A Ribosomal S-6 Kinase–Mediated Signal to C/EBP-β Is Critical for the Development of Liver Fibrosis

BACKGROUND: In response to liver injury, hepatic stellate cell (HSC) activation causes excessive liver fibrosis. Here we show that activation of RSK and phosphorylation of C/EBPbeta on Thr217 in activated HSC is critical for the progression of liver fibrosis. METHODOLOGY/PRINCIPAL FINDINGS: Chronic treatment with the hepatotoxin CCl(4) induced severe liver fibrosis in C/EBPbeta(+/+) mice but not in mice expressing C/EBPbeta-Ala217, a non-phosphorylatable RSK-inhibitory transgene. C/EBPbeta-Ala217 was present within the death receptor complex II, with active caspase 8, and induced apoptosis of activated HSC. The C/EBPbeta-Ala217 peptides directly stimulated caspase 8 activation in a cell-free system. C/EBPbeta(+/+) mice with CCl(4)-induced severe liver fibrosis, while continuing on CCl(4), were treated with a cell permeant RSK-inhibitory peptide for 4 or 8 weeks. The peptide inhibited RSK activation, stimulating apoptosis of HSC, preventing progression and inducing regression of liver fibrosis. We found a similar activation of RSK and phosphorylation of human C/EBPbeta on Thr266 (human phosphoacceptor) in activated HSC in patients with severe liver fibrosis but not in normal livers, suggesting that this pathway may also be relevant in human liver fibrosis. CONCLUSIONS/SIGNIFICANCE: These data indicate that the RSK-C/EBPbeta phosphorylation pathway is critical for the development of liver fibrosis and suggest a potential therapeutic target

C/EBPβ-Thr217 Phosphorylation Signaling Contributes to the Development of Lung Injury and Fibrosis in Mice

mice are refractory to Bleomycin-induced lung fibrosis the molecular mechanisms remain unknown. Here we show that blocking the ribosomal S-6 kinase (RSK) phosphorylation of the CCAAT/Enhancer Binding Protein (C/EBP)-β on Thr217 (a RSK phosphoacceptor) with either a single point mutation (Ala217), dominant negative transgene or a blocking peptide containing the mutated phosphoacceptor ameliorates the progression of lung injury and fibrosis induced by Bleomycin in mice. mice with a cell permeant, C/EBPβ peptide that inhibits phosphorylation of C/EBPβ on Thr217 (40 µg instilled intracheally on day-2 and day-6 after the single Bleomycin dose) also blocked the progression of lung injury and fibrosis induced by Bleomycin. Phosphorylation of human C/EBPβ on Thr266 (human homologue phosphoacceptor) was induced in collagen-activated human lung fibroblasts in culture as well as in activated lung fibroblasts in situ in lungs of patients with severe lung fibrosis but not in control lungs, suggesting that this signaling pathway may be also relevant in human lung injury and fibrosis.These data suggest that the RSK-C/EBPβ phosphorylation pathway may contribute to the development of lung injury and fibrosis

Pioglitazone Decreases Hepatitis C Viral Load in Overweight, Treatment Naïve, Genotype 4 Infected-Patients: A Pilot Study

Insulin resistance (IR) is induced by chronic hepatitis C virus (HCV) genotypes 1 and 4 infections. It is not known whether drugs that affect IR such as Pioglitazone and Prednisone also affect serum HCV RNA titers independently of PEG-Interferon-α2/ribavirin treatment. The primary aim was to assess whether Pioglitazone by improving IR and/or inflammation decreases HCV viral load independently of standard of care HCV treatment. A secondary aim was to assess whether Prednisone, a drug that induces insulin resistance and stimulates HCV viral entry and replication in replicon culture systems, increases HCV viral load in this population.We designed a two-arm, parallel Pilot Study of overweight, treatment naïve genotype 4 HCV-infected patients at a public referral Liver Clinic in Giza, Egypt. The subjects received Pioglitazone (30 mg/day for 14 days) or Prednisone (40 mg/day for 4 days) in a randomized fashion, but the two arms can be considered independent pilot studies. Only changes from baseline within each arm were assessed and no contrasts of the interventions were made, as this was not an aim of the study. Among 105 consecutive HCV genotype 4 patients, 39 were enrolled based on the optimal sample size and power analysis according to the CONSORT statement; 20 to the Pioglitazone group and 19 to the Prednisone group. Pioglitazone was effective in decreasing serum HCV RNA at day-14 (n = 10; difference of means = 205,618 IU/ml; 95% CI 26,600 to 384,600; P<0.001). Although Prednisone did increase serum HCV RNA at day-4 (n = 10; change from baseline = -42,786 IU/ml; 95% CI -85,500 to -15,700; P = 0.049), the log(10) HCV RNA titers were statistically not different from baseline day-0.This is the first documentation that Pioglitazone decreases the serum HCV RNA titers independently of PEG-Interferon-α2/ribavirin treatment. The novel findings of our Study provide the foundation for basic and clinical investigations on the molecular mechanisms responsible for the Pioglitazone-induced decrease in HCV genotype 4 RNA titers.ClinicalTrials.gov NCT01157975

Ledipasvir and sofosbuvir for untreated HCV genotype 1 infection.

