N. Dikopoulos

Hepatic dendritic cell subsets in the mouse

The CD11c+ cell population in the non‐parenchymal cell population of the mouse liver contains dendritic cells (DC), NK cells, B cells and T cells. In the hepatic CD11c+ DC population from immunocompetent or immunodeficient [recombinase‐activating gene‐1 (RAG1)–/–] C57BL/6 mice (rigorously depleted of T cells, B cells and NK cells), we identified a B220+ CD11cint subset of ‘plasmacytoid’ DC, and a B220– CD11c+ DC subset. The latter DC population could be subdivided into a major, immature (CD40lo CD80lo CD86lo MHC class IIlo) CD11cint subset, and a minor, mature (CD40hi CD80hi CD86hi MHC class IIhi) CD11chi subset. Stimulated B220+ but not B220– DC produced type I interferon. NKT cell activation in vivo increased the number of liver B220– DC three‐ to fourfold within 18 h post‐injection, and up‐regulated their surface expression of activation marker, while it contracted the B220+ DC population. Early in virus infection, the hepatic B220+ DC subset expanded, and both, the B220+ as well as B220– DC populations in the liver matured. In vitro, B220– but not B220+ DC primed CD4+ or CD8+T cells. Expression of distinct marker profiles and functions, and distinct early reaction to activation signals hence identify two distinct B220+ and B220– subsets in CD11c+ DC populations freshly isolated from the mouse liver.

Type I IFN Negatively Regulates CD8+ T Cell Responses through IL-10-Producing CD4+ T Regulatory 1 Cells

Pleiotropic, immunomodulatory effects of type I IFN on T cell responses are emerging. We used vaccine-induced, antiviral CD8+ T cell responses in IFN-β (IFN-β−/−)- or type I IFN receptor (IFNAR−/−)-deficient mice to study immunomodulating effects of type I IFN that are not complicated by the interference of a concomitant virus infection. Compared with normal B6 mice, IFNAR−/− or IFN-β−/− mice have normal numbers of CD4+ and CD8+ T cells, and CD25+FoxP3+ T regulatory (TR) cells in liver and spleen. Twice as many CD8+ T cells specific for different class I-restricted epitopes develop in IFNAR−/− or IFN-β−/− mice than in normal animals after peptide- or DNA-based vaccination. IFN-γ and TNF-α production and clonal expansion of specific CD8+ T cells from normal and knockout mice are similar. CD25+FoxP3+ TR cells down-modulate vaccine-primed CD8+ T cell responses in normal, IFNAR−/−, or IFN-β−/− mice to a comparable extent. Low IFN-α or IFN-β doses (500–103 U/mouse) down-modulate CD8+ T cells priming in vivo. IFNAR- and IFN-β-deficient mice generate 2- to 3-fold lower numbers of IL-10-producing CD4+ T cells after polyclonal or specific stimulation in vitro or in vivo. CD8+ T cell responses are thus subjected to negative control by both CD25+FoxP3+ TR cells and CD4+IL-10+ TR1 cells, but only development of the latter TR cells depends on type I IFN.

Improved Responses to Pegylated Interferon Alfa-2b and Ribavirin by Individualizing Treatment for 24–72 Weeks

BACKGROUND & AIMS Guidelines recommend that patients with chronic hepatitis C virus (HCV) infection be treated with pegylated interferon and ribavirin for 24, 48, or 72 weeks, based on their virologic response to treatment. We investigated the effects of treating patients for individualized durations. METHODS We treated 398 treatment-naïve patients who had HCV genotype 1 infections with pegylated interferon alfa-2b and ribavirin for 24, 30, 36, 42, 48, 60, or 72 weeks (mean of 39 weeks, termed individualized therapy); the duration of therapy was determined based on baseline viral load and the time point at which HCV RNA levels became undetectable (measured at weeks 4, 6, 8, 12, 24, and 30). Results were compared with those of 225 patients who received standard treatment for 48 weeks (mean of 38 weeks). RESULTS Rates of sustained virologic response (SVR) were 55% among patients who received individualized treatment and 48% among those who received standard treatment (P < .0001 for noninferiority). SVR rates, according to the time point at which HCV RNA levels became undetectable, did not differ significantly between groups. Patients with a rapid virologic response (undetectable levels of HCV RNA at week 4) who were treated for 24 to 30 weeks achieved high rates of SVR (86%-88%). Rates of SVR increased among slow responders who first tested negative for HCV RNA at week 24 and were treated for 60 to 72 weeks compared with those treated for 48 weeks (60%-68% vs 43%-44%). The CC polymorphism at IL28B rs129797860 was associated with an increased rate of SVR compared with the CT/TT polymorphism (P < .0001) at baseline but not among patients who had undetectable levels of HCV RNA following treatment. CONCLUSIONS Individualizing treatment of patients with chronic HCV genotype 1 infections for 24 to 72 weeks results in high rates of SVR among rapid responders and increases SVR among slow responders.

