bluebac expression vector (Stratagene) and expressed in SF9 cells. A single 26-kD protein, the predicted size of PAl-crystaUin, was detected on Western blots of soluble extracts of stable clones by antibodies raised to crystallin peptides. However, when the RJK 88 cells are transformed with cDNA that is the same except that codons gln7 and leu 10 have mutated in vitro to stop codons, these cells express a protein of only 24 kD, the predicted size of the (JAl -crystallin. Thus it appears that the upstream (pA3) start codon is used preferentially in cell lines, although the downstream (PAl) start codon is capable of being utilized. In SF9 cells, a protein with the same amino terminal sequence as that of the PA 1 -crystallin is produced when baculovirus-infected ceUs are grown past their prime. This is temporally correlated with disappearance of the pA3-crystallin band, suggesting that the smaller band is created by processing 122 NEI Annual Report-FY 1992 Laboratory of Mechanisms of Ocular Diseases or degradation of the larger in this system. In addi- tion, we have isolated clones for the mouse PA2-, pBl-, PB2-, and pB3-crystaUins, and we are se- quencing them in preparation for characterization of their roles in p-crystallin aggregation. 3. We have constructed an additional crystallin in which the amino terminal arm is deleted and replaced by a glycine residue, an extension identical to that found in Y2-crystallin. Expressed in RJK88 and SF9 ceUs (Blubac vector), it has an appropriate migration on Laemli gels, as weU as CD- spectrum and amino acid sequence. The activity of this |3-crystaUin in association with the typical 200- to 250-kD aggre- gates has been tested via FPLC on superdex 75 and superose columns. The normal PA3 polypeptide readily associates into homodimers while the trunc- ated PA3 associates minimally if at aU. We grew SF9 cells expressing the recombinant crystallins in ^^S-containing medium, then purified and reassociated them with an excess of lens extract containing normal crystallins (unlabeled), using limited urea denaturation followed by dialysis. Association into p-crystaUin aggregates was assessed by FPLC on sizing colimms. The recombinant full- length P-crystaUin peptide associates into both dimers and tetramers, with the dimer peak migrating slightly before the beta-light peak. However, the truncated PA3-crystallin migrates slightly behind the beta-light peak and does not form obvious tetramers. These data strongly suggest that the amino terminal arm of P-crystaUins assists the P-crystaUins' association into higher order aggregates. 4. Studies of phase transition properties of the y- crystaUin gene family have begun in collaboration with Drs. Mark Petrash (Washington University, St. Louis) and George Benedek (Massachusetts Institute of Technology, Boston). The bovine yB-crystaUin has been modified at two of the four residues proposed to be critical for phase transition behavior. 5. We also studied human geneUc diseases that have eye findings as part of complex traits. 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 con- firm the diagnostic usefulness of bilateral lens opacities in the diagnosis of myotonic dystrophy, data confirmed by examination of the trinucleotide repeat shown to be expanded in persons affected by myotonic dystrophy. The phenomenon of anticipa- tion, long controversial in myotonic dystrophy, was shown to occur with statistical significance in fami- lies witii the disorder. In addition, we showed that earlier age of onset through anticipation correlates with expansion of the trinucleotide repeat, although this correlation was not perfect A cDNA clone corresponding to the dystrophin gene product, isolated from a mouse lens library, is being charac- terized. 6. Ophthalmologic diseases have been studied in humans by linkage analysis of RFLP markers. Diseases Uiat we have mapped within the past year include myotonic dystrophy, X-linked agammaglobu- linemia, and Usher's syndrome type I. In addition, we have explored in detail the clinical and genetic heterogeneity of Usher's syndrome in the Acadian population. Genetic analysis confirms the clinical impression that botii type I and II Usher's syndrome are found in Acadians, and even within a single extended pedigree. The heterogeneity analysis described above implies that this phenomenon is due to segregation of two different, unlinked genes within this population. Two genes causing Usher's syndrome type I have been mapped. In tiie Acadian population of Louisi- ana, the genetic locus is on chromosome 1 Ip, while in British families in our study, the gene is on chromosome llq. These findings, which have been subjected to heterogeneity analysis using both the H0M0G2 program and the M-test, are significant at p < 0.01 under the most stringent analyses. This surprising finding implies that multiple genes can cause the rather specific chnical findings present in Usher's syndrome. Significance to Biomedical Research and the Program of the Institute Elucidation of the genetic defects causing visual disabiUty wiU have implications far beyond the patient populations suffering from the specific syndromes under study. Inherited diseases provide a means by which the molecular pathophysiology of the visual system may be understood; this knowledge then can be applied to a broad spectrum of diseases. This rationale, which applies to the study of inherited diseases such as visual dystrophy, have already resulted in improved diagnostic capabilities. The mechanism by which cataracts occur in this disease wiU provide insight into cataractogenesis in other 123 Laboratory of Mechanisms of Ocular Diseases NEI Annual Report-FY 1992 hereditary syndromes as well as in age-related and nonspecific cataracts. Proposed Course 1. We will continue studies on the stnicture- function relationships of lens crystaUins, concentrat- ing on the effects modifications of the terminal arms and possibly the interconnecting peptide between the two domains upon aggregation of P-crystaUins. We also will continue to explore how modification of the Greek key motifs affects crystallin stability and, where applicable, lens transparency. In addition, we will explore the effects of y-CTStallin sequence modifications on protein phase transitions and their relationship to cold cataract. 2. Sample collection and linkage analysis of a variety of human diseases will continue. The main emphasis wiU 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-Kupfer (Ophthalmic Genetics and Clinical Services Branch) and of autosomal recessive cata- racts ascertained in collaboration with Dr. J.S. Murty (Osmania University, Hyderabad, India). This woik wiU be coordinated with a new project to categorize and map expressed sequences of the human lens and the ongoing mechanistic studies on lens crystaUins described above. Together, these projects should elucidate the mechanisms of cataractogenesis 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, Ferryman MB, Epstein HF, Hejtmancik JF: Anticipation in myotonic dystrophy: Complex relationships between clinical findings and struc- ture of the GCTT repeat Neurology, in press. Ashizawa T, Dunne CJ, Dubel JR, Ferryman MB, Epstein HF, Boerwinkle E, Hejtmancik JF: Antic- ipation in myotonic dystrophy: Clinical and linkage analysis. Neurology, in press. Ashizawa T, Hejtmancik JF, Liu J, et al: Diagnostic value of ophthalmologic findings in myotonic dystrophy: Comparison with risks calculated by haplotype analysis of closely linked RFLP's. Aon J Med Genet 42:55-60, 1992. Bergen HT, Hejtmancik JF, Pfaff DW: Effects of gamma-aminobutyric acid receptor agonists and antagonist on LHRH-synthesizing neurons as detected by immunocylochemistry and in situ hybridization. Exp Brain Res 87:46-56, 1991. Dubel J, Fenwick R, Hejtmancik JF: Denaturing gradient gel electrophoresis of the al -antitrypsin gene: AppUcation to prenatal diagnosis. Am J Med Genet 41:39-43, 1991. Hejtmancik JF, Kaiser-Kupfer MI, Piatigorsky J: Inherited disorders of the eye lens, in Scriver CR, Beaudet AL, Sly WS, VaUe D (eds): The Meta- bolic Basis of Inherited Disease. New York: McGraw HiU, in press. Hejtmancik JF, Piatigorsky J: Molecular biology of the eye lens, in Raviola E, Dowling J (eds): Principles and Practice of Ophthalmology. Phila- delphia, WB Saunders, in press. Hejtmancik JF, Roberts R: Molecular genetics and the application of linkage analysis, in Roberts R (ed): Molecular Basis of Cardiology. London, BlackweU Scientific Publications, in press, ch 12, pp 355-382. Huang T, Hejtmancik JF, Hererra C, et al: Linkage of the gene for a new X-linked mental retardation disorder to a hypervariable (CTAT)n repeat motif within the human HPRT locus (Xq26). Am J Hum Ge/ier 49:1312-1319, 1991. Keats BJ, Todorov AA, Atwood LD, Pelias MZ, Hejtmancik JF, Kimberling WJ, Leppert M, Lewis RA, Smith RJ: Linkage studies of Usher syn- drome type 1: Exclusion results fiom the Usher syndrome consortium. Genomics 14:707-714, 1992. Mares A, Ledbetter DA, Ledbetter S, et al: Isolation of a human chromosome 14 somatic cell hybrid: analysis using alu and line based PCR. Genomics 11:215-218. 1991. 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 124 NEI Annual Report-FY 1992 Laboratory of Mechanisms of Ocular Diseases E (eds): Foundations of Clinical Ophthalmology. Smith RJH, Pelias MZ, Daiger SP, Keats B, Kimber- Philadelphia, JB Lippincott Co, in press. ling W, Hejtmancik JF: Qinical variability and Smith RJH. Lee EC. Kimberling WJ, Daiger SP, g^neUc heterogeneity within the Acadian Usher Pelias MZ. Keats BJB, Jay M, Bird A, Reardon populauon. Am J Med Genet 43:964-969. 1992. W, Guest M. Ayyagari R, Hejtmancik JF: Local- Steele F, Hejtmancik JF: Neurology of the visual ization of two genes for Usher's syndrome type I system, in Conn PM (ed): Neuroscience. Phila- to chromosome 11. Genomics, in press. delphia. JB Lippincott Co. in press. 125 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00237-07 LMOD PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characiars or less. Title must tit on one line between the borders.) Characterization of the Lens PRINCIPAL INVESTIGATOR (List other professional personnel beiow the Principal Investigator.) (Name, title, laboratory, and institute aftiliation) PI: Paul Russell Ph.D. Research Chemist LMOD. NET Others: Geoffrey Kidd Santa Tumminia Ph.D. Ph.D. Senior Staff FeUow Senior Staff Fellow 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: 2.8 PROFESSIONAL: 2.8 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [3 (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) We have imdertaken the development of an in vitro system to check the efficacy of anticataract agents. This work has centered on the organ culture of rat lens. To check the viability of the rat lens prior to experimental manipulation, we developed a method to determine crystaUin leakage from the lenses. This method, which provides a simple, yet accurate, indication of the integrity of the lens after dissection finm the animal, has been checked by use of a radioactive tracer to show that the organ-cultured lens acts like a lens in vivo. Analysis of the proteins surroimding the lens indicates that a process of protein modification may occur aroimd the lens in vivo. One way of investigating the lens changes occurring during cataract development is the use of two-dimensional gel electrophoresis of the parts of the lens. We have designed and developed a method to analyze small fractions of the rat lens to determine specific polypeptide changes that occur during cataract development. We have analyzed fractions firom the epithelium and found the cell components most influenced by changes that signal cataract development. The organ cultiue system has been used to look at mechanisms to protect cells against oxidative damage. Enzymes involved in the one- and two-election reduction of oxidative components have been investigated not only with whole lenses but also with cultured lens epithelial cells. It appears that the some of the enzymes involved in the detoxification of these oxidative compounds are specifically induced under conditions of oxidative stress. This woik represents an integral part of the strategic plan for the Section on Cataracts. 126 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The purpose of this project is (1) to develop model systems with which to test anticataract agents in vitro, (2) to develop additional model systems to examine how influences such as oxidation alter the tissue in the anterior chamber of the eye, and (3) to develop an in vitro system to explore basic questions concerning the cell and molecular biology of the lens epithelium. Methods Numerous biochemical and molecular biology methods used in this research include one- and two- dimensional gel electrophoresis of proteins, Western blot analysis of antigens, mRNA extraction and Northern blot analysis, slot blot analysis and quanti- tation of proteins, isoelectric focusing, the polymer- ase chain reaction, and cell culture. 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 and to obtaining an in vitro system with which to study anticataract agents. The new protocols developed for two-dimensional gel electrophoresis have enabled us to examine changes in the human epithelium with age and location in the lens. Work- ing under definable and reproducible conditions is an advantage in formulating model systems to study conditions that lead to loss of ceU function and to investigate agents that will ameliorate disease states. Proposed Course Continued efforts will be directed at obtaining an in vitro model system to test anticataract agents. NEI Research Program Cataract — Lens Development and Aging Major Findings 1. A new method for two-dimensional gel electro- phoresis allows resolution of samples of less than 2 Jig of protein. 2. An in vitro model is being developed to test anticataract drugs. Organ-cultured rat and monkey lenses have been used to study the influence of oxidative stress on the lens. 3. To detennine viability of the organ-cultured lenses, we devised a test that can rapidly screen our in vitro system for lenses that may not mimic an in vivo lens. 4. Two-dimensional gel electrophoresis has identified several polypeptides that are modified in the epitheliimi when the lens is challenged with oxidative stress. 5. In vitro analysis of enzymes involved with detoxification of reactive oxygen species has shown that some of these enzymes are induced under conditions of oxidative stress. Publications Datiles MB, Schumer DJ, Zigler JS Jr, Russell P, Anderson L, Garland D: Two dimensional gel electrophoretic analysis of human lens proteins. CurrEyeRes 11:669-677, 1992. Du X-Y, Linser P, Russell P, Zigler JS Jn Carbonic anhydrase III is expressed in bovine lens. Curr Eye Res 11:475-478, 1992. Kidd GL, Russell P: How differentiated are lentoid bodies? Invest Ophthalmol Vis Sci 33(4): 1038, 1992. Russell P, Koretz J, Epstein DL: Is primary open- angle glaucoma caused by small proteins? Med Hypothesis, in press. Russell P, Zigler JS Jn Analysis of the effects of eye bank storage conditions on primate lens epitheU- um. Exp Eye Res 54:153-155, 1992. Tumminia SJ, Rao PV, Zigler JS Jr, Russell P: Zeta- crystaUin is present in cultured mouse lens epithe- lial cells. Invest Ophthalmol Vis Sci 33(4): 1039, 1992. 127 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00252-04 LMOD PERIOD COVERED October 1. 1991 to September 30, 1992 TITLE OF PROJECT (BO characters or less. Title must tit 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 afllllation) PI: Paul Russell Ph.D. Research Chemist LMOD. NEI Others: Carolyn Chambers Ph.D. Senior Staff FeUow LMOD. NEI COOPERATING UNITS (It any) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.2 PROFESSIONAL: 1.2 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews Q (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The Philly mouse, derived from the Swiss-Webster strain, develops a cataract about 6 weeks after birth. Initial results have shown that in the lenses of these animals the epithelial ceUs fail to imdergo complete differentiation. Biochemically, a 27 kD protein appeared to be missing in the Philly lens. This 27 kD protein is the pB2-crystaIlin; in the nonnal lens it is a heat-stable protein. Investigation of the Plully mouse revealed that mRNA with approximately the same size as the normal pB2 mRNA is present in the Philly lens. Furthermore, it was shown that a protein present in the Philly lens is immunologically related to the PB2 protein in the nonnal lens. This protein shares the same amino tenninal as the normal PB2 but lacks part of the carboxyl half of the protein. The altered protein is slightly smaller and has a more acidic isoelectric point than the nonnal lens pB2-crystaUin. We cloned and sequenced cDNAs for normal and Philly mouse PB2-crystalIin. The nonnal mouse pB2 cDNA is 725 base pairs in length with 618 base pairs of open reading frame. Deduced amino acid sequences suggest that the normal mouse pB2 lacks a C-terminal phosphorylation site that is found in other manunals. The PhiUy mouse has a deletion of 12 nucleotides in the area encoding its fourth motif By polymerase chain reaction, we have shown that the deletion exists at the genomic level in the Philly mouse. The properties of the protein encoded with this deletion appear to be consistent with the earlier protein findings and may be responsible for cataract formatioa The woik on this project will be terminated to comply with the NEI strategic plan. 128 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Mechanisms of Ocular Diseases Project Description Objectives This project was designed (1) to study hereditary cataracts to learn how the alterations in the proteins of the lens affect cataract formation and (2) to map the alteration in the PhiUy mouse protein and deter- mine whether the alteration of this protein is related to the conformation and heat stability of the native protein. Methods Biochemical and molecular biological methods were used in this study. The techniques used included gel electrophoresis, Western blot analysis, mRNA extraction and Northern blot analysis, DNA sequenc- ing, isoelectric focusing, the polymerase chain reaction, and two-dimensional gel electrophoresis. Major Findings 1. The Philly mouse previously had been thought to lack a protein with a molecular weight of 27,000. That protein now has been shown to be pB2-crystal- lin, a major heat-stable crystallin in the lens. 2. The Philly mouse has an mRNA of approxi- mately the same size as the pB2-crystallin in the normal lens. It hybridizes with the pB2-crystallin probe. 3. The Philly mouse pB2-crystallin cDNA has a deletion of 12 nucleotides in the region encoding the fourth motif of the crystallin proteia 4. The deletion in the Philly mouse pB2-crystallin exists at the genomic level. 5. Both the normal mouse and the Philly mouse PB2-crystallin appear to lack a phosphorylation site on the C terminal. Significance to Biomedical Research and the Program of the Institute To understand how lens proteins are structured to permit the lens to be transparent to visible hght, we need to know how a defect in one of these proteins leads to the condition known as cataract. The PB2- crystallin appears to be present in all mammals and is the major heat-stable protein. This developmentaUy regulated crystallin may be important to the proper orientation of proteins in the inner areas of the lens. The Philly mouse cataract is a useful model because in the Philly mouse strain the pB2-crystallin is altered. The result of the alteration may be the cause of cataract. This finding will facilitate not only our understanding of the structural organization of the lens but also the discovery of properties that make a protein heat stable. This may be the first model to show that alteration of one crystallin can lead to cataract formation. Proposed Course This project will be terminated to comply with the NEI Strategic Plan. NEI Research Program Cataract — ^The Molecular Genetics 129 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00105-13 LMOD PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or loss. Title must III on one line between the borders.) Structure and Composition of Lens Crystallins with Respect to Cataractogenesis PRINCIPAL INVESTIGATOR (Ust other prolassional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute aftlllallon) PI: J. Samuel Zigler, Jr. Ph.D. Research Biologist LMOD, NEI Others: Vasantha Rao Pedro Gonzalez Mohan Rao Chuan Qin Ph.D. Ph.D. Ph.D. M.D. Visiting Associate Visiting FeUow Visiting Associate Visiting FeUow LMOD, NEI LMOD, NEI LMOD, NEI LMOD. NEI COOPERATING UNITS (II any) DepL of Ophthalmology, Univ. of Tennessee (H.M. Jemigan, Jr.); Laboratory of Molecular and Developmental Biology, NEI (G. Wistow, D. Lee); Alcon Laboratories (M. Lou); National Cancer Institute (M. Krishna); Centre for Cellular and Molecular Biology, Hyderabad, India (D. Balasubramanian) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Cataracts INSTITUTE AND LOCATION NEI, NIH, Bethesda. MD 20892 TOTAL STAFF YEARS: 4.9 PROFESSIONAL: 4.9 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 projea, directed toward elucidating the molecular mechanisms responsible for cataractogenesis, places special emphasis on the role of the structure and function of the lens crystaUins. One system under study involves a guinea pig hereditary congenital cataract that results from a mutation in the gene for a major lens protein, ^-crystallin. This protein is an enzyme/crystallin, a protein with two distinct functions. We are investigating how the mutation affects both ^-crystallin's catalytic function as a quinone reductase and its structural role as a lens crystallin. Because this protein is a crystallin in only a few species and is present at just catalytic levels in most species, it is an excellent system to use in analyzing lens-specific protein expression. Studies on the promoters for the ^ gene in several species indicate the presence of a second lens- specific promoter only in a species (i.e., guinea pig) that has high expression of ^-crystallin in the lens. A second focus of this project concerns the putative role of a-crystallin as a molecular chaperon. Studies by our group have confirmed that a-crystallin binds denaturing proteins, thus preventing their aggregation. Our current studies involve the nature and stability of the complex produced and the role such complex fonnation may play in the remaricable ability of the lens to remain transparent for decades, even though it has very limited synthesis and repair capabilities. A major new initiative of our group concerns utilization of lens organ culture as a means of identifying therapeutic agents with efficacy as anticataract agents. Our studies have involved several cataract-inducing systems and a number of potential anticataract agents, including those with antioxidant activity. 130 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 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 model systems suitable 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 Uquid chromatography, fluorescence, and circular dichroism techniques. Lens organ culture experiments use rat or monkey lenses and utilize active transport 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 electiophoretic methods, and the polymerase chain reaction. Major Findings 1. We showed that the susceptibility of lenses from different species to ultraviolet Ught-induced photodamage is dependent upon the levels of reduced pyridine nucleotide present, supporting the contention that the cellular redox potential may be a parameter which could be modified to increase resistance to cataraaogenesis. 2. We have shown that condensates produced from either tobacco or wood smoke are toxic to the lens. Membrane damage has occurred in lenses exposed in organ culture to such smoke condensates. 3. We have measured a variety of biochemical and physiological parameters in cultured lenses exposed to various cataractogenic conditions to determine the best indicators for assessment of the effects of potential anticataract drugs. 4. We have corrfirmed the "chaperone-like" activity of a-crystallin in protecting other proteins from stress-induced aggregation and have demon- strated by several methods the formation of com- plexes between a-crystallin and other proteins during thermal stress. 5. We completely charaaerized the guinea pig C- crystallin gene and showed it contains two separate promoters. One promoter accounts for high lens- specific expression while the other appears to direct low-level expression in many tissues. 6. The cDNAs for human and mouse ^-crystaUin have been sequenced and show high homology with the guinea pig sequence. The genes for these species, which are expressed only at low levels in the lens, lack the lens-specific promoter. 7. The human ^-crystallin gene has been localized to chromosome 1. 8. The ^-crystaUin present in guinea pig lens has been shown to be catalytically functional. This we proved by demonstrating stimulation of the hexose monophosphate shunt in organ-cultured lenses exposed to quinone substrates of ^-crystallia 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 studies are aimed primarily at elucidating the roles of the lens crystallins, the primary structural elements of the normally transparent lens matrix, in the processes leading to opacification. Such knowl- edge should contribute to the development of means of intervention that can prevent or delay the process of cataraa 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 underlying the high lens-specific expression of an enzyme/crystallin C^- crystallin); and (3) further investigate the chaperone- Uke function of a-crystallin and determine its physio- logical significance in the normal lens and in cataract 131 Laboratory of Mechanisms of Ocular Diseases NEI Annual Report-FY 1992 NEI Research Program Cataract — Pathogenetic Mechanisms Publications Datiles MB, Schumer DJ, Zigler JS Jr, Russell P, Anderson L, Garland D: Two-dimensional gel electrophoretic analysis of human lens proteins. Curr Eye Res 11:669-677, 1992. Du X-Y. Linser P, RusseU P, Zigler JS Jr: Carbonic anhydrase III is expressed in bovine lens. Curr Eye Res 11:475-478, 1992. Krishna CM, Uppuluri S, Riesz P, Zigler JS Jr, Balasubramanian D: A study of photodynamic efficiencies of some eye lens constituents. Photo- chem Photobiol 54:51-5%, 1991. Lee DC, Gonzalez P, Rao V, Zigler JS Jr, Wistow GJ: Carbonyl-metabolizing enzymes and their relatives recruited as structural proteins in the eye lens. Prog Clin Biol Res, in press. Rao PV, Krishna CM, Zigler JS Jr: Identification and characterization of the enzymatic activity of zeta- crystaUin firom guinea pig lens. J Biol Chem 267:96-102, 1992. Rao MC, Zigler JS Jr Levels of reduced pyridine nucleotides and lens photodamage. Photochem Photobiol 65:523-528, 1992. 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. Rao P, Zigler JS Jr: Quinone induced stimulation of hexose monophosphate shunt activity in the guinea pig lens: Role of zeta-crystallia Biochim Biophys Acta 1116:75-81, 1992. Rao PV, Zigler JS Jn Purification and characteriza- tion of zeta-crystallin/quinone reductase fi-om guinea pig liver. Biochim Biophys Acta 1117-315- 320, 1992. Rodriquez IR, Gonzalez P, Zigler JS Jr. Borras, T: A guinea pig hereditary cataract contains a splice site deletion in a crystallin gene. Biochim Biophys Acta 1180:44-52, 1992. Roquemore EP, Dell A, Morris HR, Panico M, Reason AJ, Savoy L-A, Wistow GJ, Zigler JS Jr! Earles BJ, Han GW: Vertebrate lens a-crystallins are modified by 0-linked N-acetylglucosamine. / Biol Chem 267:555-563, 1992. Russell P, Zigler JS Jr: Analysis of the effect of eye bank storage conditions on primate lens epitheli- imi. Exp Eye Res 54:153-156, 1992. Xu G-T, Zigler JS Jr, Lou MF: Establishment of a naphthalene cataract model in vitro. Exp Eye Res 54:73-81, 1992. Xu G-T, Zigler JS Jr, Lou MF: The possible mecha- nism of naphthalene cataract in rat and its preven- tion by an aldose reductase inhibitor (AL 1576). Exp Eye Res 54:63-72, 1992. Zigler JS Jr: Lens proteins, in Albert DM, Jakobiec F (eds): Principles and Practice of Ophthalmol- ogy. Philadelphia, JB Saunders Co, in press. 132 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00243-06 LMOD PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or lass. Title must til on one line between the borders.) Ocular Cells Cultured Under Normal and Diabetic Conditions PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator) (Name, title, laboratory, and Institute alflllalion) PI: Bruce A. Pfeffer Ph.D. Senior Staff FeUow LMOD, NET Others: W. Gerald Robison, Jr. Ph.D. Chief, Section on Pathophysiology LMOD, NEI COOPERATING UNITS (It any) Departments of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, MO (Christine Blazynski, Ph.D.) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Pathophysiology INSTITUTE AND LOCATION NEI, NIH, Bethesda. MD 20892 TOTAL STAFF YEARS: 0.0 PROFESSIONAL: 0.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [x| (b) Human tissues Q (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This project has been terminated. 133 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00149-19 LMOD PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or lass. Title must lit on one line between the borders.) Ultrastructure and Function of the CeUs and Tissues of the Eye PRINCIPAL INVESTIGATOR (List other prolesslonal personnel below the Principal Investigator) (Name, title, laboratory, and Institute anuialion) PI: W. Gerald Robison, Jr. Ph.D Head, Section on LMOD, NEI Pathophysiology Others: Nora Laver Anne Groome Joe Hackett Evita Bynum Joel Glover M.D. Visiting Associate LMOD, NEI Histology Technician LMOD. NEI Biologist LMOD. NEI Microbiologist LMOD, NEI Biologist LMOD, NEI COOPERATING UNITS (11 any) Alcon Laboratories, Inc. (BiUie M. York, Jr.. Ph.D.) LAB/BRANCH Laboratory of Mechanisms of Ocular Diseases SECTION Section on Pathophysiology INSTfTUTE AND LOCATION NEI, NIH. Bethesda. MD 20892 TOTAL STAFF YEARS: 5.25 PROFESSIONAL: 1.75 3.50 CHECK APPROPRIATE BOX(ES) r~1 (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.) This laboratory has developed a novel procedure which permits, in both animals and humans, isolation of the entire vasculature networic of the retina in an intact and essentially imdistorted arrangement. Although a trypsin digestion procedure was developed 30 years ago for isolation of himian retinal vessels, it has many limitations and has not been as dependable in animals. Systematic tests with rat retinas revealed that elastase, a "contaminant" of the crude trypsin mixture traditionally used, is much more effective than trypsin for the selective preservation of retinal vessels. Digestion with purified elastase permits clear identification of the degeneration of pericytes ("ghosts"), the first lesion of diabetic retinopathy. Elastase-based procedures should apply directly to retinopathy of prematurity and provide new approaches in tissues, such as brain and inner ear. This procedure was the key to obtaining success in our studies on an animal model for the very distinct group of lesions characteristic of human backgroimd diabetic retinopathy. The galactose-fed rat model, first developed in our laboratory, takes advantage of the fact that aldose reductase has a higher affinity for galactose than for glucose, which results in more intracellular polyol accumulation in galactosemia. We fed rats galactose for extended periods in an effort to produce diabetic-like retinal lesions. Galactosemia did indeed induce diabetic-like microangiopathies that were more advanced and more like himian diabetic lesions than those which develop in long-term diabetic rats. The galactose-fed rat model has distinct advantages over genetic or chemically induced models of diabetes for intervention studies. Intervention studies are under way to determine appropriate times for intervention via different aldose reductase inhibitors. 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 ceU proliferation and subsequent pathologies. 134 PHS 6040 (Rev. 5«2) NEI Annual Report-FY 1992 Laboratory of Mechanisms of Ocular Diseases Project Description Objectives The use of special diets in vivo as well as ceU culture of ocular tissues in various controlled media to mimic the diabetic state is designed to determine whether diabetic-like tissue changes can be prevented by inhibitors of aldose reductase (AR). Methods Sprague-Dawley rats were fed a normal or a 50% galactose diet with or without an AR inhibitor (tolrestat or sorbinil, 0.05% w/w) for 6 to 28 months. Mild enzyme digestion was used on the retina of one of the eyes of each rat to remove all the retinal tissues except the vessels. This digestion provided a whole mount of the retinal vasculature, thus permit- ting recognition of degenerated pericytes ("ghosts") and aU of the more advanced angiopathies by hght microscopy. The retina of the other eye of each pair was sectioned and examined by electron microscopy. Tissue culture of human, bovine, and canine retinal capillary pericytes and lens epithelial ceUs was used to investigate the mechanism underlying the diabetic angiopathies. Major Findings Elastase, not trypsin, is the most effective enzyme of the crude trypsin mixture traditionally used for preparation of retina vessels. Vascular whole mounts prepared by this new enzyme digestion procedure demonstrated multiple retinal angiopathies identical to those typical of human background diabetic retinopathy in the capillaries of rats fed galactose for 24 months. These angiopathies did not occur in the retinas of rats fed a galactose diet with an AR inhibitor. AR was shown to be present in cultured retinal pericytes (1) by immimohistochemistry involving use of antibody against human placental AR, (2) by its activity as indicated by measurements of xylitol production in ceUs grown in a medium supplemented with xylose, and (3) by the detection of messenger RNA for AR. There was a compro- mised proliferation rate in pericytes compared with that of endothelial cells with incubation in high (30 mM) sugar concentrations, suggesting toxicity of polyol at the cellular level. AR appears to be in- volved in aU the retinal complications of diabetes. from pericyte degeneration to microaneurysm formation. 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. However, 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 experi- mental studies and drug testing on diabetic retinop- athy 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. AU this has been possible because AR is involved in diabetic retinopathy. AR has been implicated in sugar cataracts, certain corneal healing defects, and peripheral neuropathy of diabetic and galactosemic animals. It now appears to be involved in all lesions of background diabetic retinopathy as well. While the normal physiological role of this enzyme in most tissues remains un- known, AR under the conditions of high plasma sugar concentrations encountered in diabetes and galactosemia converts these sugars to their respective sugar alcohols (polyols). These polyols are not readily metaboUzed, nor do they penetrate cell membranes easUy. Once formed at significant rates, they may accumulate to very high Ifevels in cells, leading to hypertonicity, alteration of ion permeabil- ity, and eventual cell death with consequent tissue changes such as cataract formation. Treatment of diabetic or galactosemic rats with potent AR inhibi- tors (eg, such as sorbinil or tolrestat) decreases polyol accumulation, 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. 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 by using a novel vessel preparation method. Pericyte loss, endotiielial ceU proliferation, microaneurysms, shunts, occlusions, dilations, and all the other micro- angiopathies that we foimd in the galactose-fed rat are identical to the histopathologies that characterize human background diabetic retinopathy. Until now. 135 Laboratory of Mechanisms of Ocular Diseases NEI Annual Report-FY 1992 the only other experimental animal model has been the diabetic or galactoseraic dog. We have shown for the first time that diabetic-like retinopathy in galacto- semic rats can be prevented with an AR inhibitor. Proposed Course The following studies are proposed for Fiscal Year 1993. We wiU extend the intervention studies to determine how late one can interrupt the disease process and still obtain beneficial results by treatment with various AR inhibitors. We also will examine u:e early formation of intracellular vacuoles, cell trans- port systems, the mechanism of basement membrane synthesis, and the relationship of these changes to AR in isolated retinal ceUs grown under diabetic conditions. We will manipulate the rat diets to shorten the time required for diabetic-Uke retinal angiopathies to appear, thus improving the rat as a model for diabetic retinopathy. NEI Research Program Retinal Diseases — Diabetic Retinopathy Publications Katz ML, Robison WG Jn Senescent alterations in the retina and retinal pigment epitheUum: Evi- dence for mechanisms based on nutritional stud- ies, in Armstrong DA, Marmor MF, Ordy JM (eds): The Effects of Aging and Environment on Vision. New York, Plenum Press, 1991, pp 195- 208. Laver N, Robison WG Jr Diabetic retinal vascular abnormalities compared to the rat model. Pro- ceedings of the Sixth Congress, US-Japan Coop- erative Cataract Research Group, Kona, HI (Nov 30-Dec 5, 1991), p 205. Laver N, Robison WG Jr, Calvin HI, Fu S-CJ: Early lesions in cataracts of GSH-depleted mouse pups retarded by ascorbate. Invest Ophthalmol Vis Sci 33(4): 1041, 1992. Laver N, Robison WG Jr, Calvin HI, Fu S-CJ: Early ultrastructural lesions in cataracts of GSH-deplet- ed mouse pups. Proceedings of the Sixth Congress, US-Japan Cooperative Cataract Re- search Group, Kona, HI (Nov 30-Dec 5, 1991), p 111. 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, in press. McCaleb ML, McKean ML, Hohman TC, Laver N, Robison WG Jr: Intervention with the aldose reductase inhibitor, tolrestat, in renal and retinal lesions of streptozotocin-diabetic rats. Diabetolo- gia 34:695-701, 1991. Robison WG Jr, Laver N: Ocular lesions in animal models of human diabetes, in Shafiir E (ed): Frontiers in Diabetes Research, Lessons from Animal Diabetes TV. London, Smith-Gordon and Company Limited, 1992, pp 145-163. Robison WG Jr, Laver N, Kador PF: Meager delay of sugar cataracts and pre-proliferative retinopathy in galactose-fed rats by the ARI ponalrestat. Invest Ophthalmol Vis Sci 33(4):878, 1992. Robison WG Jr, Laver N, Kinoshita JH: Galacto- semic rat model for diabetic retinopathy: Preven- tion of capillary dilations, microaneurysms, aceUularity, and shunt meshworks with aldose reductase inhibitors. Workshop on Aldose Reduc- tase Inhibitors. NIH Pub No 91-3114, 1991, pp 89-100. Robison WG Jr, Laver N, Kinoshita JH: Similar retinal vascular lesions in diabetic humans and rats with elevated tissue polyol. Proceedings of the Sixth Congress, US-Japan Cooperative Cata- ract Research Group. Kona, HI (Nov 30-Dec 5. 1991), p 183. Zadunaisky JA, Spring K, Sellers J, Robison WG: Contraction and drug sensitivity of cultured human trabecular meshwork cells in normal and glaucoma. Invest Ophthalmol Vis Sci 33(4):1163, 1992. 