Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon

<p>Abstract</p> <p>Background</p> <p>Alpha interferon in combination with ribavirin is the standard therapy for hepatitis C virus infection. Unfortunately, a significant number of patients fail to eradicate their infection with this regimen. The mechanisms of IFN-resistance are unclear. The aim of this study was to determine the contribution of host cell factors to the mechanisms of interferon resistance using replicon cell lines.</p> <p>Results</p> <p>HCV replicons with high and low activation of the IFN-promoter were cultured for a prolonged period of time in the presence of interferon-alpha (IFN-alpha2b). Stable replicon cell lines with resistant phenotype were isolated and characterized by their ability to continue viral replication in the presence of IFN-alpha. Interferon resistant cell colonies developed only in replicons having lower activation of the IFN promoter and no resistant colonies arose from replicons that exhibit higher activation of the IFN promoter. Individual cell clones were isolated and nine IFN resistant cell lines were established. HCV RNA and protein levels in these cells were not altered by IFN- alpha2b. Reduced signaling and IFN-resistant phenotype was found in all Huh-7 cell lines even after eliminating HCV, suggesting that cellular factors are involved. Resistant phenotype in the replicons is not due to lack of interferon receptor expression. All the cell lines show defect in the JAK-STAT signaling and phosphorylation of STAT 1 and STAT 2 proteins were strongly inhibited due to reduced expression of Tyk2 and Jak-1 protein.</p> <p>Conclusion</p> <p>This in vitro study provides evidence that altered expression of the Jak-Stat signaling proteins can cause IFN resistance using HCV replicon cell clones.</p

Obeticholic acid for the treatment of non-alcoholic steatohepatitis: interim analysis from a multicentre, randomised, placebo-controlled phase 3 trial

Background Non-alcoholic steatohepatitis (NASH) is a common type of chronic liver disease that can lead to cirrhosis. Obeticholic acid, a farnesoid X receptor agonist, has been shown to improve the histological features of NASH. Here we report results from a planned interim analysis of an ongoing, phase 3 study of obeticholic acid for NASH. Methods In this multicentre, randomised, double-blind, placebo-controlled study, adult patients with definite NASH,non-alcoholic fatty liver disease (NAFLD) activity score of at least 4, and fibrosis stages F2–F3, or F1 with at least oneaccompanying comorbidity, were randomly assigned using an interactive web response system in a 1:1:1 ratio to receive oral placebo, obeticholic acid 10 mg, or obeticholic acid 25 mg daily. Patients were excluded if cirrhosis, other chronic liver disease, elevated alcohol consumption, or confounding conditions were present. The primary endpointsfor the month-18 interim analysis were fibrosis improvement (≥1 stage) with no worsening of NASH, or NASH resolution with no worsening of fibrosis, with the study considered successful if either primary endpoint was met. Primary analyses were done by intention to treat, in patients with fibrosis stage F2–F3 who received at least one dose of treatment and reached, or would have reached, the month 18 visit by the prespecified interim analysis cutoff date. The study also evaluated other histological and biochemical markers of NASH and fibrosis, and safety. This study is ongoing, and registered with ClinicalTrials.gov, NCT02548351, and EudraCT, 20150-025601-6. Findings Between Dec 9, 2015, and Oct 26, 2018, 1968 patients with stage F1–F3 fibrosis were enrolled and received at least one dose of study treatment; 931 patients with stage F2–F3 fibrosis were included in the primary analysis (311 in the placebo group, 312 in the obeticholic acid 10 mg group, and 308 in the obeticholic acid 25 mg group). The fibrosis improvement endpoint was achieved by 37 (12%) patients in the placebo group, 55 (18%) in the obeticholic acid 10 mg group (p=0·045), and 71 (23%) in the obeticholic acid 25 mg group (p=0·0002). The NASH resolution endpoint was not met (25 [8%] patients in the placebo group, 35 [11%] in the obeticholic acid 10 mg group [p=0·18], and 36 [12%] in the obeticholic acid 25 mg group [p=0·13]). In the safety population (1968 patients with fibrosis stages F1–F3), the most common adverse event was pruritus (123 [19%] in the placebo group, 183 [28%] in the obeticholic acid 10 mg group, and 336 [51%] in the obeticholic acid 25 mg group); incidence was generally mild to moderate in severity. The overall safety profile was similar to that in previous studies, and incidence of serious adverse events was similar across treatment groups (75 [11%] patients in the placebo group, 72 [11%] in the obeticholic acid 10 mg group, and 93 [14%] in the obeticholic acid 25 mg group). Interpretation Obeticholic acid 25 mg significantly improved fibrosis and key components of NASH disease activity among patients with NASH. The results from this planned interim analysis show clinically significant histological improvement that is reasonably likely to predict clinical benefit. This study is ongoing to assess clinical outcomes

