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Biomedical subjects

H O Jauregui

Publications and source records attributed to H O Jauregui.

At least 19 recordsLinked to original sources
PubMed2001

Growth and metabolic activity of immortalized porcine hepatocytes in extracorporeal hollow-fiber liver assist devices.

The development of a cell based extracorporeal liver assist device offers a promising clinical approach to bridge individuals suffering from acute liver failure to transplant. However, a major drawback of the existing technology is the lack of a continuous supply of well differentiated hepatocytes. Although some investigators have used primary porcine cells, this approach demands costly, labor-intensive isolation procedures and yields cells with inconsistent detoxification capacity. The limitations of primary cells led us to develop the HepLiu immortalized porcine hepatocyte cell line for use in liver assist devices (LADs). HepLiu cells are nontumorigenic and exhibit multiple hepatic detoxification functions including diazepam and acetaminophen metabolism. To investigate the suitability of HepLiu cells for artificial liver support, morphology, as well as xenobiotic metabolism, was studied in perfused polysulfone hollow-fiber LADs. HepLiu cells were cultured in the intercapillary space of a prototype LAD, and the metabolism of diazepam, acetaminophen, and 7-ethoxycoumarin was evaluated over 25 days in culture. Our results indicated that HepLiu cells proliferated rapidly following inoculation of the LAD until Day 10 when proliferation appeared to cease. Ultrastructural analysis demonstrated that HepLiu cells retained many of the features of primary hepatocytes including desmosomes that sealed bile canalicular-like structures and junctional complexes (intermediate, gap junctions) that appeared concentrated in the paracanalicular areas. Unlike primary porcine hepatocytes, HepLiu cells retained drug metabolic function throughout the 25 day culture period. Diazepam metabolism by HepLiu cells was consistently higher than that of primary cells. Acetaminophen metabolism persisted throughout the 25 day period albeit at a much lower level than the primary cells exhibited on Days 1 or 2. In conclusion, we have shown that HepLiu cells proliferate to occupy the intercapillary space of perfused hollow-fiber LADs following inoculation, and retain their metabolic capacity for Phase I and Phase II detoxification reactions in perfusion culture. Our findings suggest that HepLiu cells may provide an alternative to primary porcine hepatocytes as the cellular component of bioartificial liver support systems.

Animals↗
PubMed1995

Secretion, surface localization, turnover, and steady state expression of protein disulfide isomerase in rat hepatocytes.

Protein disulfide isomerase in isolated rat hepatocytes was present at a concentration of 7 micrograms/mg cell protein, representing a approximately 2-fold enrichment compared to isolated hepatic non-parenchymal cells. Though localized mainly in microsomal fractions of hepatocytes, direct immunofluorescence and cell surface radioiodination followed by immunoprecipitation revealed the presence of M(r) 57,000 disulfide isomerase at the cell surface. Electrostatic interaction of the protein with the cell surface was suggested by susceptibility to carbonate washing. Metabolic radiolabeling and immunoprecipitation studies also indicated that some of the newly synthesized M(r) 57,000 disulfide isomerase was secreted. Treatment of cells with colchicine markedly reduced the recovery of disulfide isomerase from the media, indicating microtubular-directed secretion of the protein. Partial staphlococcal V8 proteolytic digestion of the secreted protein revealed a peptide pattern similar to that of the cellular protein. Immunoprecipitation with antibody specific to the -KDEL peptide retention sequence confirmed the presence of this sequence in the secreted protein. Studies of the turnover of disulfide isomerase revealed a half-life of approximately 96 h. Treatment of cells with tunicamycin or heat shock resulted in an increased recovery of newly synthesized disulfide isomerase from cell lysates but diminished recovery from the media. The secretion and cell surface distribution of disulfide isomerase in hepatocytes may be important for the pathogenesis of immune mediated liver injury.

Animals↗
PubMed1995

In vivo evaluation of a hollow fiber liver assist device.

