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Biomedical subjects

H O Jauregui

Publications and source records attributed to H O Jauregui.

At least 37 records · Page 2Linked to original sources
PubMed1986

Attachment and long term survival of adult rat hepatocytes in primary monolayer cultures: comparison of different substrata and tissue culture media formulations.

Long-term monolayer cultures of adult rat hepatocytes were tested for their ability to glucuronize phenol red and to maintain initial levels of cell proteins, glucose consumption, and lactic acid production. Lactate dehydrogenase leakage served as an index of culture status because a high value indicates cell death. Three tissue culture (TC) media formulations were the main variables introduced to determine ideal conditions for cell survival in vitro. Investigations of long-term cultures were preceded by studies of hepatocyte attachment to polystyrene surfaces. This attachment was influenced by the amount of substrate deposited and the number of cells seeded, but not by the uniformity of the substrate coating. A statistical analysis of our data revealed that in the absence of fetal bovine serum (FBS), air dried collagen (ADC) and Biomatrix (BMX) were superior to saline precipitated collagen and fibronectin as attachment substrates. In the presence of 10% FBS, all of the substrates performed equally. Chee's Medium (CEM) proved to be the best for preserving cell proteins over a time course of 28 d and Williams' E medium also performed adequately up to 14 d. The glucuronization of phenol red was at 50% of initial values at Day 7 in CEM-ADC hepatocytes in contrast to 30% for cells in Williams' E medium and 5% for cells grown in Waymouth's. At 14 d glucuronization was still present at 40% of original values in CEM-ADC cells but had ceased in the other two media. When BMX was used, none of the TC media supported glucuronization levels comparable to ADC cells.

Animals↗
PubMed1986

Lectin binding to parietal cells of human gastric mucosa.

A light microscopic and ultrastructural analysis of lectin receptors on parietal cells from human gastric mucosa was performed utilizing 12 biotinylated lectins in conjunction with an avidin-biotin-peroxidase complex. Peanut agglutinin conjugated directly to peroxidase was also used. Several fixatives and fixation regimens were evaluated for optimal preservation of parietal cell saccharide moieties. Formalin proved to be the most practical fixative for light microscopic studies. A periodate-lysine-paraformaldehyde (PLP) combination provided good preservation of lectin binding capacity but yielded relatively poor ultrastructure. Conversely, glutaraldehyde provided excellent preservation of ultrastructure but a somewhat diminished lectin binding activity, which was overcome by using long incubation times and high concentrations of reagents. Parietal cells reacted strongly with Bandieraea simplicifolia, Dolichos biflorus, peanut agglutinin, and soybean agglutinin (all specific for galactosyl/galactosaminyl groups) and weakly with Ulex europaeus (specific for fucose). At the light microscopic level a beaded, perinuclear staining pattern was observed which, ultrastructurally, corresponded to an intense staining of intracytoplasmic canaliculi. The membranes of the intracytoplasmic canaliculi were characterized by an abundance of galactosyl residues, a paucity of fucosyl groups, and a lack of mannosyl and glucosyl residues. The biochemical and physiological significance of these findings is discussed.

Histological Techniques↗
PubMed1984

Light and electron microscope analysis of lectin binding to adult rat liver in situ.

A comprehensive mapping of lectin receptors on adult rat liver in situ was performed at light and ultrastructural levels by using 12 biotin-labeled lectins and an avidin-biotin-peroxidase complex. In addition, concanavalin A conjugated directly to peroxidase was utilized to study intracellular membrane glycoconjugates. To achieve optimal preservation of these membrane sugar moieties, several fixatives and fixation procedures were evaluated. A periodate-lysin-paraformaldehyde combination provided the best compromise between preservation of ultrastructural details and lectin-binding reactivity. Hepatocyte cell surfaces reacted intensely with concanavalin A, Lens culinaris agglutinin, and Pisum sativum agglutinin (all specific for alpha-D-mannosyl and alpha-D-glucosyl groups) as well as Ricinus communis agglutinin type I (specific for alpha or beta-D-galactose) and wheat germ agglutinin (specific for neuraminic acid and beta-NAc-glucosaminyl groups). In addition, R. communis agglutinin and wheat germ agglutinin exhibited an extremely strong reactivity for bile canaliculi which surpassed the binding of concanavalin A, L. culinaris agglutinin, and P. sativum to these structures. Phaseolus vulgaris agglutinin (specific for beta-D-galactose-glucosyl-NAc and D-mannosyl groups), which exhibited a moderate binding to hepatocyte plasma membranes, reacted more strongly with the endothelium of sinusoids and portal vessels. Although all six of these lectins plus Bandeiraea simplicifolia stained Kupffer cells, B. simplicifolia lectin (an alpha-D-galactosyl marker) was unique in showing a strong reactivity for only this cell type. The avidin-biotin-peroxidase procedure is a sensitive method for detection of sugar moieties on cell surfaces of rat liver at both light and electron microscopic levels. In this study, the procedure was used to localize differential binding of lectins to several anatomical structures of the organ, and furthermore, we were able to map preferential localizations of carbohydrate residues in the glycocalyx of the rat hepatocyte in situ.

