Today, we're going to review the ongoing research at the Medical Neurology Branch of the National Institute of Neurological and communicative Disorders and stroke into the viral metabolic and immunologic studies on the ideology and pathogenesis of amyotrophic gland sclerosis and associated motor neuron disease. What I would like to do is review some of the evidence suggestive evidence at this point for a viral ideology to amyotrophic lateral sclerosis and outline some of the work which we are doing. In addition, we will look at some of the evidence for an immuno pathologic process involved in amyotrophic lateral sclerosis. And again, what work is being done in this field. And finally, we are going to look into the metabolic studies being performed here at NIH to see if there are any unique abnormalities, regardless of ideology, which might lead to some way to counteract the disease process. There is suggestive evidence for a viral ideology in amyotrophic lateral sclerosis for the viral ideology is the unique observation that there is a late onset progressive muscular atrophy after early childhood paralytic poliomyelitis. This has a usual delayed onset, approximately 20 to 40 years following the childhood disease and rarely involves cases that may present with the classic Charcot form of a myotropic sclerosis. Other workers have tried to see if there's a relationship between Anteed and polio virus infection through looking at serum antibodies to various polio virus types. No clear cut relationship has been noted. However, if we will remind ourselves of multiple sclerosis, we will remember that there is a unique and perhaps more dramatic rise in the antibody in the cerebral spinal fluid towards specific viruses in this disease than in the serum. We in conjunction with Dr John sever are looking at the serum and cerebral spinal fluid antibody levels which would neutralize various types of polio virus antibody in a group of patients with amyotrophic lateral sclerosis and comparing this to a group of patients with other neuromuscular disease such as peripheral nerve disease and primary myopathies. This work is ongoing and I will not report the results today. But it is important for us to conceive of a possibility that an early infection may lead to late disease. There is an animal model of marine progressive muscular atrophy. After neonatal infection with mouse C type particles, this produces an infection in wild mice and can be reproduced in the experimental situation leading to paralysis and pathological changes that are very much like what is seen in the human disease. Amyotrophic sclerosis. Specifically, there is a marked absence of inflammatory response in these animals. Now, contrary to what we cannot find in amyotrophic vito sclerosis virus particles are seen in the mice as they develop paralysis and these are localized in the anterior horn cells. However, many anterior horn cells show destruction without evidence for viral replication. The exact pathogenesis by which paralysis is produced is currently under study at the Johns Hopkins University School of Medicine. Based on the finding in the animal model. Some people including Mike Viola and Saul Spiegelman at Columbia University College of physicians and surgeons have looked at the presence of an RNA instructed DNA polymerase in the brains of patients with a myotropic sclerosis. The mouse C type virus particles and other C type virus particles are associated with an enzyme called reverse transcriptase which will make DNA from an RN A trans script. Looking at extracts of brains from patients with Guamanian Parkinson's disease. He myotropic lateral sclerosis complex. These workers have been able to find evidence for a unique RNA instructed DNA plumes in the brains of patients with this disease. However, patients without the clinical signs or pathological signs for disease also have evidence for this enzyme. More recently, there have been some reports of virus like particles occasionally seen in the extra neural tissues of two cases of acute amyotrophy out of sclerosis. The exact role of this observation in the pathology of the disease is unclear because many of these patients had intercurrent infections and it is possible that these viruses may not represent direct infection of the tissues but may be there as bystanders. The exact ideology for possible extra neural viral infection on central nervous system degeneration is unclear. However, we must keep in mind such syndromes as Ray's syndrome in which infection involving the liver leading to liver destruction can cause a severe neurological deficit. Now, there is evidence against the virus ideology in the classic sense for amyotrophic sclerosis. Primarily the patients with amyotrophic sclerosis in Guam and sporadic cases have not produced disease when inoculated into cerebrally into experimental animals in experiments similar to that which has shown a definite viral ideology for diseases such as Kuru and Creutzfeld Jakob disease in man. In fact, the absence of passage for any agent in amyotrophic sclerosis has served as an excellent control for the other studies. Highlighting that the amyotrophic out sclerosis does not behave like a simple virus infection and no clear cut virus is recoverable in animal inoculation experiments. But more importantly, in contrast to the work being done with the more sensitive methods of looking at RN A