Hepatic Hilar Lymph Node Reactivity at Kasai Portoenterostomy for Biliary Atresia: Correlations With Age, Outcome, and Histology of Proximal Biliary Remnant.

We hypothesized that if infection is the proximate cause of congenital biliary atresia, an appropriate response to antigen would occur in lymph nodes contiguous with the biliary remnant. We compared the number of follicular germinal centers (GC) in 79 surgically excised hilar lymph nodes (LN) and 27 incidentally discovered cystic duct LNs in 84 subjects at the time of hepatic portoenterostomy (HPE) for biliary atresia (BA) to autopsy controls from the pancreaticobiliary region of non-septic infants >3 months old at death. All 27 control LN lacked GC, a sign in infants of a primary response to antigenic stimulation. GC were found in 53% of 106 LN in 56 of 84 subjects. Visible surgically excised LN contiguous with the most proximal biliary remnants had 1 or more well-formed reactive GC in only 26/51 subjects. Presence of GC and number of GC/LN was unrelated to age at onset of jaundice or to active fibroplasia in the biliary remnant but was related to older age at HPE. Absent GC in visible and incidentally removed cystic duct LNs predicted survival with the native liver at 2 and 3 years after HPE, P = .03, but significance was lost at longer intervals. The uncommon inflammatory lesions occasionally found in remnants could be secondary either to bile-induced injury or secondary infection established as obstruction evolves. The absence of consistent evidence of antigenic stimulation in LN contiguous with the biliary remnant supports existence of at least 1 major alternative to infection in the etiology of biliary atresia.

De novo hepatocellular carcinoma post-multivisceral transplantation in a child.

De novo hepatocellular carcinoma (HCC) post-transplantation in patients without viral hepatitis is extremely rare, with only three reported adult cases in the English literature. Here, we present a case of de novo HCC that developed in a 7-year-old female, who at 8 months of age received a liver, small bowel, spleen, and pancreas transplantation 6.5 years ago for gastroschisis and total parenteral nutrition (TPN)-related cirrhosis. The post-transplant course was complicated by Epstein-Barr virus (EBV) infection, post-transplant lymphoproliferative disease, and subsequent development of multifocal EBV-associated post-transplant smooth muscle tumors (EBV-PTSMT) in the small bowel 1 year and 10 months after transplantation, respectively. This was managed by reducing immunosuppression with rituximab and EBV-specific cytotoxic T-cell therapy. She was noted to have a new lesion in her transplanted liver graft 6.5 years post-transplantation that was diagnosed as HCC. The HCC was resected, and the patient remained clinically stable for 7 months. At that time, recurrence of the HCC was discovered on MRI. She passed away 6 months after. To the best of our knowledge, this is the first reported occurrence of de novo HCC post-transplantation in the pediatric population that is unrelated to viral hepatitis in either recipient or donor.

Alterations in myeloid dendritic cell innate immune responses in the Galphai2-deficient mouse model of colitis.

BACKGROUND: The G protein alpha subunit type-2 (Galpha(i)2)-deficient mouse develops inflammatory bowel disease (IBD) with increased severity in mice on a 129SvEv (129) background compared to the C57BL/6 (B6) background. Since dendritic cells (DCs) are key cells of innate immunity, we determined whether Galpha(i)2(-/-) DCs have functional defects, influenced by strain background, that predispose to IBD. METHODS: By breeding these strains to homozygosity for the first time, it became possible to study innate immunity in this animal model with more precision than ever before. Immature DCs were generated using bone marrow monoblasts cultured in the presence of GM-CSF (BMDCs), DC subsets sorted and responses to TLR9 activation were assayed. RESULTS: In contrast to Galpha(i)2(-/-) B6, Galpha(i)2(-/-) 129 mice display accelerated onset and increased severity of colitis, abnormal mucosal DC distribution, accompanied by preponderance for Th1 and Th17-associated gut cytokine expression. TLR9 activation of BMDCs induces sustained p38 MAPK activation and greater Th1- and Th17-type cytokine secretion in both strains of Galpha(i)2-deficient compared to wildtype BMDCs. However, only B6 Galpha(i)2(-/-) BMDCs concomitantly produces IL-10 while Galpha(i)2(-/-) 129 BMDCs do not. CONCLUSIONS: Loss of Galpha(i)2 promotes a Th1/Th17 phenotype and relative IL-10 insufficiency in Galpha(i)2(-/-) 129 BMDCs may account for the striking difference in disease.

