Fatty liver is a common cause of liver disease in children. However, the epidemiology of pediatric fatty liver is limited to single-center case series of nonalcoholic fatty liver disease (NAFLD). ...Obesity and insulin resistance are major established risk factors for NAFLD. The role of gender, race, and ethnicity on the prevalence of fatty liver in obese children is unknown.
We recruited obese 12th-grade participants from the Child and Adolescent Trial for Cardiovascular Health in California, Louisiana, Minnesota, and Texas. Serum samples were collected at school when the participants were well. Alanine aminotransferase (ALT) was measured by kinetic enzymatic assay, and ALT >40 U/L was defined as abnormal. Causes of abnormal ALT other than NAFLD were excluded by serum testing.
A total of 127 obese students (73 female, 54 male) had a mean BMI of 35.2 kg/m2. Unexplained ALT elevation was present in 23% of participants overall. The mean ALT for participants with normal values was 28 U/L and for participants with an abnormal ALT was 56 U/L. Abnormal ALT was significantly more prevalent in boys (44%) than in girls (7%). The prevalence of abnormal ALT differed significantly by race and ethnicity (Hispanic: 36%; white: 22%; black: 14%). Serum ALT value was significantly predicted by the combination of gender, race/ethnicity, and BMI. After controlling for gender and BMI, Hispanic ethnicity significantly predicted greater ALT than black race.
In a national, school-based sample of obese adolescents, boys were 6 times more likely than girls to have an unexplained elevated ALT. Given that participants were well and causes of chronic liver disease were excluded, we speculate that obese adolescent boys have an increased prevalence of fatty liver compared with obese adolescent girls. This population-based study also supports the hypothesis that NAFLD is more common in Hispanic adolescents. These findings have implications for both disease screening and studies of fatty liver pathophysiology.
The mouse small intestinal epithelium consists of four principal cell types deriving from one multipotent stem cell: enterocytes, goblet, enteroendocrine, and Paneth cells. Previous studies showed ...that Math1, a basic helix-loop-helix (bHLH) transcription factor, is expressed in the gut. We find that loss of Math1 leads to depletion of goblet, enteroendocrine, and Paneth cells without affecting enterocytes. Colocalization of Math1 with Ki-67 in some proliferating cells suggests that secretory cells (goblet, enteroendocrine, and Paneth cells) arise from a common progenitor that expresses Math1, whereas absorptive cells (enterocytes) arise from a progenitor that is Math1-independent. The continuous rapid renewal of these cells makes the intestinal epithelium a model system for the study of stem cell regeneration and lineage commitment.
The consequences of macrophage depletion achieved by intravenous infusion of liposome-encapsulated clodronate (dichlormethylene diphosphonate (Cl2MDP)) on adenovirus-mediated transfer of a ...recombinant human alpha 1-antitrypsin (hAAT) gene were examined in 12-14-week-old male Balb/c mice. The levels of hAAT expression following tail vein infusions of 10(9) p.f.u. of Ad.RSV-hAAT were approximately four-fold higher in macrophage-depleted animals than in control animals pretreated with liposome-encapsulated phosphate-buffered saline (PBS). Clodronate pretreatment also significantly increased the survival of animals injected with high doses of viral vector. Long-term studies performed in animals receiving tail vein infusions of the adenoviral vector also indicated that clodronate pretreatment significantly attenuated the rapid loss of transgene expression usually observed in immunocompetent animals. These findings indicate that the depletion of macrophages before adenovirus-mediated gene transfer may increase the transduction efficiency and reduce the rate of immunologic elimination of the adenovirally transduced cells, thereby increasing the persistence of transgene expression in immunocompetent animals.
