HIGHER FREQUENCY OF HUMAN HERPESVIRUS-6 (HHV-6) ANTIBODY IN CEREBRAL SPINAL FLUIDS AND SERA FROM MULTIPLE SCLEROSIS PATIENTS
DV Ablashi1, 2, J Richert1, JE Whitman2, G Pearson1
1Georgetown University Medical School, Washington, DC 20007; 2Advanced Biotechnologies Inc, Columbia, MD 21046
Recently Challoner et al 1995, reported the presence of HHV-6 in plaques from patients with multiple sclerosis (MS). Based on this finding, we tested the cerebral spinal fluids (CSF) for IgG antibody to late antigen to HHV-6, Epstein-Barr Virus (EBV) and human cytomegalovirus (HCMV) by indirect immunofluorescence assay. A limited number of sera from MS patients as well as from patients with other neurological disorders were tested for IgG/IgM antibody to HHV-6 early protein (p41/38). A few of the CSF were also analyzed by PCR for HHV-6. Higher frequency of HHV-6 IgG antibody (29.2%) was detected in CSF of MS patients compared to controls (7.7%) which included patients with other neurologic disorders e.g. Guillain-Barre Syndrome, Alzheimer’s disease, diabetic neuropathy, Parkinson’s disease, leucoencephalopathy, and stroke. Out of 11 samples, one CSF sample from MS was also positive for HHV-6 DNA. Antibody to EBV-VCA was detected at a lower frequency in MS (16.7%) and Controls (7.7%). Similarly, antibody to HCMV was not detected in CSF from MS but was detected in Controls (11.5%). Five out of seven MS sera exhibited IgM antibody to p41/38, whereas five sera from Parkinson’s disease, Alzheimer’s disease, stroke and CMV neuropathy lacked IgM and IgG to p41/38 in the ELISA assay. Thus, the preliminary data support and extend the previous finding of Challoner et al. Which suggest that HHV-6 may play an important role in the pathogenesis of MS.
CORRELATION OF SCHIZOPHRENIA AND BIPOLAR DISORDER WITH CYTOKINE AND NITRIC OXIDE SYNTHASE LEVELS FROM POST-MORTEM BRAIN TISSUES
L Bobo, N Novak, N Johnston, F Leister, EF Torrey, R Yolken, the Stanley Neuropathology Consortium
The Stanley Neurovirology Laboratory, The Johns Hopkins University, Baltimore, MD 21287
Epidemiological studies suggest that infectious or immune events may play a role in the etiology of serious neuropsychiatric diseases such as schizophrenia and bipolar disease. In addition, cytokines and nitric oxide synthases (NOS) can play a pivotal role in immunoregulation, autoimmunity, inflammation, differentiation and migration during embryogenesis. We investigated the associations of serious psychiatric diseases with cytokine and NOS mRNA levels from frontal post-mortem brain tissue. The study population included n=21 with schizophrenia (SZ), n=15 with bipolar disorders (BP), and n=12 with nonpsychotic depression (NPD). Controls included n=7 with no psychiatric problems and n=6 with Huntington’s disease. cDNA amounts in samples were adjusted according to GAPdH level, and reverse transcriptase – PCR-EIA (RT-PCR-EIA) was used to measure mRNA for IL-1b , IL-1Ra, IL-2,3,4,6, TNF-a , IFN-g , TGF-b 1, CD3-d pan T cell marker, neuronal (n-), inducible (I-) and endothelial (e-) NOS. Employing analysis of variance, we found a significant elevation of IL-2 transcripts for SZ (p<0.02) and BP (p<0.001) compared to the NPD and controls. In addition, n-NOS transcripts were elevated in SZ and BP (p<0.05) compared to controls or NPD. Other cytokine and NOS transcripts did not vary significantly. Additional studies involving RNAse protection, Western blots, NOS activity and immunocytochemistry are ongoing. In conclusion, increased IL-2 mRNA in the absence of other significant cytokine alterations suggests that some individuals with serious psychiatric diseases are undergoing autoimmune or post-infectious events. PSYCHOSIS AFTER PRENATAL EXPOSURE TO RUBELLA Alan S. Brown, M.D.*, Patricia Cohen, Ph.D., Raymond Goetz, Ph.D., Ezra Susser, M.D., Dr. P.H. Columbia University We sought to examine the relation between prenatal exposure to rubella virus and psychosis in adulthood. Rubella is the first virus demonstrated to cause congenital anomalies following gestational exposure, and, over 50 years since this discovery, it remains the most frequently cited viral teratogen. Of particular relevance to psychiatric disorders, congenital rubella has prominent central nervous system manifestations. In the present study, we utilized a birth cohort of children enrolled in the Rubella Birth Defect Evaluation Project (RBDEP): all of these children were serologically documented to have been exposed to rubella virus in utero, and received a comprehensive psychiatric assessment, the Diagnostic Interview Schedule for Children, during a follow-up interview at age 22. We compared the frequency of positive responses to items on individual psychotic symptoms and the proportion of subjects meeting the A, D, and E criteria for schizophrenia between the rubella-exposed cohort (N=70) and an unexposed birth cohort (N=153), who were administered the same diagnostic interview at a comparable age. The rubella-exposed cohort had substantial and significantly increased frequencies of several psychotic symptoms, including mind reading, thought insertion, grandiose delusions, and auditory and visual hallucinations. The increased frequencies of psychotic symptoms ranged from 2 to 20-fold. The A, D, and E criteria for schizophrenia (DSM-IV) were met by 17.1% (12/70) of the rubella-exposed individuals, versus only 1.3% (12/153) of the unexposed (RR=13.1, 95% CI=3.0, 57.0). The frequencies of psychotic symptoms and of individuals meeting the A, D, and E criteria for schizophrenia did not differ between deaf and non-deaf subjects. These findings suggest that prenatal rubella exposure may be a risk factor for psychotic disorders. DNA POLYMORPHISM AND SUSCEPTIBILITY TO ENVIRONMENTALLY ACQUIRED CREUTZFELDT-JAKOB DISEASE Larisa Cervenakova Laboratory of Central Nervous System Studies, NINDS, NIH, Bethesda, MD Transmissible spongiform encephalopathy (TSE) or prion disease is a unique group of fatal neurodegenerative disorders, that are infectious and genetic in origin. The nature of the transmissible agent is controversial. Human disease may manifest as sporadic (90% of cases), familial (5-10%) and environmentally acquired cases. The last group includes kuru, that developed as a result of a ritual cannibalism in small population of highlanders in New Guinea; iatrogenic CJD resulting from transmission during different treatment procedures, and a very recently described "new variant" CJD that may have arisen from dietary exposure to bovine spongiform encephalopathy (BSE) in the United Kingdom. All of these diseases share similar neurological signs and symptoms, and pathological changes are limited to the brain, with different degrees of accumulation of an abnormal protease resistant protein (PrP). Genetic forms of TSE, inherited as discovery of amino-acid altering point and insertion mutations in the open reading frame of the PRNP gene coding for PrP, located on the short arm of chromosome 20. Common polymorphism Met129Val that occurs in the Caucasians with a frequency of 0.68 for Met allele vs. 0.32 for Val allele has been shown to play an important role in both disease susceptibility and phenotype. Remarkably, this polymorphism controls the phenotype in patients carrying a mutation at codon Asp178Asn: the mutation on the allele encoding methionine causes FFI and on the allele encoding valine causes CJD. The high prevalence of methionine homozygotes (about 90%) among sporadic CJD patients was recently established and all twelve "new variant" CJD cases were homozygous for Met Allele. Most of the iatrogenic CJD patients (90%) with intracerebral infection are also methionine homozygous. Homozygosity for either allele was observed in 88% of patients with peripheral rout of infection, and valine homozygosity was 3-fold higher in these patients compared to the normal population. The allelic distribution was similar in kuru patients (50% for each allele) compared to population controls, in which Met allele were detected in 56% and Val allele in 44%; however, homozygosity for Val allele was higher (25%) among patients compared to control individuals (14%). SUSCEPTIBILITY OF HUMAN MACROPHAGES TO CORONAVIRUS OC43 Arlene R. Collins Department of Microbiology, SUNY Buffalo, Buffalo, NY The susceptibility of adult and cord blood macrophages to human coronavirus OC43 was investigated. Adherent cells were infected at a multiplicity of 1-1.5 infectious virus per cell. After adsorption for one hour at 37ºC, cells were washed twice to remove unbound virus. In supernatant taken from both adult and fetal cells, infectious virus was detected and peaked at 2-3 days after infection at an average titer of 5+3.9 x 109 pfu/ml from seven samples. Intracellular viral nucleocapsid was detected in 15% of the adherent cells and surface staining for OC43 antigen was observed by immunocytochemistry using monoclonal antibodies specific for these viral proteins. A viral protein of ~ 80kD was detected in infected macrophages held for twelve days in culture. The susceptibility of macrophages in vitro suggests that these cells may be reservoir for persistence of the virus. INVESTIGATION OF INSERTIONAL MUTAGENESIS OF "RETROID" ELEMENTS IN THE HUMAN GENOME WITH REFERENCE TO SCHIZOPHRENIA P Deb*, TA Klempan, RL O’Reilly, SM Singh The University of Western Ontario Approximately 10% of the human genome consists of dispersed DNA sequences with homology to retroviruses. The number, sites and sequence heterogeneity of these ‘retroid’ elements is indeterminate. Most of these sequences are stable, although some retain the ability to transpose from one genomic site to another. The transposition of retroid elements is poorly understood, but has been demonstrated in a few cases of HD, NF-1 and Hemophilia. Their incorporation at different genomic sites might play a significant role in insertional mutagenesis causing disease manifestation. This project examines retroviral insertional mutagenesis as a possible cause of schizophrenia. Among the ways to study different types of mutations, randomly amplified polymorphic DNA (RAPD) and