In this study, we investigated the role of the inducible form of heat shock protein 70 (hsp70) in the presentation of the major putative autoantigen in multiple sclerosis, myelin basic protein (MBP), in the context of appropriate MHC class II. By coimmunoprecipitation, we found that MBP is associated with hsp70 in APC in an ATP/ADP-dependent manner. Additionally, using confocal microscopy, hsp70 was detected in the endocytic pathway of APC, where it colocalized with MBP and HLA-DR. The immunodominant epitopes of MBP 85–99 and 80–99 were shown to bind selectively and specifically to hsp70 by surface plasmon resonance. The functional significance of MBP interaction with hsp70 was demonstrated by the detection of enhanced responses of an MBP-specific T cell hybridoma to MBP and MBP 80–99 with increasing levels of hsp70 and reduced responses when hsp70 expression was diminished within APC-expressing DRA*0101, DRB1*1501 (DR1501). However, when MBP 85–99 was used as the stimulus, T cell hybridoma responses were not enhanced by hsp70 overexpression within APC, suggesting that hsp70 contributes to Ag processing rather than Ag presentation. The importance of a direct association between MBP and hsp70 in the presentation pathways was demonstrated by enhanced efficacy of MBP presentation by APC transfected with a plasmid vector encoding a fusion hsp70-MBP protein. This is the first report on the involvement of self-inducible hsp70 in MHC class II-dependent autoantigen processing by APC. It implicates that aberrant self hsp expression may lead to the enhancement/modulation of autoimmune responses.

Intracellular adhesion molecule-1 (ICAM-1) plays an important role in the cascade of adhesion events in the homing of inflammatory cells to the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Two single-base ICAM-1 polymorphisms have been described, in exons 4 and 6, changing codons 241 and 469 in the ICAM-1 gene, respectively. Both polymorphisms result in amino acid changes and can potentially lead to different interactions of ICAM-1 with its ligands. To detect ICAM-1 gene polymorphisms in MS, we have developed a highly sensitive and site-specific, two-stage, nested polymerase chain reaction. Genomic DNA was extracted from blood cells of 79 MS patients and 68 control subjects. The results were confirmed by direct dideoxy chain termination sequencing. The frequency of exon 6 allele T was found to be significantly higher in MS patients than in controls (68% vs 49%). Most interesting, the frequency of exon 6 homozygote K469 was significantly higher in MS patients than in controls (53% vs 34%). Higher frequency of the K469 genotype was found to be independent of possible linkage with the previously described MS susceptibility factor, the HLA class I1 DR2 allele. In the present study, we have shown for the first time the ICAM-1 gene polymorphisms in MS. The results indicate increased frequency of ICAM-1 exon 6 allele T in MS patients, which may contribute to the MS genetics background. According to structural similarities, adhesion molecules are divided into the selectins, the integrin and the Ig superfamilies, the addressins, the cadherins, and the CD44.6 During the T cell-EC interactions, the selectins are responsible for early contact and for lymphocyte "rolling" along the blood vessels.' The subsequent, firm adhesion event is mediated by the integrins expressed on activated leukocytes and Ig-like adhesion molecules expressed on ECs8 The Ig superfamily contains intercellular adhesion molecules (ICAMs), ie, ICAM-1, ICAM-2, and ICAM-3, vascular cellular adhesion molecule-1 (VCAM-I), and leukocyte functionassociated antigen-3 (LFA-3).' ICAM-1 expression was shown to be significantly up-regulated by treating brain ECs with Thl-type cytokines, ie, interferon-?, interleukin-1 a, and tumor necrosis factor, which have been shown to be involved in the pathogenesis of MS and its animal model, experimental autoimmune encephalomyelitis (EAE). 13,14 ICAM-1 interacts with leukocyte function-associated antigen-1 (LFA-1) and with another p2 integrin, Mac-l.'-'In MS and EAE it has been shown that most important in T-cell transendothelial migration are interactions of LFA-1 with ICAM-1 and of very late activation antigen-4 (VLA-4) with 5,15 Increased levels of soluble ICAM-1 have been detected in blood from MS patients, the levels tend to rise in association with the disease clinical and magnetic resonance imaging (MRI) activities.'".'' In actively induced EAE, anti-ICAM-1 antibody injections reduced the severity of clinical signs and pathological changes.I8 Moreov...

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A b s t r a c tIntroduction: TP53 and MGMT alterations play a crucial role in glioblastoma (GB) pathogenesis. TP53 and MGMT function is affected by several pathologic mechanisms, such as point mutations or promoter methylation, which are well characterized. Expression of both genes can be regulated by other mechanisms as well, e.g., microRNAs (miRNAs). Moreover, cross-talk among various pathologic processes may occur, further affecting MGMT and TP53 functionality. Material and methods: In 49 GB patients, we analyzed the possible associations between TP53 and its miRNA regulators miR-125b, miR-21, and miR34a, as well as MGMT and its miRNA regulators miR-181d and miR-648. We evaluated the possible influence of mutational and methylation status on the pre-identified associations. Results: In patients with immunohistochemistry-detected TP53 overexpression, expression levels of miR-34a and TP53 were negatively correlated (r = -0.56, p = 0.0195), and in patients with TP53 mutations, expression levels of TP53 and miR-21 were negatively correlated (r = -0.67, p = 0.0330). In patients with MGMT methylation, expression levels of MGMT were negatively correlated with miR-648 and miR-125b expression levels (r = -0.61, p = 0.0269 and r = -0.34, p = 0.0727, respectively).Conclusions: Our findings demonstrate that selected miRNAs are significantly correlated with MGMT and TP53 levels, but the extent of this correlation differs regarding the TP53 and MGMT mutational and promoter methylation status.

The molecular mechanisms that regulate the immune function of bone marrow–derived mesenchymal stem cells (BMSCs) are not known. We have shown previously that freshly isolated BMSCs when induced to express neuronal stem cell markers lose immunoregulatory function when transferred into mice sensitized to develop experimental autoimmune encephalomyelitis. Recently, microRNAs (miRs) have been shown to be involved in the regulation of several immune responses in both innate and acquired immunity. We now show that among several differentially expressed miRs, miR-146a was strongly upregulated in neuronally differentiated when compared with miR-146a expression in freshly isolated BMSCs or control BMSCs cultured in parallel but in nondifferentiating medium. Inhibition of miR-146a with a selective antagomir restored the immunoregulatory activity of nBMSCs. We mapped miR-146a to its multiple predicted target mRNA transcripts and found that miR-146a was predicted to block PGE2 synthase (ptges-2). We then showed that Ptges-2 was directly targeted by miR-146a using a luciferase reporter assay. Furthermore, increased expression of miR-146a in BMSCs correlated with inhibition of PGE synthase-2 and inhibition of PGE2 release. Accordingly, inhibition of miR-146a restored synthesis of PGE2. These data support the conclusion that miR-146a plays a critical role in the control of the immunoregulatory potential of BMSCs.