Search

La ictericia neonatal requiere el diagnóstico diferencial entre la hiperbilirrubinemia no conjugada o indirecta y la hiperbilirrubinemia directa o conjugada, característica de la colestasis neonatal, que siempre es patológica. Se recomienda en todo recién nacido de 15 días o más de vida con ictericia realizar bilirrubina diferencial para poder detectarla. La colestasis se define por bilirrubina directa > 1 mg/dl (17 µmol/L) y su frecuencia estimada durante el período neonatal es de 1:2.500 recién nacidos vivos. Las etiologías más frecuentes son: infecciosas, genéticas/metabólicas, obstructivas, endocrinológicas, tóxicas, inmunológicas. La causa más común es la atresia biliar, que se manifiesta clínicamente por ictericia y acolia, por lo que frente a estos signos siempre debe plantearse este diagnóstico como primera opción a descartar.Es fundamental que el diagnóstico de colestasis neonatal sea precoz para optimizar el manejo en las causas tratables y mejorar su pronóstico. Desafortunadamente, no siempre el diagnóstico es precoz. Es por esto que el Grupo de Trabajo de la Sociedad Latinoamericana de Gastroenterología, Hepatología y Nutrición Pediátrica realizó esta revisión de diagnóstico, tratamiento y seguimiento de la colestasis neonatal, cuyo objetivo es proporcionar una herramienta útil que permita el diagnóstico temprano y mejorar el pronóstico de los niños con colestasis neonatal.

Iceland has witnessed a dramatic decline in adolescent substance use that may be partly the result of efforts related to the Icelandic prevention model (IPM). We sought to test risk and protective factor assumptions of the IPM using a prospective cohort study with 12 months separating baseline from follow-up. Participants were students in grades 8 and 9 in the national Icelandic school system enrolled in the spring of 2018 and 2019 (N=2165). Participants self-reported their experiences of cigarette smoking, alcohol consumption, and cannabis use and seven risk and protective factors. Analyses were conducted with generalized linear modeling with extension to general estimating equations with correlated outcomes data. Both individual main-effects models and collective models including all main-effects were tested. Out of 28 individual main-effects models, 23 produced findings consistent with study premises (P

In two decades, the Icelandic prevention model (IPM) has been employed to dramatically reduce rates of adolescent substance use in Iceland. Briefly, the IPM is a multisectoral, community-based, collaborative system where researchers, policy makers, administrative leaders, and practitioners join forces to reduce the odds of adolescent substance use over time. Comparatively, Iceland now ranks among the lowest in adolescent substance use in all of Europe. Since 2005, the IPM has garnered considerable international attention, and several countries or municipalities within them have adapted, or are presently adapting, the model to their needs. In this commentary, we first briefly review the history and formation of the IPM in Iceland from a school-based survey to a fully integrated prevention system. In the second part, we present a short overview of the national consensus building and institutional collaboration that led to the implementation of the model in Chile in Latin America, as a demonstrative example. In this volume of Health Promotion Practice, we also present a series of two practice-based articles that introduce the IPM. The first article, titled “Development and Guiding Principles of the Icelandic Model for Preventing Adolescent Substance Use,” introduces the theoretical origins of the model, five guiding principles, and evidence of effectiveness to date. In the second article, titled “Implementing the Icelandic Model for Preventing Adolescent Substance Use,” we outline 10 practice-based steps to guide model implementation in other countries. Both articles are available via open access, and both are also available online in Spanish.

Hepatocyte transplantation is an investigational alternative to orthotopic liver transplantation to treat liver based inborn errors of metabolism. We report successful hepatocyte transplantation in a 4-year-old girl with infantile Refsum disease. Hepatocytes were isolated from the left liver segment of two male donors using a classic two-step perfusion method. Fresh cells were transplanted first and then cryopreserved cells, for a total of 2 billion cells. Total bile acids and abnormal dihydroxycoprostanoic acid markedly decreased in the patient's serum, indicating resolution of cholestasis and re-population of liver cells. Pipecholic acid decreased by 40% and c26:c22 fatty acid ratio by 36% after 18 months. Donor chromosomes sequences were detected on biopsy posttransplant, indicating engraftment. Hepatocyte transplantation is a safe and promising technique in the treatment of rare inborn errors of metabolism. Future improvements of cell viability and prevention of apoptosis may increase engraftment and subsequent re-population.

