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Biomedical subjects

A J Strain

Publications and source records attributed to A J Strain.

At least 73 records · Page 4Linked to original sources
PubMed1990

Biliary epithelial cells from the liver of patients with primary biliary cirrhosis: isolation, characterization, and short-term culture.

Since biliary epithelial cells of the middle-sized interlobular bile ducts are targets for lymphocyte-mediated damage in patients with primary biliary cirrhosis (PBC), we have developed a method for isolating and maintaining these cells in short-term tissue culture. Intrahepatic biliary epithelial cells were isolated from small segments of liver removed at the time of transplantation. Cells were separated from a collagenase digest by immunomagnetic separation using Dynabeads coupled to a monoclonal antibody (HEA 125) specific for a biliary epithelial cell surface antigen. The yield was approximately 3 x 10(5) cells/g of liver. The isolated cells were characterized morphologically and ultrastructurally using light and electron microscopy, and immunocytochemically using HEA 125 and anti-cytokeratin, anti-vimentin and anti-asialoglycoprotein receptor antibodies. By these criteria cells were judged to be identical to biliary epithelial cells from normal liver. The cells could be maintained in short-term tissue culture for up to 4 weeks without loss of biliary epithelial cell markers. Availability of interlobular biliary epithelial cells will be of value in future investigations of the pathogenetic mechanisms of PBC.

Bile Ducts, Intrahepatic↗
PubMed1990

Changes in sensitivity of hepatocytes isolated from regenerating rat liver to the growth inhibitory action of transforming growth factor beta.

Transforming growth factor beta (TGFB) is a potent inhibitor of DNA synthesis in adult rat hepatocytes in vitro. In the present study, the response of hepatocytes from normal or regenerating rat liver to TGFB was determined. TGFB inhibited DNA synthesis uniformly in hepatocytes from both groups in the absence of EGF. However, hepatocytes from 3 h regenerating liver maintained for 3 days in the presence of EGF were less sensitive to the growth-inhibitory action of TGFB. [3H]-thymidine incorporation was inhibited at 20 pM TGFB by only 7% in hepatocytes from 3 h regenerating liver compared with 70% in normal hepatocytes. By increasing the dose of TGFB to 100 pM, however, the full inhibitory response was restored. Reduced sensitivity was also found when the nuclear labelling index was determined, but no change was observed in cells from rats 3 h following sham hepatectomy. The change in sensitivity to TGFB required the presence of 5 ng/ml EGF or greater. Within a further 24-48 h in culture, the response to lower doses of TGFB was at least partially restored. While the present experimental design cannot directly confirm the role of TGFB as a paracrine inhibitor of liver growth in vivo, the data are compatible with this hypothesis.

Animals↗
PubMed1990

Protein-energy malnutrition induces changes in insulin sensitivity.

Early protein deprivation impairs glucose tolerance and insulin secretion, but the former generally recovers first. We have therefore investigated insulin sensitivity in vivo and in isolated muscle and cultured hepatocytes during protein-energy malnutrition and long-term follow-up in rats. Rats were weaned at 3 weeks onto normal rat chow (N rats) or onto 5% protein diet (LP rats). At 6 w of age LP rats were transferred to N chow. Insulin sensitivity was studied in the three systems at 3, 6, 12 and 24 w. At 6 w LP rats showed a greater and more prolonged fall in serum glucose in response to injected insulin than age--or weight--matched N, but by 12 w the difference was not significant. Similarly, soleus muscle from 6 w LP showed a higher basal rate of glucose transport and responded to a lower insulin dose than N, but there was no difference in sensitivity at 12 w. Hepatocytes from 6 w LP showed higher basal incorporation of glucose into glycogen than N, but insulin sensitivity was not different. Thymidine incorporation into hepatocyte DNA responded to lower insulin doses in 6 w LP than N. There was a decrease in insulin sensitivity with age in all experimental systems. Increased insulin sensitivity during malnutrition was seen in all tissues studied but differences between the two groups were not significant at 12 w and probably do not account for the normal glucose tolerance in the presence of low serum insulin levels previously reported.

Animals↗
PubMed1989

Immuno-isolation and culture of biliary epithelial cells from normal human liver.

