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Amyloid hexapeptide molecules are effective in the treatment of the murine model of neuroinflammation, known as experimental autoimmune encephalomyelitis (EAE). Efficacy however differs between two inbred mouse strains, C57BL/6J (B6) and C57BL/10SnJ (B10). Amyloid hexapeptide treatments improved the clinical outcomes of B6, but not B10 mice, indicating that genetic background influences therapeutic efficacy. Moreover, although previous studies indicated that prion protein deficiency results in more severe EAE in B6 mice, we observed no such effect in B10 mice. In addition, we found that amyloid hexapeptide treatments of B10 and B6 mice elicited differential IL4 responses. Thus, the modulatory potential of prion protein and related treatments with other amyloid hexapeptides in EAE depends on mouse strain.

Statins, inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase, are widely prescribed for their cholesterol-lowering properties to reduce atherogenesis and cardiovascular morbidity. Over recent years, statins have also been shown to exert pleiotropic immunomodulatory effects that might be of therapeutic benefit in autoimmune disorders. The primary mechanism by which statins alter immune function appears to be mediated through the inhibition of post-translational protein prenylation of small GTP-binding proteins and is largely independent of lipid-lowering. In experimental autoimmune encephalomyelitis (EAE), the mouse model for multiple sclerosis (MS), statins prevent or reverse paralysis and were recently shown to exert synergistic benefit when combined with agents approved for MS therapy. Based primarily upon the beneficial effects in EAE, statins are now being tested in patients in MS clinical trials.

Along with their established role in allergic reactions, histamine and its receptors have been implicated in the pathology of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis. In this study we analyzed the gene expression of histamine receptor 1 (HRH1), HRH2 and HRH4 in peripheral blood mononuclear cells derived from patients with clinically isolated syndrome (CIS), relapsing-remitting (RR) MS, secondary-progressive (SP) MS, primary-progressive (PP) MS, and healthy controls (HC). We found that HRH1 transcript was significantly down-modulated in SP-MS compared with HC, and HRH4 was increased in this group compared to HC, CIS and RR-MS. No other differences in the expression of histamine receptors were observed between HC, CIS and other clinical forms of definite MS.

T-cells specific for a region of human myelin basic protein, amino acids 87-99 (hMBP87-99), have been implicated in the development of multiple sclerosis (MS) a demyelinating disease of the central nervous system. Administration of soluble altered peptide ligand (APL), made by substituting native residues with alanine at either positions 91(91K > A or A91) or 97 (97R > A or A97) in the hMBP87-99 peptide, blocked the development of chronic relapsing experimental autoimmune encephalomyelitis (R-EAE), in the SJL mouse. The non-encephalitogenic APL A91, appears to induce cytokine shifts from Th1 to Th2 in the target T-cells, whereas the encephalitogenic superagonist APL A97 causes deletion of the MBP87-99 responsive cells. Thus, single amino acid changes at different positions in the same peptide epitope can lead to APL capable of controlling auto-immune disease by different mechanisms.

Oligonucleotide probes were used to investigate the role of DQβ molecules in susceptibility to multiple sclerosis. Although shared amino acid and nucleotide sequences in DQβ1 have been suggested to be critical in disease development, we find that the distribution of sequences corresponding to residues 71–77 is not greater in patients versus controls.

Classically envisioned as a key mediator in allergic reactions, histamine has been recently suggested to play a role in central nervous system (CNS) autoimmune responses occurring in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), animal model for this disease. Studies performed with histamine receptors or histidine-decarboxylase knockout mice have shown that histamine can play promoting or protective roles in EAE depending on the receptors engaged. Moreover, transcript levels of histamine receptors are significantly modulated in peripheral T cells in different stages of EAE. In order to determine whether these findings could be of relevance for the human disease, in the present study we analysed the gene expression of histamine receptor 1 (HRH1), HRH2 and HRH4 in peripheral blood mononuclear cells derived from patients with clinically isolated syndrome (CIS), relapsing–remitting (RR), secondaryprogressive (SP), primary-progressive (PP) MS, and healthy controls (HC). HRH3 has not been included in our study, as its expression is restricted to the CNS. Histamine receptors were all expressed at mRNA level and HRH2 transcripts were always more abundant than those for HRH4 and HRH1 in the analysed groups. Interestingly, we found that HRH1 transcript was significantly downmodulated in SP-MS compared with HC (p b 0.05), and HRH4 was increased in this group compared to HC (p b 0.05), CIS (p= 0.01) and SM-RR (p b 0.05). No other differences in the expression of histamine receptors were observed between HC, CIS or other clinical forms of definite MS. These results show that histamine receptors are differentially expressed in SP-MS.

