Amyloid-like Behavior of Site-Specifically Citrullinated Myelin Oligodendrocyte Protein (MOG) Peptide Fragments inside EBV-Infected B-Cells Influences Their Cytotoxicity and Autoimmunogenicity.

Multiple sclerosis (MS) is an autoimmune disorder manifested via chronic inflammation, demyelination, and neurodegeneration inside the central nervous system. The progressive phase of MS is characterized by neurodegeneration, but unlike classical neurodegenerative diseases, amyloid-like aggregation of self-proteins has not been documented. There is evidence that citrullination protects an immunodominant peptide of human myelin oligodendrocyte glycoprotein (MOG34-56) against destructive processing in Epstein-Barr virus-infected B-lymphocytes (EBV-BLCs) in marmosets and causes exacerbation of ongoing MS-like encephalopathies in mice. Here we collected evidence that citrullination of MOG can also lead to amyloid-like behavior shifting the disease pathogenesis toward neurodegeneration. We observed that an immunodominant MOG peptide, MOG35-55, displays amyloid-like behavior upon site-specific citrullination at positions 41, 46, and/or 52. These amyloid aggregates are shown to be toxic to the EBV-BLCs and to dendritic cells at concentrations favored for antigen presentation, suggesting a role of amyloid-like aggregation in the pathogenesis of progressive MS.

The MRZ reaction in primary progressive multiple sclerosis.

BACKGROUND: The MRZ reaction (MRZR), composed of the three antibody indices (AI) against measles, rubella and varicella zoster virus and found positive in the majority of relapsing-remitting multiple sclerosis (RRMS) patients, is absent in other inflammatory neurological diseases (OIND). So far, it has been uncertain whether its differential diagnostic promise extends to patients with primary-progressive multiple sclerosis (PPMS). OBJECTIVE: To investigate the prevalence of MRZR in PPMS compared to RRMS and OIND patients. METHODS: MRZR was assessed in patients with PPMS (n = 103), RRMS (n = 100) and OIND (n = 48). Both stringency levels for MRZR testing, MRZR-1 (≥1 AI positive) and MRZR-2 (≥2 AI positive), were applied. RESULTS: Prevalence of positive MRZR-1 was 83.5% in PPMS and 67.8% in RRMS (p < 0.05). A positive MRZR-2 was found in 54.4% of PPMS and in 43.0% of RRMS patients (not significant). Compared to both MS subgroups, OIND patients exhibit lower frequencies of positive MRZR (MRZR-1: 22.9%, MRZR-2: 8.3%; p < 0.0001 each). CONCLUSION: Positive MRZR was at least as frequent in PPMS as in RRMS and much less frequent in OIND, confirming its promise as a potentially useful diagnostic tool for distinguishing both MS course types from OIND.

Risk factors associated with the onset of relapsing-remitting and primary progressive multiple sclerosis: a systematic review.

Multiple sclerosis (MS) is a chronic central nervous system disease with a highly heterogeneous course. The aetiology of MS is not well understood but is likely a combination of both genetic and environmental factors. Approximately 85% of patients present with relapsing-remitting MS (RRMS), while 10-15% present with primary progressive MS (PPMS). PPMS is associated with an older onset age, a different sex ratio, and a considerably more rapid disease progression relative to RRMS. We systematically reviewed the literature to identify modifiable risk factors that may be associated with these different clinical courses. We performed a search of six databases and integrated twenty observational studies into a descriptive review. Exposure to Epstein-Barr virus (EBV) appeared to increase the risk of RRMS, but its association with PPMS was less clear. Other infections, such as human herpesvirus-6 and chlamydia pneumoniae, were not consistently associated with a specific disease course nor was cigarette smoking. Despite the vast literature examining risk factors for the development of MS, relatively few studies reported findings by disease course. This review exposes a gap in our understanding of the risk factors associated with the onset of PPMS, our current knowledge being predominated by relapsing-onset MS.

The participation of varicella zoster virus in relapses of multiple sclerosis.

