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The effect of pegylated interferon-α (IFN) add-on therapy on HBV-specific T-cell responses was evaluated in 12 patients with stable, undetectable hepatitis B virus (HBV) load under nucleos(t)ide analogue therapy. Peripheral blood mononuclear cells were isolated at week 0, 4, 8, 12, 24 and 48 of IFN add-on therapy. Quantity and quality of circulating HBV S- and core-specific CD4 and CD8 T cells were analysed ex vivo by flow cytometry. HBV S- and core-specific CD4 T-cell numbers modestly increased within 8 weeks of IFN administration (P = 0.0391 and P = 0.0195), whereas HBV-specific CD8 T cells in general showed only minor changes under IFN add-on therapy. Functionality of HBV-specific CD4 but not CD8 T cells positively correlated with serum transaminase activity. In addition, we observed an increase in CD4 T cells producing tumour necrosis factor-α (TNFα) without antigen restimulation (P = 0.0039), which correlated with elevated transaminases. During IFN add-on therapy, two patients developed an anti-HBs seroconversion, only one of whom showed a relevant increase in HBV-specific T cells. In conclusion, IFN add-on therapy of chronic hepatitis B increased HBV-specific T-cell responses and affected a previously unrecognized TNFα-monofunctional CD4 T-cell population. Although the observed T-cell responses did not correlate with HBsAg seroconversion, we expect additional insights into the immunopathogenesis of hepatitis B, following the characterization of the newly identified TNF α-monofunctional T-cell population.

In this study, administration of tremelimumab was accompanied by a tolerable toxicity profile, most frequently including skin rash (65%), fatigue (55%), and anorexia (50%). During the initial course of tremilimumab, a transient increase of serum transaminase activity was observed. Hepatotoxicity did not impair liver function, even in the context of Child-Pugh B cirrhosis and hepatitis C. Therapy was stopped in 3 patients before tumor response evaluation was completed due to tremilimumab-related diarrhea or clinical deterioration, which was not associated with the treatment regimen. The relevance of this study is strengthened by the inclusion of HCC patients, showing hepatitis C associated cirrhosis, which represents a typical clinical scenario. However, the safety profile outlined above might not apply to HCC in association with other liver diseases. Particularly chronic hepatitis B, which shows higher liver failure rates due to inflammatory flares compared to hepatitis C, might respond differently to CTLA-4 blockage.

Alternatively polarized macrophages (Mϕ) shape the microenvironment of hepatocellular carcinoma (HCC) and temper anticancer immune responses. We investigated if sorafenib alters the HCC microenvironment by restoring classical macrophage polarization and triggering tumor-directed natural killer (NK) cell responses. In vivo experiments were conducted with sorafenib (25 mg/kg)-treated C57BL/6 wildtype as well as hepatitis B virus (HBV) and lymphotoxin transgenic mice with and without HCC. Monocyte-derived Mϕ or tumor-associated macrophages (TAM) isolated from HCC tissue were treated with sorafenib (0.07-5.0 μg/mL) and cocultured with autologous NK cells. Mϕ and NK cell activation was analyzed by flow cytometry and killing assays, respectively. Cytokine and growth factor release was measured by enzyme-linked immunosorbent assay. Short-term administration of sorafenib triggered activation of hepatic NK cells in wildtype and tumor-bearing mice. In vitro, sorafenib sensitized Mϕ to lipopolysaccharide, reverted alternative Mϕ polarization and enhanced IL12 secretion (P = 0.0133). NK cells activated by sorafenib-treated Mϕ showed increased degranulation (15.3 ± 0.2% versus 32.0 ± 0.9%, P < 0.0001) and interferon-gamma (IFN-γ) secretion (2.1 ± 0.2% versus 8.0 ± 0.2%, P < 0.0001) upon target cell contact. Sorafenib-triggered NK cell activation was verified by coculture experiments using TAM. Sorafenib-treated Mϕ increased cytolytic NK cell function against K562, Raji, and HepG2 target cells in a dose-dependent manner. Neutralization of interleukin (IL)12 or IL18 as well as inhibition of the nuclear factor kappa B (NF-κB) pathway reversed NK cell activation in Mϕ/NK cocultures. Conclusion: Sorafenib triggers proinflammatory activity of TAM and subsequently induces antitumor NK cell responses in a cytokine- and NF-κB-dependent fashion. This observation is relevant for HCC therapy, as sorafenib is a compound in clinical use that reverts alternative polarization of TAM in HCC. (HEPATOLOGY 2013;57:2358–2368)

