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Further understanding of the molecular biology and pathogenesis of hepatocellular carcinoma (HCC) is crucial for future therapeutic development. SMAD4, recognized as an important tumor suppressor, is a central mediator of transforming growth factor beta (TGFB) and bone morphogenetic protein (BMP) signaling. This study investigated the role of SMAD4 in HCC. Nuclear localization of SMAD4 was observed in a cohort of 140 HCC patients using tissue microarray. HCC cell lines were used for functional assay in vitro and in immune-deficient mice. Nuclear SMAD4 levels were significantly increased in patient HCC tumors as compared with adjacent tissues. Knockdown of SMAD4 significantly reduced the efficiency of colony formation and migratory capacity of HCC cells in vitro and was incompatible with HCC tumor initiation and growth in mice. Knockdown of SMAD4 partially conferred resistance to the anti-growth effects of BMP ligand in HCC cells. Importantly, simultaneous elevation of SMAD4 and phosphorylated SMAD2/3 is significantly associated with poor patient outcome after surgery. Although high levels of SMAD4 can also mediate an antitumor function by coupling with phosphorylated SMAD1/5/8, this signaling, however, is absent in majority of our HCC patients. In conclusion, this study revealed a highly non-canonical tumor-promoting function of SMAD4 in HCC. The drastic elevation of nuclear SMAD4 in sub-population of HCC tumors highlights its potential as an outcome predictor for patient stratification and a target for personalized therapeutic development.

Objective The risk of hepatocellular carcinoma (HCC) is increased in patients with metabolic syndrome (MS), possibly related to nonalcoholic fatty liver disease (NAFLD). As histological features of NAFLD may regress in cirrhosis, we compared steatosis versus steatohepatitis in the nontumoral liver of noncirrhotic HCC patients. Patients and methods A retrospective clinicopathological analysis was carried out in 91 noncirrhotic HCC patients. Patients were divided into three subgroups: that is, patients with: (1) MS without other risk factors for underlying liver disease, (2) no underlying risk factors, or (3) other risk factors (with or without MS). The NAFLD activity score (NAS) less than 3 was classified as no steatohepatitis, NAS 3-4 as borderline steatohepatitis, and NAS 5 or more as definite steatohepatitis. Results Eleven (12%) patients had MS without other risk factors (group 1). In the nontumoral liver, significant steatosis (>= 5% of hepatocytes) was generally present (in 10/11 patients), with mild lobular inflammation and absence of ballooning in most cases. Absence of steatohepatitis, borderline steatohepatitis, and definite steatohepatitis were found in 55, 45, and 0% of cases, respectively. In groups 2 and 3, significant steatosis was frequently present (in 16/37 and 21/43 patients, respectively). Absence of steatohepatitis, borderline steatohepatitis, and definite steatohepatitis were found in 84, 16, and 0% of cases (group 2), respectively, in 77, 23, and 0% of cases (group 3). Conclusion In noncirrhotic HCC patients, histological steatosis was frequently present, whereas overt steatohepatitis did not occur. These findings may be relevant for HCC pathogenesis in NAFLD. Eur J Gastroenterol Hepatol 28:955-962 Copyright (C) 2016 Wolters Kluwer Health, Inc. All rights reserved.

summary. Antiviral treatment of patients with active chronic hepatitis B may lead to significant reduction in morbidity and mortality. However, after stopping nucleoside therapy, relapse rates are high in those without acquired specific immunity. We have treated two chronic hepatitis B patients with in vivo immunization. In vivo immunization aims to optimize conditions for an effective immune response: following rapid and profound virus suppression by interferon-lamivudine combination therapy, lamivudine is withdrawn intermittently for 4 weeks during continued interferon therapy. In both patients with profound virus suppression a rapid rebound in viral replication was observed after lamivudine withdrawal; despite continued interferon. These periods of renewed viral replication were followed by rises in hepatitis activity. After re-introduction of lamivudine HBV DNA became undetectable by PCR followed by normalization of serum ALT. These observations are a stimulus to further explore the concept of in vivo immunization as a novel therapeutic approach for chronic hepatitis B.

