IntroductionHuman bone marrow stromal cells (MSCs), more recently referred to as mesenchymal stem cells, are capable of differentiating along multiple mesenchymal lineages in addition to supporting hematopoiesis. 1,2 Due to their potential for differentiation into osteocytes, chondrocytes, myocytes, and adipocytes, MSCs have emerged as a promising tool for clinical applications such as tissue engineering and cell and gene therapy. 3,4 MSCs are of inherently low immunogenicity and, more importantly, are capable of inhibiting allogeneic T-cell responses. 5-8 These intriguing observations have prompted clinical studies to investigate cotransplantation of MSCs in allogeneic hematopoietic stem cell transplantation (HSCT) in order to promote hematopoietic engraftment by preventing host-versusgraft reactivity and to suppress graft-versus-host reactions. 9,10 As of yet, the molecular mechanisms responsible for the immunosuppressive effects of MSCs have not been unequivocally identified. The reports describing a potential role of transforming growth factor-␤1 and hepatocyte growth factor as mediators of T-cell inhibition remain controversial, but most studies agree that soluble factors are involved. [6][7][8]11 In professional antigen-presenting cells (APCs), expression of indoleamine 2,3-dioxygenase (IDO) induced by interferon-␥ (IFN-␥) and other proinflammatory cytokines catalyzes conversion from tryptophan to kynurenine and has recently been identified as a major immunosuppressive effector pathway that inhibits T-cell responses to autoantigens and fetal alloantigens in vivo. [12][13][14][15][16] Based on these findings, we investigated whether MSCs exhibit IFN-␥-inducible IDO activity and whether this mechanism contributes to T-cell inhibition mediated by MSCs. Study design Culture of human bone marrow-derived MSCsBone marrow aspirates were harvested from volunteer donors who had provided informed consent; the study was approved by the institutional review board of the University Clinic, Düsseldorf, Germany. Primary human MSCs were generated as previously described 17 except that culture medium was supplemented with 3 ng/mL basic fibroblast growth factor (R&D Systems, Minneapolis, MN). Mixed lymphocyte reactions (MLRs)Standard 5-day MLR cultures were set up with 5 ϫ 10 4 mitomycin C-treated human peripheral blood mononuclear cells (PBMCs) as stimulators and 2 ϫ 10 5 human T cells purified using sheep red blood cell rosetting as responder cells. 5,11 In MSC/MLR coculture experiments, MLRs were performed on a layer of either 5 ϫ 10 3 or 2 ϫ 10 4 MSCs seeded one day before. IFN-␥ concentration was determined in MSC/MLR coculture supernatants using a commercially available enzyme-linked immunosorbent assay (ELISA; R&D Systems) according to manufacturer's instructions. Detection of IDO expression and activityMSCs were stimulated with IFN-␥ (R&D Systems) and assayed for IDO expression and function. Standard Western blot analysis for IDO protein expression was performed. 18 IDO enzyme activity following IFN-␥ stimulation of ...

It has been postulated that antibodies specific to the hypervariable region 1 (HVR1) within the putative envelop protein E2 of hepatitis C virus (HCV) can neutralize virus. We studied such antibodies in sera of patients who were infected in a single-source outbreak by a contaminated anti-D immunoglobulin preparation (HCV-AD78). The nucleotide sequences of cDNAs encoding HVR1 of HCV-AD78 were determined. The four major variants (HVR1.A, B, C, and D) were expressed as fusion proteins in Escherichia coli. Sixty-seven percent of sera contained antibodies to HVR1.A. Sera unrelated to infection of the outbreak also recognized HVR1.A but to a lesser extent (15%), suggesting that not all HVR1-specific antibodies are absolutely isolate-specific. Antibodies directed against individual variants of HVR1 were found in sera obtained early postinfection (p.i.) (< or = 1 year) but also in sera obtained several years later. An in vitro binding assay of HCV to tissue culture cells was employed to further characterize these sera. Five of seven sera that were obtained early p.i. prevented binding of HCV to cells. Preincubation of such sera with HVR1-specific fusion proteins restored binding of HCV to cells in four of five sera. These findings suggest that the majority of neutralizing antibodies are directed against HVR1.

