Takayuki Yakushijin

Reduced Numbers and Impaired Ability of Myeloid and Plasmacytoid Dendritic Cells to Polarize T Helper Cells in Chronic Hepatitis C Virus Infection

Hepatitis C virus (HCV) infection induces a wide range of chronic liver injuries. The mechanism by which HCV evades the immune surveillance system remains obscure. Blood dendritic cells (DCs) consist of myeloid and plasmacytoid subsets that play distinct roles in the regulation of antivirus immune responses; however, their roles in the pathogenesis of HCV infection are yet to be determined. We compared the numbers and functions of myeloid and plasmacytoid DCs between 43 patients with chronic hepatitis and 26 age-matched healthy volunteers. Absolute numbers of myeloid DCs, plasmacytoid DCs, and DC progenitors in the periphery were significantly lower in patients with chronic hepatitis than in healthy volunteers. Myeloid and plasmacytoid DCs from the patients had impaired abilities to stimulate allogeneic CD4 T cells and to produce interleukin (IL)-12 p70 and interferon- alpha , compared with those from healthy volunteers. After exposure to naive CD4 T cells, myeloid DCs from the patients were less able to drive the T helper type 1 response, whereas myeloid and plasmacytoid DCs from the patients primed more IL-10-producing cells than did those from healthy volunteers. In conclusion, in chronic HCV infection, both types of blood DCs are reduced and have an impaired ability to polarize T helper cells.

Ribavirin dose reduction raises relapse rate dose-dependently in genotype 1 patients with hepatitis C responding to pegylated interferon alpha-2b plus ribavirin

Summary.  The impact of ribavirin exposure on virologic relapse remains controversial in combination therapy with pegylated interferon (Peg‐IFN) and ribavirin for patients with chronic hepatitis C (CH‐C) genotype 1. The present study was conducted to investigate this. Nine hundred and eighty‐four patients with CH‐C genotype 1 were enrolled. The drug exposure of each medication was calculated by averaging the dose actually taken. For the 472 patients who were HCV RNA negative at week 24 and week 48, multivariate logistic regression analysis showed that the degree of fibrosis (P = 0.002), the timing of HCV RNA negativiation (P < 0.001) and the mean doses of ribavirin (P < 0.001) were significantly associated with relapse, but those of Peg‐IFN were not. Stepwise reduction of the ribavirin dose was associated with a stepwise increase in relapse rate from 11% to 60%. For patients with complete early virologic response (c‐EVR) defined as HCV RNA negativity at week 12, only 4% relapse was found in patients given ≥12 mg/kg/day of ribavirin and ribavirin exposure affected the relapse even after treatment week 12, while Peg‐IFN could be reduced to 0.6 μg/kg/week after week 12 without the increase of relapse rate. Ribavirin showed dose‐dependent correlation with the relapse. Maintaining as high a ribavirin dose as possible (≥12 mg/kg/day) during the full treatment period can lead to suppression of the relapse in HCV genotype 1 patients responding to Peg‐IFN alpha‐2b plus ribavirin, especially in c‐EVR patients.

Glycomic Analysis of Alpha-Fetoprotein L3 in Hepatoma Cell Lines and Hepatocellular Carcinoma Patients

The N-glycan structures of the Lens culinaris agglutinin (LCA)-reactive fraction of alpha-fetoprotein (AFP-L3), a tumor marker of hepatocellular carcinomas (HCC), were analyzed in relationship to glycosyltransferases and LCA-affinity electrophoresis. Using HPLC and MALDI-TOF MS, we determined the N-glycan structures of AFP from HCC cell lines, and demonstrated they were affected by N-acetylglucosaminyltransferase III and fucosyltransferase VIII, but not by N-acetylglucosaminyltransferase V. Moreover, we identified the N-glycan structures of AFP in HCC patients.

Impaired cytokine response in myeloid dendritic cells in chronic hepatitis C virus infection regardless of enhanced expression of Toll-like receptors and retinoic acid inducible gene-I.

Dendritic cells utilize various sets of Toll‐like receptors (TLR) or cytosolic sensors to detect pathogens and evoke immune responses. In patients with hepatitis C virus (HCV) infection, a higher prevalence of various infectious diseases is reported; suggesting that innate immunity against pathogens is impaired. The aim of this study was to clarify whether the TLR and retinoic acid inducible gene‐I (RIG‐I) system in myeloid dendritic cells is preserved or not in chronic HCV infection. The expression of TLRs, RIG‐I and its relatives were compared in myeloid dendritic cells between 39 patients and 52 healthy volunteers. The induction of type‐I interferon (IFN) and inflammatory cytokines was examined in response to agonists for TLR2 (palmitoyl‐3‐cysteine‐serine‐lysine‐4), TLR3/RIG‐I (polyinosine–polycytidylic acid) or TLR4 (lipopolysaccharide). The relative expressions of TLR2, TLR4, RIG‐I, and LGP2 from the patients were significantly higher than those from the volunteers, whereas TLR3 and MDA‐5 expressions did not differ. In search for factors regulating TLR/RIG‐I expression, it was shown that IFN‐α, polyinosine–polycytidylic acid and lipopolysaccharide induced TLR3, TLR4 and RIG‐I, but TNF‐α, HCV core or HCV non‐structural proteins did not. For the functional analyses, myeloid dendritic cells from the patients induced significantly less amounts of IFN‐β, TNF‐α and IL‐12p70 in response to polyinosine–polycytidylic acid or lipopolysaccharide. It is noteworthy that the expression of TRIF and TRAF6, which are essential adaptor molecules transmitting TLR3 or TLR4‐dependent signals, is reduced in the patients. Thus, innate cytokine responses in myeloid dendritic cells are impaired regardless of enhanced expressions of TLR2, TLR4, and RIG‐I in HCV infection. J. Med. Virol. 80:980–988, 2008.

