Immune and Epstein-Barr virus gene expression in cerebrospinal fluid and peripheral blood mononuclear cells from patients with relapsing-remitting multiple sclerosis

Background: Gene expression analyses in paired cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMC) from patients with multiple sclerosis (MS) are restrained by the low RNA amounts from CSF cells and low expression levels of certain genes. Here, we applied a Taqman-based pre-amplification real-time reverse-transcription polymerase chain reaction (RT-PCR) (PreAmp RT-PCR) to cDNA from CSF cells and PBMC of MS patients and analyzed multiple genes related to immune system function and genes expressed by Epstein-Barr virus (EBV), a herpesvirus showing strong association with MS. Using this enhanced RT-PCR method, we aimed at the following: (1) identifying gene signatures potentially useful for patient stratification, (2) understanding whether EBV infection is perturbed in CSF and/or blood, and (3) finding a link between immune and EBV infection status. Methods: Thirty-one therapy-free patients with relapsing-remitting MS were included in the study. Paired CSF cells and PBMC were collected and expression of 41 immune-related cellular genes and 7 EBV genes associated with latent or lytic viral infection were determined by PreAmp RT-PCR. Clinical, radiological, CSF, and gene expression data were analyzed using univariate and multivariate (cluster analysis, factor analysis) statistical approaches. Results: Several immune-related genes were differentially expressed between CSF cells and PBMC from the whole MS cohort. By univariate analysis, no or only minor differences in gene expression were found associated with sex, clinical, or radiological condition. Cluster analysis on CSF gene expression data grouped patients into three clusters; clusters 1 and 2 differed by expression of genes that are related mainly to innate immunity, irrespective of sex and disease characteristics. By factor analysis, two factors grouping genes involved in antiviral immunity and immune regulation, respectively, accurately discriminated cluster 1 and cluster 2 patients. Despite the use of an enhanced RT-PCR method, EBV transcripts were detected in a minority of patients (5 of 31), with evidence of viral latency activation in CSF cells or PBMC and of lytic infection in one patient with active disease only. Conclusions: Analysis of multiple cellular and EBV genes in paired CSF cell and PBMC samples using PreAmp RT-PCR may yield new information on the complex interplay between biological processes underlying MS and help in biomarker identification

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

Journal officiel de la Guinée française

01 octobre 19131913/10/01 (A13,N296)

Additional file 6: of Transcriptional profile and Epstein-Barr virus infection status of laser-cut immune infiltrates from the brain of patients with progressive multiple sclerosis

Quantification of EBV transcripts in an EBV+ lymphoblastoid cell line by PreAmp droplet digital (dd) PCR compared to real-time PCR. The figure shows the results of an experiment to verify whether EBV gene expression data obtained using ddRT-PCR were comparable with those obtained using real-time RT-PCR. (PDF 345 kb

Osteopathic Manipulative Therapy and Multiple Sclerosis: A Proof-of-Concept Study

Context: Research on the effect of osteopathic manipulative therapy (OMTh; manipulative care provided by foreign-trained osteopaths) on chronic symptoms of multiple sclerosis (MS) is lacking. Objective: To evaluate the effect of OMTh on chronic symptoms of MS. Methods: Patients with MS who were evaluated at the neurology clinic at Genoa University in Italy were recruited for this study. Participants received 5 fortyminute MS health education sessions (control group) or 5 OMTh sessions (OMTh group). All participants completed a questionnaire that assessed their level of clinical disability, fatigue, depression, anxiety, and quality of life before the first session, 1 week after the final session, and 6 months after the final session. The Extended Disability Status Scale, a modified Fatigue Impact Scale, the Beck Depression Inventory-II, the Beck Anxiety Inventory, and the 12-item Short Form Health Survey were used to assess clinical disability, fatigue, depression, anxiety, and quality of life, respectively. Results: Twenty-two participants were included in the study (10 in the control group and 12 in the OMTh group). In the OMTh group, statistically significant improvements in fatigue and depression were found 1 week after the final session (P=.002 and P<.001, respectively). An increase in quality of life was also found in the OMTh group 1 week after the final session (P=.36). Conclusion: Results demonstrate that OMTh should be considered in the treatment of patients with chronic symptoms of M

Epstein-Barr virus latent infection and BAFF expression in B cells in the multiple sclerosis brain: implications for viral persistence and intrathecal B-cell activation

A cardinal feature of multiple sclerosis (MS) is the persistent intrathecal synthesis of antibodies. Our previous finding that a large fraction of B cells infiltrating the MS brain are infected with Epstein-Barr virus (EBV) raises the possibility that this virus, because of its ability to establish a latent infection in B cells and interfere with their differentiation, contributes to B-cell dysregulation in MS. The aim of this study was to gain further insight into EBV latency programs and their relationship to B-cell activation in the MS brain. Immunohistochemical analysis of postmortem MS brain samples harboring large EBV deposits revealed that most B cells in white matter lesions, meninges, and ectopic B-cell follicles are CD27+ antigen-experienced cells and coexpress latent membrane protein 1 and latent membrane protein 2A, 2 EBV-encoded proteins that provide survival and maturation signals to B cells. By combining laser-capture microdissection with preamplification reverse transcription-polymerase chain reaction techniques, EBV latency transcripts (latent membrane protein 2A, EBV nuclear antigen 1) were detected in all MS brain samples analyzed. We also found that B cell-activating factor of the tumor necrosis factor family is expressed in EBV-infected B cells in acute MS lesions and ectopic B-cell follicles. These findings support a role for EBV infection in B-cell activation in the MS brain and suggest that B cell-activating factor of the tumor necrosis factor family produced by EBV-infected B cells may contribute to this process resulting in viral persistence and, possibly, disruption of B-cell tolerance

Nef synergizes with IFNβ in iNOS production.

<p>(<b>A</b>) BV-2 cells were treated for 6 h with myr⁺Nef_SF2 (100 ng/ml), IFNβ (200 IU/ml) or a combination of both. iNOS mRNA was measured by real time RT-PCR as reported in the materials and methods section. (<b>B</b>) Cells were incubated for 24 h with the indicated dose of myr⁺Nef_SF2 with or without IFNβ (50 IU/ml). Cells were also incubated with heat-inactivated recNef_SF2 (inactiv. recNef, 500 ng/ml) or, as control, with LPS or pre-heated LPS (500 ng/ml each). Total cellular lysates were analyzed by Western Blot for iNOS expression. β-Tubulin expression was used as an internal loading control. (<b>C</b>) Cells were treated like in (<b>B</b>) and NO₂^- content in supernatants was measured using the Griess colorimetric assay. Empty diamond: IFNβ; filled circles: myr⁺Nef_SF2; gray empty circles: heat-inactivated myr⁺Nef_SF2; filled squares: recNef_SF2 plus IFNβ; gray empty squares: heat-inactivated myr⁺Nef_SF2 plus IFNβ.</p