Giant hepatic adenoma with atypical features in a patient on oxcarbazepine therapy.

An association between oxcarbazepine therapy and hepatic adenoma (HA) has been documented in animal models but not observed in humans. The authors report a case of a 16-year-old girl on oxcarbazepine therapy for seizure disorder who presented with a giant HA. Pathology of the HA was notable for marked periductal fibrosis and glycoprotein inclusions in the nontumor liver. The patient was not on oral contraceptives and has no other known risk factors for HA.

Mutations in bile salt export pump (ABCB11) in two children with progressive familial intrahepatic cholestasis and cholangiocarcinoma.

Fatal peripheral cholangiocarcinoma developed in 2 girls with progressive familial intrahepatic cholestasis, ABCB11 mutations, and absent bile salt export pump (BSEP) expression. BSEP deficiency may cause cholangiocarcinoma through bile-composition shifts or bile-acid damage within cells capable of hepatocytic/cholangiocytic differentiation. This observation suggests the need for hepatobiliary-malignancy surveillance and early consideration for liver transplantation.

Sustained phenotypic correction in a mouse model of hypoalphalipoproteinemia with a helper-dependent adenovirus vector.

We examined the efficacy and host response to the adenovirus (Ad)-mediated delivery of human apolipoprotein A-I (APOA1) gene to the liver of APOA1(-/-) mice. Administration of a first-generation vector (FGAd-AI) resulted in a transient appearance of APOA1 in plasma and induced an anti-APOA1 antibody titer, whereas treatment with a helper-dependent vector (HDAd-AI) resulted in sustained APOA1 expression without inducing an antibody titer. With these results, we studied the effects of FGAd vectors on APOAI expression by HDAd-AI vector. Co-treatment with an FGAd vector inhibited HDAd-AI- mediated APOA1 expression independent of transgene cassettes, but only FGAd-AI induced a humoral response. Furthermore, APOA1 mRNA levels in mice co-treated with FGAd vectors were much lower than those expected from the vector copy number, suggesting that DNA of FGAd vectors interferes with the HDAd-AI vector's APOA1 promoter. A single treatment with an HDAd-AI vector produced a supraphysiological plasma APOA1 level that gradually declined to about half the normal human level over the course of 2 years, associated with a plasma cholesterol level that is persistently higher than that in controls. This investigation provides the proof of principle that liver-directed HDAd gene delivery is effective for the long-term phenotypic correction of monogenic hypoalphalipoproteinemia.

Modulation of TNFalpha, a determinant of acute toxicity associated with systemic delivery of first-generation and helper-dependent adenoviral vectors.

Understanding the determinants of the host innate immune response to systemic administration of adenoviral (Ad) vectors is critical for clinical gene therapy. Acute toxicity occurs within minutes to hours after vector administration and is characterized by activation of innate immune responses. Our data indicate that in mice, indicators of vector toxicity include elevations of cytokine levels, liver transaminase levels and thrombocytopenia. To discern potential targets for blunting this host response, we evaluated genetic factors in the host response to systemically administered first-generation Ad vectors (FGV) and helper-dependent Ad vectors (HDV) containing beta-galactosidase expression cassettes. A preliminary screen for modulation of vector-induced thrombocytopenia revealed no role for interferon-gamma, mast cells or perforin. However, vector-induced thrombocytopenia and interleukin 6 (IL-6) expression are less evident in tumor necrosis factor alpha (TNFalpha)-deficient mice. Moreover, we also demonstrated that TNFalpha blockade via antibody or huTNFR:Fc pretreatment attenuates both thrombocytopenia (>40% increase in platelet count) and IL-6 expression (>80% reduction) without affecting interleukin 12 , liver enzymes, hematological indices or vector transduction in a murine model. Our data indicate that the use of HDV, in combination with clinically approved TNFalpha immunomodulation, may represent an approach for improving the therapeutic index of Ad gene therapy for human clinical trials.

