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Biomedical subjects

H E Soriano

Publications and source records attributed to H E Soriano.

At least 19 recordsLinked to original sources
PubMed1999

Induction of three-dimensional assembly of human liver cells by simulated microgravity.

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.

Albumins↗
PubMed1999

Risks of endoscopy in hospitalized pediatric patients with collagen vascular diseases.

BACKGROUND: The gastrointestinal manifestations of the collagen vascular diseases have been well described in the pediatric population. These patients frequently have symptoms that constitute indications for endoscopy. However, the risks and benefits of endoscopy in this population have not been examined. METHODS: A retrospective review of all patients with collagen vascular diseases hospitalized during a 7-year period was undertaken, and those patients who underwent endoscopy were identified. RESULTS: Nine patients (5%) underwent endoscopic procedures (eight upper and three lower endoscopy). Complications and outcomes were analyzed. Indications for endoscopy included abdominal pain, gastrointestinal (GI) bleeding, and/or vomiting and diarrhea. Two patients had complications that required surgery within 1 day of the endoscopic procedure. One of these patients subsequently died with GI bleeding. Five of the nine patients had changes in their management after endoscopy. Helicobacter pylori infection was identified and treated in two patients. Three patients had esophagitis or gastritis and acid suppression treatment was started or optimized. Vasculopathy was present in the patients who had complications. CONCLUSIONS: This series suggests that endoscopy can provide useful information for the management of the pediatric patient with GI symptoms and collagen vascular diseases. However, because serious and potentially life-threatening complications can occur, great care is needed in evaluating the risk/benefit ratio of endoscopy in these patients.

Adolescent↗
PubMed1999

Cultures of human liver cells in simulated microgravity environment.

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.

Albumins↗
PubMed1998

Lack of C/EBP alpha gene expression results in increased DNA synthesis and an increased frequency of immortalization of freshly isolated mice [correction of rat] hepatocytes.

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.

Adenocarcinoma↗
PubMed1998

Immunosuppressive therapy with microemulsion cyclosporine A shortens the hospitalization of pediatric liver transplant recipients.

Intravenous administration of cyclosporine (Sandimmune) to rapidly and effectively achieve therapeutic serum levels in transplant recipients has been the treatment standard in many transplantation centers. With the development of microemulsion cyclosporine (Neoral), that standard is changing. Neoral has greater bioavailability than the oral form of Sandimmune and, consequently, can be more efficacious and cost-effective. To test this hypothesis, we undertook a retrospective study of Sandimmune and Neoral in the treatment of 66 children who underwent uncomplicated orthotopic liver transplantation in the Texas Medical Center between April 1991 and December 1997. Both forms of cyclosporine were evaluated in terms of in-patient treatment cost, recuperative time in the intensive care unit and duration of hospitalization. Twenty-two patients were treated orally with Neoral, and 44 patients were treated intravenously with Sandimmune for a mean time of 14 d. Once the blood concentration of Sandimmune reached a steady state, as confirmed by daily measurements of the trough level, the patients in the Sandimmune group were converted to oral cyclosporine. None of the 22 patients treated with Neoral required intravenous treatment. The mean time spent in the intensive care unit was 4 d for the Neoral group and 5.5 d for the Sandimmune group. The mean duration of hospitalization from the date of transplantation to discharge was 12 d for the Neoral group and 20 d for the Sandimmune group (p < 0.001). Based on these results, we determined that the overall cost per patient in the Neoral group was $3598 less than that per patient in the Sandimmune group.

Adolescent↗
PubMed1995

DNA binding by C/EBP proteins correlates with hepatocyte proliferation.

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.

Animals↗
PubMed1995

Retroviral gene transfer into the intestinal epithelium.

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)

Animals↗
PubMed1993

Development of a clinical protocol for hepatic gene transfer: lessons learned in preclinical studies.

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)

Animals↗
PubMed1992

Transduction of primary human hepatocytes with amphotropic and xenotropic retroviral vectors.

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.

Animals↗
PubMed1992

The use of DiI-marked hepatocytes to demonstrate orthotopic, intrahepatic engraftment following hepatocellular transplantation.

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.

Animals↗
PubMed1992

DiI as a marker for cellular transplantation into solid organs.

Research in cellular transplantation is frequently compromised by an inability to identify transplanted cells engrafting into orthotopic sites if they exhibit normal morphology and no unique antigenic markers. A method is described for using the fluorescent dye DiI as a marker for cell transplantation studies. This dye is not metabolized or exchanged between cells in vitro or in vivo and enables identification of engrafted cells by fluorescence microscopy, flow cytometry or fluorescence-activated cell sorting. Applications are described in autologous hepatocellular and thyroid follicular cell transplantation.

Animals↗