Ronald J. Sokol

Rotavirus Infection Frequency and Risk of Celiac Disease Autoimmunity in Early Childhood: A Longitudinal Study

OBJECTIVE:Few studies have assessed the role of specific gastrointestinal infections in celiac disease. We investigated whether increased frequency of rotavirus infection, a common cause of gastrointestinal infection and inflammation, predicts increased risk of celiac disease autoimmunity.METHODS:A cohort of 1,931 children from the Denver metropolitan area who carried celiac disease human leukocyte antigen (HLA) risk alleles were followed from infancy for development of celiac disease autoimmunity, defined as positivity at two or more subsequent clinic visits for tissue transglutaminase (tTG) autoantibodies measured using a radioimmunoassay with human recombinant tTG. Blood samples were obtained at ages 9, 15, and 24 months, and annually thereafter. Rotavirus antibodies were assayed using an indirect enzyme immunoassay in serial serum samples from each case and two matched controls. Frequency of infections were estimated by the number of increases (>2 assay coefficient of variation) in rotavirus antibody between clinic visits.RESULTS:Fifty-four cases developed celiac disease autoimmunity at a median age of 4.4 yr. Thirty-six had an intestinal biopsy, of which 27 (75%) were positive for celiac disease. Frequent rotavirus infections predicted a higher risk of celiac disease autoimmunity (compared with zero infections, rate ratio 1.94, 95% confidence interval [CI] 0.39–9.56, for one infection and rate ratio 3.76, 95% CI 0.76–18.7, for ≥2 infections, rate ratio for trend per increase in number of infections = 1.94, 95% CI 1.04–3.61, p = 0.037). The result was similar after adjustment for gender, ethnic group, maternal education, breast-feeding, day-care attendance, number of siblings, season of birth, and number of HLA DR3-DQ2 haplotypes.CONCLUSIONS:This prospective study provides the first indication that a high frequency of rotavirus infections may increase the risk of celiac disease autoimmunity in childhood in genetically predisposed individuals.

Identification of a new inborn error in bile acid synthesis: mutation of the oxysterol 7alpha-hydroxylase gene causes severe neonatal liver disease.

We describe a metabolic defect in bile acid synthesis involving a deficiency in 7alpha-hydroxylation due to a mutation in the gene for the microsomal oxysterol 7alpha-hydroxylase enzyme, active in the acidic pathway for bile acid synthesis. The defect, identified in a 10-wk-old boy presenting with severe cholestasis, cirrhosis, and liver synthetic failure, was established by fast atom bombardment ionization-mass spectrometry, which revealed elevated urinary bile acid excretion, a mass spectrum with intense ions at m/z 453 and m/z 510 corresponding to sulfate and glycosulfate conjugates of unsaturated monohydroxy-cholenoic acids, and an absence of primary bile acids. Gas chromatography-mass spectrometric analysis confirmed the major products of hepatic synthesis to be 3beta-hydroxy-5-cholenoic and 3beta-hydroxy-5-cholestenoic acids, which accounted for 96% of the total serum bile acids. Levels of 27-hydroxycholesterol were > 4,500 times normal. The biochemical findings were consistent with a deficiency in 7alpha-hydroxylation, leading to the accumulation of hepatotoxic unsaturated monohydroxy bile acids. Hepatic microsomal oxysterol 7alpha-hydroxylase activity was undetectable in the patient. Gene analysis revealed a cytosine to thymidine transition mutation in exon 5 that converts an arginine codon at position 388 to a stop codon. The truncated protein was inactive when expressed in 293 cells. These findings indicate the quantitative importance of the acidic pathway in early life in humans and define a further inborn error in bile acid synthesis as a metabolic cause of severe cholestatic liver disease.

Generation of hydroperoxides in isolated rat hepatocytes and hepatic mitochondria exposed to hydrophobic bile acids

BACKGROUND & AIMS The mechanisms causing liver injury in cholestatic diseases are unclear. The hypothesis that accumulation of hydrophobic bile acids in hepatocytes during cholestasis leads to generation of oxygen free radicals and oxidative injury was tested. The aim of this study was to determine if hydrophobic bile acid toxicity is associated with increased hydroperoxide generation in isolated rat hepatocytes and mitochondria. METHODS Hepatocytes were exposed to taurochenodeoxycholic acid (TCDC; 0-2000 mumol/L) or taurocholic acid (TC; 1000 mumol/L), and cellular injury, intracellular hydroperoxide generation, and thiobarbituric acid-reacting substances (TBARS) were measured. Isolated mitochondria were incubated with 400 mumol/L chenodeoxycholic acid or 400 mumol/L cholic acid, and hydroperoxide generation was measured fluorometrically. RESULTS Hepatocyte injury, hydroperoxide generation, and TBARS increased over 4 hours on exposure to TCDC but not TC. Hydroperoxide generation preceded hepatocyte injury and accumulation of TBARS. Preincubation of hepatocytes with the antioxidant, d-alpha-tocopheryl succinate, completely abrogated cellular injury, hydroperoxide, and TBARS generation. Hydroperoxide generation was increased in mitochondria exposed to chenodeoxycholic acid. CONCLUSIONS Intracellular generation of hydroperoxides by mitochondria appears to be an early event in hydrophobic bile acid-induced hepatocyte toxicity. Antioxidants may be of benefit in cholestasis.