136 ? . •«l Laboratory of Molecular and Developmental Biology 2 Report of the Chief, Laboratory of Molecular and Developmental Biology Joram Piatigorsky, Ph.D. The Laboratory of Molecular and Developmental Biology CLMDB), completing its 11th year, comprises three sections: the Section on Molecular Genetics (headed by Joram Piatigorsky, Ph.D.), the Section on Cellular Differentiation (headed by Peggy S. Zelenka, Ph.D.), and the Section on Molecular Structure and Function (headed by Graeme J. Wistow, Ph.D.). The laboratory has grown this year, both in numbers of investigators and space. Renova- tions throughout BuUding 6 have resulted in an approximate 30% increase in laboratory space. The result is the acquisition of a cold room, a second common equipment room, another small darkroom (for autoradiography and immunofluorescence), and four additional modules for laboratory work. In addition to relieving congestion, the renovations have allowed the three sections to be collated and grouped together in a more logical and productive manner. Thus, the NEI continues to be supportive and gener- ous to us, and we are grateful. The focus of LMDB research remains to under- stand the fundamentals of gene expression and cellular differentiation in the eye, with great empha- sis on the lens. Our studies occasionally include examination of nonocular tissues, especially since the recent discovery that most crystaUins have nonrefrac- tive functions in many tissues. We have also made contributions on the retina, and we are continuing last year's initiative on the cornea. We hope that, in addition to basic research, the accessibility and particular biological attributes of the cornea wiU lead to applied contributions in the area of gene therapy. Much of our work continues to be placed in a context of evolution. This year has been very productive, as described in the detailed annual reports of the individual sections; some highlights are given below. The distinctions received by LMDB this year are numerous. Dr. Piatigorsky received a Special Recog- nition Award from ARVO, delivered the fourth Donald Abbott Lecture at Hopkins Marine Labora- tory of Stanford University, and was a Wellcome Visiting Professor in the Basic Medical Sciences at the University of Louisville. Dr. Zelenka received the NEI Director's Award for her outstanding contri- butions on proto-oncogene expression during lens differentiation; she was also elected to serve on the Visual Sciences Al Study Section. Drs. Piatigorsky, Zelenka, Wistow, and QiepeUnsky aU organized symposia in their respective fields at the International Congress of Eye Research in Stresa, Italy, indicative of their leadership roles in eye research. Finally, in the LMDB review by the Board of Scientific Coun- selors, each group was praised for its research contributions, and the LMDB was considered the leader among research laboratories on the molecular biology of the eye. Section on Molecular Genetics The Section on Molecular Genetics has ventured into the complex area of protein:DNA inter- actions involved in the expression of crystaUin genes. These experiments have opened exciting new areas of investigation. For example, recent findings indicate that although cw-regulatory elements of the mouse oA-crystallin gene are occupied by nuclear proteins, regardless of whether the gene is active, the interactions with these proteins appear subtly differ- ent, depending on whether the gene is being ex- pressed. We established that one of the key regula- tory elements (the oA-CRYBPl site) binds the cloned factor oA-CRYBPl, but that multiple forms of this putative transcription factor exisL In addition, different cell types contain different forms of otA- CRYBPl. Another intriguing finding is that the otA- CRYBPl site appears to bind ccB-crystallin, raising the possibility that this crystaUin may act to control transcription. More cjs-regulatory elements have been discovered in different crystaUin genes. Espe- ciaUy interesting is the finding that the otB-crystaUin 139 Laboratory of Molecular and Developmental Biology NEI Annual Report-FY 1992 gene has a strong muscle/weak lens enhancer up- stream of a site which may, according to footprinting analysis, be a lens-specific regulatory element. The muscle-preferred enhancer has a regulatory element which functions only in muscle cells. Other putative regulatory regions appear to be involved in the heat shock response of this gene and in expression in lung and brain, both of which use different promoters than that used in lens and other tissues. Thus, this year has clearly delineated the complexity of crystallin gene expression and has focused our attention on certain key aspects of it. The Section on Molecular Genetics has continued to examine the crystallins of invertebrates with cellular lenses. We hope that these models of convergent evolution wiU elucidate basic aspects of crystallins and their genes required for the optical properties of the transparent lens. In addition to cloning and analysis of new crystallins in cephalo- pods (squid and octopus) and jellyfish, we deter- mined that the S-crystallins, which are related to glutathione S-transferase, are recruited by the ceph- alopods as crystallins via gene duplication, without recruitment of the parental gene encoding the protein with enzymatic activity. This is an atypical situation in vertebrates: Although variations on the theme exist, the genes encoding the enzyme-crystaUins usually are identical to those encoding the active metabolic enzymes, hence we coined the term "gene- sharing." Crystallin recruitment is a fascinating portrait of molecular evolution that continues to kindle our curiosity. Dr. Chepelinsky's group in the Section on Molec- ular Genetics has advanced in its characterization of cis-acting elements involved in the regulated expres- sion of the lens-specific membrane protein (major intrinsic protein). In addition, the group is contin- uing to study in transgenic mice the effect of direa- ing growth factors and other biologically interesting proteins to the lens. Of particular interest this year was its finding that ectopic expression of interferon- gamma (IFN-y) in the lens induced major histocom- patibility complex class n gene expression in the eye and disrupted the developmental program of the lens and retina. We hope this transgenic mouse will become a useful model for the study of ocxilar autoimmune disease. It promises to provide insight into the tight control of gene expression and cell differentiation in the eye. Section on Molecular Structure and Function The Section on Molecular Structure and Function is primarily interested in the molecular basis for crystallin gene recruitment and in growth factors or lymphokines with roles in lens differentiation. The putative lens-promoter of C-crystaUin has been analyzed, showing that discrete regions confer lens preference while upstream positi . and negative elements confer improved lens specificity. In contrast to the dual promoters of ^-crystallin, this group demonstrated that 61, 52 and e-crystaUin genes use the same promoters for lens and nonlens expression. The complete duck x-crystallin gene has been intro- duced into transgenic mice to study the consequences of overexpression of an enzyme in the lens. The cDNA of p-crystallin, a mammalian taxon- specific crystallin and homologue of bacterial omi- Uiine cyclodeaminases, has been cloned. Outside the lens, p-crystallin is expressed in the retina and brain. Human retina p has been cloned. In the developing chick retina, p is found preferentially in photorecep- tors. This may indicate the presence of an unusual pathway for ornithine metabolism in the retina which could be related to the problems of gyrate atrophy. The expression of lens lOK/MIF (migratory inhibi- tory factor), previously identified as a lymphokine, has been shown to be coordinated with cellular differentiatioa Apart from any role in differentia- tion, this protein could play a role in lens-induced eye inflammations because mouse lens extract possesses MIF activity. Section on Cellular DifTerentiation Members of the Section on Cellular Differentia- tion have continued their investigations on the study of proto-oncogenes in the lens. Recent studies have revealed unexpected patterns that provide insights into the mechanisms of cell cycle arrest during differentiation. Extension of this study to include the tumor suppressor genes, p53 and Rb, and the cell cycle regulatory protein, cyclin B, has shown that all tlwee are expressed in embryonic chicken lens fiber ceUs. Whereas fiber cells are generally 140 NEI Annual Report-FY 1992 Laboratory of Molecular and Developmental Biology believed to be arrested in tlie early Gl or GO portion of the cell cycle, these proteins are usually expressed in ceUs in late Gl, S, or G2 phase. Expression of cyclin B in lens-fiber ceUs has been confirmed by sequencing of the polymerase chain reaction product and by immunoblotting with an antibody specific for cyclin B. Another insight into cell cycle regulation stems from analysis of proto-oncogene expression in the synchronized cell population of the lens epithelium, as previously described by this Laboratory. Recent experiments demonstrated that these ceUs periodically accumulate very high levels of c-fos mRNA. The possibility that high c-fos mRNA levels may be related to the mechanism that leads to synchroniza- tion of these cells is under investigation. The past year also has seen significant progress in determining the functional role of proto-oncogenes in the lens, with the identification of the x-crystaUin/a- enolase gene as a target for transcriptional regulation by c-rayc. 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 an avian retroviral veaor. Use of this vector permits transfer of the c-jun constructs to cultured cells; efficiencies approaching 100% make it possible to test for effects of proto-oncogene expression on DNA synthesis, differentiation, and expression of endoge- nous genes. Preliminary results indicate that a negative dominant mutation of c-jun has a positive effect on transcription of the endogenous otA-crystal- lin gene. Finally, insights also have been gained into the cellular mechanisms that regulate proto-oncogene expression in the lens. A collaborative study with Dr. Thomas Lysz (University of Medicine and Dentistry of New Jersey) has demonstrated that c- myc mRNA expression in the neonatal rat lens requires endogenous production of 12-hydroxyeicosa- tetraenoic acid (19-HETE), a lipoxygenase pathway metabolite of arachidonic acid. Inhibition of the 12- lipoxygenase activity of the lens blocks both c-myc expression and DNA synthesis, suggesting that this may represent a control point in the cell cycle. 141 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00238-07 LMDB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 charaaers or lass. Title must tit on one line batween the borders.) Proto-Oncogene Expression During Lens Differentiation and Development PRINCIPAL INVESTIGATOR (List other professional parsonnel below the Principal Investigator.) (Name, title, laboratory, and kistltute atliliallon) PI: Others: Peggy S. Zelenka Barbara BrewiU Jo Arm Rinaudo John Talian Ronald Warwar Chun Yun Gao Emmanuel Vacchiano Anuradha Rampalli Jaspreet Arora Ph.D. Head, Section on Cellular Differentiation LMDB. NEI Ph.D. Staff FeUow LMDB, NH Ph-D. Staff Fellow LMDB, NH Ph.D. Staff Fellow LMDB, NH M.D. Staff FeUow LMDB, NEI M.D.. Ph-D Staff Fellow LMDB, NH Ph.D. Staff Fellow LMDB, NH Ph.D. Visiting Fellow LMDB, NH Ph.D. Visiting Fellow LMDB, NH (Additional personnel listed under Program Description.) 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 NEL NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 5.15 PROFESSIONAL: 4.23 0.92 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews Q (b) Human tissues [x] (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) This projea investigates the expression of proto-oncogenes in the embryonic chicken lens and their relationship to cell growth, quiescence, and differentiation. The normal developmental profiles of four nuclear proto-oncogene mRNAs (c-myc, N-wyc, c-fos, and c-jun) have been completed, and tiie study is now being extended to include the tumor suppressors, p53 and Rb, and cell-cycle regulatory proteins such as the cyclins. Functional studies have demonstrated that c-myc is involved in the transcriptional regulation of the x-crystallin/a-enolase gene, and preliminary results implicate c-jun in the transcriptional regulation of a-crystallin. Further analysis of cell-cycle synchrony in the lens epitheliimi demonstrates that many synchronized cells are present in the central lens epithelium in the 14-day-old chicken embryo and that synchronized cells periodically express high levels of c-fos mRNA. 142 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Molecular and Developmental Biology Project Description Additional Personnel Sambath Chung VuBui Graeme Wistow B.A. Ph.D. Technician, LMDB, NEI Summer Student, LMDB, NEI Section Head, LMDB, NEI Objectives In this project we seek to determine whether the expression of specific proto-oncogenes is altered during lens ceU differentiation and to determine the function of the corresponding proto-oncogene prod- ucts in the developing lens. Methods Techniques of molecular biology are used in con- junction with traditional cell biology techniques. Conventional methods employed for protein and nucleic acid analysis include polyacrylamide gel electrophoresis, RNA and DNA isolation, polymerase chain reaction (PCR), nucleic acid hybridization, in vitro transfection, in situ hybridization, immunocyto- chemistry, and immunoblotting. DNA-protein inter- actions are studied using DNAsel footprinting, elec- trophoretic mobility shift assays, and ultraviolet (UV)-cross-linking. Studies employ lens epithelia and lens fibers of embryonic chickens, explants of embryonic chicken lens epithelia, and primary cultures of embryonic chicken lens epithelial cells. Major Findings In the past year we expanded our study of proto- oncogenes in the embryonic chicken lens to include the tumor suppressor genes p53 and Rb and the cell- cycle regulatory protein cyclin B. Development of a PCR assay for Rb mRNA is in progress. PCR assays have been developed for p53 and cyclin B, and DNA internal standards have been constructed to permit quantitation of PCR products. Expression of these proteins is known to be cell- cycle dependent in proliferating cells, p53 appearing at the Gl/S boundary and cyclin B in the S and G2 phases of the cell cycle. Interestingly, our PCR data indicate that both are expressed in embryonic chicken lens fiber cells although these cells are generally believed to be arrested in the early Gl or GO portion of the cell cycle. Expression of cyclin B in lens-fiber cells has been confirmed by sequencing the PCR product and by immunoblotting with an antibody specific for cyclin B. Immunocytochemical smdies are in progress. These studies may help to define the mechanisms of ceU-cycle arrest employed by differentiating lens fiber cells. A major goal of this project continues to be the biological function of the proto-oncogenes expressed in the lens. The past year has seen significant progress in this area with the identification of the x- crystaUin/a-enolase gene as a target for transcrip- tional regulation by c-myc. Cotransfection studies involving a c-myc cDNA expression vector and a plasmid construct consisting of 800 bp of 5'-flanking sequence from the duck x-crystaUin gene coupled to the reporter gene, chloramphenicol acetyltransferase (CAT), indicate that expression of c-myc increases transcription ftum the x-crystaUin promoter. This effect required the putative c-myc binding site at -624/-619. Nuclear extracts from both HeLa cells and embry- onic chicken lenses contained proteins which pro- tected this site from DNAsel digestion and retarded the migration of an oligonucleotide containing this site in electrophoretic mobility shift assays (EMSAs). DNAsel protection and retardation in the EMSA were abolished by mutations in the putative c-myc site. Finally, UV coupling of proteins from HeLa or lens nuclear extracts to an oligonucleotide containing this site, followed by immunoblotting with an anti- body specific for c-myc protein, confirmed that c- myc protein itself participates in binding to this site. These findings identify the x-crystallin gene as a target for transcriptional regulation by c-myc and help explain both the regional and developmental patterns of x-crystaUin expression. We have explored the function of c-jun in the embryonic chicken lens using wild-type and mutated cDNAs for chicken c-jun, cloned into the avian retroviral vector RCAS. Use of this vector permits transfer of the c-jun constructs to cultured cells with efficiencies approaching 100%, making it possible to test for effects of c-jun on DNA synthesis, differenti- ation, and expression of endogenous genes. Prelimi- nary results indicate that a negative dominant muta- 143 Laboratory of Molecular and Developmental Biology NEI Annual Report-FY 1992 tion of c-jun has a positive effect on transcription of the endogenous otA-crystallin gene. The mechanism of this effect and the possible effects on transcription of other crystallin genes are being investigated. With progress in determining how certain proto- oncogenes may play roles in regulating expression of lens crystallins, we also have gained insights into the cellular mechanisms that regulate proto-oncogene expression in the lens. In collaboration with Dr. Thomas Lysz (University of Medicine and Dentistry of New Jersey), we recently have found that c-myc mRNA expression in the neonatal rat lens involves endogenous production of 12-hydroxyeicosatetraenoic acid (12-HETE), a lipoxygenase pathway metabolite of arachidonic acid. Dr. Lysz has further shown that inhibition of the 12-lipoxygenase activity of the lens prevents DNA synthesis. These studies indicate that 12-HETE synthesis is necessary at an early step in the cell cycle, prior to expression of c-myc. Together with Dr. Lysz, we now are investigating the relationship of 12-HETE to c-fos and c-jim expres- sion and the possibility that production of this eicosanoid may be a control point in the lens epithe- lial cell cycle. Previous studies in this laboratory have demon- strated that the lens epithelium of chicken embryos at 13-16 days' development contains a population of cells that are synchronized with respect to their division cycles. We have furthered our investigation of this population using autoradiography of epithelia labeled with tritiated thymidine to localize the synchronized cells. The results indicate that the synchronized population includes nearly all the proliferating cells of the central epithelium and extends into the "proliferative zone" of the lens, where it is intermixed with a large number of asyn- chronously dividing cells. It is likely that the large proportion of synchro- nized cells in the central epithelium caused eailier investigators to underestimate the number of prolifer- ating cells in this region. Analysis of proto-onco- gene expression in the synchronized cell population demonstrated that these cells periodically accimiulate very high levels of c-fos mRNA. We currently are investigating the possibility that high c-fos mRNA levels may be related to the mechanism that leads to synchronization of these cells. Significance to Biomedical Research and the Program of the Institute The proto-oncogenes are normal cellular homologs of retroviral oncogenes. Whereas retroviral transforma- tion disrupts cell growth and differentiation, it is likely that the proto-oncogenes are involved in the normal regulation of these processes. Therefore, a study of proto-oncogene expression during lens cell differentiation may elucidate basic regulatory pro- cesses underlying lens ceU growth and differentia- tion. Many types of cataract are associated with abnormal lens epithelial ceU growth and the inhibi- tion of lens fiber cell differentiation. In addition, a number of other diseases of the eye involve loss of normal controls on cell proliferation. An understand- ing of the basic controls of ceU growth and differen- tiation would further our understanding of these disease states. Proposed Course Several studies are in progress or proposed for Fiscal Year 1993: 1. We will determine the pattern of expression of p53 and Rb mRNAs at different developmental stages and in different regions of the lens. 2. We will explore further the unexpected expres- sion of cyclin B in differentiating lens fiber cells. If initial findings are confirmed, we wiU conduct experiments to determine whether cyclin B in lens fibers is associated with the product of the cdc2 gene, p34, and whether the complex has mitosis- promoting factor activity. 3. We will explore the possible role(s) of the c- jun proto-oncogene in proliferation and differentia- tion using an avian retroviral vector to introduce wild-type and mutant versions of the c-jun cDNA into primary cultures of lens epithelial cells. 4. We will examine the effect of the N-myc proto-oncogene on transcription directed by the t- crystallin/a-enolase gene, using the techniques used to study the role of c-myc. 5. We will study the possibility that glucocorti- coids are involved in regulating the lens epithelial cell cycle, using glucocorticoid analogs and cultured lens epithelial cells. 144 NEI Annual Report-FY 1992 Labor atory of Molecular and Developmental Biology 6. We will investigate the synchronization of the Publications lens epithelial ceU cycle that we observed in 13- to Brewitt B, Talian JC. Zelenka PS: Cell cycle syn- 16-day-old chicken embryos in younger and older chiony in the developing chicken lens epithelium, embryos. Dev Biol 152:315-322. 1992. 7. We will pursue the collaborative effort with Harris LL, Talian JC, Zelenka PS: Contrasting Dr. Lysz to define the role of 12-HETE in regulating patterns of c-myc and N-myc expression in c-myc expression and DNA synthesis in lens epithe- proliferating, quiescent, and differentiating cells lial cells. of the embryonic chicken lens. Development 115:813-820, 1992. NEI Research Program ^""^^ JAS Zelenka PS. Expression of c-fos and c-jun mRNA m the developmg chicken lens: Cataract — ^The Normal Lens Relationship to cell proUferation, quiescence, and differentiaUon. Exp Cell Res 199:147-153. 1992. 145 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00251-05 LMDB PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or lass. Title must lit on one line between the borders.) Genetically Engineering the Eye with the qA-Crystallin Promoter PRINCIPAL INVESTIGATOR (Ust other prolasslonal personnel below the Principal Investigator.) (Name, title, laboratory, and Institute aHillation) PI: Ana B. Chepelinsky Ph.D. Research Biologist LMDB, NEI Others: Devonne Paiker B.S. Charles Egwuagu Ph.D. Chi-Chao Chan M.D. Robert B. Nussenblatt M.D. Jorge Sztein D.VM. Biologist Scientist, PHS Chief, Section on Immunopathology Clinical Director Visiting Associate LMDB, NEI LI, NEI LI, NEI LI, NEI VRRS, NEI COOPERATING UNITS (II any) E)epaTtmem of Cell Biology, Baylor College of Medicine, Howard Hughes Medical Institute (Paul Oveibeek, Ph.D.); Imperial Cancer Research Fund, London, England (Qive Dickson, Ph.D.); Gerontological Research Unit, National Institute of Health and Medical Research, Paris, France (Yves Courtois, Ph.D.; Maryvotme Laurent, Ph.D.) 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: 0.5 0.1 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues [xj (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Interferon-gamma (LNF-y) gene expression was directed to the lens of transgenic mice with the murine oA- crystaUin promoter. The transgenic mice obtained exhibited severe microphthalmia and microphakia; the lens architecture was disorganized, and lens-fiber cells were replaced by balloon ceUs. Differentiation of the neuroretina into inner and outer neuroblastic layers was anested at the embryonic stage. Retinal detachment and the presence of macrophages in the subretinal space was observed in the adult mouse eye. The ectopic expression of IFN-y in the transgenic mouse eye induced major histocompatibility complex (MHC) class n gene expression in the eye and arrested differentiation of lens and retina cell lineages. Our data suggest that IFN-y can also regulate gene expression in nonlymphoid tissues and provide an animal model for studying the roles of IFN-y and MHC class II in ocular autoimmxme diseases. 146 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Molecular and Developmental Biology Project Description Objectives The objective of this project is to understand how aberrant genetic expression of interferon-gamma (IFN-Y). int-2, or acidic fibroblast growth faaor (aFGF), under the control of the otA-crystallin promoter, perturbs normal eye development in transgenic mice. Methods Recombinant DNA techniques used in this smdy include plasmid construction, Southern and Northern hybridizations, DNA sequencing, primer extension, polymerase chain reaction (PCR), reversed transcrip- tion PCR (RT-PCR), and immunohistochemistry. The project involves the production and analysis of transgenic mice. Major Findings IFN-y. — ^This project is conducted in collaboration with the NEI Laboratory of Immunology. WN-y is specifically expressed by T lymphocytes and natural killer cells in response to viral or bacterial infections. 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 histocompatibility 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 oA-crystallin promoter (-366/ +46) fused to the murine IFN-y coding sequence. We obtained one FVB/N and two Balb/c transgenic lines to study the effect of different genetic back- grounds on the expression of the transgene. The transgenic lines presented a particular phenotype that segregates with the presence of the transgene: closed eyes and microphthalmia. Histological findings include serous retinal detachment, appearance of macrophages in the subretinal space, and loss of photoreceptor outer segments in adult Balb/c trans- genic mice. Developmental studies indicated that the differen- tiation of the lens was affeaed during embryonic development, with the replacement of normal pri- mary lens fibers by balloon cells. Abnormal differ- entiation of the retina also was observed, particularly in the neural retina. The retinal detachment observed in the adult due to the lack of appropriate differentia- tion of the retina and the activation of expression of MHC class II gene that is involved in antigen presen- tation render this an animal model for ocular auto- immune diseases. int-2. — In collaboration with Dr. Qive Dickson from the Imperial Cancer Research Fund (England) and Dr. Paul Overbeek from the Baylor College of Medicine, we injected the otA-crystallin promoter (-366/+46) fused to int-2 cDNA into mouse embryos. We obtained three transgenic lines that exhibited severe microphthalmia and the unusual presence of putative secretory epithelia and "dennoids" inside their eyes. The characterization of the eye pathology and the abnormal eye development produced by the aberrant expression of int-2 in the lens may shed some light on the function of this proto-oncogene. It may provide an animal model for some human ocular diseases. oFGF. — In collaboration with Dr. Overbeek and Dr. Yves Courtois from the Institute for Gerontologi- cal Research, INSERM (France), we injected into mouse embryos a recombinant DNA containing the otA-crystaUin promoter (-366/-i46) fused to the bovine aFGF cDNA. We do not yet know whether the transgene is expressed in two foimders that were obtained. Significance to Biomedical Research and the Program of the Institute The aberrant expression of DTNl-y, int-2, or aFGF will allow us to elucidate the mechanisms underlying eye development. At the same time, it will open new avenues in the development of animal models for the study of eye pathologies and gene regulation in the eye. Proposed Course Studies wiU continue during Fiscal Year 1993 with further characterization of the transgenic mice already obtained. The effect of the expression of these transgenes on lens differentiation and eye phenotype will be studied further. NEI Research Program Cataract — Molecular Genetics 147 Laboratory of Molecular and Developmental Biology NEI Annual Report-FY 1992 Publications Jaworski CJ, Chepelinsky AB, Piatigorsky J: The Donovan DM, Sax CM, Klement JF, Li X, Chepelin- oA-crystaUin gene: Conserved features of the 5'- sky AB, Piatigorsky J: Conservation of mouse flanking regions in human, mouse, and chicken. oA-crystallin promoter activity in chicken lens / Mo/ fvo/ 33:495-505, 1991. epithelial cells. J Mol Evol 35:337-345, 1992. Egwuagu CF, Sztein J, Reid W, Chan C-C, Mahdi R, Nussenblatt RB, Chepelinsky AB: Gamma inter- feron gene expression in the lens of transgenic mice directed by the ocA-crystallin gene promoter. Invest Ophthalmol ^% Sci 33(4):846, 1992. 148 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00253-04 LMDB PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or lees. Title must fit on one line between the borders.) Regulation of Expression of Lens Fiber Membrane Genes PRINCIPAL INVESTIGATOR (List other prolasslonal personnel below the Principal Investigator.) (Name, title, laboratory, and institute aHUIaUon) PI: Ana B. Chepelinsky Ph.D. Research Biologist LMDB, NEI Others: Chiaki Ohtaka-Maruyama Ph.D. Visiting Fellow LaShawn R. Drew B.S. Chemist Devonne M. Parker B.S. Biologist Kristen Ault Summer Student LMDB, NEI LMDB, NEI LMDB, NEI LMDB, NEI COOPERATING UNITS (II any) University of Southern California (Margaret McFall-Ngai, Ph.D.; Virginia Weis, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Molecular Genetics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.87 PROFESSIONAL: 0.69 1.18 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [3 (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not axceed the space provided.) This project concerns regulation of expression of genes encoding lens fiber membrane proteins involved in ceU-cell communication. We are presently studying the regulation of expression of the gene encoding the major intrinsic protein (MIP) of the lens fiber membrane, which belongs to an ancient superfamily of putative transmembrane channel proteins. We cloned 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 lens cells in transient assays and transgenic mice. We are presenUy studying the effea of several transcription factors on the in vitro transcription of the MIP gene in Drosophila and HeLa nuclear extracts. Purified human Spl activates the in vitro transcription of the MIP promoter, suggesting its involvement in the transcriptional regulation of the MIP gene. These studies will further our understanding of the role of general transcription factors on the tissue-specific expression of the MIP gene. 149 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biology NEI Annual Report-FY 1992 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 ceU-cell com- munication in the lens. The identification of the cis regulatory elements of these genes and their interac- tion with fra/w-acting factors are essential for under- standing the regulation of gene expressi'n in the lens. Methods Recombinant DNA techniques used in this study include screening genomic libraries, subcloning, plasmid construction, Southern and Northern hybrid- izations, DNA sequencing, primer extension, poly- merase chain reaction (PCR), reversed transcription PCR (RT-PCR), in vitro transcription, and tissue culture techniques, including transfection of primary lens explants and cell lines, as well as analysis of transgenic mice. Major Findings Cis regulatory sequences of the human lens major intrinsic protein (MIP) gene. — In order to study the regulation of expression of the MIP gene and identi- fy the regulatory elements imderlying its tissue specificity, we isolated and characterized 2,840 bp of the human MIP gene 5'-flanking sequence. Three complete Alu repetitive elements are found in tandem at approximately 2,000 bp upstream from the initia- tion site of transcription, while a single complete Alu sequence is present in the third intron of the gene. We focused on the 5'-flanking sequence proximal to the initiation site of transcription for the mapping of promoter regulatory elements of the MIP gene. We foimd in transfection experiments that a DNA frag- ment containing 253 bp of human MIP 5'-flanking sequence and 42 bp of exon 1 fiised to the bacterial chloramphenicol acetyltransferase (CAT) gene activates CAT gene expression in primary chicken lens explants and in a mouse lens cell line, but not in several nonlens cell lines. Therefore, the -253/+42 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'-fIaiiking sequence of the MIP gene. These include CCAAT and CACCC boxes, NF-1, Spl, and glucocorticoid receptor binding sites, which may function synergisti- cally in binding to the glucocorticoid receptor. To elucidate whether those motifs are involved in the regulation of MIP gene expression we are studying the effect of several transcription factors on the in vitro transcription of the MIP gene using Drosophila and Hela nuclear extracts. Purified human Spl activates the in vitro transcription of the MIP pro- moter, suggesting its involvement in the regulation of transcription of the MIP gene. We generated several lines of transgenic mice containing 253 bp of the human MIP gene 5'-flank- ing sequence and 42 bp of exon 1 fused to the CAT gene as a transgene. In one transgenic line, the CAT gene is expressed specifically in the ocular lens, consistent with the DNA sequence -253/+42 of the human MIP gene containing regulatory elements responsible for gene expression in the lens. In two additional transgenic lines, expression of the CAT gene is observed only in the ovaries of females; no expression is observed in any tissue of males. No MIP transcripts were detected by PCR of reverse- transcribed mouse ovary mRNA. The possibility that species-specific regulatory elements might be respon- sible for the aberrant expression of the transgene containing the human promoter cannot be excluded. Qoning the murine MIP gene will allow us to study the mouse MIP promoter in its homologous in vivo environment. 3' -Untranslated sequence of the MIP gene. — ^The 3' end of the human MIP gene was determined by comparing the nucleotide sequence of the 3'-untrans- lated region of the gene with the bovine cDNA sequence. As neither contains a classical polyadenyl- ation signal, we are continuing the sequencing of the 3'-flanking region of the human MIP gene to charac- terize more precisely the polyadenylation site(s) utilized during the processing of the MIP transcripts. MIP and connexins in the light organ lens and the ocular lens of Euprymna scolopes. — In collabora- tion with Drs. Margaret McFall-Ngai and Virginia Weis from the University of Southern California, we started investigating the possible expression of MIP, other members of the same transmembrane channel superfamily, and connexins in the octilar lens and the light organ lens of the squid, Euprymna scolopes. 150 NEI Annual Report-FY 1992 Laboratory of Molecular and Developmental Biology The light organ lens is induced during its symbiosis with the luminous bacteria Vibrio fischeri. Significance to Biomedical Research and the Program of the Institute The differentiation of lens epithelial cells into fiber cells entails extensive ceU elongation that results in a dramatic increase in the elaboration of new plasma membrane by elongating cells. Proper membrane biosynthesis and physiology are therefore 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. Lens membrane alterations, particularly those involving MIP, have been observed during cataractogenesis and aging. Studies on MIP gene expression should fiirther our understanding of not only the mecha- nisms involved in the regulation of gene expression in the normal lens but also its disruption during disease. Proposed Course The foUowing studies will continue during Fiscal Year 1993: (1) characterization of the cis regulatory elements of the human MIP gene in transient assays and in transgenic mice, (2) investigation of the interaction of the human MIP gene cis regulatory elements with trans-acting factors, (3) sequencing and characterization of the 3'-flanking region of the human MIP gene, and (4) isolation and characteriza- tion of the murine MIP gene promoter. NEI Research Program Cataract — Molecular Genetics Publications Chepelinsky AB, Drew LR, Pisano MM: Regulation of human MIP gene expression. Exp Eye Res 55(suppl 1):S25, 1992. Drew LR, Pisano MM, Chepelinsky AB: CAT gene expression in transgenic mice directed by the human MIP gene promoter. Invest Ophthalmol Vis Sci 33(4): 1043, 1992. Pisano MM, Chepelinsky AB: Genomic cloning, complete nucleotide sequence, and structure of the human gene encoding the major intrinsic protein (MCP) of the lens. Genomics 11:981-990, 1991. 151 DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00273-02 LMDB PERIOD COVERED October 1. 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or lass. Title must lit on one line between the borders.) Physiological Expression of the Retinoblastoma- Associated Gene During Lens Cell Differentiation PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute aHllialion) PI: R. Andrew Cuthbertson M.D., Ph.D. Visiting Associate LMDB, NEI Others: Joram P*iatigorsky Ph.D. Chief LMDB, NEI COOPERATING UNITS (II any) Howard Florey Institute for Experimental Physiology and Medicine, University of Melbourne, Australia (Robb Femley, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Molecular Genetics INSTITUTE AND LOCATION NEI. NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 0.0 PROFESSIONAL: 0.0 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.) This project has been terminated. 152 PHS 6040 (Rev. 5/92) DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00126-11 LMDB PERIOD COVERED October 1. 1991 to September 30, 1992 TITLE OF PROJECT (BO charaaers or less. Title must tit on one line between the borders. ) CrystaUin Genes: Structure, Organization. Expression, and Evolution PRINCIPAL INVESTIGATOR (Usi other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute allUaUon) PI: Others: Joiam Pialigorsky James Brady Ana B. Chepelinsky Sambalh Chung Ales Cvekl Peter Irederikse Cynthia J. Jaworski Marc Kanlorow Xuan Li Joan B. McDennoa Ph.D. Chief Ph.D. NRC Fellow Ph-D. Research Biologist B.A. Technician Ph.D. Visiting Fellow Ph.D. Senior Staff Fellow Ph-D. Cheniist Ph.D. Staff Fellow Ph.D. Visiting Fellow M.S. Biologist LMDB, NH LMDB. NH LMDB. NH LMDB, NH LMDB, NH LMDB, NH LMDB. NH LMDB. NH LMDB. NH LMDB. NH (Additional personnel listed under Program Description.) COOPERATING UNITS (II any) Jules Stein Eye Institute. UCLA (J. Horwitz, Ph.D.); National Institute on Child Health and Human Development, NIH (Kevin Becker. Ph.D.; K. Ozalo. Ph.D.); University of Southern California (Virginia Weis. Ph.D.; M McFall-Ngai, Ph.D.); Whimey Laboratory, University of Rorida (Russel Buono. Ph.D.; Paul Linser, Ph.D.) LAB/BRANCH Laboratory of Molecular and Developmental Biology Section on Molecular Genetics INSTrrUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 13.70 PROFESSIONAL: 11.45 2.25 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 crystaUin genes of vertebrates and invertebrates are being studied. Cw-acting control elements for lens expression have been identified in a- and P-crystaUin genes. Unexpectedly, the oA-crystallin elements are occupied in both expressing and nonexpressing cultured cells. Fooq)rint analyses have shown that ocB-crystaUin gene expression in lens and muscle involves a combination of shared and tissue-specific cw-elements. Two promoters are used in a tissue-preferred fashion for expression of the ocB-crystaUin gene; a strong muscle/weak lens enhancer is present between these promoters. This enhancer uses a muscle regulatory factor (MRF) site exclusively for muscle expression and at least two other sites for lens and muscle expression. Transgenic mouse and transfection experiments have shown that the oA-crystallin cw-acting elements are functionally redimdant. A rra/w-acting factor (ocA-CRYBPl) important for expression of the mouse oA- crystaUin gene was shown unequivocally to bind to its cognate c«-element. Immimoblotting experiments indicated tiiat this factor exists in different overlapping sizes in different tissues, and the promoter for its gene has been cloned and expressed in transfected lens cells. The two 5-crystaUin genes were shown to be under tissue-specific and developmental regulation. CrystaUin recruitment in the squid was shown to differ from that in vertebrates in that the parental enzyme gene (glutathione S-transferase) gave rise to a related family of crystallins without enhancing its own lens expression. The ALDH-like crystaUin cDNA of cephalopods has been cloned and sequenced. Qoning of the three J 1 -crystaUin genes of cubomedusan jeUyfish showed that they lack introns, encode novel proteins, and have surprisingly diverse 5' flanking sequences. 