Wilson disease is a severe genetic disorder associated with intracellular copper overload. The affected gene,
ATP7B
, has been identified, but the molecular events leading to Wilson disease remain ...poorly understood. Here, we demonstrate that genetically engineered
Atp7b
−/− mice represent a valuable model for dissecting the disease mechanisms. These mice, like Wilson disease patients, have intracellular copper accumulation, low-serum oxidase activity, and increased copper excretion in urine. Their liver pathology developed in stages and was determined by the time of exposure to elevated copper rather than copper concentration per se. The disease progressed from mild necrosis and inflammation to extreme hepatocellular injury, nodular regeneration, and bile duct proliferation. Remarkably, all animals older than 9 months showed regeneration of large portions of the liver accompanied by the localized occurrence of cholangiocarcinoma arising from the proliferating bile ducts. The biochemical characterization of
Atp7b
−/− livers revealed copper accumulation in several cell compartments, particularly in the cytosol and nuclei. The increase in nuclear copper is accompanied by marked enlargement of the nuclei and enhanced DNA synthesis, with these changes occurring before pathology development. Our results suggest that the early effects of copper on cell genetic material contribute significantly to pathology associated with
Atp7b
inactivation.
The work of liver stem cell biologists, largely carried out in rodent models, has now started to manifest in human investigations and applications. We can now recognize complex regenerative processes ...in tissue specimens that had only been suspected for decades, but we also struggle to describe what we see in human tissues in a way that takes into account the findings from the animal investigations, using a language derived from species not, in fact, so much like our own. This international group of liver pathologists and hepatologists, most of whom are actively engaged in both clinical work and scientific research, seeks to arrive at a consensus on nomenclature for normal human livers and human reactive lesions that can facilitate more rapid advancement of our field. (HEPATOLOGY 2004; 39:1739–1745.)
Background & Aims: Glucagon-like peptide-2 (GLP-2) is a nutrient-responsive hormone that exerts diverse actions in the gastrointestinal tract, including enhancing epithelial cell survival and ...proliferation, mucosal blood flow, and nutrient uptake and suppressing gastric motility and secretion. These actions are mediated by the G-protein-coupled receptor, GLP-2R. Cellular localization of the GLP-2R and the nature of its signaling network in the gut, however, are poorly defined. Thus, our aim was to establish cellular localization of GLP-2R and functional connection to vascular action of GLP-2 in the gut.
Methods: Intestinal cellular GLP-2R localization was determined with real-time, quantitative reverse-transcription polymerase chain reaction (qRT-PCR) of laser capture microdissected subtissue and fluorescence in situ hybridization and also with double and/or triple immunostaining of human and pig tissue using a validated GLP-2R polyclonal antibody. Superior mesenteric arterial blood flow and intestinal eNOS expression and phosphorylation were measured in TPN-fed pigs acutely (4 h) infused with GLP-2.
Results: We show that the porcine GLP-2R mRNA was expressed in the villus epithelium and myenteric plexus. GLP-2R protein was co-localized by confocal immunohistochemistry with serotonin in enteroendocrine cells and also with endothelial nitric oxide synthase (eNOS)-expressing and vasoactive intestinal polypeptide-positive enteric neurons. In neonatal pigs, GLP-2 infusion dose-dependently stimulated intestinal blood flow and coordinately upregulated the expression of intestinal eNOS mRNA, protein, and phosphorylation (eNOS-Ser1117).
Conclusions: We conclude that the GLP-2-induced stimulation of blood flow is mediated by vasoactive neurotransmitters that are colocalized with GLP-2R in 2 functionally distinct cell types within the gastrointestinal tract.
Published histologic studies of the hilar plate or entire biliary remnant at the time of Kasai portoenterostomy (KHPE) have not provided deep insight into the pathogenesis of biliary atresia, ...relation to age at surgery, prognosis or the basis for successful drainage. We report detailed histologic findings in 172 centrally reviewed biliary remnants with an average of 6 sections per subject. Active lesions were classified as either necroinflammatory (rare/clustered in a few subjects) or active concentric fibroplasia with or without inflammation (common). Inactive lesions showed bland replacement by collagen and fibrous cords with little or no inflammation. Heterogeneity was common within a given remnant; however, relatively homogenous histologic patterns, defined as 3 or more inactive or active levels in the hepatic ducts levels, characterized most remnants. Homogeneity did not correlate with age at KHPE, presence/absence of congenital anomalies at laparotomy indicative of heterotaxy and outcome. Remnants from youngest subjects were more likely than older subjects to be homogenously inactive suggesting significantly earlier onset in the youngest subset. Conversely remnants from the oldest subjects were often homogenously active suggesting later onset or slower progression. More data are needed in remnants from subjects <30 days old at KHPE and in those with visceral anomalies. Prevalence of partially preserved epithelium in active fibroplastic biliary atresia lesions at all ages suggests that epithelial regression or injury may not be a primary event or that reepithelialization is already underway at the time of KHPE. We hypothesize that outcome after KHPE results from competition between active fibroplasia and reepithelialization of retained, collapsed but not obliterated lumens. The driver of active fibroplasia is unknown.