Pathogenesis and outcome of biliary atresia : current concepts

Neonatal cholestatic disorders are a group of hepatobiliary diseases occurring within the first 3 months of life. Bile flow is impaired, and patients have conjugated hyperbilirubinemia, acholic stools, and hepatomegaly. Overall, 1 in 2,500 live births is affected with a neonatal cholestatic disorder (1). The two most common causes of neonatal cholestasis are biliary atresia and idiopathic neonatal hepatitis, accounting for up to 50% to 70% of cases. Other causes include a variety of neonatal infections (viral, toxoplasmosis, syphilis, bacterial), metabolic and genetic diseases, progressive familial intrahepatic cholestatic disorders (PFIC), paucity of interlobular bile duct disorders (e.g., Alagille syndrome), choledochal cyst, ischemia–reperfusion injury, association with parenteral nutrition administration, and other conditions (Table 1). Despite clinical improvement after the portoenterostomy procedure, approximately 70% to 80% of children with biliary atresia will eventually require liver transplantation; thus, biliary atresia alone accounts for almost 50% of all liver transplants performed in children (1). It should be noted that

A prospective study of the incidence of childhood celiac disease

77 million is spent each year in the United States on liver transplantation for children and the ensuing hospitalizations (2). This sum of money covers 0.2% of total health care expenditures related to children, even though these children represent 0.0006% of the total pediatric population. Importantly, this disproportionate expenditure for liver transplantation in children could be cut in half if improved therapies for biliary atresia were developed that could abrogate or further delay the need for liver transplantation. Remarkably, little is known about the etiopathogenesis of biliary atresia; consequently, there has been slow progress in developing improved therapies or preventative strategies during the past decade. The purpose of this review is to summarize recent advances in the diagnosis and management of biliary atresia, examine the clinical outcome, describe the evolving theories of the etiology and pathogenesis of this disorder, and highlight gaps in our current knowledge.

Vitamin E reduces oxidant injury to mitochondria and the hepatotoxicity of taurochenodeoxycholic acid in the rat

OBJECTIVES To estimate the frequency of celiac disease (CD) in children in the general population of Denver, Colorado. STUDY DESIGN From 22,346 newborns characterized as expressing 0, 1, or 2 HLA-DR3(DQB1*0201) alleles, 987 were selected for a prospective stratified cohort study. Participants were followed for as long as 7 years with serial testing for serum IgA anti-transglutaminase antibodies and for evidence of CD (intestinal mucosal changes or persistent seropositivity). RESULTS Of 40 children with at least one positive serologic test, 19 had evidence of CD (10 by biopsy, 9 by persistent seropositivity). Those expressing 0, 1, or 2 HLA-DR3 alleles had, respectively, 0.3% (95% CI, 0.0-2.7), 3.4% (3.0-11.7), and 3.2% (1.0-11.0) risk for evidence of CD by age 5 years. The adjusted risk estimate for evidence of CD by age 5 years for the Denver general population was 0.9% (0.4-2.0), or 1 in 104 (1:49-221). After adjusting for number of HLA-DR3 alleles expressed, risk was higher in females: RR=3.34 (1.00-10.9, P=.048). Evidence of CD was not observed before age 2.6 years. CONCLUSIONS Celiac disease may affect 0.9% of Denver children by 5 years of age. Children positive for the HLA-DR3 allele and females appear to be at increased risk.

Oxidant injury to hepatic mitochondria in patients with Wilson's disease and Bedlington terriers with copper toxicosis

BACKGROUND & AIMS Hydrophobic bile acids have been implicated in the pathogenesis of cholestatic liver injury. The hypothesis that hydrophobic bile acid toxicity is mediated by oxidant stress in an in vivo rat model was tested in this study. METHODS A dose-response study of bolus intravenous (i.v.) taurochenodeoxycholic acid (TCDC) in rats was conducted. Rats were then pretreated with parenteral alpha-tocopherol, and its effect on i.v. TCDC toxicity was evaluated by liver blood tests and by assessing mitochondrial lipid peroxidation. RESULTS Four hours after an i.v. bolus of TCDC (10 mumol/100 g weight), serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels peaked, hepatic mitochondria showed evidence of increased lipid peroxidation, and serum bile acid analysis was consistent with a cholestatic injury. Liver histology at 4 hours showed hepatocellular necrosis and swelling and mild portal tract inflammation. Treatment with parenteral alpha-tocopherol was associated with a 60%-70% reduction in AST and ALT levels, improved histology, and a 60% reduction in mitochondrial lipid peroxidation in rats receiving TCDC. CONCLUSIONS These data show that hepatocyte injury and oxidant damage to mitochondria caused by i.v. TCDC can be significantly reduced by pretreatment with the antioxidant vitamin E. These in vivo findings support the role for oxidant stress in the pathogenesis of bile acid hepatic toxicity.

