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Abstract: In renal transplantation, the presence of anti-donor HLA antibodies is associated with early rejection and accelerated graft loss. The clinical relevance of anti-HLA antibodies can be evaluated in the crossmatch assay using either a complement-dependent cytotoxicity (CDC) assay or a flow cytometric crossmatch (FCXM) method. The FCXM technique is more sensitive than CDC-based assays for detection of anti-donor antibodies and allows the simultaneous detection of antibodies against T-lymphocytes (anti-HLA class I antibodies) and B-lymphocytes (anti-HLA class I and/or HLA class II antibodies). Although the clinical relevance of a positive FCXM using T-lymphocytes in kidney graft outcome is well established, there is still debate about the clinical significance of a positive B-cell only FCXM (B+FCXM). In this review we discuss several factors to consider during the evaluation of patients with a B+FCXM and suggest ideas to improve the use of the information provided by the FCXM assay. [Copyright &y& Elsevier]

Infection as an environmental factor in autoimmunity has long been recognized. Numerous examples can be found in which pathogens express antigens that cross-react with host antigens or induce local inflammatory responses that can lead to autoimmune responses through a very complex set of circumstances. Borrowing from the relationship between chronic infection with hepatitis C virus and autoimmune hepatitis as an example, we consider the possibility that infection with an unknown virus having specific tissue tropism could lead to a perceived autoimmune process. We raise the question whether such should be considered for Type 1 diabetes. [Copyright &y& Elsevier]

Hepatitis C virus specific (HCV-specific) CD8+ cytotoxic T cells play a critical role in viral clearance. Low HCV-specific cytotoxic T lymphocyte (CTL) responses in chronic HCV infection may favor the persistence of virus, whereas stimulation and expansion of HCV-specific CTL activity may assist elimination of HCV infection. Helper T cells control the intensity of CD8+ T-cell responses and helper T-cell responses are known to be compromised in chronic carriers of HCV. In this study, we wanted to ascertain if strengthening the Th response could increase the intensity of CTL activity against HCV target antigens. We selected a synthetic CTL peptide NS31073-1081), two Th1 epitopes, peptide NS3358-375 and NS5B155-172, and one Th2 epitope, peptide NS3505-521. By using the four peptides alone or in combinations, we stimulated peripheral blood cells isolated from a chronic hepatitis C patient in vitro and then analyzed CD8 T cells specific for the NS31073-1081 CTL epitope in A2 tetramer staining and cytotoxicity assays. The results demonstrated that CTL responses could be augmented by helper T-cell epitopes NS3358-375 and NS5B155-172. Th2 epitope NS3505-521 inhibited augmentation of CTL activity by Th1 epitopes. This inhibitory effect could be overcome by combining the two Th1 epitopes NS3358-375 and NS5B155-172 together with NS3505-521. Under such conditions, CTL frequency was restored, but cytotoxic activity remained low suggesting that the help provided under these cultures was sufficient to drive proliferation of CTL, but not sufficient to drive differentiation into mature killer cells. These results may provide some insights into compromised CTL activity in HCV viral persistence. [Copyright &y& Elsevier]

Summary Hepatitis C virus (HCV) infects more than 180 million of the world’s population and causes a persistent infection that over decades can result in cirrhosis and hepatocellular carcinoma. Treatment is only partially effective and control is likely only with the development of effective vaccines. Currently, only chimpanzees can be infected with HCV and alternative animal and tissue culture models are badly needed. We have used mice transgenic for HLA-DR and human CD4 to analyse the specificity of murine responses to the HCV NS3 antigen in an effort to determine whether the epitopes recognized correspond to those recognized by human T cells. Indeed, determinants mapped in transgenic mice overlap with those in a patient exposed to HCV through infection. This result provides hope that such an animal model may be a useful tool with which to analyse particular aspects of immune responses to HCV in vivo . [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is yet another example of a pathogen that persists in the presence of a readily apparent immune response. A evidence for both humoral and cellular immune responsiveness is quite strong, our studies have begun to examine whether qualitative defects in CD4 T-cell responses to viral antigens may help to explain why HCV is not eliminated in the vast majority of infections. Direct evidence that CD4 T cells play a role in HCV persistence is lacking, but several observations are consistent with this possibility. Importantly, it does not exclude the role of antibody or killer T cells in the immunopathogenesis of HCV infection. In addition, we discuss the consequences of viral mutation and how naturally occurring variants in immunodominant viral epitopes can effectively suppress helper T-cell responses to wild type virus. [ABSTRACT FROM AUTHOR]