Orthotopic liver transplantation is the only effective form of therapy currently available for patients with fulminant hepatic failure (FHF). The use of an extracorporeal (EC) liver assist device (LAD) may result in improved presurgical clinical management. Alternatively, patients treated with LADs could avoid the transplantation procedure if they are able to regenerate a critical mass of hepatocytes that will sustain functional viability. In this study, the efficacy of a prototype hollow fiber LAD seeded with rabbit hepatocytes was assessed in vivo by the use of two different animal models: (1) normal rabbits injected with diazepam or lidocaine, and (2) a galactosamine (Gal)-intoxicated rabbit model of FHF. The EC LAD clearly decreased the blood levels of the two drugs and significantly generated diazepam and lidocaine metabolites indicating the maintenance of active P450 forms in the cellular component of the devices. A 6-hour EC treatment significantly increased the survival time and delayed the onset of hepatic encephalopathy (HE) in the Gal-intoxicated rabbits. Histological evaluations of postmortem livers showed greater hepatocyte regenerative activity in the animals treated with hepatocyte-seeded LADs than in the two control groups, e.g., rabbits not treated or treated with unseeded devices. These findings support the concept that a microporous hollow fiber LAD seeded with rabbit hepatocytes is able to sustain drug detoxification in vivo as well as to modify the course of FHF in a well-characterized animal model.

Animals↗
PubMed1994

Primary cultures of rat hepatocytes in hollow fiber chambers.

Hepatocyte culture may represent an alternative to the use of animals to study drug detoxification by the liver. An ideal in vitro system should closely mimic the in vivo environment by providing continuous media perfusion and oxygenation, and should facilitate sampling of cells and culture media. To meet these criteria, a hollow fiber bioreactor seeded with isolated rat hepatocytes was developed and tested by measuring the formation of three products of the oxidative metabolism of diazepam and the glucuronidation of phenolsulfonphthalein (PSP). To compare the performance of conventional monolayer culture to that of the bioreactor system, diazepam metabolism was studied for 45 days in both systems. The oxygen dependency of diazepam metabolism was evaluated by perfusing the bioreactor in an oxygen-rich atmosphere (30%). Total diazepam metabolism was twofold higher in the O2-rich perfused hollow fiber cultures than in the cultures perfused under normal conditions, reflecting an increase in temazepam and oxazepam production. Diazepam detoxification activity was significantly enhanced by oxygen (P < or = 0.001) over the life of the perfused cultures. PSP metabolism was similar in all three culture systems. By Day 10, diazepam metabolism in the oxygenated bioreactor system was 44% of the in vivo activity of rat hepatocytes. This activity dropped to 30% by Day 25 of culture. These results justify the use of perfused culture systems for in vitro detoxification studies as an alternative to animal use and emphasize the capacity of a culture device perfused under O2-enriched conditions to maintain long-term P450 activity of rat hepatocytes.

Animals↗
PubMed1992

Long-term cultures of adult mammalian hepatocytes in hollow fibers as the cellular component of extracorporeal (hybrid) liver assist devices.

A discussion of the treatment of liver insufficiency with extracorporeal (hybrid) liver assist devices (LADs) should address a definition of the types of liver failure susceptible to being treated by these devices as well as the modalities of in vivo and in vitro testing. Relevant to the first subject is the subject of pathogenesis of hepatic coma, which should be the target for the design of these LADs. Although this modality of therapy is new, it can be predicted that these devices will demand minimal safety conditions, i.e., the seeding with cells that are not tumorigenic or carrying viral particles. Among other topics to be considered in the development of LADs is the proper choice of hollow fiber to be used and the testing on proper animal models of hepatic failure. It is our philosophy that the long-term culture of adult mammalian hepatocytes in hollow fibers is the basis for appropriate designs of this type of temporary liver support.

Animals↗
PubMed1991

Mammalian hepatocytes as a foundation for treatment in human liver failure.

Technological advances in the separation and culture of mammalian hepatocytes have facilitated the use of these cells as the foundation for either hepatocyte transplantation or hepatocyte-seeded hollow fiber liver assist devices (LAD). To fully appreciate the practical applications of these tissue engineering solutions, it is necessary to understand the types of human liver failure as well as the corresponding animal models. The most immediate application of this type of technology is the treatment of hepatic encephalopathy (HE), an acute and highly fatal complication of fulminant hepatic failure. Although the pathogenesis of HE is unknown, failure of the detoxification function of the liver is accepted as playing an important role in this disorder. Consequently, the assaying and preservation of P450 activity in the grafted cells or in the LAD must be among the main targets of this research. This review explores the problems in hepatocyte transplantation and culture that deserve special consideration and emphasizes the conditions contributing to the in vitro maintenance of phenotypic expression of these cells.