Recommendations for Management of Liver and Biliary Tract Disease in Cystic Fibrosis

BACKGROUND/AIMS Copper overload leads to liver injury in humans with Wilsons disease and in Bedlington terriers with copper toxicosis; however, the mechanisms of liver injury are poorly understood. This study was undertaken to determine if oxidant (free radical) damage to hepatic mitochondria is involved in naturally occurring copper toxicosis. METHODS Fresh liver samples were obtained at the time of liver transplantation from 3 patients with Wilsons disease, 8 with cholestatic liver disease, and 5 with noncholestatic liver disease and from 8 control livers. Fresh liver was also obtained by open liver biopsy from 4 copper-overloaded and 4 normal Bedlington terriers and from 8 control dogs. Hepatic mitochondria and microsomes (humans only) were isolated, and lipid peroxidation was measured by lipid-conjugated dienes and thiobarbituric acid-reacting substances. In humans, liver alpha-tocopherol content was measured. RESULTS Lipid peroxidation and copper content were significantly increased (P < 0.05) in mitochondria from patients with Wilsons disease and copper-overloaded Bedlington terriers. More modest increases in lipid peroxidation were present in microsomes from patients with Wilsons disease. Mitochondrial copper concentrations correlated strongly with the severity of mitochondrial lipid peroxidation. Hepatic alpha-tocopherol content was decreased significantly in Wilsons disease liver. CONCLUSIONS These data suggest that the hepatic mitochondrion is an important target in hepatic copper toxicity and that oxidant damage to the liver may be involved in the pathogenesis of copper-induced injury.

Genetic induction of proinflammatory immunity in children with biliary atresia

Although involvement of the liver and biliary tract has been recognized as a complication of cystic fibrosis (CF) since Andersons initial description of CF in 1938 (1), the clinical significance of hepatobiliary disease in CF has not been well characterized until recently. The clinically silent development of liver complications has often been eclipsed by the obvious manifestations of pulmonary and pancreatic abnormalities. Because of improved life expectancy in the past 2 decades, now reported to be 31 years, liver disease has assumed greater importance to patients with CF and those involved in their care. Unfortunately, clinical identification of liver disease in CF has been difficult and inaccurate because of the general absence of symptoms of the developing fibrotic liver lesion. Thus, early diagnosis and therapeutic intervention has not been possible. In recent years, advances in our understanding of the function of the cystic fibrosis transmembrane conductance regulator (CFTR) in bile duct epithelia and of the mechanisms of fibrogenesis in the liver have provided a stronger scientific basis for the pathogenesis of disease, leading to insights concerning potentially novel therapeutic approaches. For these reasons, the Cystic Fibrosis Foundation has recognized the need to revise the previous Recommendations for Management of Liver and Biliary Tract Disease in Cystic Fibrosi published in 1989, to reflect the new scientific advances and clinical information in this area.

Screening and outcomes in biliary atresia: Summary of a National Institutes of Health workshop

BACKGROUND Biliary atresia is the commonest cause of pathological jaundice in infants and the leading indication for liver transplantation in children worldwide. The cause and pathogenesis remain largely unknown. Because of clinical heterogeneity and experimental difficulties in addressing molecular mechanisms underlying multifactorial disorders in human beings, we searched for genomic signatures of biliary atresia in affected infants. METHODS We generated pools of biotinylated cRNA from livers of 14 infants with biliary atresia and six with neonatal intrahepatic cholestasis (diseased controls) and hybridised the cRNA against oligonucleotide-based gene chips. Immunohistochemistry and reverse transcriptase (RT)-PCR were used to assess the specificity of the findings and functional commitment of lymphocytes in affected livers. FINDINGS Data filtering, to identify genes that are differentially expressed, and cluster analysis revealed a predominant and coordinated activation of immunity/inflammation genes within the livers of infants with biliary atresia. Most of the genes showed differential lymphocyte function, with activation of osteopontin, a regulator of cell-mediated (T-helper 1 [Th-1]) immunity in T-helper lymphocytes, and suppression of immunoglobulin genes in early stages of disease. These findings were associated with production of interferon gamma in 65% of infants with biliary atresia and no diseased control. However, histologically similar inflammatory infiltrates were present in livers of both groups, implying differential activation states of similar cell types. INTERPRETATION Livers of infants with biliary atresia have a coordinated activation of genes involved in lymphocyte differentiation. Among these genes, the overexpression of osteopontin and interferon gamma points to a potential role of Th-1-like cytokines in disease pathogenesis.