BACKGROUND: In phase 2 studies, treatment with the all-oral combination of the nucleotide polymerase inhibitor sofosbuvir and the NS5A inhibitor ledipasvir resulted in high rates of sustained virologic response among previously untreated patients with hepatitis C virus (HCV) genotype 1 infection. METHODS: We conducted a phase 3, open-label study involving previously untreated patients with chronic HCV genotype 1 infection. Patients were randomly assigned in a 1:1:1:1 ratio to receive ledipasvir and sofosbuvir in a fixed-dose combination tablet once daily for 12 weeks, ledipasvir-sofosbuvir plus ribavirin for 12 weeks, ledipasvir-sofosbuvir for 24 weeks, or ledipasvir-sofosbuvir plus ribavirin for 24 weeks. The primary end point was a sustained virologic response at 12 weeks after the end of therapy. RESULTS: Of the 865 patients who underwent randomization and were treated, 16% had cirrhosis, 12% were black, and 67% had HCV genotype 1a infection. The rates of sustained virologic response were 99% (95% confidence interval [CI], 96 to 100) in the group that received 12 weeks of ledipasvir-sofosbuvir; 97% (95% CI, 94 to 99) in the group that received 12 weeks of ledipasvir-sofosbuvir plus ribavirin; 98% (95% CI, 95 to 99) in the group that received 24 weeks of ledipasvir-sofosbuvir; and 99% (95% CI, 97 to 100) in the group that received 24 weeks of ledipasvir-sofosbuvir plus ribavirin. No patient in either 12-week group discontinued ledipasvir-sofosbuvir owing to an adverse event. The most common adverse events were fatigue, headache, insomnia, and nausea. CONCLUSIONS: Once-daily ledipasvir-sofosbuvir with or without ribavirin for 12 or 24 weeks was highly effective in previously untreated patients with HCV genotype 1 infection. (Funded by Gilead Sciences; ION-1 ClinicalTrials.gov number NCT01701401.)

Gastric adenocarcinoma inducing portal hypertension: A rare

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Blood Microbiome Quantity and the Hyperdynamic Circulation in Decompensated Cirrhotic Patients.

BACKGROUND:Recently, a complex microbiome was comprehensibly characterized in the serum and ascitic fluid of cirrhotic patients. In the current study, we investigated for the first time the induction of inflammatory pathways and Nitric Oxide, as well as the systemic hemodynamics in conjunction with the blood microbiome in a Child-Pugh class B cirrhotic cohort. METHODS AND FINDINGS:We used the Intestinal Infections Microbial DNA qPCR Array to screen for 53 bacterial DNA from the gut in the blood. Assays were designed using the 16S rRNA gene as a target, and PCR amplification primers (based on the Human Microbiome Project) and hydrolysis-probe detection. Eighteen systemic hemodynamic parameters were measured non-invasively by impedance cardiography using the BioZ ICG monitor. The inflammatory response was assessed by measuring blood cytokines, Nitric Oxide RNA arrays, and Nitric Oxide. In the blood of this cirrhotic cohort, we detected 19 of 53 bacterial species tested. The number of bacterial species was markedly increased in the blood of cirrhotic patients compared to control individuals (0.2+/-0.4 vs 3.1+/-2.3; 95% CI: 1.3 to 4.9; P = 0.0030). The total bacterial DNA was also increased in the blood of cirrhotic subjects compared to control subjects (0.2+/- 1.1 vs 41.8+/-132.1; 95% CI: 6.0 to 77.2; P = 0.0022). In the cirrhotic cohort, the Cardiac Output increased by 37% and the Systemic Vascular Resistance decreased by 40% (P< 0.00001 for both compared to control subjects). Systemic Vascular Resistance was inversely correlated to blood bacterial DNA quantity (- 0.621; 95% CI -0.843 to -0.218; P = 0.0060), blood bacterial species number (- 0.593; 95% CI -0.83 to -0.175; P = 0.0095; logistic regression: Chi Square = 5.8877; P = 0.0152), and serum Nitric Oxide (- 0.705; 95% CI -0.881 to -0.355; P = 0.0011). Many members of the Nitric Oxide signaling pathway gene family were increased in cirrhotic subjects. CONCLUSIONS:Our study identified blood bacterial DNA in ~ 90% of the cirrhotic patients without clinical evidences of infection, and suggests that the quantity of bacterial DNA in blood may stimulate signaling pathways, including Nitric Oxide, that could decrease systemic vascular resistance and increase cardiac output