OBJECTIVE: Recent studies have documented the apparent participation of varicella zoster virus (VZV) in the etiopathogenesis of multiple sclerosis (MS). The present study aimed to corroborate the possible presence of VZV during exacerbations of MS. DESIGN: Fifty-three patients with definite MS were included; of them, 31 were studied during the first week of a clinical relapse, whereas 16 were studied during remission; 6 patients with progressive MS were also studied. Genes from 5 herpes viruses: varicella zoster, herpes simplex 1 and 2, Epstein-Barr and herpes 6 were studied by polymerase chain reaction in cerebrospinal fluid and in peripheral blood mononuclear cells (PBMC). As controls 21 patients with inflammatory or functional neurological disorders were included. RESULTS: DNA from varicella zoster virus was found in the CSF from all MS patients studied during relapse (100%) and in the PBMC from 28 of them (90%). However, VZV DNA was found in the CSF only in 5 MS patients studied during remission (31%) and in the PBMC from 3 of them (19%). VZV DNA was also found, but in lower amounts, in the CSF (83%) and PBMC (33%) from patients with progressive MS. In contrast, VZV was not found either in CSF or in PBMC from controls. Results from the other herpes viruses tested were similar in MS patients and in controls. CONCLUSIONS: Our results corroborate the conspicuous, but ephemeral presence of VZV during relapses of MS and support the idea of VZV involvement in the etiopathogenesis of MS. Recent epidemiological and molecular studies as well as reports of severe VZV infections triggered by specifically induced immunosuppression during therapy of MS give additional support to this potential association.

Epstein-Barr virus-specific adoptive immunotherapy for progressive multiple sclerosis.

Defective control of Epstein-Barr virus (EBV) infection by cytotoxic CD8(+) T cells might predispose to multiple sclerosis (MS) by allowing EBV-infected autoreactive B cells to accumulate in the central nervous system. We have treated a patient with secondary progressive MS with in vitro-expanded autologous EBV-specific CD8(+) T cells directed against viral latent proteins. This adoptive immunotherapy had no adverse effects and the patient showed clinical improvement with reduced disease activity on magnetic resonance imaging and decreased intrathecal immunoglobulin production. This is the first report of the use of EBV-specific adoptive immunotherapy to treat MS or any other autoimmune disease.

B-cell enrichment and Epstein-Barr virus infection in inflammatory cortical lesions in secondary progressive multiple sclerosis.

Gray matter lesions are thought to play a key role in the progression of disability and cognitive impairment in multiple sclerosis (MS) patients, but whether gray matter damage is caused by inflammation or secondary to axon loss in the white matter, or both, is not clear. In an analysis of postmortem brain samples from 44 cases of secondary progressive MS, 26 cases were characterized by meningeal inflammation with ectopic B-cell follicles and prominent gray matter pathology; subpial cortical lesions containing dense perivascular lymphocytic infiltrates were present in 11 of these cases. Because intracortical immune infiltrates were enriched in B-lineage cells and because we have shown previously that B cells accumulating in the MS brain support an active Epstein-Barr virus (EBV) infection, we investigated evidence of EBV in the infiltrated cortical lesions. Cells expressing EBV-encoded small RNA and plasma cells expressing EBV early lytic proteins (BZLF1, BFRF1) were present in all and most of the intracortical perivascular cuffs examined, respectively. Immunohistochemistry for CD8-positive cells, granzyme B, perforin, and CD107a indicated cytotoxic activity toward EBV-infected plasma cells that was consistently observed in infiltrated cortical lesions, suggesting active immune surveillance. These findings indicate that both meningeal and intraparenchymal inflammation may contribute to cortical damage during MS progression, and that intracortical inflammation may be sustained by an EBV-driven immunopathologic response, similar to findings in white matter lesions and meninges.

Intrathecal, polyspecific antiviral immune response in oligoclonal band negative multiple sclerosis.

BACKGROUND: Oligoclonal bands (OCB) are detected in the cerebrospinal fluid (CSF) in more than 95% of patients with multiple sclerosis (MS) in the Western hemisphere. Here we evaluated the intrathecal, polyspecific antiviral immune response as a potential diagnostic CSF marker for OCB-negative MS patients. METHODOLOGY/PRINCIPAL FINDINGS: We tested 46 OCB-negative German patients with paraclinically well defined, definite MS. Sixteen OCB-negative patients with a clear diagnosis of other autoimmune CNS disorders and 37 neurological patients without evidence for autoimmune CNS inflammation served as control groups. Antibodies against measles, rubella, varicella zoster and herpes simplex virus in paired serum and CSF samples were determined by ELISA, and virus-specific immunoglobulin G antibody indices were calculated. An intrathecal antibody synthesis against at least one neurotropic virus was detected in 8 of 26 (31%) patients with relapsing-remitting MS, 8 of 12 (67%) with secondary progressive MS and 5 of 8 (63%) with primary progressive MS, in 3 of 16 (19%) CNS autoimmune and 3 of 37 (8%) non-autoimmune control patients. Antibody synthesis against two or more viruses was found in 11 of 46 (24%) MS patients but in neither of the two control groups. On average, MS patients with a positive antiviral immune response were older and had a longer disease duration than those without. CONCLUSION: Determination of the intrathecal, polyspecific antiviral immune response may allow to establish a CSF-supported diagnosis of MS in OCB-negative patients when two or more of the four virus antibody indices are elevated.