Fanconi anemia (FA) is a genetic disorder characterized by hypersensitivity to DNA damage, bone marrow failure, congenital defects, and cancer. To further investigate the in vivo function of the FA ...pathway, mice with a targeted deletion in the distally acting FA gene Fancd2 were created. Similar to human FA patients and other FA mouse models, Fancd2 mutant mice exhibited cellular sensitivity to DNA interstrand cross-links and germ cell loss. In addition, chromosome mispairing was seen in male meiosis. However, Fancd2 mutant mice also displayed phenotypes not observed in other mice with disruptions of proximal FA genes. These include microphthalmia, perinatal lethality, and epithelial cancers, similar to mice with Brca2/Fancd1 hypomorphic mutations. These additional phenotypes were not caused by defects in the ATM-mediated S-phase checkpoint, which was intact in primary Fancd2 mutant fibroblasts. The phenotypic overlap between Fancd2-null and Brca2/Fancd1 hypomorphic mice is consistent with a common function for both proteins in the same pathway, regulating genomic stability.
Recombinant adeno-associated virus (rAAV) vectors have clear potential for use in gene targeting but low correction efficiencies remain the primary drawback. One approach to enhancing efficiency is a ...block of undesired repair pathways like nonhomologous end joining (NHEJ) to promote the use of homologous recombination. The natural product vanillin acts as a potent inhibitor of NHEJ by inhibiting DNA-dependent protein kinase (DNA-PK). Using a homology containing rAAV vector, we previously demonstrated in vivo gene repair frequencies of up to 0.1% in a model of liver disease hereditary tyrosinemia type I. To increase targeting frequencies, we administered vanillin in combination with rAAV. Gene targeting frequencies increased up to 10-fold over AAV alone, approaching 1%. Fah(-/-)Ku70(-/-) double knockout mice also had increased gene repair frequencies, genetically confirming the beneficial effects of blocking NHEJ. A second strategy, transient proteasomal inhibition, also increased gene-targeting frequencies but was not additive to NHEJ inhibition. This study establishes the benefit of transient NHEJ inhibition with vanillin, or proteasome blockage with bortezomib, for increasing hepatic gene targeting with rAAV. Functional metabolic correction of a clinically relevant disease model was demonstrated and provided evidence for the feasibility of gene targeting as a therapeutic strategy.
Crigler-Najjar syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by a deficiency of uridine diphospho-glucuronosyl transferase 1A1. Current ...therapy relies on phototherapy to prevent kernicterus, but liver transplantation presently is the only permanent cure. Gene therapy is a potential alternative, and recent work has shown that helper-dependent adenoviral (HD-Ad) vectors, devoid of all viral coding sequences, induce prolonged transgene expression and exhibit significantly less chronic toxicity than early-generation Ad vectors. We used a HD-Ad vector to achieve liver-restricted expression of human uridine diphospho-glucuronosyl transferase 1A1 in the Gunn rat, a model of the human disorder. Total plasma bilirubin levels were reduced from >5.0 mg/dl to <<1.4 mg/dl for >2 yr after a single i.v. administration of vector expressing the therapeutic transgene at a dose of 3× 1012viral particles per kg. HPLC analysis of bile from treated rats showed the presence of bilirubin glucuronides at normal WT levels >2 yr after one injection of vector, and i.v. injection of bilirubins IIIα and XIIIα in the same animals revealed excess bilirubin-conjugating capacity. There was no significant elevation of liver enzymes (alanine aminotransferase) and only transient, moderate thrombocytopenia after injection of the vector. A clinically significant reduction in serum bilirubin was observed with a dose as low as 6× 1011viral particles per kg. We conclude that complete, long-term correction of hyperbilirubinemia in the Gunn rat model of Crigler-Najjar syndrome can be achieved with one injection of HD-Ad vector and negligible chronic toxicity.
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