Foroozan Mokhtarian

Molecular mimicry between a viral peptide and a myelin oligodendrocyte glycoprotein peptide induces autoimmune demyelinating disease in mice

Semliki Forest Virus (SFV) induces an encephalomyelitis followed by demyelination in the brains of C57Bl6/J (B6) mice. To investigate the role of molecular mimicry in the pathogenesis of postviral demyelination, alignment algorithms were used and amino acid homologies between immunogenic epitopes of SFV and myelin autoantigens, myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG) were identified. Immunization of B6 mice with SFV proteins induced significant lymphocyte proliferation to SFV E2 peptides and to MOG peptide, 18-32 (which had molecular mimicry with E2 115-129), but not to MBP or PLP peptides. Both MOG 18-32 and E2 115-129, induced a later-onset chronic EAE-like disease that correlated with the presence of multifocal vacuolation in the CNS white matter. This histopathology was reminiscent of the secondary demyelination seen following SFV infection. Serum antibody responses to the peptides appeared late after immunizations and some samples cross-reacted with other myelin peptides, as well as with the mimicked MOG peptides. These findings suggest that following a CNS viral infection, antibody response to an epitope of virus that exhibits molecular mimicry with a peptide of MOG may contribute to autoimmune mediated injury to CNS myelin.

A novel calpain inhibitor for the treatment of acute experimental autoimmune encephalomyelitis

Aberrant activation of calpain plays a key role in the pathophysiology of several neurodegenerative disorders. Calpain is increasingly expressed in inflammatory cells in EAE and is significantly elevated in the white matter of patients with multiple sclerosis, thus calpain inhibition could be a target for therapeutic intervention. The experiments reported here employed a myelin oligodendrocyte glycoprotein-induced disease model in C57Bl/6 mice (EAE) and a novel calpain inhibitor, targeted to nervous tissue. CYLA was found to reduce clinical signs of EAE and prevent demyelination and inflammatory infiltration in a dose- and time-dependent manner. Oral administration of the diacetal prodrug was equally effective.

Prevention of Axonal Injury using Calpain Inhibitor in Chronic Progressive Experimental Autoimmune Encephalomyelitis

Axonal injury is the major correlate of permanent disability in neurodegenerative diseases such as multiple sclerosis (MS), especially in secondary-progressive MS which follows relapsing-remitting disease course. Proteolytic enzyme, calpain, is a potential candidate for causing axonal injury. Most current treatment options only target the inflammatory component of MS. Previous work using calpain inhibitor CYLA in our laboratory showed significant reduction in clinical sign, demyelination and tissue calpain content in acute experimental autoimmune encephalomyelitis (EAE). Here we evaluated markers of axonal injury (amyloid precursor protein, Na(v)1.6 channels), neuronal calpain content and the effect of CYLA on axonal protection using histological methods in chronic EAE [myelin oligodendrocyte glycoprotein (MOG)-induced disease model of MS]. Intraperitoneal application of CYLA (2 mg/mouse/day) significantly reduced the clinical signs, tissue calpain content, demyelination and inflammatory infiltration of EAE. Similarly, markers for axonal injury were barely detectable in the treated mice. Thus, this novel drug, which markedly suppresses the disease course, axonal injury and its progression, is a candidate for the treatment of a neurodegenerative disease such as multiple sclerosis.

Production and role of cytokines in the CNS of mice with acute viral encephalomyelitis

Semliki Forest Virus (SFV) causes a more severe acute encephalomyelitis in B6 than in SJL mice despite similar T cell proliferation and antibody responses in these two strains. To determine the immunological mechanisms that may contribute to this difference, CNS tissues from SFV-infected B6 and SJL mice were analyzed for viral replication, inflammatory responses and cytokine production, by semiquantitative reverse transcriptase-PCR and immunohistochemistry. Although initially similar on day 2 p.i., SFV replicated to higher viral titers in B6 than SJL mice on days 4 and 7 p.i. Infectious virus was cleared from both strains by day 10 p.i. There were no differences in numbers of CD4+, CD8+ or MHC class I and II+ inflammatory cells at any time point. Higher levels of IL-4 mRNA, lower levels of TNF-alpha, IL-6, IL-1 beta and IL-2 mRNAs and lower IL-2+ and IFN-gamma+ cells were found in B6. These findings suggest that despite comparable immune responses, different patterns of cytokine production correlated with higher levels of virus in the brains and more severe clinical disease in B6, and more efficient clearance of virus and less severe disease in SJL mice.