Anti-HHV-6 IgG titer significantly predicts subsequent relapse risk in multiple sclerosis.

BACKGROUND: Some of the strongest associations with MS onset are for human herpesviruses, particularly Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6). Their role in MS clinical course is less clear, however. METHODS: Prospective cohort of 198 persons with clinically definite MS, followed 2002-5, and serum samples obtained from all subjects at study entry to measure anti-HHV-6 and anti-EBV (Epstein-Barr nuclear antigen [EBNA] and viral capsid antigen [VCA]) IgG titers. Association with relapse evaluated using survival analysis; association with disability/progression evaluated using linear regression or multilevel mixed-effects linear regression. RESULTS: For the 145 persons with relapsing-remitting MS followed beyond one review, anti-HHV-6 IgG titer was positively associated with the hazard of relapse with a dose-dependent trend (p = 0.003), not affected by adjustment for anti-EBV IgG titers, neither of which were independently associated with relapse. There was no significant association between anti-human herpesvirus IgG titers and baseline-measured disability scores, or change in disability scores; however, anti-HHV-6 IgG titers were 2.8 times higher among progressive-course females than progressive-course males. DISCUSSION: These findings suggest that, in addition to a potential etiological role in MS, HHV-6 infection or the immune response to HHV-6 antigens may have an effect on the risk of MS relapses and possibly on progressive courses of MS. The observed effect was directly related to anti-HHV-6 IgG titers and may indicate that either HHV-6 infection or factors associated with an altered humoral immune response to HHV-6 may have an effect on MS clinical course. Anti-HHV-6 IgG titer may be a useful prognostic factor in relapsing-remitting MS clinical course.

Human herpesvirus 6 and effectiveness of interferon ß1b in multiple sclerosis patients.

BACKGROUND: Human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV) have been repeatedly associated with multiple sclerosis (MS) pathogenesis. Also, it has been speculated that, besides its immunomodulatory properties, the efficacy of interferon beta (IFN-beta) in treating the disease may be related to its antiviral properties. The objectives of this study were to evaluate the in vivo antiviral effect of IFN-beta-1b over HHV-6 and EBV and to analyze whether such effect could be involved in its effectiveness in MS. METHODS: A total of 54 patients with MS were included in an observational, multicentric, 24-month study. HHV-6 and EBV were detected by qPCR in blood and serum samples. IFN-beta-1b effectiveness was evaluated by presence, number and severity of relapses, reduction in the relapse rate, disability progression, and response to the treatment. RESULTS: Patients with HHV-6 in blood had a higher risk of severe relapses (P=0.01) and bad response (P=0.03). HHV-6 was detected more frequently during relapses than in remission in blood (P=0.024) and in serum (P=0.0002). Patients with HHV-6 in serum had more relapses (P=0.02), lesser reduction in the relapse rate (P=0.04), and a lower proportion of responders (P=0.02) than those without HHV-6 active replication. However, any association between EBV and clinical parameters could not be found. CONCLUSIONS: We concluded that presence of HHV-6 in blood and serum during IFN-beta treatment could be a good marker of poor response.

Temporal relationship between environmental influenza A and Epstein-Barr viral infections and high multiple sclerosis relapse occurrence.

