Samar H. Ibrahim

Incidence of Gastrointestinal Symptoms in Children With Autism: A Population-Based Study

OBJECTIVE: To determine whether children with autism have an increased incidence of gastrointestinal symptoms compared with matched control subjects in a population-based sample. DESIGN/METHODS: In a previous study including all of the residents of Olmsted County, Minnesota, aged <21 years between 1976 and 1997, we identified 124 children who fulfilled criteria on the basis of Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, for a research diagnosis of autism. Two matched control subjects were identified for each case subject. Through the Rochester Epidemiology Project, all medical diagnoses, are indexed for computerized retrieval. Gastrointestinal diagnoses before 21 years of age were grouped into 5 categories: (1) constipation; (2) diarrhea; (3) abdominal bloating, discomfort, or irritability; (4) gastroesophageal reflux or vomiting; and (5) feeding issues or food selectivity. The cumulative incidence of each category was calculated by using the Kaplan-Meier method. Cox proportional hazards models were fit to estimate the risk ratios (case subjects versus control subjects) and corresponding 95% confidence intervals. RESULTS: Subjects were followed to median ages of 18.2 (case subjects) and 18.7 (control subjects) years. Significant differences between autism case and control subjects were identified in the cumulative incidence of constipation (33.9% vs 17.6%) and feeding issues/food selectivity (24.5% vs 16.1). No significant associations were found between autism case status and overall incidence of gastrointestinal symptoms or any other gastrointestinal symptom category. CONCLUSIONS: As constipation and feeding issues/food selectivity often have a behavioral etiology, data suggest that a neurobehavioral rather than a primary organic gastrointestinal etiology may account for the higher incidence of these gastrointestinal symptoms in children with autism.

Mechanisms of lipotoxicity in NAFLD and clinical implications.

ABSTRACT With the epidemic of childhood obesity, nonalcoholic fatty liver disease (NAFLD) has become the most common cause of chronic liver disease in pediatrics. NAFLD is strongly associated with insulin resistance and increased level of serum free fatty acids (FFAs). FFAs have direct hepatotoxicity through the induction of an endoplasmic reticulum stress response and subsequently activation of the mitochondrial pathway of cell death. FFAs may also result in lysosomal dysfunction and alter death receptor gene expression. Lipoapoptosis is a key pathogenic process in NAFLD, and correlates with progressive inflammation, and fibrosis. Accumulation of triglyceride in the liver results from uptake and esterification of FFAs by the hepatocyte, and is less likely to be hepatotoxic per se. To date, there are no proven effective therapies that halt NAFLD progression or unfortunately improve prognosis in children. The cellular mechanisms of lipotoxicity are complex but provide potential therapeutic targets for NAFLD. In this review we discuss several potential therapeutic opportunities in detail including inhibition of apoptosis, c-Jun-N-terminal kinase, and endoplasmic reticulum stress pathways.

A Surgical Model in Male Obese Rats Uncovers Protective Effects of Bile Acids Post-Bariatric Surgery

Bariatric surgery elevates serum bile acids. Conjugated bile acid administration, such as tauroursodeoxycholic acid (TUDCA), improves insulin sensitivity, whereas short-circuiting bile acid circulation through ileal interposition surgery in rats raises TUDCA levels. We hypothesized that bariatric surgery outcomes could be recapitulated by short circuiting the normal enterohepatic bile circulation. We established a model wherein male obese rats underwent either bile diversion (BD) or Sham (SH) surgery. The BD group had a catheter inserted into the common bile duct and its distal end anchored into the middistal jejunum for 4-5 weeks. Glucose tolerance, insulin and glucagon-like peptide-1 (GLP-1) response, hepatic steatosis, and endoplasmic reticulum (ER) stress were measured. Rats post-BD lost significantly more weight than the SH rats. BD rats gained less fat mass after surgery. BD rats had improved glucose tolerance, increased higher postprandial glucagon-like peptide-1 response and serum bile acids but less liver steatosis. Serum bile acid levels including TUDCA concentrations were higher in BD compared to SH pair-fed rats. Fecal bile acid levels were not different. Liver ER stress (C/EBP homologous protein mRNA and pJNK protein) was decreased in BD rats. Bile acid gavage (TUDCA/ursodeoxycholic acid [UDCA]) in diet-induced obese rats, elevated serum TUDCA and concomitantly reduced hepatic steatosis and ER stress (C/EBP homologous protein mRNA). These data demonstrate the ability of alterations in bile acids to recapitulate important metabolic improvements seen after bariatric surgery. Further, our work establishes a model for focused study of bile acids in the context of bariatric surgery that may lead to the identification of therapeutics for metabolic disease.