Feeding a corn oil/sucrose-enriched diet enhances steatohepatitis in sedentary rats.

The current study investigated the combined effects of feeding a high-fat/high-sucrose (HF/HS) diet to rodents rendered sedentary via hindlimb unloading (HU). For 3 wk before HU, male Wistar rats were fed chow or a diet in which 32% of calories were derived from corn oil fat and 48% of calories from sucrose. Feeding continued during an additional 3-wk period of HU. Subsequently, blood samples were collected for determination of circulating leukocyte counts, insulin levels, and portal vein endotoxin. Inflammation, necrosis, and steatosis were assessed in formalin-fixed liver sections. No biochemical or histological evidence of injury was observed in control rats fed chow or HF/HS. HU increased circulating neutrophils and resulted in hyperinsulinemia. Mild hepatic fat accumulation and minimal focal necroinflammation were observed in this group. Feeding HF/HS during HU exacerbated hyperinsulinemia, hepatic steatosis, Kupffer cell content, and cytokine expression. Significant portal endotoxemia was noted in HU rats but was not influenced by HF/HS diet. On the other hand, feeding HF/HS significantly enhanced lipid peroxidation end products in liver of HU rats by approximately threefold compared with chow-fed rats. In summary, these findings demonstrate that feeding a high-calorie diet potentiates steatosis and injury in sedentary HU rats. Mechanisms underlying enhanced injury most likely involved lipid peroxidation. Importantly, these findings suggest that dietary manipulation combined with physical inactivity can be used to model steatohepatitis.

Multiple mechanisms for oxygen-induced regulation of the Clara cell secretory protein gene.

The Clara cell secretory protein (CCSP) imparts a protective effect to the lung during oxidant injury. However, exposure to supplemental oxygen, a common therapeutic modality for lung disease, represses the expression of CCSP in the adult mouse lung. We investigated the mechanisms of hyperoxia-induced repression of the mouse CCSP promoter. Deletion experiments in vivo and in vitro indicated that the hyperoxia-responsive elements are localized to the proximal -166 bp of the CCSP promoter. Electrophoretic mobility shift and supershift analyses demonstrated increased binding of c-Jun at the activator protein-1 site, increased binding of CCAAT/enhancer binding protein (C/EBP) beta at the C/EBP sites, and decreased binding at the Nkx2.1 sites. Western analyses revealed that hyperoxia exposure induced an increase in the expression of the C/EBPbeta isoform liver-inhibiting protein (LIP) and an increase in cytoplasmic Nkx2.1. Cotransfection of LIP or c-Jun expression plasmids decreased the transcriptional activity of the proximal -166-bp CCSP promoter. These observations suggest that hyperoxia-induced repression of the CCSP gene is mediated, at least in part, at the level of transcription and that multiple mechanisms mediate this repression. Moreover, these novel observations may provide insights for generation of therapeutic interventions for the amelioration of oxidant-induced lung injury.

Long-term therapy with NTBC and tyrosine-restricted diet in a murine model of hereditary tyrosinemia type I.

In human patients with hereditary tyrosinemia type I (HT1) a combination therapy of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexane dione (NTBC) and dietary restriction of phenylalanine and tyrosine is currently widely used. We previously reported that the use of NTBC in a murine model of HT1 abolished acute liver failure but did not prevent the development of hepatocellular carcinoma (HCC) in the setting of nonrestricted protein intake. Here we present the results obtained with higher doses of NTBC plus dietary tyrosine restriction on long-term follow up (>2 years). Liver function tests and succinylacetone levels were completely corrected with this regimen and cancer-free survival was improved when compared to historical controls. However, while no HT1 animals had HCC at age 13 months, the incidence was 2/16 (13%) at age 18 months and 1/6 (17%) after 24 months. Thus, even the most stringent therapy could not prevent the emergence of HCC in the mouse model of HT1, even when initiated prenatally.

