Hepatocyte proliferation is the possible mechanism for the transient decrease in liver injury during steatosis stage of alcoholic liver disease

Steatosis is a frequent pathologic stage in alcoholic liver disease (ALD). Although the mechanisms for increased susceptibility of steatotic liver to injury have been postulated, the ability of these hepatocytes to proliferate and withstand injury is unknown. There are conflicting reports on the status of hepatocyte regeneration following chronic alcohol ingestion. Hence, the objective of this study was to investigate the temporal dynamics between the pattern of liver injury and hepatocyte proliferation during the steatosis stage of ALD. Alcoholic steatosis was induced in male Sprague-Dawley rats by feeding an ethanol (EtOH)-containing Lieber-DeCarli liquid diet for a period of 5 weeks. Microvesicular steatosis was evident in H&E sections by three weeks in the EtOH-treated rats, which further developed into panlobular macrovesicular steatosis by 5 weeks. Plasma transaminase activities indicated progressive increase in liver injury peaking at 3 weeks with significant but mild decrease at 4 and 5 weeks. CYP2E1 protein and activity was significantly increased in EtOH-fed rats as measured by Western blot and pNP hydroxylation assay. PCNA analysis of liver sections indicated that EtOH-treated rats had a significantly higher number of cells in S phase of cell division at weeks 1 (3.20 +/- 0.19), 2 (7.03 +/- 0.92), and 3 (4.23 +/- 1.41) when compared to controls (1.5 +/- 0.22). NF-kappaB DNA binding and Cyclin D1 proteins increased significantly in the EtOH-treated rats corresponding with enhanced hepatic proliferation. These data suggest the transient decline in liver injury during alcoholic steatosis is due to enhanced NF-kappaB-dependent hepatocyte proliferation.     

Dual β-Catenin and γ-Catenin Loss in Hepatocytes Impacts Their Polarity through Altered Transforming Growth Factor-β and Hepatocyte Nuclear Factor 4α Signaling

Hepatocytes are highly polarized epithelia. Loss of hepatocyte polarity is associated with various liver diseases, including cholestasis. However, the molecular underpinnings of hepatocyte polarization remain poorly understood. Loss of β-catenin at adherens junctions is compensated by γ-catenin and dual loss of both catenins in double knockouts (DKOs) in mice liver leads to progressive intrahepatic cholestasis. However, the clinical relevance of this observation, and further phenotypic characterization of the phenotype, is important. Herein, simultaneous loss of β-catenin and γ-catenin was identified in a subset of liver samples from patients of progressive familial intrahepatic cholestasis and primary sclerosing cholangitis. Hepatocytes in DKO mice exhibited defects in apical-basolateral localization of polarity proteins, impaired bile canaliculi formation, and loss of microvilli. Loss of polarity in DKO livers manifested as epithelial-mesenchymal transition, increased hepatocyte proliferation, and suppression of hepatocyte differentiation, which was associated with up-regulation of transforming growth factor-β signaling and repression of hepatocyte nuclear factor 4α expression and activity. In conclusion, concomitant loss of the two catenins in the liver may play a pathogenic role in subsets of cholangiopathies. The findings also support a previously unknown role of β-catenin and γ-catenin in the maintenance of hepatocyte polarity. Improved understanding of the regulation of hepatocyte polarization processes by β-catenin and γ-catenin may potentially benefit development of new therapies for cholestasis.