Impaired Function of Dendritic Cells Circulating in Patients Infected with Hepatitis C Virus Who Have Persistently Normal Alanine Aminotransferase Levels

Hepatitis C virus (HCV) induces chronic liver disease in hosts which can eventually progresses to liver cirrhosis and hepatocellular carcinoma. However, progression of liver disease is slower in patients with persistently normal levels of alanine aminotransferase (ALT) than in those with active hepatitis. Although distinct immune responses against HCV have been proposed in asymptomatic infection, the role of circulating dendritic cells (DC) in the pathogenesis of these patients remains obscure. To address this issue, we compared the number and function of myeloid DC (MDC) and plasmacytoid DC (PDC) between uninfected individuals and HCV-infected patients with or without elevated ALT levels. Numbers of DC and DC progenitors were significantly lower in patients with chronic active hepatitis than in control subjects. However, no differences were found in the number of DC between normal controls and HCV-infected patients with persistently normal ALT levels. MDC from patients with active hepatitis were less able to polarize naive CD4 T cells into the Th1 phenotype, while their MDC and PDC primed more CD4 T cells producing IL-10 than those from normal controls. Such dysfunction of DC was also observed in patients with persistently normal ALT levels. In conclusion, circulating DC decrease in number predominantly in HCV-infected patients with active hepatitis, and the function of DC is impaired even in those with normal ALT levels.

Post-treatment Levels of α-Fetoprotein Predict Incidence of Hepatocellular Carcinoma After Interferon Therapy

BACKGROUND & AIMS In patients with chronic hepatitis C virus (HCV) infection, lack of sustained virologic response (SVR) 24 weeks after the end of interferon therapy is a significant risk factor for hepatocellular carcinoma (HCC). Although many pretreatment factors are known to affect HCC incidence, less is known about post-treatment factors-many change during the course of interferon therapy. METHODS We performed a prospective study, collecting data from 2659 patients with chronic hepatitis C without a history of HCC who had been treated with pegylated interferon (Peg-IFN) plus ribavirin from 2002 through 2008 at hospitals in Japan. Biopsy specimens were collected before treatment; all patients received Peg-IFN plus ribavirin for 48 to 72 weeks (HCV genotype 1) or 24 weeks (HCV genotype 2). Hematologic, biochemical, and virologic data were collected every 4 weeks during treatment and every 6 months after treatment. HCC was diagnosed based on angiography, computed tomography, and/or magnetic resonance imaging findings. RESULTS HCC developed in 104 patients during a mean observation period of 40 months. Older age, male sex, lower platelet counts and higher levels of α-fetoprotein at baseline, and lack of an SVR were significant risk factors for HCC. The cumulative incidence of HCC was significantly lower in patients without SVRs who relapsed than those with no response to treatment. Levels of α-fetoprotein 24 weeks after the end of treatment (AFP24) were significantly lower than levels of α-fetoprotein at baseline in patients with SVRs and those who relapsed, but not in nonresponders. Post-treatment risk factors for HCC among patients with SVRs included higher AFP24 level and older age; among those without SVRs, risk factors included higher AFP24 level, integrated level of alanine aminotransferase, older age, and male sex. AFP24 (≥10 ng/mL, 10-5 ng/mL, and then <5 ng/mL) was a better predictor of HCC incidence than pretreatment level of AFP among patients with and without SVRs. CONCLUSIONS In patients with chronic HCV infection, levels of α-fetoprotein decrease during interferon therapy. High post-treatment levels of α-fetoprotein predict HCC, regardless of whether patients achieve an SVR. University Hospital Medical Information Network Clinical Trials Registry: C000000196, C000000197.