arbitrarily primed PCR (AP-PCR) are two methods of detecting polymorphisms that have been extensively used to search for genetic alterations in many organisms. By contrast, little research has been conducted using these techniques, in the analysis of polymorphisms in humans, especially diseases. In an attempt to isolate sequences that differ between genomes (schizophrenic individuals and their matched controls), towards testing the retroviral hypothesis for schizophrenia, we have used primers specific to box 4 and 5 of the pol gene. The large number of sequences that are reproducibly amplified using single and combinations of primers, represent the various priming sites present in the genome and allow the detection of quantitative and qualitative differences. One primer set has yielded a distinct polymorphism with a 344 bp, individual-specific band present in approximately 30% of the samples (normal and schizophrenic) studied. The significance of this polymorphism must be established using a larger number of samples. Further characterization of this band will answer questions such as the type of variation present in these sequences, chromosomal locations etc. Use of these approaches would be particularly useful in the analysis of monozygotic twins discordant for schizophrenia. MODULATION OF CYTOKINE EXPRESSION AND IL-2Ra RECEPTOR BY MEASLES VIRUS INFECTION OF T LYMPHOCYTES AF Bell, JB Burns & RS Fujinami* University of Utah Measles virus (MV) suppressed specific functions in cells of the immune system and causes a generalized immunosuppression by a mechanisms(S) which remains undefined. It has been previously established that mitogen induced proliferation of peripheral blood mononuclear cells (PBMC) is suppressed by infection with MV. Our current study demonstrates that MV infection inhibits antigen specific proliferation of T lymphocytes. IL-2 production in cultures of infected and uninfected antigen specific T cells was similar, indicating that IL-2 generation was not affected by MV. Further, addition of exogenous IL-2 did not overcome the suppression of proliferation induced by MV, attesting that IL-2 was not a limiting factor. In contrast, we found that the IL-2Ra subunit was decreased in mitogen stimulated, MV infected PBMC and antigen stimulated T lymphocytes. However, the IL-2 Rb subunit was not altered in MV infected T cells. We also studied the influence of MV infection on the production of the cytokines IL-4, IL-6, IL-10 and IFN-g secreted by T lymphocytes. By comparing infected versus uninfected antigen specific T cell lines, we found that MV infection of antigen specific activated T cells caused no change in IFN-g levels, a significant decrease in IL-4 production and an increase in IL-6 and IL-10. These data indicate that the immunosuppression by MV infection is not associated with a broad based pan cytokine defect. One mechanism may be a block in the expression of the IL-2Ra subunit limiting signal transduction. DETECTION OF VIRAL PARTICLES IN GLIAL CELLS INOCULATED WITH BRAIN TISSUE FROM INDIVIDUALS WITH SCHIZOPHRENIA AND BIPOLAR DISEASE I Dé, LM Melson, L Brando, R Yolken, EF Torrey, The Stanley Neuropathology Consortium Stanley Neurovirology Laboratory, The Johns Hopkins University, Baltimore, MD 21287 Tissue culture studies were used to identify any replication viral agents in brains from individuals with schizophrenia and bipolar disorder. Identification of such an agent would provide support for the theory that viruses may play an important role in the etiopathogenesis of these neuropsychiatric disorders. Material extracted from the frontal cortex of postmortem brains of four cases and one control was used to inoculate monolayers of A172, a human gliobastoma cell line. Five days post-inoculation, cells and supernatant were harvested and analyzed initially by electron microscopy for evidence of viral replication. Cells inoculated with brain extracts from the three cases showed 150-200 nm sized, enveloped virus-like particles budding from cell membranes and/or structures resembling nucleocapsids of paramyxoviruses. Such particles or structures were not seen in cells inoculated with brain tissue from the unaffected individual. Initial evaluation of the samples of PCR indicated that the viral particles were not identical to know members of the paramyxovirus family. These particles may represent novel viral agents. Further molecular and antigenic characterization of these agents will elucidate their role in the pathogenesis of schizophrenia and bipolar disease. NEONATAL INFECTION INCREASES NEUROTROPHIC FACTOR mRNA Gilmore JH*, Jarskog LF, Watson N, Xiao H Department of Psychiatry, University of North Carolina School of Medicine Exposure to infection during early brain development has been associated with increased risk of schizophrenia and other neurodevelopmental disorders. We have hypothesized that general factors related to an immune response to infection, specially cytokines, play a role in this association, as cytokines can regulate neuronal development. In an effort to understand how the immune response to infection can impact the developing brain, we examined the effect of neonatal infection on the mRNA expression of the neurotrophic factors brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3). Day 6 rats (n=6 per condition) received intraperitoneal injections of 0.0, 0.05, 0.5, or 2.5 m g of e, coli lipopolysaccaride (LPS). 4 hours after injection, rats were sacrificed and cortex and hippocampus were dissected. Changes in mRNA expression were measured in a semi-quantitative way using RT-PCR with oligonucleotide standards (MIMICS). In the hippocampus, LPS caused a significant increase in the expression of BDNF mRNA (repeated measures ANOVA, p=0.02, max effect 30%) and NT-3 mRNA (repeated measures ANOVA, p=0.03, max effect 63%). Expression of BDNF and NT-3 mRNA was not significantly changed in the cortex. Exposures to LPS causes an acute increase in expression of the neurotrophic factors BDNF and NT-3 in the hippocampus of day 6 rats. Alteration of neurotrophic factor expression may be a mechanism by which the immune response to infection can alter brain development. THE INCIDENCE AND PREVALENCE OF BIPOLAR AFFECTIVE DISORDER IN JAMAICA Frederick Hickling* and Pamela Rodgers-Johnson Psychotherapy Associates and Department of Medicine, UWI, Mona, Jamaica Objective: To establish the incidence and period prevalence of bipolar affective disorders in Jamaica. Method: Data was collected in a national mental health Case Register from the Community Mental Health Services, the Bellevue Mental Hospital and the University Hospital of the West Indies. Data from patients seen in the year 1992 with the diagnoses of manic depressive psychosis was examined. The prevalence rate for this condition was established on the basis of total population data for that year. The incidence rate for 1993 was established by identifying the total numbers of patients appearing in those years for the first time with this condition. Results: The incidence rate for manic-depressive psychosis in 1993 was 0.286 per 10,000 while the period prevalence rate for the same condition in 1992 was 0.16 per 1000, and the age corrected (15+) prevalence rate was 0.23 per 1000. Conclusions: Compared with rates of this condition from other parts of the world, the Jamaican rates were found to be significantly lower. Some possible reasons for these extremely low rates are discussed. RATES OF FLU-LIKE ILLNESS IN PATIENTS WITH AFFECTIVE DISORDERS Hornig-Rohan M§*, Amsterdam JD§,, Rybakowski J# §Depression Research Unit, University of Pennsylvania, Philadelphia, PA; #Department of Psychiatry, Medical Academy of Bydgoszcz, Bydgoszcz, Poland Background: A close association of infectious and psychiatric disorders has long been recognized. In this regard, depressed patients demonstrate immune changes which are similar to those found in infectious, chronic inflammatory, or autoimmune disease. Furthermore, evidence is accumulation that certain psychotropic drugs may influence immunomodulations and have antiviral activity that might play a role in their efficacy in neuropsychiatric disorders. We previously showed a reduced rate of oral/labial herpes (HSV) infections in depressed patients on chronic lithium therapy and subsequently showed that low dose lithium carbonate reduced rates of genital HSV-2 infections in nondepressed women with highly recurrent outbreaks. The present study explored the possibility that psychotropic drugs may influence the rates of flu-like illness. Methods: Rates of illness episodes consistent with colds or influenza were retrospectively assessed in 236 patients before and during treatment with chronic psychotropic medications: 177 who were receiving lithium carbonate (68 men, 109 women) and 59 who were taking other antidepressants (20 men, 39 women). The mean age was 46 + 13 years for the lithium group and 48 + 15 years for the group taking other antidepressants. Results: A small but highly statistically significant decrease in the mean yearly rate of colds and flu-like illnesses was reported by all patients receiving lithium (1.48 + 1.14 episodes of flu-like illnesses per year pretreatment vs. 1.14 + 1.20 episodes during treatment, p < 0.0001). Additionally, patients receiving other antidepressant drugs showed a similar but nonsignificant decrease in flu-like episode frequency (2.15 + 2.19 vs. 1.75 + 1.84, p = 0.08). There was an increasingly significant effect in conjunction with longer administration of lithium: patients on lithium for less than 5 years had a mean difference of 0.32, vs. 0.33 and 0.40 for those on lithium for 5 to 10 years or greater than 10 years, respectively. Men in the other antidepressant group had a significant decrease in illness rate during treatment (p = 0.03) while women demonstrated no significant change (p = 0.14). Conclusions: Prior work has shown immune disturbances during affective illness and its treatment. Our results suggest that psychotropic drugs such as lithium and other agents with antidepressant efficacy may