MicroRNA (miR) as part of the epigenetic machinery regulates cellular fate by binding to target messenger RNA (mRNA) and thus inhibiting gene expression and their deregulation has been associated with various diseases. Due to their presence in several body fluids miRs are possible candidates to serve as diagnostic markers. We have previously identified upregulated miR-181c in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients in contrast to patients with other neurologic diseases (OND). However, the effects of this deregulation on central nervous system (CNS) tissue remain unknown. In this study we examined the effect of miR181c overexpression on astroglia. Therefore a pure culture of astrocytes was established from newborn wild-type C57/Bl6 mice. For stable expression cells were transduced with vectors containing the sequence of MS-related miR-181c, control miR-21, which is upregulated in glioma and a non-coding vector. We quantified cell proliferation over one week with and without activation of overexpression (n = 3) using a real-time cell analyser. The comparison between the transduced but not activated astrocytes and the tetracycline-treated and therefore miR-overexpressing cells shows no difference in cell proliferation for all vectors used. Our findings indicate that the overexpression of MS-associated miR-181c has no influence on the proliferation of murine astrocytes in vitro. Although we show no direct effect on astrocyte proliferation under normal culture conditions, possible effects either could become visible under disease-like stress signaling conditions or could be mediated indirectly by other CNS cells in vivo. Additional investigations of effects on remaining CNS cells and other MSassociated miR are warranted to determine potential therapeutic approaches. Although the role of miR-181c in MS pathomechanism is yet to be determined we considered it as a potential diagnostic marker.

The expression of leukocyte adhesion molecules in the intact brains of mice with experimental autoimmune encephalitis (EAE) was visualized by Magnetic Resonance Imaging (MRI) through the use of a new, target-specific MR contrast agent. Antibody-conjugated paramagnetic liposomes (ACPLs) were designed to achieve in vivo targeting of molecules expressed on vascular endothelium, while providing sufficient signal enhancement at these sites for detection by MRI. ACPLs targeted to intercellular adhesion molecule-1 (ICAM-1), an endothelial leukocyte receptor upregulated on cerebral microvasculature during EAE, were administered to diseased mice. Fluorescence microscopy confirmed that fluorescently-tagged ACPLs were localized to central nervous system (CNS) microvasculature in a pattern consistent with ICAM-1 upregulation described immunohistochemically. High resolution MRI of mouse brains ex vivo demonstrated that ACPL binding conferred significant enhancement of signal intensity (SI) as compared to control images. These results suggest that ACPLs can be used as MRI contrast agents to visualize specific molecules expressed on vascular endothelium during disease.

We used competitive polymerase chain reaction to quantify messenger RNA for the lymphocyte antigens CD4 and CD8, the adhesion molecules ICAM-1 and VCAM-1, and the MHC class II I-A molecule in the spinal cords of SJL/J mice at multiple times during the development and resolution of experimental allergic encephalomyelitis (EAE). CD4 and CD8 were not quantifiable at baseline, became detectable at 5 days after immunization, and increased steadily to a peak during clinical disease. I-A increased after CD4 and CD8, but before onset of disease. ICAM-1 and VCAM-1 did not increase until after onset of clinical disease. CD4, CD8, and I-A remained elevated long after recovery from disease. These results suggest that infiltration of CD4 and CD8 cells into the spinal cord and subsequent upregulation of I-A mRNA play an important role in the development of EAE, but reversal of these processes is not necessary for recovery. Upregulation of ICAM-1 and VCAM-1 mRNA does not appear to be important for development of disease.

The identification of activated T cells in the brains of patients with multiple sclerosis (MS) suggests that these cells are critical in the pathogenesis of this disease. Recently we have used the PCR method to analyse rearrangements of V alpha and V beta genes of the T cell receptor (TCR) in samples of MS and control brains. The results of these studies showed that TCR V gene usage in MS brains may be restricted and in particular that V beta genes may be preferentially rearranged in certain HLA haplotypes associated with susceptibility to MS. In view of the recent evidence that T lymphocytes bearing the gamma delta chains may have autoreactive potential, we have assessed whether or not such TCR-bearing lymphocytes were also present in chronic MS lesions. TCR V gamma and V delta were analysed by the PCR method using a panel of V gamma and V delta primers paired with C gamma or C delta primers in 12 MS brains, as well as in brain samples of ten normal post-mortem cases and three neurological controls. TCR V gamma-C gamma and V delta-C delta rearrangements were confirmed using Southern blotting and hybridisation of the PCR products with specific C gamma and C delta probes. Only one to four rearranged TCR V gamma and V delta transcripts were detected in each of the 23 brain samples obtained from 12 MS patients, with the majority of gamma delta T cells expressing the V gamma 2 and V delta 2 chains. In marked contrast, V gamma and V delta transcripts could only be found in one of the ten non-neurological control brains analysed. To assess the clonality of V gamma 2 and V delta 2 T cell receptor chains in the brain samples of MS patients, we have sequenced the junctional regions of the TCR V gamma-N-J gamma-C gamma and V delta-N-D delta-N-J delta-C delta segments amplified from brain tissues, CSF and spleens of two MS patients and from the spleen of two control subjects. The sequence analysis obtained so far shows no compelling evidence of an MS specific expansion of one or more clones expressing particular types of gamma delta T cell receptors. In contrast, a clonal expansion of a different population of TCR gamma delta-bearing T cells was found in the spleen of both an MS patient and one of the control individuals.(ABSTRACT TRUNCATED AT 400 WORDS)

Experimental allergic encephalitis (EAE) can be prevented with the in vivo administration of monoclonal anti I-A antibody. A radiometric assay was developed to measure the accumulation of lymphocytes in the central nervous system of EAE animals. A direct correlation was observed between severity of clinical disease and the amount of radiolabelled lymph node cells (LNC) in the central nervous system. Injection of anti I-A antibody in vivo prevented clinical EAE and decreased the accumulation of radiolabelled LNC in spinal cord after immunization with mouse spinal cord homogenate and adjuvants.