BACKGROUND: Multiple sclerosis (MS) relapses have been associated with viral and bacterial infection epidemics in MS patients who have not used interferon. OBJECTIVES: We studied whether environmental viral infections in the general population can be associated with increased MS relapse occurrence using retrospective data from 1986 to 1995 when interferons were not yet available. METHODS: Logistic regression modelling was used to compare retrospectively the monthly relapse occurrence from 407 MS patients in Turku University hospital archives and data on ten different specifically diagnosed viral infection epidemics in the general population of Southwestern Finland from 1986 to 1995. The outcome was the odds ratio (OR) of very high relapse occurrence versus low relapse occurrence, or moderate versus low relapse occurrence. RESULTS: After a peak in diagnosed influenza A cases in the general population, the MS relapse occurrence was 6.5 times more likely to be very high (95% CI 1.8-24.0) and 7.1 times more likely to be moderately high (95% CI 1.5-33.2). An increase in MS relapse counts also followed Epstein-Barr virus (EBV) infections (OR 4.4, 95% CI 1.3-15.1), but we found no significant association with adenovirus infections and MS relapses. The MS relapse occurrence was lowest in the summer months July-August (Chi-square test, p<0.01). CONCLUSIONS: Our findings suggest that influenza A and EBV viral infections in the general population are associated with a higher occurrence of exacerbations in MS patients, and thus environmental infection data should be included in epidemiological models on MS relapses.

Varicella zoster virus in progressive forms of multiple sclerosis.

OBJECTIVE: The apparent association between varicella zoster virus (VZV) and multiple sclerosis (MS) has been described. In patients with relapse/remission (R/R) MS we have found high loads of VZV DNA in lymphocytes and in cerebrospinal fluid (CSF), as well as abundant viral particles in CSF visualized by electron microscopy at the time of relapse. Both, the molecular and the ultrastructural evidence of VZV became negative in the same patients at the time of remission. METHODS: In the present study we analyzed the presence of VZV in patients with progressive forms of MS; DNA from VZV was searched by real-time PCR in blood lymphocytes and in CSF of 20 patients with progressive MS. Ultrastructural study searching for viral particles in CSF was made with transmission electron microscopy. RESULTS: VZV DNA was found in the CSF from 65% of cases with progressive MS- and VZV-like viral particles were found in 30% of these patients. Nonetheless, the amount of DNA and the number of viral particles were lower than those that have been found in MS patients with R/R at the time of relapse, but higher than those found during remission. CONCLUSION: Similar to findings in patients with R/R MS, VZV might be associated to progressive MS, but in minor quantity. In these cases, the virus may produce a chronic, relentless infection or trigger a process of immune-mediated demyelination.

JCV detection in multiple sclerosis patients treated with natalizumab.

Natalizumab therapy is associated with an increased risk of progressive multifocal leukoencephalopathy (PML). Because the prognosis of established PML is uniformly dismal, identification of highly susceptible patients to the disease may improve outcomes. We wanted to investigate whether serial plasma and cerebrospinal fluid (CSF) screening for polyomavirus would identify patients with laboratory evidence of viral infection prior to the development of clinical PML. Two hundred MS patients had pre-treatment CSF/plasma screening for JC virus (JCV) and BK virus (BKV) DNA, and thereafter every six treatments of natalizumab. In all positive patients treatment is stopped (due to potential risk of PML), they have follow-up clinical examinations and plasma/CSF JCV/BKV tests until all evaluations are normal. No patient developed clinical evidence of PML. Eight of the 200 patients had detectable JCV or BKV DNA. Five patients were positive for BKV DNA in the CSF and three patients were positive for JCV DNA (one in plasma, two in CSF). After cessation of natalizumab treatment, all patients converted to undetectable viral DNA. Screening for JCV in CSF in natalizumab-treated patients could help identify those at heightened risk for developing PML and discontinuing treatment in these patients may abort development of the clinical illness.

Association of a history of varicella virus infection with multiple sclerosis.

OBJECTIVE: To analyze the association of a previous history of varicella virus infection with multiple sclerosis (MS) and its subtypes. MATERIAL AND METHODS: We performed a case-control study including 126 cases and 157 controls. Subjects were divided into subgroups according to MS subtype and the history of varicella virus infection along with other variables was assessed. RESULTS: History of varicella zoster virus (VZV) infection was positive in 42% of controls and 66% of MS cases (p

Quantitative PCR for Epstein-Barr virus DNA and RNA in multiple sclerosis.