Cell therapy, in particular liver cell transplantation, holds great therapeutic potential and is partially hindered by the high rate of apoptosis during cell isolation, cryopreservation, and engraftment. Apoptosis occurring due to cell detachment from the extracellular matrix is a phenomenon termed “anoikis. ” The purpose of this review is to describe signaling mechanisms pertinent to anoikis in both immortalized cell lines, but particularly in primary normal epithelial cells. The mechanisms described include integrin signaling and survival molecules, caspase activation, and the role of mitochondrial proteins in anoikis. Strategies to prevent anoikis during isolation and cryopreservation of hepatocytes are discussed.

Background Efficiency of engraftment after liver cell transplantation is less than 1% under conventional conditions. Our aim was to develop a high-efficiency, nonsurgical, no-genetic-advantage mouse model of liver repopulation with transplanted cells. Methods Mice were conditioned with nonlethal doses of a cell cycle inhibitor, retrorsine, 70 mg/kg, to irreversibly block proliferation of native hepatocytes. After the drug was eliminated, 2 million freshly isolated beta-galactosidase-labeled liver cells were transplanted into the spleens of C57BL/6J recipient mice. To stimulate donor cell proliferation, three doses of carbon tetrachloride (CCl4), 0.5 ml/kg, were given. Several control groups were studied to evaluate the contribution of each treatment to liver repopulation. Results Repopulation, as measured by cell isolation from recipient livers 1-7 months after transplantation, was on average 20%. Repopulation was 10% if CCl4 was given only once, between 0.5% and 1% if only retrorsine or CCl4 were used, and 0.05% if no conditioning was used. Phenotypically, whole livers turned blue on exposure to X-gal staining, whereas negative (control) livers remained pale brown. More than 55% of liver repopulation resulted from clusters containing 21 or more cells, some of which contained more than 200 cells, suggesting seven or more rounds of cell division in a subset of transplanted cells. Conclusion This murine study demonstrates high levels of repopulation after liver cell transplantation into nongenetically modified livers, using a cell cycle inhibitor and chemical liver injury to provide transplanted cells a proliferative advantage. Liver repopulation was effected mostly by a small fraction of transplanted cells. Analogous nonsurgical liver cell transplantation strategies, but with clinically applicable drugs, could be devised for the treatment of liver-based metabolic diseases.

Isolation and cryopreservation of freshly isolated hepatocytes is considered a standard procedure for the long-term storage of liver cells. However, most existing methods for banking hepatocytes do not allow sufficient recovery of viable cells to meet the needs of basic research or clinical trials of hepatocyte transplantation. The mechanisms underlying this poor rate of hepatocyte recovery are unknown. Although much of the cellular damage in freezing is caused by formation of ice crystals within the cells, this is largely prevented by the use of dimethyl sulfoxide (DMSO) and controlled rate freezing. As we demonstrated recently, necrosis does occur in primary hepatocytes following isolation and cryopreservation. In the present study, we explored the contribution of apoptosis, another form of cell death, in primary hepatocytes banked for transplantation. We evaluated apoptosis of C57BL/6J mouse primary hepatocytes using several different methods. Annexin binding and the TUNEL assay, in conjunction with flow cytometry and confocal laser scanning microscopy, revealed that the percentage of apoptotic cells was dramatically elevated in cryopreserved cells compared with that in the control group of unfrozen cells. DNA laddering detected by DNA electrophoresis in agarose gel also supported the presence of apoptosis in isolated and banked liver cells. Moreover, we found that the addition of glucose (from 10 to 20 mM) into the freezing solution (University of Wisconsin Solution) decreased the rate of apoptosis by 84% and improved the cell attachment at least fourfold in cryopreserved cells. These results suggest that apoptosis might contribute to cell death in isolated and banked primary hepatocytes.

