Eishiro Mizukoshi

Hepatic ISG Expression Is Associated With Genetic Variation in Interleukin 28B and the Outcome of IFN Therapy for Chronic Hepatitis C

BACKGROUND & AIMS Multiple viral and host factors are related to the treatment response to pegylated-interferon and ribavirin combination therapy; however, the clinical relevance and relationship of these factors have not yet been fully evaluated. METHODS We studied 168 patients with chronic hepatitis C who received pegylated-interferon and ribavirin combination therapy. Gene expression profiles in the livers of 91 patients were analyzed using an Affymetrix genechip (Affymetrix, Santa Clara, CA). The expression of interferon-stimulated genes (ISGs) was evaluated in all samples by real-time polymerase chain reaction. Genetic variation in interleukin 28B (IL28B; rs8099917) was determined in 91 patients. RESULTS Gene expression profiling of the liver differentiated patients into 2 groups: patients with up-regulated ISGs and patients with down-regulated ISGs. A high proportion of patients with no response to treatment was found in the up-regulated ISGs group (P=.002). Multivariate logistic regression analysis showed that ISGs (<3.5) (odds ratio [OR], 16.2; P<.001), fibrosis stage (F1-F2) (OR, 4.18; P=.003), and ISDR mutation (>or=2) (OR, 5.09; P=.003) were strongly associated with the viral response. The IL28B polymorphism of 91 patients showed that 66% were major homozygotes (TT), 30% were heterozygotes (TG), and 4% were minor homozygotes (GG). Interestingly, hepatic ISGs were associated with the IL28B polymorphism (OR, 18.1; P<.001), and its expression was significantly higher in patients with the minor genotype (TG or GG) than in those with the major genotype (TT). CONCLUSIONS The expression of hepatic ISGs is strongly associated with treatment response and genetic variation of IL28B. The differential role of host and viral factors as predicting factors may also be present.

A liver-derived secretory protein, selenoprotein P, causes insulin resistance.

The liver may regulate glucose homeostasis by modulating the sensitivity/resistance of peripheral tissues to insulin, by way of the production of secretory proteins, termed hepatokines. Here, we demonstrate that selenoprotein P (SeP), a liver-derived secretory protein, causes insulin resistance. Using serial analysis of gene expression (SAGE) and DNA chip methods, we found that hepatic SeP mRNA levels correlated with insulin resistance in humans. Administration of purified SeP impaired insulin signaling and dysregulated glucose metabolism in both hepatocytes and myocytes. Conversely, both genetic deletion and RNA interference-mediated knockdown of SeP improved systemic insulin sensitivity and glucose tolerance in mice. The metabolic actions of SeP were mediated, at least partly, by inactivation of adenosine monophosphate-activated protein kinase (AMPK). In summary, these results demonstrate a role of SeP in the regulation of glucose metabolism and insulin sensitivity and suggest that SeP may be a therapeutic target for type 2 diabetes.

Cross-Reactivity between Hepatitis C Virus and Influenza A Virus Determinant-Specific Cytotoxic T Cells

ABSTRACT The cellular immune response contributes to viral clearance as well as to liver injury in acute and chronic hepatitis C virus (HCV) infection. An immunodominant determinant frequently recognized by liver-infiltrating and circulating CD8+ T cells of HCV-infected patients is the HCVNS3-1073 peptide CVNGVCWTV. Using a sensitive in vitro technique with HCV peptides and multiple cytokines, we were able to expand cytotoxic T cells specific for this determinant not only from the blood of 11 of 20 HCV-infected patients (55%) but also from the blood of 9 of 15 HCV-negative blood donors (60%), while a second HCV NS3 determinant was recognized only by HCV-infected patients and not by seronegative controls. The T-cell response of these healthy blood donors was mediated by memory T cells, which cross-reacted with a novel T-cell determinant of the A/PR/8/34 influenza A virus (IV) that is endogenously processed from the neuraminidase (NA) protein. Both the HCV NS3 and the IV NA peptide displayed a high degree of sequence homology, bound to the HLA-A2 molecule with high affinity, and were recognized by cytotoxic T lymphocytes with similar affinity (10−8 M). Using the HLA-A2-transgenic mouse model, we then demonstrated directly that HCV-specific T cells could be induced in vivo by IV infection. Splenocytes harvested from IV-infected mice at the peak of the primary response (day 7 effector cells) or following complete recovery (day 21 memory cells) recognized the HCV NS3 peptide, lysed peptide-pulsed target cells, and produced gamma interferon. These results exemplify that host responses to an infectious agent are influenced by cross-reactive memory cells induced by past exposure to heterologous viruses, which could have important consequences for vaccine development.