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Hepatitis C virus (HCV) is a major cause of parenterally Antibodies directed to hypervariable region 1 (HVR1) transmitted acute and chronic hepatitis. 1 Upon infection, paof hepatitis C virus (HCV) have recently been shown to tients develop a chronic persistent infection in more than neutralize the corresponding HCV isolate in vitro. We 50% of these cases. 2 Antibody responses 3 and T-cell-medianalyzed the appearance of antibodies directed to HVR1 ated immune responses to various regions of the HCV polyduring the course of infection in a large group of paprotein 4,5 have been studied, but so far a prognostic marker tients who have been infected by the same isolate of a associated with acute self-limited or persistent infections has HCV contaminated anti-D immunoglobulin (HCV-AD78).not been found. An enzyme-linked immunosorbent assay (ELISA) was esVaccination studies of chimpanzees have shown the importablished using a synthetic peptide to detect antibodies tance of the immune response against the putative HCV enagainst the main HVR1 variant of HCV-AD78. 207 sera velope proteins E1/E2. 6 However, protection of animals deobtained at different time points post infection (p.i.) of pends on the HCV isolate used for challenge of immunized 51 patients having either acute self-limiting (n Å 28) or animals. In addition, reinfection occurred in chimpanzees 7 chronic infection (n Å 23) were studied. Antibodies diand was suggested for patients. [8][9][10] Isolate-specific, neutralizrected to HVR1 were found at least at one time point ing antibodies directed against E1 and E2 may be involved during the infection course in 15 of 28 patients (53%) in determination of the course of infection. Antibodies present having acute self-limiting infections and in 17 of 23 pain chronically infected patients after several years postinfectients (74%) with chronic disease. The time of appeartion (p.i.) fail to neutralize the viral variant present in the ance of anti-HVR1 was significantly different between infectious source 11 most probably because of an immune esthese two patient groups (P õ .025) although appearance cape of newly arising viral variants of hypervariable region and titers of other HCV-specific antibodies were found 1 (HVR1). 12 Sequences of HVR1 found in earlier time points to be similar at early time points p.i. In acute self-limof infection are rapidly mutated during chronic infection and iting infections 9 of 21 sera (43%) of respective patients followed by respective, HVR1-specific antibody response. 13,14with sera available within the first 6 months p.i. were A monoclonal antibody directed against HVR1 was capable anti-HVR1 positive. The highest prevalence of antiof HCV in vitro neutralization. 15 Antibodies that neutralize HVR1 in this group of patients was within month 6 to HCV have also been observed in sera of HCV-infected pa-12 p.i. (64%). None of the sera available after 24 months tients. 11,16 Such neutralizing antibodies in patient sera are p.i. had such antibodies. In contrast, only 2 of 15 sera mainly direc...

In foot-and-mouth disease virus (FMDV)-infected cells, the disappearance of nuclear protein histone H3 and the simultaneous appearance of a new chromatin-associated protein termed Pi can be observed (P. R. Grigera and S. G. Tisminetzky, Virology 136:10-19, 1984). We sequenced the amino terminus of protein Pi and showed that Pi derives from histone H3 by proteolytic cleavage. The 20 N-terminal amino acid residues of histone H3 are specifically cleaved off early during infection. Truncated histone H3 remains chromatin associated. In addition, we showed that the histone H3-Pi transition is catalyzed by the FMDV 3C protease. The only known function of the viral 3C protease was, until now, the processing of the viral polyprotein. The viral 3C protease is the only FMDV protein required to induce the histone H3-Pi transition, as well as being the only viral protein capable of cleaving histone H3. No viral precursor fusion protein is needed for this specific cleavage as was reported for the processing of the poliovirus P1 precursor polyprotein by 3C/D protease. As the deleted part of the histone H3 corresponds to the presumed regulatory domain involved in the regulation of transcriptionally active chromatin in eucaryotes, it seems possible that this specific cleavage of histone H3 is related to the host cell transcription shutoff reported for several picornaviruses.