Abstract: Whether sensitized patients wait for a compatible crossmatch with a deceased donor, enter a paired exchange program with the hope of finding a compatible living donor, or go through a desensitization protocol depends on a number of factors, not the least of which is the overall philosophy of the transplant center. Centers such as ours take the position that donor-directed antibodies detected by solid phase assays (even those that are “weak”) present an unacceptable risk factor to the patient. This philosophy is predicated on the biologic role of the immune system, specifically that antibodies were generated in response to a non-self (allo) antigen and that a successful immune response eliminates that which caused its stimulation. Although obviously an oversimplification, this philosophy mandates a comprehensive evaluation of HLA antibodies in sensitized recipients. This article addresses the challenges and conundrums associated with human leukocyte antigen antibody identification. [Copyright &y& Elsevier]

Abstract: The premise of this work is that within a given hepatitis C virus (HCV) protein there exists an array of Th1 and Th2 epitopes, each of which can provide synergistic (positive or negative) effects upon other epitopes by intramolecular, cytokine-mediated immunoregulation of helper T-cell responses. To address this question, we constructed minigene plasmids pHCVTh1, pHCVTh1X3 and pHCVThR, and HCV NS3 full-length plasmid pHCVNS3. 293T cells were transfected with these plasmids and cell lysates from the transfected cells were used to stimulate PBMC from a patient with chronic HCV infection. IL-2 and IFN-γ in the supernatant of the cultured PBMC were tested and proliferation of the PBMC was measured. The results demonstrate that interactions exist among helper T-cell epitopes; the synergistic effects of suppressive Th2 epitopes upon Th1 epitopes will inhibit the responses induced by Th1 epitopes, which may contribute to chronic infection by HCV; synergistic effects among Th1 epitopes induce higher levels of IFN-γ, which may suggest a new strategy for HCV vaccine development. Further, stimulation of an HCV NS3 specific clone with cell lysates from 293T cells transfected with different constructs shows that the HCV NS3 clone could respond to all suggesting that the epitope-specific suppression may be due to an imbalance of Type 1 and Type 2 cytokines or regulatory T-cells. [Copyright &y& Elsevier]

A disturbing feature of hepatitis C virus (HCV) is its long-term persistence in roughly 85% of those infected. Escape mutants may play a major role in HCV persistence. Our previous studies have identified a human leukocyte antigen DRB1*15 (HLA-DRB1*15) restricted Th1 epitope in the HCV NS3 protein, NS3358-375, and escape variants of this epitope that may emerge under immune selection. Such variants attenuate or fail to stimulate T-cell proliferation. Here we provide data from peripheral blood mononuclear cells derived from four HLA-DRB1*15 patients chronically infected with HCV, and report that naturally occurring single amino acid substitutions in the Th1 epitope NS3358-375 fail to stimulate proliferation, which is accompanied by a shift in cytokine secretion patterns from one characteristic of a Th1 antiviral responses to a Th2 form. Further, in one patient, we demonstrate that HCV variant peptides can effectively inhibit host polyclonal peripheral T-cell proliferation. We speculate that this phenomenon may be a factor in chronic HCV infection. [Copyright &y& Elsevier]

Background Immune allosensitization can be triggered by continuous-flow left ventricular assist devices (CF LVAD). However, the effect of this type of allosensitization on post-transplant outcomes remains controversial. This study examined the post-transplant course in a contemporary cohort of patients undergoing transplantation with and without LVAD bridging. Methods We included consecutive patients who were considered for cardiac transplant from 2006 to 2015. Serum alloantibodies were detected with single-antigen beads on the Luminex platform (One Lambda Inc., Canoga Park, CA). Allosensitization was defined as calculated panel reactive antibody (cPRA) > 10%. cPRA was determined at multiple times. LVAD-associated allosensitization was defined as development of cPRA > 10% in patients with cPRA ≤ 10% before LVAD implantation. Post-transplant outcomes of interest were acute cellular rejection (ACR), antibody-mediated rejection (AMR), and survival. Results Allosensitization status was evaluated in 268 patients (20% female). Mean age was 52 ± 12 years, and 132 (49.3%) received CF LVADs. After LVAD implant, 30 patients (23%) became newly sensitized, and the level of sensitization appeared to diminish in many of these patients while awaiting transplant. During the study period, 225 of 268 patients underwent transplant, and 43 did not. A CF LVAD was used to bridge 50% of the transplant recipients. Compared with patients without new sensitization or those already sensitized at baseline, the patients with LVAD-associated sensitization had a higher risk of ACR ( p = 0.049) and higher risk of AMR ( p = 0.018) but a similar intermediate-term post-transplant survival. The patients who did not receive a transplant had higher level of allosensitization, with a baseline cPRA of 20% vs 6% in those who received an allograft and a high risk (40%) of death during follow-up. Conclusions New allosensitization takes place in > 20% of patents supported with CF LVADs. Among patients who undergo transplant, this results in a higher risk of ACR and AMR, but survival remains favorable, likely due to the efficacy of current management after transplant. However, mortality in sensitized patients who do not reach transplant remains high, and new approaches are necessary to meet the needs of this group of patients. [ABSTRACT FROM AUTHOR]