Treatment with interferon-alpha (IFN-alpha) leads to a response in only a minority of patients with chronic hepatitis B virus (HBV) infection, but the reasons for this are poorly understood. It was recently shown that in patients with chronic HBV infection, CD4+CD25+ regulatory T-cells (Treg) can suppress the HBV-specific immune response. We aimed to investigate whether in non-responders to IFN-alpha therapy Treg contribute to treatment failure by downregulating the HBV-specific T-cell responses.Fourteen patients positive for hepatitis B e antigen received pegylated IFN-alpha monotherapy for 52 weeks and were followed for 26 weeks.Compared with non-responders, responders displayed an increased HBV-specific T-helper cell proliferation. At the start of treatment there was no difference in the frequencies of CD4+CD25+ Treg between responders and non-responders. During therapy, the frequency of CD4+CD25+ Treg increased in non-responders, but not in responders. In contrast to the responders, the non-responders showed a significant increase in the frequency of interleukin-10-producing cells. Treg depletion resulted in increased proliferation capacity, but did not affect the frequency of interleukin-10-producing cells measured during the course of the treatment.This study indicates that Treg might have an important role in HBV persistence during and after pegylated IFN-alpha therapy.

In liver cancer tumor-infiltrating regulatory T cells (Ti-Treg) are potent suppressors of tumor-specific T-cell responses and express high levels of the Treg-associated molecules cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and glucocorticoid-induced tumor necrosis factor receptor (GITR). In this study, we have evaluated the capacity of GITR-ligation, CTLA-4-blockade and a combination of both treatments to alleviate immunosuppression mediated by Ti-Treg. Using ex vivo isolated cells from individuals with hepatocellular carcinoma (HCC) or liver metastases from colorectal cancer (LM-CRC) we show that treatment with a soluble form of the natural ligand of GITR (GITRL), or with blocking antibodies to CTLA-4, reduces the suppression mediated by human liver tumor-infiltrating CD4+Foxp3+ Treg, thereby restoring proliferation and cytokine production by effector T cells. Importantly, combined treatment with low doses of both molecules exhibited stronger recovery of T cell function compared with either treatment alone. Our data suggest that in patients with primary and secondary liver cancer both GITR-ligation and anti-CTLA-4 mAb can improve the antitumor immunity by abrogating Ti-Treg mediated suppression.

Based on virological and biochemical parameters patients with chronic hepatitis B virus (HBV) are divided into distinct clinical phases: the immune-tolerance phase, the immune-clearance phase, and the inactive carrier state. Unclear is whether these phases have characteristic intrahepatic immune responses. The composition of liver-derived lymphocytes in patients with chronic HBV infection was studied. In 47 patients the composition of liver-derived lymphocytes was analyzed by flow cytometry of fine needle aspiration biopsies of the liver. The proportion natural killer (NK) cells in the liver was significantly higher in immune-tolerant than in immune-clearance patients and inactive carriers. No differences were found in proportion CD4+ T-cells and CD8+ T-cells, in these phases. However, when patients in the immune-clearance phase, with similar alanine transaminase (ALT), were grouped according to viral load, the proportion CD8+ T-cells was higher in those with high viral load. In contrast, the proportion CD4+ T-cells was increased in patients with low HBV-DNA. These differences were absent in the peripheral blood (PB). Intrahepatic HBV-specific CD8+ T-cells were mainly found in immune-clearance patients with low viral load. In conclusion, clear differences in the intrahepatic cellular infiltrate were found between the various clinical phases of chronic HBV infection. These findings are relevant to the design of new, individualized anti-viral strategies.

Background/Aims: Characteristics of the intrahepatic virus-specific T-cell response in patients with acute hepatitis B virus (HBV) infection have not been studied due to the risk of complications associated with standard liver biopsies. In this study we aimed to characterize the virus-specific CD8 + T-cell response in the liver of patients with acute HBV infection using fine-needle aspiration-biopsy (FNAB). Methods: In HLA-A2 positive patients with acute HBV infection a FNAB was performed at first presentation, at the time of HBsAg-seroconversion and 3 months after HBsAg-seroconversion. HLA-A2 tetramers were used to identify HBV-specific CD8 + T-cells in FNAB-cytology and peripheral blood (PB). Results: At first presentation there was a correlation between the frequency of intrahepatic CD8 + T-cells and the degree of liver damage. At all time points there was sequestering of HBV-specific CD8 + T-cells in the liver, and the percentage of intrahepatic HLA-DR expressing HBV-specific CD8 + T-cells was higher than in PB. Three months after HBsAg-seroconversion the frequency of intrahepatic HBV-specific CD8 + T-cells remained high. Conclusions: HBV-specific CD8 + T-cells are compartmentalized in the liver during acute HBV infection. Their presence in the liver may suggest a role in the resolution of the infection. Intrahepatic HBV-specific CD8 + T cells remain detectable at high frequencies after HBsAg-seroconversion.

