Biomedical subjects

T J Caperna

Publications and source records attributed to T J Caperna.

42 records · Page 3Linked to original sources

PubMed1985

Isolation and culture of parenchymal and nonparenchymal cells from neonatal swine liver.

Procedures for the isolation and monolayer culture of hepatocytes and nonparenchymal (Kupffer and endothelial) cells from livers of neonatal pigs (1 to 15 d of age) are described. Cell suspensions were obtained by a modification of the two-step collagenase perfusion technique. Hepatocytes were collected by low-speed centrifugation and nonparenchymal cell populations were purified by centrifugal elutriation. Hepatocytes were readily maintained in arginine-free medium fortified with either fetal calf serum or bovine serum albumin and oleate for periods as long as 6 d. The ability of cultured hepatocytes to incorporate 3H-leucine and 3H-thymidine into protein and DNA, respectively, demonstrated that cells were metabolically active for at least 3 d in culture. The 3H-leucine incorporation into total cell protein was constant regardless of animal age at the time of cell isolation, while incorporation of 3H-thymidine was influenced by animal age. Incorporation of both precursors was dependent upon duration of culture period in vitro and the type of medium (serum-free vs serum-containing) in which the cells were maintained. Morphological observation and analysis of the DNA and protein levels of hepatocyte monolayers suggest that cells did not replicate during the 3-d incubation period. The ability to isolate and culture metabolically active, nonreplicating hepatocytes from neonatal pigs in a serum-free medium affords opportunities for investigation of the influence of specific hormones and specific growth factors on the uptake and metabolism of nutrients by the liver. Similarly, the neonatal pig will serve as a useful model for the characterization of hepatic nonparenchymal cell metabolism during the neonatal period.

Animals↗

PubMed1984

Cadmium metabolism by rat liver endothelial and Kupffer cells.

The metabolism of cadmium was investigated in Wistar-rat liver non-parenchymal cells. Kupffer and endothelial cells, the major cell populations lining the sinusoidal tracts, were isolated by collagenase dispersion and purified by centrifugal elutriation. At 20 h after subcutaneous injection of the metal salt (1.5 mg of Cd/kg body weight), endothelial cells accumulated 2-fold higher concentrations of Cd than did Kupffer or parenchymal cells. Most of the Cd in non-parenchymal cells was associated with cytosolic metallothionein (MT), the low-Mr heavy-metal-binding protein(s). When MT was quantified in cytosols from cells isolated from control rats by a 203Hg competitive-binding assay, low levels were found to be present in Kupffer, endothelial and parenchymal cells. Cd injection significantly increased MT levels in all three cell types. The induction of MT synthesis was investigated in vitro by using primary monolayer cultures. The incorporation of [35S]cysteine into MT increased 47% over constitutive levels in endothelial-cell cultures after the addition of 0.8 microM-Cd2+ to the medium for 10 h. MT synthesis in Kupffer cells was not observed. The lack of MT synthesis by monolayer cultures of Kupffer cells in vitro was associated with a decreased capacity of these cells to accumulate heavy metals from the extracellular medium. This apparent decreased ability to transport metals did not reflect a general defect in either cellular function or metabolic activity, since isolated Kupffer cells incorporated [3H]leucine into protein at rates comparable with those shown by liver parenchymal cells and readily phagocytosed particles.

Animals↗

PubMed1982

Antigen handling in aging. II. The role of Kupffer and endothelial cells in antigen processing in Fischer 344 rats.

A method has been developed for the purification of Kupffer and endothelial cells from rat liver by collagenase enzyme perfusion followed by centrifugal elutriation. After intravenous injection of a soluble antigen, [3H]azoaniline bovine serum albumin ( [3H]BSA), its distribution was studied in isolated cell populations from liver and spleen tissue of two aged groups of male F-344 rats. In young adult rats (6-8 months) both sinusoidal cell types contained the same amount of [3H]BSA; however, in older rats (22-24 months) the amount of antigen in the endothelial cells was significantly decreased. In comparison to the liver, the spleen retained only a small fraction of the injected dose. In order to assess the catabolic properties of both Kupffer and endothelial cells, supernatants obtained from in vitro cell culture were evaluated for both biological and physiochemical properties. Antigen was almost completely degraded by both cell types as determined by gel filtration and did not directly stimulate BSA-primed lymphocytes in vitro; however, these supernatants were shown to enhance the lymphoproliferative response of primed lymphocytes to additional antigen exposure. Kupffer cell receptors, Fc and C3, assayed by direct rosetting, did not vary with age; endothelial cells also possessed Fc receptors that were found to be unchanged with age. These studies are an initial attempt to better define our previous finding of defective antigen handling with aging by use of isolated pure cell populations.