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SummaryWilson disease (WD) is an autosomal recessive disorder resulting from mutations in the ATP7B gene, with over 600 mutations described. Identification of mutations has made genetic diagnosis of WD feasible in many countries. The heterogeneity of ATP7B mutants is, however, yet to be identified in the Indian population. We analyzed the mutational pattern of WD in a large region of Western India. We studied patients (n = 52) for ATP7B gene mutations in a cohort of families with WD and also in first-degree relatives (n = 126). All 21 exon-intron boundaries of the WD gene were amplified and directly sequenced. We identified 36 different disease-causing mutations (31 exonic and five intronic splice site variants). Fourteen novel mutations were identified. Exons 2, 8, 13, 14, and 18 accounted for the majority of mutations (86.4%). A previously recognized mutation, p.C271 * , and the novel mutation p.E122fs, were the most common mutations with allelic frequencies of 20.2% and 10.6%, respectively. Frequent homozygous mutations (58.9%) and disease severity assessments allowed analysis of genotype-phenotype correlations. Our study significantly adds to the emerging data from other parts of India suggesting that p.C271 * may be the most frequent mutation across India, and may harbor a moderate to severely disabling phenotype with limited variability.

Mouse fibroblast cell lines were transfected with truncated forms of the human poliovirus receptor (PVR) cDNA and tested for the expression of functional receptors for poliovirus. Several receptor constructs, all containing the coding region of the first 143 amino acids of PVR, were able to render mouse cells susceptible to poliovirus infection. A deletion of 65 amino acids in the first extracellular domain of PVR prevented virus attachment and infection. These data suggest that domain 1 is necessary and sufficient for the virus-receptor interaction. A PVR/intercellular adhesion molecule 1 hybrid receptor, expressing the PVR variable domain on a truncated receptor molecule for human rhinovirus 14, was shown to be a functional receptor for poliovirus. This observation indicates that, subsequent to attachment to the PVR-binding domain, poliovirus can use the same pathway as the major receptor group rhinoviruses to enter cells.The cellular receptor for poliovirus (PVR) has recently been identified as a protein belonging to the immunoglobulin supergene family (1,2). The sequence of cDNA encoding PVR revealed a polypeptide with a predicted molecular mass of 46 kDa prior to glycosylation. This polypeptide consists of three extracellular domains characteristic for certain immunoglobulin-like proteins (3) in the order V-C2-C2 (where V = variable and C = constant) that are followed by a hydrophobic membrane-spanning segment and a C-terminal tail ( Fig. 1; PVR dl + 2 + 3). The extracellular portion ofthe PVR carries eight potential N-glycosylation sites (see Fig. 2), and, indeed, the protein migrates as a 67-kDa polypeptide when HeLa cell membranes are subjected to Western blot analyses (4) or when PVR-specific cDNA is expressed in vivo by means of a vaccinia virus vector (4) or in a baculovirus expression system (5) and in vitro by translation in a rabbit reticulocyte extract in the presence of dog pancreas microsomes (4). The molecular parameters of the interaction between poliovirus and its receptor, however, have not been elucidated.Poliovirus is a small RNA virus belonging to the genus Enterovirus of the family Picornaviridae. Another genus of this family is Rhinovirus, which encompasses two groups: the minor receptor group and the major receptor group rhinoviruses, of which the latter represents >80% of all known rhinoviruses (6, 7). Although entero-and rhinoviruses are very closely related with respect to their structure (8) and molecular biology (9), they use a variety of receptors most of which remain unknown (reviewed in ref. 10). However, the receptor for the major group rhinoviruses has recently been identified to be intercellular adhesion molecule 1 (ICAM-1) (11)(12)(13). Like PVR, it is a protein of the immunoglobulin supergene family, but it differs from PVR in that ICAM-1 consists of five C2-like folds (Fig. 1).The pathway of penetration and uncoating of all picornaviruses is poorly understood. Evidence has been presented suggesting that after poliovirus and rhinovirus attach to their respective re...