Interferon signaling in chronic hepatitis C : mechanisms and implications for therapy

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide and can lead to liver cirrhosis and hepatocellular carcinoma. The current standard therapy of chronic hepatitis C (CHC) consists of a combination of pegylated interferon alpha (pegIFNα) and ribavirin. However, sustained viral clearance is achieved in only 50-60% of patients. The underlying mechanism of failure of pegIFNα based therapy remains unknown and no molecular or genetic markers have been identified that could predict the treatment outcome. The overall aim of the study described in this thesis is to understand the molecular basis for failure of IFNα based therapies in patients with CHC. The study has focused on the IFNinduced Jak-STAT (janus kinase-signal transducer and activator of transcription) signaling pathway. To address the molecular basis of treatment response to IFN therapy, three experimental approaches have been employed. The first approach involved the analysis of IFNα signaling and expression of interferon stimulated genes (ISGs) in liver biopsies and peripheral blood mononuclear cells (PBMCs) of HCV patients undergoing pegIFNα treatment. Paired liver biopsies and PBMCs from 16 patients were collected before andhours after the first injection of pegIFNα, and were subjected to analysis of global gene expression using Affymetrix arrays. Further, activation of the IFN-induced Jak-STAT signaling pathway was analyzed by immunoblotting, immunohistochemistry and gel shift assays. The correlation of these biochemical and molecular data with the clinical response to treatment demonstrated that in the liver of patients with a rapid response pegIFNα induced a strong upregulation of ISGs, whereas in patients that did not respond to therapy, induction of IFN-dependent gene expression was impaired. Surprisingly, the non-responders had maximally induced ISG expression already before treatment with pegIFNα. Furthermore, the analyses of STAT1 phosphorylation, nuclear localization and DNA binding confirmed that the endogenous IFN signaling pathway in non-responders is pre-activated and refractory to further stimulation. In contrast to liver samples, ISG expression in PBMCs was stimulated by pegIFNα in both responders and nonresponders, indicating that PBMCs are not a good surrogate marker for IFNα responses in the liver and that chronic HCV infection has strong local effects on the IFN system in liver. Our findings support an interesting concept that activation of the endogenous IFN system in CHC not only is ineffective in clearing the infection, but may also impede the response to therapy, most likely by inducing a refractory state of the IFN signaling pathway in the liver. In the second approach we addressed the mechanisms underlying the pre-activation of the endogenous IFN system in a defined group of HCV patients (future non-responders). For this purpose, we analyzed ISG expression by quantitative RT-PCR and nuclear localization of STAT1 by immunohistochemistry in a cohort of 112 patients with CHC. By subdividing this cohort according to the HCV genotype (GT), we discovered that patients infected with HCV GT 1 and 4 more often show hepatic ISG preactivation than GT 2 and 3 patients, thus providing an explanation for the poor response to IFN therapy seen in GT 1/4 patients. We analyzed the possible involvement of viral sensory pathways in type I IFN production and ISG upregulation. Previously, the viral HCV NS3-4A protease was shown to interfere with viral sensory pathways by cleaving and thereby inactivating an important adaptor molecule, Cardif. We therefore assessed Cardif cleavage in liver biopsies of HCV patients and found that cleavage more often occurred in patients infected with HCV GTs 2 and 3. Our findings support a concept that the success of the virus in preventing the induction of the endogenous IFN system in the livers of these patients would, however, come at the cost of being more susceptible to IFNα therapies as is the case with GT 2/3 patients. In the third approach we designed an experimental model to study the molecular basis of refractoriness of IFN signaling in vivo. Previously, cell culture experiments demonstrated a long lasting desensitization period, which followed the initial activation of the IFNα signaling pathway. In the approach used here, we established a mouse model in which continuous presence of IFNα in vivo was achieved by multiple subcutaneous injections, mimicking the constitutively high serum levels achieved by pegIFNα in patients. Interestingly, this resulted in refractoriness of IFNα signaling. Activation of STAT1 and STAT2, but not STAT3, in the mouse liver was desensitized by continuous IFNα stimulation. To elucidate the mechanism of this refractoriness, the role of negative regulators of the Jak- STAT signaling pathway was investigated. IFN signaling remained refractory in mice deficient in suppressor of cytokine signaling (SOCS) 3 and persisting refractoriness was also observed in mice deficient in IL-10, a strong inducer of SOCS3. Ubiquitin specific peptidase 18 (USP18/UBP43) was recently identified as novel negative regulator of IFNα signal transduction. Interestingly, refractoriness could be overcome in USP18/UBP43 knockout mice. These data strongly indicate that UBP43 is the decisive factor in inducing a refractory state in the IFNα signaling pathway in vivo