Kinetics of CD4+ and CD8+ memory T-cell responses during hepatitis C virus rechallenge of previously recovered chimpanzees.

ABSTRACT The immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood. Antibodies to HCV structural proteins do not appear to play a key role in clearance of the virus and do not persist after recovery. Here, we studied the kinetics of the cellular immune responses of three HCV-recovered chimpanzees during rechallenge with increasing doses of homologous HCV. Although HCV envelope antibodies remained undetectable throughout the rechallenge, all animals mounted rapid HCV-specific T-cell responses. The pattern of the cellular immune response in blood and liver correlated with the virological outcome. The animal that most rapidly cleared circulating HCV as determined by nested reverse transcription-PCR (RT-PCR) displayed the most vigorous and sustained response of gamma interferon (IFN-γ)-producing and proliferating CD4+ T cells in the blood. Vigorous CD4+ T-cell proliferation during viremia was followed by an increased frequency and a phenotypic and functional change of the tetramer+ CD8+ T-cell population. The second animal cleared HCV initially with strong peripheral and intrahepatic CD4+ T-cell responses but experienced low-level HCV recrudescence 12 weeks later, when HCV-specific T cells became undetectable. The third animal maintained minute amounts of circulating HCV, detectable only by nested RT-PCR, in the face of a weak IFN-γ+ T-cell response. Collectively, the results suggest protective rather than sterilizing immunity after recovery from hepatitis C. The rate of HCV clearance following reexposure depends on the cellular immune response, the quality and quantity of which may vary among chimpanzees that recovered from HCV infection.

Effects of antiviral therapy on the cellular immune response in acute hepatitis C

Spontaneous recovery occurs in a minority of patients with acute hepatitis C but is associated with vigorous and long‐lasting cellular immune responses. Treatment‐induced recovery can be achieved in the majority of patients who are treated in the acute phase, but the kinetics and mechanisms of viral clearance and immune responsiveness are not known. Both direct antiviral effects and indirect immune‐mediated effects, such as immune modulation of Th2 to Th1 responses and prevention of exhaustion of cellular responses by rapid reduction of viral titer, have been proposed. To investigate how early antiviral therapy affects hepatitis C virus (HCV)‐specific T cell responses, we performed detailed prospective clinical, virological, and immunological studies on 7 patients with acute hepatitis C who received antiviral therapy and were followed at 2 to 4 week intervals for 1 to 2 years. The total CD4+ and CD8+ cell response was analyzed with 600 overlapping HCV peptides and 6 proteins by ex vivo enzyme‐linked immunospot (ELISpot), intracellular cytokine staining, and proliferation assays. In contrast to earlier studies with selected HCV epitopes, this extended analysis detected multispecific interferon γ+ (IFN‐γ+) responses in each patient, even in the absence of T‐cell proliferation. After initiation of antiviral therapy (at a mean of 20 weeks after infection), all sustained responders demonstrated gradually decreasing, then nearly absent HCV‐specific T‐cell responses, whereas the sole patient who developed viral breakthrough after initial HCV control maintained cellular immune responses. In conclusion, a sustained response to antiviral therapy was not associated with a lasting enhancement of HCV‐specific T‐cell responsiveness in the blood. (HEPATOLOGY 2004;40:87–97.)