instructed DNA polymerase. It is important to note that the brains of patients with sporadic cases of amyotrophic lateral sclerosis show no evidence for the presence of this enzyme. Thus, on the balance, there is no clear cut evidence for a viral ideology. But some animal models suggest that it is possible for a progressive muscular atrophy type syndrome, similar to amyotrophic sclerosis and associated motor neuron diseases to occur following a viral infection early in childhood. We are searching for other possible leads in this regard. The possibility that there may be an immuno pathologic process involved in amyotrophic lateral sclerosis is less clearly established and evidence sort of lines up on the side against an immune process. For this disease. Serum cytotoxic against cultured embryo anterior horns and culture was thought to be specific for amyotrophic lao sclerosis serum in the past few years, groups at the NIH and elsewhere have shown that the serum cytotoxic against heterologous mouse embryo neurons in culture is non specific and most likely related to heterologous antibodies directed toward blood group specific antigens which cross react in the urine system at the present time. There's no definite histocompatibility linkage. Some groups have suggested that there is an increase in H three and seven in paralytic poliomyelitis patients and in patients with amyotrophic lateral sclerosis. Two other groups including Dr Paul Terrasa, who is probably the father of the histocompatibility method have shown that there is no clear cut statistically significant linkage. However, some groups have shown an increase in the L 2-21 haplotype in patients with severe paralytic poliomyelitis and amyotrophic lateral sclerosis. These findings are suggestive that there might be a unique susceptibility to some pathological process that it is linked to the his compatibility antigens or to the haplotype is not clear yet, but perhaps further studies with more specific delineations of histocompatibility linkages might give us some idea as to what is going on at the present time, there is no specific lymphocyte response as measured by macrophage inhibition factor or lymphocyte transformation to brain antigens or neuromuscular junction antigens in the lymphocytes from patients with amyotrophic lateral sclerosis as compared to controlled patients. Some recent evidence has suggested that there may be a role for complement in the pathogenesis of atrophy sclerosis in the medical neurology branch. Several groups have demonstrated that there might be borderline increase in C three levels of patients with amyotrophic sclerosis compared to other disease groups. Recently, one group has shown an increased C one Q binding in patients with Amyotrophy out sclerosis compared to controls, it is not clear. However, if the controls are adequately age or sex matched, but there is some suggestive evidence that there may be an abnormality and complement binding in patients with aosis, whether this is due to intercurrent infection or related directly to the pathogenesis of the disease is unclear. These same people found evidence for some immune complex deposition in the renal glome in nine of 18 patients with a sclerosis suggesting that there might be some intercurrent infection. Most recently, a paper from the same group has shown that there is no evidence for circulating immune complexes in these patients. So exactly why there is increased evidence for deposition without the presence of circulating complexes is unclear. And this issue remains to be decided by future research, regardless of whether there is a virus or immunologic ideology. For the disease. There might be some metabolic pathway which is affected, which can then be understood and perhaps changed by drug therapy. The best example of this would be Parkinson's disease in man where there is very good evidence that the post encephalitic Parkinson's is due to a virus infection. However, the exact way that we treat patients is based on metabolic abnormalities attended in the dopamine colergic system, we are searching for a similar metabolic abnormality in amyotrophic sclerosis patients because some patients with hyper parathyroidism may present with neurological abnormalities and occasionally be differentially diagnosed as having amyotrophic sclerosis. We have conducted a longitudinal study of calcium metabolism in patients with amyotrophic lateral sclerosis, peripheral neuropathy and primary muscle disease. We have looked at the seven day whole body calcium retention in patients with als compared to other controls. This is a measure of the absorption of calcium by the body. Its distribution into the metabolically active pools of bone and muscle and its excretion from the body. In a fixed length of time. Normally, patients will maintain after a given dose on a given diet of calcium. A calcium retention ranging between 30 to 44 patients with neuromuscular disease are statistically below normal. But patients with amyotrophic lao sclerosis are lower yet and lower. Compared to patients with other neuromuscular disease. We have been able to show by measuring the vitamin D level that is not due to an abnormality in the vitamin D level. In the blood as measured by 25 hydroxy vitamin D and serum calcium is similar in all three groups. Although in the low normal range, interestingly, when we looked at the parathyroid hormone