A hallmark of epithelial cells is polarization, which is achieved by the orchestration of external cues, such as cellular contact, extracellular matrix, signal transduction, growth factors, and spatial organization.¹ Hepatocytes in the liver show a unique polarity by forming several apical and basolateral poles within a cell.² The apical poles of adjacent hepatocytes form a continuous network of bile canaliculi into which bile is secreted, whereas the basolateral membrane domain forms the sinusoidal pole, which secretes various components, such as proteins or drugs, into the blood circulation.³ Loss of hepatic polarity has been associated with several cholestatic and developmental disorders, including progressive familial intrahepatic cholestasis (PFIC) and primary sclerosing cholangitis (PSC).^4,5 Although the molecular mechanisms governing hepatocyte polarity have been extensively studied in the in vitro systems, there is still a significant gap in our understanding of how polarity is established within the context of tissue during development or maintained during homeostasis.^6,7 Similarly, the molecular pathways contributing to hepatic polarity are not entirely understood, and a better comprehension of hepatic polarity regulation is thus warranted.Previous studies have confirmed the role of hepatocellular junctions, such as tight and gap junctions, in the maintenance of hepatocyte polarity.^8,9 Studies done in vitro and in vivo have shown that loss of junctional proteins, such as zonula occludens protein (ZO)-1, junctional adhesion molecule-A, and claudins, lead to impairment of polarity and distorted bile canaliculi formation.10, 11, 12, 13 In addition, proteins involved in tight junction assembly, such as liver kinase B1, are also involved in polarity maintenance.¹⁴ Among adherens junction proteins, various in vitro cell culture models have confirmed the role of E-cadherin in the regulation of hepatocyte polarity, possibly through its interaction with β-catenin.^15,16 However, there is a lack of an in vivo model to study the role of adherens junction proteins in hepatocyte polarity and their misexpression contributing to various liver diseases.β-Catenin plays diverse functions in the liver during development, regeneration, zonation, and tumorigenesis.17, 18, 19 The relative contribution of β-catenin as part of the adherens junction is challenging to study because like in other tissues, γ-catenin compensates for the β-catenin loss in the liver.^20,21 To address this redundancy, we previously reported a hepatocyte-specific β-catenin and γ-catenin double-knockout (DKO) mouse model was reported.²² Simultaneous deletion of β-catenin and γ-catenin in mice livers led to cholestasis, partially through the breach of cell-cell junctions. However, more comprehensive understanding of the molecular underpinnings of the phenotype is needed.In the current study, prior preclinical findings of dual β-catenin and γ-catenin loss were extended to a subset of PFIC and PSC patients. In vivo studies using the murine model with hepatocyte-specific dual loss of β-catenin and γ-catenin showed complete loss of hepatocyte polarity compared to the wild-type controls (CONs). Loss of polarity in DKO liver was accompanied by epithelial-mesenchymal transition (EMT), activation of transforming growth factor (TGF)-β signaling, and reduced expression of hepatocyte nuclear factor 4α (HNF4α). Our findings suggest that β-catenin and γ-catenin and in turn adherens junction integrity, are critical for the maintenance of hepatocyte polarity, and any perturbations in this process can contribute to the pathogenesis of cholestatic liver disease.     

Type 1 diabetic mice are protected from acetaminophen hepatotoxicity.

Streptozotocin (STZ)-induced diabetic (DB) mice challenged with single ordinarily lethal doses of acetaminophen (APAP), carbon tetrachloride (CCl4), or bromobenzene (BB) were resistant to all three hepatotoxicants. Mechanisms of protection against APAP hepatotoxicity were investigated. Plasma alanine aminotransferase, aspartate aminotransferase, and liver histopathology revealed significantly lower hepatic injury in DB mice after APAP administration. HPLC analysis of plasma and urine revealed lower plasma t1/2, increased volume of distribution (Vd), and increased plasma clearance (CLp) of APAP in the DB mice and no difference in APAP-glucuronide, a major metabolite in mice. Interestingly, covalent binding of 14C-labeled APAP to liver target proteins; arylation of APAP to 58, 56, and 44 kDa acetaminophen binding proteins (ABPs); and glutathione (GSH) depletion in the liver did not differ between nondiabetic (non-DB) and DB mice in spite of downregulated hepatic microsomal CYP2E1 and 1A2 proteins in the DB mice, known to be involved in bioactivation of APAP. Compensatory cell division measured via 3H-thymidine pulse labeling and immunohistochemical staining for proliferating cell nuclear antigen (PCNA) indicated earlier onset of S-phase in the DB mice after exposure to APAP. Antimitotic intervention of liver cell division by colchicine (CLC) after administration of APAP led to significantly higher mortality in the DB mice suggesting a pivotal role of liver cell division and tissue repair in the protection afforded by diabetes. In conclusion, the resistance of DB mice against hepatotoxic and lethal effects of APAP appears to be mediated by a combination of enhanced APAP clearance and robust compensatory tissue repair.     