153 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biology NEI Annual Report-FY 1992 Project Description Additional Personnel Rashmi Gopal-Srivastava Ph.D. Barbara Norman H. John Roth Ph.D. Christina M. Sax Ph.D. Stanislav Tomarev Ph.D. Peggy S. Zelenka Ph.D. Rina Zinovieva Ph.D. NRC Fellow, LMDB, NEI Chemist, LMDB, NEI Senior Staff Fellow, LMDB, NEI Senior Staff Fellow, LMDB, NEI Visiting Scientist, LMDB, NEI Geneticist, LMDB, NEI Research Worker, LMDB, NEI Objectives The objectives of this project are to understand the structure, organization, expression, and evolution of the gene families encoding the lens crystallins. Particular attention is given to the regulation of crystaUin gene expression in the developing lens and, in the case of multifunctional crystallins and enzyme- crystaUins, in nonlens tissues. Methods Conventional methods for analysis of proteins and nucleic acids used include polyacrylaraide and agarose gel electrophoresis, RNA and DNA isolation, molecular hybridization (Southern and Northern blots), cDNA and gene cloning, DNA sequencing, recombinant DNA construction, in situ hybridization, expression of recombinant DNAs in transfected ceUs and transgenic mice, polymerase chain reactions (PCRs), site-directed mutagenesis of recombinant DNAs, in vitro and in vivo footprinting, gel mobility shift analysis, and chromatographic purification of proteins. Major Findings Vertebrates. — ^Last year we reported on the identifi- cation of numerous cw-acting regulatory elements for expression of the mouse and chicken oA-, mouse oB-, and chicken PA3/Al-crystallin genes. Site- specific mutagenesis experiments have established that these are indeed important control elements. DNAse I and in vivo dimethylsulfate interference footprinting experiments have shown that three major control elements in the mouse oA-crystallin promoter (DEI, oA-CRYBPl, and a CAT sequence) are all occupied in aTN4-l ceUs that express the gene and in L929 cells that do not express the gene although the details of the footprints indicate subtle differences in binding specificity and affinity. The oA-CRYBPl site has been investigated in greater detail and shown in immunoblots to bind a cloned factor called oA- CRYBPl. cxA-CRYBPl u i ubiquitous zinc finger protein. This year we showed that this transcription factor has multiple sizes and that different tissues show different patterns of oA-CRYBPl sizes. Each of the sizes binds its cognate site in the oA-crystaUin promoter. Preliminary immunoblotting experiments also suggest that oB-crystallin joins in the complex formed with the ocA-CRYBPl site. The 5'-fianking sequence for the ocA-CRYBPl gene has been cloned and shown to be active in transfected lens cells; the production of transgenic mice containing this pro- moter has been initiated. Taken together, these experiments suggest that tissue-specific processing (either at the level of RNA or protein modifications) of the oA-CRYBPl factor and its interaction with other proteins play a signifi- cant role in transcriptional regulation of the olA- crystaUin gene. In addition, these experiments raise the possibility that crystallins themselves contribute to the regulation of crystallin genes. These results have obvious significance for the cascade of crystal- lin gene expression that occurs during lens develop- ment. Comparison of the activity of baaerial chlor- amphenicol acetyltransferase (CAT) genes driven by various mutant promoters of the mouse oA-crystallin gene in transfection and transgenic mice have shown that each of the control elements mentioned above functions redundantly, ie, at least two must be inacti- vated before the promoter loses its function. Experiments on the expression of the mouse oB- crystallin gene in lens and muscle have continued. We have used cultured C2C12 cells to study muscle expression and the SV40 T-antigen-transfonned aTN4-l cells to study lens expressioa The enhancer responsible for strong muscle and weak lens expres- sion identified last year has been studied in detail this year. Site-specific mutagenesis and DNAse I footprinting experiments have demonstrated that at 154 NEI Annual Report-FY 1992 Laboratory of Molecular and Developmental Biology least two separate sites in the enhancer are function- ally important in cultured muscle and lens cells. An additional site, the muscle regulatory function (MRF) site is only used in the muscle cells. Cotransfection experiments with cloned MyoD and myogenin cDNAs showed that the MRF site is capable of activating the enhancer in NIH 3T3 ceUs, which normally do not express the otB-crystallin gene. In contrast to the muscle-specific control element, DNAse I footprinting experiments showed that the -148/-118 sequence of the ocB-crystallin gene, which is downstream from the -427/-259 enhancer, is occupied only in the lens cells and not in the muscle ceUs, suggesting that it comprises a lens-specific regulatory element. Thus, the expression of the oB- crystallin gene in lens and muscle appears to be controlled by a combination of shared and tissue- specific control elements. Experiments performed last year showed that the oB-crystaUin gene uses an upstream (^70) transcrip- tion initiation site in lung and brain and a down- stream (+1) initiation site in lens, muscle, and other tissues. No RNA splicing occurs when the upstream site is used, so the control elements in between the two start sites are represented in the 5'-untranslated sequence of the mRNA. This year both sites were shown to be recognized in a HeLa cell extract. Deletion constructs of the promoter fused to the CAT gene have been integrated into NIH 3T3 ceUs, forming a set of transformed ceUs. Preliminary experiments have indicated that the upstream promot- er is required for activity of the fiision gene. More- over, heat shock experiments have been Initiated to identify the sequences required for this stress response. Last year we identified a number of control elements in the chicken pBl-crystaUin gene and a minimal functional promoter in the chicken PA3/A1- crystallin gene. This year in vivo dimethylsulfate interference experiments demonstrated that the PBI- crystallin control elements are occupied in the lens, indicating that the in vitro experiments and transfec- tion experiments are directiy relevant to the natural situation in chromatin. Fimction of the |3A3/A1 promoter was shown by deletion and site-specific mutagenesis experiments to depend on an AP-1 site and a T-rich tract. This finding fits with our finding that AP-1 sites are prevalent throughout crystallin genes, even in invertebrates. However, further experiments are clearly necessary to identify the basis for lens specificity of this gene. We have been investigating the expression of the two chicken 5-crystaUin genes for many years. These fascinating genes are closely Unked, the 5' 51 gene being specialized for lens expression and the 3' 52 gene encoding the active argininosuccinate lyase (ALS) gene. Thus, 5-crystaUin is an enzyme -crystal- lin. In the past several years we have shown by PCR experiments that both of the chicken 5-crystaUin genes are expressed in many tissues, including the heart, brain, and lens. These experiments indicated that the 61 gene is predominantiy expressed in the lens, while the 52 gene is preferentially expressed in the heart and brain of the chicken embryo. Despite the difference in the ratio of the two 6-crystaUin mRNAs, the embryonic lens always has virtually thousands of times more 52 mRNA than other tissues, consistent with 52-crystallin having a refrac- tive function in lens and an enzymatic role in non- lens cells. This year, in collaboration with Dr. Peggy Zelenka (LMDB, NEI), we reinvestigated more quantitatively with internal controls the relative expression of the two 5-crystaIlin genes. Particular attention was given to 5-crystaLlin expression in the cornea and retina, which, like the lens, are trans- parent ocular structures. The results showed that each embryonic tissue examined (cornea, retina, heart, brain, lens) has a characteristic ratio of expres- sion of the two 5-crystaIlin genes. Moreover, we found unexpectedly that embryonic cornea, retina, and lens all contain greater amounts of 51 mRNA than 52 mRNA. By 1 week after hatching, however, 52 exceeded 51 mRNA in the cornea and retina. In contrast to the eye tissues, the embryonic heart had a trace more 62 than 61 mRNA and the embryonic brain had much more 62 than 61 mRNA. The absolute amount of 6-crystallin mRNAs was lower in the 1 -year-old chicken in aU tissues examined and, in all tissues, there was more 62 than 61 mRNA. Thus, there is clearly a complex tissue-specific and developmental regulation of the two 5-crystaUin genes in the chick- en. One intriguing possibility under investigation is that although the 5l-crystaUin polypeptide lacks ASL activity, it modulates enzymatic activity of the native ASL by interaction with the 62 -crystallin polypeptide in the tetrameric proteins. 155 Laboratory of Molecular and Developmental Biology NEI Annual Report-FY 1992 This year we completed a project examining the developmental expression of carbonic anhydrase-U (CA-II) in the chicken lens. The results showed that, in contrast to its prevalence in the retina (3% of the protein), CA-II makes up only about 0.1% of the protein of the lens, an amoimt insufficient to make it an enzyme-crystaUin in this species. Transfection of embryonic chicken lens epitheUal ceUs with deletion mutants of the CA-II promoter fused to the CAT gene led to a gradual loss of promoter activity, consistent with an additive effect of putative cis regulatory elements foimd in many crystallin genes. Invertebrates. — Within the past few years we have studied the crystallins of invertebrates with cellular eye lenses for several reasons. First, while the vertebrate crystallins have been studied extensive- ly, virtually nothing was known of invertebrate crystallins, despite the abundance of invertebrate species with complex eyes. In addition, invertebrate eyes are a model of convergent evolution, and many have eyes and lenses that were apparently derived independently from those of vertebrates. Conse- quently, we reasoned that the most fundamental characteristics of crystallins for transparency and of their genes for high expression in the lens might be shared. Our initial studies on squid and octopus showed that their crystallins, called S-crystallins, were derived from the detoxification enzyme glutathione S-transferase and consist of a family of at least 10 genes. We also showed last year that octopus contains a minor crystallin related or identical to aldehyde dehydrogenase (ALDH), called i2-crystallin. This year we cloned this cDNA and established its structure. Whereas ALDH is the major crystallin of the mammalian elephant shrews, Q-crystaJlin is the first crystallin common to both vertebrates and invertebrates. Another cormnon feature of vertebrate and invertebrate lenses is their use of detoxification enzymes for refractive purposes. This year we cloned the squid gene that encodes the active GST enzyme and showed that it is highly expressed in the digestive gland and barely expressed in other tissues, including lens. Thus, in contrast to vertebrate enzyme-crystallin recruitment, which depends on modification of gene expression to produce high concentrations of protein in the lens (often without gene duplication), the cephalopod S- crystallins recruitment involved multiple gene dupli- cations of the parental enzyme. Furthermore, ceph- alopod recruitment did not use the ancestral gene for active GST for refractive purposes in the lens. A similar situation might exist for the p/y-crystallins in vertebrates, although their nonrefractive function has not yet been discovered. We also have explored noncrystallin proteins of squid and octopus lens by cloning, and those being analyzed include actin, tubulins, and ferritin. Of special interest is the finding that some actins and tubulins appear to be expressed selectively in these invertebrates' lenses. Collaborating with Dr. Margaret McFall-Ngai (University of Southern California) and Dr. Ana Chepelinsky (LMDB, NEI), we have cloned abundant cDNAs of the squid light organ. The original idea was to clone the ALDH-like crystallin that is ex- tremely abundant in the muscle-derived cellular lens of this unique organ. To our surprise, however, we found a number of myeloperoxidase-encoding cDNAs not derived from the light organ lens. Whereas the light organ is infected with limiinescent bacteria, it is possible that the abimdant myeloperoxi- dases are used for this symbiotic relationship. A little-known fact is that cubomedusan jellyfish have extremely well-developed eyes with ceUular lenses. Our previous studies have shown that these lenses contain at least three different crystallins called J1-, J2-, and J3-crystallin. Last year we reported cloning three members of the 35-kD Jl- crystallin genes of cubomedusae. Their coding sequences are 85-98% identical and lack introns; by contrast, their 5'- and 3'-untranslated sequences are extremely divergent, consistent with an ancient duplication of their genes. This year, we established, by primer extension, PCR, and Northern blotting experiments, the initia- tion site for transcription of the J 1 -crystallin genes. Their putative promoters each have TATA and putative CCAAT boxes; however, alignment of their 5'-flanking sequences has provided little clue of what they have in common for their shared high expres- sion in the lens. One short sequence was foimd to be repeated in the Jl A-crystallin 5'-flanking sequence and also partially present further 3' in the S'-flanking region of the JlB-crystallin gene. Thus, it is pos- sible, as we recognized earlier for the oA-crystallin gene promoter of vertebrates, that regulatory sequence motifs are arranged in a modular fashion in the J 1 -crystallin genes. Functional studies have been initiated by tising Jl promoter/CAT constructs in 156 NEI Annual Report-FY 1992 Laboratory of Molecular and Developmental Biology transfected lens cells of vertebrates and cell-free transcription of the cloned genes in HeLa extracts. So far we have been unable to obtain clear evidence of promoter activity in these heterologous systems. Finally, analysis of the deduced sequences for the Jl-crystallin polypeptides has not revealed any striking homology with any other proteins in the database. Thus, die jellyfish crystallins fit the taxon- specific character of crystallins among vertebrates and other invertebrates. Heat denaturation studies showed that Jl-crystallin is stable at 50°C but precipitates at 60°C, making it unlikely that it is a heat-shock protein. Interestingly, the smaller-molec- ular-weight J2/J3-crystallins appeared to be much more stable at 60°C than Jl-crystallin. They will be studied further next year. Significance to Biomedical Research and the Program of the Institute The lens crystallins, which comprise several families of conserved proteins that are differentially expressed in the developing lens, are necessary for lens trans- parency. Understanding the structure, function, and evolution of these protein families and their genes contributes to our knowledge of embryonic develop- ment, eukaryotic gene expression, cell differentiation, molecular evolution, the visual system, and cataract. The finding that many crystallins are multifunctional proteins that are expressed in nonlens tissues gives these genes another dimension of general interest and has implications for metabolism, cell biology, and drug and gene therapy. Proposed Course The following are proposed for Fiscal Year 1993: 1. We will continue studies on identified cis- acting control elements of the crystallin genes, focusing on both their mechanism of action and interactions and seeking additional elements. 2. We will continue studies on oA-CRYBPl and will attempt to clone other trans-acting factors that regulate the crystallin genes. 3. We will modify the squid's active GST and S- crystallin genes in an effort to identify the active sites for this ubiquitously important detoxification enzyme. 4. We will continue to test the idea that 51- crystallin modulates enzymatic activity of native ASL. 5. We will clone and characterize the J2- and J3- crystallin cDNAs and their genes and continue functional studies on the jellyfish crystallin gene promoters. 6. Time permitting, we wiU continue efforts to obtain a ceU-free transcription system for studying lens-preferred crystallin gene expression. NEI Research Program Molecular Biology Publications Barbosa P, Wistow GJ, Cialkowski M, Piatigorsky J, O'Brien WE: Expression of duck lens 5-crystallin cDNAs in yeast and bacterial hosts. J Biol Chem 266:22319-22322. 1991. Brady JP, Piatigorsky J: Qoning and characterization of a novel zinc-finger protein-encoding cDNA from the mouse eye lens. Gene, in press. Buono RJ, Linser PJ, Cuthbertson RA, Piatigorsky J: Molecular analyses of carbonic anhydrase-U expression and regulation in the developing chicken lens. Dev Dynamics 194:33-42, 1992. Dubin RA, Gopal-Srivastava R, Wawrousek EF, Piatigorsky J: Expression of the murine oB- crystallin gene in lens and skeletal muscle: Identi- fication of a muscle-preferred enhancer. Mol Cell fijo/ 11:4340-4349, 1991. Donovan DM, Sax CM, Klement JF, Li X, Chepelin- sky AB, Piatigorsky J: Conservation of mouse ctA-ciystallin promoter activity in chicken lens epitiielial cells. /Mo/ Evol 35:337-345, 1992. 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 Yoik, McGraw-Hill Publishing Co, in press. Hejtmancik JF, Piatigorsky J: Molecular biology of the eye lens, in Albert DM, Jakobiec FA (eds): Principles and Practice of Ophthalmology: The Harvard System. Philadelphia, WB Saunders Co, in press. Jaworski CJ, Chepelinsky AB, Piatigorsky J: The oA-crystallin gene: Conserved features of the 5' flanking regions in human, mouse, and chickea J Mol Evol 33:495-505, 1991. Kim RY, Liemian T, Piatigorsky J, Wistow GJ: Structure and expression of the duck a-enolase/T- 157 Laboratory of Molecular and Developmental Biology NEI Annual Report-FY 1992 crystallin-encoding gene. Gene 103:193-200, 1991. McDermott JB, Peterson CA, Piatigorsky J: Structure and lens expression of the gene encoding chicken pA3/Al-crystaIlin. Gene 117:193-200, 1992. Piatigorsky J: Lens ciystallins: Irmovation associated with changes in gene regulation. / Biol Chem 261 Mil -Aim, 1992. Sax CM, Klement JF, Piatigorsky J: Role of the oA- CRYBPl site in lens-specific expression of the oA-crystallin gene, in Loveh PS, Mongkolsuk S, Trempy JS (eds): Biotechnology and Environ- mental Science. New Yoik, Plenum Press, in press. Tomarev SI, Zinovieva RD. Piatigorsky J: Character- ization of squid crystallin genes: Comparison with mammalian glutathione S-transferase gems. J Biol Chem 267:8604-8612, 1992. Tomarev SI, Zinovieva RD, Piatigorsky J: Crystallins of the octopus lens: Recruitment finom detoxifica- tion enzymes. / Biol Chem 266:24226-24231, 1991. 158 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00259-03 LMDB PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (BO characters or lass. Title must lit on one Una betwaan the borders.) Molecular Biology of the Cornea PRINCIPAL INVESTIGATOR (List other prolesslonal personnel below the Principal Investigator,) (Name, title, laboratory, and Institute afliliation) PI: Joram Piatigorsky Ph.D. Chief LMDB, NEI Others: R. Andrew Cuthberson Xuan Li M.D., Ph.D. Ph.D. Visiting Associate Visiting Associate LMDB. NEI LMDB. NEI COOPERATING UNITS (il 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: 0.9 PROFESSIONAL: 0.9 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.) Last year we initiated a program on the molecular biology of cornea. The major finding was that many of the abimdant proteins of the corneal anterior epithelial cells of the mouse, human, kangaroo, chicken, and squid are either related or identical to metabolic enzymes, as are the lens crystaUins. Indeed, many of these abundant corneal proteins are the same as those used as lens crystaUins. Moreover, as with the lens crystallins. a taxon specificity was observed. In addition, we cloned the cDNA and gene for the most abundant protein in the epithelial cells of the mammalian cornea, class 3 aldehyde dehydrogenase (ALDH). This year we made transgenic mice that carry the 5' flanking sequence of the class 3 ALDH gene fused to a reporter gene to test whether this promoter preferentially directs gene expression in the cornea. Ten founder mice have been bred. Five ALDH and two 60 kD protein cDNAs have been cloned from the mouse cornea. The 60 kD protein is an abundant corneal protein that is highly conserved between mouse and human. A chicken corneal cDNA library also has been made in order to clone cyclophilin. an enzyme that last year was shown to comprise approximately 10% of the protein of the epithelial cells of the chicken cornea. Interestingly, this enzyme does not seem especially abundant in the mammalian cornea. 159 PHS 6040 (Rev. 5/92) Laboratory of Molecular and Developmental Biology NEI Annual Report-FY 1992 Project Description Objectives The objectives of this project are to identify and characterize 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 tissue. Methods Conventional molecular biology methods of cloning, sequencing, recombinant DNA construction, transfec- tion, and transgenic mice production are used. Major Findings Last year we reported the cloning of a 6.5 kbp fragment of the mouse class 3 aldehyde dehydroge- nase (ALDH) gene. This year we sequenced about 2 kbp of putative 5'-flanking sequence of the class 3 ALDH gene and made recombinant DNAs containing about 1.1 kbp of 5'-flanking sequence of the gene inserted in both orientations into pNASSp. This promoterless vector contains the p-galactosidase gene as a reporter gene. Transgenic mice have been made to test the functional ability of this putative promoter and to examine whether it may be corneal preferred. In addition to the gene, we have isolated five class 3 ALDH cDNA clones that remain to be sequenced. We have isolated two cDNAs selected by hybrid- ization to oligodeoxynucleotides that encode two different peptides obtained from an abimdant novel 60-kD proteia Our peptide analyses indicate that this protein is highly conserved between mouse and man. One of the cDNAs is 1.5 kbp; the other, approximately 5 kbp. The shorter cDNA has been sequenced and its coding region for the peptide encoded in the oligodeoxynucleotide identified; the longer cDNA is now being sequenced. 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 needs to be investigated. The cornea is a transparent ectodermaUy derived tissue like the lens; thus, comparative studies between it and the lens are of special interest with respect to transparency. More- over, because of our finding that corneal epithelial cells use a similar principle of taxon-specific gene sharing of metabolic enzymes, comparative studies on the cornea and lens, our major tissue of research, are of obvious importance from developmental and evolutionary viewpoints. Finally, the cornea is particularly accessible for gene therapy on account of its exposure to the surface and its association with numerous hereditary diseases. Proposed Course Several projects are proposed for Fiscal Year 1993: (1) continued analysis of the class 3 ALDH promot- er, to determine its tissue preference and to identify its cw-acting regulatory elements; (2) cloning and characterization of the chicken cyclophUin cDNA and gene(s); (3) completion of cDNA analysis for the 60- kD mouse corneal epithelial cell protein; and (4) time permitting, cloning and characterization of the squid corneal cDNAs to determine whether they are identi- cal to those expressed in the lens, as suggested by the protein data obtained last year. NEI Research Program Molecular Biology Publications Cuthbertson RA, Tomarev SI, Piatigorsky J: Taxon- specific recruitment of enzymes as major soluble proteins in the corneal epitheliimi of three mam- mals, chicken and squid. Proc Natl Acad Sci USA 89:4004^008, 1992. 160 DEPARTMENT OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00274-02 LMDB PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Gene Expression in the Retinal Pigment Epithelium PRINCIPAL INVESTIGATOR (Ust other proleesional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atliliallon) PI: Robert Y. Kim M.D. Senior Staff FeUow LMDB. NEI Others: Graeme J. Wistow Ph.D. Visiting Scientist LMDB. NEI COOPERATING UNITS (II any) Department of Ophthalmology, University of CaUfomia. San Francisco (Ge Ming Lui, Ph.D.) 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: 0.0 PROFESSIONAL: 0.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews □ (b) Human tissues |x] (c) Neither SUMMARY OF WORK ^Uss standard unreduced type. Do not exceed the space provided.) This project has been terminated. 161 PHS 6040 (Rev. 5«2) DEPARTMEm' OF HEALTH AND HUMAN SERVICES • PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00255-()4 LMDB PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must fU on one line belweert the borders.) Molecular Biology and Functions of Lens Proteins PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute aHlliation) LMDB. NEI PI: Others: Graeme J. Wistow Robert Kim Douglas Lee Michael Shaughnessy Jason Hodin Vishwas Paralkar Caroline Graham L^enzo Segovia Pc^gy Zelenka Ph.D. M.D. Ph-D. B.S. B.S. Ph-D. B.S. Ph-D. Ph.D. Chief, Section on Molecular Structure and Function Staff Fellow NRC FeUow Biologist Special Volunteer Visiting Associate Biologist Visiting Fellow Chief, Section on Cellular Differentiation LMDB. NH LMDB, NH LMDB, NEI LMDB, NEI LMDB. NEI LMDB. NEI LMDB. NEI LMDB, NEI COOPERATING UNITS (if any) The George Washington University (F. Noonan, Ph.D.); Harvard University (S. Bruhn, B.S.; C. Cepko, Ph.D.); University of Melbourne, Australia (R. Gasser, PltD.) LAB/BRANCH Laboratory of Molecular and Developmental Biology SECTION Section on Molecular Structure and Function INSTITUTE AND LOCATION NEI. NTH. Bethesda, MD 20892 TOTAL STAFF YEARS: 5.41 PROFESSIONAL: 1.00 4.41 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.) Crystallins. which have been recruited from stress proteins and enzymes, generally maintain dual roles without gene duplication. The gene recruitment of orthoquinone reductase/C-crystallin has occurred through the use of alternative promoters. We have analyzed the putative lens promoter of this gene showing that discrete regions confer lens preference, while upstream positive and negative elements confer improved lens specificity. The rapid amplification of cDNA ends (RACE) polymerase chain reaction (PCR) technique has been used to show tfiat 61, 52. and e-crystaUins use the same promoters for lens and nonlens expression. The duck x- crystallin gene has been introduced into transgenic mice to study expression in lens. The cDNA for p- crystallin, a mammalian taxon-specific crystallin. has been cloned. p-Crystallin is a mammalian homologue of bacterial ornithine cyclodeaminases. Outside the lens, p is expressed in the retina and brain, and we have cloned himian retina p. In the developing chick retina, p is preferentially associated with photoreceptors. We have continued to analyze the expression and function of growth factors and other proteins that have roles in lens differentiatioa The coordination of lens lOK/major intrinsic factor (MIF) v«th differentiation has been established. Furthennore, we have found that total mouse lens extract has MIF activity, suggesting that it could play a role in lens-induced eye inflammation. The cloning of the himian lOK/MIF gene is imder way. We have also found that transfonning growth faaor beta (TGF-P) may play a role in regulating lOK/MIF expression. 162 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Molecular and Developmental Biology Project Description Objectives We are investigating some of the most basic charac- teristics of the normal lens. In particular, we continue to identify the major structural components of the lens and their functions in both lens and nonlens tissues. We also are identifying protein maiicers of the differentiation process which is characteristic of the normal lens and wh'ch may be disrupted in cataract and in eye inflammation. Apart from its significance for eye research, this work is of consid- erable importance in the growing field of molecular evolution. Methods We use a wide range of modem molecular biology techniques, including RNA analysis, gene and cDNA cloning and sequencing, and expression of genes in bacterial hosts. The polymerase chain reaction (PCR) is becoming increasingly important to our work. We continue to analyze proteins by gel electrophoresis and contract microsequencing. We make extensive use of computers for analysis and molecular model- ing. For gene promoter studies, we are using electro- phoretic mobility shift analysis, "footprinting," transfection into cultured cells, and construction of transgenic mice. Major Findings Gene recruitment: enzyme crystallins. — ^The putative lens promoter for guinea pig ^-crystallin has been cloned and expressed. Promoter fragments up to -180 bp from the transcription start site have no activity in lens-derived N1003A cells or in NIH 3T3 cells. However the -380 bp promoter, which contains T- rich stretches and other elements common to other crystallin promoters, is an active promoter in N 1003 A cells although it is still inactive in fibroblast 3T3 cells. Increasing the promoter to -527 bp causes a drop in activity, revealing a negative element. A further increase to -751 bp causes a very large increase in activity although it is again inactive in 3T3 cells. A -1500 bp construction is somewhat less active. The -380 and -751 bp promoters were tested in transgenic mice. The shorter promoter is highly lens preferred, but it has weak expression in the brain. The longer fragment is apparently lens specif- ic. Thus the ^-crystallin gene has been recruited through a lens-specific promoter that comprises several positive and negative elements working together. The lens promoter of ^-crystaUin lies in what otherwise would be the first intron of the gene. This is an appealing model for recruitment because it does not disrupt the existing enzyme promoter, however, it does not appear to be a general model. We used the RACE PCR method to clone the 5' ends of mRNA for three taxon-specific crystallins, 51, argininosuccinale lyase/52, and lactate dehydrogenase B/e, from lens and nonlens tissues of embryonic duck. In all three cases, identical 5' ends were found in lens and in the brain and liver (for 51 and 52) or heart (for e). Thus, these three genes, hke x-crystaUin described earlier, use the same promoter for both lens and nonlens expression. We constructed a line of transgenic mice contain- ing the entire duck a-enolase/x-crystaUin gene with 5'- and 3'-flanking regions and aU introns. Full-sized duck a-enolase mRNA was expressed in the trans- genic mice with the same pattern as the endogenous mouse a-enolase isozyme. Although there was no evidence of tissue preference, the concentration of enolase increased markedly in transgenic lens as well as in other tissues. Nonetheless, transgenic lenses were transparent, and the animals were normal in appearance. The increase in enolase levels in the transgenic lens mimics the stepped increase that might occur in the early stages of enzyme-crystallin recruitment. These results demonstrate that lens transparency is sufficiently robust to be refractory to some increase in metabolic enzyme concentration without the need for compensatory adaptation. Circumstantial evi- dence has linked a-enolase expression with the oncogene c-myc. We found a possible c-myc binding site in the a-enolase/x-crystallin gene promoter. Collaborating with Dr. Zelenka's group, we showed that c-myc is a positive regulator of the duck t- crystaUin gene, the first example of a target gene for this proto-oncogene. The major component of the eye lens in several Australian marsupials is p-crystallin. The complete sequence of kangaroo p-crystallin has now been obtained by cDNA cloning. The predicted amino-acid sequence shows similarity with ornithine cyclo- deaminases (OCD) encoded by the tumor-inducing (Ti) plasmids of Agrobacterium tumefaciens. Until now, neither OCD nor any strucmrally related enzymes have been observed in eukaiyotes. Northern 163 Laboratory of Molecular and Developmental Biology NEI Annual Report-FY 1992 analysis of kangaroo tissues shows that p-crystallin is abundantly expressed in lens; but outside the lens it is preferentially expressed in neural tissues, retina, and brain. This pattern of expression and relationship to an enzyme involved in unusual amino-acid metab- olism raises the possibility that mammalian p-crystal- lin may have a role in the metabolism of excitatory amino acids, while an alternative or additional role in lens could lie in osmoregulation. Human retinal p- crystaUin has been cloned and sequenced. In the developing chicken retina, p-crysi llin detected by immunofluorescence is preferentially associated with the photoreceptors, pertiaps indicating an unusual pathway for ornithine metabolism in the retina and possibly related to the problems of gyrate atrophy. Growth factors and lymphokines in the lens. — A discrete 10-kD polypeptide (lOK) is expressed from early stages in the embryonic chicken lens. Because of its potential as a marker for lens cell development, we identified chicken lOK and its homologues from mouse and human lenses by protein sequencing and cloning. Surprisingly, the lens lOK protein appears to be identical to a lymphokine, macrophage migration inhibitory factor (MIF), which was originally identi- fied in activated himian T cells. Using microdissec- tion and PCR techniques, we found that expression of lOK/MIF is strongly correlated with cell differen- tiation in the developing chicken lens. This finding suggests that proteins with MIF activity may have roles beyond the immune system, perhaps as intercel- lular messengers or as part of the machinery of differentiation itself. Indeed, partial sequencing of other small lens proteins has identified another MIF- related protein (MRP8) in calf lens. The relatively abundant expression of MIF in lens may have clinical significance, with the possibility of involvement in ocular inflammations such as endoph- thalmitis and uveitis that may follow damage to the lens. Preliminary data suggest that total lens extracts do indeed have MIF activity. Recombinant mouse lOK/MIF is being produced in Escherichia coli. and the human gene is being cloned in our Section. The important growth factor TGF-pi has been cloned from mouse lens. In cell culture experiments and in isolated rat lens epithelium we have demon- strated that TGF-pi can regulate the expression of lOK/MIF, which we have shown to be a ma±er for lens cell differentiation. We are investigating the interplay of these and other growth factors in normal lens and in several transgenic and gene-knockout models, including mice carrying most of the HIV-l genome and others in which the TGF-pi gene has been eliminated. Significance to Biomedical Research and the Program of the Institute We are discovering fundamental mechanisms in the processes by which complex tissues are organized and develop. Along the way, we have discovered a novel enzyme that has possible significance in neural tissues, particularly retina, and which may be a useful pharmacological target. We also have discov- ered important markers for cellular differentiation that may have clinical relevance in ocular inflamma- tion. Proposed Course Work on this project will continue with (1) examina- tion of the molecular mechanisms for lens preferred expression and for gene recruitment, (2) engineering of crystaUins and other lens components in trans- genic animals to mimic gene recruitment and con- structing specific cataract models, and (3) exploration of the molecular biology and function of lympho- kines and growth faaors expressed in lens and examination of their role(s) in eye disease. NEI Research Program Cataract — ^Molecular Genetics Publications Barbosa P, Wistow GJ, Cialkowski M, Piatigorsky J, O'Brien WE: Expression of duck lens 5-crystallin cDNAs in yeast and bacterial hosts: 52-crystallin is an active argininosuccinate lyase. J Biol Chem 266:22319-22322, 1991. Doniger J, Landsman D, Gonda MA, Wistow G: The product of unr, the highly conserved gene up- stream of N-ras, contains multiple repeats of the cold-shock domain (CSD), a putative DNA-binding motif. New Biologist 4:1-7, 1992. Kim RY, Gasser R, Wistow GJ: p-crystallin is a mammalian homologue of Agrobacterium orni- thine cyclodeaminase and is expressed in himian retina. Proc Natl Acad Sci USA 89:9292-9296, 1992. Kim RY, Wistow GJ: The cDNA RPEl and mono- clonal antibody HMB-SO define gene products 164 NEI Annual Report-FY 1992 Laboratory of Molecular and Developmental Biology preferentially expressed in retinal pigment epithe- Earles BJ, Hart GW: Vertebrate lens a-crystailins lium. E:q) Eye Res 55:657-662, 1992. are modified by 0-linked N-acetylglucosamine. J Lee DC, Gonzalez P, Rao PV, Zigler JS Jr, Wistow ^'^' ^^^^ 267:555-563. 1992. GJ: Carbonyl-metabolizing enzymes and their Shaughnessy M, Wistow G: Absence of MHC gene relatives recruited as structural proteins in the eye expression in lens and cloning of dbpBA'B-l, a lens, in 'WemerH (ed): Enzymology and Molecu- DNA-binding protein expressed in mouse lens. lar Biology of Carbonyl Metabolism, Vol 4, in Curr Eye Res 11:175-181, 1992. P'^ss- Wistow G: Lens crystallins: A model system for Roquemore EP, Dell A, Morris HR, Panico M, gene recruitment. Methods Enzymol, Vol 224, in Reason AJ, Savoy L-A, Wistow GJ, Zigler IS Jr, press. 165 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 (ARI's) and anti- cataraa agents. The development of clinical ARI's has evolved primarily around analogs derived from related carboxylic acids and spirohydantoins. Unfor- tunately, these compounds have demonstrated poten- tially significant problems, including low biological half-lives, poor tissue penetrations, and nonspecific side effects that range from skin rash to the induction of liver enzymes. Pursuing more effective and less toxic therapies, LOT scientists discovered an inhibitor unrelated to previously reported ARI's. Studies are being con- ducted to characterize this inhibitor by biochemical, pharmacological, and computer molecular design techniques. In addition, drug distribution studies in ocular tissues are being conducted using noninvasive magnetic resonance imagine (MRI) techniques. Other studies are designed to elucidate the specific mechanism(s) by which aldose reductase initiates diabetic complications. In studies utilizing galactose-fed dogs, LOT investigators have estabhshed the progression of retinal changes associated with diabetic retinopathy and their dose-dependent arrest with ARI's. Studies are now focused on the observation that long-term galactose-fed dogs develop advanced retinal changes similar to clinical proliferative retinopathy. In addition, investigators are analyzing the specific role of aldose reductase in neuropathy, thyroid changes, and immunological system responses. 169 DEPARTMEtfT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1. 1991 to September 30, 1992 PROJECT NUMBER ZOl EY 00003-19 LOT TITLE OF PROJECT (BO charaaers or lass. Title must fit on one line between the borders.) Pharmacology of Octilar Complications PRINCIPAL INVESTIGATOR (Ust other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) LOT. NH LOT. NEI LOT. NH LOT. NEI LOT. NEI LOT. NH LOT. NH LOT..,H LOT. NH PI: Peter F. Kador Others: Julia Dot Jun Inoue Yong Lee Anita Bartoszko-Malik Kazuhiko Mori Maneo Schaffliauser Yukio Takahashi Tomoyuki Terada Ph.D. Chief B.A. Biologist M.S. Pharmacy, Special Volunteer Ph.D. Staff Fellow Ph.D. Visiting Fellow M.D.. Ph.D. Visiting Fellow Ph.D. Visiting Fellow M.D.. Ph.D. Visiting Associate Ph.D. Special Volunteer COOPERATING UNITS (If any) National Heart, Limg and Blood Institute, Bethesda, MD (Robert Balaban, Ph.D.); Ohio State University College of Pharmacy, Columbus, OH G^uane Miller, Ph.D.) Laboratory of Ocular Therapeutics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 PROFESSIONAL: 6.6 OTHER: 1.0 TOTAL STAFF YEARS: T6_ CHECK APPROPRIATE BOX(ES) D (a) Human subjects (b) Human tissues □ (c) Neither n (al) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Events leading to the onset of various ocular compUcations are being investigated. Specifically, the studies focus on the role of the enzymes aldose reductase and aldehyde reductase in the onset and progression of retinopathy, cataraa, keratopathy. pupQ function changes, and iris and cQiary process structural 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 interventioa 170 PHS 6040 (Rev. S/92) NEI Annual Report-FY 1992 Laboratory of Ocular Therapeutics Project Description Objectives This research is designed to gain insight into the mechanisms by which polyol-induced ocular diabetic comphcations and cataracts are formed and to develop methods for their regulation. Methods Diabetes can be experimentally induced in animals through the injection of streptozotocin. Diabetes- related complications linked to the sorbitol pathway can also 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 studies used to purify enzymes include column chromatography, polyacrylamide gel electrophoresis (PAGE), isoelectric focusing, chro- matofocusing, and high-pressure liquid chromatogra- phy (HPLC). Polyol levels were determined by gas- liquid chromatography (GLC). Immunological analyses include the use of enzyme-linked immuno- sorbent assay (ELISA), radioimmunoassay (RIA), Western blots, and immunohistochemical techniques employing the coupled antibody DAB-PAP tech- nique. 