Animals↗
PubMed1991

Xenobiotic induction of P-450 PB-4 (IIB1) and P-450c (IA1) and associated monooxygenase activities in primary cultures of adult rat hepatocytes.

1. The long-term maintenance of metabolism of representative drugs and steroid hormone substrates by cytochromes P-450, and their inducibility, was investigated in primary cultures of adult rat hepatocytes. Collagenase-isolated cells were seeded on collagen-coated tissue culture dishes and cultured in Chee's essential media in the presence or absence of phenobarbital (PB, 0.75 mM, 96 h or continuously) and 3-methylcholanthrene (MC, 5 microM, 48 h) for up to 45 days. 2. Hepatic P-450-dependent metabolism of diazepam to its primary oxidized metabolite was inducible by PB both in vivo (monitored in isolated liver microsomes) and in cultured cells (up to 100% and 400% increases in the formation of temazepam and nordiazepam, respectively, after 25 days in culture). Hepatocyte microsomal androstenedione 16 beta-hydroxylase activity was also induced by PB treatment of the hepatocytes (350-650% increase in 20-day-old cells). 3. Western blot analysis revealed that immunoreactive P-450 form PB-4 (IIB1), which catalysed the N-demethylation of diazepam to yield nordiazepam as well as androstenedione 16 beta-hydroxylation when assayed in a purified enzyme system, was substantially elevated following PB treatment of the cultured cells. Similarly, MC induced 7-ethoxycoumarin O-deethylase activity (up to 2000% increase from 5 to 45 days) as well as immunoreactive P-450c (IA1) in the hepatocyte cultures. 4. These studies demonstrate that cytochrome P-450 activities can be maintained, and also induced, after extended periods of time in hepatocytes cultured using a simple collagen mixture as substrate and a commercially available tissue culture media. This culture system should provide an important tool for further studies of P-450-dependent xenobiotic metabolism in a well-defined, liver-derived cellular system.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1990

Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove valuable for more detailed investigations of the molecular and mechanistic basis of the response to PB and its modulation by endogenous hormones.

Animals↗
PubMed1990

Gastric adenocarcinoma: prognostic significance of several pathologic parameters and histologic classifications.

Considerable controversy exists about the value of histologic classifications of gastric adenocarcinoma in the prediction of patient survival. Histologic sections of 75 consecutive gastrectomies were used to compare Lauren and Ming classifications with emphasis on clinical stage, size, location of tumor, desmoplasia, inflammatory reaction, and 5-year survival. Both classifications generally correlated and, when combined, proved helpful in defining certain cases. At surgery, about one third of the total cases of intestinal (INT, Lauren) and expanding (ET, Ming) were in early stages, whereas almost all the diffuse (DT, Lauren) and infiltrative (INF, Ming) types were in late stages. When the Lauren classification was applied to preoperative endoscopic biopsies, a 72% diagnostic correlation with the surgical specimens was found. Followup revealed no survivors of the DT and INF and 12 and 11 survivors of INT and ET, respectively, regardless of stage. Inflammatory response was associated with good prognosis. Desmoplasia and size had no prognostic significance. Tumors of the cardia had worse prognoses than those in the body or antrum. Both Lauren and Ming classifications, and especially the degree of inflammation, were significant in predicting survival. Lauren INT and Ming ET should be declared only when they are the sole or predominant features.

Adenocarcinoma↗
PubMed1988

A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces.

