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.

C/EBPß-Thr217 Phosphorylation Stimulates Macrophage Inflammasome Activation and Liver Injury.

Amplification of liver injury is mediated by macrophages but the signaling by which the macrophage inflammasome enhances liver injury is not completely understood. The CCAAT/Enhancer Binding Protein-ß (C/EBPß) is a critical signaling molecule for macrophages because expression of a dominant inhibitor of C/EBPß DNA-binding sites or a targeted deletion of C/EBPß results in impaired macrophage differentiation. We reported that expression of the phosphorylation-mutant C/EBPß-Glu217, which mimics phosphorylated C/EBPß-Thr217, was sufficient to confer macrophage survival to Anthrax lethal toxin. Here, using primary hepatocytes, primary liver macrophages, dominant positive and negative transgenic mice of the C/EBPß-Thr217 phosphoacceptor, macrophage ablation, and an inhibitory peptide of C/EBPß-Thr217 phosphorylation, we determined that this phosphorylation is essential for the activation of the inflammasome in liver macrophages and for the hepatocyte apoptosis induced by hepatotoxins that results in liver injury. Similar findings were observed in the livers of patients with acute injury induced by Toxic Oil Syndrome.

Novel inflammatory biomarkers of portal pressure in compensated cirrhosis patients.

UNLABELLED: The rationale for screening inflammatory serum biomarkers of the hepatic vein pressure gradient (HVPG) is based on the fact that portal hypertension is pathogenically related to liver injury and fibrosis, and that in turn these are associated with the activation of inflammatory pathways. This was a nested cohort study in the setting of a randomized, clinical trial to assess the development of gastroesophageal varices (GEV) (N Engl J Med 2005;353:2254). Patients had cirrhosis and portal hypertension but did not have GEV. A total of 90 patients who had baseline day-1 sera available were enrolled in the present study. The objective of this study was to determine whether inflammatory biomarkers in conjunction with clinical parameters could be used to develop a predictive paradigm for HVPG. The correlations between HVPG and interleukin (IL)-1ß (P=0.0052); IL-1R-α (P=0.0085); Fas-R (P=0.0354), and serum VCAM-1 (P=0.0007) were highly significant. By using multivariate logistic regression analysis and selected parameters (transforming growth factor beta [TGFß]; heat shock protein [HSP]-70; at-risk alcohol use; and Child class B) we could exclude HVPG ≥ 12 mmHg with 86% accuracy (95% confidence interval [CI]: 67.78 to 96.16%) and the sensitivity was 87.01% (95% CI: 69.68 to 96.34%). Therefore, the composite test could identify 86% of compensated cirrhosis patients with HVPG below 12 mmHg and prevent unnecessary esophagogastroduodenoscopy with its associated morbidity and costs in these patients. Our diagnostic test was not efficient in predicting HVPG ≥ 12 mmHg. CONCLUSION: A blood test for HVPG could be performed in cirrhosis patients to prevent unnecessary esophagogastroduodenoscopy.

Pioglitazone decreases hepatitis C viral load in overweight, treatment naïve, genotype 4 infected-patients: a pilot study.

BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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. TRIAL REGISTRATION: ClinicalTrials.gov NCT01157975.

C/EBPß-Thr217 phosphorylation signaling contributes to the development of lung injury and fibrosis in mice.

BACKGROUND: Although C/EBPß(ko) 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)-ß 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. METHODOLOGY/PRINCIPAL FINDINGS: Mice expressing the non-phosphorylatable C/EBPß-Ala217 transgene had a marked reduction in lung injury on day-13 after Bleomycin exposure, compared to C/EBPß(wt) mice, judging by the decrease of CD68(+) activated monocytes/macrophages, bone marrow-derived CD45(+) cells and lung cytokines as well as by the normal surfactant protein-C expression by lung pneumocytes. On day-21 after Bleomycin treatment, C/EBPß(wt) mice but not mice expressing the dominant negative C/EBPß-Ala217 transgene developed severe lung fibrosis as determined by quantitative collagen assays. All mice were of identical genetic background and back-crossed to the parental wild-type inbreed FVB mice for at least ten generations. Treatment of C/EBPß(wt) mice with a cell permeant, C/EBPß peptide that inhibits phosphorylation of C/EBPß 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/EBPß 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. CONCLUSIONS/SIGNIFICANCE: These data suggest that the RSK-C/EBPß phosphorylation pathway may contribute to the development of lung injury and fibrosis.

Decreased Jun-D and myogenin expression in muscle wasting of human cachexia.

Muscle wasting is a critical feature of patients afflicted by acquired immune deficiency syndrome (AIDS), cancer, or chronic inflammatory diseases. In a mouse model of muscle wasting, TNF-alpha induces oxidative stress and nitric oxide synthase-2 (NOS2) and decreases myogenin, Jun-D, and creatinine kinase muscle isoform (CKM) expression. Here, we studied 12 patients with muscle wasting due to cancer (N = 10) or AIDS (N = 2) and 4 control subjects. We show that in skeletal muscle of cachectic patients there is 1) increased expression and activity of the TNF-alpha signaling, including TNF-alpha mRNA, activation of TNFR1, and TNF-alpha-associated to TNFR1; 2) increased oxidative stress, as determined by the presence of malondialdehyde-lysine adducts; 3) increased NOS2 mRNA and protein; 4) decreased expression of Jun-D, myogenin, myosin, and CKM mRNA and protein; 5) impaired CKM-E box binding activities, associated with decreased Jun-D/myogenin activities; and 6) oxidative modification and ubiquitination of Jun-D. These studies show that these molecular pathways are modulated in association with muscle wasting in patients with cancer or AIDS, and whether or not they cause muscle wasting remains to be determined.