We used microgravity-simulated bioreactors that create the unique environment of low shear force and high-mass transfer to establish long-term cultures of primary human liver cells (HLC). To assess the feasibility of establishing HLC cultures, human liver cells obtained either from cells dissociated by collagenase perfusion or minced tissues were cultured in rotating vessels. Formation of multidimensional tissue-like spheroids (up to 1.0 cm) comprised of hepatocytes and biliary epithelial cells that arranged as bile duct-like structures along newly formed vascular sprouts were observed. Electron microscopy revealed clusters of round hepatocytes and bile canaliculi with multiple microvilli and tight junctions. Scanning EM revealed rounded hepatocytes that were organized in tight clusters surrounded by a complex mesh of extracellular matrix. Also, we observed that co-culture of hepatocytes with endothelial cells stimulate albumin mRNA expression. In summary, a simulated microgravity environment is conducive for the establishment of long-term HLC cultures and allows the dissection of the mechanism of liver regeneration and cell-to-cell interactions that resembles in vivo conditions.

The CCAAT/enhancer binding protein alpha (C/EBP alpha) binds to specific promoter sequences and directs transcription of many genes expressed in the liver. Overexpression of C/EBP alpha in established cell lines inhibits cell proliferation. Primary hepatocytes from newborn C/EBP alpha(-/-) mice and normal littermates were used to determine whether the absence of C/EBP alpha increased proliferation and/or transformation of these cells in vitro. DNA synthesis, as measured by bromodeoxyuridine (BrdU) incorporation 24 hours postharvest, was fourfold higher in cells from C/EBP alpha(-/-) pups. Established cell lines were derived from 7 of 8 hepatocyte cultures initiated from null mutants, 4 of 23 cultures from heterozygotes, and 0 of 12 cultures from wild-type animals. C/EBP alpha(-/-) cultures had epithelial morphology, showed bile canaliculi, and expressed albumin messenger RNA (mRNA). When cultured on Matrigel, which promotes differentiation, cell lines derived from C/EBP alpha(-/-) mice formed cords and increased albumin mRNA expression by 1.7- to 3.8-fold. C/EBP alpha(-/-) cell lines exhibited rapid growth and rapid accumulation of chromosomal abnormalities, and were capable of forming nodules when inoculated into the abdominal subcutaneous tissue of nude mice. Our data show that C/EBP alpha is an important regulator of hepatocyte proliferation and participates in the maintenance of the nontransformed hepatic phenotype in vitro.

Strategies for hepatic gene therapy have been proposed that involve isolation of primary hepatocytes and introduction of recombinant genes into these cells in culture, followed by autologous hepatocellular transplantation (HCT). Consideration of clinical applications requires data suggesting that HCT can be performed safely in human subjects in addition to data indicating that recombinant gene expression can reverse a disease process. This report describes preclinical studies that underlie a clinical trial of HCT in which hepatocytes would be labeled with a marker gene to facilitate assessment of engraftment in the recipient. Human hepatocytes were harvested from liver segments preserved in Belzar's solution and transduced with an amphotropic retroviral vector carrying a recombinant marker gene (neomycin phosphotransferase II). Human hepatocytes were recovered from monolayer culture, stained with the fluorescent dye 1,1'-dioctadecyl-3,3,3,3'-tetra-methylindo-carbocyanine perchlorate (DiI) and transplanted into severe combined immunodeficient mice by splenic injection. Engrafted hepatocytes were identified in the liver and spleen of severe combined immunodeficient mice but not immunocompetent controls. Two large animal models of HCT are described. In a dog model, neomycin phosphotransferase II-containing hepatocytes were identified in the liver 7 wk after transplantation. In a baboon model, autologous HCT with DiI-stained cells demonstrated that transplanted cells assume a normal morphology and constitute up to 5% of hepatocytes. These data demonstrate transduction and transplantation of human hepatocytes and the feasibility of HCT in large animals. On the basis of these studies, the proposed clinical trial for gene transfer and transplantation in human subjects has been approved by the National Institutes of Health and the Food and Drug Administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Apoptosis occurs during the isolation and even short-term storage and culture of hepatocytes, and in the pathogenesis of liver diseases, such as hepatic failure and hepatitis. Therapeutic hypothermia has beneficial effects in experimental models of fulminant hepatic failure. The mechanisms underlying the potential benefits of mild hypothermia on the liver have not been well investigated. We examined the effects of temperature on soluble Fas ligand-induced apoptosis in freshly isolated mouse hepatocytes. Decreasing the culture temperature from 37 degrees C to 32 degrees C produced significant suppression of Fas-mediated apoptosis in cultured hepatocytes over a 12-h period. This observation was supported by cell morphology, flow cytometry analysis of cellular DNA content, and Annexin V-FITC staining of membrane phosphatidylserine translocation. In hypothermic conditions, Fas-mediated cytochrome c release from mitochondria of hepatocytes and the proximate downstream activation of caspase-9 were suppressed under mild hypothermic conditions. Effector caspase-7 activity was also inhibited at 32 degrees C. In contrast, the activation of initiator caspase-8 and cleavage of Bid were not affected after Fas-ligand stimulation. These findings suggest that mild hypothermia suppresses Fas-mediated apoptosis of liver cells by the partial inhibition of signaling events including mitochondrial damage, cytochrome c release, and subsequent apoptosome formation and effector caspase activation.