SUMMARY: Information about the character and grade of the intrahepatic immune response in viral hepatitis is important for the evaluation of disease stage and effect of therapy. Complications like haemorrhage limit the frequent performance of tissue-needle biopsies (TB), and the cells of peripheral blood have to be used as surrogate markers instead. Fine-needle-aspiration biopsy (FNAB) of the liver represents a safe and atraumatic method that allows frequent cytological sampling. Our aim was to investigate whether flow cytometry of FNAB specimens allows co-analysis of phenotype, function and specificity of key populations of liver-infiltrating lymphocytes (LIL). In 20 consecutive patients with chronic viral hepatitis [10 hepatitis B virus (HBV), 10 hepatitis C virus (HCV)], flow cytometry was performed on FNAB cytology, and simultaneously on lymphocytes isolated from a TB and peripheral blood mononuclear cells (PBMC). The ratio of CD8+/CD4+ lymphocytes in FNAB correlated well with LIL from TB (r =0.78, P < 0.05) but differed from PBMC (mean ratio: 2.6, 2.1 and 0.7, respectively). Similarly, a correlation was observed for percentage CD56+ natural killer (NK) cells (mean %: 29.9, 32.3 and 14.5, respectively; r = 0.69, P < 0.05). The percentage of interferon (IFN)-gamma-producing CD3+ lymphocytes in both FNAB and TB was higher than in PBMC (mean %: 41, 44 and 22, respectively; P < 0.05). Furthermore, tetrameric complexes allowed analysis of HBV-specific T cells in FNAB specimens. In conclusion, flow cytometry of FNAB allows easy, atraumatic and reliable analysis of lymphocytes obtained from the intrahepatic compartment. Therefore, the FNAB is a valuable tool in the study of the immunopathology of viral hepatitis, and it may contribute to the improved clinical evaluation of chronic viral liver disease.

Hepatitis B virus (HBV) is one of the most prevalent viral pathogens of man with around 350 million chronically infected patients. It has been postulated that in persistently infected individuals the HBV-specific immune response is too weak to eliminate HBV from all infected hepatocytes, but sufficiently strong to continuously destroy HBV-infected hepatocytes and to induce chronic inflammatory liver disease. The primary aim in the treatment of chronic hepatitis B is to induce sustained disease remission and prevent serious complications like liver failure and/or hepatocellular carcinoma. The recent emergence of drug-resistant HBV mutants and post-treatment relapse as a consequence of nucleoside analogue monotherapy emphasizes that the principal goal should be to stimulate a successful immune response. In this paper we will focus on the immune response to HBV and we will review reported data on immunotherapeutic strategies like immunomodulatory drugs (cytokines and Thymic derivates) and vaccine therapies using currently available recombinant anti-HBV vaccines, lipopeptide-based T cell vaccine and newly developed genetic vaccines.

SUMMARYGerm-free HLA-B27 transgenic (TG) rats do not develop colitis, but colonization with specific pathogen-free (SPF) bacteria induces colitis accompanied by immune activation. To study host-dependent immune responses to commensal caecal bacteria we investigated cytokine profiles in mesenteric lymph node (MLN) cells from HLA-B27 TG versus nontransgenic (non-TG) littermates after in vitro stimulation with caecal bacterial lysates (CBL). Supernatants from CBL-stimulated unseparated T- or B- cell-depleted MLN cells from HLA-B27 TG and non-TG littermates were analysed for IFN-γ, IL-12, TNF, IL-10 and TGF-β production. Our results show that unfractionated TG MLN cells stimulated with CBL produced more IFN-γ, IL-12 and TNF than did non-TG MLN cells. In contrast, CBL-stimulated non-TG MLN cells produced more IL-10 and TGF-β. T cell depletion abolished IFN-γ and decreased IL-12 production, but did not affect IL-10 and TGF-β production. Conversely, neither IL-10 nor TGF-β was produced in cultures of B cell-depleted MLN. In addition, CD4+ T cells enriched from MLN of HLA-B27 TG but not from non-TG rats produced IFN-γ when cocultured with CBL-pulsed antigen presenting cells from non-TG rats. Interestingly, IL-10 and TGF-β, but not IFN-γ, IL-12 and TNF were produced by MLN cells from germ-free TG rats. These results indicate that the colitis that develops in SPF HLA-B27 TG rats is accompanied by activation of IFN-γ-producing CD4+ T cells that respond to commensal bacteria. However, B cell cytokine production in response to components of commensal intestinal microorganisms occurs in the absence of intestinal inflammation.

