Anné Møller-Larsen

The implications of Epstein‐Barr virus in multiple‘ sclerosis ‐ a review

The objective of this article is to bring together knowledge about Epstein‐Barr virus (EBV) in relation to multiple sclerosis (MS) in order to evaluate its implications in this disease. All MS patients are EBV seropositive, but EBV is not normally detected in the brain. EBV can explain many of the epidemiological dogmas known in MS. In addition, other studies point towards the involvement of EBV in MS. Despite this, other co‐actors seem also to be involved. We still need to know whether EBV may be an initiating factor in MS or whether it is a factor in the pathogenesis. Possible ways of EBV involvement are discussed: direct involvement, an autoimmune inducing factor or a transactivating factor. A current treatment study of MS patients with a specific herpes antiviral drug may add further information to the etiology and pathogenesis of MS.

The effect of interferon on lymphocyte-mediated effector cell functions: Selective enhancement of natural killer cells

Abstract The effect of human interferons on different types of lymphocyte-mediated killer assays was explored. Killing by T cells generated through mixed lymphocyte cultures as well as antibody-dependent lymphocyte-mediated cytotoxicity was not influenced by the addition of interferon. Enhancement of cytolysis produced by natural killer cells was observed when interferon was added during the assay, but enhancement could also be induced if the effector cells were pretreated with interferon for 2 hr prior to the lytic reaction. Killing of a cell line susceptible to natural killing was increased and a cell line which is normally relatively resistant to this type of killing became a susceptible target.

B cells and monocytes from patients with active multiple sclerosis exhibit increased surface expression of both HERV-H Env and HERV-W Env, accompanied by increased seroreactivity

BackgroundThe etiology of the neurogenerative disease multiple sclerosis (MS) is unknown. The leading hypotheses suggest that MS is the result of exposure of genetically susceptible individuals to certain environmental factor(s). Herpesviruses and human endogenous retroviruses (HERVs) represent potentially important factors in MS development. Herpesviruses can activate HERVs, and HERVs are activated in MS patients.ResultsUsing flow cytometry, we have analyzed HERV-H Env and HERV-W Env epitope expression on the surface of PBMCs from MS patients with active and stable disease, and from control individuals. We have also analyzed serum antibody levels to the expressed HERV-H and HERV-W Env epitopes. We found a significantly higher expression of HERV-H and HERV-W Env epitopes on B cells and monocytes from patients with active MS compared with patients with stable MS or control individuals. Furthermore, patients with active disease had relatively higher numbers of B cells in the PBMC population, and higher antibody reactivities towards HERV-H Env and HERV-W Env epitopes. The higher antibody reactivities in sera from patients with active MS correlate with the higher levels of HERV-H Env and HERV-W Env expression on B cells and monocytes. We did not find such correlations for stable MS patients or for controls.ConclusionThese findings indicate that both HERV-H Env and HERV-W Env are expressed in higher quantities on the surface of B cells and monocytes in patients with active MS, and that the expression of these proteins may be associated with exacerbation of the disease.

A Putative New Retrovirus Associated with Multiple Sclerosis and the Possible Involvement of Epstein-Barr Virus in This Diseasea