Severe hepatocellular injury with apoptosis induced by a hepatitis C polymerase inhibitor.

GOALS: To describe the mechanisms of severe hepatocellular injury with apoptosis in 2 patients receiving hepatitis C virus (HCV)-796. BACKGROUND: HCV-796 is a hepatitis C polymerase inhibitor approved by the US Food and Drug Administration for a phase 2 study of the treatment of hepatitis C in combination with PEG-Interferon and ribavirin. RESULTS: The injury occurred after more than 12 weeks of treatment, with a >20-fold increase in serum alanine aminotransferase and aspartate aminotransferase, and a marked increase in total (and direct) bilirubin in the absence of cholestasis. There was no evidence of autoimmune or viral hepatitis. Involvement of the mitochondrial apoptotic pathway was demonstrated by (1) release of cytochrome C into the cytosol; (2) association of cytochrome C with apoptotic protease activating factor-1 in the cytosol; (3) activation of initiator caspase 9; (4) activation of effector caspase 3; (5) increased serum caspase-3 cleaved cytokeratin-18 peptide; (6) nuclear fragmentation; (7) mitochondrial structural abnormalities; (8) expression of light chain 3 B, an indicator of autophagy; (9) probable autophagy of mitochondria by autophagosomes; and (10) probable phagocytosis of apoptotic hepatocytes by activated macrophages. Immunoglobulin G immune complexes were identified in the hepatocytes and localized to the endoplasmic reticulum and Golgi of these patients after the drug-induced liver disease, reflecting a primary or secondary target. Hepatitis C treatment was discontinued at weeks 15 and 19 in patients 1 and 2, respectively. After more than 6 months off the medication, both patients normalized the serum alanine aminotransferase, aspartate aminotransferase, and total bilirubin with undetectable HCV RNA. CONCLUSIONS: HCV-796 may cause severe hepatocellular injury and apoptosis, with a marked immune reaction in susceptible patients.

Targeting ribosomal S-6 kinase for the prevention and treatment of liver injury and liver fibrosis.

Activation of hepatic stellate cells (HSC) is responsible for the development of liver fibrosis in chronic liver diseases of all causes, and remarkably, HSC clearance by apoptosis may allow recovery from liver injury and reversal of liver fibrosis. Because in preclinical studies it has been shown that activation of ribosomal S-6 kinase (RSK) and phosphorylation of the CCAAT/enhancer-binding protein (C/EBP) beta in activated HSC is critical for the progression of liver fibrosis, RSK has been considered as a therapeutic target for the liver fibrosis. Unexpectedly, preclinical studies documented a strong antiinflammatory effect of RSK inhibitors, decreasing liver injury induced by hepatotoxins. Therefore, RSK inhibitors reduce liver fibrosis directly by inducing apoptosis of activated HSC, and indirectly by preventing liver injury and inflammation. The activation of RSK and C/EBPbeta phosphorylation also occurs in human liver fibrosis. Thus, appropriate RSK inhibitors may be beneficial in the prevention and treatment of liver injury and liver fibrosis. These issues will be discussed in this review.

Direct infection and replication of naturally occurring hepatitis C virus genotypes 1, 2, 3 and 4 in normal human hepatocyte cultures.

BACKGROUND: Hepatitis C virus (HCV) infection afflicts about 170 million individuals worldwide. However, the HCV life cycle is only partially understood because it has not been possible to infect normal human hepatocytes in culture. The current Huh-7 systems use cloned, synthetic HCV RNA expressed in hepatocellular carcinoma cells to produce virions, but these cells cannot be infected with naturally occurring HCV obtained from infected patients. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe a human hepatocyte culture permissible to the direct infection with naturally occurring HCV genotypes 1, 2, 3 and 4 in the blood of HCV-infected patients. The culture system mimics the biology and kinetics of HCV infection in humans, and produces infectious virions that can infect naïve human hepatocytes. CONCLUSIONS/SIGNIFICANCE: This culture system should complement the existing systems, and may facilitate the understanding of the HCV life cycle, its effects in the natural host cell, the hepatocyte, as well as the development of novel therapeutics and vaccines.

A novel domain of BRCA1 interacts with p53 in breast cancer cells.