Small cell undifferentiated histology in hepatoblastoma may be unfavorable.

BACKGROUND: Chemotherapy has improved the prognosis of hepatoblastoma demonstrably. It renders seemingly unresectable primary tumors amenable to surgery and can cure metastases. Some authors advocate preoperative chemotherapy for patients with tumors that are deemed resectable, relying solely on percutaneous biopsy or even on diagnostic imaging and elevated serum alpha-fetoprotein levels for diagnosis. However, certain cytologic features of hepatoblastoma appear to have important prognostic consequences. Well differentiated fetal hepatoblastomas that are confined to the liver and that have minimal mitotic activity may not require additional therapy if they are resected totally. METHODS: In the current study 16 completely resected hepatoblastomas that exhibited partial or predominant small cell undifferentiated histology were identified retrospectively and correlated with patient outcome. RESULTS: Ten of 16 patients with completely resected tumors exhibiting small cell undifferentiated histology developed a disease recurrence. Five of these recurrences were fatal. CONCLUSIONS: Small cell undifferentiated histology may have an unfavorable effect on outcome in patients with completely resected hepatoblastoma. The focal distribution of small cell histology in the majority of these tumors suggests that treating hepatoblastoma based on limited preexcision biopsies may deprive some patients of appropriate therapy.

Requirement of Math1 for secretory cell lineage commitment in the mouse intestine.

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.

Neutrophil-rich anaplastic large cell lymphoma of the skull presenting after head trauma.

The presentation of anaplastic large cell lymphoma in bone is uncommon. We report a case of anaplastic large cell lymphoma of the skull that was diagnosed after head trauma. Biopsy revealed significant destruction of the outer table of the frontal bone. Histopathologically, the initial evaluation suggested osteomyelitis because of a mixed inflammatory infiltrate with large numbers of neutrophils. However, several clusters and individual mononuclear cells were atypical. The tumor cells had large, pleomorphic nuclei; these cells stained positively with antibodies to Ki-1 (CD 30), ALK-1, and EMA. Fluorescence in situ hybridization (FISH) showed rearrangement of the ALK gene, which usually results from the t(2;5) translocation, present in most anaplastic large cell lymphomas. There was no evidence of systemic disease. The patient has tolerated chemotherapy and is free of disease 12 months later.

Outcome of early hepatic portoenterostomy for biliary atresia.

BACKGROUND: The outcome of the hepatic portoenterostomy (Kasai) procedure for biliary atresia is improved when it is performed before 90 days of age. However, it is not known whether intervention before 30 days is better than intervention between 30 and 90 days. METHODS: The authors reviewed the records of all patients seen by the Pediatric Gastroenterology Service at St. Louis Children's Hospital from 1984-1999 to ascertain the outcome of patients who underwent Kasai procedure before or after 30 days of age. RESULTS: Of 92 patients with biliary atresia treated at St. Louis Children's Hospital over 15 years, 9 underwent the Kasai procedure before 30 days of age. Liver transplantation was necessary in 77.8% of these patients at a mean age of 11.0 +/- 4.26 months, as compared with 53.4% at 32.14 +/- 7.14 months for the remainder of the patients who underwent the procedure after 30 days of age. CONCLUSIONS: Although these data suggest that outcomes are worse for patients who undergo the procedure before 30 days of age, they may reflect a difference in the pathogenesis of biliary atresia that brings it to clinical attention earlier and may provide further evidence that biliary atresia is a phenotype for a number of distinct underlying disease processes.

Phenotypic consequences of lung-specific inducible expression of FGF-3.