Animals↗
PubMed1982

Phenolsulfonphthalein (phenol red) metabolism in primary monolayer cultures of adult rat hepatocytes.

The sulfonic acid dye, phenolsulfonphthalein (PSP or phenol red), has been incorporated as a pH indicator in many tissue culture media formulations since the emergence of tissue culture methodologies. The present study was designed to examine the pathway, time course, and degree of metabolism of this anionic dye in monolayer cultures of adult rat hepatocytes. Thin layer chromatographic studies coupled with beta-glucuronidase studies show that glucuronidation is the major metabolic pathway for PSP in vitro. About 20% of the dye is metabolized in the first 24 h, but this functional activity is decreased by approximately half at 48 h, and even further at 72 h of culture. This metabolic activity was not affected by continuous exposure to the dye. The effect of PSP concentration on its rate of metabolism by the adult rat hepatocyte in culture seemed to be biphasic, and at concentrations of less than 100 microM there was indication of a saturable process. Although PSP seemed not to be toxic to hepatocyte cultures, it is partially metabolized by these cells (as opposed to no observed metabolism in human fibroblasts or HeLa cells). Therefore, its incorporation into tissue culture media formulations for use in hepatocyte cultures should be avoided, especially when studying the mechanism(s) of glucuronidation or metabolic pathways thought to be affected by this anionic dye.

Animals↗
PubMed1981

Trypan blue dye uptake and lactate dehydrogenase in adult rat hepatocytes--freshly isolated cells, cell suspensions, and primary monolayer cultures.

Leakage of lactate dehydrogenase and staining by the vital dye trypan blue were investigated in adult rat hepatocytes at the time of isolation, in suspensions up to 3 h and in primary monolayer cultures up to 3 d. These two parameters of plasma membrane integrity were found to correlate closely in hepatocyte suspensions, but to a lesser degree in monolayer cultures. Functional activity was demonstrated in culture by glucose consumption and lactic acid production. There was a balance of total lactate dehydrogenase (LDH) activity over time for both hepatocyte suspensions and cultures. Loss of LDH activity in the cell fraction was accompanied by a corresponding increase in enzyme activity in the media fraction. Lactate dehydrogenase activity per dye-excluding hepatocyte was calculated to be 9.2 +/- 1.5 X 10(-6) IU assayed at 37 degrees C for 25 preparations of isolated hepatocytes. The results suggest that leakage of cytoplasmic enzyme and vital dye staining are of comparable sensitivity in evaluating hepatocyte preparations. Measurement of LDH leakage offers a less subjective alternative to cell counting procedures and is applicable to both attached and suspended cells.

Animals↗
PubMed1978

Lymphomatoid granulomatosis after immunosuppression for pemphigus.

Lymphomatoid granulomatosis developed in a patient with pemphigus vulgaris who had been undergoing immunosuppressive therapy for three years. At autopsy, typical angiodestructive and angiocentric lesions with large lymphoid cells (immunoblasts) were found in the lungs and mediastinal nodes. Although a direct causal relationship between immunosuppression and lymphomatoid granulomatosis is lacking, the association of the two has been described. Immunoblastic sarcoma is also seen in immunosuppressed patients, and the role of the immunoblast in both of these disorders indicates that they may be related. This report suggests that, like immunoblastic sarcoma, lymphomatoid granulomatosis may develop under conditions of diminished immunologic surveillance, whether iatrogenic or naturally occurring.