Biliary epithelial cells (BEC) lining the intra-hepatic biliary ducts are the site of damage in several immunologically mediated liver diseases. BEC are difficult to isolate since they represent only 5% of the total cell number in normal liver. In this communication, a novel method for their isolation from normal liver is presented using a monoclonal antibody (HEA125) with specificity for an epithelial cell surface glyco-protein reported to be expressed in liver only by biliary epithelium. By combining differential density centrifugation and immuno-magnetic separation using HEA125 pure BEC (10(5) cells/g fresh tissue) were prepared routinely. These cells were maintained in culture for up to 4 weeks with significant increases in cell numbers. The ability to prepare BEC from human liver offers an opportunity to develop In Vitro models to investigate the aetiology of diseases of intra-hepatic biliary epithelium.

Antibodies, Monoclonal↗
PubMed1989

Immunological distribution of one form of insulin-like growth factor (IGF)-binding protein and IGF peptides in human fetal tissues.

Insulin-like growth factors (IGFs) are expressed by, and are biologically active on, human fetal cells. The mitogenic actions of IGF-I are modulated by the 21-41 kDa class of IGF-binding proteins (IGF-BPs). Using a rabbit anti-human IGF-BP antibody raised against a highly pure 26 kDa IGF-BP derived from amniotic fluid, we have compared the cellular location of IGF-BP and IGF peptides in tissue sections from prostaglandin-induced human abortuses of 14-16 weeks of gestation. The monoclonal and polyclonal antibodies used were raised against human IGF-I, but did not distinguish between IGF-I and IGF-II. Positive staining for IGF-BP was seen in every tissue except brain, spleen and thyroid. With the exception of skin, the cellular distribution of IGF-BP was similar to that of IGF peptides. Strong immunostaining was found in hepatocytes, hepatic erythropoietic cells, pulmonary epithelium, the tubular epithelium of kidney, intestinal epithelia, the fetal adrenal cortex and cardiac and skeletal muscle fibres. In skin, IGF-BP was located throughout the dermis and in the germinal layer of the epidermis. IGF peptide in skin was restricted to the deeper dermal layers. In the tibial epiphyseal growth plate both IGF-BP and IGF peptide were located in chondrocytes throughout the proliferation and hypertrophic zones. The similarity in distribution of IGF-BP and IGF peptides in fetal tissues suggests that the latter may exist predominantly complexed to IGF-BP in or on the surfaces of cells in vivo. The distribution of IGF-BP may define the sites of biological action of IGF peptides.

Adrenal Cortex↗
PubMed1989

Insulin-like growth factor (IGF)-binding protein release by human fetal fibroblasts: dependency on cell density and IGF peptides.

Insulin-like growth factors (IGFs) are bound to specific binding proteins in extracellular fluids in vivo and when released by cells in vitro. One class of binding protein (IGF-BP), a peptide of 26 kDa purified from amniotic fluid, has been shown to modulate IGF bioactivity on isolated human fibroblasts. We have determined the factors that control release of IGF-BP from monolayers of human fetal fibroblasts using a radioimmunoassay, and have compared this with the effects of these factors on the release of IGF-I and -II. Separation of cell-conditioned cultured medium on SDS-PAGE, and subsequent immunoblotting with antibody against IGF-BP showed that fibroblasts released a single species of immunoreactive protein of an estimated molecular weight of 30 kDa. This was not the predominant binding protein released by cells since major bands of approximately 42 kDa and 39 kDa were visualized following separation by SDS-PAGE and ligand blotting with 125I-labelled IGF-I. The 30 kDa IGF-BP was released in parallel with radioimmunoassayable IGF-I and -II over 48 h. However, a significant inverse correlation was found between the release of IGF-BP, IGF-I, IGF-II and cell density. The exposure of fibroblasts to 1.3 nmol/l or greater of IGF-I or -II caused a significant release of IGF-BP. Maximum release was seen in sparse cultures with little or no release from confluent cultures. IGF-I and -II were approximately equipotent with a fourfold increase in IGF-BP release at 19.7 nmol/l. Insulin caused a release of IGF-BP and IGF-I and -II from fibroblasts at supraphysiological concentrations (16.7 nmol/l) which again was maximal on sparse cell cultures. Increases in IGF-BP, IGF-I and -II release were also found in the presence of human placental lactogen (23.3 nmol/l), but human GH, epidermal growth factor, fibroblast growth factor and platelet-derived growth factor were without effect. The results show that human fetal fibroblasts released an IGF-BP immunologically similar to that seen in amniotic fluid together with IGF-I and -II, that IGF-BP release was enhanced by exogenous addition of IGF peptides, and that the release of all three peptides was a property of sparsely plated, rapidly growing cells. These findings strengthen the hypothesis that the cellular expression of IGF-binding proteins may represent an important level of control in IGF physiology.