Lipid-Induced Signaling Causes Release of Inflammatory Extracellular Vesicles From Hepatocytes

BACKGROUND & AIMS Hepatocyte cellular dysfunction and death induced by lipids and macrophage-associated inflammation are characteristics of nonalcoholic steatohepatitis (NASH). The fatty acid palmitate can activate death receptor 5 (DR5) on hepatocytes, leading to their death, but little is known about how this process contributes to macrophage-associated inflammation. We investigated whether lipid-induced DR5 signaling results in the release of extracellular vesicles (EVs) from hepatocytes, and whether these can induce an inflammatory macrophage phenotype. METHODS Primary mouse and human hepatocytes and Huh7 cells were incubated with palmitate, its metabolite lysophosphatidylcholine, or diluent (control). The released EV were isolated, characterized, quantified, and applied to macrophages. C57BL/6 mice were placed on chow or a diet high in fat, fructose, and cholesterol to induce NASH. Some mice also were given the ROCK1 inhibitor fasudil; 2 weeks later, serum EVs were isolated and characterized by immunoblot and nanoparticle-tracking analyses. Livers were collected and analyzed by histology, immunohistochemistry, and quantitative polymerase chain reaction. RESULTS Incubation of primary hepatocytes and Huh7 cells with palmitate or lysophosphatidylcholine increased their release of EVs, compared with control cells. This release was reduced by inactivating mediators of the DR5 signaling pathway or rho-associated, coiled-coil-containing protein kinase 1 (ROCK1) inhibition. Hepatocyte-derived EVs contained tumor necrosis factor-related apoptosis-inducing ligand and induced expression of interleukin 1β and interleukin 6 messenger RNAs in mouse bone marrow-derived macrophages. Activation of macrophages required DR5 and receptor-interacting protein kinase 1. Administration of the ROCK1 inhibitor fasudil to mice with NASH reduced serum levels of EVs; this reduction was associated with decreased liver injury, inflammation, and fibrosis. CONCLUSIONS Lipids, which stimulate DR5, induce release of hepatocyte EVs, which activate an inflammatory phenotype in macrophages. Strategies to inhibit ROCK1-dependent release of EVs by hepatocytes might be developed for the treatment of patients with NASH.

Vismodegib Suppresses TRAIL-mediated Liver Injury in a Mouse Model of Nonalcoholic Steatohepatitis

Hedgehog signaling pathway activation has been implicated in the pathogenesis of NASH. Despite this concept, hedgehog pathway inhibitors have not been explored. Thus, we examined the effect of vismodegib, a hedgehog signaling pathway inhibitor, in a diet-induced model of NASH. C57BL/6 mice were placed on 3-month chow or FFC (high saturated fats, fructose, and cholesterol) diet. One week prior to sacrifice, mice were treated with vismodegib or vehicle. Mice fed the FFC diet developed significant steatosis, which was unchanged by vismodegib therapy. In contrast, vismodegib significantly attenuated FFC-induced liver injury as manifested by reduced serum ALT and hepatic TUNEL-positive cells. In line with the decreased apoptosis, vismodegib prevented FFC-induced strong upregulation of death receptor DR5 and its ligand TRAIL. In addition, FFC-fed mice, but not chow-fed animals, underwent significant liver injury and apoptosis following treatment with a DR5 agonist; however, this injury was prevented by pre-treatment with vismodegib. Consistent with a reduction in liver injury, vismodegib normalized FFC-induced markers of inflammation including mRNA for TNF-α, IL-1β, IL-6, monocyte chemotactic protein-1 and a variety of macrophage markers. Furthermore, vismodegib in FFC-fed mice abrogated indices of hepatic fibrogenesis. In conclusion, inhibition of hedgehog signaling with vismodegib appears to reduce TRAIL-mediated liver injury in a nutrient excess model of NASH, thereby attenuating hepatic inflammation and fibrosis. We speculate that hedgehog signaling inhibition may be salutary in human NASH.