Since tropical spastic paraparesis in 1985 was found to be associated with HTLV-I infection, it has been suggested that a retrovirus might be involved in multiple sclerosis (MS). Our group has studied long-term cultures of cerebrospinal fluid cells and peripheral blood mononuclear cells from MS patients and controls with the purpose of elucidating the possible involvement of a retrovirus in MS. For an extended period electron microscopical analysis (EM) of T-cell lines, derived from MS patients and controls and cultured for 4 weeks was performed. In two cultures obtained 8 months apart from a patient with progressive MS, retrovirus-like particles were observed in 1-2% of the cells examined. Recently a B-lymphoblastoid cell line (LCL) producing retrovirus-like particles and EBV was established from a 30-year-old male patient with a chronic progressive myelopathy, clinically resembling multiple sclerosis. Similar cell lines have now been established from two MS patients. The retrovirus-like particles produced by the LCL have been purified by gradient ultracentrifugation. In the purified material reverse transcriptase assays are clearly positive in the gradients where EM shows retrovirus-like particles. Antigen characterization, nucleic acid sequence analysis and antibody studies are now being performed. The retrovirus found is definitively different from other known human retroviruses. It has previously been found that 100% of patients with MS have antibodies against EBV, in contrast to controls where only 86-95% have antibodies against this virus. Previous epidemiological studies have pointed toward a post-pubertal primary EBV infection as an important event in the induction of MS disease. These studies have now been substantiated by our group. Though it is still unknown whether EBV infection is a prerequisite for development of MS or whether the 100% EBV seropositivity is a consequence of the MS disease, we have put forward the hypothesis that the etiological agent for development of MS and MS-like diseases is a new hitherto uncharacterized retrovirus, whereas development of neurologic disease is related to or even dependent on a delayed infection with a virus from the herpes group, most likely EBV. This dual infection hypothesis has been analyzed and was found to be in accordance with the most consistent epidemiological characteristics of MS. We have previously, also from epidemiological data, negated retroviruses, behaving as the known human retroviruses, as an independent cause of MS.

Is Multiple Sclerosis Caused by a Dual Infection with Retrovirus and Epstein-Barr Virus?

Although the etiology of multiple sclerosis is as yet unknown, epidemiological observations strongly point toward one or more infectious agent(s) being involved in the disease. In recent years some studies have indicated involvement of retrovirus in multiple sclerosis (MS). However, an intrafamilial epidemiological study revealed that MS and the known human retroviruses had a divergent epidemiology. Some studies have shown the association of Epstein-Barr virus (EBV) with MS and one recent study revealed dual infection by retrovirus and EBV in a cell line established from a patient with an MS-like disease. Our hypothesis for the development of MS and MS-like diseases is that a hitherto uncharacterized retrovirus is the etiological agent, but development of neurologic disease is related to or even dependent on a delayed EBV infection. The dual infection hypothesis is analyzed and found to be consistent with the epidemiological characteristics of MS.

The Etiology of Multiple Sclerosis: Genetic Evidence for the Involvement of the Human Endogenous Retrovirus HERV-Fc1

We have investigated the role of human endogenous retroviruses in multiple sclerosis by analyzing the DNA of patients and controls in 4 cohorts for associations between multiple sclerosis and polymorphisms near viral restriction genes or near endogenous retroviral loci with one or more intact or almost-intact genes. We found that SNPs in the gene TRIM5 were inversely correlated with disease. Conversely, SNPs around one retroviral locus, HERV-Fc1, showed a highly significant association with disease. The latter association was limited to a narrow region that contains no other known genes. We conclude that HERV-Fc1 and TRIM5 play a role in the etiology of multiple sclerosis. If these results are confirmed, they point to new modes of treatment for multiple sclerosis.

The significance of Epstein—Barr virus seropositivity in multiple sclerosis patients?

The objective of this study was to evaluate and investigate the significance of the previously found 100% seropositivity toward Epstein–Barr virus (EBV) found in multiple sclerosis (MS) patients in contrast to healthy controls. Using a commercially available ELISA‐test (Biotest), which differentiates infections with EBV into previous infections, primary infections, reactivated infections and no previous infection, we found 137 of 138 MS patients and 124 of 138 healthy controls seropositive. A primary infection in 4 of the 124 EBV seropositive healthy controls in contrast to no primary infections in the MS EBV seropositive group was significant (P=0.049652, Fishers exact test). This may be suggestive of a lack of primary infections in MS patients, and thus strengthens the idea that MS patients are infected with EBV before development of MS. Further studies are in progress to analyse whether EBV infection is a prerequisite for the development of this disease.