Indications and limitations for aged patients with chronic hepatitis C in pegylated interferon alfa-2b plus ribavirin combination therapy

BACKGROUND & AIMS This study investigated the efficacy and adverse effects of pegylated interferon (Peg-IFN) plus ribavirin therapy in aged patients with chronic hepatitis C (CH-C). METHODS A total of 1040 naïve patients with CH-C (genotype 1, n=759; genotype 2, n=281), of whom 240 (23%) over 65 years old (y.o.), were treated with Peg-IFN alfa-2b plus ribavirin and assessed after being classified into five categories, according to age. RESULTS The discontinuance rate was higher for patients over 70 y.o. (36%), the most common reason being anemia. In the presence of genotype 1, the SVR rate was similar (42-46%) among patients under 65 y.o. and declined (26-29%) among patients over 65 y.o. For patients over 65 y.o., being male (Odds ratio, OR, 3.5, p=0.035) and EVR (OR, 83.3, p<0.001) were significant factors for SVR, in multivariate analysis. The Peg-IFN dose was related to EVR, and when EVR was attained, 76-86% of patients over 65 y.o. achieved SVR. SVR was not achieved (0/35, 0/38, respectively) if a 1-log decrease and a 2-log decrease were not attained at week 4 and week 8, respectively. In the presence of genotype 2, the SVR rate was similar (70-71%) among patients under 70 y.o. and declined among patients over 70 y.o. (43%). CONCLUSIONS Aged patients up to 65 y.o. with genotype 1 and 70 y.o. with genotype 2 can be candidates for Peg-IFN plus ribavirin therapy. The response-guided therapy can be applied for aged patients with genotype 1.

Effect of interferon α-2b plus ribavirin therapy on incidence of hepatocellular carcinoma in patients with chronic hepatitis.

Aim:  The objective of this study was to elucidate the long‐term effects of interferon (IFN)α‐2b plus ribavirin combination therapy and to clarify whether this therapy can reduce the incidence of hepatocellular carcinoma (HCC) in patients with chronic hepatitis C.

Factors affecting efficacy in patients with genotype 2 chronic hepatitis C treated by pegylated interferon alpha-2b and ribavirin: reducing drug doses has no impact on rapid and sustained virological responses.

Summary.  Reducing the dose of drug affects treatment efficacy in pegylated interferon (Peg‐IFN) and ribavirin combination therapy for patients with hepatitis C virus (HCV) genotype 1. The aim of this study was to investigate the impact of drug exposure, as well as the baseline factors and the virological response on the treatment efficacy for genotype 2 patients. Two‐hundred and fifty patients with genotype 2 HCV who were to undergo combination therapy for 24 weeks were included in the study, and 213 completed the treatment. Significantly more patients who achieved a rapid virological response (RVR), defined as HCV RNA negativity at week 4, achieved a sustained virological response (SVR) (92%, 122/133) compared with patients who failed to achieve RVR (48%, 38/80) (P < 0.0001). Multivariate logistic‐regression analysis showed that only platelet counts [odds ratio (OR), 1.68; confidence interval (CI), 1.002–1.139] and RVR (OR, 11.251; CI, 5.184–24.419) were independently associated with SVR, with no correlation being found for the mean dose of Peg‐IFN and ribavirin for RVR and SVR. Furthermore, in the stratification analysis of the timing of viral clearance, neither mean dose of Peg‐IFN (P = 0.795) nor ribavirin (P = 0.649) affected SVR in each group. Among the patients with RVR, the lowest dose group of Peg‐IFN (0.77 ± 0.10 μg/kg/week) and ribavirin (6.9 ± 0.90 mg/kg/day) showed 100% and 94% of SVR. Hence, RVR served as an important treatment predictor, and drug exposure had no impact on both SVR and RVR in combination therapy for genotype 2 patients.

Pegylated interferon alpha-2b (Peg-IFN α-2b) affects early virologic response dose-dependently in patients with chronic hepatitis C genotype 1 during treatment with Peg-IFN α-2b plus ribavirin.

Summary.  Chronic hepatitis C (CH‐C) genotype 1 patients who achieved early virologic response have a high probability of sustained virologic response (SVR) following pegylated interferon (Peg‐IFN) plus ribavirin therapy. This study was conducted to evaluate how reducing drug doses affects complete early virologic response (c‐EVR) defined as hepatitis C virus (HCV) RNA negativity at week 12. Nine hundred eighty‐four patients with CH‐C genotype 1 were enrolled. Drug doses were evaluated independently on a body weight base from doses actually taken. From multivariate analysis, the mean dose of Peg‐IFN α‐2b during the first 12 weeks was the independent factor for c‐EVR (P = 0.02), not ribavirin. The c‐EVR rate was 55% in patients receiving ≥1.2 μg/kg/week of Peg‐IFN, and declined to 38% at 0.9–1.2 μg/kg/week, and 22% in patients given <0.9 μg/kg/week (P < 0.0001). Even with stratified analysis according to ribavirin dose, the dose‐dependent effect of Peg‐IFN on c‐EVR was observed, and similar c‐EVR rates were obtained if the dose categories of Peg‐IFN were the same. Furthermore, the mean dose of Peg‐IFN during the first 12 weeks affected HCV RNA negativity at week 24 (P < 0.0001) and SVR (P < 0.0001) in a dose‐dependent manner. Our results suggest that Peg‐IFN was dose‐dependently correlated with c‐EVR, independently of ribavirin dose. Thus, maintaining the Peg‐IFN dose as high as possible during the first 12 weeks can yield HCV RNA negativity and higher c‐EVR rates, leading to better SVR rates in patients with CH‐C genotype 1.