Semliki Forest virus-induced demyelination and remyelination--involvement of B cells and anti-myelin antibodies.

Semliki Forest virus (SFV) infection induces a demyelinating encephalomyelitis in the central nervous system (CNS) of mice and serves as a model for multiple sclerosis (MS). This study investigated CNS immune responses at different stages of infection and during SFV-induced demyelination and remyelination. Following the initial CNS inflammation, pathology and viral clearance on days 6-10 post-infection (pi), primary demyelination was observed in cerebellar, brainstem and corpus collosal white matter by days 15-21 pi, with plasma cells and microglia as main participants, and this was followed by remyelination. By day 35 pi, the tissue appeared almost normal. Fluorescent antibody cell sorter (FACS) analysis showed that brain CD8(+) T cells increased during the initial inflammatory response and gradually decreased thereafter. Brain B cell (B220(+)CD19(+)) numbers did not change significantly during the course of infection; however, from days 14 to 35 pi, they matured and produced antibodies to viral and myelin proteins (and peptides) during the period of demyelination and remyelination. The proportion of CD3(-)B220(-)CD11b(+) cells also progressively increased throughout the periods of de- and remyelination. Our results suggest that CD8(+) T cells are involved in the initial destruction of CNS tissue during the first weeks of SFV infection, while B cells, antibodies and microglia may contribute to the myelin pathology seen after recovery.

Influenza virus vaccination of patients with multiple sclerosis

Prior to vaccination with a trivalent influenza vaccine (AT/Texas, AB/Beijing, and BP/Panama), sera from 19 MS patients had a significantly higher mean level of antibody than 9 normal subjects to AT strain of influenza, but not to AB or BP strains. After Flu vaccination, the mean anti-AT and anti-AB antibody titers significantly increased 4-fold in I I MS patients and 9 normal subjects. The ratio of MS responders (6II1), however, was lower than normal (8/9). The mean PBL proliferative response to the Flu antigens increased after vaccination significantly more in MS patients than in normal subjects, and increased in 9 of I I MS patients and 3 of 9 normal subjects. Although MS patients responded to Flu antigens with higher antibody levels and proliferative responses of PBL, than normal subjects, a clinical protective effect of the vaccine against Flu was not dearly demonstrated in these patients, and vaccination did not cause or protect against exacerbation of MS.

Phenotypic and functional characterization of T cells from patients with myasthenia gravis.

A study of cell surface phenotypes of PBL of myasthenia gravis (MG) patients showed that their T cells had a significantly higher percentage of 4B4+ T cells (the helper/inducer subset) than age- and sex-matched controls. The PBL of MG patients proliferated significantly higher than those of normal subjects (NS) in response to the purified alpha chain of the acetylcholine receptor (AChR). Anti-AChR antibody was present in sera of 88% of MG and none of the NS. The PBL B cells from MG only, when cultured with autologous T cells and stimulated with either pokeweed mitogen (69%), or AChR-alpha chain (38%), secreted antibody to AChR-alpha chain, whereas T and B cells alone secreted no antibody. T cells from PBL of MG patients were more readily cloned than T cells of NS, by limiting dilution, in the presence of recombinant IL-2 and in the absence of AChR-alpha chain. About 50% of T cell clones from MG patients, compared to none from NS, proliferated to AChR-alpha chain. This response was HLA-DR restricted. MG T cell clones did not display significant cytotoxic activity, as compared to control T cell clones. Our results indicate that in MG, 4B4+ regulatory T cells play their role in the pathogenesis of MG, not by cytotoxicity, but more likely by their ability to stimulate specific antibody production by B cells.