concentrations, we found that all three groups showed evidence that the parathyroid hormone concentration was borderline elevated with approximately 40 to 50% of the various groups being above two standard deviations from the mean for normal. Suggesting that there might be some role of secondary hyper parathyroidism in these patients in response to abnormalities in calcium metabolism or in the regulators of calcium metabolism. In order to determine whether there was an abnormality in absorption early of calcium. In patients with amyotrophic sclerosis, we looked at the uptake of radioactive calcium into the plasma pool in patients with amyotrophic lateral sclerosis. Compared to those patients with nerve and muscle disease. Patients with neuromuscular disease, exclusive of als show quite normal uptake. Patients with als show an increased uptake into the plasma pool and a more heterogeneous distribution. Thus, we were able to show that the decreased retention is not due to abnormalities in the absorption acutely of calcium. In patients with amyotrophic lateral sclerosis. The reason for the delayed decreased retention must have some other basis. We know that these, we know that these patients have normal vitamin D levels, but we do not know whether they respond adequately to this level. In order to determine this, we gave patients with als dihydrotachysterol and a control group with similarly decreased retentions and followed these patients before and after treatment with the seven day whole body retention, we were able to show that patients with amyotrophic lao sclerosis showed an increase in the whole body retention of calcium 47 at seven days, similar to the controls and into the normal range. This was maintained as long as they were on the dihydrotachysterol therapy and showed that there was no vitamin D resistance in these patients. The exact role that calcium metabolism plays in amyotrophic sclerosis is unclear. Is it an epi phenomena or is it related to the underlying pathogenesis? Further studies will have to be done to determine whether patients with A LS respond adequately to the normal stimulation of parathyroid hormone. Those studies are currently in progress at the medical neurology branch because we were unable to show an abnormality in calcium levels in the serum or spinal fluid between patients with als and other neurological disease. We looked at other contaminants of calcium metabolism in these patients cyclic nucleotide, cyclic amp and cyclic GMP are present in the human body in large amounts, but more specifically in the central nervous system in extraordinarily high amounts compared to other tissues. In addition, the enzymes for their synthesis and degradation are also present in these tissues. Cyclic amp and cyclic GMP play an important role in the metabolic mediation of certain reactions such as phosphorylase. In addition, there is increasing evidence for a role of cyclic amp and electrochemical mediation of certain events in the central nervous system. In human studies of patients with als and other diseases, we use the radio immunoassay to measure cyclic nucleotides and show here the unique specificity in distinguishing between the two nucleotides using our different rabid antibiotics. When we looked at cerebral spinal fluid cyclic A MP levels in the same patient at two different times under identical pharmacological and physiological situations, we showed an excellent correlation with excellent linear regression for cyclic amp and cyclic GMP. The black dots indicate those patients who have not had proven acid treatment and the white squares show those patients who have had proven acid treatment. There is a clear correlation regardless of pharmacological manipulations between the CSF levels of these cyclic nucleotides in the same patients on two different occasions. And we were able to show that there is no clear cut difference in the plasma cyclic amp P level between those patients with motor neuron disease and those without motor neuron disease. However, there is a significant decrease in the CSF cyclic amp P compared to these patients. And there's a more grouping of these patients compared to a more wide range seen in other groups. In order to determine whether the cerebral spinal fluid cyclic nucleotide level was completely representative of what was going on in the central nervous system and did not reflect any changes in the plasma pool. We performed the following experiment in which we gave Glucagon infusions and raised the plasma cyclic A MP level 40 fold without changing the plasma cyclic GMP level. There was no significant difference between the elevations in patients with ALS compared to those without ALS, the CSF levels did not change at all during the whole period of observation significantly. However, the CSF glucose levels rose significantly in both groups to similar levels indicating that there was transfer of glucose into the cerebral spinal fluid at a time when there was no similar transfer of cyclic A MP. Thus, there is a significant barrier to cyclic A MP entry into the cerebral spinal fluid under physiological conditions such as we employed here, the main mode of egress for cyclic A MP from the plasma pool is to be excreted in the kidney. Now, this clearly established that the level of cyclic nucleotides was unique in our hands. And if we look at our formal data here, correlating the CSF level of