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 Desiga Major Findings Biochemical studies. — ^Recent studies have demon- strated that the dog is an excellent animal model for investigating ocular diabetic complications. Cataracts in the dog develop in a manner similar to that of cataracts in humans with diabetes. They are charac- terized by formation of anterior and posterior super- ficial cortical opacities, with the posterior polar region more advanced. Furthermore, the dog devel- ops advanced retinal changes similar to those ob- served clinically in himian diabetics. Polyol forma- tion iiutiated by the NADPH-dependent enzyme aldose reductase has been demonstrated to initiate these lens and retinal changes. Investigations of NADPH-dependent reductases in the dog lens, retina, and thyroid have revealed the presence of a third, labile NADPH-dependent en- zyme in dog tissues that is not inhibited by aldose reductase inhibitors. This enzyme has not been observed in either human or rat lens and kidney. Its stability increases with 0.2 to 0.3 M sodium chloride, has a higher molecular weight than either aldose and aldehyde reductases on gel filtration. Kinetic study indicates that it primarily utilizes DL-glyceraldehyde as substrate and that its activity with either D-gluc- uronate or aldose sugars (D-glucose, D-galactose, and D-xylose) is negligible. It is not inhibited by aldose reductase inhibitors. On the basis of these studie:. the enzyme has been assigned the tentative name "glyceraldehyde reductase." Interest in the enzyme stems from the fact that its utilization of NADPH may contribute to changes in ceUular redox potentials. Studies on defining the inhibitor site(s) of aldose reductase and aldehyde reductase continue with the evaluation of a number of Michael addition affinity- labeled aldose reductase inhibitors. In addition, the pharmacophore requirements of the inhibitor site and the location of reactive nucleophilic and electrophilic sites are being refined through molecular modeling. New, potent aldose reductase inhibitors have been uncovered, and patent application has been made. Lens studies. — ^Posterior migration of lens epithe- lial cells, believed to be a common phenomenon in cataracts, has been observed in human lenses from diabetics with posterior subcapsular cortical opacities. However, similar epithelial cell migration has not been reported in the various rodent sugar cataraa models that form via a common osmotic mechanism linked to the aldose reductase-associated excess lenticular production of sugar alcohols. Unlike what occurs in human lenses, sugar cataract formation in young rats occurs rapidly; their lens opacities form predominantiy in the anterior cortical regions. In addition, rat lenses contain higher levels of aldose reductase. Recent studies in the dog, an animal model whose lenses contain levels of aldose reduc- tase activity more similar to those of humans than of rats, indicate that dog lenses also form aldose reduc- tase-linked sugar cataracts. However, their formation in the dog is slower than in the rat, and they progress to form anterior and posterior superficial cortical opacities, with the posterior polar region more advanced. Histological studies were conducted on 44 cata- ractous lenses obtained from young male beagle dogs fed a 30% galactose diet for 19-60 months. Exami- 171 Laboratory of Ocular Therapeutics NEI Annual Report-FY 1992 nation of toluidine blue-stained sections (2.5 pm) of methacrylate-embedded lenses revealed epithelial ceU migration to the posterior pole in 14 of 44 lenses. AH lenses with migration displayed a separation of the posterior Y suture and liquefaction. In addition, we observed a liigher incidence of lens-fiber ceU nuclei disarrangement and separation of the anterior Y suture than in lenses from similar dogs not demon- strating migration. These lesions are similar to those observed in humans, supporting the contention that the dog is a better animal model than the rat for investigating human sugar cataracts. In previous studies we established through in vitro dog lens experiments that aldose reductase activity can be accurately assessed by measuring the conver- sion of 3-deoxy-3-fluoroglucose to 3-deoxy-3-fluoro- sorbitol by nuclear magnetic resonance spectroscopy ("F NMR). This work has now been extended to the in vivo evaluation of aldose reductase activity in rabbit lenses. Initial spatial coordinates for lenses were calculated firom 'H-images determined on a 2.0 Tesla GE Omega-CSI spectrometer of the eyes of normal rabbits (2.7 kg) anesthetized with halothane. The spectral localization with optimal pointspread function (SLOOP) technique was then used with a proton decoupler to measure the accumulation of sorbitol in the lens. We used a double spin-echo sequence with selective excitation and refocusing pulses and with optimized phase-encoding gradient pulses that had 1 sec repetition times and 25 msec echo times. Fluorinated glucose was administered to the rabbit either by infusing 40 ml of 150 mM 3-deoxy-3- fluoroglucose into the right carotid artery at a rate of 10 ml per hour or by topical application of soft contact lenses hydrated in saturated 3-deoxy-3- fluoroglucose over a 48-hour time period. In SLOOP experiments, 3-deoxy-3-fluorosorbitol was only observed in spectra of the anterior portion of the lens, not in the nucleus or anterior chamber. These results demonstrate the good localization properties of the SLOOP technique and the useful- ness of this method for in vivo determination of aldose reductase activity. Retinal studies. — We have observed that vascular changes associated with diabetic retinopathy can be produced experimentally in beagle dogs fed a 30% galactose diet. In addition, we have reported in prevention studies in which dogs were fed a galac- tose diet for up to 36 months that the concomitant administration of the aldose reductase inhibitors can affect the onset and progression of the appearance of pericyte ghosts (19-21 months), acellular capillaries (24 months), microaneurysms (27 months), and intraretinal hemorrhages (33 months) in a dose- dependent manner. These changes, observed in isolated trypsin-digested preparations, are initiated by the aldose reductase-catalyzed reduction of galactose to galactitol. To determine whether the progression of retinal changes can also be arrested through reduction of galactitol production at early stages of retinal lesion development, we conducted an intervention study using 60 yoimg male beagles. Ten dogs fed a control diet containing 30% non-nutrient filler served as controls while 50 dogs were fed a 30% galactose diet. From the galactose-fed group, 15 dogs were switched to normal diet (Purina chow) after 24 months (a period during which pericyte ghosts and aceUular capillaries are present); an additional 15 dogs were switched after 30 months (a period in which microaneurysms are present). Twenty-four months after the initiation of galactose feeding, four to five eyes from each experimental group and two to three eyes from the control group were enucleated at 6-month intervals. Investigations of those eyes' retinal vessels, isolated by trypsin digestion, revealed no apparent reversal of retinal lesions. Nevertheless, differences in the progression of retinal lesions between the galactose-fed and reversed groups became evident 12-18 months after reversal. Asteroid hyalitis (hyalosis) is characterized by minute particles considered to be liquid crystals of lipids suspended without orderly arrangement in the vitreous. Although rarely causing visual impairment, they have recently been incriminated in hindering visualization of the retina for diagnostic purposes and in predisposing to refractive errors in the selection of intraocular lenses for use following cataract extrac- tion. The etiology of these bodies has not been definitively identified; however, 27-70% of patients with asteroid hyalitis also have diabetes meUitus. Humans with hypercholesterol also have a greater incidence of asteroid hyalitis than an at-risk population. Systemic disease has also been incrimi- nated as a cause of asteroid hyalitis. In dogs fed a long-term 30% galactose diet, we have observed that the fonnation of asteroid hyalitis begins 42-48 months after the oiiset of galactose feeding. Ten dogs fed 30% galactose for 72 months 172 NEI Annual Report-FY 1992 Laboratory of Ocular Therapeutics were more closely examined and compared with five age- and sex-matched dogs fed a control diet contain- ing 30% fiber. Nine of the ten dogs whose lenses allowed visualization of the fundus demonstrated severe asteroid hyalitis, particularly in the dependent part of the vitreous. In the dog whose lens precluded ophthalmoscopic examination, we found severe asteroid hyalitis when the eye was examined after euthanasia. None of the control dogs manifested asteroid hyalitis. All of the dogs on the galactose diet had various degrees of retinopathy identified as those seen in humans with ocular complications of diabetes mellitus. We believe this model will provide an opportimity to study the etiopathogenesis of asteroid hyalitis and possible methods for its treatment. 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 needed. We have developed an animal model that demonstrates advanced retinal vessel changes that are virtually clinically and histologically identical to those observed in advanced diabetic retinopathy. Our present studies in dogs demonstrate for the first time that loss of retinal pericytes, associated with aldose reductase, initiates retinal changes associated with both background and advanced diabetic retinop- athy. The results show that administration of aldose reductase inhibitors in prevention studies can amelio- rate the loss of pericytes and subsequent microaneu- rysms and retinal hemoniiages in a dose-dep)endent manner. The successful development of noninvasive methods for monitoring aldose reductase activity by NMR procediuTes may have direct impact on ongoing and plaimed clinical trials in which this procedure could serve as a quantitative indicator of drug efficacy. Cataract is also one of the major causes of blindness in the developing world; loss of vision due to cataract is one of the major health problems of both diabetic and aging populations in the United States. Proposed Course These studies will be continued. Recently discovered aldose reductase inhibitors will be pharmacologically evaluated and developed. The inhibitor site will be further probed through the use of affinity labe's so that more potent and specific inhibitors may be developed. Studies of the mechanisms through which aldose reductase induces diabetic complica- tions in various tissues will be continued. NEI Research Program Retinal Disease — Diabetic Retinopathy, Sickle Cell Retinopathy, and Other Vascular Abnormalities Publications Abdel-Ghany Y, Donkor I, Kador PF, Mizoguchi T, Malik A, MiUer D: Novel alrestatin analogs with potent reversible and irreversible inhibitory activity for the enzyme aldose reductase. MEDI 135, 1992. Ceckler TL, Karino K, Kador PF, Balaban RS: Magnetic resonance imaging of the rabbit eye: Improved anatomical detail using magnetization transfer contrast Invest Ophthalmol Vis Sci 32:3109-3113, 1991. Kador PF: Update on aldose reductase inhibitors. Endocrine Soc 10A:15, 1992. Kador PF: Intermediary metabolism of the lens, in Raviola E, Dowling J (eds): Principles and Practice of Ophthalmology. New York, John Wiley & Sons, in press. Kador PF, Takahashi Y, Sato S, Wyman M: Aldose reductase, retinal vessel changes, and cataracts in galactose fed dogs, in Rifkin H, ColweU JA, Tayler SI (eds): Diabetes 1991. International Congress Series 1000. 14th International Diabetes Society Congress, Washington, DC, Jime 23-28, 1991. Amsterdam, Elsevier Science Publishing, 1991, pp 373-378. Kador PF, Takahashi Y, Schaffhauser M: Vortieug- img diabetischer Komplikationen im Auge mit Aldosereduktase-Hemmem. Diabetes und Stoff- wechsel, in press. Kador PF, Takahashi Y, Wyman M: Retinal vessel changes in galactose-fed dogs: Intervention studies. Diabetes 41:169A, 1992. Kador PF, Takahashi Y, Wyman M: Retinal vessel changes in galactose-fed dogs: Intervention studies. Invest Ophthalmol Vis Sci 33(4):878. 1992. 173 Laboratory of Ocular Therapeutics NEI Annual Report-FY 1992 Kador PF, Takahashi Y, Wyman M, Kinoshita JH: The role of aldose reductase in retinal vessel changes associated with diabetic retinopathy in galactose-fed dogs. National Eye Institute- Juvenile Diabetes Foundation Workshop on Aldose Reductase Inhibitors. US DHHS Pub No (NIH) 91-3114, 1991, pp 75-88. Karino K, Kador PF, Bericowitz B, Balaban R: ^'-F NMR quantitation of lens aldose reductase activ- ity using 3-deoxy-3-fluoro-D-glucose. J Biol Chem 266:20970-20975, 1991. Lin L, Giblin F, Kador PF, Kinoshita J, Lou M: The efficacy of aldose reductase inhibitors on polyol accumulation in human lens and retinal pigment epithelium in tissue culture. Invest Ophthalmol Vis Set 33(4):1376, 1992. Mori K, von Klenlin M, Balaban R, Kador PF: In vivo measurement of aldose reductase activity in the rabbit lens by ''F NMR spectroscopy with 3- deoxy-3-fluoroglucose. Invest Ophthalmol Vis Sci 33(4):1377, 1992. PoweU HC, Garrett RS, Kador PF, Mizisin AP: Fine- structural localization of aldose reductase and ouabaine-sensitive K*-dependent p-nitro-phenyl- phosphatase in rat peripheral nerve. Acta Neuro- pathol 81:529-539, 1991. Reddy VN, Lin LR, Giblin FJ, Lou M, Kador PF. Kinoshita JH: The efficacy of aldose reductase inhibitors on polyol accumulation in human lens and retinal pigment epithelium in tissue culture. Ocular Pharmacology, in press. Robison WG, Laver N, Kador PF: Meager delay of sugar cataracts and pre-proliferative retinopathy in galactose-fed rats by the ARI ponalrestaL Invest Ophthalmol Vis Sci 33(4):878, 1992. Schaffhauser M, Sato S, Terada T, Kador PF: NADPH-dependent reductases in dog lens, retina and thyroid: The presence of a third enzyme "glyceraldehyde reductase." Invest Ophthalmol Vis Sci 33(4):1377, 1992. Smar MW, Ares J, Nakayama T, Itabe H, Kador PF, MUler DD: Synthesis and biological evaluation of affinity labels for aldose reductase. / Med Chem 35:1117-1120, 1992. Takahashi Y, Wyman M, Ferris F, Kador PF: Retinal vessel changes associated with advanced diabetic retinopathy in galactose-fed dogs. Arch Ophthal- mol 110:1295-1302, 1992. Takahashi Y, Wyman M, Kador PF: Posterior migra- tion of lens epithelial cells in cataracts of galac- tose-fed dogs. Invest Ophthalmol Vis Sci 33(4):137, 1992. von Kienlin M, Karino K, Mori K, Kador PF, Despres D, Fleming J, Balaban R: Localized "F spectroscopy using SLOOP to measure aldose reductase activity in the eye. Society of Magnetic Resonance in Medicine 10th Annual Meeting, August 10-16, 1991, San Francisco, CA, 1991. p 1057. Waldbillig RJ, Jones BE, Schoen TJ, Heidersbach S, Bitar MS, Van Kuijk FJGM, de Juan E, Kador PF, Chader GJ: Vitreal insulin-like growth faaor binding proteins (IGFBPs) are increased in human and animal diabetics: Implications for understand- ing diabetic retinopathy. / Clin Invest, in press. Wyman M. Takahashi Y. Raber J, Kador PF: Aster- oid hyalitis (hyalosis) in galactose-fed dogs. Invest Ophthalmol Vis Sci 33(4):858, 1992. 174 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00275-01 LOT PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 charaaers or laes. Title must tit on one line between the borders.) Role of NADPH-Dependent Reductases in Ocular Complications PRINCIPAL INVESTIGATOR (List other proleesional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute atlltlaUon) PI: Sanai Sato M.D. Visiting Scientist LOT, NEI Others: Shigeru Fukase M.D. Special Volunteer LOT, NEI COOPERATING UNITS (11 any) LAB/BRANCH Laboratory of Ocular Therapeutics INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 1.5 PROFESSIONAL: 1.5 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects [3 (b) Human tissues □ (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) In diabetes, the incidence and progression of complications associated with hyperglycemia have been linked to the production of sugar alcohols (polyols). The relationship between aldose reductase, other closely related NADPH-dependent reductases, and polyol production is being investigated to define the biochemical mechanism(s) that initiate the onset of these complications and to develop specific pharmacological methods of intervention. 175 PHS 6040 (Rev. 5/92) Laboratory of Ocular Therapeutics NEI Annual Report-FY 1992 Project Description Objectives This project is designed to assess the importance of aldose reductase versus aldehyde reductase in polyol accumulation in various tissues in which diabetic changes occur. The research wiU provide inforaia- tion important for the development of aldose reduc- tase inhibitors. Methods Biochemical techniques include gel filtration and affinity chromatography, electrophoresis, immuno- blotting, and isoelectric focusing on high-pressure liquid chromatography (HPLC). Gas chromatogra- phy is used to identify and quantitate sugars. In vitro techniques include the culture of retinal capil- lary pericytes and endothelial cells, leukocytes, and fibroblasts. Results, including enzyme kinetic evaluations, are calculated using the NIH PROPHET computer system. Major Findings Kidney. — The production of polyols by both aldose and aldehyde reductases has been confirmed by in vitro experiments in which the newly produced polyol peaks were demonstrated by gas chromatogra- phy after the incubation of purified rat kidney enzymes with aldoses in the presence of an NADPH- generating system containing citric acid and citrate dehydrogenase. Polyol production by aldehyde reductase was confirmed in vivo in galactosemic rats by demonstrating that aldose reductase-specific inhibitors reduced the polyol level in the aldehyde reductase-rich kidney cortex less than nonspecific inhibitors. These results strongly suggest that the inhibition of both aldose reductase and aldehyde reductase is required to ameliorate diabetic nephrop- athy. Comparison of rat and human kidneys has shown a similar aldose reductase versus aldehyde reductase distribution. The kinetic properties of the human enzymes are similar to those of the rat. Retina. — ^Evaluation of cultured human retinal pigment epithelial (RPE) cells has revealed that these cells possess both aldose and aldehyde reductases. Aldose reductase predominates in this tissue whereas the amount of aldehyde reductase is negligible. The data indicate that, as in the lens, only aldose reduc- tase contributes to polyol accumulation, which causes several dysfunctions of the RPE associated with diabetes. Leukocytes. — Both polymorphonuclear cells and mononuclear cells contain aldose and aldehyde reductases. Examination of dog leukocytes revealed that aldehyde reductase is the predominant enzyme in these cells. Although aldose reductase activity was minimal, polyol accumulation was detected in both polynuclear and mononuclear cells. The sorbitol pathway in neutrophils was confirmed through nuclear magnetic resonance (NMR) studies with 3- fluorodeoxyglucose (3FDG) in which 3FDG was converted to 3-fluorodeoxyfructose through 3-fluoro- deoxysorbitol. These results suggest that aldehyde reductase contributes to polyol accumulation in these cells under hyperglycemic and/or galactosemic conditions. Significance to Biomedical Research and the Program of the Institute Despite the establishment of insulin therapy, signifi- cant numbers of persons with diabetes are at risk for debilitating complications associated wdth this dis- ease. These complications can lead to loss of vision, neuropathy, and kidney failure. Although experi- mental studies demonstrate that excess polyol pro- duction is linked to the onset and progression of many of these complications, understanding the interrelationship of NADPH-reductases and polyol production is essential for the development of specif- ic pharmacological methods of intervention. Proposed Course These studies will be continued. The presence of NADPH-dependent enzymes and their ability to produce polyols wiU be evaluated in target cells at sites of diabetic complications. Targets for investiga- tion will be retinal capillary pericytes and endothelial cells, fibroblasts, key cells associated with prolifera- tive changes, both mesangial and endothelial cells of the kidney glomerulus, and leukocytes. NEI Research Program Diabetic Complications and Cataract Research 176 NEI Annual Report-FY 1992 Laboratory of Ocular Therapeutics Publications Carper D, Sato S, Old S, Chung S, Kador PF: In vitro expression of human placental aldose reduc- tase in Escherichia coli. Adv Exp Med Biol 284:129-139, 1991. Sato S: Naphthalene diol dehydrogenase in rat lens. Invest Ophthalmol Vis Sci 33(4):1377, 1992. Sato S: Purification of aldose and aldehyde reduc- tases from dog kidney. Adv Exp Med Biol 284:153-163. '991. Sato S: Rat kidney aldose reductase and aldehyde reductase and polyol production in rat kidney. Am J Physiol 263(Renal Fluid Electrolyte Physiol 32): F799-F805. 1992. Schaffhauser M, Sato S, Terada T, Kador PF: NADPH-dependent reductases in dog, retina and thyroid. Invest Ophthalmol Vis Sci 33(4):3420, 1992. 177 Laboratory of Retinal Cell and Molecular Biology Report of the Chief, Laboratory of Retinal Cell and Molecular Biology Gerald J. Chader, Ph.D. The mission of the Laboratory of Retinal Cell and Molecular Biology (LRCMB) is to uncover new aspects of functioning of the retinal pigment epithe- lium (RPE) complex in health and disease. The focus is on elucidating new genes and biochemical mecha- nisms and learning the imderlying causes of ocular diseases. Most of the approaches taken are molecular biological and/or candidate gene approaches. Where possible, the LRCMB wiU seek clinical collaboration to translate the findings from laboratory to chnic. Most of the work of the lab members is within the following NIH Strategic Initiatives and NEl Priorities: (1) molecular medicine, (2) gene research and gene therapy, and/or (3) research of high clinical relevance. Within the Laboratory, three areas are empha- sized: • Immunopathology of uveoretinitis • Establishment of an ocular gene center focus- ing on genetic research and candidate genes for retinal diseases • Transgenics and gene therapy for retinal diseases. Immunopathology of Uveoretinitis Several LRCMB investigators are involved in immu- nogenetic and immimopathogenic studies. Most if not aU of the work is perfonned in collaboration with investigators in the Laboratory of Immunology G..I); its focus is on the induction of experimental auto- immune uveitis (EAU). The areas of concentration are several. Immunopathology. — Collaborative woiic with Dr. Igal Gery (LI) has established an excellent animal model for studying human uveitis. Continued dissec- tion of the immunopathological site(s) of the inter- photoreceptor retinoid-binding protein (IRBP) in the Lewis rat and in the human has the final goal of comrolling or preventing the disease pix)cess in man. Immunogenetics. — Collaborative work with Dr. Rachel Caspi (LI) has established the IRBP-mouse model for EAU as very useful for studying the genetics of the disease and its relapsing character- istics, for example. Antigen presentation. — Collaborative work with Drs. Gerj' Marc de Smet, and Robert Nussenblatt (LI) has demonstrated the presence of a 70-kD B-ceU surface protein that specifically binds the major 23- amino acid immunopathological determinant of IRBP. This cell surface protein may function as a molecular chaperon in antigen presentation. S-antigen (S-Ag)-responsive element. — A major new discovery is that the S-Ag promoter has a steroid hormone consensus site. This, the first re- ported visual cycle protein, opens a new aspect of vision research involving the effects of steroid and thyroid hormones on the expression of visual pro- teins. Furthermore, it has direct clinical impUcations (eg, glucocorticoid treatment), which wUl be pursued in collaboration with Drs. Nussenblatt and Chi-Chao Chan. Genetic Research and Genes for Retinal Diseases The main focus of most of the LRCMB scientists is the identification of specific genes that cause ocular disease. To accomphsh this goal, a consortium of LRCMB investigators has been established to form an NEI Gene Center. Our purpose is to develop a chromosomal map of eye-specific genes involved in ocular diseases. There are four areas of concentra- tion. Fatty acid and tubulin defects in retinal degenera- tion. — ^Fatty acid uptake and metabolism in Bietti's crystalline retinopathy and a tubulin acetylation defect in a form of Usher's syndrome are being investigated in collaboration with Dr. Muriel Kaiser- Kupfer (Ophthalmic Genetics and Clinical Services Branch) in the hope of elucidating the specific defects. Significant progress has been made in pinpointing the metabolic problems. Candidate genes for hereditary retinal diseases. — Genes expressed predominantly or exclusively in ocular tissues are being identified by subtractive cloning. Retina-specific genes and genes located on 181 Laboratory of Retinal Cell and Molecular Biology NEI Annual Report-FY 1992 the short ami of the X chromosome are of specific interest. These genes are being chroraosomally localized to see whether they are disease linked. Concurrent genomic cloning and sequencing is being done to generate appropriate polymorphisms and/or microsatellite repeats are generated. Using patient material, Dr. Bruce Pfeffer, an expert in tissue culture, will immortalize cell lines to facilitate subsequent laboratory experiments. RPE-specific genes. — Although the RPE is absolutely essential in the photoreceptive process, very little is known about the specific complement of genes in the RPE. In our major effort to clone genes unique to RPE and its functioning, the highlight is the isolation of a new, specific 65-kD protein from the human RPE. Cloning of the gene for the new protein, which has potential immimologic impor- tance, allows the study of tissue-specific expression. This gene is the first RPE-specific gene to be report- ed and characterized. Unique photoreceptor processes. — Diseases unique to vision are probably caused by problems with processes that are unique to the eye. Several Laboratory members have focused on identifying and characterizing proteins and enzymes that seem to be critical in functioning of the retina/RPE complex. The molecular biology (eg, expression control) of two proteins of the phototransduction cascade, S-Ag and phosducin, is under intensive study. Both pro- teins are rod outer segment-specific and interact with visual cycle components (eg, opsin), but their actual function remains controversial. cDNAs and genomics for S-Ag and phosducin have been cloned and thoroughly analyzed, allowing for current advances in our understanding of the expression, function, and pathology of the gene products. IRBP is also a critical link in the chain of enzymes and proteins that make up the visual cycle and visual transduction but, because of the huge size of the gene, it would be very difficult to sequence in potential disease cases. In an attempt to get around this problem, we are cloning the Drosophila (fruit fly) homolog of the hiunan IRBP gene. It has now been m^ped to an area of the Drosophila genome that is rich in ocular mutants. The idea of this woik is to see whether the fly IRBP maps to a known eye- disease locus within the Drosophila genome. Then, knowing the characteristics (eg, erg) of the disease in the fly, we can pinpoint a specific human population with similar characteristics and examine the gene for defects in specific human families. Many retinal degenerations are manifest in very early development and thus may involve autocrine or paracrine growth factor defects. We have identified a novel neurotrophic protein, pigment epithelium- derived factor (PEDIO, that is synthesized by fetal human RPE cells. PEDF may be critical in the development of retinal neurons. At very low concen- tration, PEDF causes the extension of elaborate neuronal processes from cultured retinoblastoma ceUs in culture. An important possibility is that, as with the Royal CoUege of Surgeons rat, a defect in the PEDF of the RPE could cause a retinal degeneration. Also being evaluated is the clinical use of PEDF in retinal transplantation in a rabbit model system with Dr. Manuel del Cerro. The molecular biology of this potentially very important neurotrophic agent is now being studied for application to retinal dysfunctions. Transgenics and Gene Therapy of Retinal Diseases A growing focus of the LRCMB is on the establish- ment of transgenic models of retinal disease and on possible modalities of therapy, such as the use of ribozymes. Transgenic studies. — ^The IRBP and S-Ag genes are the best studied retinal genes other than riiodop- sin. The cw-acting elements that control IRBP and S- Ag promoters, and thus their protein expression, are being studied in transfected human cells and in transgenic mice. Transgenic studies, in particular, will uncover factors controlling gene activation in the embryonic period, specifically in the photoreceptor cell. Gene analysis systems in transgenic mice and in transient transfections in cultured human retinoblasto- ma cells using a chloramphenicol acetyltransferase or CAT assay have been established for IRBP. Almost 2 kb of IRBP 5' flanking region has been thoroughly examined to date; similar progress has been made with the S-Ag. Promoter (+ or -) and enhancer elements necessary for normal and abnormal expres- sion are being defined through target mutagenesis studies. Tissue- and stage-specific elements, includ- ing TATA and CAAT boxes, will be precisely defined as to retinal and/or pineal expression. This woric is important because specific molecules can be precisely gene-taigeted to the retina. 182 NEI Annual Report-FY 1992 Laboratory of Retinal Cell and Molecular Biology Gene therapy. — Ribozymes are specifically constructed RNA species that can control protein expression within ceUs. 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 for the treatment of now immanageable autosomal dominant disorders. Following the design of ribozyme constructs for IRBP, they are being studied in a transfected human retinoblastoma cell system, concurrently with rearing transgenic mice to determine whether an retinitis pigmentosa-like condition can be mimicked through downregulating IRBP synthesis. Once perfected, ribozymes should be useful not only with IRBP- related retinopathies but in conditions such as diabet- ic retinopathy and retinopathy of prematurity in which the disorders probably involve overexpression of normal proteins such as growth factors. 183 DEPARTMErrr OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00070-15 LRCMB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or less. Title must lit on one ling between the borders.) Vitamin A and Ocular Tissues PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and Institute aflillallon) PI: Barbara Wiggert Ph.D. Head, Section on LRCMB, NEI Biochemistry Others: Kalpana Rengarajan R. Krishnan Kutty Todd Duncan Ph.D. Visiting Fellow LRCMB, NEI Ph.D. Senior Staff FeUow LRCMB, NEI M.S. Biologist LRCMB, NEI COOPERATING UNITS (II 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.); U. Penn. Sch. Vet Med., PhUadelphia (G. Aguirre, D.V.M.. Ph.D.); Med. U.S.C. (R. Crouch, Ph.D., S. Hazard, Ph.D.); SLU InsL F. Kir. Sweden (K. Narfsliom, 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.) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology SECTION Section on Biochemistry INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 3.1 PROFESSIONAL; 1.1 2.0 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.) Studies on factors regulating interphotoreceptor retinoid-binding protein (IRBP) gene expression in the mouse eye demonstrated that light deprivation markedly downregulated IRBP message in developing and adult mice. However, IRBP protein levels were not decreased, suggesting regulation by some sort of feedback control mechanism. Studies on retinoid levels in the eyes of the CSVvitA'it mouse model of retinal degeneration showed markedly elevated levels of retinyl palmitate as well as some elevation of levels of 11-cis retinal, all-rra/w retinal and aH-trans retinol at 6 weeks of postnatal development, suggesting a malfunction in some aspect of the visual cycle in this mutant. An effect of IRBP on the process of l\-cis retinal formation has been suggested by studies of retinoid processing in the toad retinal pigment epithelium (RPE) eye cup which show that both the molar amount and the specific radioactivity of 1 l-cis retinal increase with increasing IRBP concentration. Analysis of endogenously bound ligands of Drosophila head retinoid-binding protein demonstrated the presence of a possible isomer of retinol and both covalent and noncovalently bound fatty acids. We also characterized the binding affinities of retinol and fatty acid. 184 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Retinal Cell and Molecular Biology Project Description Additional Personnel Igal Gery Ph.D. Head, Section on Experimental Immunology, LI, NEI Rachel Caspi Ph.D. Visiting Associate, LI, NEI Cooperating Units Medical College of Georgia (S. Smith, Ph.D.); Emory Eye Center (J. Nickerson, Ph.D.) 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, high-performance liquid chromatography (HPLC), 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 In a study investigating the possibility that light or the lack of light stimulation may play a role in regulating gene and/or protein expression of IRBP, we foimd an 80-90% reduction in IRBP message in the eyes of mice reared fi"om birth to postnatal day 14 (P14) under dim red lighting conditions, com- pared with normal control animals reared under cyclic light. At the same time, immunochemical quantitation of IRBP showed no reduction in protein levels, and immunocytochemistry demonstrated normal interphotoreceptor matrix of mice reared under dim red lights. When adult mice were placed imder dim red light for 14 days, there was also a marked decrease in IRBP message in their eyes and no reduction in IRBP. These results may indicate both a lower IRBP turnover rate when bleaching of rhodopsin does not occur and a regulation of IRBP gene expression by some sort of feedback control mechanism. In a study of retinoid processing in the toad retinal pigment epithelium (RPE) eyecup as a func- tion of the amount of extracellular IRBP available to receive U-cis retinal from the RPE, both the molar amount and the specific radioactivity of 1 1-ds retinal in the extracellular medium increased with increasing IRBP concentration. These results suggest an effect of IRBP on the process of 11-cis retinal formation during the visual cycle. We studied the C57vii/vit mouse, which has a progressive retinal degeneration in which photorecep- tor cells are lost slowly over a year's time and RPE cells are unevenly pigmented. HPLC analysis of its retinoids demonstrated levels of retinyl palmitate approximately 2.5 times greater in the eyes of 6- week affected mice compared with those of congenic controls. Levels of l\-cis retinal, ail-trans retinal, and ail-trans retinol were elevated to levels more than 1.5 times greater than controls. Whereas in the normal retina, photoreceptor cells and RPE cells function cooperatively to regulate retinoids involved in the visual cycle, one or more aspects of the visual cycle may malfunction, thus contributing to the progressive retinal degeneration in the C57vit/vit mouse. In studies of ligand binding by a retinoid-binding glycoprotein from Drosophila melanogaster heads, analysis of endogenously bound retinoids by HPLC and mass spectrometry identified a component with a molecular ion m/z at 286, indicating a possible isomer of retinol. Analysis of endogenously bound fatty acids demonstrated both covalent and noncova- lent binding of several fatty acids (including lauric, myristic, palmitic, oleic, and linolenic acids) charac- teristic of vertebrate IRBP, which is also a farty acid- binding protein. Binding affinities of retinol and 16- (9-anthroyloxy) palmitic acid were charaaerized by fluorometric titrations. 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. Significance to Biomedical Research and the Program of the Institute Because of its importance in normal photoreceptor cell physiology, ie, in facilitating the transport of retinoids during the visual cycle as well as transport of fatty acids that are essential to normal function, 18S Laboratory of Retinal Cell and Molecular Biology NEI Annual Report-FY 1992 abnormalities in IRBP function resulting fnam changes in concentration, distribution, or affinity for retinoids or fatty acids could be important, either directly or indirectly, in visual ceU pathogenesis. Proposed Course We will continue to analyze factors controlling IRBP gene expression, using polymerase chain reaction quantitation of IRBP message and comparison with other retinal proteins such as opsin and S-antigen. Further studies wiU be conducted on the physiologi- cal role of IRBP in the visual cycle and how remov- ing IRBP during retinal detachment affects regenera- tion of visual pigment. Studies on the C57vit/vit mouse model of retinal degeneration will include the quantitation of IRBP and message and further analy- ses of retinoid levels at different stages of postnatal development. Further studies on Drosophila retinoid- binding protein will include peptide analysis and antibody production, toward the goal of cloning and sequencing this protein for comparison to IRBP. Both retinol and fatty acid-binding sites on IRBP will be analyzed further via fluorescence studies. Large-scale purification of IRBP protein for studies on EAU wiU continue. NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications Caspi RR, Chan C, Fujino Y, Oddo S, Najafian F, Bahmanyar S, Wilder RL, Wiggert B: Genetic factors in susceptibility and resistance to experi- mental autoimmime uveoretinitis. Curr Eye Res ll(suppl):81-86,1992. Caspi RR, Grubbs BG, Chan CC, Chader GJ, Wiggert B: Genetic control of susceptibility to experimental autoimmune uveoretinitis in the mouse model. Concomitant regulation by MHC and non-MHC genes. J Immunol 148:2384-2389, 1992. Crouch RK, Hazard S, Lind T, Wiggert B, Chader G, Corson DW: Interphotoreceptor retinoid- binding protein and a-tocopherol preserve the isomeric and oxidation state of retinol. Photochem Photobiol 56:251-255.1992. Hara Y, Caspi, RR, Wiggert B, Chan CC, Wilbanks GA, Streilein JW: Suppression of experimental autoimmune uveitis in mice by induction of anterior chamber associated immune deviation with inteiphotoreceptor retinoid-binding protein. J Immunol 148:1486-1685, 1992. Hara Y, Caspi RR, Wiggert B, Dorf M, Streilin JW: Analysis of an in vitro generated signal that induces systemic immune deviation similar to that elicited by antigen injected into the anterior chamber of the eye. J Immunol 149:1531-1538, 1992. Kawano Y, Sasamoto Y, Kotake S, Thurau SR, Wiggert B, Gery I: Trials of vaccination against experimental autoimmime uveoretinitis with a T- cell receptor peptide. Curr Eye Res 10:789-795, 1991. Korf HW, Korf B, Schachenmayr W, Chader GJ, Wiggert B: Immunocytochemical demonstration of interphotoreceptor retinoid-binding protein in cerebellar medulloblastoma. Acta Newopathol 83:482^87, 1992. Kotake S, Sasamoto Y, Kawano Y, Sanui H, Wiggert B, Chader GJ, Gery I: The existence of two completely distinct antigenic sites within a deca- peptide. Cell Immunol 140:123-129, 1992. Kutty G, Hayden B, Osawa Y, Wiggert B, Chader GJ, Kutty RK: Heme oxygenase: Expression in human retina and modulation by stress agents in a human retinoblastoma cell model system. Curr Eye Res 11:153-160, 1992. Pepperberg DR, Okajima TL, Ripps H, Chader GJ. Wiggert B: Functional properties of interphoto- receptor retinoid-binding protein. Photochem Photobiol 54:1057-1060, 1991. Robbins SG, Wiggert B, Kutty G, Chader GJ. Detrick B, Hooks J J: Redistribution and reduction of interphotoreceptor retinoid-binding protein during ocular coronavirus infection. Invest Oph- thalmol Vis Sci 33:60-67, 1992. Rodrigues MM, Rajagopalan S. Lee L, Nair CN. Advani SH, Donoso L, Chader GJ, Wiggert B: Retinoblastoma: Messenger RNA for inteiphoto- receptor retinoid-binding protein. Curr Eye Res 11:425^33, 1992. 186 NEI Annual Report-FY 1992 Laboratory of Retinal Cell and Molecular Biology Smith SB, Lee L, Nickerson J, Si JS, Chader GJ, Wiggert B: Synthesis and secretion of interphoto- receptor retinoid-binding protein (IRBP) and developmental expression of IRBP mRNA in normal and rd mouse retinas. Exp Eye Res 54:957-963, 1992. Wiggert B, Kutty G, Long KO, Inouye L, Gery I, Chader GJ, Aguirre GD: Interphotoreceptor retinoid-binding protein (IRBP) in progressive rod-cone degeneration (prcd) — Biochemical, immunocytochemical and immunologic studies. Exp Eye Res 53:389-398. 1991. Yamamoto JH, Okajima O, Mochizuki M, Shinohara T, Wiggert B, Chader GJ, Gery I, Nussenblatt RB: Cellular immune responses to retinal antigens in retinitis pigmentosa. Graefes Arch Clin Exp Ophthalmol 230A\9-\23, 1992. 187 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00196-09 LRCMB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or lees. Title must fit on one line between the borders.) Molecular Genetics of the Eye and Ocular Diseases PRINCIPAL INVESTIGATOR (List ottter professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: Diane E. Borst Ph.D. Staff FeUow LRCMB, NEI Steven Bernstein Ph.D., M.D. Senior Staff Fellow LRCMB, NEI COOPERATING UNITS (if any) Emory University, Atlanta, GA (J.M. Nickerson, Ph.D., J-S. Si, M.D.); University of Michigan, Ann Arbor (E. Farr, M.D.) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology SECTION Section on Gene Regulation INSTITUTE AND LOCATION NEI, NIH. Bethesda, MD 20892 TOTAL STAFF YEARS: 2.0 PROFESSIONAL: 2.0 0.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (al) Minors □ (a2) interviews [x| (b) Human tissues Q (c) Neitlier SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Inteiphotoreceptor retinoid-binding protein (IRBP) is an abundant glycolipoprotein expressed in the retina and pineal gland. Photoreceptor cells in the retina contain IRBP mRNA. We are characterizing the cw-elements regulating IRBP expression using a transient transfection assay and transgenic mice. There are two conserved areas of sequence in the 5' flanking regions of the bovine, human, and mouse IRBP genes, one from -1 to -350 and another from -1200 to -1410. The 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. 188 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 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 define the c«-acting elements and trans-acting factors that regulate interphotoreceptor retinoiu-binding protein (IRBP) gene expression in a tissue-specific and/or develop- mentally specific manner and to downregulate eye- specific genes by exogenously derived genetic elements introduced either as antisense/catalytic RNA or as antisense DNA. In addition to the obvious gene therapeutic possibilities, this approach also can be used when total deletion of a target gene would be deleterious to survival 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. Chloram- phenicol acetyltransferase (CAT) activity is measured by an enzyme-linked immunosorbent assay or the biphase assay. Catalytic RNA/antisense (ribozyme) constructs are used for permanent transfection of ceU 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 quantitatively measured by polymerase chain reaction (PCR)-based techniques. Major Findings Gene expression. — Constructions containing pre- sumptive elements of the IRBP promoter joined to an indicator gene (CAT) were made with both the bovine and mouse IRBP promoters for study of the expression of the IRBP gene. Two sites in the 5' flanking (promoter) region show significant homol- ogies across species, and we have made constructions containing both conserved blocks, only one of the two blocks, or neither. Tests of these constructions in several systems, including retinoblastoma cells (Y-79 and WERI), frog oocytes, mixed pinealocyte primary cultures, transformed pinealocytes, and normal mouse fibroblasts show promoter activity in Y-79 cells transfected with the ERBP promoter-CAT constructs containing both conserved 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 trans-acling 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 protein of 120,000 MW that binds to the -300 region of the promoter. This binding activity is not unique to the retina, being present in the heart, kidney, and lung; however, it is not ubiquitous. Downregulation of gene expression. — IRBP-based ribozymes have been developed which exhibit activity in vitro against an IRBP mRNA substrate. These ribozymes have been selected and modified to yield molecules with high activity. Constructions that use ribozymes in tandem with promoters from both human P-actin (Kedes et al., 1987) and mouse phos- phoglycerokinase have been made and transfected into human Y-79 retinoblastoma cells. Because of the extremely low level of IRBP mRNA in these cells, even when induced with butyrate, IRBP mRNA levels were not detectable following transfection with the ribozymes, in either the sham-transfected controls or transfected ceU fines. Significant levels of the ribozyme construct were detected in the experimental fines. Because of this difficulty, PCR-based detection methods are under development, and we anticipate the methodology will permit mRNA detection at much lower levels, as well as provide quantitative estimation of IRBP mRNA inhibition. We now are utilizing a mouse retinoblastoma cell line derived from mice transfected with SV-40 large T antigea This ceU line, which we have shown expresses high levels of IRBP mRNA, secretes IRBP in moderately high levels. We expect that transfec- tion of these cells will provide a better model for evaluating IRBP downregtilation, as well as correlat- ing direcUy with die mouse transgenic animals now being developed. Sequence analysis of the mouse IRBP genome. — Sequencing the genomic clones encoding the mouse IRBP gene has shown that 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 189 Laboratory of Retinal Cell and Molecular Biology NEI Annual Report-FY 1992 (2.4 kb) and human (0.7 kb) orthologs. We are examining tlie sequence to detemiine alternative splice sites that may explain the unique apjjearance 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 fimda- mental to understanding normal retuial development and functioa 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 Y-79 cells. However, fewer than 205 bases of the 5'-flanking region are needed for basal promoter activity in the Y-79 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 wiU compare the expression of these constructions in transgenic animals and Y-79 cells under different culture conditions. Preliminary studies show that the transgene is active in development as eariy as embry- onic day 9, but high levels of expression coincide with the beginning of outer segment elongation, and steady-state adult levels are not reached until about posmatal day 30. In future studies, we will examine other transgenic mouse lines in the retina and several other tissues during development We will character- ize the c«-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 expres- sion libraries by our established Southwestern blot- ting procedure. Preliminary screenings have yielded two potential clones. NEI Research Program Retinal Diseases — ^Photoreceptors and Retinal Pig- ment Epithelium Publications Humayun M, Bernstein SL, Gould HL, Chavis RM: Orbital childhood acute lymphoblastic leukemia as the initial presentation. / Pediatr Ophthalmol Strabismus 29:252-255. 