Breaking tolerance in hepatitis B surface antigen (HBsAg) transgenic mice by vaccination with cross‐reactive, natural HBsAg variants

Processing exogenous hepatitis B surface antigen (HBsAg) of the hepatitis B virus (HBV) generates the Kb‐binding S208–215 epitope 1; processing endogenous HBsAg generates theKb‐binding S190–197 epitope 2. Cross‐reactive CD8+ T cell responses were primed to epitope 1 but not epitope 2 when mice were immunized with natural HBsAgayw, orHBsAgadw2 variants differing within both epitopes by one or two residues. Expression of HBsAgayw from a transgene in the liver renders (HBs‐tg) mice tolerant to epitope 1 of HBsAgayw. CD8+ T cells specific for epitope 1 could be primed in HBs‐tg mice by HBsAgadw2; these specific CD8+ T cells cross‐reacted with epitope 1 processed from the transgene‐encoded HBsAgayw. The liver of vaccinated HBsAgayw transgenic mice showed severe histopathology and contained functional (IFNγ‐producing), cross‐reactive CD8+ T cells, and vaccinated HBs‐tg mice showed reduced antigenemia. Hence, vaccination with natural HBsAg variants from different HBV sero/genotypes can prime cross‐reactive, specific CD8+ T cellimmunity that breaks tolerance to HBsAg.

Rapid and strong antiviral activity of the non-nucleosidic NS5B polymerase inhibitor BI 207127 in combination with peginterferon alfa 2a and ribavirin

BACKGROUND & AIMS BI 207127 is a potent non-nucleoside hepatitis C virus (HCV) NS5B polymerase inhibitor in vitro. METHODS In this double-blind, placebo-controlled study, 57 HCV genotype (GT)-1 patients (n=27 treatment-naïve [TN]; n=30 treatment-experienced [TE]) with compensated liver disease were randomised for 28-day treatment with 400, 600, or 800 mg BI 207127 three times daily (TID) or placebo (only TN) in combination with peginterferon alfa 2a and ribavirin (PegIFN/RBV). Plasma HCV RNA was measured by Roche COBAS TaqMan assay. RESULTS HCV RNA decreased in a dose-dependent manner with little difference between 600 mg (TN 5.6 log(10), TE 4.2 log(10)) and 800 mg (TN 5.4 log(10), TE 4.5 log(10)). Rapid virological response (RVR; HCV RNA <15 IU/ml) at day 28 occurred in 11/19 TN and 4/30 TE patients treated with BI 207127. GT-1b patients had stronger reductions in HCV RNA than GT-1a (RVR: TN 64% vs. 43%; TE 33% vs. 5%). There were no breakthroughs (HCV RNA rebound >1 log(10) from nadir) in the TN groups, whereas 3/30 TE patients experienced breakthrough due to P495-mutations. Gastrointestinal adverse events (AEs) and rash were the major AEs and most frequent at higher doses. One and four patients discontinued due to AEs in the 600 and 800 mg groups, respectively. Overall, tolerability was good and better at 600 mg than 800 mg. CONCLUSIONS BI 207127 in combination with PegIFN/RBV demonstrated strong antiviral activity with a favourable safety and tolerability profile. The best benefit/risk ratio was observed at 600 mg.

Inhibition of nitric oxide synthesis by aminoguanidine increases intestinal damage in the acute phase of rat TNB-colitis

The role of nitric oxide (NO) in the pathophysiology of inflammatory bowel diseases (IBD) is controversially discussed. The aim of the present study was to investigate the role of NO inhibition in the acute phase of rat 2,4,6‐trinitrobenzenesulphonic acid (TNB)‐colitis. To inhibit NO synthesis we used aminoguanidine (AG) as a selective inhibitor of inducible nitric oxide synthase (iNOS).

Efficacy and tolerability of peginterferon alfa-2a or alfa-2b plus ribavirin in the daily routine treatment of patients with chronic hepatitis C in Germany: the PRACTICE study.

Summary.  In randomized clinical trials, treatment with peginterferon plus ribavirin (RBV) results in a sustained virological response (SVR) in around half of hepatitis C virus genotype 1‐infected and 80% of genotype 2/3‐infected individuals. This study aimed to evaluate efficacy and tolerability of peginterferon alfa‐2a plus RBV compared with peginterferon alfa‐2b plus RBV for the treatment of chronic hepatitis C in routine clinical practice. The intent‐to‐treat cohort consisted of 3414 patients treated with either peginterferon alfa‐2a plus RBV (Group A) or peginterferon alfa‐2b plus RBV (Group B) in 23 centres participating in the large, multicentre, observational PRACTICE study. Collected data included baseline characteristics, treatment regimen, RBV dose and outcome. Rates of early virological response, end of treatment response and SVR were 76.6%, 75.7% and 52.9% in Group A, and 70.2%, 65.6% and 50.5% in Group B, respectively. In patients matched by baseline parameters, 59.9% of patients in Group A and 55.9% in Group B achieved an SVR (P ≤ 0.051). In genotype 1‐infected patients matched by baseline parameters and cumulative RBV dose, SVR rates were 49.6% and 43.7% for Group A and Group B, respectively (P ≤ 0.047); when matched by baseline parameters and RBV starting dose, SVR rates were 49.9% and 44.6%, respectively (P = 0.068). Overall, 21.8% of group A and 29.6% of group B patients discontinued treatment (P ≤ 0.0001). The efficacy and tolerability of peginterferon plus RBV in this large cohort of patients treated in routine daily practice was similar to that in randomized clinical trials. In matched pairs analyses, more patients achieved an SVR with peginterferon alfa‐2a compared with peginterferon alfa‐2b.

