Recurrent hepatic encephalopathy (HE) is a leading cause of readmission despite standard of care (SOC) associated with microbial dysbiosis. Fecal microbiota transplantation (FMT) may improve dysbiosis; however, it has not been studied in HE. We aimed to define whether FMT using a rationally-derived stool donor is safe in recurrent HE compared to SOC alone. An open-label, randomized clinical trial with a 5 month follow-up in outpatient cirrhotic men with recurrent HE on SOC was conducted with 1:1 randomization. FMT-randomized patients received 5-days of broad-spectrum antibiotic pre-treatment then a single FMT enema from the same donor with the optimal microbiota deficient in HE. Follow-up occurred on days 5, 6, 12, 35 and 150 post-randomization. The primary outcome was safety of FMT compared to SOC using FMT-related serious adverse events (SAE). Secondary outcomes were AEs, cognition, microbiota and metabolomic changes. Participants in both arms were similar on all baseline criteria and were followed till study-end. FMT with antibiotic pre-treatment was well-tolerated. Eight (80%) SOC participants had a total of 11 SAE compared to two (20%) FMT participants with SAEs (both FMT-unrelated, p=0.02). Five SOC and no FMT participants developed further HE (p=0.03). Cognition improved in FMT, but not SOC group. MELD score transiently worsened post-antibiotics, but reverted to baseline post-FMT. Post-antibiotics, beneficial taxa and microbial diversity reduction occurred with Proteobacteria expansion. However, FMT increased diversity and beneficial taxa. SOC microbiota and MELD score remained similar throughout. Conclusions: FMT from a rationally selected donor reduced hospitalizations, improved cognition and dysbiosis in cirrhosis with recurrent HE.

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Background/aimsIntestinal permeability with systemic distribution of bacterial products are central in the immunopathogenesis of alcoholic liver disease (ALD), yet links with intestinal immunity remain elusive. Mucosa-associated invariant T cells (MAIT) are found in liver, blood and intestinal mucosa and are a key component of antibacterial host defences. Their role in ALD is unknown.Methods/designWe analysed frequency, phenotype, transcriptional regulation and function of blood MAIT cells in severe alcoholic hepatitis (SAH), alcohol-related cirrhosis (ARC) and healthy controls (HC). We also examined direct impact of ethanol, bacterial products from faecal extracts and antigenic hyperstimulation on MAIT cell functionality. Presence of MAIT cells in colon and liver was assessed by quantitative PCR and immunohistochemistry/gene expression respectively.ResultsIn ARC and SAH, blood MAIT cells were dramatically depleted, hyperactivated and displayed defective antibacterial cytokine/cytotoxic responses. These correlated with suppression of lineage-specific transcription factors and hyperexpression of homing receptors in the liver with intrahepatic preservation of MAIT cells in ALD. These alterations were stronger in SAH, where surrogate markers of bacterial infection and microbial translocation were higher than ARC. Ethanol exposure in vitro, in vivo alcohol withdrawal and treatment with Escherichia coli had no effect on MAIT cell frequencies, whereas exposure to faecal bacteria/antigens induced functional impairments comparable with blood MAIT cells from ALD and significant MAIT cell depletion, which was not observed in other T cell compartments.ConclusionsIn ALD, the antibacterial potency of MAIT cells is compromised as a consequence of contact with microbial products and microbiota, suggesting that the ‘leaky’ gut observed in ALD drives MAIT cell dysfunction and susceptibility to infection in these patients.

The impact of prolonged direct antiviral therapy on hepatitis B surface antigen (HBsAg) levels in patients with chronic hepatitis B is poorly understood. We quantitatively assessed serum HBsAg levels during 3 years of telbivudine treatment, as well as their relationship with virologic and biochemical characteristics in 162 hepatitis B e antigen–positive patients who maintained undetectable serum hepatitis B virus (HBV) DNA long‐term. Telbivudine treatment progressively reduced serum HBsAg levels (mean ± SD) from baseline (3.8 ± 0.6 log10 IU/mL) to treatment week 24 (3.4 ± 0.7 log10 IU/mL), treatment year 1 (3.3 ± 0.8 log10 IU/mL), and treatment year 3 (3.0 ± 1.4 log10 IU/mL) (P <0.0001). In this patient population, HBsAg loss was observed in nine (6%) of 162 patients through year 3. During the first year of treatment, three patterns of HBsAg decline were observed: rapid (≥1 log10 IU/mL) in 32 patients, slow (0‐1 log10 IU/mL) in 74 patients, and steady levels in 56 patients. These findings were associated with different likelihoods of HBsAg loss during long‐term telbivudine therapy. Eight of 32 patients with rapid HBsAg decline versus none of 56 patients with steady HBsAg levels achieved HBsAg loss at year 3 (P = 0.0024). HBV genotype was a significant determinant for HBsAg kinetics, with the fastest decline in genotype A patients. In patients with subsequent HBsAg loss, viral antigens were already undetectable in liver biopsy samples after 1 year of treatment. This was associated with markedly enhanced antiviral T cell reactivity. Conclusion: In patients who have effective suppression of viral replication during telbivudine treatment, a rapid decline in serum HBsAg levels during the first year may identify those with a greater likelihood of achieving HBsAg clearance. (HEPATOLOGY 2010