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon-7

<p><b>Copyright information:</b></p><p>Taken from "Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon"</p><p>http://www.virologyj.com/content/4/1/89</p><p>Virology Journal 2007;4():89-89.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2075494.</p><p></p>sferase downstream of the HCV IRES. The second cistern in the same RNA contains EMCV-IRES sequences for efficient translation of HCV non-structural proteins and this chimeric RNA terminates with the HCV 3'UTR. This sub-genomic clone has adaptive mutation S1179I that allows high level replication of RNA in Huh-7 cells and cell colonies develop that are resistant to neomycin. (B) pISRE-Luc reporter plasmid construct containing firefly luciferase reporter gene used to measure IFN-promoter activation in replicon cell lines. This construct has four copies of ISRE sequences positioned up stream of Herpes simplex virus thymidine kinase promoter TATA box, which drives the expression of firefly luciferase. (C) Represents monocistronic reporter constructs that express green fluorescent protein translated by HCV IRES by using a T7 inducible system

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon-5

<p><b>Copyright information:</b></p><p>Taken from "Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon"</p><p>http://www.virologyj.com/content/4/1/89</p><p>Virology Journal 2007;4():89-89.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2075494.</p><p></p>7°C for one hour. Non-specific binding was measured in presence of 100-fold excess of unlabeled IFN-α2b for each dilution. Cells were washed twice in PBS. Radioactivity (cpm) of each cell pellet was measured. The amount of interferon specifically bound was determined by subtracting non-specific binding from total binding. Specific binding of I-IFN-α2b to three groups of resistant cell lines and one sensitive cell line are shown (A-D)

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon-1

<p><b>Copyright information:</b></p><p>Taken from "Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon"</p><p>http://www.virologyj.com/content/4/1/89</p><p>Virology Journal 2007;4():89-89.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2075494.</p><p></p>eplicon cell lines (9/13, 5/15 and KR) with high ISRE promoter activation were treated with or without interferon alpha 2b (1000 IU/ml) for 4 weeks in a growth medium containing G-418 (1 mg/ml). The ability of replicon cells to develop G-418 resistant cell colonies was examined. Cell colonies were developed only in low ISRE replicons. Three individual colonies from each dish were picked up and nine stable cell lines were generated

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon-4

<p><b>Copyright information:</b></p><p>Taken from "Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon"</p><p>http://www.virologyj.com/content/4/1/89</p><p>Virology Journal 2007;4():89-89.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2075494.</p><p></p>stant Huh-7 cell lines. Cells were transfected with IRES-GFP plasmid and treated with 1000 IU/ml interferon alpha. After 24 hours, GFP expression was recorded. Cured cells prepared from resistant clones unable to activate interferon signaling and no inhibition of HCV IRES was seen

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon-8

<p><b>Copyright information:</b></p><p>Taken from "Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon"</p><p>http://www.virologyj.com/content/4/1/89</p><p>Virology Journal 2007;4():89-89.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2075494.</p><p></p>eplicon cell lines (9/13, 5/15 and KR) with high ISRE promoter activation were treated with or without interferon alpha 2b (1000 IU/ml) for 4 weeks in a growth medium containing G-418 (1 mg/ml). The ability of replicon cells to develop G-418 resistant cell colonies was examined. Cell colonies were developed only in low ISRE replicons. Three individual colonies from each dish were picked up and nine stable cell lines were generated

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon-3

<p><b>Copyright information:</b></p><p>Taken from "Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon"</p><p>http://www.virologyj.com/content/4/1/89</p><p>Virology Journal 2007;4():89-89.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2075494.</p><p></p>n of IFN promoter in the replicon cell lines was determined in the presence or absence of IFN-α2b (1000 IU/ml) after 24 hours. All nine interferon resistant cell lines have lower activation of IFN-promoter as compared to interferon sensitive cell lines

Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon-0

<p><b>Copyright information:</b></p><p>Taken from "Reduced expression of Jak-1 and Tyk-2 proteins leads to interferon resistance in Hepatitis C virus replicon"</p><p>http://www.virologyj.com/content/4/1/89</p><p>Virology Journal 2007;4():89-89.</p><p>Published online 18 Sep 2007</p><p>PMCID:PMC2075494.</p><p></p>sferase downstream of the HCV IRES. The second cistern in the same RNA contains EMCV-IRES sequences for efficient translation of HCV non-structural proteins and this chimeric RNA terminates with the HCV 3'UTR. This sub-genomic clone has adaptive mutation S1179I that allows high level replication of RNA in Huh-7 cells and cell colonies develop that are resistant to neomycin. (B) pISRE-Luc reporter plasmid construct containing firefly luciferase reporter gene used to measure IFN-promoter activation in replicon cell lines. This construct has four copies of ISRE sequences positioned up stream of Herpes simplex virus thymidine kinase promoter TATA box, which drives the expression of firefly luciferase. (C) Represents monocistronic reporter constructs that express green fluorescent protein translated by HCV IRES by using a T7 inducible system