Cytotoxic T cell responses to human telomerase reverse transcriptase in patients with hepatocellular carcinoma

Human telomerase reverse transcriptase, hTERT, has been identified as the catalytic enzyme required for telomere elongation. hTERT is expressed in most tumor cells but seldom expressed in most human adult cells. It has been reported that 80% to 90% of hepatocellular carcinomas (HCCs) express hTERT, making the enzyme a potential target in immunotherapy for HCC. In the current study, we identified hTERT‐derived, HLA‐A*2402–restricted cytotoxic T cell (CTL) epitopes and analyzed hTERT‐specific CTL responses in patients with HCC. Peptides containing the epitopes showed high affinity to bind HLA‐A*2402 in a major histocompatibility complex binding assay and were able to induce hTERT‐specific CTLs in both hTERT cDNA‐immunized HLA‐A*2402/Kb transgenic mice and patients with HCC. The CTLs were able to kill hepatoma cell lines depending on hTERT expression levels in an HLA‐A*2402–restricted manner and induced irrespective of hepatitis viral infection. The number of single hTERT epitope‐specific T cells detected by ELISPOT assay was 10 to 100 specific cells per 3 × 105 PBMCs, and positive T cell responses were observed in 6.9% to 12.5% of HCC patients. hTERT‐specific T cell responses were observed even in the patients with early stages of HCC. The frequency of hTERT/tetramer+CD8+ T cells in the tumor tissue of patients with HCC was quite high, and they were functional. In conclusion, these results suggest that hTERT is an attractive target for T‐cell–based immunotherapy for HCC, and the identified hTERT epitopes may be valuable both for immunotherapy and for analyzing host immune responses to HCC. (HEPATOLOGY 2006;43: 1284–1294.)

Cellular Immune Responses to the Hepatitis B Virus Polymerase

CD4+ T cells play an important role in hepatitis B virus (HBV) infection by secretion of Th1 cytokines that down-regulate HBV replication, and by promoting CD8+ T cell and B cell responses. We have identified and characterized 10 CD4+ T cell epitopes within polymerase and used them to analyze the immunological effects of long-term antiviral therapy as compared with spontaneous recovery from HBV infection. Candidate epitopes were tested for binding to 14 HLA-DR molecules and in IFN-γ ELISPOT and cytotoxicity assays using peripheral blood lymphocytes from 66 HBV-infected patients and 16 uninfected controls. All 10 epitopes bound with high affinity to the most prevalent HLA-DR Ags, were conserved among HBV genomes, and induced IFN-γ responses from HBV-specific CD4+ T cells. Several epitopes contained nested MHC class I motifs and stimulated HBV-specific IFN-γ production and cytotoxicity of CD8+ T cells. HBV polymerase-specific responses were more frequent during acute, self-limited hepatitis and after recovery (12 of 18; 67%) than during chronic hepatitis (16 of 48 (33%); p = 0.02). Antiviral therapy of chronic patients restored HBV polymerase and core-specific T cell responses during the first year of treatment, but thereafter, responses decreased and, after 3 years, were no more frequent than in untreated patients. Decreased T cell responsiveness during prolonged therapy was associated with increased prevalence of lamivudine-resistant HBV mutants and increased HBV titers. The data provide a rationale for the combination of antiviral and immunostimulatory therapy. These newly described HBV polymerase epitopes could be a valuable component of a therapeutic vaccine for a large and ethnically diverse patient population.

Impact of diabetes on recurrence of hepatocellular carcinoma after surgical treatment in patients with viral hepatitis.