Characterization of a T suppressor cell line that downgrades experimental allergic encephalomyelitis in mice

A T-suppressor (Ts) cell line of CD8 phenotype was isolated from spleens of SJL/J mice that had recovered from experimental allergic encephalomyelitis (EAE) induced by injection of MBP-activated T cells. The Ts cell line inhibited the proliferation of MBP-sensitized T cells in vitro. Addition of recombinant IL-2 enhanced the Ts-mediated suppression. Adoptively transferred Ts line was able to downgrade EAE in mice subsequently challenged with MBP-activated T cells. The mechanism of suppression appeared to involve neither direct cytolysis of the effector T cells nor the production of a soluble suppressor factor. The findings suggest an in vivo role for suppressor T cells in the regulation of EAE.

Needlestick injury: Blood, mononuclear cells, and acquired immunodeficiency syndrome*

INTRODUCTION Because transmission of HIV to health care workers after needlestick injury has occurred mainly a result of deep insertion of large gauge needles, blood and viable mononuclear cells transferred after needlestick injury were measured. METHODS Needles of 20 to 27 gauge were filled with HIV-1 seropositive blood and inserted through extracorporeal human skin or parafilm covering physiologic saline solution modified Drabkins solution, or culture medium, or inserted directly into one of these fluids, to a depth of one third of the needle length (0.5 inch) for 1 second. Volume of blood transferred was measured by both modified Drabkins method and by chromium 51 labeling of red blood cells. Transfer of viable mononuclear cells was measured by growth in culture medium containing autologous feeder cells. RESULTS The volume of blood transferred from a needle passed through skin varied from 312 +/- 69 nl from a 20-gauge needle to 14 +/- 4 nl from a 27-gauge needle, as measured by modified Drabkins technique, and from 404 +/- 80 nl to 12 +/- 3.1 nl, as measured by chromium 51 labeling of red blood cells. The volume of blood transferred from a needle passed through parafilm was twice that transferred through skin. The volume of blood transferred through skin was 40% that transferred directly into fluid not covered by any barrier; blood transferred through parafilm was 80% of that transferred directly. When needles containing blood were inserted into culture medium for 1 second in the absence of a barrier, at least one viable mononuclear cell was almost always transferred to fluid from all gauges of needle tested. Insertion of needles through skin prevented transfer of all viable mononuclear cells from only 3% to 5% of 20- to 23-gauge needles, and from 12% to 32% of 26- and 27-gauge needles. Parafilm was an even less effective barrier than skin. Insertion of needles through parafilm completely prevented transfer of viable mononuclear cells from no 20- to 23-gauge needles and from only 5% to 10% of 26- and 27-gauge needles. CONCLUSION The volume of blood transferred after needle insertion through skin for 1 second varied with the gauge of the needle and was 30-fold higher from a 20-gauge than from a 27-gauge needle. Variable mononuclear cells were transmitted after insertion through skin from more than 95% of 20- to 23-gauge needles and from 68% to 88% of 26- and 27-gauge needles. Parafilm was less effective than skin in reducing transmission of blood and viable mononuclear cells.

Role of γδ T cells in antibody production and recovery from SFV demyelinating disease

Semliki Forest Virus (SFV) encephalomyelitis has been used to study the pathogenesis of virus-induced demyelination and serves as a model for multiple sclerosis. SFV-infection of mice invariably leads to clinical weakness accompanied by CNS inflammation, viral clearance and primary demyelination by day 21 postinfection (pi), followed by recovery and remyelination by day 35 pi. We have applied this model to the examination of the effects of γδ T cells in antibody production and the pathogenesis of demyelinating lesions. SFV-infection of γδ T cell KO mice resulted in more severe clinical signs than in wild type (WT) B6 mice. SFV-infected WT and γδ KO mice both cleared virus by day 10 pi and inflammation was comparable. Demyelination also appeared to be similar in both groups except that KO mice did not exhibit extensive remyelination which was seen in WT mice by day 21. SFV-infected WT mice showed widespread remyelination by day 35 pi, whereas KO mice still displayed some demyelination through day 42 pi. Both WT and KO mice developed serum antibodies to SFV. However, the reactivity of WT sera with the SFV epitope, E2 T(h) peptide₂, was significantly higher than in KO sera. Immunization with E2 T(h) peptide₂ resulted in elevated antibody production to this peptide (p<0.05) and earlier remyelination (day 28 pi) in KO mice. Thus, our study has shown for the first time that immunization of SFV-infected γδ T cell KO mice with a viral peptide, E2 T(h) peptide₂ led to enhanced recovery and repair of the CNS.