1992. 190 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00124-12 LRCMB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (BO characters or less. Title must lit on one line between the borders.) Metabolism of the Retina and Pigment Epithelium PRINCIPAL INVESTIGATOR (List other prolesslonal personnel below the Principal Investigator.) (Name, lltle, laboratory, and Institute alllliallon) 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. Ph.D. Ph.D. Ph.D. P.:D. B.S. Expert Senior Staff Fellow Staff FeUow Staff FeUow Visiting Scientist Biologist LRCMB, NEI LRCMB. NEI LRCMB. NEI LRCMB, NEI LRCMB, NEI LRCMB, NEI COOPERATING UNITS (II any) Laboratory of Retinal CeU and Molecular Biology SECTION Section on Gene Regulation INSTITUTE AND LOCATION NEI. NIH, Bethesda. MP 20892 TOTAL STAFF YEARS: 6.5 PROFESSIONAL: 5.5 1.0 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Interviews [3 (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The retina and pigment epithehum are neuroepitheUal tissues that woric in close cooperation, as do other ocular tissues. Specific growth and differentiating factors found in the eye guide the development and interactions of individual ocular tissues to form a functional visual system. For example, ocular tissues synthesize insulin- hke growth factor (IGF-I) and its binding proteins (BP), thus may self-regulate activity of this important growth factor in the vitreous and surrounding tissues. In addition to IGFBP. we have identified a unique differentiating protein secreted firom fetal human pigment epitheUal ceUs called pigment-epitheUal-derived factor that is neurotropic 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. 191 PHS 6040 (Rev. 5/92) Laboratory of Retinal Cell and Molecular Biology NEI Annual Report-FY 1992 Project Description Objectives Our objective is to obtain a better understanding of the growth, development, and general metabolism of ocular tissues in health and disease. Study of growth and differentiation factors, be they protein (eg, pigment epithelium -derived factor [PEDF], polypep- tide (eg, insulin-like growth factor [IGPH-I), or low- molecular-weight compoimds (eg, cyclic AMP), is critical in obtaining a view of the events that control the early development of the eye and also in main- taining normal function in the adult. Methods Standard biochemical, molecular biological, and immunocytochemical techniques are used. Tissue culture is performed using various cells, in particular, the human retinoblastoma cell line, Y-79, which is used as a test system for differentiating agents. Major Findings PEDF. — Purified from conditioned medium of cultured fetal human pigment epithelial cells, PEDF also appears to be present in nonnal adult interphoto- receptor matrix (IPM). The protein, which migrates at approximately 540 kD on SDS-polyacrylamide gels, causes marked differentiation of human Y-79 retinoblastoma cells in culture. This differentiation is characterized by an extensive elongation of neurite- like processes and a gathering of cells into "rosette- like" aggregates. Immunocytochemically, the expres- sion of specific neuronal markers is also enhanced. PEDF is a imique protein, synthesized and secreted by retinal pigment epithelial cells, that could direct early development, even in early embryogenesis. It thus may be that PEDF is also present after the important developmental period and may help to maintain retinal cell viability in the adult retina. Cloning of the PEDF gene. — We now have cloned the cDNA for the PEDF gene. The protein is a member of the serine protease inhibitor superfamily of genes. Some members of this family are known to promote cellular differentiation, so it is more prob- able that PEDF has a major, similar role in the retina. Localization of the PEDF gene. — Using fluores- cent in situ hybridization, polymerase chain reaction and Southern blotting, we have localized the PEDF gene to the short arm of human chromosome 17. By 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 same chromosomal area as the Li-Fraumeni cancer gene; thus, PEDF may be part of an important cluster of genes involved in ceU prohferation and cancer. IGF-I. — IGF-I is involved in the development and function of many tissues. In the eye, IGF-I probably participates in attaining overall eye size and in the function of individual ocular tissues and cell types. Specific IGF-binding proteins (IGFBPs) control the bioavailability of the IGFs, thus are important regula- tors of IGF activity in health and disease. The vitreous and several ocular tissues, including the cornea, contain high levels of IGFBPs not derived from extraocular sources. The ciliary body is the probable site of synthesis for at least one of the vitreal BPs, specifically IGF-BP2. Other ocular tissues, such as the neural retina or pigment epitheli- um, do not contain IGF-BP2, thus the ciliary body probably secretes the BP into the vitreous where it could be a major factor in regulating developmental programs in the eye. It is interesting that the cornea exhibits exceptionally high amounts of binding protein activity. Their role in corneal metabolism is yet unknown but, because of their growth-regulating potential, BPs could be involved in such important processes as wound healing and corneal compli- cations of diabetes. Significance to Biomedical Research and the Program of the Institute Determining the factors that affect normal ocular growth, differentiation, and fimction will aid us in understanding diseases of the eye, especially those of a hereditary or early developmental nature. Proposed Course The effects of growth factors on ocular development will be further examined. The factors that affect normal and abnormal growth of the sclera wiU be further investigated, with particular reference to myopia. The fiill PEDF gene (genomic) will be examined and analyzed to help to elucidate its presumptive role(s) in retinal development The generality of the PEDF gene in other tissues will be examined, specificaUy in relation to its possible role in cancer growth. 192 NEI Annual Report-FY 1992 Laboratory of Rednal Cell and Molecular Biology NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications 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 famQy. Proc Natl Acad Sci USA, in press. Tombran-Tink J, Chader GJ, Johnson LV: PEDF: A pigment epithelium-derived factor with potent neuronal differentiative activity. Exp Eye Res 53:411-414, 1991. Tombran-Tink J, Li A, Johnson MA, Johnson LV, Chader GJ: Neurotrophic activity of interphoto- receptor matrix on human Y-79 retinoblastoma cells. / Comp Neurol 317:175-186, 1992. Viczian A, Sanyal S, Toffenetti J, Chader GJ, Farber DB: Photoreceptor-specific mRNAs in mice carrying different allehc combinations at the rd and rds loci. Exp Eye Res 54:853-860, 1992. Waldbillig RJ, Arnold DR, Hetcher RT, Chader GJ: Insulin and IGF-I binding in developing chick neural retina and pigment epithelium: A charac- terization of binding and structural differences. Exp Eye Res 53:13-22, 1991. Waldbillig RJ, Chader GJ, Pfeffer BA: Monkey retinal pigment epithelial ceUs in vitro synthesize, secrete and degrade insulin-like growth factor binding proteins. J Cell Physiol 150:76-83, 1992. WaldbiUig RJ, Pfeffer BA, Schoen TJ, Adier AA, Shen-Orr Z, Scavo L, LeRoith D, Chader GJ: Evidence for an insulin-like growth factor auto- crine-paracrine system in the retinal photorecep- tor-pigment epithehal ceU complex. J Neurochem 57:1522-1533, 1991. 193 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PERIOD COVERED October 1, 1991 to September 30. 1992 PROJECT NUMBER ZOl EY 00148-19 LRCMB TITLE OF PROJECT (80 characters or lass. Title must lit on one line between the borders.) Visual Control Mechanisms PRINCIPAL INVESTIGATOR (Lis, other professional personnel below the Principal Investigator.) (Name, title, laboratory, and insUtute allillation) PI: Gerald J. Oiader Ph.D. Chief LRCMB, NEI Others: Paul Wong Ph.D. Tatiana Putilina Ph.D. Ignacio Rodriguez Ph.D. Jun Li M.D. R. Theodore Fletcher M.S. Visiting Fellow Visiting Associate Staff Fellow Visiting Associate Chemist LRCMB, NEI LRCMB, NEI LRCMB, NEI LRCMB, NEI LRCMB. NEI COOPERATING UNITS (If any) ~ -^ School of V«erina,y Medicme, Universiq, of Penn^lvania (G. Aguure. D.V.M.. Ph.D.); Department of Amuomy. Erasmus University. Ronerdam. Tie Netherlands (S. Sanyal. Ph.D.); Department of Zoology. University of Lund. Lund. Sweden (T. van Veen. Ph.D.); InsUtuto Nazionale per la Ricera sul Ca«:ro Genova, Italy (A. Albini. Ph.D., D. Noonan, Ph-D.) LAB/BRANCH Laboratory of Retinal Ce ll and Molecular Biology SECTION Section on Gene Regulation INSTITUTE AND LOCATION NEI, NIH. Bethesda. MP 20892 TOTAL STAFF YEARS: 5.5 CHECK APPROPRIATE BOX(ES) n (a) Human subjects n (a1) Minors n (a2) Interviews PROFESSIONAL: 4.5 OTHER: 1.0 S (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) The ftmction of neural tissues such as the retina depends upon the presence of normal complements of tissue- h^i^' ^T' Tu" *' "°™^ expression of the protein gene products. If these mechanisms fail hereditaiy diseases of the retina, such as retinoblastoma or retinitis pigmentosa, will result. We have found mat lamimn, an miportant extracellular matrix protein, slows retinoblastoma ceU growth and promotes differenuauoa Speafically, it switches development from a photoreceptor pathway to a conventional neuronal-bke Pathway via a low affinity "differentiative" binding activity, which we have called laminin- ?n S,T ^^r'^i^^^'?- "-^"^-'^ "'^'^ ""''' ' •"^j^^ ^ff«« o" ^^ly '^tinal cell develoTmrt. ^Sy "^^ developed new techniques to clone and sequence retina-specific genes at a higher 194 PHS 6040 (Rev. Sfl2) NEI Annual Report-FY 1992 Laboratory of Retinal Cell and Molecular Biology Project Description Objectives Normal development and expression of genes in the retinal photoreceptor neuron is crucial to visual function in the adult; thus, the factors that code for normal gene control and expression in human retina and in animal models of retinal degeneration are of primary interest. As a coroUary, we have mounted a major effort to develop new molecular biological techniques such that unique retinal and retinal pigment epithelium (RPE) 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 routinely employed. Histochemical techniques are used when necessary. Major Findings Laminin-binding molecule-100. — A ubiquitous extracellular matrix protein, laminin has profound effects on a variety of cell types. For example, both gene and protein expression in cultured human Y-79 retinoblastoma cells are switched from a photorecep- tor to a conventional neuronal pathway by the addition of this basement membrane glycoprotein in culture. Unlike other cell systems in which laminin influences differentiation, Y-79 cells can neither attach to nor chemotactically respond to laminin. Affinity chromatography of radiolabeled Y-79 surface proteins revealed a single 100-kD laminin- binding protein. We have named the protein laminin- binding molecule 100 (LBM-100) and propose that it serves as an embryonic and/or tumor "differentia- tion receptor" through which laminin influences gene expression and development. Advances in molecular biology. — ^Each tissue of the body expresses a unique complement of genes that are transcribed and translated at a high level. In the retina, for example, opsin and interphotoreceptor retinoid-binding protein (IRBP) 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 degeneration such as retinitis pigmentosa. We are interested in developing new molecular biological techniques that will allow for more efficient identifi- cation of highly expressed genes of the RPE com- plex. In this, we have had two significant advances. First, we developed a new method for rapid polymerase chain reaction-based construction of specifically enriched Ubraries from very small retinal samples. This development wiU be especially impor- tant when tissue samples are limited, eg, early development and rare pathology ^amples. Our second methodological advance involves subtractive cloning on an immobilizing Dynabead® base. One of the most common problems in subtraction cloning is that specific mRNAs or cDNAs of a particular tissue are subtracted by common sequences (eg, gene families) or repetitive elements (eg, Alu repeats), leaving little or no product for use as a probe. We synthesized single-strand antisense cDNA from retina on magnet- ic Dynabeads such that the mRNA could be easily destroyed and short, labeled sense fragments could be generated. We validated the technique by generat- ing a nimiber of clones, among which are opsin, IRBP, green visual pigment, and several RPE-specif- ic genes not previously described. Significance to Biomedical Research and the Program of the Institute To understand and control or even reverse a heredi- tary disease process in a tissue, one must identify the normal complement of unique genes expressed in that tissue and on the functioning of the protein products. This is especially true in an eariy degenera- tive process, eg, retinitis pigmentosa, and in other hereditary diseases, such as retinoblastoma, in which abnormal changes are subtle and can be masked by normal developmental switches in gene expression. Thus, studying the molecular biological development and metabolic changes in retina-specific systems wiU lead to a better understanding of disease processes unique to the neural retina. Proposed Course Molecular biological and developmental control mechanisms in the retina and pigment epithelium wiU continue. In particular, we will investigate gene expression in normal retinas and in retinas affected with specific genetic diseases. 195 Laboratory of Retinal Cell and Molecular Biology NEI Annual Report-FY 1992 NEI Research Program Retinal Diseases — Retinitis Pigmentosa and Other Inherited Disorders Publications Albini A, Noonan DM, Melchiori A, Fassina GF, Percario M, Gentleman S, Toffenetti J, Chader GJ: Laminin switches retinoblastoma ceU differ- entiation from a photoreceptor- to a neuronal-like pathway via a low affinity "differentiative" binding activity. Proc Natl Acad Sci USA 89:2257-2261, 1992. Aresu O, Nicolo G, Allavena G, Melchiori A, Schmidt J, Kopp JB, d'Amore E, Chader GJ, Albini A: Invasive activity, spreading on and chemotactic response to laminin are properties of high but not low metastatic mouse osteosarcoma cells. Invasion Metastasis 11:2-13, 1991. Boje KM, Skohiick P, Raber J, Fletcher RT, Chader GJ: l-AminocyclopropanecarboxyUc acid attenu- ates NMDA excitotoxicity in embryonic chick retina. Neurochem Int, in press. Caspi RR, Grubbs EG, Chan CC, Chader GJ, Wiggert B: Genetic control of susceptibility to experimental autoimmune uveoretinitis in the mouse model. Concomitant regulation by MHC and non-MHC genes. J Immunol 148:2384-2389, 1992. Crouch RH, Hazard ES, Lind T, Wiggert B, Chader GJ, Corson DW: Inteiphotoreceptor retinoid- binding protein and a-tocopherol preserve the isomeric and oxidative state of retinol. Photochem Photobiol 56:251-255, 1992. del Cerro M, Notter MF, Seigel G, Lazar E, Chader G, del Cerro C: Intraretinal xenografts of differen- tiated himian retinoblastoma cells integrate with the host retina. Brain Res 583:12-22, 1992. del Cerro M, Siegel GM, Lazar ES, Grover DA, del Cerro C, DiLoreto D, Chader GJ: Transplantation of Y-79 cells into rat eyes: A useful in vivo model for the study of human retinoblastoma. Brain Res, in press. Hershfield B, Inouye L, Chader GJ, Ripps H, Aguirre G: Organization and transcription of canine (CAC)n sequences. / Hered 82:251-254, 1991. Korf HW, Korf B, Schachenmayr W, Chader GJ, Wiggert B: Immunocytochemical demonstration of interphotoreceptor retinoid-binding protein in cerebellar medulloblastoma. Acta Neuropathol 83:482-487, 1992. Kotake S, Sasamoto Y, Kawano Y, Sanui H, Wiggert B, Chader GJ, Gery I: The existence of two completely distinct antigenic sites with a deca- peptide. Cell Immunol 140:123-129, 1992. Kutty G, Hayden B, Osawa Y, Wiggen B, Chader GJ, Kutty RK: Heme oxygenase: Expression in human retina and modulation by stress agents in a human retinoblastoma cell model system. Curr Eye Res 11:153-160, 1992. Kutty RK, Fletcher RT, Chader GJ, Krishna G: Expression of guanylate cyclase-A mRNA in the rat retina: Detection using polymerase chain reaction. Biochem Biophys Res Commun 182:851- 857, 1992. Pepperberg DR, Okajima TI, Ripps H, Chader GJ, Wiggert B: Functional properties of interphoto- receptor retinoid-binding protein (IRBP). Photo- chem Photobiol 54:1051-1060, 1991. Robbins SG, Wiggert B, Kutty G, Chader GJ, E>etrick B, Hooks JJ: Redistribution and reduction of interphotoreceptor retinoid-binding protein during ocular coronavirus infection. Invest Oph- thalmol Vis Sci 33:60-67, 1992. Rodriguez IR, Chader GJ: A novel method for the isolation of tissue specific genes. Nucleic Acids Res 20:3528, 1992. Rodrigues MM, Rajagopalan S, Lee L, Nair CN, Advani SH, Donoso L, Chader GJ, Wiggert BN: Retinoblastoma: Differentiation and mRNA for interphotoreceptor retinoid-binding protein. Curr Eye Res 11:425-433, 1992. Sasamoto Y, Kawano YI, Bouligny R, Wiggert B, Chader GJ, Gery I: Immunomodulation of experi- mental autoimmime uveoretinitis by intravenous injection of uveitogenic peptides. Invest Ophthal- mol Vis Sci 33:2641-2649, 1992. Smith SB, Lee L, Nickerson J, Si JS, Chader GJ, Wiggert B: Synthesis and secretion of interphoto- receptor retinoid-binding protein (IRBP) and developmental expression of IRBP mRNA in normal and rd mouse retinas. E^ Eye Res 54:957-963, 1992. 196 NEI Annual Report-FY 1992 Laboratory of Retinal Cell and Molecular Biology Wiggert B, Kutty G, Long KO, Inouye L, Gery I, Chader GJ, Aguirre GD: Interphotoreceptor retinoid-binding protein (IRBP) in progressive rod-cone degeneration (prcd) — Biochemical, immunocytochemical and immunologic studies. Exp Eye Res 53:389-398. 1991. 197 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00260-03 LRCMB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or /ass. Titia must tit on one line between the borders.) Molecular Biology of Outer Retina-Specific Proteins PRINCIPAL INVESTIGATOR (Lisl other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute affiliation) PI: T. Michael Redmond Ph.D. Research Biologist LRCMB, NEI Others: Christian P. Hamel Ekaterina Tsilou M.D. M.D. Visiting Associate Visiting FeUow LRCMB, NEI LRCMB, NEI COOPERATING UNITS (If any) LAB/BRANCH Laboratory of Retinal Cell and Molecular Biology Section on Gene Regulation INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 3.0 PROFESSIONAL: 3.0 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.) Retinal pigment epithelial (RPE) cells and photoreceptor (PR) cells are functionally and developmentaUy closely integrated. We are characterizing a 65 kD RPE-specific protein that is membrane-associated, hydrophobic, and dependent upon detergent solubilization for its extraction. In cell fractionation experiments this protein is found in the microsomal pellet. We have obtained its amino acid analysis and the sequences of several tryptic and chymotryptic peptides. Its amino terminus is blocked. We have cloned cDNA for this protein, which encodes a 533-residue protein unlike any others in the data base. The interphotoreceptor retinoid-binding protein (IRBP) is involved in the transport of retinoids, a functional relationship between the RPE and the PR. We have subcloned DNA fragments corresponding to integral repeats of bovine IRBP into a bacterial expression vector. The resultant expressed protein fragments were tested for their retinoid-binding functions. One of the fragments, which comprises ttie central two repeats, bound retinol. This finding indicates that aU four repeats of IRBP are not required for retinoid-binding activity. The binding appears to be localized to the central region of the IRBP protein. 198 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Retinal Cell and Molecular Biology Project Description Objectives The retinal pigment epithelium (RPE) is essential for continued maintenance of the photoreceptor (PR) rod outer segments (ROS). These two components of the outer retina share a close developmental and func- tional relationship; however, knowledge of derange- ments of the RPE known to be involved in certain retinal dystrophies is, at the molecular level, lacking. We are trying to understand better the RPE/PR relationship by investigating the structure and func- tion of outer retina-specific proteins: one, a 65-kD RPE-specific protein; another, interphotoreceptor retinoid-binding protein (IRBP) is involved in the transport of retinoids between the RPE and the ROS. In addition, we are isolating RPE-expressed cDNAs to generate other molecular probes. Methods Molecular cloning and biochemical and protein chemistry techniques are employed in this study. We also are using automated fluorescent DNA sequenc- ing and gene mapping techniques. Major Findings A 65-kD outer retina-specific protein isolation and characterization. — ^The membrane-associated 65-kD protein is most effectively solubilized by the use of various detergents. We have foimd that the protein preferentially fractionates with the microsomal pellet when it is the major protein. Its affinity for phospho- lipids has facilitated partial purification of the pro- tein. Approximately 40% of the protein has been sequenced by way of tryptic and chemotryptic peptides. The protein is not expressed by RPE cells in culture. Cloning of the 65-kD protein. — We have cloned the 65-kD protein. Using peptide sequence-directed oligonucleotide probes we have isolated two overlap- ping clones covering 2.4 kb. The remaining 5' sequence was obtained by polymerase chain reaction (PCR), coupled to reverse transcription of mRNA. The final mRNA is 3,115 bp long with a 1,602 bp open reading frame coding for 533 amino acids that match perfectly with the sequenced peptides. The amino acid sequence does not match any other in the database, definitively proving that it is a novel molecule. Characterization of IRBP .—PCR-Atn\eA frag- ments of IRBP corresponding to repeats 1 + 2 and 2 + 3 have been characterized. It appears that only the 2 + 3 repeat fragment binds all-o-a«j-retinol. Significance to Biomedical Research and the Program of the Institute There is a paucity of reliable RPE-specific proteins that have been characterized. This is esf)ecially onerous in view of the indispensable function of the RPE in such specialized functions as retinoid metab- ohsm, outer segment phagocytosis, and various transport mechanisms. The 65-kD protein identified is a conserved, RPE-specific molecule found in mammal and bird RPE. Furthermore, it first appears in rat just prior to the appearance of the ROS sug- gesting a temporal or developmental control of expression. The loss of the protein in culture sug- gests that a continuous in vivo signal is required for its expression. Sequencing of the cDNA clones demonstrate that it is a novel protein. Elucidation of the function of the 65-kD protein may help our understanding of the RPE. Using this molecular probe, we will be able to dissea further the specific role of the RPE in the development and maintenance of processes important to vision. Similarly, the relationship of structure to the function of IRBP, which is thought to transport retinoids between the RPE and the ROS, has not been fully elucidated. In particular, in the relation- ship of the fourfold repeat strucmre of IRBP to the hgand-binding properties is of major importance. A bacteriaUy expressed fragment of IRBP consisting of the control two repeats waU bind retinol, indicating that the whole protein is not required for binding, whereas a fragment consisting of 1 + 2 repeats does not appear to bind retinol. This finding wiU be important in understanding the trafficking of reti- noids between the RPE and the ROS. The identifica- tion of further RPE-expressed cDNAs will markedly enhance our knowledge of the RPE in health and disease. Proposed Course 1 . Further characterization and purification of the 65-kD protein for structural, immunological, and biochemical analysis will continue. Elucidation of its function v«U involve a variety of approaches. 199 Laboratory of Retinal Cell and Molecular Biology NEI Annual Report-FY 1992 2. We will screen human and mouse genomic libraries for the respective genes, which will be sequenced and characterized by chromosomal local- izations and possible association with disease. Trans- genic studies will be undertaken. 3. The 65-kD protein will be overexpressed to provide material for structural, functional, and immunological studies. 4. Human RPE libraries will be screened using the expressed sequence tag approach to identify other RPE-specific genes. 5. We will continue using PCR techniques to produce defined sequences for expression of the repeats of IRBP. The expressed proteins will be purified and studied with respect to their Ugand- binding properties. Certain expressed proteins wUl be supplied for analysis of uveitogenicity in the mouse model by Dr. Rachel Caspi (Laboratory of Immunol- ogy). NEI Research Program Retinal Diseases — ^Photoreceptors and Retinal Pig- ment Epithelium Publications Redmond TM, Nickerson JM: Retinoid binding to a recombinant control two-repeat segment of IRBP. Invest Ophthalmol Vis Sci 33(4):2446. 1992. Tsilou ET, Hamel CP, Harris E, Detrick B, Hooks JJ, Redmond TM: Partial purification, character- ization and cloning of a RPE-specific protein. Invest Ophthalmol Vis Sci 33(4):1109, 1992. 200 DEPARTMErfT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00132-11 LRCMB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (BO charaaars or lass. Title must til on one line between the borders.) Molecular Biology of Phototransduction PRINCIPAL INVESTIGATOR (List other professional personnel below the Pnnclpal Investigator.) (Name, title, laboratory, and institute atlUlatlon) PI: Toshimichi Shinohara Ph.D. Head, Section on LRCMB, NEI Molecular Biology Others: Thoru Abe Takanobu Kikuchi Shirley Yu M.D., Ph.D. Ph.D. B.S. Visiting Associate Visiting Associate Biologist LRCMB, NEI LRCMB, NEI LRCMB, NEI COOPERATING UNITS (It 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 Cell and Molecular Biology SECTION Section on Molecular Biology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 3.8 PROFESSIONAL: 2.8 1.0 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.) We have characterized the human and mouse S-antigens and 33K protein genes and a human Shuzin gene, and we have deteraiined the gene sequences of the human and mouse S-antigens. The S-antigen genes were approximately 50 kbp in length, contained 16 exons and 15 introns, and comprised of 97% intron and 3% exon. The 5'-flanking regions of the genes, approximately 1.5 kbp long, had no known regulatory elements for transcription, such as TATA, GC, or CCAAT boxes. Interestingly, the 5' flanking regions of the human, bovine, and mouse genes expressed tissue-specific promoter activity in both in vitro and in vivo transcription assays as well as in transgenic mice. Isolation of two 33K protein genes and determination of their DNA sequences revealed genes of approximately 10 kbp in length, containing four exons and three introns. The 33K protein gene seems to represent a family of genes: At least three genes have similar sequences. The functional role of the retinal {5rotein Shuzin is unknown. We isolated and sequenced several cDNAs each from humans and cows. The entire human Shuzin gene sequence also was determined. This gene, composed of two introns and three exons, has a highly repetitive sequence in the 5' noncoding region. We have constructed fusion genes containing a 5'-flanking S-antigen gene sequence upstream of the bacterial gene chloramphenicol acetyl transferase (CAT). A hybrid gene containing the 5'-flanking region of the mouse S-antigen gene and the CAT gene was microinjected into transgenic mice. The mice expressed CAT activity in the retina and pineal gland, suggesting tiiat die 1,300 bp S-antigen promoter segment contains sufficient information to direct appropriate tissue-specific gene expression in transgenic mice. In addition, S-antigen was found in lens fiber and epithelial ceUs, the cerebellum, and the cerebral cortex. These results indicate that S- antigen is expressed in a wider spectrum of the cell types than previously recognized. 201 PHS 6040 (Rev. 5«2) Laboratory of Retinal Cell and Molecular Biology NEI Annual Report-FY 1992 Project Description Objectives The objective of this project is to understand the basic mechanism of phototransduction in the retina and to understand the structure, function, and evolu- tion of the proteins present in photoreceptor rod cells and pinealocytes. Metho'is Conventional methods for analysis of proteins and nucleic acids used include protein purification and RNA and DNA isolation, characterization, and sequence determination. Recombinant DNA tech- niques used include a baculovirus expression vector system, synthesis of point mutation clones, character- ization of promoters, and transgenic animals. We also have synthesized and used purified oligopeptides and oligonucleotides. Major Findings 1 . We determined the gene sequences of S-antigen (S-Ag) from human and mouse. S-Ag is 50 kbp in length and has 15 introns and 16 exons. The smallest exon encodes for three amino acids. 2. We foimd that the intron-exon map sequence of the mouse S-Ag gene has been well conserved. Approximately 97% of this gene is intron; 3% is exon. 3. We subcloned the hiunan and mouse S-Ag cDNAs into two expression vectors and expressed. The products of S-Ag cDNA were purified by column chromatography and prepared for crystalliza- tion. 4. We determined the 5'-flanking sequence of the himian and mouse S-Ag genes and demonstrated promoter activity in in vivo and in vitro transcrip- tional assays. 5. Although the S-Ag promoter sequences are highly conserved between himian and mouse, we found promoter activity at different locations of the 5'-flanking region in human and mouse genes. This result suggests that promoter activity is highly specific to tissues and species. 6. We fused the 1,300-bp mouse S-Ag promoter with the chloramphenicol acetyltransferase (CAT) gene and introduced it into transgenic mice. The transgenic animals expressed CAT activity only in the retina and pineal gland, indicating that the promoters have tissue-specific enhancer and promoter activity. 7. We fused the opsin promoter with a diphtheria toxin gene and introduced it into transgenic mice, which subsequently lost only their photoreceptor rod cell layers. 8. We isolated several cDNAs of Shuzin, a retinal photoreceptor protein, from human and cow retinal cDNA libraries (X-gtll) and determined the entire DNA sequences. The deduced protein has sequence similarity with TFIID. We isolated its gene from a genomic library and determined the DNA sequence, showing it is composed of two introns and three exons. 9. We isolated two genes of 33-kD rod outer segment (ROS)-specific proteins from the retinal libraries of human and mouse and determined the entire DNA sequence of these genes. They have four exons and three introns. Significance to Biomedical Research and the Program of the Institute Eyes have remarkable properties in functioning efficiently over a wide range of illuminations. Rod cells having photosensitive rtiodopsin, are more sensitive to dim light, and adapt in the dark to increase their sensitivity. However, rod cells cease their sensitive phototransduction in bright light. In contrast, cone cells do not operate in dim light but are operative in bright light. Rhodopsin, transducin, phosphodiesterase, ihodop- sin kinase, and S-Ag have been known to be associ- ated with the phototransduction cascade. Rhodopsin kinase and S-Ag are considered to be the important proteins for light-dependent modulation of photo- transduction. To understand this light-dependent modulatory mechanism in ROS, we have character- ized S-Ag, Shuzin, and 33-kD protein as well as their genes. Interestingly, other signal transduction systems have cascades similar to that of phototransduction (one of the best characterized receptor-mediated signal transduction processes). In the phototransduc- tion cascade, the shutoff mechanism appears to be modulated by the phosphorylation and dephosphoryl- ation of rtiodopsin. Studying this modulation mecha- nism is important for understanding phototransduc- tion as well as for understanding signal transduction in general In addition, we think that the night 202 NEI Annual Report-FY 1992 Laboratory of Retinal Cell and Molecular Biology blindness of vision may in part be associated with light adaptatioa 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 c/s-acting factors of the S-Ag and 33-kD protein promoter; (3) the crystallization of S-Ag and 33-lcD protein; and (4) investigation of a functional role foi o-Ag, 33-kD protein, and Shuzin: the homologous recombination between a mutant gene and a normal gene wiU be induced in ES cell culture. The recombinant ES cells will be introduced into a transgenic animal system to produce a mutant mouse. NEI Research Program Retinal Diseases — ^Photoreceptors and Retinal Pig- ment Epithelium 5' flanking sequences. J Biol Chem 266:15505- 15510. 1991. Dancinger M, Kozak CA, Abe T, Shinohara T, Farber DB: The gene for retinal rod 33-kDa protein is on mouse chromosome 1 near Iambi. Genomics, in press. Schaad NC, Shinohara T, Abe T, Klein DC: Photo- neural control of the synthesis and phosphoryla- tion of pineal MEKA (phosducin). Endocrinology 129:3289-3298, 1991. Shinohara T, Kikuchi T, Tsuda M, Yaraaki K: A family of retinal S-antigen (Arrestin) and their genes: Comparative analyses of human, mouse, rat, bovine, and Drosophila. Comp Biochem Physiol, in press. Tsuda M, Kikuchi T, Yamaki K, Shinohara T: The mouse S-antigen gene; comparison with human and Drosophila. Eur J Biochem 200:95-101, 1991. Publications Breitman ML, Tsuda M, Kikuchi T, Zucconi A, Khoo W, Shinohara T: Expression of S-antigen in retina, pineal gland, lens and brain is directed to 203 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00250-05 LRCMB PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (BO characters or loss. Title must lit art one line between the borders.) Molecular Biology of Experimental Autoimmune Uveitis PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute afliliatlon) PI: Toshimichi Shinohara Ph.D. Head, Section on LRCMB, NEI Molecular Biology Others: Kotaro Eto Santhanakrishnan Simil M.D., Ph.D. M.D. Visiting Associate Visiting Fellow LRCMB. NEI LRCMB, NEI COOPERATING UNITS (It 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 CeU and Molecular Biology SECTION Section on Molecular Biology INSTITUTE AND LOCATION NEI, NIH, Bethesda, MD 20892 TOTAL STAFF YEARS: 2.2 PROFESSIONAL: 2.2 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.) We previously determined the amino acid sequences of himian, mouse, rat. and bovine retinal S-antigen and rat pineal gland S-antigen. Immunogenic sites and four uveitopathogenic sites of S-antigen also were determined. Two of the immimogenic sequences were highly conserved among these species. Many proteins in the National Biomedical Research Foundation data base have sequences similar to that of a uveitopathogenic site. We chemically synthesized many peptides, some of which induced experimental autoimmime uveitis (EAU) and experimental autoimmune pinealitis (EAP) in Lewis rats. The peptides include synthetic peptides from yeast (Saccharomyces cerevisiae) histone H3, Escherichia coli hypothetical protein, potato proteinase inhibitor, hepatitis virus protein, Moloney murine sarcoma virus protein, and Moloney murine leukemia virus protein. In addition, native yeast histone H3 was capable of inducing EAU. The animals administered yeast histone by the oral route suppressed the induction of EAU and EAP by either the yeast histone H3 peptide or an S-antigen peptide. Thus, the peptides that have molecular mimicry cross- induced the tolerance. These findings provide a basis for autoimmune inflammatory diseases of the eye in humans. 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, either with or without incomplete Freund's adjuvant (BFA). The rats injected with IFA developed EAU, but most rats injected without IFA did not develop EAU. To assess the impact of infection by live microorganisms, we injected the rats several times with low doses of live E. coli expressing S-antigen and baker's yeast with a cross-reactive antigen. The rats injected with either live E. coli or live yeast developed EAU. We conclude that infection by microorganisms that have cross-reactive antigens can break immune tolerance to self-antigens and induce inflammatory autoimmune diseases. 204 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Retinal Cell and Molecular Biology Project Description Objectives The objectives of this project are to understand the basic etiology of autoimmune inflammation, includ- ing uveitis, and to find possible treatments for human uveitis. Methods Conventional methods for the analysis of proteins and nucleic acids used in this research include protein purification, RNA and DNA isolation, charac- terization and sequencing, molecular cloning, screen- ing of clones, in situ hybridization, immunocyto- chemistry, and chromosome mapping. We also synthesized and used oligopeptides and oligonucleo- tides. Bovine, murine, primate, and himian materials are used; animal experiments were conducted with Lewis rats and monkeys. T-cell response and adop- tive transfer were done with lymph node or spleen cells of rat Major Findings 1. We found local sequence homology between peptide M and several other foreign proteins, includ- ing potato proteinase inhibitor Ua, 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 Lewis rats with pathology similar to that of EAU induced by peptide M or native S-antigen (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 autoimmime diseases in general. 4. Oral administration of histone H3 peptide suppressed EAU in the Lewis rats. 5. We also foimd the suppression of EAU by histone H3 in the EAU induced by S-Ag; thus, the tolerance also cross-reacted between the peptide which has molecular mimicry. 6. The T lymphocytes obtained from rats immu- nized with peptide M or yeast histone H3-transferred disease (EAU) in the naive rats (adoptive transfer) when stimulated with either peptide M or histone H3. In addition, oral tolerance was adoptively transferred from rats fed peptide M or histone H3 to the naive rats. 7. Infection by microorganisms that have cross- reactive antigens can break immune tolerance to a self antigen and induce inflammatory autoimmune 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 world. Many physicians have suspected for decades that some types of uveitis are induced by bacterial and viral infections; however, there is no clear link between infection and disease. Autoimmime 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 non-self protein, cross- reacts with a normal host protein, may play a role in autoimmimity. Here, we have proposed the idea of molecular mimicry and provided evidence that molecular mimicry plays a role in the pathogenesis of EAU. In addition, we have offered evidence that infection is a cause of autoimmune inflammation. These findings provide an important clue for under- standing the etiology of autoimmune inflammatory diseases, in human. Proposed Course The following studies are in progress or proposed for Fiscal Year 1993: (1) further evaluation of foreign proteins similar to S-Ag that induce EAU; (2) char- acterization of peptide M, with respect to the mini- mum nimiber of amino acids required for induction of EAU; (3) study of the induction of EAU in transgenic mice that express foreign proteins in the photoreceptor cells; and (4) further characterization of molecular mimicry and its role in EAU and hiunan uveitis. NEI Research Program Retinal Diseases — Inflammatory Diseases 205 Laboratory of Retinal Cell and Molecular Biology NEI Annual Report-FV 1992 Publications Babila T, Schaad NC, Simonds WF, Shinohara T, Klein DC: Development of MEKA (phosducin), Gb, Gg and S-antigen in the rat pineal gland and retina. Brain Res 585:141-148, 1992. Eto K, Suzuki S, Singh VK, Shinohara T: Immuniza- tion with recombinant E. coli expressing retinal S-antigen induced experimental autoimmime uveitis (EAU) in Lewis rats. Cell Immunol, in press. 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- gea Exp Eye Res, in press. Ni M, Yamaki K, Kikuchi T, Ferrick M, Shinohara T, Nussenblatt RB, Chan C-C: Developmental expression of S-antigen in fetal human and rat eye. Curr Eye Res 11:219-229, 1992. Sai S, Usukura J, Shinohara T, Wakabayashi T, Awaya S: S-antigen localization in developing rds mouse retina. J Ophthalmol 36:331-341, 1992. Shinohara T: S-antigen: Molecular mimicry may play a role in autoimmune uveitis. Clin Digest Ser Sept, 1992. 