Abstract: Shellfish allergy affects approximately 2% of the population and can cause immediate hypersensitivity reactions such as urticaria, swelling, difficulty breathing, and, in some cases, anaphylaxis. Tropomyosin is the major shrimp allergen and binds IgE in two-thirds of patients. A total of 38 shrimp-allergic patients and 20 negative control subjects were recruited and evaluated on the basis of history, skin prick testing, specific immunoglobulin E (IgE) levels, and peripheral blood mononuclear cell proliferation in response to shrimp tropomyosin or shrimp tropomyosin–derived peptides. Of the classically allergic patients by history, 59% tested positive for serum shrimp IgE antibodies. Of patients with shrimp-specific IgE in sera, 70% also had significant IgE levels specific for shrimp tropomyosin. Peripheral blood mononuclear cells from classically shrimp-allergic patients proliferated in a dose-dependent manner in response to to tropomyosin. In addition, a T-cell line derived from a shrimp-allergic patient proliferated specifically in response to tropomyosin-derived peptides. These studies suggest a strategy for immunotherapy using a tropomyosin-derived T-cell epitope vaccination. [Copyright &y& Elsevier]

Abstract: Immune-mediated diseases [multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE)] are driven by proliferating, highly activated autoreactive T-cells that are unresponsive to in vivo immunoregulatory mechanisms. The compound Lenaldekar (LDK) was identified in a zebrafish screen by inhibiting T-cell expansion. By monitoring mitogen- and antigen-driven proliferation, we found that LDK inhibited human and murine T-cell expansion in a non-cytolytic manner. This suppressive activity directly correlated with the degree of activation/proliferation of the T-cells. In testing LDK in an EAE model of MS, exacerbations were suppressed in treated animals. Therefore, LDK represents a novel therapeutic approach to T-cell-mediated autoimmune diseases. [Copyright &y& Elsevier]

Abstract: Hepatitis C virus (HCV) has a high rate of replication and lacks RNA-proofreading capabilities, thereby leading to variant or mutant viruses circulating within the host as a quasispecies. Previous work in our laboratory identified viral variants that emerged in a class-II immunodominant epitope NS3358-375 of the non-structural-3 (NS3) protein region of HCV, the sequence of which is based on genotype 1A, the most prevalent genotype in the United States. Further work suggested that positive immune selection pressure was driving viral variation. Paradoxically, viral variants account for only a small percentage of the circulating virus in human beings and in chimpanzees, suggesting that passive evasion is not the only means of escape by HCV. This observation suggests a unique pathogenesis for HCV as it persists in the host. In the current study, we hypothesize that viral variants are acting as altered peptide ligands (APLs). To test this hypothesis, we used cloned T cells specific for NS3358-375 peptide, which demonstrated attenuated T-cell and interferon-γ (IFN-γ) responses to individual variant peptides, when compared with the NS3358-375 stimulated T-cell clones. Furthermore, such variants could act as APLs, based on their ability to antagonize the IFN-γ proliferative responses of clones specific for NS3358-375. In addition, major histocompatibility complex (MHC) class II tetramer staining demonstrated that variant peptide–MHC complexes were able to specifically bind to NS3358-375 T-cell clones and that both the variant and NS3358-375 tetramers were able to bind to the same CD4+ T cells. Taken together, the results suggest that viral variants may act as APL to effectively blunt the T-cell response to an important HCV epitope. [Copyright &y& Elsevier]

Regulatory T cell markers are increased in chronically infected individuals with the hepatitis C virus (HCV), but to date, the induction and maintenance of Tregs in HCV infection has not been clearly defined. In this paper, we demonstrate that naturally occurring viral variants suppress T cell responses to cognate NS3358-375 in an antigen-specific manner. Of four archetypal variants, S370P induced regulatory T cell markers in comparison to NS3358-375-stimulated CD4 T cells. Further, the addition of variantspecific CD4 T cells back into a polyclonal culture in a dose-dependent manner inhibited the T cell response. These results suggest thatHCV is able to induce antigen-specific regulatory T cells to suppress the antiviral T cell response in an antigen-specific manner, thus contributing to a niche within the host that could be conducive to HCV persistence. [ABSTRACT FROM AUTHOR]