Background and aims: Bacteroides vulgatus induces colitis in gnotobiotic HLA-B27 transgenic (TG) rats while broad spectrum antibiotics prevent and treat colitis in specific pathogen free (SPF) TG rats although disease recurs after treatment ends. Lactobacilli treat human pouchitis and experimental colitis. We investigated if Lactobacillus rhamnosus GG (L GG) can prevent colitis in TG rats monoassociated with B vulgatus and if L GG or Lactobacillus plantarum 299v (LP 299v) can treat established colitis in SPF TG rats and prevent recurrent disease after antibiotics were stopped. Methods: Germfree B27 TG rats were monoassociated with B vulgatus for four weeks following two weeks of colonisation with L GG or no bacteria. SPF B27 TG rats received oral vancomycin and imipenem for two weeks, or water alone, followed by four weeks of treatment with oral L GG, LP 299v, or water only. Disease activity was quantified by blinded gross and histological scores, caecal myeloperoxidase (MPO) activity, and levels of interleukin (IL)-1β, tumour necrosis factor (TNF), transforming growth factor β, and IL-10. Results: L GG did not prevent colitis in B vulgatus co-associated TG rats or treat established disease in SPF rats. However, L GG but not LP 299v prevented colitis relapse in antibiotic treated rats with reduced gross and histological scores, caecal MPO, IL-1β, and TNF whereas caecal IL-10 was increased. Conclusions: L GG does not prevent colitis in gnotobiotic TG rats or treat established disease in SPF rats, but is superior to LP 299v in the prevention of recurrent colitis. These studies suggest that antibiotics and probiotic agents provide synergistic therapeutic effects, perhaps mediated by altered immunomodulation with selective activity of different lactobacillus species.

Purpose/objective(s) Patients with perihilar cholangiocarcinoma (pCCA) can only be cured by surgery. The standard of care for those with inoperable tumors is palliative chemotherapy. In an attempt to control the disease locally without adding severe toxicity in patients with unresectable pCCA, we investigated the safety and feasibility of adding stereotactic body radiation therapy (SBRT) after chemotherapy. Materials/methods Patients were eligible with a diagnosis of unresectable pCCA, stage T1-T4N0-N1M0 (AJCC staging 7th edition), ECOG performance status of 0-1, and having finished 6-8 cycles chemotherapy treatment (cisplatin and gemcitabine). SBRT was planned in 15 fractions of 3.0-4.5Gy, aiming for a biologically effective dose > 80.5Gy, while not exceeding organs at risk (OAR) constraints. The primary endpoints were feasibility, defined as completing SBRT as planned, and toxicity evaluated within 3 months of treatment. A conventional '3+3'-design was used, corresponding to a sample size of 6 patients. Toxicity was scored with the CTCAE v4.03 grading system. Dose-limiting toxicity (DLT) was defined as grade ≥4 hepatobiliary or grade ≥3 gastrointestinal toxicity. Secondary endpoints, measured from start of radiotherapy, were local control, progression-free survival (RECIST 1.1), overall survival, and quality of life (QoL). QoL was measured using the QLQ-C30, EQ-5D-5L, and QLQ-BIL21 questionnaires. Results Six patients were enrolled between 1 November 2017 and 30 March 2020. All patients completed 6-8 cycles of chemotherapy. They were staged T4N0-1M0, each with unresectable disease due to vascular involvement. SBRT was completed as planned. A dose of 60Gy (15×4.0Gy) was prescribed at the 80% isodose. Due to OAR constraints, especially duodenum, the median PTV coverage was limited to 82.4% (75.7%-89.6%). No SBRT related DLT was observed. The most common grade ≥3 toxicity was cholangitis (n = 5), which could be resolved by improving biliary drainage. The 12-month local control rate was 80%. Metastases emerged in the form of peritoneal metastases/ascites (n = 3) and/or liver metastases (n = 1). Three of the 4 patients who developed distant metastases had lymph-node-positive disease at baseline. After a median follow up of 14 months, 4 of 6 patients were still alive. We observed no substantial changes in QoL. Median QLQ-C30 'global health status' scores were 75.0 at baseline, 3 months and 12 months. Conclusion Fractionated SBRT following standard chemotherapy was a feasible and safe local therapy option for unresectable pCCA, albeit with the use of hard constraints for OAR. Cholangitis was often observed, but was reversible and QoL was maintained. Based upon the observed safety and the promising local control, additional evaluation of effectiveness in larger studies is warranted.