Hepatocyte transplantation is an investigational alternative to orthotopic liver transplantation to treat liver based inborn errors of metabolism. We report successful hepatocyte transplantation in a 4-year-old girl with infantile Refsum disease. Hepatocytes were isolated from the left liver segment of two male donors using a classic two-step perfusion method. Fresh cells were transplanted first and then cryopreserved cells, for a total of 2 billion cells. Total bile acids and abnormal dihydroxycoprostanoïc acid markedly decreased in the patient's serum, indicating resolution of cholestasis and re-population of liver cells. Pipecholic acid decreased by 40% and c26:c22 fatty acid ratio by 36% after 18 months. Donor chromosomes sequences were detected on biopsy posttransplant, indicating engraftment. Hepatocyte transplantation is a safe and promising technique in the treatment of rare inborn errors of metabolism. Future improvements of cell viability and prevention of apoptosis may increase engraftment and subsequent re-population.

Transplanted hepatocytes can engraft, proliferate, and function permanently in host animals. After one cell infusion, however, engrafted hepatocytes constitute only between 1 in 200 to 1 in 3,000 host liver cells. Although transplanted cells can be identified using biochemical and molecular techniques, more accurate methods are needed to evaluate interventions that could improve cell engraftment rates. Real-time polymerase chain reaction (PCR) was done using primers and probes complementary to human testis determining gene (SRY) and mouse testis-specific Y-encoded protein (TSPY) pseudogene. Tissue samples from human or mouse recipients of liver cell transplantation were used to determine the test ability to detect transplanted cell DNA. Real-time PCR for the human SRY and mouse TSPY were species- and sex-specific. These two tests were sensitive in the detection of male DNA. Test sensitivity was consistently found at minimum 1:10,000 of male and female DNA mixing curve in both human SRY and mouse TSPY assays. The optimal amount of sample DNA per reaction to produce the highest sensitivity was 300 ng to 1 microg. Real-time PCR gave similar results whether standard male-female mixtures were prepared from liver cells or mononuclear cells. Engraftment of male liver cells in female liver tissues in mice and humans ranging from 0.125% to 0.257% was successfully measured using this method. Real-time PCR for SRY and TSPY affords a specific, sensitive, and reproducible tool for chimerism analysis in transplanted human and mouse liver tissues. This method could be used to optimize current models of cell transplantation.

Liver cell isolation and transplantation have been successfully performed in animal models and in humans. However, lack of initial engraftment due to cell death is a major roadblock to achieving clinical significance. Apoptosis was recently identified as an important cause of freshly isolated and banked hepatocyte cell death [Cell Transplant. 10 (2001) 59]. Pathways involving detachment-induced apoptosis (anoikis) are well characterized in other cell types. Loss of cell anchorage occurs during the hepatocyte isolation procedure prior to cell transplantation, but little is known about the role of this pathway in the survival of isolated hepatocytes. We report early occurrence of anoikis in primary mouse hepatocytes cultured under detached conditions on glass plates as compared to under attached conditions on plastic plates. Apoptosis in detached cells was determined using complementary techniques (DNA laddering, cell death ELISA assay, TUNEL assay and morphological analysis) and was detected as early as 15 min after culture under detached conditions. Further analysis of the mechanisms inducing apoptosis during liver cell isolation and transplantation and of ways to prevent them could lead to improved clinical protocols of liver cell therapies.

