[show abstract][hide abstract] ABSTRACT: It has long been known that multiple sclerosis (MS) is associated with an increased Epstein-Barr virus (EBV) seroprevalence and high immune reactivity to EBV and that infectious mononucleosis increases MS risk. This evidence led to postulate that EBV infection plays a role in MS etiopathogenesis, although the mechanisms are debated. This study was designed to assess the prevalence and magnitude of CD8+ T-cell responses to EBV latent (EBNA-3A, LMP-2A) and lytic (BZLF-1, BMLF-1) antigens in relapsing-remitting MS patients (n = 113) and healthy donors (HD) (n = 43) and to investigate whether the EBV-specific CD8+ T cell response correlates with disease activity, as defined by clinical evaluation and gadolinium-enhanced magnetic resonance imaging. Using HLA class I pentamers, lytic antigen-specific CD8+ T cell responses were detected in fewer untreated inactive MS patients than in active MS patients and HD while the frequency of CD8+ T cells specific for EBV lytic and latent antigens was higher in active and inactive MS patients, respectively. In contrast, the CD8+ T cell response to cytomegalovirus did not differ between HD and MS patients, irrespective of the disease phase. Marked differences in the prevalence of EBV-specific CD8+ T cell responses were observed in patients treated with interferon-β and natalizumab, two licensed drugs for relapsing-remitting MS. Longitudinal studies revealed expansion of CD8+ T cells specific for EBV lytic antigens during active disease in untreated MS patients but not in relapse-free, natalizumab-treated patients. Analysis of post-mortem MS brain samples showed expression of the EBV lytic protein BZLF-1 and interactions between cytotoxic CD8+ T cells and EBV lytically infected plasma cells in inflammatory white matter lesions and meninges. We therefore propose that inability to control EBV infection during inactive MS could set the stage for intracerebral viral reactivation and disease relapse.
[show abstract][hide abstract] ABSTRACT: Sir, despite the association between Epstein–Barr virus infection and multiple sclerosis being supported by an increasing number of epidemiological, serological and immunological studies (Ascherio and Munger, 2010; Maghzi et al., 2011; Pender, 2011), the mechanisms linking increased immune reactivity to this ubiquitous herpes virus to CNS pathology in patients with multiple sclerosis remain to be demonstrated (Lü nemann and Mü nz, 2009; Giovannoni, 2011). A point of contention is whether an active and persistent Epstein–Barr virus infection in the CNS of patients with multiple sclerosis may sustain a detrimental immu-nopathological response (Pender, 2009; Ascherio and Bar-Or, 2010). In support of this scenario, we have shown high frequen-cies of Epstein–Barr virus latently infected B cells and more spor-adic signs of viral reactivation in post-mortem brain tissue from patients with multiple sclerosis but not patients with other inflam-matory neurological diseases (Serafini et al., 2007, 2010; Aloisi et al., 2010). Presence of Epstein–Barr virus latently infected cells in active multiple sclerosis brain lesions has been confirmed recently (Tzartos et al., 2012). In contrast, other groups did not find Epstein–Barr virus in all or the majority of post-mortem mul-tiple sclerosis brain samples analysed (Hilton et al., 1994; Opsahl and Kennedy, 2007; Willis et al., 2009; Peferoen et al., 2010; Sargsyan et al., 2010; Torkildsen et al., 2010). As recently discussed in Brain (Lassmann et al., 2011), no con-sensus has been reached on technical and interpretation issues relating to the detection of Epstein–Barr virus in the multiple sclerosis brain. Although a variety of techniques [in situ hybrid-ization for Epstein–Barr virus-encoded small RNA (EBER), immuno-histochemistry for Epstein–Barr virus latent and lytic proteins and PCR assays for Epstein–Barr virus nucleic acids] have been used to search for Epstein–Barr virus in multiple sclerosis brain, comparing and interpreting the contrasting results from different studies has been difficult owing to the lack of common procedures and tools. EBER in situ hybridization is considered the most powerful standard method to study the anatomical localization of Epstein– Barr virus–infected cells (Gulley and Tang, 2008). EBERs (EBER1 and EBER2) are small untranslated Epstein–Barr virus transcripts that are abundantly expressed during all phases of Epstein–Barr virus latent infection and generally localize in the cell nucleus. Despite high target specificity and sensitivity of EBER probes, false negative results due to RNA degradation or presence of non-specific staining can invalidate the conclusions of in situ hybridization studies (Gulley and Tang, 2008). Regarding Epstein–Barr virus presence in the multiple sclerosis brain, criticism has been raised toward the high number of EBER-positive cells we found in multiple sclerosis white matter lesions and meninges (par-ticularly inside B cell follicles) (Serafini et al., 2007) and the pres-ence of both nuclear and cytoplasmic EBER signals observed using a peptide nucleic acid (PNA) probe and detection kit from DakoCytomation (Lassmann et al., 2011). The reported absence of EBER signals in brain tissues from cases with other inflammatory neurological diseases or in non-pathological brain and lymphoid tissues (Serafini et al., 2007, 2010) was not deemed sufficient to interpret the EBER signals observed in the multiple sclerosis brain as specific (Lassmann et al., 2011). We reasoned that a sound strategy to confirm or disprove the findings obtained with the commercial EBER in situ hybridization kit (Serafini et al., 2007) was to take advantage of the high sen-sitivity of a radioactive in situ hybridization protocol routinely used by one of the authors (L.M.) at S. Raffaele Hospital in Milan (Muzio et al., 2002, 2005a, b, 2010; Centonze et al., 2009) and the availability of non-commercial EBER probes validated in previous studies (Niedobitek et al., 1991) to analyse EBER doi:10.1093/brain/aws315
[show abstract][hide abstract] ABSTRACT: 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 of neuropathology and experimental neurology. 12/2012;
[show abstract][hide abstract] ABSTRACT: 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.