Aging↗

PubMed1981

Bovine serum albumin humoral immune response in aged Fischer 344 rats.

The circulating antibody titers to bovine serum albumin (BSA) were determined for two groups of aged Fischer 344 rats obtained form the National Institute on Aging. One group received a single immunization of BSA in complete Freund's adjuvant, and the second group was immunized three times at two-week intervals with twice the antigen dosage. An age difference was observed during the peak response in both groups, the younger rats having higher titers; however, upon multiple injections the older rats show an enhanced response although significantly lower than the younger rats. All antibody titers increased at the same rate, yet measurable antibody always appeared first in younger animals.

Aging↗

PubMed1980

Antigen handling in relation to ageing.

The distribution of a soluble antigen, 3H-BSA was studied in tissues (blood, liver, spleen, kidneys, lymph nodes, thymus and lungs) after intravenous injection into cohorts of the Fischer-344 rat colony of the National Institute on Aging that is maintained at Charles River Breeding Laboratories. The ten groups for which results are reported were maintained during the experimental procedures as the cohorts received from the "colony". Both the primary and secondary antigen injection were administered as 1 mg of protein per 50 g body weight. All the highly vascularized tissues of those studied showed the same relative distribution of antigen with age as the blood; i.e. a lower percentage of the injection at both extremes of the age span studied, 2 and 24 months of age. These age-related differences are more pronounced with primary than secondary immunization. Because of the higher level of antigen in the liver than blood, it is concluded that a specific localization of antigen occurs in the liver. Products of in vivo metabolism of 3H-BSA are isolated as liver nucleopeptides and are also co-isolated with liver polysomes. These particular forms of metabolized antigen have been shown to be immunogenic in other studies. The co-isolated polysome antigen in the present findings is reduced quantitatively in the older rats (for example, in 24 months compared to 12 month). The study of the changes in antigen parceling at the cellular and subcellular level, which occurs with ageing, is likely to provide insight into antigen induction.

Aging↗

PubMedDate not supplied

Selective and organotypic culture of intrahepatic bile duct cells from adult pig liver.

Secondary culture of nontransformed bile duct epithelium has been difficult to achieve. STO feeder cell-dependent secondary cultures of adult pig bile duct cells were established from primary cultures of adult pig liver cells. Adult pig hepatocytes exhibited limited or no replication and were lost from the secondary culture at Passage 3 or 4. In contrast, adult pig bile duct cells replicated and were carried for 4-8 passages in secondary culture. A simple method to produce nearly pure pig intrahepatic bile duct cultures was first to freeze a relatively crude liver cell preparation. Upon subsequent thawing, all hepatocytes and most macrophages were lysed. Bile duct cells composed 95% of the surviving cells after the freeze/thaw, and they grew out rapidly. The bile duct cells grew on top of the STO feeder cells as closely knit epithelial, colonial outgrowths. Histocytochemical and biochemical analyses demonstrated high levels of gamma-glutamyltranspeptidase activity and low levels of P450 activity in the bile duct cultures. The bile duct cells spontaneously adopted a multicellular ductal morphology after 7-10 d in static culture which was similar to that found in in vivo pig liver. Transmission electron microscopic examination revealed complex junctions and desmosomes typical of epithelium, and lumenally projecting cilia typical of in vivo intrahepatic bile ductules. This simple method for the coculture of pig intrahepatic bile duct cells which adopt in vivo-like structure may facilitate biological studies of this important, but difficult to culture, cell type.

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