Aged↗
PubMed1976

Pigmented squamous cell carcinoma of cornea and conjunctiva: A light microscopic, histochemical, and ultrastructural study.

A light microscopic, histochemical, and ultrastructural study was performed on tissue obtained from a pigmented squamous cell carcinoma of the cornea and conjunctiva. To the best of our knowledge this is the first time that one of these rare pigmented squamous cell carcinomas of the ocular mucous membrane has been described in detail. The occurrence of this neoplasm, which needs to be differentiated from other pigmented lesions, and particularly epibulbar melanomas, underscores the fact that all pigmented tumors are not melanomas. The study revealed that melanin granules, which accounted for the pigmentation of the neoplasm, were present in variable numbers in neoplastic squamous cells, Langerhans cells, macrophages, and stellate cells identified as melanocytes with mature melanosomes. Premelanosomes were not observed in any of these cells types. Current concepts concerning the common association of Langerhans cells with their characteristic intracytoplasmic "Birbeck granules" and melanocytes are discussed.

Carcinoma, Squamous Cell↗
PubMed1975

Prevention of Ozonide-Induced Heinz Bodies in human Erythrocytes by Vitamin E.

Methyl oleate ozonide, a proposed intermediary in ozone intoxication, produced Heinz body inclusions in human erythrocytes at concentrations of from 10-4 to 2 x 10-3 M. Daily oral supplementation with either 100 mg or 200 mg of D-alpha-tocopheryl acetate prevented Heinz body formation by methyl oleate ozonide. These observations suggest that the protective effects of vitamin E against ozone-produced toxicants occurs in man as well as in animals.

Environmental Exposure↗
PubMed1975

Heinz bodies formed in erythrocytes by fatty acid ozonides and ozone.

Ozonides of the methyl esters of oleic, linoleic, linolenic and arachidonic acids were found to produce Heinz body inclusions in human and mouse erythrocytes. No simple relationships between structure and activity were noted. Concomitant with Heinz body formation, methemoglobin and loss of cellular thiols were observed. Methyl ozonides readily oxidized glutathione and 1 mole of oxidized glutathione was formed per mole of methyl oleate ozonide. Methyl ozonides catalyzed the formation of disulfide-linked interchain polymers between hemoglobin and ovalbumin. Heinz bodies were not produced with ozone in the absence of unsaturated lipids. Heinz bodies were observed in the blood of mice exposed to ozone (0.85 ppm) for 48 hours. These observations suggest that fatty acid ozonides could serve as a toxic chemical species formed on ozone inhalation and could explain the divergent protective effects of lipid antioxidants and thiol generating systems in vivo.

Animals↗
PubMed1975

Iron metabolism and cell membranes. III. Iron-induced alterations in HeLa cells.

The morphologic characteristics of acute iron loading were studied in HeLa cells incubated in an iron-enriched Eagle's medium containing 500 mug/ml of iron. Chemical studies showed that ferritin synthesis was rapidly induced and the concentration of intracellular ferritin increased up to 72 hours. Closely coupled with an increase in HeLa cell ferritin was a marked decrease in the rate of cell multiplication. The significant ultrastructural findings of iron-induced HeLa cell injury are characterized by the appearance of both autophagic multivesicular and residual bodies over the first 72 hours of iron incubation. The prominence of multivesicular bodies was noted after only 4 hours' incubation, with iron and myelin figures first appearing after 6 hours. Thus, the partial arrest of cell multiplication was associated with an increase in cytoplasmic residual bodies containing iron and other debris. The distribution of intracellular ferritin within HeLa cells differs significantly from the distribution described previously in hepatic parenchymal cells. In HeLa cells, ferritin particles were confined to lysosomal vesicles and were not identified in cell sap, endoplasmic reticulum, or Golgi apparatus.

Acid Phosphatase↗
PubMedDate not supplied

Isolation and culture of porcine hepatocytes for artificial liver support.