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. Comp Biochem Physiol, in press. Singh VK, Kalra HK, Yamaki K, Shinohara T: Suppression of experimental autoimmune uveitis in rats by the oral administration of the uveito- pathogenic S-antigen fragment or a cross-reactive homologous peptide. Cell Immunol 139:81-90, 1992. Singh VK, Usukura J, Shinohara T: Molecular mimicry: Uveitis induced in Macaca fasicularis by a microbial protein having sequence homology with retinal S-antigen. Jpn J Ophthalmol 36:108- 117, 1992. Sunil S, Ito K, Shinohara T: Immunogenic determi- nants common to cross-reactive self and nonself antigens in experimental autoimmune uveitis (EAU). Cell Immunol, in press. 206 Laboratory of Sensorimotor Research Report of the Chief, Laboratory of Sensorimotor Research Robert H. Wurtz, Ph.D. The generation of movement and its sensory control are among the brain's most critical functions. In primates, including humans, the visual sense predominates and visually controlled move- ments are the most precise. The Laboratory of Sensorimotor Research concentrates its research effort on the brain mechanisms underlying these critical functions. We study neither the receptor input nor the muscular output but rather the intricate systems in between that lead to visual perception and the generation of movement. In this 14th annual report of the Laboratory of Sensorimotor Research one salient investigation in each of the five sections within the laboratory is used to illustrate the range of our studies on the visuomotor system. Section on Neural Modeling Dr. Lance M. Optican and his colleagues in the Section on Neural Modeling concentrated on the processing of visual input to the brain. Their studies indicate that different visual areas in the brain may communicate via temporally modulated messages. Their previous work had shown that neurons in different areas of the monkey brain encode and transmit information about stationary, two-dimen- sional pictures that vary in form, brightness, and duration. These investigators also had shown that information about remembered visual features is carried by a temporal code. Now they have extended those studies to show that neurons in visual areas of the cerebral cortex (areas VI, V2, V3, and V4) carry information about the form and color of a stimulus in a temporally modulated code. The results suggest that cortical neurons can 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. Section on Neuro-Ophthalmologic Mechanisms The effects of selective attention on visual proc- essing have been investigated by Dr. Michael Goldberg and his colleagues in the Section on Neuro-Ophthalmologic Mechanisms. They studied neurons in the posterior cingulate cortex of the frontal lobe of the cerebral cortex during visuomotor tasks to determine their activity with respect to visual stimuli that were usually salient themselves or that were used as the targets for saccades. Neurons responding to salient visual stimuli such as checker- boards did not respond to small spots of light, even when those spots were used as targets for saccadic eye movements or as stimuli in a luminance-detec- tion task. This result suggests that the brain treats voluntary attention differently from involuntary attention to salient and novel stimuli. The cingulate cortex is important in the generation of involuntary but not voluntary attention. Section on Oculomotor Control The influence of a motor control system on visual input has been the focus of Dr. Frederick Miles and his colleagues in the Section on Oculomotor Control. Primates have excellent binocular vision and use vergence movements to align the two eyes on a common object, thereby facilitating stereopsis, the visual perception of depth. Binocular disparity, which refers to the slight difference in the locations of the images on the two retinas resulting from the slight difference in the viewpoint of the two eyes, is known to provide an important drive for these vergence eye movements. Using stimuli that extend over a much greater region of the visual field than had been employed in 209 Laboratory of Sensorimotor Research NEI Annual Report-FY 1992 most previous studies, Dr. Miles' group discovered that sudden clianges in binocular disparity induce vigorous, machine-like vergence eye movements with a roughly exponential time course (time constant, 100-150 msec), at ultra-short latencies of approxi- mately 60 msec. The obligate nature of these eye movements was evident from the fact that the ani- mals were neither trained to make such responses nor reinforced for doing so. The latency is 100 msec or so less than values obtained using smaller visual targets reported in the literature. Such reflex-like vergence responses at ultra-short latencies in re- sponse to pure disparity stimuli have not been previously described; they are all the more surprising because the neural decoding of disparity is known to be a cortical function. These experiments suggest that large-field disparity stimuli provide a powerful yet simple new approach for probing the neural processing of disparity information and its role in the cortical control of vergence eye movements. Section on Visuomotor Integration In the Section on Visuomotor Integration, Dr. Robert H. Wurtz and his collaborators studied the generation of an eye movement, ie, the rapid or saccadic eye movements that move the eye from one object of interest in the visual field to another. Many neurons in the superior colliculus, a nucleus in the brain stem, give a burst of ceU discharges before the onset of these saccades. Work over the last 20 years has progressively fit these neurons into models of a brain system for the control of this movement. This year Dr. Wurtz and coworkers found that another type of cell lying just below these burst cells is also related to the generation of saccades. These cells' behavior illustrates a method of motor process- ing only recently recognized. Instead of increasing their discharge before a given movement via the activity of a single group of cells (as burst cells do), these deeper cells support a moving front of activity that slides across the layer of their coUiculus. This spread of activity may represent a type of neural processing not previously recognized in the brain. Section on Visual Behavior The control of movement centers not only on the eye but on the head as well. Dr. David Lee Robinson and his colleagues in the Section on Visual Behavior, studying the role of the brain stem reticu- lar formation in the coordination of head and eye movements, have discovered that electrical stimula- tion of this region evokes brisk movements of the head to the ipsilateral side. These head movements are never associated with shifts in the direction of gaze although they are influenced by the starting position of the head, active fixation, and level of alertness. These observations suggest that this brain region is the central integrative site for head move- ments. To enhance understanding of the global nature of this region, Dr. Robinson and coworicers have injeaed tracer substances into physiologically identi- fied portions of this head movement area and dis- covered anatomical coimections between the reticular formation and (1) those portions of the superior coUiculus that drive head movements, (2) parts of the motor and premotor cortices that initiate head move- ments, and (3) regions of the midbrain that have limbic/emotional functions. In addition, the group has discovered efferent projections to the upper cervical spinal cord that end in the region of the neck motoneurons. These studies provide important information for understanding the neural mechanisms and sites for the control of head movements. 210 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00256-04 LSR PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 charaaers or less. Title must til on one line between the borders.) Infonnation Processing by Visual System Neurons PRINCIPAL INVESTIGATOR (UsI other professional personnel below the Pnncipal Investigator.) (Name, title, laboratory, and Institute affiliation) PI: Lance M. Optican Ph.D. Chief, NMS LSR, NEI Others: John W. McQurkin Arthur V. Hays Brad J. Zoltick Jennifer A. Zarbock Merk Na Chee-Orts Marc H. Cohen Ph.D. Staff FeUow LSR, NEI B.A. Electronics Engineer LSR, NEI M.A. Computer Programmer LSR, NEI B.A. Electronics Engineer LSR, NEI Ph.D. Visiting Associate LSR, K "I M.S.E. Visiting Associate LSR, NEI COOPERATING UNITS (II any) Laboratory of Sensorimotor Research Neural Modeling Section INSTfTUTE AND LOCATION NEI, NIH, Bethesda. MP 20892 TOTAL STAFF YEARS: 5.6 PROFESSIONAL: 4.0 1.6 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.) The brain requires many functions to span the gap from visual sensation to perception. Our studies indicate that dilTerent visual areas in the brain may communicate via temporally modulated messages. These findings suggest that understanding temporally encoded messages may give clues to the types of functional processes involved in visual infonnation processing. New evidence suggests one such functional role for neurons in inferior temporal (IT) cortex during a short-term visual memory task. In all visual areas studied, neurons encode information about pictures in a multidimensional temporal code. We are recording from individual neiuDns diuing a color-versus-form discrimination task. It appears that neurons in the IT cortex send four messages during a pattern recognition task: Two messages describe the color and form of the visual cue, and two describe the color and form of the visual target These results lead to a new hypothesis concerning the role of visual neurons in discrimination tasks. The multiplex-code hypothesis states that neurons carry separate color and form messages, multiplexed together. The temporal waveform of the response of visual neurons is formed by multiplying these two waveforms together. 211 PHS 6040 (Rev. 5/92) Laboratory of Sensorimotor Research NEI Annual Report-FY 1992 Project Description Objectives Perception and recognition of complex visual pic- tures depend on tiie normal function of a hierarch- ically connected system of 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 neurons encode infoimation in these successive stages of processing. If we could under- stand this neuronal code, it might become possible to distinguish between information related to the physi- cal properties of a stimulus (eg, form, luminance, color) and that related to its behavioral significance (eg, leading to a reward). Individual neurons in aU the visual areas studied (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 infonnation 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 stimulus-dependent messages. We have shown that visual neurons convey complex messages about both a stimulus' physical parameters and its behavioral significance. Using information theory, we can begin to explore how physical and behavioral components of a neuron's response contribute to such higher visual cognitive functions as perception, attention, and memory. Major Findings We have developed a new approach to studying single neurons in which they are treated as communi- cation charmels that transmit information about visual pictures in their responses. This approach has allowed us to apply methods from signal processing, statistics, systems analysis, and information theory to understand 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, eg, orientation or color. If response strength were the only parameter a neuron could use to encode information, different stimulus features would be confoimded by individual neurons. Using informational analysis, we have shown that information about different stimulus parameters is not confounded but is carried across the different parts of the multidimensional neuronal code. In recent experiments, responses of neurons in four visual cortical areas (VI, V2, V3, and V4) were recorded ftom 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. Informa- tion about stimulus features developed continuously, but not uniformly, throughout the time course of neiuonal responses. Most of the infonnation 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 tiie first. These results show that neurons in VI -V4 carry information about the color and pattern of both current and remembered stimuli. Furthermore, the relative amount of infonnation carried by a neuron depends upon the behavioral task. Finally, the development of information over time in different areas suggests that temporally modulated waves of activity may form a code for visual information. In fact, the response to each stimulus could be repre- sented as the product of two waveforms, one for color and one for pattern. Feature-specific wave- forms for each color and each pattern were isolated from the neuronal responses by a neural net. The product of these feature waveforms predicted the neuronal responses to stimuli with color and pattern combinations not used to train the neural net. Feature waveforms were often similar for all neurons within a cortical area. To compare these waveforms across cortical areas, we pooled aU the responses from neurons within each area. Wave- forms encoding pattern were strikingly similar across aU areas, irrespective of the behavioral task; those encoding color differed between cortical areas, depending on the behavioral task. The color wave- forms VI and V4 were different and showed no task dependence; those in V2 and V3 were different fi"om those in VI and V4 when pattern was the cue but identical to those in VI when color was the cue. These results suggest that neurons convey informa- tion about compound visual features by multiplexing feature-specific messages. The invariance of pattern 212 NEI Annual Report-FY 1992 Laboratory of Sensorimotor Research waveforms suggests — at least for these stimuli — that the processing of pattern information is completed in VI. 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 occur in amblyopia, and for developing visual prosthetic devices to compensate for field defects or blindness. Proposed Course Discovering that the responses of visual system neiu-ons 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 from the influence of feedback. 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. The structures of the spatial filters seen in lateral geniculate nuclei have already generated new ideas about the properties of encoded information there. Both issues are being pursued. In addition, our findings suggest previously imconsidered principles as the basis for interactions among neurons. To investigate them, 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. We will relate the simultaneously recorded responses to each other using recent extensions to methods of signal identification that should allow us to develop models to 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 used for cognitive functions such as pattern perception and recognition. 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 need to unconfound stimulus features. However, we propose that the simultaneous messages about different features can be used as tags, so that messages arising in different processing regions of the visual system can be reunited in a unified percept, thus providing the mechanism to buUd a whole perception across many processing regions. Using 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 Eskandar EN, Hertz JA, Richmond BJ, Optican LM, Kjaer T: Decoding of neuronal signals in visual pattern recognition, in Moody JE, Hanson SJ, Lippmann RP (eds): Neural Information Process- ing Systems 4. San Mateo, Morgan Kaufmarm, 1992, pp 356-363. Eskandar EN, Optican LM, Richmond BJ: Role of inferior temporal neurons in visual memory: n. Comparing temporal waveforms arising from 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. Gawne TJ, McQurkin JW, Richmond BJ, Optican LM: Lateral geniculate neurons in behaving primates: HI. Predictive power of a multi-charmel model. J Neurophysiol 66:809-823, 1991. Gawne TJ, Richmond BJ, Optican LM: Interactive effects among several stimulus parameters on the responses of striate cortical complex cells. J Neurophysiol 66:379-389. 1991. Mcauricin JW, Gawne TJ, Optican LM, Richmond BJ: Lateral geniculate neurons in behaving pri- mates: II. Encoding of visual information in the 213 Laboratory of Sensorimotor Research NEI Annual Report-FY 1992 temporal shape of the response. / Neurophysiol 66:794-808, 1991. McClurkin JW, Gawne TJ, Richmond BJ, Optican LM, Robinson DL: Lateral geniculate neurons in behaving primates: I. Responses to two-dimen- sional stimuli. J Neurophysiol 66:111-193,, 1991. McCluricin JW, Optican LM, Richmond BJ, Gawne TJ: Concurrent processing and complexity of temporally encoded neuronal messages in visual perception. Science 253:675-677, 1991. Optican LM, Gawne TJ, Richmond BJ, Joseph PJ: Unbiased measures of transmitted information and channel capacity from multivariate neuronal data. Biol Cyber 65:305-310, 1991. Richmond BJ, Optican LM: The structure and inter- pretation of neuronal codes in the visual system, in Wechsler H (ed): Neural Networks for Human and Machine Perception. London, Academic Press, 1992, pp 105-131. Waitzman DM, Ma TP, Optican LM, Wurtz RH: Superior coUiculus neurons mediate the dynamic characteristics of saccades. / Neurophysiol 66:1716-1737, 1991. Zee DS, FitzGibbon EJ, Optican LM: Saccade vergence interactions in human beings. / Neuro- physiol 68:1624-1646, 1992. 214 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00049-14 LSR PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 charaaers or lass. Title must lit on one line between the borders.) Cerebral Cortical Mechanisms for Eye Movements and Visual Attention PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Pnncipal Investigator.) (Name, title, laboratory, and institute allillation) PI: Michael E. Goldberg M.D. Chief, NMS LSR, NEI Others: Edmond J. FitzGibbon Carol L. Colby Suzanne Y. Musil M.D. Medical Officer LSR, NEI Ph.D. Senior Staff FeUow LSR, NEI Ph.D. NRSA Fellow LSR, NEI COOPERATING UNITS (II any) LAB/BRANCH Laboratory of Sensorimotor Research Neuro-Ophthalmologic Mechanisms Section INSTITUTE AND LOCATION NEI, NIH, Bethesda, MP 20892 TOTAL STAFF YEARS: 4.6 PROFESSIONAL: 3.3 1.3 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects □ (a1) Minors □ (a2) Intervievifs □ (b) Human tissues [xj (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Two lines of inquiry were foUowed to determine how the cerebral cortex and its efferent regions control eye movements and visuospatial attention. In one, we studied the activity of neurons in the posterior cingulate during various visuomotor tasks to determine their activity with respect to visual stimuli that were either spontaneously salient or used as the targets for saccades. Neurons that responded to salient visual stimuh, such as checkerboards, did not respond to small spots of light, even when those spots were used as targets for saccadic eye movements or as stimuli in a luminance-detection task. These results suggest that the brain treats volimtary attention differently from involimtary attention to salient and novel stimuli. The cingulate cortex is important in the generation of voluntary but not involimtary attentioa The second line of inquiry was the study of visually responsive neurons in the superior coUiculus. We used the visual stimulation engendered by eye movements to understand the effect of motor plarming on the visual responsiveness of these neurons. For a distinct subpopulation of visuomovement neurons in the intermediate layers of the superior coUiculus, the neurons would respond to visual stimuli that were about to be brought into their visual receptive fields by a saccadic eye movement. This predictive visual response resembled that previously described by this group for the lateral intraparietal area of the parietal cortex. It may well describe a general mechanism by which the brain achieves spatially accurate behavior. 215 PHS 6040 (Rev. 5/92) Laboratory of Sensorimotor Research NEI Annual Report-FY 1992 Project Description Objectives This Section has concentrated on two aspects of the physiology and phenomenology of higher visual and oculomotor processing in monkey and man, especial- ly 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 neurons which, paradoxically, maintain spatially accurate coding in a retinotopic framewori: by demonstrating presaccadic predictive responses: When a monkey makes a saccade that wiU bring a stimulus into the receptive field of a neuron, that neuron can start discharging in response to the stimulus before the saccade starts. In effect, this is a transient shift of visual receptive field, a projection target of the parietal cortex. We studied the superior colliculus to see whether it contains the presaccadic predictive responses discovered in the parietal cortex. In a second series of experiments the cingulate cortex was shown to have neurons that discharged after eye movements and had visual responses. Work in the laboratory for the past year was concen- trated on various aspects of neural function related to these earlier findings, ie, study of the physiology of the cingulate cortex in tasks designed to probe the nature of oculomotor and visuospatial processing in this region. Methods Monkeys implanted with magnetic search coUs for the measurement of eye position, along with devices for temporary restraint and electrophysiological recording and stimulation, were trained to perform a number of visuomotor tasks, including fixation, saccades, and smooth pursuit. Microelectrodes were placed in the superior colliculus or posterior cingu- late cortex, and single neurons were studied while the monkeys performed various visuomotor tasks. Major Findings Neurons in the posterior cingulate discharge after eye movements of a certain direction and amplitude. This discharge is modulated by target orbital posi- tion, saccade amplitude, saccade direction, and ambient Ulumination. Neurons that have no presacc- adic response to saccade targets but are affected by ambient illumination can be shown to respond to the appearance of large, salient visual targets such as a black and white checkerboard. To determine whether voluntary attention is a component of the responsiveness of such neurons, we used two atten- tional tasks: (1) a peripheral attention task in which the monkey makes a limiinance discrimination on a dim peripheral light and (2) a saccade task in which the monkey makes a saccade to the same target. Neurons that respond to the checkerboard fail to respond to these nonsalient but behaviorally signifi- cant targets. Targets that had the same luminance and total flux of the checkerboard but were dispersed and lacked the dramatic contrast of the checkerboard also failed to drive the neurons. These results suggest that the cingulate cortex is important in the analysis of the visual environment for novel, atten- tion-grabbing stimuli but not for maintaining atten- tion on behaviorally significant but visually non- salient stimuli. Neurons in the superficial and intermediate layers of the intermediate coUiculus have visual responses. To see whether these visual responses had a presacc- adic predictive study, we examined the neurons using a paradigm in which a saccade brought either a preexisting stimulus or the spatial location of a recently flashed stimulus into the neuron's receptive field. A special population of neurons in the inter- mediate layers demonstrated such predictive re- sponses. Predominating in these neurons are partial- ly clipped movement responses and large contralat- eral receptive fields. These neurons lie in the deeper parts of the intenmediate layers of the colliculus, at least 0.5 mm below the stratum opticum. Neurons in the superficial layers of the colliculus do not show a predictive response. Significance to Biomedical Research and the Program of the Institute Understanding how the cerebral cortex and its afferent regions guide eye movements and modulate visual attention and learning is useful both as a model for the neural control of other, more compli- cated behaviors and as a key to understanding and developing treatments for disorders of the neural control of vision, eye movements, and attention. 216 NEI Annual Report-FY 1992 Laboratory of Sensorimotor Research Proposed Course The examination of frontal eye fields will show whether they have predictive responses. We will examine the nature of the parietal cortical predictive responses to see if the responses occur in association with smooth pursuit eye movements as well as saccades, how they interact with orbital position planar gain fields, and whether the presaccadic predictive responses move continuously or discontin- uously through the cortex. 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: Neurons in the lateral intraparietal area (LIP) of the monkey remap visual space in connection with saccadic eye movements: II. Remapping of a visual memory trace. Sac Neurosci Abstr 17:1282, 1991. 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, in press. Duhamel JR, Colby CL, Goldberg ME: The updating of the representation of visual space in parietal cortex by intended eye movements. Science 255:90-92, 1992. Duhamel JR, Colby CL, Goldberg ME: Congruent representations of visual and somatosensory space in single neurons of monkey ventral intraparietal cortex (area VIP), in PaUlard J (ed): Brain and Space. Oxford. Oxford University Press, 1991, pp 223-236. Duhamel JR, Colby CL, Goldberg ME: Neurons in the lateral intraparietal area of the monkey remap visual space in connection with saccadic eye movements: 1. Presaccadic events. Soc Neurosci Abstr 17:1282, 1991. 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. FitzGibbon EJ, Inchingolo P, Optican LM, Goldberg ME: The effect of botulinum toxin on saccadic eye movements in riiesus monkeys. Soc Neurosci Abstr 17:459, 1991. Goldberg ME, Colby CL: Oculomotor control and spatial processing. Curr Opinion Neurobiol 2:198-202, 1992. Musil SY, Olson CR, Goldberg ME: Posterior cingulate cortex of rhesus monkey: Mechanisms of orbital-position and postsaccadic sensitivity. Soc Neurosci Abstr 17:442, 1991. Segraves MA, Goldberg ME: Properties of eye and head movements evoked by electrical stimulation of the monkey superior colliculus, in Berthoz A, Graf W, Vidal PP (eds): The Head-Neck Sensory- Motor System. New York, Oxford University Press. 1992, pp 292-295. 217 DEPARTMEKT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00153-10 LSR PERIOD COVERED October 1, 1991 lo September 30, 1992 TITLE OF PROJECT (80 characters or less. Title must lit on one line between the borders.) Visual Motion and the Stabilization of Gaze PRINCIPAL INVESTIGATOR (Ust other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute atflllallon) PI: Frederick A. Miles D.Phil. Senior Research LSR, NEI Physiologist Others: Urs Schwarz Qaudio Busettini M.D. Ph.D. Visiting Associate Visiting Fellow LSR, NEI LSR, NEI COOPERATING UNITS (II any) LAB/BRANCH Laboratory of Sensorimotor Research SECTION Oculomotor Control Section INSTITUTE AND LOCATION NEI, NIH, Bethesda. MP 20892 TOTAL STAFF YEARS: 3.5 PROFESSIONAL: 2.3 1.2 CHECK APPROPRIATE BOX(ES) ixl (a) Human subjects □ (al) Minors □ (a2) Interviews □ (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Primates have excellent binocular vision and use vergence eye movements to align the two eyes on a conunon object, thereby facilitating stereopsis, the visual perception of depth. Binocular disparity, which refers to the slight difference in the locations of the images on the two retinas resulting from the slight difference in the viewpoint of the two eyes, is known to provide an important drive for these vergence eye movements. Disparity-induced vergence eye movements have traditionally been studied using an optical arrangement that allows identical stimuh to be presented independently to the two eyes, thus allowing the application of pure disparity stimuli. In these experiments we studied disparity-induced vergence eye movements in monkeys by placing a red filter over one eye and a green one over the other. The animal faced a tangent screen onto which were projected two large, textured patterns that were identical except in color — one being red (therefore seen only by the eye with the red filter) and the other green (seen only by the eye with the green filter). Sudden changes in the horizontal ahgnment of the two images (disparity steps) induced vigorous, machine-like vergence eye movements with a roughly exponential time course (time constant, 100 to 150 msec), despite the fact that the animals were neither trained to make such responses nor reinforced for doing so. The latency of such vergence eye movements was typically about 60 msec, which is about 100 msec less than previous values reported in the Uterature for experiments using smaller visual targets. One possibility was that these ultra-short-latency vergence responses were actually akin to the short-latency ocular-foUowing responses that we have previously reported in response to the motion of large, textured scenes. However, this possibility is unlikely because the short-latency vergence responses did not show postsaccadic enhancement, one of the characteristic features of short-latency ocular following. Whereas neiu"al encoding of disparity is known to occur in the cortex, these vergence responses must be cortically mediated, despite their ultra-short latency. 218 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Laboratory of Sensorimotor Research Program Description Objectives The discovery reported here was entirely serendip- itous and not the result of any prior hypothesis. The objective of the original study was concerned with the question of whether early ocular following is generated by a neuronal system that is sensitive to the binocular disparity of visual motion. Existing evidence that human optokinetic responses are better when the moving visual scene is binocularly fused than when disparate supported the idea that the visual stabilization mechanism can respond selectively to the motion of objects in the plane of fixation and can ignore the motion of objects that are nearer or further. However, the visual stimuli in those experi- ments were prolonged (ie, closed loop) and the apparent sensitivity to disparity might have been secondary to changes in the subject's attention level, a factor known to be very important for human optokinetic responses. The disparate images (double vision) in this situation simply could have failed to capture the subject's attention so that it was not necessary to invoke any sensitivity to disparity in the neurons decoding the visual motion. To eliminate this attentional problem, we used several disparities and introduced them randomly just prior to moving the visual scene so there would be insufficient time for the subject — a monkey in our experiments — to alter his/her attentional level in accordance with the disparity. We were surprised to find that the sudden changes in the disparity of the scene resulted in vergence eye movements at unexpectedly shori latencies, severely contaminating the short-latency ocular following responses under study. At that point, we turned our attention to short-latency vergence responses. Methods The movements of both eyes were recorded in response to sudden changes in the disparity of the visual scene in three monkeys. The animals faced a tangent screen onto which were projected two independent images that were identical except in color, one being red and the other green. (When viewed through goggles with a green gelatine filter in front of one eye and a red one in front of the other, the observer sees a single fused image that can be positioned anywhere in front or behind the plane of the screen by merely offsetting the two images horizontally.) At the start of a trial, the two images were exactly superimposed and so were seen in the plane of the screen at a distance of 3 reciprocal meters. Each monkey was required to fixate a target spot projected onto the center of the scene, and, at a variable time after the spot was extinguished, the horizontal alignment of the two images was suddenly changed, introducing disparity steps of ±0.5, 1, 2, or 3 meter angles. We computed vergence eye move- ments by subtracting the position of the right eye from the position of the left eye. Major Findings Disparity steps evoked vigorous and machine-like vergence eye movements with a roughly exponential time course (time constant, 100-150 msec) at laten- cies of approximately 60 msec. These vergence responses were obtained despite the fact that the monkeys were neither trained to produce them nor reinforced for doing so: Reinforcement was based solely on appropriate prior fixation of the centered target spot. The short latency and consistency of the responses raised the possibility that they represent a disconjugate tracking response akin to short-latency ocular following. A charaaeristic of short-latency ocular following is that stimuli apphed in the immed- iate wake of a saccadic eye movement are much more effective than identical stimuli applied some time later, ie, post-saccadic enhancement. However, the vergence responses induced by disparity steps were not subject to post-saccadic enhancement, pointing to an independent neural mechanism. Significance to Biomedical Research and the Program of the Institute The latency of the vergence eye movements reported here was approximately 100 msec less than the values reported in the literature. We attribute this difference to the fact that the disparity stimuli used in our experiments extended over a considerably larger area of the visual field than those employed in any previous experiment. Further, a clear difference between the present and all previous studies was that in our experiments training or reinforcement of the animal was unnecessary; vergence eye movements appeared to be obligate. Previously undescribed, such reflex-like vergence responses to pure disparity stimuli are aU the more surprising because neural 219 Laboratory of Sensorimotor Research NEI Annual Report-FY 1992 decoding of disparity is known to be a cortical function. Our experiments suggest that large-field disparity stimuli provide a powerful yet simple new approach for probing the neural processing of dispar- ity information and its role in the cortical control of vergence eye movements. Proposed Course Future experiments will examine visual and vestibu- lar ocular stabilization in the presence o^ computer- generated optic flow patterns that simulate those experienced by the moving observer. NEI Research Program Strabismus, Amblyopia, and Visual Processing — Image Formation and Stabilization (Ocular Motility) Publications Busettini C, Miles FA, Schwarz U: Ocular responses to translation and their dependence on viewing distance: n. Motion of the scene. J Neurophysiol 66:865-878, 1991. Kimmig HG, Miles FA, Schwarz U: Effects of stationary textured backgrounds on the initiation of pursuit eye movements in monkeys. J Neuro- physiol, in press. Miles FA: The sensing of rotational and translational optic flow by the primate optokinetic system. Rev Oculomot Res, in press. Miles FA, Busettini C: Ocular compensation for self motion: Visual mechanisms, in Cohen B, Tomko DL, Guedry F (eds): Sensing and Controlling Motion: Vestibular and Sensorimotor Function. Ann NY Acad Sci 656:220-232. 1992. 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, in press. Miles FA, Schwarz U, Busettini C: The decoding of optic flow by the primate optokinetic system, in Berthoz A, Graf W, Vidal PP (eds): The Head- Neck Sensory-Motor System. New York, Oxford University Press, 1992, pp 471-478. Miles FA, Wallman J: Prologue. Rev Oculomot Res, in press. Schwarz U, Miles FA: Ocular responses to transla- tion and their dependence on viewing distance: I. Motion of the observer. J Neurophysiol 66:851-864, 1991. 220 DEPARTMENT OF HEALTH AND HUMAN SERVICES ■ PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00045-14 LSR PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must in on or)e line between the borders.) Visuomotor Properties of Neurons in the Thalamus PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator.) (Name, title, laboratory, and institute alliliation) PI: David Lee Robinson Ph.D. Section Chief LSR, NEI COOPERATING UNITS (11 any) Department of Anatomy, Howard University (Robert J. Cowie, Ph.D.) Laboratory of Sensorimotor Research SECTION Visual Behavior Section INSTITUTE AND LOCATION NEL MH. Bethesda, MD 20892 TOTAL STAFF YEARS: 2.4 PROFESSIONAL; 1.0 1.4 CHECK APPROPRIATE BOX(ES) □ (a) Human subjects Q (b) Human tissues [x] (c) Neither □ (a1) Minors □ (a2) Interviews SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Throughout our daily lives, there is continual movement of the head and eyes, and their coordination, which is essential for proper vision, requires complex interactions to deal with changes in the position of the head and shifts of gaze. We have discovered a region in the pontine and medullary reticular formation that, when electrically stimulated, leads to brisk head movements. The vast majority of these movements are in the ipsilateral direction. Horizontal rotatory movement predominates, but tilting and stabilizing movements also are elicited. Although eye movements frequently occur with head movements, stimulation of this region with the head restrained does not produce eye movements. Movements elicited from this area never change the direction of gaze, which is stablized by the vestibulo-ocular reflex. However, the elicited head movements are modulated by the initial position of the head, attentive fixation, and level of alertness. To understand the role of this region of the brainstem in the control of head movements further, we injected tracer compounds to reveal afferent and efferent connections. Areas that send axons to our head movement area include our previously identified head movement portions of the superior colliculus, those parts of the motor and premotor cortices that initiate head movements, the periaqueductal gray (extensively connected to limbic/emotional areas), and the interstitial nucleus of Cajal Oong suggested to have head movement function). Oiu- data demonstrate a central role for this part of the brainstem reticular formation in the integration of head movements. We also studied the efferent projections from the reticular formation head movement area by means of localizing terminal fibers, learning that the reticular formation sends axons to the motoneuronal regions of the upper cervical spinal cord. These connections provide direct access to the motoneurons that move the head. Furthermore, axonal endings within the caudal medulla, vestibular nuclei, and parvocellular reticular fields integrate head movements with the vestibulo-ocular reflex and other movements of the eyes. Together these studies demonstrate for the first time the critical role that the medullary and pontine reticular formation plays in the control of head movement They show how it integrates influences from voluntary, stabilizing, emotional, and orienting systems. 221 PHS 6040 (Rev. 5«2) Laboratory of Sensorimotor Research NEI Annual Report-FY 1992 Project Description Objectives Primates are continually moving, which requires constant control of the position of the head and eyes. Precise coordination of the head and eyes is needed to maintain stable vision. Within the past two decades, there has been considerable progress in understanding the neural control of eye movements, but littie attention has been directed toward head movements. The experiments described here have been directed toward understanding the ways in which the brain stem controls movements of the head and toward learning the afferent and efferent connec- tions of the major centers for control of head move- ments. Methods To localize specific regions of the brain and reliably test certain regions of the central nervous system, we trained adult monkeys to enter a primate chair, sit quietiy, and fixate on a spot of Ught. After the monkeys' adaptation to these situations, we per- formed sterile surgery to implant them with several devices for recording eye and head movements, electrical activity within the brain, and instruments for head restraint and recording. Once the monkeys had recovered from the surgery, we positioned fine wire electrodes into specific brain sites and electric- ally excited these loci. The evoked head movements, eye movements, and other behavioral responses were recorded and quantified. At the sites fiiom which we could elicit head movements, we injected a tracer compound (horse- radish peroxidase [HRP]). Examination of histologi- cal sections of the prepared brains allowed us to localize those neurons that project to the injected head movement center and to identify the efferent target sites of this head movement area. Major Findings We have discovered within the pontine and medul- lary reticular formation regions that have significant relations to head movement. When we electrically stimulate these areas, we obtain brisk movements of the head in the ipsilateral direction. These head movements are found within a topographically organized map of the whole upper body. The predominant movement is a horizontal, rotary motion of the head, but flexion-extension, tilting, and stabi- lizing movements can also be evoked. Since eye movements fiiequentiy accompany movements of the head, we wished to determine whether this region also had some involvement with eye movements. At the loci fit)m which we could elicit head movements, we tested the stimulation procedures with the head restrained. In none of these situations was an eye movement ever produced. We f'onclude from these observations that this area is related to head motion, not eye movements. Whereas the superior coUiculus is a major afferent to this region and it is related to shifts of gaze, our goal was to determine how the reticular area related to gaze shifts. We tested reticular stimulation in a variety of conditions while the monkey was actively fixating or scanning in the light, as well as during its relaxed behavior in total darkness. In no situation did stimulation of the reticular formation evoke a gaze shift associated with the head movement. These observations led to our conclusion that this part of the reticular formation mediates movements of the head and that the vestibulo-ocular reflex compensates for the movements unless the reflex is suppressed. We found several conditions that modulate the amplitude of the evoked head movement. The initial position of the head had a significant impact on evoked head movements. When the head was pointed in the contralateral direction, the elicited movement was larger than when the head pointed in the ipsi- lateral direction. Furthermore, when the monkey was actively fixating a spot of light, the amplitude and velocity of the movement was larger than when the animal sat quietiy in total dailoiess. We also ob- served that the animal was able to suppress the effectiveness of the electrical stimulation voluntarily. From these observations we conclude that this reticular region is a central site for the integration of head movements and that many other neural systems interact with the head movement system at this site. Because this portion of the brain stem appears to play such a critical role in the control and integration of head movements, we sought to determine the afferent and efferent connections of this part of the brain. To study such questions, we injected a tracer compoimd (HRP) into reticular formation sites we had physiologically characterized as head movement sites. Some of the HRP was transported orthogradely. 