Herpes simplex virus colitis mimicking acute severe ulcerative colitis: a case report and review of the literature

A 60-year-old female patient with longstanding left-sided ulcerative colitis presented with symptoms mimicking an acute flare and developed a colonic perforation shortly after starting steroid treatment. Following left hemicolectomy and Hartmann's procedure, rescue treatment with infliximab was started. Within a few days, the patient developed hepatic failure. Histology and immunohistochemistry of the specimen revealed extensive necrotizing herpes simplex virus colitis, and liver biopsy demonstrated herpes simplex virus hepatitis. Sixteen days after admission, the patient died from multiorgan failure. This compelling case of severe herpes simplex virus colitis raises awareness of a rare but potentially detrimental infection in patients with inflammatory bowel disease

Mild hypothermia provides Treg stability

Regulatory T cells (Tregs) play crucial role in maintenance of peripheral tolerance. Recent clinical trials confirmed safety and efficacy of Treg treatment of deleterious immune responses. However, Tregs lose their characteristic phenotype and suppressive potential during expansion ex vivo. Therefore, multiple research teams have been studding Treg biology in aim to improve their stability in vitro. In the current paper, we demonstrate that mild hypothermia of 33 °C induces robust proliferation of Tregs, preserves expression of FoxP3, CD25 and Helios, and prevents TSDR methylation during culture in vitro. Tregs expanded at 33 °C have stronger immunosuppressive potential and remarkably anti-inflammatory phenotype demonstrated by the whole transcriptome sequencing. These observations shed new light on impact of temperature on regulation of immune response. We show that just a simple change in temperature can preserve Treg stability, function and accelerate their proliferation, responding to unanswered question- how to preserve Treg stability in vitro

S-Adenosyl-Methionine and Betaine Improve Early Virological Response in Chronic Hepatitis C Patients with Previous Nonresponse

Treatment of chronic hepatitis C (CHC) with pegylated interferon (pegIFN ) and ribavirin results in a sustained response in approximately half of patients. Viral interference with IFN signal transduction through the Jak-STAT pathway might be an important factor underlying treatment failure. S-adenosyl-L-methionine (SAMe) and betaine potentiate IFN signaling in cultured cells that express hepatitis C virus (HCV) proteins, and enhance the inhibitory effect of IFN on HCV replicons. We have performed a clinical study with the aim to evaluate efficacy and safety of the addition of SAMe and betaine to treatment of CHC with pegIFN /ribavirin

Stimulatory MAIT cell antigens reach the circulation and are efficiently metabolised and presented by human liver cells.