Journal of Neuropathology and Experimental Neurology 07/2010; 69(7):677-93. · 4.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Increasing evidence supports a link between Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic human herpesvirus, and common B-cell-related autoimmune diseases. We sought evidence of EBV infection in thymuses from patients with myasthenia gravis (MG), an autoimmune disease characterized by intrathymic B-cell activation.
Seventeen MG thymuses (6 follicular hyperplastic, 6 diffuse hyperplastic, 5 involuted) and 6 control thymuses were analyzed using in situ hybridization for EBV-encoded small RNAs (EBERs), immunohistochemistry for EBV latent and lytic proteins, and polymerase chain reaction for EBV DNA and mRNA.
All 17 MG thymuses showed evidence of active EBV infection, whereas none of the control thymuses were infected. Cells expressing EBERs (12 of 17) and EBV latency proteins (EBNA2, LMP1, and LMP2A) (16 of 17) were detected in medullary infiltrates and in germinal centers. Cells expressing early (BFRF1, BMRF1) and late (p160, gp350/220) lytic phase EBV proteins were present in 16 MG thymuses. Latency (EBNA1, LMP2A) or lytic (BZLF1) transcripts (often both) were present in all MG thymuses, and EBV DNA (LMP1 gene) was detected in 13 MG thymuses. We also found CD8+ T cells, CD56 + CD3-natural killer cells, and BDCA-2+ plasmacytoid dendritic cells in immune infiltrates of MG thymuses, but not germinal centers, suggesting an attempt of the immune system to counteract EBV infection.
Dysregulated EBV infection in the pathological thymus appears common in MG and may contribute to the immunological alterations initiating and/or perpetuating the disease.
Annals of Neurology 06/2010; 67(6):726-38. · 11.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Objective
Increasing evidence supports a link between Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic human herpesvirus, and common B-cell–related autoimmune diseases. We sought evidence of EBV infection in thymuses from patients with myasthenia gravis (MG), an autoimmune disease characterized by intrathymic B-cell activation.Methods
Seventeen MG thymuses (6 follicular hyperplastic, 6 diffuse hyperplastic, 5 involuted) and 6 control thymuses were analyzed using in situ hybridization for EBV-encoded small RNAs (EBERs), immunohistochemistry for EBV latent and lytic proteins, and polymerase chain reaction for EBV DNA and mRNA.ResultsAll 17 MG thymuses showed evidence of active EBV infection, whereas none of the control thymuses were infected. Cells expressing EBERs (12 of 17) and EBV latency proteins (EBNA2, LMP1, and LMP2A) (16 of 17) were detected in medullary infiltrates and in germinal centers. Cells expressing early (BFRF1, BMRF1) and late (p160, gp350/220) lytic phase EBV proteins were present in 16 MG thymuses. Latency (EBNA1, LMP2A) or lytic (BZLF1) transcripts (often both) were present in all MG thymuses, and EBV DNA (LMP1 gene) was detected in 13 MG thymuses. We also found CD8+ T cells, CD56 + CD3-natural killer cells, and BDCA-2+ plasmacytoid dendritic cells in immune infiltrates of MG thymuses, but not germinal centers, suggesting an attempt of the immune system to counteract EBV infection.InterpretationDysregulated EBV infection in the pathological thymus appears common in MG and may contribute to the immunological alterations initiating and/or perpetuating the disease. ANN NEUROL 2010;67:726–738
[show abstract][hide abstract] ABSTRACT: Di-(2-ethylhexyl)-phthalate (DEHP), the plasticizer employed in the fabrication of polyvinyl chloride, is known to be released by many medical devices, namely endotracheal tubes currently utilised for pulmonary ventilation of pre-term newborns. When experimentally administered, especially to rodents, the phthalate reportedly causes alterations to several tissues, immature animals being even more responsive targets than adult ones. In the present research, female rats were fed with DEHP in the last week of pregnancy and after delivery, and lung of their pups was morphologically and immunohistochemically analysed. We detected significant alveolar simplification (larger but fewer alveoli with decreased septation), with consequent sensible reduction of gas-exchange surface, at several stages of postnatal development, in distal lung parenchyma of DEHP-treated rats. Moreover, the quantification of PCNA-expressing cells demonstrates that in treated pups the proliferation rates of epithelial and mesenchymal cells progressively increased during the first two postnatal weeks, at difference with controls animals, where the highest proliferation levels were reached at postnatal day 7. The obtained results strongly support the hypothesis that DEHP profoundly affects the alveolarization process in mammalian lung.