Wnt/beta-catenin signaling mediates oval cell response in rodents

Adult hepatic stem cells or oval cells are facultative stem cells in the liver that are activated during regeneration only during inhibition of innate hepatocyte proliferation. On the basis of its involvement in liver cancer, regeneration, and development, we investigated the role of the Wnt/beta-catenin pathway in oval cell response, which was initiated in male Fisher rats with 2-acetylaminofluorine and two-third partial hepatectomy (PHX). Extensive oval cell activation and proliferation were observed at 5 and 10 days post-PHX, as indicated by hematoxylin-eosin and proliferating cell nuclear antigen analysis. A noteworthy increase in total and active beta-catenin was observed at this time, which was localized to the oval cell cytoplasm and nuclei by immunohistochemistry and confirmed by double immunofluorescence. A concomitant increase in Wnt-1 in hepatocytes along with increased expression of Frizzled-2 in oval cells was observed. This paracrine mechanism coincided with a decrease in Wnt inhibitory factor-1 and glycogen synthase kinase-3beta down-regulation leading to beta-catenin stabilization. To strengthen its role, beta-catenin conditional knockout mice were treated with 3,5-diethoxycarbonyl-1,4-dihydrocollidine to induce oval cell activation. A dramatic decrease in the A6-positive oval cell numbers in the absence of beta-catenin demonstrated a critical role of beta-catenin in oval cell biology. CONCLUSION: The Wnt/beta-catenin pathway plays a key role in the normal activation and proliferation of adult hepatic stem cells.     

EFFECTS OF BICYCLE HELMET LAWS ON CHILDREN'S INJURIES

In recent years, many states and localities in the USA have enacted bicycle helmet laws. We estimate the effects of these laws on injuries requiring emergency department treatment. Using hospital‐level panel data and triple difference models, we find helmet laws are associated with reductions in bicycle‐related head injuries among children. However, laws also are associated with decreases in non‐head cycling injuries, as well as increases in head injuries from other wheeled sports. Thus, the observed reduction in bicycle‐related head injuries may be due to reductions in bicycle riding induced by the laws. Copyright © 2013 John Wiley & Sons, Ltd.     

Prevalence and correlates of depression among HIV-infected and -affected older people in rural South Africa

Background

Little is known about depression in older people in sub-Saharan Africa, the associated impact of HIV, and the influence on health perceptions.

Objectives

Examine the prevalence and correlates of depression; explore the relationship between depression and health perceptions in HIV-infected and -affected older people.

Methods

In 2010, 422 HIV-infected and -affected participants aged 50+ were recruited into a cross-sectional study. Nurse professionals interviewed participants and a diagnosis of depressive episode was derived from the Composite International Diagnostic Interview (Depression module) using the International Classification of Diseases diagnostic criteria and categorised as major (MDE) or brief (BDE).

Results

Overall, 42.4% (n=179) had a depressive episode (MDE: 22.7%, n=96; BDE: 19.7%, n=83). Prevalence of MDE was significantly higher in HIV-affected (30.1%, 95% CI 24.0–36.2%) than HIV-infected (14.8%, 95% CI 9.9–19.7%) participants; BDE was higher in HIV-infected (24.6%, 95% CI 18.7–30.6%) than in HIV-affected (15.1%, 95% CI 10.3–19.8%) participants. Being female (aOR 3.04, 95% CI 1.73–5.36), receiving a government grant (aOR 0.34, 95% CI 0.15–0.75), urban residency (aOR 1.86, 95% CI 1.16–2.96) and adult care-giving (aOR 2.37, 95% CI 1.37–4.12) were significantly associated with any depressive episode. Participants with a depressive episode were 2–3 times more likely to report poor health perceptions.

Limitations

Study limitations include the cross-sectional design, limited sample size and possible selection biases.

Conclusions

Prevalence of depressive episodes was high. Major depressive episodes were higher in HIV-affected than HIV-infected participants. Psycho-social support similar to that of HIV treatment programmes around HIV-affected older people may be useful in reducing their vulnerability to depression.     

Role of Bile Acids in Liver Injury and Regeneration following Acetaminophen Overdose