OBJECTIVE Mucosal-associated invariant T (MAIT) cells are the most abundant T cells in human liver. They respond to bacterial metabolites presented by major histocompatibility complex-like molecule MR1. MAIT cells exert regulatory and antimicrobial functions and are implicated in liver fibrogenesis. It is not well understood which liver cells function as antigen (Ag)-presenting cells for MAIT cells, and under which conditions stimulatory Ags reach the circulation. DESIGN We used different types of primary human liver cells in Ag-presentation assays to blood-derived and liver-derived MAIT cells. We assessed MAIT cell stimulatory potential of serum from healthy subjects and patients with portal hypertension undergoing transjugular intrahepatic portosystemic shunt stent, and patients with inflammatory bowel disease (IBD). RESULTS MAIT cells were dispersed throughout healthy human liver and all tested liver cell types stimulated MAIT cells, hepatocytes being most efficient. MAIT cell activation by liver cells occurred in response to bacterial lysate and pure Ag, and was prevented by non-activating MR1 ligands. Serum derived from peripheral and portal blood, and from patients with IBD stimulated MAIT cells in MR1-dependent manner. CONCLUSION Our findings reveal previously unrecognised roles of liver cells in Ag metabolism and activation of MAIT cells, repression of which creates an opportunity to design antifibrotic therapies. The presence of MAIT cell stimulatory Ags in serum rationalises the observed activated MAIT cell phenotype in liver. Increased serum levels of gut-derived MAIT cell stimulatory ligands in patients with impaired intestinal barrier function indicate that intrahepatic Ag-presentation may represent an important step in the development of liver disease

4C Mortality Score correlates with in-hospital functional outcome after COVID-19-associated ischaemic stroke

Aim of the study. The 4C Mortality Score was created to predict mortality in hospitalised patients with COVID-19 and has to date been evaluated only in respiratory system disorders. The aim of this study was to investigate its application in patients with COVID-19-associated acute ischaemic stroke (AIS).Clinical rationale for study. COVID-19 is a risk factor for AIS. COVID-19-associated AIS results in higher mortality and worse functional outcome. Predictors of functional outcome in COVID-19-associated AIS are required.Materials and methods. This was a retrospective observational study of patients with AIS hospitalised in seven neurological wards in Małopolska Voivodship (Poland) between August and December 2020. We gathered data concerning the patients’ age, sex, presence of cardiovascular risk factors, type of treatment received, and the presence of stroke-associated infections (including pneumonia, urinary tract infection and infection of unknown source). We calculated 4C Mortality Score at stroke onset, and investigated whether there was a correlation with neurological deficit measured using the National Health Institute Stroke Scale (NIHSS) and functional outcome assessed using the modified Rankin Scale (mRS) at discharge.Results. The study included 52 patients with COVID-19-associated AIS. The 4C Mortality Score at stroke onset correlated with mRS (rs = 0.565, p < 0.01) at discharge. There was also a statistically significant difference in the mean 4C Mortality Score between patients who died and patients who survived the stroke (13.08 ± 2.71 vs. 9.85 ± 3.47, p = 0.04).Conclusions and clinical implications. 4C Mortality Score predicts functional outcome at discharge in COVID-19-associated AIS patients

Morbus Wilson oder hepatolentikuläre Degeneration

Wilson's disease or hepatolenticular degeneration Abstract. Wilson's disease, or hepatolenticular degeneration, is a rare inherited disorder of copper metabolism. The most common clinical presentations are liver disease and / or neuro-psychiatric manifestations. Pathophysiologically, Wilson's disease is caused by mutations in the ATP7B gene, which lead to defective biliary excretion of copper and subsequent accumulation of copper in the liver and in other organs. Its prevalence is approximately 1:30 000, however its penetrance, clinical presentation and disease severity vary widely, ranging from asymptomatic elevation of liver enzymes to cirrhosis or acute liver failure with or without neuro-psychiatric symptoms. For this reason, Wilson's disease should be suspected and ruled out in cases of indeterminate liver disease or neuropsychiatric disturbances. The diagnostic algorithms are complex and involve clinical tests, ophthalmologic examination (Kayser-Fleischer rings in split-lamp examination), blood and urine tests, genetic testing, imaging and histology. In compensated liver disease, treatment of Wilson's disease by copper depletion (chelators, zinc) is usually effective. In case of liver failure liver transplantation may be needed, which corrects the underlying error of copper metabolism. New drugs with improved efficacy and tolerability are in clinical development