Citrullination of Histone H3 Interferes with HP1-Mediated Transcriptional Repression

Multiple Sclerosis (MS) is an autoimmune disease associated with abnormal expression of a subset of cytokines, resulting in inappropriate T-lymphocyte activation and uncontrolled immune response. A key issue in the field is the need to understand why these cytokines are transcriptionally activated in the patients. Here, we have examined several transcription units subject to pathological reactivation in MS, including the TNFα and IL8 cytokine genes and also several Human Endogenous RetroViruses (HERVs). We find that both the immune genes and the HERVs require the heterochromatin protein HP1α for their transcriptional repression. We further show that the Peptidylarginine Deiminase 4 (PADI4), an enzyme with a suspected role in MS, weakens the binding of HP1α to tri-methylated histone H3 lysine 9 by citrullinating histone H3 arginine 8. The resulting de-repression of both cytokines and HERVs can be reversed with the PADI-inhibitor Cl-amidine. Finally, we show that in peripheral blood mononuclear cells (PBMCs) from MS patients, the promoters of TNFα, and several HERVs share a deficit in HP1α recruitment and an augmented accumulation of histone H3 with a double citrulline 8 tri-methyl lysine 9 modifications. Thus, our study provides compelling evidence that HP1α and PADI4 are regulators of both immune genes and HERVs, and that multiple events of transcriptional reactivation in MS patients can be explained by the deficiency of a single mechanism of gene silencing.

A single subtype of Epstein–Barr virus in members of multiple sclerosis clusters

Objectives – Epidemiological studies strongly indicate an infectious involvement in multiple sclerosis (MS). Epstein–Barr virus (EBV), to which all multiple sclerosis patients are seropositive, is also interesting from an epidemiological point of view. We have reported a cluster of MS patients with 8 members from a small Danish community called Fjelsø. To further evaluate the role of EBV in MS we have investigated the distribution of EBV subtypes in cluster members and in control cohorts. Materials and methods – Blood mononuclear cells were isolated from cluster members, unrelated MS patients, healthy controls, including healthy schoolmates to the Fjelstø cluster patients and finally from persons with autoimmune diseases in order to investigate the number of 39 bp repeats in the EBNA 6‐coding region in the EBV seropositive individuals. Results – We observed a preponderance of the subtype with 3 39 bp repeats in the EBNA 6‐coding region both in the MS patients and the healthy controls. In the Fjelsø cluster all 8 cluster members were harbouring this subtype, which is significantly different from the finding in healthy controls (n = 16), which include 8 schoolmates to the cluster members and 8 randomly selected healthy persons (Fischers exact test P=0.0047), and also compared to all non‐clustered individuals studied (P=0.017). Conclusion – Infection with the same subtype of EBV links together the 8 persons from the Fjelso cluster who later developed MS. This finding adds to the possibility that development of MS is linked to infection with EBV.

Expression of HERV-Fc1, a human endogenous retrovirus, is increased in patients with active Multiple Sclerosis

ABSTRACT Multiple sclerosis (MS) is considered to be an autoimmune disease with an unknown cause and with immune system dysregulation. Among environmental factors, viruses are most often connected with the etiology of MS. Human endogenous retroviruses (HERVs) constitute 5 to 8% of human genomic DNA and have been detected as transcripts and proteins in the central nervous system (CNS) and peripheral blood, frequently in the context of neuroinflammation. HERV-Fc1, which belongs to the HERV-H/F family, has received our attention largely because of the genetic association with MS. We studied the expression of a capsid (Gag) protein of HERV-H/F origin by flow cytometry in peripheral blood mononuclear cells (PBMCs) from healthy controls and from MS patients with nonactive or active disease. There was a significant increase in HERV-H/F Gag expression in CD4+ (P < 0.001) and CD8+ (P < 0.001) T lymphocytes and in monocytes (P = 0.0356) in PBMCs from MS patients with active disease. Furthermore, we have undertaken the first rigorous SYBR green-based absolute quantitative PCR (Q-PCR) evaluation approach to quantify extracellular HERV-Fc1 RNA viral loads in plasma from MS patients and healthy controls. We found a 4-fold increase in extracellular HERV-Fc1 RNA titers in patients with active MS compared with healthy controls (P < 0.001). These findings strengthen the link between HERV-Fc1 and the pathology of MS. The cause and biological consequences of these differential expression levels will be the subject of further investigation. HERV-Fc1 biology could be a compelling area for understanding the pathology of MS and possibly other autoimmune disorders.