222 NEI Annual Report-FY 1992 Laboratory of Sensorimotor Research demonstrating the efferent sites of our head motion area; other aspects of the HRP were transported anterogradely, illustrating the afferent regions. When we evaluated the sites that receive efferent projections from our reticular head movement area, there were four major targets. The most prominent pathway was to the upper levels of the cervical spinal cord. Axon terminals were localized in the ventral grey matter of the cord in the region of the motoneurons that would effect head movements. A second efferent pathway ftom the reticular region traveled medially in the brain stem, entered the ipsilateral medial longitudinal fasciculus, and ended in the caudal medullary supraspinal nucleus as well as the upper cervical ventral gray matter. A third projection pathway traveled laterally within the brain stem to end in the ipsilateral parvoceUular reticular field, the vestibular nuclei, and the deep cerebellar white matter. Finally, some terminal labeling was found in the interstitial nucleus of Cajal and the caudal fields of Forel. Together, these efferent projections from the head movement area end in those sites that actually move the head or in sites that other data suggest have integrative functions in head movement. To understand what types of information are integrated within the head movement area, we localized neurons that had accumulated HRP by means of retrograde transport. One of the major sites of labeled cells was the superior colliculus; labeled neurons in the intermediate and deep layers were numerous, located mainly in the caudal portions of the colliculus. Some of our previous studies of the colliculus demonstrated that coordinated move- ments of the head and eyes are elicited by stimula- tion of these regions of the colliculus. These then represent precise anatomical and physiological correlations of the tectoreticular spinal route for orienting movements; they suggest that the head movements evoked from the superior colliculus are mediated by the pontomedullary reticular formation. A second important site of retrogradely labeled neurons was within the cortex; we localized neurons in the ipsilateral motor and premotor cortices. The neurons containing the HRP were large-diameter, pyramidal cells which were located in layer V. The sites of these labeled cells were contained appropri- ately within the neck representations of the motor maps. These connections provide pathways through which voluntary processes can influence movements of the head. Perhaps this is the pathway through which the monkeys were able to reduce the effects of reticular stimulation intentionally. In addition to these major afferents to our head movement area, there is input from the ipsUateral interstitial nucleus of Cajal, the nucleus of the posterior commissure, and the nucleus subcoeruleus. Contralateral to the HRP injections, we observed labeled neuroas in the vestibular nuclei, nucleus prepositus hypoglossi, and the paramedian reticular formation. Also of great interest is the observation of a substantial number of labeled neurons in the dorsal, lateral, and ventrolateral subnuclei of the periaque- ductal gray. This region of the brain stem is power- fully connected with various limbic sites. Finally, there were small-diameter neurons labeled within laminae VII and VIII of tiie ventral horn of the cervical levels of the spinal cord on the ipsilateral side. These observations demonstrate that our reticu- lar head movement area is richly interconnected with several neural systems such that it can participate in head movements within the context of orienting, stabilization, or emotive behaviors. Significance to Biomedical Research and the Program of the Institute There is a continual interplay between movements of the head and eyes. When the coordination of these two functional systems deteriorates, there is a dimi- nution of vision. Losses in this coordination can result fk>m various brain lesions and certain degener- ative diseases. Understanding each region of the brain that controls movements of the head and its specific contributions facilitates precise diagnosis of central nervous system disorders. In addition, fuller understanding of the mechanisms of control of the head and eyes could lead to new strategies in rehabil- itating humans with this and similar motor disorders. Proposed Course Data from a variety of laboratories have led to the suggestion that there are several different mecha- nisms for initiating saccadic eye movements. Under certain conditions, which manipulate the state and direction of attention, saccadic eye movements can begin with extremely short latencies, termed "express saccades." Our previous studies have demonstrated 223 Laboratory of Sensorimotor Research NEI Annual Report-FY 1992 that certain regions of the parietal cortex and pulvi- nar are related to visuospatial attention. In future studies, we will attempt to determine the contribu- tions of these portions of the brain to express sac- cades. We will train riiesus monkeys in a variety of eye movement tasks that reliably evoke express saccades. Subsequently, we wiU electrically excite or chemically inactivate the pulvinar and parietal cortex to learn 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 McClurkin JW, Gawne TJ, Richmond BJ, Optican LM, Robinson DL: Lateral geniculate neurons in behaving primates: I. Responses to two-dimen- sional stimuli. J Neurophysiol 66:777-793. 1991. Robinson DL: Functional contributions of the pri- mate pulvinar. Prog Brain Res, in press. Robinson DL, McQuricin JW, Kertzman C, Petersen SE: Visual responses of pulvinar and collicular neiu"ons during eye movements of awake, trained monkeys. J Neurophysiol 66:4^5-496, 1991. Robinson DL, Petersen SE: The pulvinar and visual salience. Trends Neurosci 15:127-132, 1992. 224 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00109-12 LSR PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 charaaars or loss. Title must lit 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 alfUialion) PI: Robert H. Wurtz Ph.D. Chief LSR, NEI Others: Charles J. Duffy Hiroshi Aizawa Gregg H. Recanzone Douglas Munoz M.D., Ph.D. Staff Fellow Ph.D. Visiting Fellow Ph.D. Guest Researcher Ph.D. Guest Researcher LSR, NEI LSR, ^fEI LSR, NEI LSR, NEI COOPERATING UNITS (it any) LAB/BRANCH Laboratory of Sensorimotor Research SECTION Visuomotor Integration Section INSTITUTE AND LOCATION NEI, NIH. Bethesda. MD 20892 TOTAL STAFF YEARS: 3.5 CHECK APPROPRIATE BOX(ES) 13 (a) Human subjects □ (a1) Minors □ (a2) Interviews PROFESSIONAL: 2.0 OTHER: 1.5 n (b) Human tissues □ (c) Neither SUMMARY OF WORK (Use standard unreduced type. Do not exceed the space provided.) Among experiments concentrating on the visual motor system of monkeys and humans, the first set involved analysis of the mechanisms controlling the generation of rapid or saccadic eye movements in the superior coUiculus of awake-behaving monkeys. Previous experiments had demonstrated that a subset of cells within the superior colliculus discharges with a burst of activity before the onset of saccadic eye movements. Cells just deeper to these burst cells also discharge in relationship to saccadic eye movements but without the crisply delineated high-frequency bursts. These cells, characterized by anticipatory discharges in addition to saccade- related bursts, have been called preparatory cells. In the preparatory cell layer, and once the saccade began, there was a continuous change in the spatial distribution of activity. Preparatory cells located progressively more rostral to the initially active zone were progressively activated until the activity reached the rostral pole of the superior colliculus. This moving edge of activity represents a potentially new mechanism for controlling the amplitude of the generated saccadic eye movement. The second set of experiments concerned the use of optic flow stimuli, those stimuli that are generated as we move through the visual environment. We made several psychophysical observations on the human response to the simulated optic flow pattern that we had previously used to test the visual response of neurons in the monkey brain. We observed a striking illusion in the optic flow pattern. When we superimposed a field of moving dots on the pattern of radially moving dots, we found that human subjects mislocated the center of this radial pattern; they consistently indicated that the center was shifted in positioa We think this illusion reveals a strategy used in separating the location of the center of the optic fiow pattern from the confounding movement of other objects in the visual field as one moves through the visual environment 225 PHS 6040 (Rev. 5/92) Laboratory of Sensorimotor Research NEI Annual Report-FY 1992 Project Description Objectives The work performed this year centered on two functions of the visual-oculomotor system. The first is the saccadic system that moves the eye from one object of interest in the visual field to another. This is probably the best understood of aU oculomotor systems, and our research has expanded on previous work in the monkey superior coUiculus. The second experimental area concerns human behavior and the use of optic flow information, the visual motion that falls on the retina as a result of the observer moving through the environment. This higher order visual motion processing is likely to be used to provide visual information on the direction in which the observer heads through the environment. Methods In studies of the saccadic system, we have continued to use the liiesus monkey, Macaca mulatta. It is an ideal model of the visual-movement systems in humans, given that its eye movements and visual processing are so close to those in humans. In our experiments, the monkeys are awake and trained to perform visual motor tasks; their cooperation is an essential ingredient of the experiments. The entire experiment must be painless and stressless for the monkey because the visual tasks performed require a high level of cooperation that would deteriorate were any pain present. A monkey was trained to look forward at a small spot on a screen to obtain a liquid reward. A computer system measured the monkey's eye movements, registered the activity recorded from single ceUs in the brain, entirely controlled the experiment, and displayed experimen- tal online progress. In our work on himians, the subjects looked directly forward at a screen on which an optic flow stimulus was projected. Their task was to indicate with a light pointer where the center of this flow stimulus appeared to be located. Major Findings In the first set of experiments on the monkey, we studied the mechanisms controlling the generation of rapid or saccadic eye movements in the colliculus in awake behaving monkeys. Previous experiments had demonstrated that a subset of cells within the superi- or colliculus discharge with a burst of activity before the onset of saccadic eye movements. Analysis of these cells over the past 20 years has shown that they are critical for the generation of saccadic eye move- ments. CeUs just deeper than these burst cells also discharge in relationship to saccadic eye movements but without the crisply delineated high-frequency bursts. These preparatory cells' characteristic antici- patory discharges occur in addition to the saccade- related bursts of the cells just above them in the superior colliculus. We observed that the ceUs also have large move- ment fields tiiat lead to activity of the ceU preceding all saccades greater than a given amplitude. In preparatory cells, activity builds up at a specific point in the motor map that codes the amplitude and direction of the impending saccadic eye movement. Once the saccade begins, there is a continuous change in the spatial distribution of activity in these neurons across this motor map. Preparatory ceUs located progressively more rostral to the initially active zone are active until that activity reaches the rostral pole of the superior coUiculus. Their activity locus moves across the collicular movement-related layer from the area representing large saccades to the area representing small saccades, in contrast to that of the burst cells, whose activity increases and then decreases with a saccade, aU at only one location within the superior coUiculus. This moving hiU of activity represents a potentiaUy new mechanism for controUing .the amplitude of the generated saccadic eye movement. The second area of investigation summarized here is related to the analysis of large-field visual motion, optic flow, that simulates the visual motion generated as an observer moves through the environment We had previously foimd that neurons in an area of the cerebral cortex referred to as extrastriate area medial superior temporal (MST) discharge in response to large field stimulation and have other characteristics that would make them appropriate for the analysis of optic flow stimulation. This year, while making several psychophysical observations on the human perception of the simu- lated optic flow pattern we had previously used to test the visual response of neurons in monkey brain, we noted a striking iUusion in the optic flow pattern. When we superimposed a field of moving dots on the pattern of radiaUy moving dots, we found that 226 NEI Annual Report-FY 1992 Laboratory of Sensorimotor Research human subjects mislocated the center of this radial pattern; they consistently indicated that the position of the center shifted in the direction in which the plane of dots moved. The iUusory shift of this center, which persisted even when the observer was free to make eye movements, indicated that it was independent of any particular following eye move- ment. We think this illusion reveals a strategy used in separating the location of the focus of expansion from the confounding movement of other objects in the visual field as one moves through the visual environment. Significance to Biomedical Research and the Program of the Institute Saccades are necessary to move the eye ftum one point in the visual field to another. These move- ments must also be coordinated with head move- ments. Our experiments have revealed a new set of cells that may contribute to this control and coordi- nation. The contribution of optic flow stimulation to the guidance of our movement through the environment has been the subject of increasingly active psycho- physical and neural modeling investigations in other laboratories. Our work on psychophysics this year suggests how the brain may separate the information contained in the optic flow stimulus that results from movement of the observer, as opposed to rotation of the observer's head and eyes. Both the saccadic and the optic flow systems are functions essential to visual performance and the control of movement in normal humans and those with diseases or damage to the nervous system. Proposed Course Further experiments on saccadic eye movements will detemiine whether the preparatory cell function is related to head movement as weU as eye movement. In further analysis of optic flow, we wiU return to the analysis of the response of neurons in MST in the monkey, in part to see whether the stimuli used in humans reveal more of the functional organization of cells in the brain. NEI Research Program Strabismus, Amblyopia, and Visual Processing — Visual Processing and Functional Organization (Structure and Function of Central Visual Pathways) Publications Yamasaki DS, Wurtz RH: Recovery of function after lesions in the superior temporal sulcus in the monkey. J Neurophysiol 66:651-673, 1991. Wurtz RH, Duffy CJ: Neuronal correlates of optic flow stimulation, in Cohen B, Tomko DL, Guedry F (eds): Sensing and Controlling Motion: Vestibu- lar and Sensorimotor Function. Ann NY Acad Sci 656:205-219, 1992. Wurtz RH, Duffy CJ, Roy J-P: Motion processing for the control of movement in primate extra- striate area MST, in Ono T, Squire LR, Raichle ME, Perrett D, Fukuda M (eds): Brain Mecha- nisms of Perception and Memory: From Neuron to Behavior. New York, Oxford University Press, in press. Roy J-P, Komatsu H, Wurtz RH: Disparity sensitiv- ity of neurons in monkey extrastriate area MST. / Neurosci 12:2478-2492, 1992. 227 Ophthalmic Genetics and Clinical Services Branch Report of the Chief, Ophthalmic Genetics and Clinical Services Branch Muriel I. Kaiser-Kupfer, M.D. The Ophthalmic Genetics and Qinical Services Branch within the National Eye Institute Intra- mural Research Program has been operational since February 1989. The Branch is organized into four sections: Ophthalmic Genetics, Acting Qiief Muriel I. Kaiser-Kupfer, M.D.; Cataract and Comeal Dis- eases, Acting Qiief Manuel B. Datiles, M.D.; Oph- thalmic Pathology, Acting Chief W. Gerald Robison, M.D. Jr., Ph.D.; and Qinical 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 comeal and retinal diseases, cataract, and visual pathway abnoimalities. The Branch is responsible for the essential psychophysical and electrophysiologic 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-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 Comeal Diseases continued to pursue research on the anterior segment, especially the short-terai 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-comea interaction, the risk to comeal tissues, and how systemic or local ocular disorders may increase the risk of wearing contact lenses. Comeal 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 the analysis of data, which when performed by hand is laborious and time consuming. This Section has been particularly productive in studies using different systems to develop objective and subjective methods of monitoring and doounent- ing opacities in the human lens. Reproducibility studies on objective systems include the use of the Scheimpflug cameras (Zeiss and Oxford) and the retroillumination camera (Neitz and Oxford). Subjec- tive systems or methods such as the LOCS II grading system and the effects of cataracts on visual percep- tion, contrast sensitivity, and glare may be useful in identifying additional parameters. These systems are used to study the natural history of various cataracts, eg, pre-senile, senile, or age-related, steroid-induced, radiation, diabetic, retinitis pigmentosa, gyrate atrophy (GA), and neurofibromatosis 2. Genetic Unkage studies are under way to pursue the gene(s) of congenital cataracts. Monitoring and documenting human cataract development is a cmcial step toward the ultimate testing of several medications that might be helpfiil in preventing or reversing himian cataracts. Research on cataractogenesis has been hampered by the extreme scarcity of tissue and an abmpt shift in surgical technique from intracapsular (intact lens) to extracapsular (fragmented lens). Through the collaborative efforts of cataract surgeons and basic researchers, we have made efforts to develop and modify techniques to study materials that become available at surgery and can be well docimiented clinically. We are now carefully docimienting cata- racts in preoperative patients using clinical and photographic LOCS II grading, Zeiss Scheimpflug and Oxford retroillumination videophotography. and 231 Ophthalmic Genetics and Clinical Services Branch NEI Annual Report-FY 1992 image analysis. Cataracts are extracted extracapsular- ly with implantation of an intraocular lens. The specimens obtained are examined histologically via light and electron microscopy and biochemically by 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. With aging there is an acidic shift of proteins, and an increasv i number of polypeptide species are found in the molecular weight range of the crystallins. These aging changes need to be differentiated from changes occurring in cataract formatioa Investigators in this Section have been in the forefront 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 the definition of genetic abnormalities has been unique. Confirmation of evidence for biochemical, clinical, and molecular heterogeneity continues. There appear to be many different single-point mutations in the ornithine aminotransferase gene in GA patients. Dietary intervention studies that use an arginine deficient diet have been very promising, especially in young patients, in whom a delay in the onset of pathologic changes has been demonstrated. Foveal cone sensitivity (assessed by measure- ments of increment thresholds) and orientation (estimated with measurements 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 orientation and sensitivity before the encroachment on the foveal area by the atrophic lesions of GA. Albinism in animals has been associated 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 claim VHP asymmetry is diagnostic; they propose a uniform type of asymmetry. We used the same recording conditions to determine the diagnostic value of VHP in albinism and to attempt correlation of the VEP results with clinical features. This study shows that two different patterns of VEP asymmetry in albinism. They may be explained by differences in the reorganization of the geniculo- cortical pathway. Although VEP asymmetry is very frequent in this condition, it may not be constant. 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 saw approximately 200 individuals representing approxi- mately 60 different disease categories. Because of the high frequency of ocular involvement in these cases, almost all of these patients were evaluated by the Ophthalmic Genetics staff Neurofibromatosis 2 (NF2), otherwise known as bilateral acoustic neuroma, is inherited as an auto- somal dominant disorder. Multiple members of several large pedigrees as weU as a large number of unrelated families have been studied in collaboration with Dr. Roswell Eldridge (National Institute of Neurologic Diseases) and Dr. Dilys Parry (National Cancer Institute). An important original observation was the striking frequency of posterior capsular cataract in patients with NF2 (80-85%). In addition, 30% of patients have shown an associated cortical cataracts. These findings are helpful in establishing a diagnosis of NF2 in at-risk patients. The etiology of the cataract is unclear; however, it is interesting that the gene locus for bilateral acoustic neuromas is on chromosome 22 as is the gene for ^-crystalline. In a collaborative study with Dr. Larry Chamas of the National Institute of Child Health and Human Development, data from patients with Lowe's syn- drome and the patients' mothers have confirmed the 232 NEI Annual Report-FY 1992 Ophthalmic Genetics and Clinical Services Branch usefulness of detecting lens changes in making the diagnosis of the carrier state. Finally, the results from the continuing double- masked control clinical trial of topical cysteamine in patients with nephropathic cystinosis are very excit- ing. After confirming the usefulness of 0.5% cyste- amine eye drops in the young patients, we expanded our study to include older patients, and the results were similarly striking. Particularly important is the fact that these patients have shown dramatic relief from their ocular sy.-^ptoms, with a decrease in crystals in the treated eye and a significant improve- ment in quality of life. Section on Clinical Services The Qinical Services Section has been very active in characterizing psychophysical and electrodiag- nostic 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 electrodiag- nostic information is particularly helpful in under- standing the pathogenesis of disease, as weU as being available for use as a maricer in various treatment modalities. The results of specially designed studies have demonstrated a strong positive correlation between electroretinogram (ERG) amplitude and electrooculogram (EOG) results in both patients with retinal diseases and normal subjects. These findings confirm that EOG results are not independent of ERG amplitude and emphasize the need for a diag- nostic test of retinal pigment epithelium (RPE) integrity that does not depend on light-induced RPE responses. Other studies described a reduction in the ampli- tude of the ERG responses correlated with an in- crease in ocular length. These findings suggest that the effect of ocular length on ERG amplitude should be taken into consideration with ERG results from patients with high refractive error. Section on Ophthalmic Pathology The Section on Ophthalmic Pathology has pro- vided technical support services to investigators involved in clinical and basic research as weU as to those performing routine pathology. Careful 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 fit>m the elimination of costly contract services. 233 DEPARTMEhTT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00187-09 OGCSB PERIOD COVERED October 1. 1991 to September 30, 1992 TITLE OF PROJECT (80 charaaers or less. Title must lit on one Una between the borders.) The Effects of Corneal Contact Lenses on the Cornea PRINCIPAL INVESTIGATOR (List other prolassional personnel below the Principal Investigator.) (Name, title, laboratory, and institute aHillalion) PI: Manuel B. Datiles M.D. Medical Officer OGCSB, NET Others: Lessie McCain R.N. LoueUa Lx)pez M.D Sailaja Chintalagiri M.S. Nurse Specialist Visiting Associate Visiting Associate OGCSB. NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (it any) Image Processing and Analysis Laboratory, Division of Computer Research and Technology, NIH (Mark Vivino, B.S.) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Cataract and Corneal Diseases INSTFTUTE AND LOCATION NEI, NIH, Bethesda. MD 20892 TOTAL STAFF YEARS: 0.225 PROFESSIONAL; 0.200 0.025 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 investigation of short-term as well as long-term effects of contact lens wear on the cornea includes specular microscopy studies of changes in corneal curvature, corneal epithelial morphology, and corneal endothelial ceU morphology. Analysis of the data obtained will help us understand the dynamics involved in the interaction between a contaa 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 in wearers of soft compared with hard contact lenses. Animal models showing corneal endothelial abnormalities similar to those in long-term contact lens wearers are also being explored in diabetic and galactosemic animal models. Treatment with aldose reductase inhibitors helps prevent these corneal abnormalities. 234 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 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 come J tissues by contact lens wear. Methods Each patient's complete history, ophthalmologic examination, photography, keratotometry, pachyme- try, and specular microscopy of the corneal endothe- lium arc used. 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 pleomorphisra. Furthermore, 2 years after some of our patients discontinued wearing contact lenses, we foimd 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 weU as for therapy. 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 also pooriy 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 firom one type of contact lens to another; and (3) development of automated computer analysis of all types to facilitate the data analysis (under way). NEI Research Program Corneal Diseases — Corneal Edema, Endothelial Dysfunction, Dystrophies and Inherited Disease Publications Sibug M, Datiles M, McCain LM, Kashima K, Kracher G: Specular microscopic studies on the corneal endothelium after cessation of contact lens wear. Cornea 10:395^01, 1991. 235 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00188-09 OGCSB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or lass. Title must lit art one line between the borders.) Documentation and Monitoring of Opacities in the Human Lens PRINCIPAL INVESTIGATOR (List other professional personnel below the Principal Investigator) (Name, title, laboratory, and institute afliliatlon) PI: Manuel B. Datiles M.D. Medical Officer OGCSB, NEI Others: Rafael C. Caruso M.D Benjamin Magno M.D Susan M. Lasa M.D Sailaja Chintalagiri M.S. Lessie McCain R.N. LoueUa Lopez M.D. Visiting Scientist Visiting Associate Visiting Associate Visiting Scientist Nurse Specialist Visiting Associate OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB. NEI COOPERATING UNITS (11 any) Image Processing and Analysis Laboratory, Division of Computer Research and Technology (DCRT), NIH (Benes Trus, Ph.D.; Mark Vivino, M.S.; Hal Frederickson, B.S.); Biomedical, Engineering and Instrumentation Branch, DCRT, NIH (Michael Unser, Ph.D.); Epidemiology Branch, NEI, NIH (Robot Sperduto, M.D.; Marvin Podgor, Ph.D.; Valeria Friedlin, Ph-D.; Nancy Remaley, B.S.; Roy Milton, Ph.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: 2.305 PROFESSIONAL: 2.280 0.025 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 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 LOCS n 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, retinitis pigmentosa, gyrate atrophy, and neiuufibromatosis 2 cataracts. This study will prepare the way for eventual clinical trials of anticataract drugs. Genetic linkage studies now under way are pursuing the gene(s) of congenital cataract. 236 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Ophthalmic Genetics and Clinical Services Branch Project Description Additional Personnel Maria A. Drews- Bankiewicz Yvonne Duglas-Tabor Marvin Podgor Rita Hiller Robert Sperduto Doretha Leftwood Anup Mahurkar Laura Wozencraft Mark H. Scott J. Fielding Hejtmancik M.D. Visiting Fellow, OGCSB, NEI Biologist, OGCSB, NEI Epidemiologist, NEI Epidemiologist, NEI Qiief , Epidemiology Section, NEI Computer Specialist, OGCSB, NEI B.S.E. Computer Engineer, OGCSB, NEI Genetic Counselor, OGCSB, NEI Senior Staff Fellow, OGCSB, NEI B.S. M.D. M.S. M.D. B.S. M.D. 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, retroiUumination photography, specular microscopy, and laser hght- scanning spectroscopy. Major Findings We have found that clinical grading of cataracts using the LOGS II system can quantitatively detea the progression of age-related cataracts within 1 year. In addition, we found that in various types of cata- racts, glare and contrast sensitivity testing shows abnormal results only in the severe or more advanced grades. The only exception was in posterior subcap- sular cataracts, which showed an abnormality in contrast and glare sensitivity in the eariy stages. In a study of pure nuclear cataracts, we found a signifi- cant correlation between lens nuclear density (mea- sured by either LOGS II grading or Scheimpflug photography) and contrast sensitivity loss of interme- diate and high spatial frequencies. In our continued development of objective, semiautomated methods of detecting and following cataracts, we now are able to quickly perform densi- tometry of Scheimpflug nuclear cataract images and compare them to previous images to detect signifi- cant 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 millimeters using retroiUimiination photographs. Signijicance to Biomedical Research and the Program of the Institute Monitoring and documenting himian cataract progres- sion 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 the physical and genetic factors within specific geographic regions. Subjective methods of determining visual function are also important to determine the degree of handi- cap cataract patients have in coping with daily activities. Since in our studies none of the subjective methods could demonstrate subjective experiences in eariy cataracts, research is needed to develop more sensitive techniques. Proposed Course We win continue the study and development of subjective and objective methods of documenting and monitoring human cataracts. We wiU pursue the improvement and automation of present systems of lens photography (eg, such as Scheimpflug, retroiUu- 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. 237 Ophthalmic Genetics and Clinical Services Branch NEI Annual Report-FY 1992 NEI Research Program Cataracts — Epidemiology of Cataract Publications Datiles M: Qinical evaluation of cataracts, in Tasman W, Jaeger E (eds): Duane's Clinical Ophthalmology Philadelphia, Lippincott Co., in press. Datiles M, Kinoshita J: Pathogenesis of cataracts, in Tasman W, Jaeger E (eds): Duane's Clinical Ophthalmology. Philadelphia, Lippincott Co. 1:72B:M4, 1991. Drews-Bankiewicz MA, Caruso RC, Datiles MB, Kaiser-Kupfer MI: Contrast sensitivity in patients with nuclear cataracts. Arch Ophthalmol 1 10:953- 959, 1992. Lasa S, Datiles M, Podgor M, Magno B, Lee J: Contrast and glare sensitivity: Association with the type and severity of the cataract. Ophthalmol- ogy 99:1045-1049, 1992. Magno B, Datiles M, Sperduto R, Podgor M, Lasa S, Lee J: Catar'^t progression rates using the lens opacity classification system. Invest Ophthalmol Vis Sci, in press. 238 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00212-07 OGCSB PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (80 cheraaers or less. Title must lit 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, laborelorjf, and Institute anUlaUon) PI: Manuel B. Datiles M.D. Medical Officer OGCSB. NEI Others: Susan M. Lasa M.D. Benjamin Magno M.D Yvonne Tabor B.S. Pushpa Sran M.D Louella Lopez M.D Miguel Bumier, Jr. M.D Visiting Associate Visiting Associate Biological Technician Medical Officer Visiting Associate Visiting Scientist OGCSB. NEI OGCSB. NEI OGCSB. NEI OGCSB. NEI OGCSB. NEI LI. NEI COOPERATING UNITS (il any) Laboratory of Mechanisms of Ocular Disease, NEI. NIH (Donita Garland. Ph.D.; J. Samuel Zigler. Jr., Ph.D.; Paul RusseU. Ph.D.); Image Processing and Analysis Laboratory. Division of Computer Research and Technology. NIH (Benes Trus. Ph.D.; Mark Vivino, M.S.) Ophthalmic Genetics and Clinical Services Branch Section on Cataract and Corneal Diseases INSTfTUTE AND LOCATION NEI. NIH, Bethesda. MP 20892 TOTAL STAFF YEARS: 2.55 PROFESSIONAL; 1.55 1.00 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 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 lens. 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 carefuUy documenting the cataracts in patients preoperatively using clinical and photographic LOGS n grading and Zeiss Scheimpflug and Oxford retroillumination video photography and image analysis. Cataracts are extracted extracapsulaily with implantation of an intraocular lens. Specimens obtained are examined histologically using light and electron microscopy and biochemically using two-dimensional gel electrophoresis (PHAST and LSB systems). Cataractous specimens are compared to normal tissues obtained from eye bank eyes. Abnormal proteins are identified using immunoblotting techniques as well as protein sequencing. 239 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report-FY 1992 Project Description Additional Personnel Samuel Zigler Ph.D. Clinical Protocol Number 84-EI-194 Head, Section on Cataracts, LMOD, NEI 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 the project Documentation and Monitoring of Opacities in the Human Lens (ZOl EY 00188). After extracapsular cataract extraction, fragmented lens materials — including the anterior lens capsule, lens nucleus, and aspirated lens cortical material — undergo protein and biochemical analyses. Some of the specimens are frozen for future 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 needs a close professional relationship between the clini- cian-surgeon and the basic researcher. Although the two professionals have different immediate priorities (one being patient care; the other, adequacy of tissue sample 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 time (300 meg). Aging results in acidic shifting of proteins, an increased number of polypeptide species in the molecular weight range of the crystaUins, and cova- lent cross-linking of crystaUins — 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 crystaUins; optimum samples are those we obtain separately, thus avoiding oxida- tion. Significance to Biomedical Research and the Program of the Institute A severe setback is being dealt many cataract pro- jects because of the lack of human cataract material available for basic research studies. Whereas the current technique, which involves fragmenting the cataract during extraction, is extremely successful and effective, there is no foreseeable change back to utiUzation of the intracapsular method (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 the various basic lens research projects that deal with human materials. Proposed Course We wiU continue to pursue development of the use of fragmented lens material in basic research experi- ments. Using two-dimensional gel electrophoresis, we wiU study ways in which surgeons can modify their surgical techniques without compromising patient care whUe providing scientists with critical lens tissue for basic research. In addition, we wiU investi- gate ways in which scientists can work with surgeons in handUng lens materials to maximize the quaUty of specimens for basic research. NEI Research Program Cataract — ^Treatment of Cataract and Correction of Aphakia Publications Datiles M, Schumer J, Zigler S, RusseU P, Anderson L: Two dimensional gel electrophoretic analysis of human lens proteins. Curr Eye Res 1 1 :669-677, 1992. 240 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00084-14 OGCSB PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or lees. Title must lit on one line between the borders.) Anterior Chamber Anomalies Associated with Glaucoma or Ocu lar Hypertension PRINCIPAL INVESTIGATOR (Usi other prolassional personnel below the Principal Investigator.) (Name, title, laboratory, and msUtule aflHiaUon) PI: CarlKupfer M.D. Director NEI Others: Muriel I. Kaiser-Kupfer M.D. Chief Lessie McCain R.N. Nurse Specialist Manuel B. Datiles M.D. Medical Officer Maria Susan M. Lasa M.D. Visiting Associate Benjamin V. Magno M.D. Visiting Associate Louella Lopez M.D. Visiting Associate OGCSB. NEI OGCSB. NEI OGCSB. NEI OGCSB. NEI OGCSB, NLi OGCSB. NEI COOPERATING UNITS (II 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; 0.45 CHECK APPROPRIATE BOX{ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interview/s PROFESSIONAL: 0.35 0.10 □ (b) Human tissues Q (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 connective tissues anterior to the lens epitheUum. Therefore, the group of developmental anomalies of the anterior chamber with glaucoma or ocular hypertension is being reviewed. 241 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report-FY 1992 Project Description Clinical Protocol Number 77-EI-119 Objectives The object of this study is to determine whether congenital 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 the involvement of cornea, trabecu- lar 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 indicated, that procedure is performed and the corneal specimen is examined histologically. Major Findings It appears that in this gnaup 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 endothehum, anterior iris stroma, Descemet's mem- brane, and trabecular meshwork endothelium. We recently performed trabeculectomies on patients with the irido-comeal-endothelial syndrome. HistopathologicaUy, we found a membrane covering the trabecular meshwork. That membrane may have caused 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 win continue to study cases of congenital corneal disorders to imcover 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, Qian C-C, Bumier M Jr, Kaiser-Kupfer MI: Histopathology of the ICE syndrome. Trans Am Ophthalmol Sac, in press. 242 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00123-12 OGCSB PERIOD COVERED October 1. 1991 to September 30. 1992 TITLE OF PROJECT (BO characters or lass. Title must fit on one line between the borders.) Clinical Psvchophysics of the Visual System PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute atlUlalion) PI: Rafael Caruso M.D. Visiting Scientist OGCSB, NEI Others: Muriel I. Kaiser-Kupfer M.D. Doris J. Collie A.A. Maria Bankiewicz M.D. Patricia A. Mercer M.P.A. Leanne M. Reuter B.S. Chief Ophthalmic Technician Visiting Associate Ophthalmic Technician Ophtiialmic Technician OGCSB, NEI OGCSB. NfEl OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (It any) LAB/BRANCH Ophtiialmic Genetics and Clinical Services Branch SECTION Eye Services Section INSTITUTE AND LOCATION NEI. NIH. Betiiesda, MD 20892 TOTAL STAFF YEARS: 1.31 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) intervievi^s PROFESSIONAL: 0.68 OTHER: 0.63 □ (b) Human tissues D (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 tiie visual patiiways and of normal subjects is measured using psychophysical techniques. These data are correlated witii those obtained with elecUDphysiological tests of visual function. The results obtained contiibute to tiie diagnosis of ocular and neural disorders tiiat affect vision and are needed to characterize their nauire and evolution. They are also valuable in the assessment of how different forms of tieatinent affect tiie outcome of tiiese diseases. 