possibly be associated with a decreased frequency of rhinovirus- and influenza-like infections. The data further demonstrate that the antiviral effect of lithium may become more robust over time. We speculate that psychotropic drugs may directly influence viral replication and/or exert an antiviral effect by inducing immunomodulatory substances that limit the symptoms of flu-like infections. ASSOCIATION OF HHV-6 AND MULTIPLE SCLEROSIS Rossana Berti, Samantha Soldan, Paola Sechiero, Peter Calabresi, Henry McFarland, Madumita Patnaik, Steven Jacobson NIH, Bethesda, MD; Institute of Human Virology, Baltimore, MD; Specialty Labs, Santa Monica, CA Recently, the association of human herpes virus type 6 (HHV-6) in multiple sclerosis (MS) plaques has been reported (PNAS 1995;92:7440). This was based on the immunocytochemical demonstration of HHV-6 early antigen in MS oligodendrocytes compared to more neuronal localization in certain controls. To extend these observations, we have investigated the presence of antibodies to HHV-6 antigens in serum and CSF of a well defined MS population consisting of patients with relapsing/remitting and chronic progressive neurologic disease evaluated at the clinical center at NIH. As controls, patients with other neurologic disease, autoimmune disease, and normal individuals were also screened. As a marker of active infection, the presence of HHV-6 cell-free viral DNA from MS sera was detected using a highly sensitive and specific nested PCR procedure. RESULTS: No difference was observed in the serum IgG response to HHV-6 from all groups. By contrast, preliminary evidence suggests that IgM responses from relapsing/remitting MS patients were significantly elevated to an HHV-6 early antigen. We have begun to correlate these elevated anti-HHV-6 IgM responses in MS patients with magnetic resonance imaging analysis of MS disease activity and the effect of immunotherapeutic treatment of this disorder. HHV-6 cell-free viral DNA was detected from MS patients sera compared to healthy adults and sera from patients with other neurologic disease. CONCLUSION: Collectively, these results support an association of HHV-6 in a subset of MS patients and raise the possibility that this virus and/or immune responses to this agent are involved in the pathogenesis of multiple sclerosis. COMPARISON OF cDNA LIBRARIES FROM THE BRAINS OF NORMAL AND SCHIZOPHRENIC INDIVIDUALS SHOW THAT AN UNUSUALLY HIGH NUMBER OF NOVEL SEQUENCES ARE PRESENT IN THE BRAIN OF THE SCHIZOPHRENIC INDIVIDUAL Nancy Johnston, Robert Yolken, E. Fuller Torrey, The Stanley Neuropathology Consortium The Stanley Neurovirology Laboratory, The Johns Hopkins University, Baltimore, MD 21287 The molecular basis of schizophrenia might be examined in detail through the use of cDNA libraries constructed from the RNA of human brains, obtained from cases and controls. We have constructed cDNA libraries from the frontal lobe tissue of normal schizophrenic individuals through random and dT priming of poly A+ RNA, and subsequent cloning to a l AP II vector (Stratagene). Sequencing of randomly selected clones from each primary library, as well as an equal number of clones from the amplified schizophrenic library, revealed a striking difference. In the case of the library from the normal brain, 50% of the clones were homologous to sequences of known function and 28.6% of the sequences were homologous to expressed sequence tags (EST’s) or other sequences derived from the human genome. Only 7.1% had no homologies with previously characterized sequences. In contrast, the library derived from the brain of a schizophrenic individual, had only 23% clones of known function and more than 42% clones without significant levels of homology at the nucleotide or amino acid level to know human, animal or prokaryotic sequences. Similar to the library from the normal brain, 23% of clones were homologous to EST’s and related sequences. Our studies indicate that the brains of individuals with schizophrenia may express large numbers of previously uncharacterized RNA species. These species may represent novel genetic or infectious elements. The characterization of these RNAs might lead to a greater understanding of the etiology and pathogenesis of schizophrenia. MEASUREMENT OF RNA FROM 89 POSTMORTEM HUMAN BRAINS; A MULTIVARIATE STATISTICAL ANALYSIS OF PRE- AND POST MORTEM EFFECTS ON THE YIELDS OF GAPdH AS MEASURED BY RT-PCR N Johnston, J Cervenak, R Yolken, A Shore, EF Torrey and The Stanley Neuropathology Consortium The Stanley Neurovirology Laboratory, The Johns Hopkins University, Baltimore, MD 21287 The study of mRNA from the brains of individuals with serious mental illnesses relies heavily on the use of postmortem brain tissue. Previous studies have indicated that RNA from postmortem brain is remarkably stable, but its levels are variable between individuals. To uncover the cause for this variability, we measured glyceraldehyde 3-phosphate dehydrogense (GAPdH RNA) by RT-PCR from the total RNA from 89 human occipital lobes, and compared the levels to several pre- and post mortem measures. We also looked at the effects of diagnosis, suicide, carbon monoxide poisoning, hospitalization with resuscitative efforts, and the pH of the postmortem tissue. By comparing reactions primed with oligo dT, random hexamers or GAPdH-specific primers in the RT reaction, we found that the 3’ end of the molecule was particularly sensitive to pre- and postmortem factors. We performed a multiple regression on the data to develop a model to describe the variability in RNA yields. We found pH to correlate most highly with RNA levels in all forms of priming (p<0.001 for GAP specific and oligo dT primed reactions, p<0.02 for random primed reactions), and for freezer interval (p<0.05) and suicide (p<00.01) to affect oligo dT priming. Specific priming from the 3’ end of GAPdH showed that postmortem intervals may have a minor effect on levels (p<0.08). These studies indicate that usable RNA may be purified from postmortem brain tissue that has undergone a range of pre- and postmortem conditions. The 3’ ends may be more sensitive to postmortem factors than other regions of the message, so some methods of analysis may also be differentially affected by variations in postmortem conditions. RETROVIRUS SEQUENCE AND REVERSE TRANSCRIPTASE ACTIVITY IN BRAIN TISSUE FROM AN INDIVIDUAL WITH SCHIZOPHRENIA L Jones-Brando, I Dé, R Yolken, EF Torrey, and the Stanley Neuropathology Consortium The Stanley Neurovirology Laboratory, The Johns Hopkins University, Baltimore, MD 21287 Retroviruses are plausible candidates for an etiological agent of schizophrenia. Using reverse transcriptase (RT) these viruses insert the viral genome into the host genome and then either go through a productive infection or become latent only to reappear months or years later. Our investigation found that a cell-free extract from the brain of a schizophrenic individual, whose mother experienced an acute episode of psychosis during pregnancy, had a small amount of RT activity. When human neuroblastoma cells (A172) were inoculated with this extract we found increased RT activity relative to the inoculum (normal brains negative). We infer that the RT and thus possibly a retrovirus, had been amplified by the cell culture passage thereby indicating the presence of an infectious retrovirus in the original brain extract. Further the supernatant from PBL’s inoculated with the schizophrenic brain extract contained a sequence amplified by PCR primers to retrovirus polymerase genes. The sequence of the amplicon was highly homologous to a known endogenous retrovirus. Our data indicate that this individual may have had a latent retrovirus infection of the brain. We propose that such an infection may have been instrumental in the development of schizophrenia. PREVALENCE OF BORNA DISEASE VIRUS RNA IN PERIPHERAL BLOOD MONONUCLEAR CELLS FROM PATIENTS WITH AFFECTIVE DISORDERS M. Kishi1*, T. Izumi2, Y. Shoya1, T. Kobayashi1, T. Koda1, P. K. Lai2, T. Koyama3, K. Ikuta4, and M. Kakinuma1 Sections of 1Bacterial Infection and 4Serology, Institute of Immunological Science, and 3Department of Psychiatry, School of Medicine, Hokkaido University, Sapporo, Japan, 2Molecular Biology and Biotechnology Program, Salem-Teikyo University, Salem, West Virginia, USA Purpose: Borna disease virus (BDV) is as yet an unclassified agent which causes a neurologic disease in horses and sheep. Serological evidence suggested an association of BDV or related agent with human psychiatric diseases, in particular, major depression. However, prevalence of BDV RNA in patients with major depression has never been examined. Here we show prevalence of BDV RNA in depressed inpatients. Methods: A nested RT-PCR was performed to detect BDV RNA (p24) in total RNA of peripheral blood mononuclear cells (PBMC) from 25 inpatients with affective disorders (19 unipolar disorder-patients and 6 bipolar disorder-patients). Plasma antibodies to BDV p24 antigen was detected by Western blot analysis using a GST-BDV p24 fusion protein as test antigen. Results: Among the 25 patients with affective disorders, BDV RNA carrier was found at high rate (28%), as compared to that (4.65%) in 172 blood donors. Seroprevalence to BDV p24 was 44%, which was higher than that (1%) in 100 blood donors. Of interest, a significantly higher prevalence of BDV RNA was found in patients with unipolar disorders who had relatively shorter clinical histories after the first episode. Furthermore, BDV RNA was found at significantly high rate in the PBMC of patients with relatively severe depressive episode. Conclusion: Our study demonstrated high prevalence of BDV RNA in PBMC from inpatients with major depression, suggesting a possible correlation between BDV and major depression in humans. THE APPLICATION AND ADAPTATION OF REPRESENTATIONAL DIFFERENCE ANALYSIS IN THE EVALUATION OF RETROVIRAL HYPOTHESIS FOR SCHIZOPHRENIA TA Klempan*, P Deb, RL O’Reilly, SM Singh The University of Western Ontario Concordance between monozygotic twins (44%), dizygotic twins (17%), full siblings (95) and the parents (6%) and offspring (13%) of schizophrenic individuals demonstrate a significant heritable and environmental component to this disease. Such familial transmission and variations in relative risk have long argued for a complex genetic etiology to schizophrenia. Insertional mutagenesis involving retroviral sequences and disruption of a neurodevelopmental gene(s), as postulated by Crow (1984), provide a possible explanation for these findings. This hypothesis remains largely untested due to multiple possible mechanisms for action such as an infective cycle, genomic integration of retroviral DNA (random or directed), or germ-line transmission of retroid elements. Representational difference analysis (RDA) is a powerful molecular DNA-based methodology capable of distinguishing small differences between complex genomes. Subtractive hybridization and kinetic enrichment through exponential PCR amplification of unique self re-annealed sequences form the basis for this technique. We have examined the potential of this technique to isolate individual-specific retroviral sequences that may be associated with schizophrenia. Driver and tester populations (amplicons) constitute a segment of the retroviral reverse transcriptase (Pol) amplified from genomic DNA’s from unrelated individuals using degenerate primers. The extensive individual-specific heterogeneity seen with these sequences necessitates the use of discordant monozygotic twins for application of RDA in the evaluation of the retroviral hypothesis of schizophrenia. Preliminary results show a considerable difference in the amplification efficiency between ligated and unligated samples and temperature dependence on final outcome. Critical parameters expected to affect the identification of a singular tester sequence include: ratio of driver to tester during hybridization, efficient ligation of linkers, purity, stringency and specificity of PCR amplification, and total rounds of RDA carried out. Continual evaluation of these factors are essential in any application of RDA in the search for retroviral sequences associated with Schizophrenia, Multiple Sclerosis, and other neurological diseases. IS ACTIVE BORNA DISEASE VIRUS INFECTION REALLY A CONTRIBUTOR TO HUMAN PSYCHIATRIC DISORDERS? D. Craig Hooper, Ph.D. and Hilary Koprowski, M.D. Center for Neurovirology, Department of Microbiology and Immunology, Thomas Jefferson University, Philadelphia, PA The presence of antibodies reactive with Borna disease virus (BDV) in the sera of some psychiatric patients has been interpreted as indicating that infection with this veterinary neurotrophic virus may cause psychiatric disorders in humans. We report here the results of our attempts to correlate the presence of BDV-reactive antibodies with evidence of BDV-specific RNA in blood cells from patients with psychiatric diseases. Two centers, one in Germany and one in the USA, were involved in this study and aliquots of the human material to be assayed for BDV-RNA were tested independently in both countries in at least two different classes of laboratories: one routinely involved in BDV work and a second without prior exposure to BDV. We obtained positive results for the presence of BDV-RNA subsequent analysis of replicate samples in other laboratories where there had been no prior work on BDV yielded negative results. We conclude that contamination may seriously influence the outcome of studies using RT-PCR methodology to probe for BDV-sequences in PBLs of psychiatric patients, and stringent criteria must be met to control against false positive results. Ongoing or prior work with BDV sequences in the assay environment, together with the exquisite sensitivity of RT-PCR, may account for the sporadic appearance of false positive evidence that BDV-specific RNA is present in human blood cells. Contrary to the results obtained by others, we have found no evidence for the presence of BDV-specific RNA in such cells. TRYPANOSOMA BRUCEI DYSREGULATES THE MAMMALIAN CIRCADIAN CLOCK J. Christensson, G. Lundkvist, Z.-P. Peng, M. Bentivoglio, K. Kristensson* Department of Neuroscience, Karolinska Institutet, Sweden; Department of Anatomy, University of Verona, Italy The extracellular parasite Trypanosoma brucei causes African sleeping sickness, a severe neuropsychiatric disease with marked disturbances of sleep-wake alternation. Electrophysiological properties of the hypothalamic suprachiasmatic nuclei, which play a role as circadian oscillators entraining biological rhythms, were analyzed in slice preparations from trypanosome-infected rats. The spontaneous activity of suprachiasmatic nuclei neurons was markedly altered, displaying a reduced firing rate and a phase-advance of its peak during 24 h. Tract tracing showed normal organization of the retino-hypothalamic nerve fibers terminating in the suprachiasmatic nuclei, while immunohistochemistry revealed a diminished expression of glutamate AMPA glu R2/3 and NMDA R1 receptors that gate retinal afferents in the suprachiasmatic nuclei. Thus, molecules released by trypanosomes, and/or by the host in response to the infection, affect the circadian rhythmicity and excitatory neurotransmission in the biological clock. Together, these findings provide the first direct, unequivocal evidence of functional disturbances in the biological clock in African trypanosomiasis and provide a model to unravel functional and molecular mechanisms behind dysregulation of circadian oscillator. INSTABILITY OF SCMV-DERIVED STEALTH VIRAL GENOME W. John Martin Center for Complex Infectious Diseases, Rosemead, CA Atypical cytopathic (stealth) viruses have been cultured from blood, cerebrospinal fluid and brain biopsies of patients with a spectrum of neuropsychiatric dysfunctional brain syndromes. Genetic sequence analyses of the stealth viruses isolated from a patient with chronic fatigue syndrome (CFS) and from a patient with severe encephalopathy following a bipolar psychotic illness, indicate that they were probably both derived from African green monkey simian cytomegalovirus (SCMV). Additional sequencing of multiple EcoRI and SACI clones of the virus isolated from the CFS patient confirms that it has undergone significant genetic changes from SCMV including point mutations, deletions, recombinations and duplications. These changes result in a fragmented viral genome which shows marked microheterogeneity. Genetic diversification is consistent with impaired fidelity of viral replication. Mutations and/or loss of genes coding viruses generally evoke little or no inflammatory reactions within the parenchyma of the brain. Furthermore, the production of mutated, inhibitory viral products offers a potential explanation for the clinical recovery seen in several patients with stealth viral encephalopathy and in animals inoculated with these viruses. CYTOLOGICAL IMMUNITY AND SCHIZOPHRENIA. THREE TRAILS OF ABNORMALITY IN THE CSF Nikkila H*, Müller K, Ahokas A, Miettinen K, Rimón R, Andersson LC Section of Clinical Neurosciences, Institute of Occupational Health, Topeliuksenkatu 41 aA, 00250 Helsinki, Finland There are several reports on organic/structural alterations in the central nervous system (CNS) of patients with schizophrenia. Given that pathological conditions in the CNS are frequently reflected in the cerebrospinal fluid (CSF), we analyzed the lymphocyte distributions and mononuclear cell cytology of the CSF from patients with acute untreated schizophrenia. The CSF findings of the patient group were in many respects aberrant as compared to healthy controls. Abnormalities in the distribution of T lymphocyte subsets were detected in the majority of schizophrenic patients (23 out of 31). The cytological examination revealed significantly (p<0.001) elevated proportions of macrophages (microglia derived?), and the appearance of morphologically distinct atypical lymphocytes in the CSF from schizophrenic patient group. The dominant cell type in the CSF of schizophrenic patients, the monocyte, was established in pleomorphic maturational states: from juvenile mononuclear phagocytes with quite compact nucleus and homogeneous cytoplasm to mature amoebic macrophages with irregular nuclear shape and a voluminous cytoplasm with numerous small vacuoles. The morphological features of atypical lymphocytes comprised of an irregularly stained nucleus with sporadic intendations breaking the contour of nuclear wall, and a strongly basophilic staining of the cytoplasm. The findings of this study point out, that in the CSF of schizophrenic patients there is a remarkable deviation of cell profile, that may have reference to some active degenerative and/or immunological process in the CNS of schizophrenic patients. MEASLES VIRUS INVASION THROUGH LIMBIC STRUCTURES IN MICE WITH DISRUPTED GENE FOR THE TRANSPORTER ASSOCIATED WITH ANTIGEN PRESENTATION E Norrby, E Urbanska, BJ Chambers, H-G Ljunggren, and K Kristensson Microbiology and Tumor Biology Center and Department of Neurosciences, Karolinska Institute In its natural host, measles virus can give three different forms of infections in the central nervous system. These are acute progressive infectious encephalitis, acute postinfectious encephalitis and subacute sclerosing panencephalitis (SSPE). The acute progressive form of brain disease, also referred to as inclusion body encephalitis, reflects a direct attack by the virus under conditions of yielding cell-bound immunity. The post-infectious acute disease is interpreted to reflect an autoimmune reaction, the nature of which has been poorly studied. The late progressive form of encephalitis has been extensively analyzed. Recent molecular genetic studies have unraveled a range of mechanisms by which a defective expression of either the matrix (M), the fusion (F) or the hemagglutinin (H) proteins may lead to viral persistence in brain cells under conditions not allowing identification by immune surveillance mechanisms. Rodents have been used to establish experimental systems in which phenomena, pivotal to the evolution of acute as well as persistent measles virus