Animal Models of Nonalcoholic Steatohepatitis: Eat, Delete, and Inflame

With the obesity epidemic, nonalcoholic fatty liver disease (NAFLD) has become a public health problem with increasing prevalence. The mechanism of disease progression remains obscure and effective therapy is lacking. Therefore, there is a need to understand the pathogenic mechanisms responsible for disease development and progression in order to develop innovative therapies. To accomplish this goal, experimental animal models that recapitulate the human disease are necessary, especially, since causative mechanistic studies of NAFLD are more difficult or unethical to perform in humans. A large number of studies regarding the pathophysiology and treatment of nonalcoholic steatohepatitis (NASH) have been undertaken in mice to model human NAFLD and NASH. This review discusses the known dietary, genetic, and inflammation-based animal models of NASH described in recent years, with a focus on the major advances made in this field.

Glycogen synthase kinase-3 (GSK-3) inhibition attenuates hepatocyte lipoapoptosis.

BACKGROUNDS & AIMS Saturated free fatty acids induce hepatocyte lipoapoptosis, a key pathologic feature of non-alcoholic steatohepatitis. The saturated free fatty acid palmitate induces hepatocyte lipoapoptosis via an endoplasmic reticulum stress pathway resulting in c-Jun-N-terminal (JNK) activation. Glycogen synthase kinase (GSK)-3 is a serine/threonine kinase which may also promote JNK activation. Thus, our aim was to determine if GSK-3 inhibition suppresses palmitate induced JNK activation and lipoapoptosis. METHODS For these studies, we employed mouse primary hepatocytes, Huh-7 and Hep3B cell lines. RESULTS Palmitate-induced GSK-3 activation was identified by phosphorylation of its substrate glycogen synthase. GSK-3 pharmacologic inhibition, by GSK-3 inhibitor IX and enzastaurin, significantly reduced PA-mediated lipoapoptosis. More importantly, Huh-7 cells, in which either GSK-3α or GSK-3β isoforms were stably and selectively knocked down by shRNA, displayed resistance to palmitate-induced cytotoxicity. GSK-3 pharmacological inhibitors and shRNA-targeted knockdown of GSK-3α or GSK-3β also suppressed JNK activation by palmitate. JNK activation, in part, promotes lipoapotosis by inducing expression of the pro-apoptotic effector p53-upregulated modulator of apoptosis (PUMA). Consistent with this concept, GSK-3 pharmacologic inhibition also reduced PUMA cellular protein levels during exposure to palmitate. On the other hand, the GSK-3 inhibitors did not prevent PA induction of ER stress. CONCLUSIONS Our results suggest that GSK-3 activation promotes a JNK-dependent cytotoxic signaling cascade culminating in lipoapoptosis.

Mixed lineage kinase 3 mediates release of C-X-C motif ligand 10–bearing chemotactic extracellular vesicles from lipotoxic hepatocytes

Mixed lineage kinase 3 (MLK3) deficiency reduces macrophage‐associated inflammation in a murine model of nonalcoholic steatohepatitis (NASH). However, the mechanistic links between MLK3 activation in hepatocytes and macrophage‐driven inflammation in NASH are uncharted. Herein, we report that MLK3 mediates the release of (C‐X‐C motif) ligand 10 (CXCL10)‐laden extracellular vesicles (EVs) from lipotoxic hepatocytes, which induce macrophage chemotaxis. Primary mouse hepatocytes (PMHs) and Huh7 cells were treated with palmitate or lysophosphatidylcholine (LPC). Released EVs were isolated by differential ultracentrifugation. LPC treatment of PMH or Huh7 cells induced release of EVs, which was prevented by either genetic or pharmacological inhibition of MLK3. Mass spectrometry identified the potent chemokine, CXCL10, in the EVs, which was markedly enriched in EVs isolated from LPC‐treated hepatocytes versus untreated cells. Green fluorescent protein (GFP)‐tagged CXCL10 was present in vesicular structures and colocalized with the red fluorescent protein (RFP)‐tagged EV marker, CD63, after LPC treatment of cotransfected Huh‐7 cells. Either genetic deletion or pharmacological inhibition of MLK3 prevented CXCL10 enrichment in EVs. Treatment of mouse bone‐marrow–derived macrophages with lipotoxic hepatocyte‐derived EVs induced macrophage chemotaxis, an effect blocked by incubation with CXCL10‐neutralizing antisera. MLK3‐deficient mice fed a NASH‐inducing diet had reduced concentrations of total plasma EVs and CXCL10 containing EVs compared to wild‐type mice. Conclusions: During hepatocyte lipotoxicity, activated MLK3 induces the release of CXCL10‐bearing vesicles from hepatocytes, which are chemotactic for macrophages. (Hepatology 2016;63:731–744)

