DCs Pulsed with Novel HLA-A2-Restricted CTL Epitopes against Hepatitis C Virus Induced a Broadly Reactive Anti-HCV-Specific T Lymphocyte Response

OBJECTIVE: To determine the capacity of dendritic cells (DCs) loaded with single or multiple-peptide mixtures of novel hepatitis C virus (HCV) epitopes to stimulate HCV-specific cytotoxic T lymphocyte (CTL) effector functions. METHODS: A bioinformatics approach was used to predict HLA-A2-restricted HCV-specific CTL epitopes, and the predicted peptides identified from this screen were synthesized. Subsequent IFN-γ ELISPOT analysis detected the stimulating function of these peptides in peripheral blood mononuclear cells (PBMCs) from both chronic and self-limited HCV infected subjects (subjects exhibiting spontaneous HCV clearance). Mature DCs, derived in vitro from CD14(+) monocytes harvested from the study subjects by incubation with appropriate cytokine cocktails, were loaded with novel peptide or epitope peptide mixtures and co-cultured with autologous T lymphocytes. Granzyme B (GrB) and IFN-γ ELISPOT analysis was used to test for epitope-specific CTL responses. T-cell-derived cytokines contained in the co-cultured supernatant were detected by flow cytometry. RESULTS: We identified 7 novel HLA-A2-restricted HCV-specific CTL epitopes that increased the frequency of IFN-γ-producing T cells compared to other epitopes, as assayed by measuring spot forming cells (SFCs). Two epitopes had the strongest stimulating capability in the self-limited subjects, one found in the E2 and one in the NS2 region of HCV; five epitopes had a strong stimulating capacity in both chronic and self-limited HCV infection, but were stronger in the self-limited subjects. They were distributed in E2, NS2, NS3, NS4, and NS5 regions of HCV, respectively. We also found that mDCs loaded with novel peptide mixtures could significantly increase GrB and IFN-γ SFCs as compared to single peptides, especially in chronic HCV infection subjects. Additionally, we found that DCs pulsed with multiple epitope peptide mixtures induced a Th1-biased immune response. CONCLUSIONS: Seven novel and strongly stimulating HLA-A2-restricted HCV-specific CTL epitopes were identified. Furthermore, DCs loaded with multiple-epitope peptide mixtures induced epitope-specific CTLs responses

Consequences of Cold-Ischemia Time on Primary Nonfunction and Patient and Graft Survival in Liver Transplantation: A Meta-Analysis

Introduction: The ability to preserve organs prior to transplant is essential to the organ allocation process. Objective: The purpose of this study is to describe the functional relationship between cold-ischemia time (CIT) and primary nonfunction (PNF), patient and graft survival in liver transplant. Methods: To identify relevant articles Medline, EMBASE and the Cochrane database, including the non-English literature identified in these databases, was searched from 1966 to April 2008. Two independent reviewers screened and extracted the data. CIT was analyzed both as a continuous variable and stratified by clinically relevant intervals. Nondichotomous variables were weighted by sample size. Percent variables were weighted by the inverse of the binomial variance. Results: Twenty-six studies met criteria. Functionally, PNF%=-6.678281+0.9134701*CIT Mean+0.1250879*(CIT Mean-9.89535) 2 - 0.0067663*(CIT Mean-9.89535) 3, r2=.625, p<.0001. Mean patient survival: 93 % (1 month), 88 % (3 months), 83 % (6 months) and 83 % (12 months). Mean graft survival: 85.9 % (1 month), 80.5 % (3 months), 78.1 % (6 months) and 76.8 % (12 months). Maximum patient and graft survival occurred with CITs between 7.5-12.5 hrs at each survival interval. PNF was also significantly correlated with ICU time, % first time grafts and % immunologic mismatches. Conclusion: The results of this work imply that CIT may be the most important pre-transplant information needed in the decision to accept an organ. © 2008 Stahl et al