The primary requirement of cells in a liver support system is the preservation of the in vivo metabolic functions that prevent or decrease the progress of hepatic encephalopathy (HE) by providing interim support to liver failure patients. While rodent hepatocytes offer a model for liver assist device (LAD) research, their limited number per animal prohibits direct scale up to human devices. Healthy human liver cells are seldom available in adequate numbers to support clinical LAD use; consequently, a large animal source of liver cells is needed. The study presented here explored the potential of porcine hepatocytes to proliferate and maintain metabolic function in vitro. Porcine hepatocytes were isolated from approximately 12 kg swine by a modification of Seglen's method. Hepatocytes cultured up to 10 days were shown to metabolize ammonia and maintain both Phase I and II detoxification functions. In addition, the cultures showed proliferative activity both as an increase in total protein content and by thymidine incorporation. Immunocytochemical staining identified cell proliferation through Day 4 to be primarily hepatocytes while Days 6 and 10 showed nonparenchymal cells to be increasing. The detoxification functions measured showed peak activity on Day 4 and gradually declined through Day 10. The ability of porcine hepatocytes to proliferate and maintain a diversity of hepatic functions in culture strongly suggests their potential for use as the biological component of artificial LADs.

Acetaminophen↗
PubMedDate not supplied

Use of mammalian liver cells for artificial liver support.

Advances in orthotopic liver transplantation have improved the survival rate of both acute and chronic liver failure patients to nearly 70%. However, the success of this treatment modality has created an international organ shortage. Many patients die while awaiting transplantation in part due to the minimal capacity to store viable transplantable livers beyond 24 h. Additionally, for many areas of the world, routine use of whole liver transplantation to treat liver disease is impractical due to the demands on both financial and technical resources. Potentially, these issues may be alleviated, at least in part, by the use of liver cell transplantation or cellular-based liver assist devices. The well-documented regenerative capacity of the liver may obviate the need for whole organ transplantation in some instances of acute failure, if the patient may be provided temporary metabolic support. Although other patients ultimately may require transplantation, a longer period of time to find a suitable organ for transplantation may be gained by that supportive therapy. The field of liver cell transplantation may offer solutions to patients with inherited metabolic deficiencies or chronic liver disease. The potential to treat an hepatic disorder by using only a fraction of the whole liver would increase the number of whole organs available for orthotopic liver transplantation. Research in the fields of hepatocyte based intra- and extra-corporeal liver support is providing evidence that these therapeutic modalities may ultimately become routine in the treatment of severe liver disease. A historic overview of that technology along with its current status is discussed.

Animals↗
PubMedDate not supplied

Functional recovery of porcine hepatocytes after hypothermic or cryogenic preservation for liver support systems.

The provision of an immediate supply of isolated porcine hepatocytes for artificial liver support requires preservation techniques that will allow maintenance of cell viability and detoxification functions. By means of a simple and cost-effective cryopreservation system, porcine hepatocytes can be available for both local and distant medical treatment facilities. Additionally, cryopreservation provides an adequate period for quality control testing to be completed prior to use of any specific cell lot. We are reporting a dual approach, namely the preservation of porcine hepatocytes, at 4 degrees C and at -196 degrees C in liquid nitrogen (LN2). Using a combination of cryoprotectant agents with Chee's modified Eagle's culture media (CEM), collagenase isolated hepatocytes stored at 4 degrees C for 24 h maintained 80% of the initial diazepam metabolism measured in freshly isolated cells and nearly 100% of initial function was preserved in hepatocytes stored up to 6 mo at -196 degrees C. University of Wisconsin solution (UW) was also tested and while adequate for 4 degrees C storage, it certainly did not match the performance of the CEM formulations for preservation of metabolic function of cells stored in liquid nitrogen. Based on our results of viability and detoxification function the combination of CEM with DMSO, polyethylene glycol and serum provided optimal protection for LN2 frozen cells. Other findings in these studies underlined the importance of the gradual introduction of DMSO in the prefreezing process, the period of osmotic equilibration, and the rapid postthaw withdrawal of this agent to minimize cytotoxic effects at these critical stages. Our freezing methodology provides the foundation for further technological developments in the cryopreservation of the large numbers of cells (billions) that are necessary for extracorporeal liver assist devices.

Animals↗
PubMedDate not supplied

Characterization and evaluation of detoxification functions of a nontumorigenic immortalized porcine hepatocyte cell line (HepLiu).

Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TAI (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.

Animals↗