Extracellular Vesicles in Liver Pathobiology: Small Particles with Big Impact

Extracellular vesicles (EVs) are nanometer‐sized, membrane‐bound vesicles released by cells into the extracellular milieu. EVs are now recognized to play a critical role in cell‐to‐cell communication. EVs contain important cargo in the form of proteins, lipids, and nucleic acids and serve as vectors for delivering this cargo from donor to acceptor or target cell. EVs are released under both physiologic and pathologic conditions, including liver diseases, and exert a wide range of effects on target cells. This review provides an overview on EV biogenesis, secretion, cargo, and target cell interactions in the context of select liver diseases. Specifically, the diverse roles of EVs in nonalcoholic steatohepatitis, alcoholic liver disease, viral hepatitis, cholangiopathies, and hepatobiliary malignancies are emphasized. Liver diseases often result in an increased release of EVs and/or in different cargo sorting into these EVs. Either of these alterations can drive disease pathogenesis. Given this fact, EVs represent a potential target for therapeutic intervention in liver disorders. Because altered EV composition may reflect the underlying disease condition, circulating EVs can be exploited for diagnostic and prognostic purposes as a liquid biopsy. Furthermore, ex vivo modified or synthesized EVs can be engineered as therapeutic nano‐shuttles. Finally, we highlight areas that merit further investigation relevant to understanding how EVs regulate liver disease pathogenesis. (Hepatology 2016;64:2219‐2233).

A 3-year-old with immunoglobulin G4-associated cholangitis.

JPGN Volume 53, N I mmunoglobulin G4 (IgG4)-associated cholangitis (IAC) is recognized in the adult population as a steroid-responsive biliary disease, often associated with autoimmune pancreatitis (1). IAC has been reported in some instances in the absence of pancreatic involvement (2). IAC is characterized by the elevation of serum IgG4 and infiltration of IgG4-positive plasma cells in bile ducts (3). We report on a 3-year-old female patient with features of IgG4associated cholangitis (markedly elevated serum IgG4 and heavy hepatic infiltration with IgG4-positive plasma cells), without evidence of pancreatic involvement. To the best of our knowledge, our patient is the first reported pediatric patient with IAC. A 3-year-old female patient presented in February 2009 to the outpatient pediatric gastroenterology clinic with a 6-month history of dark urine and intermittent episodes of acholic stools. Three months before presentation she was noted to have an enlarged abdomen. She had not been noticeably jaundiced nor did she have scleral icterus until about 3 months before presentation. There were no reported fevers, joint pains, abdominal complaints, and no obvious episode of hepatitis. The patient denied diarrhea, vomiting, hematemesis, hematochezia, or lethargy. Family history is not suggestive of liver or autoimmune diseases. Physical examination revealed a 14.7-kg child (weight at the 63rd percentile), body mass index 17.8 kg/m (at the 93rd percentile), with jaundice and scleral icterus. The liver extended 5 cm below the right costal margin and across the midline to the left midclavicular line. There was no splenomegaly, obvious ascites, or signs of chronic liver disease. The cardiovascular and respiratory examinations were normal. The liver tests at her initial evaluation revealed an alanine aminotransferase (ALT) of 407 U/L (normal 7–45 U/L), an aspartate aminotransferase (AST) of 297 U/L (normal 8–50 U/L), a total protein of 8.6 g/dL, an albumin of 3.4 g/dL, total bilirubin of 4.2 mg/dL with a direct fraction of 2.8 mg/dL, alkaline phosphatase 1990 U/L (normal 169–372 U/L), gamma-glutamyl transpeptidase (GGT) 969 U/L (normal 6–29 U/L), international normalized ratio 1.3, and partial thromboplastin time of 42 seconds. Coagulation factor analysis revealed an isolated decrease in factor V (39% activity) with otherwise normal coagulation factors including a normal factor VII activity of 122%. Total IgG was 3122 mg/dL