[show abstract][hide abstract] ABSTRACT: The expression patterns of tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a pleiotropic cytokine with proinflammatory and cell death-inducing activities, and its receptor, fibroblast growth factor-inducible 14 (Fn14), were examined in postmortem brain tissue samples from patients with multiple sclerosis (MS) and controls. Immunohistochemical analysis and real-time reverse transcription-polymerase chain reaction demonstrated that both TWEAK and Fn14 were upregulated in the MS compared with control unaffected brain samples. Perivascular and meningeal macrophages and astrocytes and microglia associated with lesions were identified as the main sources of TWEAK in the MS brains. The highest frequency of TWEAK+ cells was found at edges of chronic active white matter lesions and in subpial cortical lesions inMS cases with abundant meningeal inflammation and ectopic B-cell follicles. Neurons and reactive astrocytes expressing Fn14 were mainly localized in the cerebral cortex in highly infiltrated MS brains. Numerous TWEAK-expressing microglia were associated with the extensive loss of myelin and astrocytosis, neuronal damage, and vascular abnormalities in subpial cortical lesions; this suggests that TWEAK could synergize with other cytotoxic factors diffusing from the inflamed meninges to promote cortical injury. Taken together, these findings indicate that the TWEAK/Fn14 pathway contributes to inflammation and tissue injury and is, therefore, a potential therapeutic target in MS.
Journal of Neuropathology and Experimental Neurology 12/2008; 67(12):1137-48. · 4.35 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lymphoid chemokines play an essential role in the establishment and maintenance of lymphoid tissue microarchitecture and have been implicated in the formation of tertiary (or ectopic) lymphoid tissue in chronic inflammatory conditions. Here, we review recent advances in lymphoid chemokine research in central nervous system inflammation, focusing on multiple sclerosis and the animal model experimental autoimmune encephalomyelitis. We also highlight how the study of lymphoid chemokines, particularly CXCL13, has led to the identification of intrameningeal B-cell follicles in the multiple sclerosis brain paving the way to the discovery that these abnormal structures are highly enriched in Epstein-Barr virus-infected B cells and plasma cells.
Journal of Neuroimmunology 07/2008; 198(1-2):106-12. · 3.03 Impact Factor
[show abstract][hide abstract] ABSTRACT: Epstein-Barr virus (EBV), a ubiquitous B-lymphotropic herpesvirus, has been associated with multiple sclerosis (MS), an inflammatory disease of the central nervous system (CNS), but direct proof of its involvement in the disease is still missing. To test the idea that MS might result from perturbed EBV infection in the CNS, we investigated expression of EBV markers in postmortem brain tissue from MS cases with different clinical courses. Contrary to previous studies, we found evidence of EBV infection in a substantial proportion of brain-infiltrating B cells and plasma cells in nearly 100% of the MS cases examined (21 of 22), but not in other inflammatory neurological diseases. Ectopic B cell follicles forming in the cerebral meninges of some cases with secondary progressive MS were identified as major sites of EBV persistence. Expression of viral latent proteins was regularly observed in MS brains, whereas viral reactivation appeared restricted to ectopic B cell follicles and acute lesions. Activation of CD8+ T cells with signs of cytotoxicity toward plasma cells was also noted at sites of major accumulations of EBV-infected cells. Whether homing of EBV-infected B cells to the CNS is a primary event in MS development or the consequence of a still unknown disease-related process, we interpret these findings as evidence that EBV persistence and reactivation in the CNS play an important role in MS immunopathology.
Journal of Experimental Medicine 12/2007; 204(12):2899-912. · 13.21 Impact Factor