infection in the brain, can be studied. A wide range of potentially important mechanisms have been highlighted and will be discussed. More recently mice with genetic defects in immune functions were used to evaluate consequences as to initiation and dissemination of infection in the brain. Recent studies have demonstrated that intranasally administered measles virus in TAPI-/- mice leas to an accentuated spread of virus in the brain, emphasizing the role of cell-mediated immune responses in containment of virus dissemination, of axonal spread of virus and effects of targeting of virus infections to the limbic system and monoaminogic neurons and ensuing symptomatic consequences. THE IMMUNE RESPONSE IN THE DISRUPTION OF HIPPOCAMPAL GABA CIRCUITS FOLLOWING NEONATAL INFECTION WITH LYMPHOCYTIC CHORIOMENINGITIS VIRUS (LCMV) BD Pearce*, C Po, TL Pisell, AH Miller Emory University School of Medicine, Atlanta, GA 30322 Accumulating evidence suggests that viral infections during development may be responsible for the neuropathological alterations in schizophrenia. Recent postmortem studies have focused on the loss of cortical hippocampal GABAergic interneurons. The relationship between these neuropathological findings, and the cellular and immunological events engendered by perinatal viral infections have not been defined. An understanding of these mechanisms through human studies are hampered because schizophrenia may be manifested only after the causative virus is cleared, and the neuropathogenic sequence is complete. Thus we have turned to an animal model, using neonatal infection of rats with LCMV, to investigate possible mechanisms. Our previous studies suggested this infection caused a loss of inhibitory interneurons in the hippocampal dentate gyrus, and resulted in the gradual death of dentate granule cells. To examine the role of GABAergic interneurons in this process, we immunostained for LCMV and a GABA cell marker (GAD). GABAergic interneurons co-staining for LCMV were detected in the dentate gyrus of rats 12 days after neonatal infection. This supports the hypothesis that the disruption of inhibitory circuits may occur early, and could initiate a pathological cascade in which dentate granule cells die because of unbalanced excitatory neurotransmission. We also investigated the contribution of T-cells to the loss of dentate granule cells by treating rats with anti-T-cell serum during the LCMV infection. Rats (90 days p.i.) were protected from cerebellar but not hippocampal cell loss suggesting that an early T-cell-mediated attack of infected hippocampal neurons cannot explain the hippocampal pathology. Conversely, local production of interleukin-1, which we detected in glia at 12 days p.i., may disrupt GABAergic circuits indirectly. Developing GABAergic interneurons are dependent on brain derived neurotrophic factor, which is known to be inhibited by IL-1. Supported by the Theodore and Vada Stanley Foundation. NEURAL DEVELOPMENT PATTERNS IN RATS INFECTED WITH BORNA DISEASE VIRUS AS NEONATES MC Zinc, JM Pyper* Johns Hopkins University School of Medicine, Division of Comparative Medicine Borna disease virus (BDV) causes severe neurological disease in several species (e.g., horses, sheep, cattle, and cats). Experimental infections lead to disease of varying severity in additional species, including rats and primates. Infected tree shrews (Tupaia glis) exhibit subtle behavioral deficits, including altered social interactions. BDV is currently being intensively studied as a potential etiologic agent for schizophrenia both because of the nature of clinical disease in animal models and because of some immunological and PCR data suggesting a possible involvement of BDV in human disease. There are two forms of disease in the rat model system. Infection of immunocompetent rats leads to a massive immunopathological response in which infected neurons are destroyed. In contrast, infection of neonates leads to a persistent tolerant infection (PTI) without immunopathological damage to neurons. Previous work has shown that these rats show subtle behavioral deficits and retarded growth compared to uninfected controls. PTI animals lose neurons in the dentate gyrus of the hippocampus, presumably as a direct result of the infection since there are no inflammatory lesions. We undertook this study to examine whether there are more subtle neurological changes and whether normal neurological developmental patterns are altered. One day old rats were infected by intranasal inoculation. Pairs of rats were sacrificed every 3-4 days for 6 weeks following infection. BDV RNA and antigen expression, infectious virus titers, and anti-BDV antibody titers were monitored during the study. During the course of infection we observed progressive changes in the neuroanatomy of the PTI animals. At 11 days p.i. we observed hyperplastic and activated astrocytes in the brain. Hippocampal abnormalities were apparent by 2 1/2 weeks p.i. The neurons of the dentate gyrus lost their regular organization and began to decrease in number. By 4 weeks the dentate gyrus was nearly gone and the cerebral cortex was reduced in thickness by 40%. Our observations of neuroanatomical changes in the brains of PTI rats suggest a physical basis for the behavioral deficits that have been described for these animals. EVIDENCE OF DIFFERENCES IN IMMUNE FUNCTION IN OLDER AND YOUNGER SCHIZOPHRENIC PATIENTS: IS THIS AGE OR A FUNCTION OF SEVERITY OF ILLNESS MH Rapaport, MD*, Dilip Jeste, MD Department of Psychiatry UCSD and Psychiatric Service San Diego Veteran’s Affairs Medical Center There is diverse but convincing evidence that changes in immune function may be associated with certain individuals with schizophrenia. There are a variety of mechanisms for investigating the mean function of schizophrenia, two of the most commonly used ones are measurements of peripheral cytokine levels and measurements of phenotypic lymphocyte markers. Our group has consistently found elevations in serum cytokine levels and in particular soluble interleukin-2 receptor levels (SIL-2Rs). In this submission we present data investigating SIL-2R and phenotypic lymphocyte surface marker levels in older schizophrenic patients and matched controls. We were unable to demonstrate differences from matched normal volunteers in serum SIL-2Rs for older schizophrenic patients (370(230) U/ml vs 346 (239) U/ml; p=ns). We did demonstrate interesting shifts in peripheral phenotype markers in older schizophrenic patients with decreased numbers of CD3 and CD19 bearing cells. We will discuss our findings in the context of whether we are seeing an age effect or whether these differences represent differences due to differences in severity of illness within these populations. This is the first study to carefully investigate immune changes in older schizophrenic patients. A PILOT STUDY OF CYTOKINE AND SOLUBLE INTERLEUKIN-2 RECEPTOR LEVELS IN SYMPTOMATIC BIPOLAR PATIENTS MH Rapaport, MD*, L Guylai, MD Department of Psychiatry UCSD and University of Pennsylvania, USA There have been case reports and small studies suggesting that immune activation is associated with acute mania. Our group has investigated immune function in euthymic unmedicated and medicated bipolar patients and found that immune activation or depression are not trait markers of bipolar illness. We have also demonstrated that lithium carbonate may have mild in vivo immune stimulating effects in normal volunteers. This study investigated peripheral markers of immune activation in unmedicated bipolar patients and then reexamine those markers of immune activation after acute treatment. At a Time 1 serum SIL-2R levels were 808 (533) U/ml for the unmedicated patients and 648.8 (495.6) U/ml for the controls (p=ns). At time 2 the serum SIL-2R levels were 753.5 (476.1) U/ml for the medicated patients and 849 (410) U/ml for the controls (p=ns). Preliminary analyses demonstrate normal levels of serum soluble interleukin-2 receptors in patients before and after treatment. We will present data on a larger sample of subjects, of other cytokines, and correlations with clinical ratings. If these preliminary findings are correct, then immune activation associated previously found in smaller studies may be an epiphenomenon of stress. GENETICS OF HUNTINGTON’S DISEASE: LESSONS FOR PSYCHIATRY Christopher Ross The Johns Hopkins University School of Medicine, Baltimore, Maryland Huntington’s Disease (HD) has been considered a classic Mendelian disorder, with autosomal dominant transmission and complete penetrance. Positional cloning strategies were developed in part by the groups searching for the HD gene. The identification of the gene which causes HD, IT-15 or huntingtin, has revealed additional complexities. The mutation is an expanded CAG repeat. HD, at least in retrospect, shows clear anticipation. The age of onset in HD is strongly correlated with the length of the triplet repeat, though other uncharacterized influences clearly operate. Both new mutations and incomplete penetrance (which formerly were thought not to exist) are present, though fairly rare. Finally, in occasional cases, other genes can cause an HD-like phenotype. One of these, DRPLA, is also caused by an expanding CAG repeat. At least some subtypes of major psychiatric disorders have a strong genetic component. This observation is fully compatible with the viral hypothesis-there could be several independent subtypes of illness, or more interestingly, genes could influence viral infectivity or pathogeneity. However, the positional cloning approach may be even more difficult to apply than in HD because of difficulty in specifying phenotypes, genetic heterogeneity, and likely incomplete penetrance. A complementary approach to finding disease genes by positional cloning is the identification of candidate genes based on hypotheses about pathogenesis. We have proposed that triplet repeats may be candidates for some forms of psychiatric disorders based on the observation of anticipation. Additional candidate gene approaches could arise from other hypotheses such as the neurodevelopmental hypothesis for the pathogenesis for schizophrenia. VIROLOGICAL ASPECTS OF NEUROPATHOGENESIS OF HIV-1 DISEASE Shizuko Sei* and Philip A. Pizzo National Cancer Institute, National Institutes of Health, Bethesda, MD Human Immunodeficiency virus type 1 (HIV-1) is often detected in brain tissues and cerebrospinal fluid (CSF) obtained from patients with AIDS, especially those with HIV-encephalopathy. To examine whether the level of viral burden was important determinant for the progression of HIV-encephalopathy, we evaluated the amount of HIV-1 DNA in brain tissues, HIV-1 RNA and the presence of drug-resistant HIV-1 variants in the CSF and plasma obtained from HIV-1 infected individuals with or without HIV-encephalopathy. The brains from patients with HIV-encephalopathy (n=8) not only contained higher levels of HIV-1 DNA assessed by quantitative polymerase chain reaction (PCR) methodology (cerebrum: P<.01, cerebellum: P<0.05), but also showed higher rate of viral pol gene mutations suggestive of zidovudine or didanosine resistance than brains from patients who had no neurological abnormalities (n=5) or documented central nervous system (CNS) opportunistic conditions (n=7) (P<001). The levels of HIV-1 RNA were evaluated in CSF and plasma obtained from 41 HIV-1 infected children, ranging in age from 6 months to 12 years (median 2.2 years old). Children with acute onset of neurological symptoms were excluded from the study. 