cyclic amp and cyclic GMP in patients with muscle disease, peripheral nerve disease. To those patients with amyotrophic lateral sclerosis, we show here a significant decrease in both cyclic amp and cyclic GMP compared to patients with these other diseases. Indeed, patients with other central nervous system diseases such as multiple sclerosis, the level might be slightly increased compared to normal. This is important because of our recent studies involving the Wobbler mutant, which is a urine progressive spinal muscular atrophy, which is genetically based. We were able to show in conjunction with Dr David loss of the laboratory of neuro neuropathology that there is an 80% decrease in cyclic GMP in the spinal cord and brain stem of these patients of these mice. Compared to their literate controls. At the same time, there was no decrease in a neurotransmitter such as Gaba and high energy phosphate compounds showed no difference between control animals and those affected by the disease. Thus, motor neuron disease with anterior horn loss is associated uniquely with an abnormality in cyclic GMP concentration both in the tissue of animals and in the cerebral spinal fluid of man. To highlight this further, we employed a technique where we gave probenecid infusions to block the escape of cyclic nucleotide from the cerebral spinal fluid. Using an intravenous technique as recently described by doctors Ebert and Cartel. We were able to show that there is a increase in cyclic A MP at four hours after the infusion, which is maintained at eight hours after the infusion. In comparing patients with als to those with other neuromuscular disease, we can show that there is a decrease in the cyclic A MP turnover and in the cyclic GMP turnover. Although this is much more marked in the case of cyclic GMP turnover. In order to see whether we could change this in any manner, we employed a fossil dia inhibitor called the lasino, which does not block the adeno receptor. The Ayin and papaverine, which are well known drugs do block the adenosine receptor and do not allow positive feedback for the increase in cyclic amp and central nervous system tissues. By giving thol to patients with als, we were able to show a dose related increase in CSF cyclic amp but no significant change in C GMP plasma levels show no significant change. And we believe that it is because we're able to show that there is a rapid elimination of plasma cyclic amp from the plasma pool. After this drug is being given to see if there were any other effect of this drug on the transport of cyclic amp. We did probe infusion studies on patients before and after therapy. As we can see here, there is no significant alteration from the pre treatment level and those scraps that I showed previously. However, after treatment with thain, there is a significant increase in the CSF level of cyclic amp, cyclic GMP increases slightly but not statistically at this dose of 40 mg per kilogram. Thus, we're able to show a significant effect on cyclic A MP metabolism but not on cyclic GMP metabolism. And this possibly relates to the fact that the drug has a 10 fold greater inhibition of fossil dira for cyclic amp than GP. The exact locus for the drug action is unclear. Although when we take cerebral spinal fluid from patients with amy sclerosis and allow hydrolysis to proceed at room temperature at four degrees, it proceeds very rapidly for both cyclic GMP and cyclic cyclic amp and cyclic GMP. And the presence of thain is a temperature gradient and decreased hydrolysis. For cyclic A MP but not for cyclic GMP. In summary, then we are able to show that diseases affecting the anterior horn cell are correlated with decreased cyclic amp and cyclic GMP in the cerebral spinal fluid diseases affecting the nerve accent or the muscle are not similarly correlated with changes in CSF levels. And diseases affecting upper motor neurons may actually enhance or increase the level of the cyclic nucleotide. If I may spend a moment on hypothesis and exact locus of action for some of these things. I'd like to relate the observations of doctor Engle who has shown that a Neil cycles is correlated best with the vessels in the spinal cord and is much less prominent in the actual neurons. Most of the previous data showing that there is increased adniel cyclo and cyclic nucleotides in gray matter compared to white matter has been based on dissection studies and homogenates without clear cut histo chemical localization. As you can see here, the distribution of the anterior spinal artery is such that the anterior horn and the lateral column are in the distribution of this artery. If there is some way that disease affecting the vessels may somehow affect nutrition to the anterior horns, we may have some pathological substrate for the disease, amyotrophic lateral sclerosis, the distribution is the same and the findings would correlate with our biochemical findings. It is of interest that most recently we have been able to show there is increased Norine in the spinal fluid of patients with amyotrophic lao sclerosis. And how this relates to any possible small vessel abnormality is unclear. So these metabolic studies may point the way to some pathogenesis of the disease. The exact ideology is unclear, but we must remember that viruses can attack vessels and perhaps we can tie everything together in a similar fashion to Parkinson's disease. Thank you.