Children with cholestatic liver disease have been thought to develop hepatic osteodystrophy resulting from vitamin D and calcium malabsorption, resulting in secondary hyperparathyroidism and osteomalacia or rickets. However, treatment with vitamin D has not always proven successful in improving the bone disturbance. The aim of our study was to determine the role of vitamin D deficiency in the pathogenesis of hepatic osteodystrophy. We studied five patients, three female and two male, ages 0.9-19 yr, with biopsy-proven chronic cholestatic liver disease and previously low serum levels of vitamin D despite oral intake of vitamin D preparations. Patients were admitted to the Clinical Research Center for 8 days for sunlight deprivation and ultraviolet light substitution and for determinations of serum 25-hyroxyvitamin D(25(OH)) D2 and -D3, osteocalcin, and type I collagen telopeptide (ICTP), the last two being markers of bone formation and resorption, respectively. Samples were taken on admission, at discharge, and 1 month later. Results demonstrated low serum levels of osteocalcin and normal circulating levels of ICTP. Admission serum 25(OH)D2 levels were uniformly low or undetectable and remained so. Admission levels of circulating 25(OH)D3 were normal or low and did not rise during ultraviolet light therapy or subsequent resumption of oral vitamin D therapy and remained low 1 month later. These results indicate that in the face of low-normal to low total 25(OH)D levels, the low osteocalcin and normal ICTP levels suggest that decreased bone formation and not increased bone resorption is the main determinant of bone loss in a subset of children with chronic cholestatic liver disease.

The establishment of long-term cultures of functional primary human liver cells (PHLC) is formidable. Developed at NASA, the Rotary Cell Culture System (RCCS) allows the creation of the unique microgravity environment of low shear force, high-mass transfer, and 3-dimensional cell culture of dissimilar cell types. The aim of our study was to establish long-term hepatocyte cultures in simulated microgravity. PHLC were harvested from human livers by collagenase perfusion and were cultured in RCCS. PHLC aggregates were readily formed and increased up to 1 cm long. The expansion of PHLC in bioreactors was further evaluated with microcarriers and biodegradable scaffolds. While microcarriers were not conducive to formation of spheroids, PHLC cultured with biodegradable scaffolds formed aggregates up to 3 cm long. Analyses of PHLC spheroids revealed tissue-like structures composed of hepatocytes, biliary epithelial cells, and/or progenitor liver cells that were arranged as bile duct-like structures along nascent vascular sprouts. Electron microscopy revealed groups of cohesive hepatocytes surrounded by complex stromal structures and reticulin fibers, bile canaliculi with multiple microvilli, and tight cellular junctions. Albumin mRNA was expressed throughout the 60-d culture. A simulated microgravity environment is conducive to maintaining long-term cultures of functional hepatocytes. This model system will assist in developing improved protocols for autologous hepatocyte transplantation, gene therapy, and liver assist devices, and facilitate studies of liver regeneration and cell-to-cell interactions that occur in vivo.

Mouse lines carrying specific mutations in their genomes can be obtained using targeted recombination in murine embryonic stem (ES) cells. This is a powerful method for studying the significance of gene products both in vivo, in the whole animal, and in vitro, at the cellular level using cultures derived from the knockout mice. In addition, ES cells can be made that are homozygous for a particular mutation. Because ES cells can differentiate into various cell types in vitro, a variety of developmental and differentiated genes can be examined in mutant ES cell lines. Both loss-of-function (ablation of gene function) and gain-of-function (acquisition of a new activity conferred by mutation) are possible using this technique of gene targeting by homologous recombination in ES cells.