29 of 41 patients had been receiving zidovudine, as a single drug (n=21) or in combination (n=8), for more than 6 weeks at the time of lumbar puncture (LP). Three patients were naive to any antiretroviral treatment. Each patient’s CNS status at the lime of LP was classified by the age-appropriate neuropsychometric testing to either severely encephalopathic (n=25), moderately encephalopathic (n=7), or encephalopathy (n=9). Viral RNA obtained from CSF or plasma was subjected to a reverse transcription followed by documented mutations for zidovudine resistance. Children with severe encephalopathy had the highest level of HIV-1 RNA in CSF (median: 430 copies/ml, range: 0~2.2 x 105 copies/ml) followed by moderately encephalopathic (median: 330, range: 0~1130) and non-encephalopathic group (median: 0, range: 0~566) (P=0.007). There was no correlation between CSF and plasma HIV-1 RNA levels. Higher levels of CSF HIV-1 RNA were associated with more severe cortical atrophy by computed tomographic brain scan (P=0.008). Treatment with zidovudine was in general associated with lower levels of CSF HIV-1 RNA as compared to other drugs or no treatment (P<0.005). All fifteen paired CSF/plasma specimens had consistent codon 215 types. Among the children who received zidovudine, 5 of 7 with codon 215 mutation in CSF had a progression of encephalopathy despite therapy, while all 8 children with wild type codon 215 had an improved or stable disease (P=0.007). Increased level of viral burden within CNS appears to be linked to the development of HIV-encephalopathy. Emergence of drug-resistant HIV-1 variants, which could permit an increased viral replication despite therapy, may contribute to a poor neurologic outcome during the antiretroviral treatment. Our data may also underscore the importance of therapeutic strategy that includes potent antiretroviral agents with a good CNS penetration for HIV-infected patients with encephalopathy. ANTIBODIES TO NEURAL TISSUE PROTEINS IN SCHIZOPHRENIC PATIENTS Pinkhas Sirota*, Doron Mazeh, Abraham Novogrodsky Abarbanel Mental Health Center, Bat-Yam, Felsenstein Medical Research Institute, Rabin Medical Center, Petah Tiqwa, Sackler school of Medicine, Tel Aviv University, Tel Aviv Israel Different immunological abnormalities were reported in schizophrenia and major depression. Heat shock proteins are produced in every celltype in reaction to different stressors. They are found in cells in normal conditions in minor quantities. In autoimmune diseases they were found in large quantities. Kilidries et al. Reported an increase of hsp60 antibodies in the serum of patients with schizophrenia. The serum of 26 patients with schizophrenia, 8 patients with major depression and 22 normal subjects, were tested for the presence of hsp antibodies. The serum samples were tested for antibody binding to protein extracts of 1.M.R-32 neuroblastoma cell line on Western Blots. In Western Blots IgG in the Serum of all patients but one, and of normal subjects, reacted with a protein of 60 kDa. The intensity of each band in the 60 kDa region was quantified in a scale of four groups. No significant differences were found among the three groups. On the other hand, IgG of 8 patients with schizophrenia (30.71%) formed a band in the region of 85kDa. This band was not formed with the serum of other groups. Hsp 60 kDa is an antigen of many pathogens and antibodies against it can be a result of an infection and can thus not be a good indicator of an autoimmune process. The presence of antibodies against Hsp 90 kDa might be a much more specific indicator. DETECTION AND QUANTITATION OF GENOMIC DIFFERENCES IN MICROSATELLITES OF MONOZYGOTIC TWINS Natalia E. Broude1, Sergei A. Lukyanov2, Joel H. Graber1, Eugene D. Sverdlov1, 2 and Cassandra L. Smith1* 1Center for Advanced Biotechnology and Departments of Biomedical Engineering, Biology and Pharmacology, Boston University, Boston, MA 02215. 2Shemyakin-Ovchinnikov Institute for Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia. Identical twins are the most genetically similar systems in humans. However, they do not have identical genomes. Most twins discordance studies have focused on a particular gene, chromosome, phenotypic trait or stochastic epigenetic factors. A technique which could search for differences en masse between similar genetic systems would be highly desirable. Recently we described a technique which uses a solid phase capture procedure to display many repeat-containing sequences simultaneously . Here a simple and enhanced technique for differential display of repeat-containing sequences is described. The technique takes advantage of suppression PCR. It involves cleavage of genomic DNA with a restriction enzyme and ligation of resulting restriction fragments with 40-nucleotide adaptors, which allows subsequent amplification and labeling of fragments by nested PCR. Fluorescein labeled PCR products are fractionated on a denaturing polyacrilamid gel using automatic DNA sequencing instrument (Pharmacia). Sequence analysis of the PCR product showed that the technique allows the amplification of the repeat, or at lease its portion and one of the flanking sequences. The method was applied to the analysis of differences between monozygotic twins. We focus on trinucleotide repeats known to expand in several human neurological diseases. A preliminary quantitation of differences showed that relative variation between co-twins is 0.13 to 0.17, while differences between pairs are much higher (relative variation 0.5 - 2.6). The differences between co-twins have been cloned and are currently being characterized. DO THE ANATOMY AND COURSE OF SCHIZOPHRENIA ASSIST IN THE SEARCH FOR ETIOLOGY? Janice R. Stevens, MD Oregon Health Sciences University Although introduction of increasingly specific criteria for diagnosis of schizophrenia has improved the reliability of diagnosis between raters it has not greatly decreased the variability of morphologic, pathologic and neurochemical changes reported between various studies. In fact, the larger the study, the greater the variability in the anatomical findings and overlap with controls. Ventricular enlargement, diffuse or focal neocortical atrophy, smaller size of mesial temporal structures or of specific subcortical nuclei and a host of unusual histologic findings have been reported in the brains of varying numbers of patients diagnosed with schizophrenia. None of these findings is, however, ubiquitous in, necessary or restricted to, nor sufficient for the diagnosis of schizophrenia. This suggests that schizophrenia may be, like epilepsy, a response to a number of different causes, only certain types of which are associated with specific morphologic or other pathologic changes. Current DSM, ICD and positive-negative classification systems have not succeeded in sorting out these different pathologies. I propose two new strategies to disaggregate some of the reported clinical and morphologic findings by: replacing the traditional schizophrenic subtypes with subdivision by course or trajectory of illness and by correlation of outliers in the distribution of morphologic and other pathologic findings with clinical and demographic data. These proposals may avoid some of the overlap of morphologic and other biologic measures present in comparisons of pooled average data from schizophrenics of different course and, thus perhaps of varying etiology. SERIAL ANALYSIS OF GENE EXPRESSION OF BRAIN TISSUE FROM SCHIZOPHRENIA PATIENT Y Sun, RH Yolken, the Stanley Neuropathology Consortium Stanley Neurovirology Laboratory, The Johns Hopkins University School of Medicine, Baltimore, MD 21287 Schizophrenia is a clinically heterogeneous syndrome of unknown etiology. Both clinical and basic research data point to possible structural and functional abnormalities within the central nervous system. From a molecular point of view it is hypothesized that a perturbation of normally well-regulated gene expression may be associated with the pathogenesis of schizophrenia. Many factors including virus infection and immune response are known to influence gene expression of the host resulting in a variety of disorders. To test this hypothesis, we employed the technique of serial analysis of gene expression (SAGE) to analyze postmortem tissue from brains of schizophrenia patients and controls. Messenger RNA was extracted from human brain and converted to cDNA by reverse transcriptase. The cDNA was then digested with a selected restriction enzyme so that a pool of genomic fragments with the same end were generated and ligated to a pair of linkers. The unique