OBJECTIVES: Consensus has been reached that diabetes is a risk factor for development of HCC, but the impact on postoperative recurrence is still controversial. To clarify this point, we analyzed the relationship of postoperative recurrence rate of HCC and coexistence of diabetes in the patients with viral hepatitis.METHODS: A total of 90 patients who had undergone curative resection for HCC were analyzed. They were divided into two groups with and without diabetes, and the recurrence-free survival rates after surgical treatment and the factors contributing to recurrence were examined.RESULTS: Kaplan-Meier survival analysis showed the recurrence-free survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P = 0.005) and overall survival rates in the diabetic group were significantly lower than those in the nondiabetic group (P = 0.005). These results were emphasized in the analysis of patients infected with hepatitis C virus. Univariate and multivariate analyses showed diabetes was a significant factor contributing to HCC recurrence after treatment. Furthermore, multivariate analysis in HCC patients with diabetes showed Child-Pugh classification B (P = 0.001) and insulin therapy (P = 0.049) were significant factors contributing to HCC recurrence after treatment.CONCLUSIONS: The results of the present study suggest that diabetes is a risk factor for the recurrence of HCV-related HCC and decreases the overall survival rates after surgical treatment. HCV-related HCC patients with diabetes should be closely followed for postoperative recurrence.

Hepatitis C Virus (HCV)-Specific Immune Responses of Long-Term Injection Drug Users Frequently Exposed to HCV

BACKGROUND Injection drug users (IDUs) who successfully clear hepatitis C virus (HCV) have a reduced risk of developing chronic reinfection, despite their continuing exposure to the virus. To identify immunological correlates for this apparent protection, we studied HCV-specific immune responses in long-term IDUs (duration, >10 years). METHODS HCV-specific T cell responses were assessed in proliferation, enzyme-linked immunospot (ELISPOT), interferon (IFN)-gamma secretion, and cytotoxicity assays, whereas HCV-specific antibodies were assessed in enzyme immunoassays (EIAs), chemiluminescent assays, and in vitro neutralization assays. RESULTS HCV-specific T cell proliferation and IFN-gamma production were more common in nonviremic EIA-positive IDUs (16 [94%] of 17 IDUs) than in viremic EIA-positive IDUs (9 [45%] of 20 IDUs) (P= .003). They were also noted in 16 (62%) of 26 nonviremic EIA-negative IDUs. In contrast, 19 (90%) of 21 viremic IDUs displayed neutralizing antibodies (nAbs), compared with 9 (56%) of 16 nonviremic EIA-positive IDUs (P= .04) and 0 of 24 nonviremic EIA-negative IDUs. Nonviremic IDUs with nAbs were older (P= .0115) than those without nAbs, but these groups did not differ in terms of either injection drug use duration or HCV-specific T cell responses. CONCLUSION The reduced risk of HCV persistence in IDUs previously recovered from HCV infection correlated with T cell responses, and prolonged antigenic stimulation appears to be required to maintain humoral responses.

Identification of α‐fetoprotein‐derived peptides recognized by cytotoxic T lymphocytes in HLA‐A24+ patients with hepatocellular carcinoma

α‐Fetoprotein (AFP) has been proposed as a potential target forT‐cell‐based immunotherapy for hepatocellular carcinoma (HCC), but the number of its epitopes that have been identified is limited and the status of AFP‐specific immunological responses in HCC patients has not been well‐characterized. To address the issue, we examined the possibility of inducing AFP‐specific cytotoxic T cells (CTLs) using novel HLA‐A*2402‐restricted T‐cell epitopes (HLA, human leukocyte antigen) derived from AFP and then analyzed the relationship between its frequency of occurrence and clinical features associated with patients having HCC. Five AFP‐derived peptides containing HLA‐A*2402 binding motifs and showing high binding affinity to HLA‐A*2402 induced CTLs to produce IFN‐γ and kill an AFP‐producing hepatoma cell line. The frequency of AFP‐specific CTLs was 30–190 per 1 × 106 peripheral blood mononuclear cells, which was the same as that of other immunogenic cancer associated antigen‐derived epitopes. Analyses of the relationships between AFP‐specific CTL responses and clinical features of patients with HCC revealed that AFP epitopes were more frequently recognized by CTLs in patients with advanced HCC correlating to tumor factors or the stage of TNM classification. The analyses of CTL responses before and after HCC treatments showed that the treatments changed the frequency of AFP‐specific CTLs. In conclusion, we identified five HLA‐A*2402‐restrictedT‐cell epitopes derived from AFP. The newly identified AFP epitopes could be a valuable component of HCC immunotherapy and for analyzing host immune responses to HCC.