A quantitative evaluation of lectin binding to adult rat hepatocyte cell surfaces was done using cells isolated by two different collagenase perfusion methodologies and cultured as monolayers with two different tissue culture media formulations (protocol I vs. protocol II). The presence of alpha-D-mannosyl and alpha-D-glucosyl groups was detected by the binding of Concanavalin A (Con A), Lens culinaris agglutinin (LCA), and Pisum sativum agglutinin (PSA) to freshly isolated cells. Furthermore, beta-D-galactose [Ricinus communis agglutinin (RCA)] and sialic acid residues [wheat germ (WGA)] were also found. Protocols I and II served as models for evaluation of: a) the stripping effect of collagenase separation procedures, b) the restoration in culture of collagenase-stripped sugar residues, c) the effect of the culture environment on cell viability [as measured by lactic acid dehydrogenase (LDH) leakage] and the protein content of hepatocytes, and d) the presence of cell surface sugar residues as a function of culture duration. The ultrastructural morphology of freshly isolated and cultured hepatocytes was also evaluated. These studies indicated that a decline in lectin binding invariably occurred earlier than a massive leakage of LDH and a decrease in the protein content of the cells in culture. Ultrastructurally, autophagocytosis was an early phenomenon in cells isolated and cultured by protocol I, which was also inferior to protocol II regarding the preservation of hepatocyte glycocalyces. Sugar residues lost due to the collagenase-stripping effect were restored, as shown by lectin binding, within the first 24 h of culture. This stripping effect was confirmed by quantitative evaluations of lectin binding to hepatocytes in culture after an incubation with collagenase. This study shows that the binding of peroxidase-labeled lectins is a useful tool for quantitative evaluation of the sugar composition of hepatocyte cultures.

Animals↗
PubMed1988

Hepatocyte cell surface polarity as demonstrated by lectin binding.

We performed an investigation at the ultrastructural level of the differential distribution of lectin-binding sites among sinusoidal, lateral, and bile canalicular domains of adult rat hepatocytes. Lectin binding to hepatocyte glycocalices was studied in situ or after cellular dissociation by enzymatic (collagenase), chemical (EDTA), and mechanical methods, as well as during cell culture. Using thirteen biotinylated lectins and an avidin-biotin-peroxidase complex (ABC), we have identified lectin-binding sites that are predominantly localized in the bile canalicular [Ricinus communis agglutinin (RCA)] or sinusoidal [Phaseolus vulgaris (PHA)] domains in situ and in mechanically dissociated cells. Lens culinaris (LCA) staining was prominent on sinusoidal surfaces, slight along lateral surfaces, and completely absent in the bile canalicular domain. Concanavalin A (ConA) was unique in binding equally to all domains. Triticum vulgaris [wheat germ agglutinin (WGA)] was also bound to all domains, but most intensely to the bile canalicular region. Cells dissociated via collagenase or EDTA treatment exhibited a spherical morphology characterized by many surface microvilli and absence of morphological domains. Lectin binding to dissociated cells was uniformly distributed over the entire cell surface, suggesting a redistribution of lectin receptors that was independent of the separation procedure. Hepatocytes in culture exhibited a partial restoration of morphological domains, but lectin binding polarity was not re-established.

Animals↗
PubMed1988

Mucin histochemistry of intestinal metaplasia in Barrett's esophagus.

Histological and histochemical evaluation of 33 biopsies and 8 distal esophagectomy specimens revealed specialized columnar epithelium with intestinal features [intestinal metaplasia (IM)] to be the most common type (91%) of metaplasia in Barrett's esophagus (BE). Junctional epithelium was found in only 3 of the 33 biopsies. The type III subvariety of IM (TIII-M), characterized by the presence of sulfomucins in the non-goblet columnar cells, was found in 58% of all our biopsies and 62% of operative specimens. Six of the 7 cases of epithelial dysplasia were associated with TIII-M; one of them subsequently developed an adenocarcinoma. The transitional epithelium adjacent to adenocarcinomas in the operative specimens also showed TIII-M in five of six cases. Our findings indicate that TIII-M is almost as common in Barrett's-associated carcinoma as in nonneoplastic cases of BE, thereby limiting the usefulness of this histological marker as an indicator of neoplastic change (P = 0.5). On the other hand, TIII-M seems to be significantly associated with mild dysplasia in BE. The value of TIII-M as a prognostic indicator regarding the subsequent development of esophageal carcinoma remains in doubt and could be more precisely assessed by a prospective study.

Adenocarcinoma↗