Background Preclinical data suggested all-trans retinoic acid (tretinoin) as a potential antiviral agent against chronic hepatitis C infection. Aims To assess efficacy, safety, and tolerability of tretinoin in combination with peg-interferon and ribavirin in genotype-1 infected patients with prior non-response. Method We performed an open-label multicentre clinical trial. Patients were randomised to either receive additional tretinoin (45 mg/m 2 /day) for 12 weeks (arm A), or peg-interferon and ribavirin alone (arm B). Primary endpoint was the slope of the third phase of viral decline (M δ ) as determined in an established kinetic model known to correlate with treatment outcome. Secondary endpoints were additional kinetic parameters, viral response rates, safety, and tolerability. Results 27 patients in arm A and 30 patients in arm B were treated per protocol until week 12. Viral kinetic parameters did not differ. Rates of early virological response (>2 log 10 drop at week 12) were similar (10/27 versus 11/30 patients). In arm A, patients experienced a higher rate and intensity of adverse events, most commonly skin and mucosal dryness, and headache. Conclusion Addition of tretinoin was safe and acceptably well tolerated. However, it did not influence viral kinetics and thus cannot be further considered as a treatment option.

The metabolic syndrome (MetS) might contribute to morbidity after orthotopic liver transplantation (OLT). For this reason, we searched for MetS-associated risk factors and analyzed the link with nonalcoholic fatty liver disease (NAFLD) in OLT recipients. De novo MetS affected 32.9% of our cohort (n = 170) within 2 years after OLT. Multivariate analysis identified glycosylated hemoglobin (HbA1c) levels ≥5% [odds ratio (OR) = 3.5; 95% confidence interval (CI) = 1.56-8.13, P = 0.003], diabetes mellitus (OR = 4.31, CI = 1.69-10.99, P = 0.002), and arterial hypertension (OR = 4.59, CI = 1.46-14.49, P = 0.009) as independent risk factors for de novo MetS. MetS incidence correlated with steroid dosage after OLT (5.2 ± 2.4 mg/day vs. 7.1 ± 4.7 mg/day, P = 0.014), and was linked to NAFLD (P = 0.001) via obesity (OR = 4.67, CI = 1.55-14.1, P = 0.006) and dyslipidemia (OR = 4.23, CI = 1.35-13.3, P = 0.013) post-OLT. In conclusion, we were able to identify low threshold HbA1c as a novel risk factor for MetS after OLT and described a link of MetS with NAFLD in transplant organs. This study also indicated that steroid treatment is associated with MetS rates after OLT.

Human hepatitis B virus (HBV) infects the liver of humans or humanoid primates. In humans, HBV infection often causes an inflammatory liver disease – hepatitis B. The virus is transmitted by perinatal, percutaneous and sexual exposure, as well as by close person-to-person contact. The latter occurs especially among young children, presumably by open cuts or sores. Vertical transmission from mothers to their neonates, or infection during the first year of life, results in persistent often lifelong infection in >90% of cases. In contrast, infection during adulthood is cleared in >95% of cases, and results in lifelong protective immunity. While a correlation between the strength of HBV-specific T cell responses and virus clearance has been established, factors determining the strength of a T cell response as well as factors shifting the balance from immune tolerance to immune clearance are hardly understood. The innate immune response, early adaptive B and T cell responses, regulatory T cells, the liver microenvironment, and the peculiar properties of hepatocytes and nonparenchymal liver cells to present antigen seem to play a role. Understanding this complex interplay requires systematic immune monitoring of well characterized human cohorts, but also experimental approaches using primary human cells and genetically modified mouse models. Using these models, we begin to understand the immune recognition of HBV and how it influences the outcome of HBV infection. In this paper we review the current knowledge about virus-host interactions and how it influences the outcome of HBV infection and describe the immune signatures associated with clinical recovery and/or persistent infection.