Members of the fibroblast growth factor (FGF) family play a critical role in embryonic lung development and adult lung physiology. The in vivo investigation of the role FGFs play in the adult lung has been hampered because the constitutive pulmonary expression of these factors often has deleterious effects and frequently results in neonatal lethality. To circumvent these shortcomings, we expressed FGF-3 in the lungs under the control of the progesterone antagonist-responsive binary transgenic system. Four binary transgenic lines were obtained that showed ligand-dependent induction of FGF-3 with induced levels of FGF-3 expression dependent on the levels of expression of the GLp65 regulator as well as the dose of the progesterone antagonist, RU486, administered. FGF-3 expression in the adult mouse lung resulted in two phenotypes depending on the levels of induction of FGF-3. Low levels of FGF-3 expression resulted in massive free alveolar macrophage infiltration. High levels of FGF-3 expression resulted in diffuse alveolar type II cell hyperplasia. Both phenotypes were reversible after the withdrawal of RU486. This system will be a valuable means of investigating the diverse roles of FGFs in the adult lung.

Hepatitis B virus X protein acts as a tumor promoter in development of diethylnitrosamine-induced preneoplastic lesions.

Chronic infection with hepatitis B virus (HBV) is one of the major etiological factors in the development of human hepatocellular carcinoma. Transgenic mice that express the HBV X protein (HBx) have previously been shown to be more sensitive to the effects of hepatocarcinogens. Although the mechanism for this cofactor role remains unknown, the ability of HBx to inhibit DNA repair and to influence cell cycle progression suggests two possible pathways. To investigate these possibilities in vivo, we treated double-transgenic mice that both express HBx (ATX mice) and possess a bacteriophage lambda transgene with the hepatocarcinogen diethylnitrosamine (DEN). Histological examination of liver tissue confirmed that DEN-treated ATX mice developed approximately twice as many focal lesions of basophilic hepatocytes as treated wild-type littermates. Treatment of mice with DEN resulted in a six- to eightfold increase in the mutation frequency (MF), as measured by a functional analysis of the lambda transgene. HBx expression was confirmed by immunoprecipitation and Western blotting and was associated with a modest 23% increase in the MF. Importantly, the extent of hepatocellular proliferation in 14-day-old mice, as measured by the detection of proliferating cell nuclear antigen and by the incorporation of 5-bromo-2'-deoxyuridine, was determined to be approximately twofold higher in ATX livers than in wild-type livers. These results are consistent with a model in which HBx expression contributes to the development of DEN-mediated carcinogenesis by promoting the proliferation of altered hepatocytes rather than by directly interfering with the repair of DNA lesions.

Long-term stable correction of low-density lipoprotein receptor-deficient mice with a helper-dependent adenoviral vector expressing the very low-density lipoprotein receptor.

BACKGROUND: Familial hypercholesterolemia (FH) that results from LDL receptor (LDLR) deficiency affects approximately 1 in 500 persons in the heterozygous state and approximately 1 in 1 million persons in the homozygous state. We tested a novel gene therapy strategy for the treatment of FH in a mouse model. METHODS AND RESULTS: We delivered the VLDL receptor (VLDLR) to the liver of LDLR-deficient mice and compared the effect of a helper-dependent adenoviral vector with all viral coding sequences deleted (HD-Ad-mVLDLR) with a first-generation vector (FG-Ad-mVLDLR), an HD-Ad (HD-Ad-0) that contained no expression cassette, and dialysis buffer (DB). A single intravenous injection of HD-Ad-mVLDLR led to a lowering of plasma cholesterol that lasted >/=6 months. Acute liver toxicity (as measured with liver enzyme elevation) occurred after FG-Ad-mVLDLR but not after HD-Ad-mVLDLR, HD-Ad-0, or DB treatment. At 6 months, VLDLR was detected in the liver with Western blotting and with immunofluorescence staining only in HD-Ad-mVLDLR-treated mice. Aortic atherosclerosis was almost completely prevented in these animals. CONCLUSIONS: HD-Ad-mediated intravenous delivery of VLDLR to hepatocytes is well tolerated. It produces long-term lowering of plasma cholesterol and prevents atherosclerosis development in LDLR-deficient mice. These data provide support for the feasibility and safety of this approach for therapy of human subjects.

Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells.