Bile acids play a critical role in liver injury and regeneration, but their role in acetaminophen (APAP)–induced liver injury is not known. We tested the effect of bile acid modulation on APAP hepatotoxicity using C57BL/6 mice, which were fed a normal diet, a 2% cholestyramine (CSA)–containing diet for bile acid depletion, or a 0.2% cholic acid (CA)–containing diet for 1 week before treatment with 400 mg/kg APAP. CSA-mediated bile acid depletion resulted in significantly higher liver injury and delayed regeneration after APAP treatment. In contrast, 0.2% CA supplementation in the diet resulted in a moderate delay in progression of liver injury and significantly higher liver regeneration after APAP treatment. Either CSA-mediated bile acid depletion or CA supplementation did not affect hepatic CYP2E1 levels or glutathione depletion after APAP treatment. CSA-fed mice exhibited significantly higher activation of c-Jun N-terminal protein kinases and a significant decrease in intestinal fibroblast growth factor 15 mRNA after APAP treatment. In contrast, mice fed a 0.2% CA diet had significantly lower c-Jun N-terminal protein kinase activation and 12-fold higher fibroblast growth factor 15 mRNA in the intestines. Liver regeneration after APAP treatment was significantly faster in CA diet–fed mice after APAP administration secondary to rapid cyclin D1 induction. Taken together, these data indicate that bile acids play a critical role in both initiation and recovery of APAP-induced liver injury.Bile acids are versatile biological molecules that regulate energy homeostasis, activate nuclear receptors and cell signaling pathways, and control cell proliferation and inflammatory processes in the liver and gastrointestinal tract.^1,2 Bile acids maintain their own homeostasis by activating a complex signaling network involving hepatic and intestinal farnesoid X receptor (FXR), small heterodimer partner, and intestinal fibroblast growth factor (FGF) 15 (FGF19 in human) expression, culminating in inhibition of the primary bile acid–synthesizing enzyme, CYP7A1.^3–6 Although bile acids are potent signaling molecules at pathophysiological concentrations, they cause apoptosis, necrosis, and oxidative stress.^3,7–10 Bile acids have also been implicated in stimulation of liver regeneration.^11–14 Studies in recent years indicate that the bile acid–mediated gut-liver signaling axis may play a critical role in regulation of liver homeostasis.^6,15,16Acetaminophen (APAP) is the most commonly used analgesic and antipyretic agent.¹⁷ An overdose of APAP is the major cause of acute liver failure in the United States.^18,19 The mechanisms of APAP-induced liver injury and subsequent liver regeneration are the focus of intense investigation.^20–22 In an overdose situation, excess APAP is mainly metabolized by CYP2E1 to a reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). In hepatocytes, conjugation of NAPQI to GSH is the key mechanism for detoxification of NAPQI. Once the GSH is depleted, NAPQI attacks cellular proteins, especially mitochondrial proteins, to form protein adducts. This triggers a cascade of intracellular signaling events involving c-Jun N-terminal protein kinase (JNK) activation and mitochondrial permeability transition, finally culminating in necrotic cell death.²⁰ Liver injury is followed by compensatory liver regeneration, which is a critical determinant of final outcome of liver injury.²³ Despite decades of research, how these intracellular events are affected by extracellular signaling is not known.The current study was designed to explore the role of bile acids in initiation of liver injury and stimulation of liver regeneration after APAP overdose. These studies are highly significant because the data reveal a novel role of bile acids in cellular protection and liver regeneration after APAP overdose, and these studies investigate the effect of resin-mediated bile acid depletion, a commonly used therapy, on APAP toxicity.     

Cyclophilin and NS5A Inhibitors,but Not Other Anti-Hepatitis C Virus (HCV) Agents,Preclude HCV-Mediated Formation of Double-Membrane-Vesicle Viral Factories

Although the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.     

Peripheral venous and arterial blood gas analysis in adults: are they comparable? A systematic review and meta‐analysis

Peripheral venous blood gas (PVBG) analysis is increasingly being used as a substitute for arterial blood sampling; however, comparability has not been clearly established. To determine if the pH, PCO₂ and PO₂ obtained from PVBG analysis is comparable with arterial blood gas (ABG) analysis. A search was conducted of electronic databases as well as hand‐searching of journals and reference lists through December 2012 to identify studies comparing PVBG with ABG analysis in adult subjects. A systematic review was conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta‐Analyses statement. A meta‐analysis using a random effects model was used to calculate the average difference (bias) and the limits of agreement for the venous and arterial pH, PCO₂ and PO₂. A total of 18 studies comprising 1768 subjects were included in the meta‐analysis. There was considerable heterogeneity between studies with I² approaching 100%. There was little difference between the pH obtained from the PVBG and the ABG, with the arterial pH typically 0.03 higher than the venous pH (95% confidence interval 0.029–0.038). The venous and arterial PCO₂ were not comparable because the 95% prediction interval of the bias for venous PCO₂ was unacceptably wide, extending from ?10.7 mm Hg to +2.4 mm Hg. The PO₂ values compared poorly, the arterial PO₂ typically 36.9 mm Hg greater than the venous with significant variability (95% confidence interval from 27.2 to 46.6 mm Hg). PVBG analysis compares well with ABG analysis for pH estimations in adults but not to the PCO₂ or PO₂. These differences are sufficiently large to be of clinical significance.