Estimating the likelihood of sustained virological response in chronic hepatitis C therapy

Summary.  The likelihood of a sustained virological response (SVR) is the most important factor for physicians and patients in the decision to initiate and continue therapy for chronic hepatitis C (CHC) infection. This study identified predictive factors for SVR with peginterferon plus ribavirin (RBV) in patients with CHC treated under ‘real‐life’ conditions. The study cohort consisted of patients from a large, retrospective German multicentre, observational study who had been treated with peginterferon alfa‐2a plus RBV or peginterferon alfa‐2b plus RBV between the years 2000 and 2007. To ensure comparability regarding peginterferon therapies, patients were analysed in pairs matched by several baseline variables. Univariate and multivariate logistic regression analyses were used to determine the effect of nonmatched baseline variables and treatment modality on SVR. Among 2378 patients (1189 matched pairs), SVR rates were 57.9% overall, 46.5% in HCV genotype 1/4‐infected patients and 77.3% in genotype 2/3‐infected patients. In multivariate logistic regression analysis, positive predictors of SVR were HCV genotype 2 infection, HCV genotype 3 infection, low baseline viral load and treatment with peginterferon alfa‐2a. Negative predictors of SVR were higher age (≥40 years), elevated baseline gamma‐glutamyl transpeptidase (GGT) and low baseline platelet count (<150 000/μL). Among patients treated with peginterferon plus RBV in routine clinical practice, genotype, baseline viral load, age, GGT level and platelet levels all predict the likelihood of treatment success. In patients matched by baseline characteristics, treatment with peginterferon alfa‐2a may be a positive predictor of SVR when compared to peginterferon alfa‐2b.

Recently primed CD8+ T cells entering the liver induce hepatocytes to interact with naïve CD8+ T cells in the mouse

Large number of T cells traffic through the liver. In order to examine the effects of such traffic on the phenotype of hepatocytes, we vaccinated mice using DNA vaccines encoding antigens with MHC class I‐binding epitopes. Small numbers of activated CD8+ T blasts (105–106/liver) changed the surface phenotype and cytokine expression profile of hepatocytes (HCs). HCs upregulate surface expression of major histocompatibility complex (MHC) class I molecules and CD1d but not MHC class II molecules Qa‐1, CD80, CD86, CD54, or CD95; in addition, they expressed/secreted interleukin (IL)‐10 and IL‐4 but not IL‐1, IL‐6, IL‐13, interferon (IFN)‐γ, tumor necrosis factor (TNF), IL‐4, or IL‐27 (i.e., they acquire the HC* phenotype). HCs* (but not HCs) induced specific activation, proliferation, and IFN‐γ, TNF, and IL‐13 release of cocultured naïve CD8+ T cells. In contrast to the specific activation of naïve CD8+ T cells by dendritic cells (DCs), specific CD8+ T cell activation by HC* was not down‐modulated by IFN‐αβ. Only recently activated CD8+ T blasts (but not recently activated CD4+ T blasts or activated cells of the innate immune system, including natural killer T [NKT] cells) induced the HC* phenotype that is prominent from day 10 to day 20 postvaccination (i.e., time points at which peak numbers of recently primed CD8+ T blasts are found in the liver). In conclusion, recently activated CD8+ T blasts that enter the liver postimmunization in small numbers can transiently modulate the phenotype of HC, allowing them to activate naïve CD8+ T cells with unrelated specificities. (HEPATOLOGY 2004;39:1256–1266.)

Specific, functional effector/memory CD8+ T cells are found in the liver post-vaccination

BACKGROUND The liver efficiently eliminates activated CD8+ T blasts. It is unknown if vaccine-primed CD8+ T blasts migrate to and establish functional CD8+ T cell immunity in the liver post-immunization. AIMS We tested, if functional CD8+ T cell populations can be detected in the liver post-vaccination. METHODS Murine CD8+ T cells with different epitope/restriction specificities were primed by intramuscular injection of protein- or DNA-based vaccines. The kinetics of appearance in the liver, as well as the surface phenotype and functional competence of intrahepatic, specific CD8+ T cell populations was tested. RESULTS High numbers of specific CD8+ T cells appear in the liver after vaccination that are activated (CD69+ CD44+), express effector functions (CD27lo/CD28lo phenotype, interferon gamma secretion, specific cytolytic reactivity), but show no evidence of apoptosis (annexin V-, B220lo, similar numbers/kinetics in primed, congenic lpr/lpr mice). Specific CD8+ T cells from the liver adoptively transferred into a naïve, syngeneic host successfully reconstitute specific CD8+ T cell immunity. CONCLUSIONS Specific, functionally competent CD8+ effector/memory T cell populations are established in the liver for months post-vaccination.