Background: Organ transplant candidates with serum antibodies directed against human leukocyte antigens (HLA) face longer waiting times and higher mortality while awaiting transplantation. This study examined the accuracy of virtual crossmatch, in which recipient HLA-specific antibodies, identified by solid-phase assays, are compared to the prospective donor HLA-type in heart transplantation. Methods: We examined the accuracy of virtual crossmatch in predicting immune compatibility of donors and recipients in heart transplantation and clinical outcomes in immunologically sensitized heart transplant recipients in whom virtual crossmatch was used in allograft allocation. Results: Based on analysis of 257 T-cell antihuman immunoglobulin complement-dependent cytotoxic (AHG-CDC) crossmatch tests, the positive predictive value of virtual crossmatch (the likelihood of an incompatible virtual crossmatch resulting in an incompatible T-cell CDC-AHG crossmatch) was 79%, and the negative predictive value of virtual crossmatch (the likelihood of a compatible virtual crossmatch resulting in a compatible T-cell CDC-AHG crossmatch) was 92%. When used in a cohort of 28 sensitized patients awaiting heart transplantation, 14 received allografts based on a compatible virtual crossmatch alone from donors in geographically distant locations. Compared with the other 14 sensitized patients who underwent transplant after a compatible prospective serologic crossmatch, the rejection rates and survival were similar. Conclusion: Our findings are evidence of the accuracy of virtual crossmatch and its utility in augmenting the opportunities for transplantation of sensitized patients. [Copyright &y& Elsevier]

Left ventricular assist device (LVAD) implantation before heart transplantation has been associated with formation of antibodies directed against human leukocyte antigens (HLA), often referred to as sensitization. This study investigated whether prior sensitization or LVAD type affected the degree of post-implantation sensitization. The records of consecutive HeartMate (HM) I and HM II LVAD patients were reviewed. Panel reactive antibody (PRA) was assessed before LVAD implantation and biweekly thereafter. Sensitization was defined as PRA > 10%, and high-degree sensitization was defined as PRA > 90%. An HM LVAD was implanted in 64 patients, and 11 received a HM II LVAD as a bridge to transplant. Ten HM I patients (16%) were sensitized before LVAD implantation (HM I-S), and 54 (84%) were not (HM I-Non-S). Nine HM I-S patients (90%) became highly sensitized (PRA > 90%) compared with 9 HM I-Non-S patients (16.7%; p < 0.001). The PRA remained elevated (> 90%) in 8 of the 9 (88.9%) highly sensitized HM I-S patients vs 5 of the 9 (55.6%) HM I-Non-S highly sensitized patients. The PRA levels in the rest of the HM I-S highly sensitized patients declined from 93% ± 4% to 55% ± 15% (p = 0.01). Among the 11 HM II patients, 1 (9%) was sensitized before LVAD implantation (PRA, 40%) and the PRA moderately increased to 80%. No other HM II patient became sensitized after implantation. Thus, 1 of 11 (9%) HM II patients became sensitized compared with 29 of 64 (45%) HM I patients (p = 0.04). Pre-sensitized patients are at higher risk for becoming and remaining highly HLA-allosensitized after LVAD implantation. The HeartMate II LVAD appears to cause less sensitization than HeartMate I. [Copyright &y& Elsevier]

Abstract: Hepatitis C virus (HCV) causes non-A, non-B hepatitis and infects an estimated 170 million people worldwide. The treatment for HCV infection is often unsuccessful with high costs and many side-effects. There is a great need for alternative therapies including preventive and therapeutic vaccination for HCV infection. The experiments in this study were carried out to elucidate whether endogenously expressed antigen can be presented to helper T-cells restricted by class II molecules and to determine whether responses to plasmid-derived antigen resemble those that we have reported for recombinant antigens or synthetic peptides. To address these issues, a multi-epitope minigene was expressed in 293T-cells and Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cells (BLCL). The transfected BLCLs were employed as APCs to stimulate epitope-specific T-cell hybridomas (THC). The results demonstrated that the endogenously expressed minigene antigens could be processed and presented to T-cell hybridomas by HLA matched BLCL. Five out of seven incorporated epitopes were recognized. Blockade of HLA DR could abolish the release of IL-2, which demonstrated that the endogenously expressed minigene antigens were presented by MHC class II molecules. The presentation of endogenously expressed antigens was much more efficient than that of exogenous antigens, at least in the present study. The findings obtained here have important significance for the development of an HCV DNA vaccine. [Copyright &y& Elsevier]