An in vitro IgG secretion assay was developed to investigate the regulation of the humoral immune response in humans. Pokeweed mitogen (PWM), a plant lectin derived from Phytolacca americana stimulates human peripheral blood mononuclear cells (PBMNC) to divide and resting B-lymphocytes to differentiate into immunoglobulin secreting cells (ISC). This differentiation requires that both monocytes and T-lymphocytes be present in the culture system. The amount of IgG secreted by these differentiated B-lymphocytes in response to PWM appears to be the net result of a balance between the functional activity of the regulatory T-helper and T-suppressor cells. Alterations, qualitative or quantitative in any of these leukocyte subsets could conceivably alter the amount of IgG secreted by the B-lymphocyte subpopulation. We have employed this assay to investigate the immune status in a group of chronic progressive multiple sclerosis (MS) patients and to assess the immunoregulatory effects of interferon (Wellferon R, INF) administered in vivo to this selected group. Their mononuclear cells (MNC) were studied in this PWM induced IgG secretion assay before INF treatment and again after 7 days of daily sub-cutaneous injections (5 X 10⁶ u/day). Twenty patients received the interferon (INF) preparation and eighteen received normal saline. The study was carried out in a double blind manner and the code was broken only after individual results had been calculated. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

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In patients with multiple sclerosis (MS), microglia become activated due to the autoimmune inflammatory response which is directed against the central nervous system (CNS). Following the first disease relapse, microglia remain activated and do not return to a resting state during remissions. Chronically-activated microglia release inflammatory mediators that cause CNS tissue damage, and as such, MS progression has been associated with widespread, chronic microglial activation that correlates with neurodegeneration. To date, only one histone demethylase, Jmjd3, has been described to have a role in inflammation. In agreement with this, up-regulation of Jmjd3 expression was observed following microglial treatment with several pro-inflammatory stimuli, including a range of toll-like receptors ligands and cytokines, suggesting a universal role of Jmjd3 during microglial activation. Subsequent ChIP-qPCR assays revealed that Jmjd3 was recruited to the promoters of Il6, Ccl3, Ccl5 and Nos2 following activation, which, in turn, presented a decrease in their H3K27me3 levels. Using an experimental autoimmune encephalomyelitis (EAE) mouse model of MS, Jmjd3 expression was shown to be increased in activated microglia from mice in the acute and late phases of disease. Immunization with complete Freud’s adjuvant (CFA) alone, also caused microglial activation with Jmjd3 induction, indicating a CFA-mediated TLR2 and TLR4 stimulation of microglia. Further investigation, in which primary microglia were isolated from mice deficient in Jmjd3 (Jmjd3-/-), however demonstrated that the absence of Jmjd3 alone had no resultant effect on the expression of a subset of immune response and inflammation related genes, including the Jmjd3 target genes Il6, Ccl3, Ccl5 and Nos2, before or after activation. This suggested that Jmjd3 acts in concert with a repertoire of other demethylases to facilitate microglia activation, and as such was rendered redundant in this setting. Deciphering the epigenetic profile of microglia in MS and determining whether it is involved in the maintenance of chronic microglial activation in the progressive phase of the disease remains an important line of investigation, and through a clearer understanding of its role in MS pathophysiology, could lead to the development of novel therapeutic interventions in the future.