243 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report-FY 1992 Project Description Clinical Protocol Number 81 -EI- 108 Objectives The aims of this project are to apply and develop psychophysical techniques for the study of vision in the clinical setting, to characterize the human visual system's normal function, and to analyze the patterns of its alteration in ocular diseases and lesions of the visual pathways. Methods Several psychophysical techniques are employed: (1) Perimetry: Visual fields are explored with kinetic quantitative perimetry and static quantitative perime- try. (2) Color vision: Central color vision is esti- mated using HRR pseudoisochromatic plates, Ishi- hara pseudoisochromatic plates, Famsworth's Tritan plate, Famsworth-Munsell D-15 panel, Lanthony's desaturated D-15 panel, Famsworth-Munsell 100 hue test, and the Nagel anomaloscope. (3) Adaptometry: Daric-adapted rod and cone thresholds are measured with a modified Goldmann-Weekers adaptometer. (4) 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-fiiee judgement of threshold visibility. (5) Luminance and chromatic increment thresholds 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 We described abnoiroalities of foveal cone thresholds and directional sensitivity in eight patients (five females and three males, ages 12-48 years). The diagnostic criteria were biochemical (hyperomithin- emia) and ophthalmoscopic (presence of classical chorioretinal atrophic lesions). We conducted a complete ophthalmological examination and evalua- tion of visual function (ie, visual acuity, contrast sensitivity, visual fields, color vision, dark adapta- tion, Ganzfeld and focal electroretinogram, and electrooculogram). The SCE of the first kind was used as an indicator of foveal cone orientation, and increment threshold (IT) measurements were used to asses foveal cone sensitivity. Both measurements were obtained using a two-channel MaxweUian-view instrument, varying background intensity for IT detennination, and background position for SCE measurements. All patients showed abnormal IT results. In three of them, we observed only a sensitivity loss, while the remaining five showed both sensitivity loss and alteration in effective adaptation status. The shape of the SCE function (estimated by Stiles' iho parameter) was normal for the same three patients whose IT measurements showed only sensitivity loss. Four patients' data showed flat SCE functions (low riio values), results that agree with the previous report on a gyrate atrophy (GA) patient (Tasuma T, et al: Clin Vis Sci 1:93-102, 1986). The remaining patient, who had the most severe chorioretinal atrophy, failed to show directional sensitivity. 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. Signijicance 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 Qinical psychophysical studies of visual fimction in diseases of the eye and visual pathways wiU 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 Strabis- mus Amblyopia, and Visual Processing — Visual Processing and Amblyopia Publications Drews-Bankiewicz MA, Caruso RC, Datiles MB, Kaiser-Kupfer MI: Contrast sensitivity in patients with nuclear cataracts. Arch Ophthalmol 1 10:953- 959, 1992. 244 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00144-11 OGCSB PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must til on one line between the borders.) Clinical Electrophysiology of the Visual System PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Inyesllgalor) (Name, tale, laboratory, and Insllwie aftUlatlon) PI: Rafael Caruso M.D. Visiting Scientist OGCSB, NEI Others: Muriel I. Kaiser-Kupfer M.D. Maria 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. NTH, Bethesda, MD 20892 TOTAL STAFF YEARS: 1.85 PROFESSIONAL: 0.68 1.17 CHECK APPROPRIATE BOX(ES) fx] (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 visual function of both patients with ocular diseases or lesions in the visual pathways and normal subjects is measured objectively with electrophysiological techniques. The data are correlated with those obtained with psychophysical tests of visual function. The results obtained 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 effect of different forms of treatment on the outcome of these diseases. 245 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report-FY 1992 Project Description Clinical Protocol Numbers 91-EI-26 82-EI-55 Objectives The aims of this project are to apply and develop electrophysiological techniques for the study of v.- jal function in the clinical setting, to characterize the normal electrical activity of the human visual system, and 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 (RPE) (electrooculogram [EOG]), the neural retina (electroretinogram [ERG]), and the central visual pathways (visually evoked potentials [VEPs]). These potentials are elicited by unstructured stimuli (Ganzfeld full-field or focal stimulation) and spatially structured stimuli (sinusoidal gratings or checkerboard panems). Major Findings The ERG is used as the standard electrophysiological test of RPE function. Unfortunately, EOG results also are affected by the functional status of photorecep- tors, which can be assessed by the ERG. We found a strong positive correlation between ERG amplitude and EOG results in both patients with retinal diseases and normal subjects. Five groups (83 subjects) were included in the study: patients with retinitis pigmen- tosa (RP, 21), gyrate atrophy (GA, 21), juvenile macular dystrophy (JMD, 12), Best's vitelliform dystrophy (BVD, 2), and normal subjects (27). We analyzed the results obtained in one eye chosen at random for each subject We recorded ERGs using the international stan- dard protocol and analyzed EOGs using the light peak/daric trough (Arden) ratio. Correlation analysis showed a statistically significant relationship between the Arden ratio and the amplitude of the rod-medi- ated b-wave (r:0.822), the maximal retinal response (r:0.841), the cone mediated b-wave (r:0.827) when patients with RP, GA, JMD, and normal subjects were included in the analysis. For these subjects, the ratio between nonnalized ERG amplitude and nor- malized Arden ratio was approximately constant As expected, BVD patients (with normal ERGs and subnormal EOGs) fell outside the "main sequence" of the EOG versus ERG relationship, and their ERG/EOG ratio was unusually high. ERG results were more useful than EOG results to assign a given patient to a diagnostic group (nearest neighbor analysis). When ERG and EOG results were used simultaneously to classify a patient, the improvement in diagnostic specificity over the ERG alone was not meaningful. These findings confirm that EOG results are not independent of ERG amplitude and emphasize the need for a diag- nostic test of RPE integrity that does not depend on light-induced RPE responses. We described a reduction in the amplitude of ERG responses correlated with an increase in ocular length. Previous studies have yielded contradictory results regarding tiiis relationship. One of these reports used a nonstandard ERG recording technique, and another included only seven ametropic subjects. To minimize the effect of known sources of ERG variance (age and sex), we included in the study only women in the 20-49 age range; 28 normal women were evaluated. A complete ophthalmologic examina- tion was performed to rule out ocular disease. Axial length and vitreous segment length were measured using A-mode ultrasonography. An expanded version of the international standard ERG protocol was used for Ganzfeld ERG recordings in one eye, and an intensity-response function was obtained in the fellow eye. Regression analysis was performed to describe and quantify the relationship between ERG findings and ocular measurements. Our principal finding was a statistically significant inverse correlation between ocular length and the amplitude of rod-mediated b-waves, daric-adapted cone-mediated b-waves, maximal retinal responses, light-adapted cone-mediated b-waves, and 30 Hz flicker responses. In contrast, no significant associa- tion was foimd between b-wave implicit time and ocular length. This change in amplitude without variation in implicit time suggests that our results are due to the difference in change in retinal sensitivity. Our findings suggest that when ERG results ft-om patients with high refractive error are analyzed, the effect of ocular length on ERG amplitude should be taken into consideration and the normative data adjusted. 246 NEI Annual Report-FY 1992 Ophthalmic Genetics and Clinical Services Branch Albinism in animals has been associated 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 himians, indirect evidence of the same anomaly is demonstrated by asymmetry in VEPs elicited by pattern reversal stimulation. Recent studies using appearing-disappearing patterns claim VEP asymmetry is diagnostic; they propose a uni- form type of asymmetry. We used the same record- ing conditions To determine the diagnostic value of VEP in albinism and to attempt correlation of VEP results with clinical features. Eighteen subjects (6-42 years of age) with differ- ent types of albinism (oculocutaneous and ocular) underwent complete ocular examination and VEP recording. VEPs were generally of low amplitude, interfering with the interpretation in two cases. One patients with albinism without nystagmus had sym- metric responses; 15 showed asymmetry of two distinct types. In eight, ttie major positivity of the response was distributed in the channels contralateral to the stimulated eye and, in seven, it was ipsilateral to the stimulated eye. There was no correlation of either of these two types of asymmetry with race, sex, type of albinism, or any particular clinical finding. This study shows that there are two different patterns of VEP asymmetry in albinism. They may be explained by differences in the reorganization of the geniculocortical pathway. Although VEP asym- metry is very frequent in this condition, it may not be constant. However, its value is decreased in some cases in which the low amphtude of the responses makes interpretation difficult. Furthermore, there is no correlation of the type of asymmetry with any other feature of albinism. 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 validated 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 Retinal and Choroidal Diseases — Noninvasive Techniques in the Study of Retinal Disorders Strabismus, Amblyopia, and Visual Processing — Visual Processing and Amblyopia 247 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00257-04 OGCSB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characlars or lees. Title must lit 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 attiliation) PI: Rafael Caruso M.D. Visiting Scientist OGCSB, NEI Others: Muriel I. Kaiser-Kupfer Tracy T. Nolan Dessie Koutsandreas Donna M. Chandler Lorraine Kcidra M.D. Chief OGCSB. NEI M.A. Health Technician OGCSB, NEI B.S. Ophthalmic Technician OGCSB, NEI Health Technician OGCSB, NEI B.A. Health Technician OGCSB, NEI COOPERATING UNITS (It any) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Eye Services Section INSTITUTE AND LOCATION NEI, NIH. Bethesda, MD 20892 TOTAL STAFF YEARS: 3.5 PROFESSIONAL: 0.1 3.4 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 National Eye Institute 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. 248 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Ophthalmic Genetics and Clinical Services Branch Project Description Additional Personnel Linda Goodman Antoinnette Laren R. Patrick McDaniel Anne Randall Sueli Mueller Ophthalmic Technician OGCSB, NEI Ophthalmic Technician OGCSB, NEI Ophthalmic Technician OGCSB, NEI Ophthalm'" Technician OGCSB, NEI 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 (ie, determining whether visual loss is present). The second step is quantification of a detected deficit. The third is 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 pathways (visually evoked potentials). Major Findings During the period October 1, 1991, through Septem- ber 30, 1992, we performed the following tests: Kinetic perimetry 216 Static perimetry 276 Screening perimetry Manifest refi"action Color vision tests Adaptometry Contrast sensitivity tests Glare disabiUty tests Ganzfeld electroretinography Focal electroretinography Electrooculography Visually evoked potentials 109 450 146 46 226 294 110 33 93 30 This represents an increase of 57% over the tests performed during the same period in Fiscal Year 1991. 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 the progress of a disease or the effect of a treatment. Therefore, sensitive and accurate measurements of visual function are essential for these clinical re- search projects. Proposed Course The provision of clinical electrophysiologic 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; Stra- bismus, Amblyopia and Visual Processing — Visual Processing and Amblyopia Publications The nature of this project is such that the results obtained are included in publications listed under other projects. 249 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00011-18 OGCSB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characters or less. Title must lit on one line between ttie borders.) Pigment Dispersion With and Without Glaucoma PRINCIPAL INVESTIGATOR (List ottier prolassional personnel below the Principal Investigator.) (Name, title, laboratory, and Institute attiliation) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEI Others: Lessie McCain R.N. Nurse Specialist OGCSB, NEI COOPERATING UNITS (II any) LAB/BRANCH Ophthalmic Genetics and Clinical Services Branch SECTION Section on Ophthalmic Genetics INSTfTUTE AND LOCATION NEI. NIH, Bethesda. MP 20892 TOTAL STAFF YEARS: 0.2 PROFESSIONAL: 0.1 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 detennination of pigment dispersion syndrome patients' risk of developing glaucoma, as well as adding to the understanding of the pathology of the disease. 250 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Ophthalmic Genetics and Clinical Services Branch Project Description Additional Personnel Marvin Podgor M.S. Statistician Biometry and Epidemiology Program, NEI 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 included the following: complete family history with detailed pedigree; best- corrected visual acuity with manifest refraaion; slit- lamp biomicroscopy; visual field examination (Goldmann Ije and l4e); applanation 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 and sixty-four patients were classified into three groups: (1) pigment dispersion syndrome (PDS) without abnormal ocular pressure, (2) PDS with ocular hypertension, and (3) PDS with glau- coma (PDS+GL). Analysis of baseline characteristics with respect to anatomical and physiological parame- ters 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 ffecting 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 pigment dispersion syndrome with httle difference between the measurements of the two eyes is being investigated in followup studies. 6. In our series, retinal 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 the 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 pigment dispersion syndrome. NEI Research Program Glaucoma — Other Glaucomas (Developmental, Congenital, and Infantile Glaucomas) 251 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00060-14 OGCSB PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must lit 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 alllllatlon) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NET Others: Lessie McCain Rafael Caruso Evrydiki Bouza Malina Bankiewicz R.N. M.D. M.D. M.D. Nurse Specialist Visiting Scientist Visiting Scientist Visiting Associate OGCSB, NEI OGCSB, NEI OGCSB, NEI OGCSB, NEI COOPERATING UNITS (11 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: 0.40 PROFESSIONAL: 0.35 0.05 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects [x] (a1) Minors □ (a2) Interview's □ (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, Hennansky-Pudlak syndrome, and iris transillumination defects, are being recruited to determine visual function with these conditions and to evaluate the changes in visual function over time. Family members are evaluated to attempt to determine factors which may identify the heterozygous state. 252 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Ophthalmic Genetics and Clinical Services Branch Project Description Cttnical 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 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 abnomialities 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 For each patient, we compile a complete family history with detailed pedigree and perform the following procedures: best-corrected visual acuity near and at distance with refraction; sUt-lamp exami- nation; psychophysical testing, including D-15 and Munsell 100 hue as well as rod and cone thresholds; dilated ophthalmoscopic examination; photography to docimient hair color, eye color, iris transillumination, and the status of the disc and macula; visually evoked response testing; and contrast sensitivity measurements, in selected patients. Information on family members is collected by examination of best- corrected visual acuity, slit-lamp examination of iris, photography of iris transillumination, and fundus examination when vision is not correaed to 20/20. Major Findings 1. Examination of patients and family members indicated that transillumination of the iris may be seen 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. Three patients presented with marked iris transillumination, reduced pigmentation of the fundus, and no nystagmus, but they had decreased visual acuity, which has improved in conjimction with an increase of the pigmentation of the fundae. 3. Visually evoked responses were normal in some patients, but in a subset of albinos, there was evidence of abnormalities, such as crossing of optic nerve fibers of the chiasm. These findings are being pursued with more refined and updated equipment. 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 coimseling. Determination 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 li^t 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 verify the reported findings of abnormalities of the crossing fibers, as measured by visually evoked responses, contrast sensitivity, degree of nystagmus, and amount of pigmentation. Proposed Course This project wiU 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, Bankiewicz MA, Kaiser- Kupfer MI: Two different VEP patterns demon- strated in albinism unrelated to clinical type. Invest Ophthalmol Vis Sci 33(4):964, 1992. 253 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00083-15 OGCSB PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 characters or less. Title must tit on one line between the borders.) Gyrate Atrophy of the Qioroid and Retina and Other Retinal Degenerations PRINCIPAL INVESTIGATOR (Usl other protesslonal personnel below the Principal Investigator.) (Name, title, laboratory, and Institute atllllatlon) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB. NEI Others: Evrydiki Bouza Lessie McCain Rafael Caruso Pushpa K. Sran Doris CoUie M.D. Visiting Scientist OGCSB, NEI R.N. Nurse Specialist OGCSB. NEI M.D. Visiting Scientist OGCSB. NEI M.D. Medical Officer OGCSB. NEI A. A. Ophthalmic Technician OGCSB. NEI COOPERATING UNITS (II any) The Howard Hughes Medical Institute Laboratory and the Department of Pediatrics. The Johns Hopkins University. School of Medicine. Baltimore. MD (David L. Valle. M.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: 1.1 PROFESSIONAL: 0.9 0.2 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 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 the disease. If patients are not considered eligible for the diet, or if they appear 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 are also examined. The courses of their diseases are compared with those of gyrate atrophy patients. 254 PHS 6040 (Rev. 5/92) NEI Annual Report-FY 1992 Ophthalmic Genetics and Clinical Services Branch Project Description Additional Personnel Laura Wozencraft J. Fielding Hejtmancik Susan Gentleman Ph.D. Ph.D. Genetic Counselor OGCSB, NEI Medical Officer LMOD, NEI 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 wiU 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 foUow 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; ornithine aminotransferase activity is measured, and patient molecular defect is characterized. Complete evalua- tion of ocular function in these patients includes 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 GA, a rare autosomal recessive disorder, is associated with hyperomitiiinemia, overflow omithinuria, and a deficiency of activity of the mitochondrial enzyme ornithine-5-aminotransferase (OAT). Although rare, the condition has been described worldwide in aU races. Thirty-six patients have been recruited and evaluated in this study. The patients' ethnic origins vary, including Scottish, English, Welsh, Portuguese, Finnish, Lebanese, American Black, Asian Indian, German, and Israeh. In this study, the 19 females and 17 males range in age from 2.5 to 65 years, with 9 children less than 12 years old at the time of recruitment. Observations of these patients have enabled docimientation of both clinical evidence and laboratory heterogeneity. Analysis of the mutation that causes GA of the choroid and retina has been imdertaken by Drs. David VaUe, Grant Mitchell, and colleagues of The Johns Hopkins University. They have analyzed probands from 72 GA pedigrees. No gross structural alterations of the OAT gene have been detected; 85% of the probands express nearly normal amounts of nonnal-sized OAT mRNA. The remainder express littie or no OAT mRNA (n = 5) or an mRNA with an altered size (n = 2). Western blot studies showed the OAT antigen was absent in 67% of the mRNA+ mutants and all of the mRNA- mutants. A total of 14 mutations have been delineated 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-frame three- nucleotide deletion of A la- 184 (A185F0), and a nonsense mutation at a CpG dinucleotide (R396ter). The fimctional 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. 255 Ophthalmic Genetics and Clinical Services Branch NEI Annual Report-FY 1992 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 B^. Only one of our patients showed an in vivo response to this treatment. Whereas arginine is the precursor of ornithine in the metabolic pathway of ornithine metabolism, we have undertaken a dietary intervention smdy limiting arginine. Of 23 patients placed on a low-protein Oow-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 compUance and in 7 other patients because of a variety of factors. Of 13 patients remaining on the diet, 4 have excellent control; 4, fair control; 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 6-12 months, travel penmitting. 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 daiic adaption, averaged ERG, and color vision. This improvement was sus- tained for 30 months, then the ERG amplitude showed a small but definite reduction. The second patient with lowered plasma ornithine levels, who had been on the diet for 1 1 years, showed progres- sive improvement in visual field and color vision and has since remained stable. A tiiird patient, despite fair control, was stable for 36 months but has deteri- orated 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 appear stable. Of particular interest are the children who were ages 2.5 to 9 years at the outset of the low-arginine 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. Studies with Usher's syndrome are continuing for the pur- pose of identifying the gene for Usher's syndrome through molecular genetic applications. Significance to Biomedical Research and the Program of the Institute GA of the choroid and retina is the first of the genetically determined isolated severe retinal de- generations for which a specific biochemical marker and concomitant enzyme defect have been demon- strated. This study to test the efficacy of treatment for this blinding eye disease will serve as a model for the investigation of other genetically determined retinal degenerations. Study of the two yoimg patients is the best opportunity for the evaluation of diet control. Proposed Course This project will be continued for 3 more years to assess further the knowledge concerning reduced ornithine in halting chorioretinal degeneration. NEI Research Program Retinal and Choroidal Disease — ^Development and Hereditary Disorders Publications Kaiser-Kupfer MI, Caruso RC, Valle D: Gyrate ad-ophy of the choroid and retina: Long-term reduction of ornithine slows retinal degeneration. Arch Ophthalmol 109:1539-1548. 1991. 256 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00163-10 OGCSB PERIOD COVERED October 1, 1991 to September 30, 1992 TITLE OF PROJECT (80 characlars or less. Title must tit on one line between the borders.) NTH Interinstitute Genetics Program: The Genetics Oinic PRINCIPAL INVESTIGATOR (List other prolessional personnel below the Principal Investigator.) (Name, title, laboratory, and PI: Muriel I. Kaiser-Kupfer M.D. Chief Others: Evrydiki Bouza M.D. Visiting Scientist Mark Scott M.D. Senior Staff Fellow Lessie McCain R.N. Nurse Speciahst Anren Li M.D. Visiting Associate Laura Wozencraft Genetic Counselor Steve Bernstein M.D. Senior Staff FeUow Robert Kim M.D. Senior Staff Fellow Institute BttUlalion) OGCSB. NEI OGCSB. NEI OGCSB, NEI OGCSB. NEI OGCSB. NEI OGCSB. NEI LRCMB. NEI LMDB. NEI COOPERATING UNITS (II any) Interinstitute Medical Genetics Program, NIH Ophthalmic Genetics and Clinical Services Branch Section on Ophthalmic Genetics INSTnXITE AND LOCATION NEI. NIH. Bethesda. MP 20892 TOTAL STAFF YEARS: 0.55 PROFESSIONAL: 0.30 0.25 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.) 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, abnost all the patients were evaluated by Clinical Branch staff or were discussed in consulution. The clinic serves as a source of interesting case material concerning patients with inherited or developmental abnormalities of the visual system. 257 PHS 6040 (Rev. 5/92) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report-FY 1992 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; (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 woricup is recommended. Major Findings 1. Iris nodules were seen commonly in the classic cases of neurofibromatosis (NFl) and less frequent in patients with less-weU-defined disease. They were seen rarely in patients with bilateral acoustic neuro- ma (BAN or NF2). Patients with NF2 showed increased frequency of posterior capsular cataracts, which serve as an excellent maricer, being present in 29 of 30 patients with NF2. A new finding is the association of congenital cortical cataracts in NF2 patients. 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 5 years. 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, and they are responsible for about 35% of the cases of blindness 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 Retinal and Choroidal Disease — Development and Hereditary Disorders Publications Bouzas EA, Kransewich D, Koutroumanidis M, Papadimitriou A, Marini JC, Kaiser-Kupfer MI: Ophthalmological examination in the diagnosis of Proteus syndrome. Ophthalmology, in press. Bouzas EA, Parry DM, Eldridge R, Kaiser-Kupfer MI: Familial occurrence of combined pigment epithelial and retinal hamartomas associated with neurofibromatosis 2. Retina 12(2): 103-107, 1992. Parry DM, Kaiser-Kupfer MI, Sherman JL, Pikus A, Eldridge R: Neurofibromatosis 2 (bilateral acous- tic or central neurofibromatosis), a treatable cause of deafiiess, in Ruben, RJ, de Water TR, Steel KP (eds): Genetics of Hearing Impairment. Ann NY AcadSci 630:305-307, 1991. 258 DEPARTMENT OF HEALTH AND HUMAN SERVICES - PUBLIC HEALTH SERVICE NOTICE OF INTRAMURAL RESEARCH PROJECT PROJECT NUMBER ZOl EY 00211-07 OGCSB PERIOD COVERED October 1, 1991 to September 30. 1992 TITLE OF PROJECT (80 charadars or lass. Tilla must lit on one ling batwaen tha bordars.j A Double-Masked Controlled Randomized Clinical Trial of Topical Cysteamine PRINCIPAL INVESTIGATOR (List othar profassional personnal below tha Principal Investigator.) (Name, title, laboratory, and institute aimiation) PI: Muriel I. Kaiser-Kupfer M.D. Chief OGCSB, NEl Others: Lessie McCain Manuel Datiles Evrydiki Bouza Mark Scott R.N. M.D. M.D. M.D. Nurse Specialist Medical Officer Visiting Scientist Senior Staff Fellow OGCSB, NEI OGCSB, NEl OGCSB, NEI OGCSB. NEI COOPERATING UNITS (It any) Human Genetics Branch. National Institute of ChUd 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: I PROFESSIONAL; 0.50 CHECK APPROPRIATE BOX(ES) [x] (a) Human subjects □ (a1) Minors □ (a2) Interviews 0.25 OTHER; 0.25 □ (b) Human tissues n (c) Neither SUMMARY OF WORK ^Lfee standard unraducad type. Do not exceed the space provided.) Nephropathic cystinosis is an autosomal, recessively inherited storage disease in which nonprotein 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. Systemic complications include the Fanconi syndrome and renal failure. 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 studies to test for compUcations revealed none, we began a double-masked cUnical trial to test the efficacy of topical cysteamine (0.1%) in humans. Fourteen patients of ages less than 3 years were enroUed and randomized to 0.1% cysteamine. Five patients showed a significant decrease in crystals in the cysteamine-treated eyes. To test the effects of increasing the concentration of cysteamine eye drops in humans, a toxicity smdy was perfonned in rabbits. It showed no adverse reactions. The results permitted an increase in concentration to 0.5% for human use. and all patients receiving 0.1% cysteamine were switched to 0.5%. An additional five young patients showed a significant decrease in treated eyes. Thus, of 28 young patients. 14 successftilly had the code broken; of the 14 remaining. 2 died, 1 discontinued medication. 2 are still in the trial with poor compUance, and 9 have been receiving treatment too short a time to tell. 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-one patients have shown a significant decrease in crystals in treated eyes as well as improvements in comfort, i.e.. reUef of pain and photophobia. This study has resulted in significanUy improved quality of life for the successfully treated patients. 259 PHS 6040 (Rev. 5fl2) Ophthalmic Genetics and Clinical Services Branch NEI Annual Report-FY 1992 Project Description Additional Personnel Ernest M. Kuehl Chief, Photography Section OGCSB, NfEI Clinical Protocol Number 86-EI-62 Objectives The purpose of this project is to test the efficacy of topical cystearaine 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 weU 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. 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 is planned for 1- to 3-years' continuation. NEI Research Program Corneal Diseases — Ocular Surface Problems (Drug Delivery and Toxicity) 260 Index Index The authors of the reports in this volume contributed to the index by submitting their own lists of descriptor terms. Acetazolamide 82 Acyclovir (ACV) 76 Acyclovir, oral 3, 9 Adhesion molecules 32, 91 cell 87 endothelial leukocyte adhesion molecule 1 (ELAM-1) 45 intercellular adhesion molecule 1 (ICAM-1) 45,87 Agammaglobulinemia, X-linked 123 Age-Related Eye Disease Study (AREDS) 3, 8, 15 Age-related macular degeneration (AMD) 3, 7, 15 Aggregation 131 Aging 31 and cataract 240 AIDS (acquired immunodeficiency syndrome) 8, 29, 31, 43, 83 HIV infection 85 pediatric 98 therapeutic agents 98 Albinism 31,232,247,253 VEP 31 Aldehyde dehydrogenase class 3 160 Aldehyde reductase 171, 176 Aldose reductase 115, 117, 169, 171, 176 inhibitors 29, 135, 169, 176 Animal models 61 cephalopod 156 chicken 160 embryonic 143 cubomedusan jellyfish 156 dog 171 experimental ocular autoimmune 91 monkey 219 macaque 34, 36 mouse 160 oACry-IFN-Y 55 C3H-Hen 87 C57vit/vit 185 EAU 91 MHC-deficient 45 Philly 129 transgenic 8, 30, 55, 140, 147, 182, 189. 202 rat EAU 91 EIU 82 galactosemic 135 Lewis 87 Royal CoUege of Surgeons (RCS) 38 squid 156, 160 transgenic 55 Anterior chamber 127 anomahes 242 congenital anomalies 242 Anterior chamber-mediated immune deviation (ACAID) 92 Anticataract agents 29, 30. 115. 127, 131 Antigens lymphocyte function-associated (LFA-1) 45, 87 MHC class II 101 uveitogenic 32, 112 Antioxidants 131 Antiviral therapy 75 Aqueous humor dynamics 251 Arginine-deficient diet 31 Asteroid hyalitis 172 Attention involuntary 30, 209 visospatial 216 voluntary 30, 209 Autoimmune inflammation 205 B Barbados Eye Smdy 19 Barbados Incidence Study 19 Behcet's disease 109,112 Bilateral acoustic neuroma 232, 258 263 Index NEI Annual Report-FY 1992 Binocular disparity 209, 219 Binocular vision 219 Biochemistry, nutritional 38 Blue-sensitive cone pathway 36 Brain 30, 163, 210 Cataract 3, 9, 19, 31, 38, 105, 115, 171, 231, 258 age-related 15, 237 congenital 131 congenital gene 237 formation 30, 129, 237 hereditary 129 surgery 9, 240 Cataractogenesis 131, 231 Caudal medulla 223 cDNA 160 Cell differentiation 144 Cell cycle 31 regulation 141 regulatory protein cyclin B 140, 143 Cell(s) cultures, RPE 79 development, RPE 72 differentiation 144 discharges 210 growth 144 infiltrating immunocompetent 64 quiescence 144 synchronized 144 Cerebral cortex 30, 216 Chloramphenicol acetyltransferase (CAT) 150, 189 Choroid 255 Chromosomal localization 182 Chromosomal map, eye-specific genes 181 Cis-acting elements 154 Cis-regulatory elements 139 Clinical research protocols 249 Clinical services 233 Clinical trials 3 Cloning genomic 182 molecular 199 subtractive 182, 195 CMV Retinitis Retreatment Trial 8 Collaborative Corneal Transplantation Study (CCTS) 3 Collaborative Initial Glaucoma Treatment Trial 10 Color-opponent ganglion cells 34 Cones, blue 36 Contact lens wear 235 Cornea 139, 160, 260 Corneal disease 8, 231 Corneal graft failure 88 Corneal grafts 258 Corneal tissues 235 Corneal transplant 3, 9 Coronavirus model 31 Corticosteroids 67, 83 Crystallins 163 a- 3, 10, 131 oA 55 oA- promoter 147 oAins 55 ^ 122 pB2- 129 Y- 119, 123 51- 163 51- gene 140 52- 163 62- gene 140 e- 163 e- gene 140 C- 131. 163 p- 140, 163 T- 163 T- gene 140 enzyme/ 131 gene, ocB- 154 gene, P- 154 gene, 5- 155 invertebrate 30, 140, 156 J- 156 lens 4,30 recruitment 140 taxon-specific 163 vertebrate 156 Cyclophilin 160 Cyclosporine 83, 109 Cysteamine 31. 233. 260 Cystinosis 31 Cytokines 67. 69, 76, 79, 87 264 NEI Annual Report-FY 1992 Index D Diabetes 117 complications 29, 115, 169, 171 Diabetic eye disease campaign 3 Diagnosis of visual loss 249 Diagnostic testing 31 Diagnostics 244 Dideoxyinosine (ddl) 85 Diet, low-arginine 256 Dietary intervention 232 Differentiation ceU 31 ceUular 30, 139, 140 lens cell 164 DNA automated fluorescent sequencing 199 DNA, antisense 189 Drosophila (fruit fly) 182 Drosophila melanogaster, heads 185 Dynabead® 195 E Education kits 25 Elastase 30, 135 Electrodiagnostic information 233 Electrophoresis, two-dimensional gel 119, 127 Electrophysiological techniques 246, 249 Enzyme-linked immunosorbent assay 189 Evolution 139 Extramural research activities 7 Eye development 30 embryonic 55 Eye movement 210 saccadic 30, 226 vergence 219 visually controlled 30 143 Galactosemia 171 Gancyclovir 29, 31,43 Gaze 210 Gaze shift 222 Gene T-crystallin/a-enolase CAT 150 crystallin 30, 139 expression 139 major intrinsic protein (MIP) 55 regulation 29 RPE-specific 29. 182 T-ceU receptor (TCR) 92 therapy 29, 32, 45, 139, 183 timior suppressors, p53 and Rb 140, 143 Usher's syndrome 256 Gene expression 30, 231 crystallin in lens 154 in muscle 154 IRBP 185, 189 lens fiber membrane 150 MHC class II 30, 55, 140. 147 regulation 150 Genetic counseling 253, 258 Genetic engineering 147 Genetic markers 123, 251 Genetics, ophthalmic 231 Geniculo-cortical pathway 31 Glaucoma 10, 19, 105, 251 juvenile 10 Glaucoma campaign 3 Glaucoma implant Moiteno 31, 43, 105 Glutathione S-transferase 156 Growth faaor TGF-pl 164 Gyrate atrophy 31, 32, 45, 231, 244, 255 H Fixation, attentive 222 FK506 106 5-fluorouracil 31, 43, 105 Foscamet 31 Herpes simplex stromal keratitis 3 Herpetic Eye Diseases Study (HEDS) Human population studies 15 3.9 Galactose-fed dogs 29, 169 Galactose-fed rat 135 IGF-binding proteins GGFBPs) Immune responses 64 Immunology 43 192 26S Index NEI Annual Report-FY 1992 Immunosuppressants noncytotoxic 47 Immunosuppression 31 Immunosuppressive medications 61 Indian Council of Medical Research (ICMR) 19 Inferior temporal cortex 212 Insulin-like growth factor (IGF)-I 192 Interferon 30 -gamma (TFN-y) 55, 69, 91, 140 Interinstitute Genetics Program 232, 258 Interleukin 2 (IL-2) 69, 91 imerleukin 3 (IL-3) 91 Interleukin 4 (IL-4) 67, 91 Interleukin 6 (JL-6) 44, 69, 79, 91 International activities 19 Interphotoreceptor retinoid-binding protein (IRBP) 29, 44. 47, 52, 57, 91, 112, 181, 189, 199 protein expression 185 Intraocular pressure (lOP) 10 Irido-comeal-endothelial (ICE) syndrome 242 K Laminin 195 Laminin-binding molecule- 100 195 Lateral geniculate nucleus 34 Lens 38, 119 -promoter, ^-crystallin 140 enzyme overexpression 140 epithelium 127. 141, 143 fiber membrane 150 materials for research 240 opacities 231, 255 organ culture 131 proteins 129 Leukoregulin 76 Ligand binding 185 Light stimulation, lack of 185 Lipid peroxidation 38 Lymphokine lOK/MIF 140 macrophage migration inhibitory factor (MIF) 164 Lymphokines 64 Lymphoma intraocular 64, 105 non-Hodgkin's, CNS 82 orbital 64 M Macular edema uveitis-associated 82 Major intrinsic protein 140, 150 ...essages temporally encoded 212 temporally modulated 30, 209 Metal-catalyzed oxidation 30, 115, 119 Microphakia 147 Microphthalmia 147 Molecular biology 160, 182 Molecular chaperone 131 Molecular interactions 231 Molecular mimicry 205 Molecular mutations 255 Monoclonal antibodies mouse IgG3 72 Motor processing 210 Movement, head 210 control 222 mRNA, c-fos 144 Mutagenesis studies 182 Mutation mechanisms 122 Mycophenolate mofetil 57 Myotonic dystrophy 123 N National Eye Health Education Program (NEHEP) 24 NEHEP Partnership 24 Nephropathic cystinosis 233, 258, 260 Neural crest 242 Neural discharge 30 Neural modeling 209 Neurofibromatosis 231, 258 Neuroprocessing 30 Neurotrophic agent 182 O Ocular diseases autoimmune 147 inflammatory 31 266 NEI Annual Report-FY 1992 Index inherited 258 Ocular herpetic infection 9 Ocular Hypertension Treatment Study (OHTS) 10 Ocular inflammation 64, 87 Ocular toxoplasmosis 19 Oculomotor control 209 Onchocerciasis 67 Optic flow stimuli 226 Optic neuritis 1 1 Optic Neuritis Treatment Trial (ONTT) 3, 11 Oral prednisone 3 Ornithine aminotransferase 32, 45 gene 232 Omithine-5-aminotransferase (OAT) 255 Orthoguinone reductase 163 Oxidative modification systems human 119 primate 1 19 Oxidative stress 127 Parplanitis 112 Parvocellular 223 Pathology ophthalmic 233 Pericyte degeneration 135 Photoreceptor 140, 199 Phototransduction 202 Pigment dispersion syndrome (PDS) 25 1 Pigment epitheliimi-derived factor (PEDF) 182, 192 Pigmentation, ocular 253 Polymerase chain reaction 163, 192 Polyol pathway 30, 117 Polyols 171, 176 Posterior cingulate 216 Posttranscriptional mechanisms 70 Precursor frequency 101 Primate visual system 36 Promoter IRBP 189 lens-specific 131, 163 opsin 202 Protein(s) 65-kD, novel 199 outer retina-specific 199 rod outer segment (ROS)-specific, 33-kD 202 Proto-oncogenes 30 c-fos 143 c-jun 143 c-myc 143 lens 141 N-myc 143 Psychophysical diagnostic information 233 Psychophysical techniques 244, 249 Public service campaigns 3, 24 Q R Rapamycin 31, 44, 47 Refractive error 246 Renal toxicity 109 Research Contracts 7 Research Grants 7, 25 Research Training Awards 7 Reticular 223 Reticular fomiation region medullary 222 pontine 222 Retina 30, 38, 139, 164, 255 Retinal atrophy 85 Retinal degeneration 31,38,44,75 genetic 232 Retinal detachment 79, 147 Retinal disease 231 Retinal diseases age-related 79 hereditary 29, 79 Retinal formation 11-cis 185 Retinal pigment epithelial (RPE) cells 3, 69, 72, 79, 185 Retinal pigment epitiieUum (RPE) 29, 38, 44, 85, 199 cell transplants 31 Retinitis cytomegalovirus (CMV) 8, 29, 31, 43, 98 Retinitis pigmentosa 8, 195, 231 Retinoblastoma cells 69 Y-79 192, 195 Retinoid-binding glycoprotein 1 85 Retinopathy Bietti's crystallin 29 diabetic 7, 29, 36, 115, 135, 169 Ribozymes 29, 189 267 Index NEI Annual Report-FY 1992 S-antigen (S-Ag) 44, 47, 52. 67, 91. 101. 112. 202 feeding protocol 45 Saccades 30 Sarcoidosis 82 Serine protease inhibitor (SERPIN) 193 SightFirst 20 Silicone Oil Study 8 Sodium^tassium ATPase 117 Sorbitol dehydrogenase 30, 115, 117 Spleen 112 Studies of the Ocular Complications of AIDS (SOCA) 8 Superior coUiculus 216, 226 Surgery in ocular inflammation 105 T-cell lines 101 receptor 52 TFUD 202 Tissue typing 3 Toxoplasmosis 43 Trabeculectomy 43 Trans-acting factor oA-CRYBPl 154, 157 Transcriptional mechanisms 70 Transcriptional regulation 141, 143, 150 Transforming growth factor-^ (TGF-P) 91 Transgenic studies 182 Transient expression 189 Transplantation RPEcell 72 Tumor necrosis factor (TNF) 69, 91 Uveitis genicity in vitro system 31 Uveitogenic peptide 57 Uveoretinitis experimental autoimmune 57, 61 Vergence responses 210 Vestibular 223 Vestibular-ocular rtJex 12 Vestibulo-ocular reflex 222 Virology 31, 44 Virus infections 75 coronaviruses 75 cytomegalovirus 75 herpes simplex virus type 1 (HSV-1) 75 Vision Research— A National Plan: 1994-1998 23 Visual behavior 210 Visual cortex 34 Visual cycle 185 Visual diseases inherited 122 Visual information processing 212 Visual loss 249 Visual neurons 212 Visual pathway abnormalities 231 Visual pathway lesions 244 Visual processing 30, 209 Visual-oculomotor system human 226 monkey 226 Visually evoked potentials (VEP) 31, 232, 246 Visuomotor integration 210 Visuomotor neurons 216 Vitamin A 19 Vitrectomy, diagnostic 105 U w Usher's syndrome 29, 30, 115. 123. 256 Uveitis 31, 44. 79. 82, 105, 205 endotoxin-induced (EIU) 45, 61. 87 experimental autoimmune (EAU) 44. 47, 52. 87, 91, 185. 205 noninfectious 112 posterior 109 Y Z 268 ^'flltuiy •-■■-err