AIM: To investigate the efficacy and safety of cape-citabine plus irinotecan ± bevacizumab in advanced or metastatic colorectal cancer patients. METHODS: Forty six patients with previously untreated, locally-advanced or metastatic colorectal cancer (mCRC) were recruited between 2001-2006 in a prospective open-label phase II trial, in German community-based outpatient clinics. Patients received a standard capecitabine plus irinotecan (CAPIRI) or CAPIRI plus bevacizumab (CAPIRI-BEV) regimen every 3 wk. Dose reductions were mandatory from the first cycle in cases of > grade 2 toxicity. The treatment choice of bevacizumab was at the discretion of the physician. The primary endpoints were response and toxicity and secondary endpoints included progression-free survival and overall survival. RESULTS: In the CAPIRI group vs the CAPRI-Bev group there were more female than male patients (47% vs 24%), and more patients had colon as the primary tumor site (58.8% vs 48.2%) with fewer patients having sigmoid colon as primary tumor site (5.9% vs 20.7%). Grade 3/4 toxicity was higher with CAPIRI than CAPIRI-Bev: 82% vs 58.6%. Partial response rates were 29.4% and 34.5%, and tumor control rates were 70.6% and 75.9%, respectively. No complete responses were observed. The median progression-free survival was 11.4 mo and 12.8 mo for CAPIRI and CAPIRI-Bev, respectively. The median overall survival for CAPIRI was 15 mo (458 d) and for CAPIRI-Bev 24 mo (733 d). These differences were not statistically different. In the CAPIRI-Bev, group, two patients underwent a full secondary tumor resection after treatment, whereas in the CAPIRI group no cases underwent this procedure. CONCLUSION: Both regimens were well tolerated and offered effective tumor growth control in this outpatient setting. Severe gastrointestinal toxicities and thromboembolic events were rare and if observed were never fatal.

There is a variety of differential diagnoses of acute and chronic human hepatitis regularly seen in clinical praxis. Risk factor assessment for specific entities provides important information and should guide individual diagnostic procedures. Liver screening tests often remain the first indicator for hepatic pathologies and should include quantification of liver enzymes, liver function parameters and cholestatic parameters. Nevertheless, virus serology should always be done during further laboratory evaluation. To estimate the parenchymal liver damage and to exclude biliary obstruction or hepatic lesions an abdominal ultrasound scan is essential. A liver biopsy may complete the diagnostic approach, if either the underlying hepatic pathology remains obscure or parenchymal liver damage needs to be defined.

BACKGROUND The efficacy of consensus interferon (CIFN), a synthetic IFN with optimised in vitro activity, was assessed in chronic hepatitis C virus (HCV) patients who had failed the pretreatment with interferon-alpha (IFNalpha) and ribavirin. METHODS One hundred and three patients after non-response (n=69) or relapse (n=34) to IFNalpha+/-ribavirin were randomly assigned to high-dose induction (CIFN 27-->9 microg daily for 24 weeks, 9 microg t.i.w. for 24 weeks) or low-dose treatment (CIFN 18 microg t.i.w. for 12 weeks, 9 microg t.i.w. for 36 weeks); each with ribavirin 800 mg/day. Follow-up was 24 weeks. RESULTS Non-responder patients treated with high-dose induction had higher early virological response rates (63% vs. 39%, P

For the study of hepatitis B virus infection, no permissive cell line or small animal is available. Stably transfected cell lines and transgenic mice which contain hepadnavirus genomes produce virus, but—unlike in natural infection—from an integrated viral transcription template. To transfer hepadnavirus genomes across the species barrier, we developed adenovirus vectors in which 1.3-fold-overlength human and duck hepatitis B virus genomes were inserted. The adenovirus-mediated genome transfer efficiently initiated hepadnavirus replication from an extrachromosomal template in established cell lines, in primary hepatocytes from various species, and in the livers of mice. Following the transfer, hepatitis B virus proteins, genomic RNA, and all replicative DNA intermediates were detected. Detection of covalently closed circular DNA in hepatoma cell lines and in primary hepatocytes indicated that an intracellular replication cycle independent from the transferred linear viral genome was established. High-titer hepatitis B virions were released into the culture medium of hepatoma cells and the various primary hepatocytes. In addition, infectious virions were secreted into the sera of mice. In conclusion, adenovirus-mediated genome transfer initiated efficient hepatitis B virus replication in cultured liver cells and in the experimental animals from an extrachromosomal template. This will allow development of small-animal systems of hepatitis B virus infection and will facilitate study of pathogenicity of wild-type and mutant viruses as well as of virus-host interaction and new therapeutic approaches.

Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer related mortality world wide and therapeutic options are limited. Therefore we aimed to generate cytotoxic T lymphocytes (CTL), which are capable to eliminate AFP-presenting tumor cells and could be useful for an alternative immunotherapy of HCC. Material and Methods: CTL were cloned from the naive T cell repertoire of HLA-A2.1 negative donors by in vitro stimulation with HLA-A2.1and AFP-pulsed dendritic cells (DC) derived from the same donor. After CTL expansion in presence of AFP peptidepulsed DC (AFP158–166, AFP325–334), AFP-specific clones were stained and enriched via AFP-streptamers. After single cell cloning, effector function was tested by chromium release assays against AFP peptide-pulsed target cells (T2 cells). Alloreactivity was excluded by testing against HLA-A2.1 positive lymphoblastoid cell lines. Results: Naive T cells were stimulated in presence of HLA-A2.1/AFPpulsed DCs, as indicated by an IFNg-positive CTL frequency of 5.7%. Unspecific stimulation reached IFNg-positive CTL frequencies of 1.2%, shown by a control experiment in presence of non-pulsed DCs. Streptamer staining identified AFP158 and AFP325 specific CTL populations in 24.98% and 4.36%, respectively. CTL clones derived from these populations developed an AFP-dependant cell lysis of up to 91% (AFP158) and 56% (AFP325) against AFP-pulsed T2 cells. Control peptide-pulsed T2 cells or HLA-A2.1 positive lymphoblastoid cell lines showed no significant lysis in presence of the same CTL clones. Conclusion: Allorestricted stimulation was able to expand AFP specific CTL from the naive T cell repertoire with high proliferation and killing function, which might be useful for immunotherapy of HCC. Nevertheless, further detailed analysis will be required to unravel the off-target effects in context of AFP-directed immunotherapy via studies in vivo.

Introduction Despite many efforts to develop new chemotherapies for metastatic upper gastrointestinal (GI) cancer, overall prognosis continues to be fatal, particularly in gastric and pancreatic cancer. Many of these patients deserve second-or third-line treatment after failure of first-line chemotherapy. Therefore, we analysed toxicity and response rate of weekly docetaxel after failed upfront regimes in these upper GI cancer patients. Patients and Methods Between 2001 and 2006, 18 patients received docetaxel based regimes (35 mg/m2 weekly) after informed consent. Response rates were determined using RECIST criteria or tumor progression if clinically evident. Toxicities were graded based on NCI CTC criteria (version 2). Most patients had gastric cancer (13/18). The remaining entities comprised of bilio-pancreatic cancer (5/18). Results Docetaxel was administered as 2nd line therapy in 28% (5/18), 3rd line therapy in 56% (10/18) and 4th or 5th line therapy in 17% (3/18). The average docetaxel dose was 38 mg/m2 (Median: 35 mg/m2) once weekly. Over a treatment duration of 14.7 weeks, the average dosage was 58 gr per patient and week. Overall, docetaxel was well tolerated with only few chemotherapy-associated toxicities (Grade 3/4), including nausea (17%), polyneuropathy (17%), anorexia (11%), neutropenia (6%) and leukopenia (17%). Docetaxel administration did not achieve any complete responses (CR) and one (5.6%) partial response (PR) was seen (1/18). In addition 5 patients (27.8%) had stable disease (SD), thus inducing a tumor control rate of 33.3% (6/18). Median progression-free survival was 2.4 months for all patients, 2.1 months in the gastric-cancer and 2.4 months in the bilio-pancreatic cancer subgroups respectively. After first docetaxel administration median survival for all patients was 4.5 months, patients with gastric cancer survived for 4.9 months whereas patients suffering from bilio-pancreatic carcinoma survived for 4.2 months. However, taken together 27% (5/18) had a remarkable overall survival of more than 2.5 years. Discussion In severely pretreated patients, with documented chemoresistant GI tumors, weekly docetaxel was well tolerated, presented good tumor control rate and overall survival. Therefore, this regimen may be used as salvage treatment in individual patients with upper GI cancers.

We evaluated the safety and efficacy of an escalating dose regimen of pegylated interferon alpha-2a (PEG-IFN(alpha-2a)) and ribavirin in the early phase of recurrent hepatitis C after orthotopic liver transplantation (OLT). In this prospective study, 26 patients transplanted for hepatitis C virus cirrhosis with recurrent hepatitis C were treated 3.4 +/- 3.6 months after OLT and compared with an untreated historical control. PEG-IFN(alpha-2a) was initiated as monotherapy, following stepwise dose escalation up to 180 mug/week and the addition of ribavirin up to 1200 mg/day or maximally tolerated doses for 48 weeks. In the intent-to-treat analysis, 38% showed an early virological response (EVR), 35% an end of treatment response (ETR) and 19% a sustained virological response (SVR). SVR was associated with EVR (P = 0.0001) and cumulative PEG-IFN(alpha-2a) dose (P = 0.04). There was no significant histological improvement compared with untreated patients. There were no treatment-related serious adverse events. Adverse events included leucopenia (77%) and thrombocytopenia (46%). Three patients discontinued therapy due to side effects, fourteen were nonresponders and four relapsers. Treatment with PEG-IFN(alpha-2a) and ribavirin in the acute phase of post-transplant recurrent hepatitis C yielded an EVR of 38% and an SVR of 19%. The combination was safe and well tolerated.