The interactions between BRCA1 and p53 are relevant for understanding hereditary breast and ovarian cancer. Although in vitro studies reported that BRCA1 (amino acids 224-500) and the second BRCT domain of the BRCA1 C-terminus may interact with p53, quantitative biophysical measurements indicate that these regions of BRCA1 do not bind efficiently to p53. Here we show that BRCA1 interacts with p53 in vivo in breast cancer cells, through another BRCA1 domain (amino acids 772-1292). Expression of a truncated BRCA1 (amino acids 772-1292) stimulated p53 DNA-binding and transcription activities and apoptosis, recapitulating some effects of DNA damage. These results suggest that a novel domain of BRCA1 may interact with p53 in breast cancer cells.

C/EBPbeta associates with caspase 8 complex proteins and modulates apoptosis in hepatic stellate cells.

GOALS: To analyze the role of C/EBPbeta phosphorylation on hepatic stellate cell survival/cell death. BACKGROUND: Activation and survival of stellate cells is critical for the development of liver fibrosis. C/EBPbeta phosphorylation regulates stellate cell survival by affecting caspase 8 activation. The mechanisms responsible for these effects are unknown. STUDY: We study the effects of caspase 8 activators signaling through death receptors. In addition, we assess the role of C/EBPbeta phosphorylation on the susceptibility of stellate cells to apoptotic stimuli. Finally, we investigated whether C/EBPbeta is associated with the caspase 8 complex protein FLIP, a critical inhibitor of caspase 8. RESULTS: Primary mouse stellate cells from C/EBPbeta wild type and the phosphorylation mimic C/EBPbetaGlu transgenic mice were treated with lipopolysaccharide [an inducer of tumor necrosis factor-alpha (TNF-alpha)], FAS, or TNF-alpha. Stellate cell apoptosis was determined by assessing the binding of annexin-V to exposed phosphatidylserine of plasma membranes. TNF-alpha and FAS, but not lipopolysaccharide, induced annexin-V binding at 6 hours in C/EBPbeta wild type stellate cell. However, the stimulation of apoptosis by TNF-alpha and FAS was markedly blocked in C/EBPbetaGlu stellate cells (P<0.001). Stellate cells activated on a collagen type 1 matrix expressed both C/EBPbeta and FLIPL. Treatment of stellate cells with a MAP kinase kinase1 (MEK1) inhibitor blocked FLIPL cellular localization, suggesting that MEK1 signaling through C/EBPbeta modulates FLIP activity. The colocalization of C/EBPbeta and FLIPL was disrupted by activation of the FAS receptor, by blocking the association of C/EBPbeta with the long form of FLIP, FLIPL. CONCLUSIONS: The MAPK-RSK-C/EBPbeta signaling may modulate stellate cell survival through caspase 8-associated protein FLIPL. This step is critical for liver fibrosis and if blocked with competitor peptides may prevent fibrogenesis.

A ribosomal S-6 kinase-mediated signal to C/EBP-beta 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/EBPbeta phosphorylation rescues macrophage dysfunction and apoptosis induced by anthrax lethal toxin.

Bacillus anthracis lethal toxin (LT) impairs innate and adaptive immunity. Anthrax lethal factor stimulates cleavage of MAPK kinases, which prevents the activation of antiapoptotic MAPK targets. However, these MAPK targets have not been yet identified. Here, we found that LT induces macrophage apoptosis by enhancing caspase 8 activation and by preventing the activation of ribosomal S6 kinase-2 (RSK), a MAPK target, and the phosphorylation of CCAAT/enhancer binding protein-beta (C/EBPbeta) on T(217), a RSK target. Expression of the dominant positive, phosphorylation mimic C/EBPbeta-E(217) rescued macrophages from LT-induced apoptosis by blocking the activation of procaspase 8. LT inhibited macrophage phagocytosis and oxidative burst and induced apoptosis in normal mice but not in C/EBPbeta-E(217) transgenic mice. These findings suggest that C/EBPbeta may play a critical role in anthrax pathogenesis, at least in macrophages.

Ecdysone and a dietary alkaloid interact in the development of the pheromone gland of a male moth (Creatonotos, Lepidoptera: Arctiidae).

Hair-covered scent organs of the male arctiid moth Creatonotos produce and dissipate the volatile pheromone hydroxydanaidal. The biosynthesis of this substance depends quantitatively upon the uptake of pyrrolizidine alkaloids (PA) with the larval foodplant. The size of the tubular, eversible scent organ (corema) is also positively correlated with the ingested amount of the same alkaloid, which acts like a specific growth factor. After an assessment of the corema normogenesis by Rick-Wagner (PhD thesis, University of Cologne, 1986) we injected PA into PA-free raised larvae, prepupae, and pupae. We found that the PA competence (sensitivity) of the corema anläge terminates with the first prepupal day. Ecdysone titer determinations (radioimmunoassay) are in agreement with those in other moth species. Ligated (ecdysone-free) pupal abdomina never developed imaginal structures, with or without earlier PA application. Ecdysone injection into ligated pupal abdomina of PA-fed specimens initiated the development of imaginal structures and also of coremata of more than (ecdysone-free) control size. Pupal abdomina without PA pre-treatment only developed very small coremata. With these experiments we have separated and identified two morphogenetic control agents of corema development: the dietary PA specifies the size of the organ whereas ecdysone induces the anlage to proliferate within these PA-dependent ranges and to differentiate adult structures, as it does with other imaginal anlagen.