linker contains a recognition site for a type II restriction enzyme which exerts its enzymatic activity at a distance of 9-base pair from the recognition site. A short fragment of nucleic acid was thus generated form each genomic fragment. These 9-base-pair fragments were ligated and amplified by PCR. The PCR products were then ligated by T4 ligase to form concatemers, cloned into plasmids, sequenced. The sequence information was analyzed using a computerized program which utilizes data base information extracted from the GenBank. Genes that are abnormally expressed are identified and analyzed by cDNA library screening. Our preliminary results indicate that this method can be utilized to identify novel brain sequences and to obtain a quantitative representation of gene expression. ANALYSIS OF GENOMIC DNA FROM MONOZYGOTIC TWINS DISCORDANT FOR SCHIZOPHRENIA Yeping Sun, Jhy-Jhu Lin, Shuojia Li, Flora Leister, Robert Yolken, The Stanley Neuropathology Consortium Stanley Neurovirology Laboratory, Johns Hopkins University, Baltimore, MD It is postulated that interaction of genetic components and environmental factors play a role in the etiology of schizophrenia and related disorders. The pathogenesis may involve changes in the genes occurring as a result of either genetic inheritance or viral integration. Identical twins discordant for these disorders provide the opportunity for direct assessment of this hypothesis. We applied the technique of representational difference analysis (RDA) and restriction fragment length polymorphism (RFLP) to analyze differences in genomic DNA obtained from lymphocytes of monozygotic twins discordant for schizophrenia or bipolar disorder. Restriction fragments of DNA from 12 different primer combinations in the RFLP and representations of 3 different restriction endonucleases for the RNA showed polymorphism among the twin pairs. Screening of different products isolated from RDA resulted in the identification of sequences highly homologous to a range of known human structural and functional genes as well as genes of unknown identity. However, it has been difficult to identify polymorphic bands that consistently differentiate the affected and unaffected twins. The result of RDA and RFLP suggests that the difference in genomic DNA of affected and unaffected twins, if any, may have occurred as a result of point mutation, deletion or insertion of nucleotides that are too small to be detected by RDA or RFLP. PANDAS: IS THERE A NEW "SPECIES" OF PEDIATRIC NEUROPSYCHIATRIC DISORDERS? S Swedo, H Leonard, M Garvey, B Mittleman Section on Behavioral Pediatrics, Child Psychiatry Branch, NIMH The first case of strep-related autoimmune neuropsychiatric symptoms were actually described by Sir William Osler over 100 years ago, but his observations were virtually ignored until the late 1980’s when we began a series of investigations to test the hypothesis that some children with neuropsychiatric symptoms have exacerbations related to strep-triggered autoimmune dysfunction, in a manner similar to Sydenham’s chorea, a variant of rheumatic fever1. Longitudinal observations revealed that a subgroup of children was distinguished by the following criteria: 1) presence of OCD and/or a tic disorder; 2) pediatric onset; 3) episodic course of symptom severity; 4) association with group-A b -hemolytic strep. (GABHS) infection; and 5) association with neurological abnormalities. These patients are now identified by the acronym PANDAS (Pediatric Neuropsychiatric Disorders Associated with Streptococcal infections). We will describe the clinical presentation of the first 50 children with PANDAS, including their young age at onset and presentation, high rates of psychiatric co-morbidity, and details of their clinical course. Results will also be presented for D8/17 measurements - D8/17 is a biological marker which is known to identify rheumatic fever susceptibilities and appears to identify PANDAS patients, as well2. These clinical and biological findings will then be discussed in terms of a possible pathophysiologic model for PANDAS. References: 1) Swedo SE. Sydenham’s chorea: A model for childhood autoimmune neuropsychiatric disorders. JAMA 1994;272(22):1788-1791. 2) Swedo SE, Leonard HL, Mittleman BB, Allen AJ, Rapoport JL, Dow SP, Kanter ME, Chapman F, Zabriskie J. Children with PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Strep. Infections) are identified by a marker associated with rheumatic fever. American Journal of Psychiatry, in press. BORNA DISEASE VIRUS IN HUMANS Daniel Gonzalez-Dunia, Christian Sauder, Beatrice Cubitt, and Juan C. De la Torre* Department of Neuropharmacology, Division of Virology, The Scripps Research Institute Borna disease virus (BDV), the prototype of a new group of animal viruses, causes central nervous system (CNS) disease in several vertebrate species accompanied with diverse behavioral abnormalities. Seroepidemiological data has suggested an association of BDV with certain human mental disorders. This hypothesis has been further supported by the detection of both BDV antigen and RNA in peripheral blood mononuclear cells (PBMC) of patients with psychiatric disorders. We have conducted serological and molecular epidemiological studies on psychiatric patients from the area of Homburg (Germany). We found a BDV seroprevalence of 9.6% among 416 neuropsychiatric patients which is significantly higher than the 1.4% found among 203 healthy control individuals. RT-PCR analysis of RNA extracted from PBMCs of a subset of 26 of the neuropsychiatric patients revealed 50% BDV RNA positives. BDV p40 and p24 sequences derived from human PBMCs exhibited both a high degree of inter- and intrapatient conservation and a close genetic relationship to animal derived BDV sequences. Recently, using co-cultivation procedures we have isolated infectious BDV derived from PBMC of three hospitalized psychiatric patients, two with major depressive illness and one with chronic mental disorders. Molecular characterization revealed that human BDV isolates displayed a high degree of sequence conservation with respect to BDV isolates from naturally infected animals of different species. With the hypothesis that BDV could contribute to the pathophysiology of certain human mental disorders, it would be reasonable to predict the presence of markers of BDV in the CNS of such patients. Therefore, we searched for human brain autopsy cases with histopathlogical features resembling those of BDV infection in animals. Five out of 600 USA cases examined were identified as having hippocampus sclerosis and astrocytosis characteristically observed in BDV-infected animals. Using immunocytochemistry, RT-PCR and in situ hybridization, we detected both BDV antigen and RNA in autopsy brain samples from four of these five patients, who presented with a clinical history of mental disorders involving memory loss and depression. Our results provide strong support of the hypothesis that BDV is associated with certain human neuropsychiatric disorders. More comprehensive studies are warranted to rigorously evaluate a possible contribution of BDV to the pathophysiology of human mental disorders. THE ROLE OF IMMUNE MEASURES IN PSYCHOSIS AND STRESS SENSITIVITY IN SCHIZOPHRENIA van Kammen, DP*, McAllister CG, Kelley ME, Mathé AA, Brown WA, Yao JK, Gurklis JA Department of Veterans Affairs Medical Center, Pittsburgh, PA Recent studies have raised the issue as to whether the reported immune disturbances in schizophrenia may be the result of a stress induced dysregulation of CNS cytokine production, rather than a permanent immunological disorder, I.E., autoimmunity. Our group has previously shown that the cytokine interleukin-2 (IL-2), but not interleukin-1 (IL-1), in the CSF was elevated in haloperidol treated patients who relapsed within 6 weeks following drug withdrawal. This indicated a possible stress sensitivity in the relapsed patients which could be identified using CSF immune measures. IL-2 is produced primarily by the Th1 subset of CD4+ T lymphocytes, which mediate cellular immune responses, i.e. those that initiate an autoimmune-type reaction to environmental insults. In contrast, the primary mechanism of action for Th2 cells in the production of antibodies. However cytokines have also been shown to be produced by cells within the CNS including microglia and astrocytes. Thus, they may have more direct effects on the brain than previously thought. In an attempt to uncover evidence of a direct effect of the immune system on brain function, we first looked for associations with both Th1 (IL-2) and Th2 (IL-10, TGFb ) cytokines with measures of behavior, specifically psychosis, in 36 haloperidol treated schizophrenic patients. The only significant association was found with IL-10 (r=0.51, df=34, p=0.001). To demonstrate a possible direct association we controlled for elements associated with the two proposed mediums of the immune system and brain, the HPA axis and the sympathetic nervous system. For the elements of the HPA axis, we used measures of the hypothalamic (CSF CRF), pituitary (CSF prolactin), and adrenal (CSF cortisol) systems. For an indicator of sympathetic nervous system activity, we used CSF NE. When all variables were entered into a multiple regression predicting psychosis levels, IL-10 remained significant after adjusting for the levels of the other substances [t=3.463, p=0.0016], indicating a possible direct effect on behavior. ACTH was also tested as the measure of pituitary function in this model; IL-10 again remained a significant predictor of psychosis [t=3.354, p=0.0022]. BORNA DISEASE VIRUS SEROLOGY IN A FIRST EPISODE SCHIZOPHRENIA COHORT 1,2Waltrip RW*, 3Lieberman J, 4Robinson D, 4Bilder RM, 4Alvir JM, 1,2King LR, 2Rubin SA, 2Carbone KM 1Maryland Psychiatric Research Center, Baltimore, Maryland; 2Laboratory of Pediatric Viral Diseases and Respiratory Viruses, FDA/CBER/OVRR/DVP; 3University of North Carolina Department of Psychiatry; 4Long Island Jewish Medical Center, Hillside Hospital, Department of Psychiatry Borna disease virus (BDV) is a