The leucine zipper transcription factors C/EBP alpha and C/EBP beta exhibit growth-related variations of expression and DNA binding during liver regeneration. We examined the expression of C/EBP proteins in relation to hepatocyte proliferation by studying their DNA-binding activity in primary mouse hepatocytes in vitro. Mouse hepatocytes were dissociated by collagenase perfusion and cultured in a serum-free, defined medium containing a variety of growth factors and hormones. Cell protein extracts were collected every 24 h for up to 10 d and examined for DNA-binding activity by gel retardation analysis using a C/EBP consensus sequence oligomer (bZIP). C/EBP alpha is the major bZIP-binding protein present in the dissociated cells prior to plating. With the culture conditions we employed, little or no binding of C/EBP proteins was observed in the first 24 to 48 h of cultivation. After 48 h, C/EBP beta binding activity was elevated relative to the level seen in freshly dissociated cells. In contrast, C/EBP alpha binding continued to be greatly reduced and no C/EBP delta binding was observed. C/EBP beta binding remained elevated for the duration of the experiment. Additional growth factor treatment (EGF, FGF, TGF alpha, and HGF) of the hepatocytes did not appreciably alter the pattern of C/EBP binding. However, TGF beta treatment, known to decrease hepatocyte proliferation, increased C/EBP beta binding activity earlier and more actively than in control cells. This study confirms a negative correlation between DNA binding by the C/EBP transactivator proteins and the proliferation of primary mouse hepatocytes in vitro.

The epithelial cells of the gastrointestinal tract may be attractive targets for somatic gene therapy. In these studies, we have used rats and mice to explore the feasibility of gene transfer into the small intestinal epithelium using retroviral vectors. The first series of experiments was conducted in mature Sprague-Dawley rats using an ecotropic retroviral vector that has bacterial beta-galactosidase (beta-Gal) as the reporter gene. The vector was introduced into the lumen of ligated segments of terminal ileum. After a 4-hr exposure period, the ligatures were removed. Sham-operated animals were subjected to the same ligation procedure but received only tissue culture medium in the ligated segment. All animals were sacrificed 6 days later, and tissue from both the experimental segment and an upstream control segment was assessed for cytoplasmic beta-Gal activity using X-Gal histochemistry. Expression of the reporter gene was observed in the crypt epithelium of tissue exposed to the vector. In the villus epithelium, high background staining precluded accurate assessment of reporter gene expression. To obviate the latter problem, we sought an alternative reporter gene for which there would be no background staining in control animals. We repeated the experiments with beta-glucuronidase as the reporter gene in MPS VII mutant mice, which are devoid of this enzyme. In these studies, ileal segments exposed to the vector demonstrated expression of the reporter gene in both the crypt and villus epithelium 4 days after exposure. These results indicate that genes can be transferred into the intestinal epithelium using retroviral vectors introduced luminally.(ABSTRACT TRUNCATED AT 250 WORDS)

Hepatocellular transplantation has previously been performed in experimental animals by infusion of hepatocyte suspensions into the spleen or portal venous system. Cells injected into these sites flow to the liver and engraft within the hepatic parenchyma. We designed this study to evaluate the feasibility of hepatocellular transplantation through the umbilical vein in the prenatal or perinatal periods. Allogeneic sheep hepatocytes were harvested, stained with the vital fluorescent dye DiI, and injected into the umbilical vein of fetal lambs at 85% gestation and term. Hemodynamic studies performed to assess the physiological impact of transplantation on the recipient animal demonstrated that the procedure was well tolerated. No significant short-term complications were encountered and no lesions were found by conventional histological examination at necropsy 1-17 days after transplantation. Engrafted cells were identified within the liver by fluorescent microscopy and flow cytometry in 4/7 animals constituting 1.2-5% of the hepatocyte population. Fluorescent cellular material with the morphology of hepatocytes, noncellular material, and fluorescent phagocytic cells were seen occasionally in other organs including lung, brain, adrenal, and placenta. These studies demonstrate the feasibility of performing hepatocellular transplantation in the fetus via the umbilical vein in experimental animals.

A novel method is described for marking primary hepatocytes with the fluorescent dye DiI prior to hepatocellular transplantation and identifying these cells within the hepatic parenchyma of recipient animals by fluorescence microscopy and flow cytometry. Optimal conditions are described for marking cells with DiI in suspension or in monolayer cultures prior to transplantation. DiI is shown to be nontoxic to hepatocytes and not to be exchanged between adjacent cells in vitro. Histological analysis of transplanted tissues shows DiI staining of engrafted hepatocytes and phagocytotic cells (Kupffer cells). This analysis shows that hepatocytes engraft within the hepatic parenchyma and exhibit a histological appearance indistinguishable from normal by conventional hematoxylin and eosin staining. Many previous reports of hepatocellular transplantation have been limited by their inability to unequivocally identify transplanted cells within the liver. These data illustrate the importance of having specific markers for transplanted cells that engraft in an orthotopic location and assume a normal morphological appearance.