The emergence of cells with hepatocellular properties in the adult pancreas has been described in several experimental models. To determine whether adult pancreas contains cells that can give rise to therapeutically useful and biochemically normal hepatocytes, we transplanted suspensions of wild-type mouse pancreatic cells into syngeneic recipients deficient in fumarylacetoacetate hydrolase and manifesting tyrosinemia. Four of 34 (12%) mutant mice analyzed were fully rescued by donor-derived cells and had normal liver function. Ten additional mice (29%) showed histological evidence of donor-derived hepatocytes in the liver. Previous work has suggested that pancreatic liver precursors reside within or close to pancreatic ducts. We therefore performed additional transplantations using either primary cell suspensions enriched for ducts or cultured ducts. Forty-four mutant mice were transplanted with cells enriched for pancreatic duct cells, but only three of the 34 (9%) recipients analyzed displayed donor-derived hepatocytes. In addition, 28 of the fumarylacetoacetate hydrolase-deficient mice were transplanted with cultured pancreatic duct cells, but no donor-derived hepatocytes were observed. Our results demonstrate for the first time that adult mouse pancreas contains hepatocyte progenitor cells capable of significant therapeutic liver reconstitution. However, contrary to previous reports, we were unable to detect these cells within the duct compartment.

Purified hematopoietic stem cells can differentiate into hepatocytes in vivo.

The characterization of hepatic progenitor cells is of great scientific and clinical interest. Here we report that intravenous injection of adult bone marrow cells in the FAH(-/-) mouse, an animal model of tyrosinemia type I, rescued the mouse and restored the biochemical function of its liver. Moreover, within bone marrow, only rigorously purified hematopoietic stem cells gave rise to donor-derived hematopoietic and hepatic regeneration. This result seems to contradict the conventional assumptions of the germ layer origins of tissues such as the liver, and raises the question of whether the cells of the hematopoietic stem cell phenotype are pluripotent hematopoietic cells that retain the ability to transdifferentiate, or whether they are more primitive multipotent cells.

Randomized comparison of cisplatin/vincristine/fluorouracil and cisplatin/continuous infusion doxorubicin for treatment of pediatric hepatoblastoma: A report from the Children's Cancer Group and the Pediatric Oncology Group.

PURPOSE: Previous studies demonstrated that chemotherapy with either cisplatin, vincristine, and fluorouracil (regimen A) or cisplatin and continuous infusion doxorubicin (regimen B) improved survival in children with hepatoblastoma. The current trial is a randomized comparison of these two regimens. PATIENTS AND METHODS: Patients (N = 182) were enrolled onto study between August 1989 and December 1992. After initial surgery, patients with stage I-unfavorable histology (UH; n = 43), stage II (n = 7), stage III (n = 83), and stage IV (n = 40) hepatoblastoma were randomized to receive regimen A (n = 92) or regimen B (n = 81). Patients with stage I-favorable histology (FH; n = 9) were treated with four cycles of doxorubicin alone. RESULTS: There were no events among patients with stage I-FH disease. Five-year event-free survival (EFS) estimates were 57% (SD = 5%) and 69% (SD = 5%) for patients on regimens A and B, respectively (P =.09) with a relative risk of 1.54 (95% confidence interval, 0.93 to 2.5) for regimen A versus B. Toxicities were more frequent on regimen B. Patients with stage I-UH, stage II, stage III, or stage IV disease had 5-year EFS estimates of 91% (SD = 4%), 100%, 64% (SD = 5%), and 25% (SD = 7%), respectively. Outcome was similar for either regimen within disease stages. At postinduction surgery I, patients with stage III or IV disease who were found to be tumor-free had no events; those who had complete resections achieved a 5-year EFS of 83% (SD = 6%); other patients with stage III or IV disease had worse outcome. CONCLUSION: Treatment outcome was not significantly different between regimen A and regimen B. Excellent outcome was achieved for patients with stage I-UH and stage II hepatoblastoma and for subsets of patients with stage III disease. New treatment strategies are needed for the majority of patients with advanced-stage hepatoblastoma.