neurotropic and nonlytic agent that causes an immunopathological meningoencephalitis in a rapidly expanding range of recognized natural hosts (cattle, rabbits, goats, deer, llamas, alpacas, horses, sheep, ostriches, and domestic cats). BDV sequences have been detected, infectious isolates recovered from humans, viral antibodies have been associated with neuropsychiatric diseases, but BDV is not conclusively demonstrated to be a human pathogen. In previous studies we found anti-BDV antibodies at a high rate in patients with schizophrenia, an association between BDV seropositivity and both neuroanatomical abnormalities on MRI. Deficit subtype, and strongly correlated with neurological soft signs. In the present study of first episode schizophrenic patients, we compared anti-BDV seropositive rates between newly admitted schizophrenic patients and controls. The use of first episode patients reduces the likelihood that seropositive patients have anti-BDV antibody recognition because of nosocomial infection with BDV or cross-reactive antibodies resulting from exposure to psychopharmacological agents. Using a criterion of antibodies to 2 or more BDV proteins for seropositivity (5+), we found 10/82 (12.2%) first episode schizophrenic patients and 0/22 controls to be seropositive. Clinical, quantitative MRI and neurological data will be presented. PPARs AND ITS POTENTIAL INVOLVEMENT IN SCHIZOPHRENIA Guoqiang Xing*, Lixin Zhang, Lei Zhang, Takeo Yoshikawa, Zoran Brkanac, Sevilla Detera-Wadleigh and Mark A. Smith NIMH/NIH There is clinical evidence to suggest that schizophrenia (SZ) is linked with a defect in fatty acid (FFA) metabolism. So far, the underlying mechanism remains unknown. We propose that peroxisome proliferator activated receptor (PPARs ) is a candidate gene for the defect FFA metabolism in SZ. Peroxisome proliferator activated receptors (PPARs) are orphan nuclear hormone receptors of the steroid/thyroid hormone superfamily involved in the mediation of peroxisome proliferation, lipid metabolism and adipocyte differentiation. We have recently isolated a rat PPAR homolog (PPARs ) by low stringency screening. PPARs contains a 14 CGG triplet repeat on the 5’untranslated region. Unlike other PPARs, rat PPARs mRNA is highly expressed in the CNS since early embryonic stage. Furthermore, brain in situ hybridization analysis revealed that PPARs is prominently expressed in the thalamus, particularly in the posterior part of the ventral medial nucleus, a site responsive to pain and temperature sensation. We further mapped the human PPARs (NUCI) to chromosome 6 and the distal chromosome 6p21.1-21.2 by somatic hybrid PCR and fluorescence in situ hybridization. As PPARs is activated by polyunsaturated fatty acids (PUFA) and possibly by prostaglandin, these results indicate that PPARs is a candidate gene potentially involved in the defect fatty acid metabolism in schizophrenia. This putative conclusion is also based on the facts: 1) PPARs is mapped to 6p21.1-21.2 nearby which loci for schizophrenia have recently been reported by several groups; 2) PPARs regulate lipid homeostasis: PPAR response elements have been found in the promoter region of the genes coding the enzyme that control FFA metabolism and lipid homeostasis; 3) Schizophrenia patient shows defect lipid metabolism in the brain: with reduced PUFA content and prostaglandin (Pgs) content in the neocortex. Supplement of dietary PUFA improves the outcome of schizophrenia; 4) PPARs is expressed in the thalamus nuclear for pain sensation; Schizophrenia patients show decreased sensitivity to pain indicating abnormality of the pain sensation; 5) Several genetic disorders are known to be linked with the defect of peroxisome metabolism; 6) The CGG triplet repeat of the rat PPARs is also interesting as mutational expansion of trinucleotide repeats is known to cause several neurological disorders, including fragile-X mental retardation. Taken together, our results suggest that PPARs may be candidate gene for schizophrenia. We are planning to investigate if human PPARs (NUCI) gene contains mutations in the SZ population, and to examine the pattern of the PPARs (NUCI) gene expressed in the brains of the SZ postmortem which will be available from the Stanley Foundation’s brain bank. VIRAL AND VIRUS-RELATED RNA TRANSCRIPTS ARE DIFFERENTIALLY EXPRESSED IN THE BRAINS OF INDIVIDUALS WITH SCHIZOPHRENIA AND BIPOLAR DISORDER F Yee*, Johnston NL, Leister F, Li S, Ross CA, Torrey EF, Yolken RH, The Stanley Neurovirology Consortium Stanley Neurovirology Laboratory, Johns Hopkins University School of Medicine In this study, we have examined the hypothesis that the expression of brain RNAs may be altered in schizophrenia. A modified version of the arbitrarily primed-polymerase chain reaction (AP-PCR) method (Welsh et al, Nucl. Acids Res. 20:4965, 1992) was used to determine whether RNAs are differentially expressed in cortical regions obtained post-mortem from individuals with schizophrenia and normal controls. Several RNA transcripts had higher expression levels in the frontal cortex of an individual with schizophrenia compared to the normal control. One of the candidate RNAs displayed 77% amino acid homology to the Pol polyprotein of simian retrovirus (SRV-2), which is a primate type-D retrovirus. A second transcript was nearly identical to the DB1 zinc finger protein (>99% predicted amino acid similarity), which interacts with the tax protein of human T-cell leukemia virus to increase interleukin-3 transcription. The third candidate RNA was similar (64% nucleotide homology) to a simian paramyxovirus (SV-5).
These candidate clones were then screened for disease association using semi-quantitative reverse transcription-PCR on frontal cortical RNAs extracted post-mortem from individuals with schizophrenia (n=21), bipolar disorder (n=19) and controls without history of psychiatric illness (n=10). Initial evaluation of the DB1 and SV-5-like transcripts indicate that there are significant differences between the cases and controls (p<0.03 and p<0.04, respectively, by ANOVA). Northern blots will be used to confirm the differential expression of these candidate RNAs. Our preliminary findings indicate that viral and virus-associated RNA transcripts are expressed in the brains of some individuals with schizophrenia and bipolar disorder, and may play an important role in disease pathogenesis. INTERLEUKIN-6 SENSITIZES RATES TO THE LOCOMOTOR-ACTIVATING EFFECTS OF AMPHETAMINE Steve Zalcman*, Loulia Savina, Roy A. Wise Center for Studies in Behavioral Neurobiology, Concordia Univ., Montreal, Canada Cytokines are potent modulators of central nervous system (CNS) activity. Various macrophage and T lymphocyte-derived cytokines, may alter central neurochemical activity in regions that mediate brain-immune interactions. These changes, in turn regulate peripheral immunity via sympathetic outflow to immune organs and/or hypothalamic-pituitary-adrenal axis activity. As well, it appears that cytokines have particular sites of CNS action and hence, influence activities of specific neurotransmitter pathways. Interleukin (IL)-6 is a macrophage- and Th2-derived glycoprotein that has also been detected centrally. It has been implicated in various CNS pathologies, including schizophrenia, which is associated with alterations of meso-corticolimbic and striatal activity. Zalcman et al. (1995) reported that IL-6 induced profound alternations of serotonin and dopamine activity in these pathways. Paralleling these findings, behavioral-activating effects (including increases in ambulatory exploration, rearing and digging behavior) were rapidly induced following IL-6 administration in mice. Given this profile of central monoamine and behavioral alterations, coupled with evidence indicating a link between IL-6 and schizophrenia, we assessed (I) if IL-6 would increase locomotion in rats, (ii) if IL-6-induced increases in locomotion would increase with repeated injection and, (iii) if repeated administered IL-6 (0, 0.5, 1.0 and 2.0 m g/rat, ip) and immediately thereafter were placed in locomotor test boxes for two hours. Locomotor counts of rats receiving 1.0 or 2.0 m g of IL-6 exceeded those of controls and of rats that received s 0.5m g dose. Interestingly, repeated IL-6 treatment sensitized rats to the locomotor-activating effects of amphetamine. In an initial treatment phase, rats received five daily injections of saline or IL-6 (1.0 m g). Immediately after each injection, rats were exposed to the test box for two hours and locomotor counts were recorded. Although daily counts of rats in both groups declined over the five day period, those of IL-6-treated rats were consistently higher than those of controls (about 20%). As well, disfiguration of cardboard sheets placed beneath the test boxes were observed in IL-6-treated animals but not in controls. Moreover, all rats showed moderate weight gains during the course of this and subsequent experiments. Two weeks after the last injection, rats were administered saline or amphetamine (0.5 mg/kg, ip). Locomotor counts of rats that received IL-6 during the treatment phase were approximately 30% higher than those of rats that initially received saline injections. In a separate experiment, an identical treatment phase was employed. However, rats received a 1.0 mg/kg dose five days later. Locomotor counts of rats that initially received IL-6 were about 45% in a slightly prone position, staring in one direction, with eyelids widely opened at the end of the test session (whereas rats that initially received saline were simply moving around the test box). Hence, (I) locomotion was increased following acute IL-6 administration in rats, (ii) locomotor responses did not increase with repeated injection and, (iii) repeated intermittent administration of IL-6 sensitized rats to the locomotor-activating effects of amphetamine. It is suggested that IL-6 might influence responding associated with schizophrenic-like states. Accordingly, psychopathology could develop in conditions wherein IL-6 is overproduced and/or dysregulated.