Experiments in animal models suggest that it is feasible to consider hepatic gene therapy using a strategy in which hepatocytes would be isolated by partial hepatectomy, transduced with recombinant retroviral vectors containing genes of therapeutic importance, and then transplanted back into the patient by autologous hepatocellular transplantation. The application of this strategy in clinical trials will require adapting these methods to human cells. We describe the transduction of primary human hepatocytes with two forms of retroviral vectors: amphotropic vectors, which have been used previously in clinical trials, and xenotropic vectors, which have a different host range. Human hepatocytes were harvested from organs preserved in Belzer's solution and were cultivated in a serum-free, tyrosine-free, hormonally defined medium. These cells proliferated for 3-5 days in culture, exhibited characteristic hepatocyte morphology, and expressed liver-specific functions, including phenylalanine hydroxylase, alpha 1-antitrypsin, and glutamine synthase. Transduction with an amphotropic LNL6 retroviral vector resulted in stable incorporation of the provirus into 1% of the cells as estimated by semiquantitative PCR. Consistently higher transduction efficiencies (as much as 10% of the cells) were observed with a xenotropic N2 vector. These data support the feasibility of using LNL6 as a marker gene in clinical trials of hepatocellular transplantation. These data also suggest that the efficiency of transducing hepatocytes with amphotropic vectors in animal models may not accurately reflect the utility of these vectors for human applications. Consideration should be given to the use of xenotropic vectors for optimizing the efficiency of transduction for human applications.

Results of a pilot study suggest that bismuth subsalicylate (BSS) favorable altered the course of rotavirus-associated diarrhea in children. This was more evident in those who also had a bacterial pathogen. Subsequently 123 infants and children with acute diarrhea were admitted to a randomized parallel double-blind placebo- controlled clinical trial. Patients received either BSS at a dosage of 100 mg/(kg x d) for 5 days or a placebo. Patients in the 2 groups were comparable with respect to age sex weight height and baseline disease parameters. Compared with subjects treated with placebo those who received BSS had significantly lower stool weight sooner improved stool consistency sooner shorter hospital stay fewer number of stools decreased need for intravenous fluids and better evolution of clinical condition. The maximum mean serum level of salicylate occurred on day 3 and the mean blood level of bismuth on the last day of dosing (day 5) was 5.8 ppb. (authors)

HEPATOCELLULAR TRANSPLANTATION (HCT) VIA PORTAL VEIN CATHETER IN A PATIENT WITH FULMINANT LIVER FAILURE. † 746

The gastrointestinal manifestations of collagen vascular diseases, such as systemic lupus erythematosus and polymyositis, have been well described in the pediatric population. These patients frequently have symptoms which are indications for endoscopic studies. However, the risks and benefits of gastrointestinal endoscopy in this population have not been examined. A complete review for all rheumatologic patients admitted to Texas Children's Hospital during the past five years was performed and those patients undergoing endoscopy were identified. Ten patients (5%) underwent endoscopic procedures (nine upper and three lower endoscopies) which were analyzed with emphasis on resultant complications and therapeutic benefit. Indications for endoscopy included pain in six of ten patients, bleeding in three of ten patients and vomiting and diarrhea in one patient. Three patients had severe complications, two of which required surgery within one day of the endoscopic procedure. One of these patients subsequently died with GI bleeding. Five of the ten patients had changes in management following their endoscopy. Two patients had Helicobacter pylori and treatment was initiated. Three patients had esophagitis or gastritis and acid suppression treatment was started or optimized. Vasculopathy was present in the patients who had complications and may have contributed to their development. This study provides evidence that endoscopy can provide useful information for the management of the pediatric patient with GI symptoms and rheumatologic disease. However, since serious and potentially life-threatening complications occur, great care is needed in evaluating risk/benefit for endoscopic procedures in these high risk patients.