Resolution of hepatitis B virus (HBV) infection was believed to be attributed to the cytotoxic T cell–mediated killing of infected hepatocytes. However, studies in HBV transgenic mice and HBV-infected chimpanzees revealed that T cell control of HBV replication also involves cytokine-mediated noncytolytic mechanisms. The relative role of cytolytic and noncytolytic functions of virus-specific CD8+ T cells during interaction with HBV-producing hepatocytes is not well understood. By using HLA-A2 matched effector cells (CD8+ T cell line or clone) and target cells supporting full HBV replication, we demonstrate that virus-specific CD8+ T cells can inhibit HBV replication in HBV-producing hepatocytes with minimal cell lysis. Although CD8+ T cells kill a fraction of infected cells, this effect is minimal, and most of the viral inhibition is mediated by noncytolytic mechanisms. CD8+ T cells produce an array of cytokines, among which IFN-γ and TNF-α are responsible for HBV inactivation in the target cells. Blockade of IFN-γ and TNF-α abrogated the noncytolytic inhibition of HBV, indicating that these two cytokines mediate the control of HBV by noncytolytic mechanisms. Furthermore, treatment of the HBV-producing hepatocytes with rIFN-γ and rTNF-α resulted in an efficient suppression of viral replication without cytotoxicity. In contrast, coculture of the same target cells with activated HLA-mismatched mitogen-activated lymphomononuclear cells caused a marked cytolytic effect and was less effective in HBV control. These results provide direct evidence that virus-specific CD8+ T cells efficiently control HBV replication by noncytolytic mechanisms, and this effect is mediated by IFN-γ and TNF-α.

Tumours have at least two mechanisms that can alter dendritic cell (DC) maturation and function. The first affects the ability of haematopoietic progenitors to differentiate into functional DCs; the second affects their differentiation from CD14 þ monocytes, promoting an early but dysfunctional maturation. The aim of this study was to evaluate the in vivo relevance of these pathways in breast cancer patients. For this purpose, 53 patients with invasive breast cancer were compared to 68 healthy controls. To avoid isolation or culture procedures for enrichment of DCs, analyses were directly performed by flow cytometry on whole-blood samples. The expression of surface antigens and intracellular accumulation of regulatory cytokines upon LPS stimulation were evaluated. The number of DCs, and in particular of the myeloid subpopulation, was markedly reduced in cancer patients (Po0.001). Patient DCs were characterized by a more mature phenotype compared with controls (P ¼ 0.016), and had impaired production of IL-12 (Po0.001). These alterations were reverted by surgical resection of the tumour. To investigate the possible role of some tumourrelated immunoactive soluble factors, we measured the plasmatic levels of vascular endothelial growth factor, IL-10 and spermine. A significant inverse correlation between spermine concentration and the percentage of DCs expressing IL-12 was found. Evidence was also obtained that in vitro exposure of monocyte-derived DCs to spermine promoted their activation and maturation, and impaired their function. Taken together, our results suggest that both the above-described mechanisms could concomitantly act in breast cancer to affect DC differentiation, and that spermine could be a mediator of dysfunctional maturation of DCs.

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Background Chronic liver injury triggers a progenitor-cell repair-response, and liver fibrosis occurs when repair becomes de-regulated. Previously, we reported that reactivation of the Hedgehog (Hh) pathway promotes fibrogenic liver-repair. Osteopontin (OPN) is a Hh-target, and a cytokine that is highly upregulated in fibrotic tissues, and regulates stem-cell fate. Thus, we hypothesized that OPN may modulate liver progenitor-cell response, and thereby, modulate fibrotic outcomes. We further evaluated the impact of OPN-neutralization on murine liver fibrosis. Methods Liver progenitors (603B and BMOL) were treated with OPN-neutralizing aptamers in the presence or absence of TGF–β, to determine if (and how) OPN modulates liver progenitor function. Effects of OPN-neutralization (using OPN-aptamers or OPN-neutralizing antibodies) on liver progenitor-cell response and fibrogenesis were assessed in three models of liver fibrosis (carbon tetrachloride, methionine-choline deficient diet, 3, 5,-diethoxycarbonyl-1,4-dihydrocollidine diet) by qRTPCR, Sirius-Red staining, hydroxyproline assay, and semi-quantitative double-immunohistochemistry. Finally, OPN expression and liver progenitor response were corroborated in liver tissues obtained from patients with chronic liver disease. Results OPN is over-expressed by liver progenitors in humans and mice. In cultured progenitors, OPN enhances viability and wound-healing by modulating TGF-β signaling. In vivo, OPN-neutralization attenuates the liver progenitor-cell response, reverses epithelial-mesenchymal-transition in Sox9+ cells, and abrogates liver fibrogenesis. Conclusions OPN upregulation during liver injury is a conserved repair-response, and influences liver progenitor-cell function. OPN-neutralization abrogates the liver progenitor-cell response and fibrogenesis in mouse models of liver fibrosis.