BACKGROUND: Epstein-Barr virus (EBV) is associated with MS, but it is not clear whether EBV plays a role in the pathogenesis of MS. HYPOTHESIS: We hypothesized that the immune control of EBV might be defective in MS, and that reactivation of EBV might drive the immune response in MS. METHODS: We collected blood from controls and patients with MS, and measured the amounts of EBV DNA and RNA using quantitative PCR. RESULTS: We found that EBV DNA and RNA were frequently detectable in peripheral blood leukocytes from both patients with MS and normal controls. There was no significant difference between patients with MS or controls. Paired samples from a small number of subjects suggest that EBV DNA may increase before and during clinical relapse. CONCLUSIONS: We conclude that the immune control of EBV infection is similar in MS and controls, and that reactivation of EBV may correlate with MS disease activity.

Analysis of cerebrospinal fluid and cerebrospinal fluid cells from patients with multiple sclerosis for detection of JC virus DNA.

OBJECTIVE: 1) To determine whether JC virus (JCV) DNA was present in the cerebrospinal fluid (CSF) and blood from patients with multiple sclerosis (MS) in comparison with controls and 2) to find out if our clinical material, based on presence of JCV DNA, included any patient at risk for progressive multifocal leukoencephalopathy (PML). METHODS: The prevalence of JCV DNA was analyzed in CSF and plasma from 217 patients with MS, 86 patients with clinically isolated syndrome (CIS), and 212 patients with other neurological diseases (OND). In addition, we analyzed CSF cells, the first report of JCV DNA in CSF cells in a single sample, and peripheral blood cells in a subgroup of MS (n = 49), CIS (n = 14) and OND (n = 53). RESULTS: A low copy number of JCV DNA was detected in one MS cell free CSF sample and in one MS CSF cell samples. None of these had any signs of PML or developed this disease during follow-up. In addition, two OND plasma samples were JCV DNA positive, whereas all the other samples had no detectable virus. CONCLUSION: A low copy number of JCV DNA may occasionally be observed both in MS and other diseases and may occur as part of the normal biology of JC virus in humans. This study does not support the hypothesis that patients with MS would be at increased risk to develop PML, and consequently screening of CSF as a measurable risk for PML is not useful.

Strong EBV-specific CD8+ T-cell response in patients with early multiple sclerosis.

Epstein-Barr virus (EBV) has been associated with multiple sclerosis (MS), however, most studies examining the relationship between the virus and the disease have been based on serologies, and if EBV is linked to MS, CD8+ T cells are likely to be involved as they are important both in MS pathogenesis and in controlling viruses. We hypothesized that valuable information on the link between MS and EBV would be ascertained from the study of frequency and activation levels of EBV-specific CD8+ T cells in different categories of MS patients and control subjects. We investigated EBV-specific cellular immune responses using proliferation and enzyme linked immunospot assays, and humoral immune responses by analysis of anti-EBV antibodies, in a cohort of 164 subjects, including 108 patients with different stages of MS, 35 with other neurological diseases and 21 healthy control subjects. Additionally, the cohort were all tested against cytomegalovirus (CMV), another neurotropic herpes virus not convincingly associated with MS, nor thought to be deleterious to the disease. We corrected all data for age using linear regression analysis over the total cohorts of EBV- and CMV-infected subjects. In the whole cohort, the rate of EBV and CMV infections were 99% and 51%, respectively. The frequency of IFN-gamma secreting EBV-specific CD8+ T cells in patients with clinically isolated syndrome (CIS) was significantly higher than that found in patients with relapsing-remitting MS (RR-MS), secondary-progressive MS, primary-progressive MS, patients with other neurological diseases and healthy controls. The shorter the interval between MS onset and our assays, the more intense was the EBV-specific CD8+ T-cell response. Confirming the above results, we found that EBV-specific CD8+ T-cell responses decreased in 12/13 patients with CIS followed prospectively for 1.0 +/- 0.2 years. In contrast, there was no difference between categories for EBV-specific CD4+ T cell, or for CMV-specific CD4+ and CD8+ T-cell responses. Anti-EBV-encoded nuclear antigen-1 (EBNA-1)-specific antibodies correlated with EBV-specific CD8+ T cells in patients with CIS and RR-MS. However, whereas EBV-specific CD8+ T cells were increased the most in early MS, EBNA-1-specific antibodies were increased in early as well as in progressive forms of MS. Our data show high levels of CD8+ T-cell activation against EBV--but not CMV--early in the course of MS, which support the hypothesis that EBV might be associated with the onset of this disease.