Carrier Proteins↗
PubMed1988

Placental lactogen and growth hormone receptors in human fetal tissues: relationship to fetal plasma human placental lactogen concentrations and fetal growth.

The specific binding of human placental lactogen (hPL) and human GH (hGH) to particulate cell membranes from human fetal liver and skeletal muscle at 12-19 weeks gestation was examined. Fetal liver and muscle specifically bound [125I]hPL. This binding was inhibited by increasing concentrations of unlabeled hPL (half-maximal concentrations, 2.2 and 3.4 nmol/L, respectively). Scatchard analysis of the hepatic membrane binding revealed curvilinear plots with higher (Kd, 2.2 nmol/L) and lower (Kd, 24 nmol/L) affinity sites, while binding to muscle involved a single receptor class of Kd 5.6 nmol/L. The binding capacities for the two hepatic sites correlated positively with fetal body weight. [125I]hGH specifically bound to liver, but not muscle, with higher (Kd, 1.6 nmol/L) and lower (Kd, 8.6 nmol/L) affinity sites. [125I]PRL bound to hepatic membranes, but was preferentially displaced by hPL or hGH. Between 4 and 500 micrograms/L (mean, 82 micrograms/L, 3.8 nmol/L) hPL were present in fetal plasma. The findings identify distinct hPL receptors in human fetal liver and skeletal muscle and a hepatic hGH receptor in midgestation.

Embryonic and Fetal Development↗
PubMed1988

Transforming growth factor beta and inhibition of hepatocellular proliferation.

Transforming growth factor beta (TGF beta) is a recently characterized polypeptide that elicits diverse biologic actions in a wide range of cell types in vitro. TGF beta is a bifunctional growth regulator of fibroblasts with either growth stimulation or growth inhibition but inhibits the growth of most epithelial cells. In addition, TGF beta can either block or induce the differentiation of certain cells. TGF beta reversibly inhibits DNA synthesis in normal adult rat hepatocytes and in cells isolated from regenerating liver 12 h and 18 h after partial hepatectomy. However, at 3 h and 6 h after hepatectomy there is a decrease in sensitivity of hepatocytes to growth inhibition by TGF beta. Recent data from other laboratories indicate that TGF beta expression increases substantially in liver after partial hepatectomy and that administration of purified TGF beta in vivo inhibits DNA synthesis in regenerating rat liver. Together with our observations, these findings suggest that TGF beta may play a central role as a negative paracrine growth regulator in adult rat liver.

Animals↗
PubMed1987

Transforming growth factor beta inhibits DNA synthesis in hepatocytes isolated from normal and regenerating rat liver.

The inhibitory action of transforming growth factor beta (TGF beta) on DNA synthesis in hepatocytes isolated from the liver of normal rats or from the liver remnant of rats 18 h following partial hepatectomy was compared. Continuous exposure to TGF beta inhibited DNA synthesis of cultured hepatocytes to a similar degree in both groups when labelled with 3H thymidine from 24-48 h or 48-72 h. At 20 pM TGF beta, 3H-thymidine incorporation was reduced by 64-78% in hepatocytes from normal liver and by 60-73% in cells from 18 h regenerating liver. The nuclear labelling index was reduced by 70-80% in all cells. Exposure to TGF beta at concentrations up to 500 pM from 0-24 h had no effect on 3H-thymidine incorporation, but exposure at 20 pM for 24 h periods thereafter was uniformally effective. These results indicate that there is no change in sensitivity of hepatocytes from 18 h regenerating liver to TGF beta, compared with normal cells, and that TGF beta may act at some point in the G1 phase of the cell cycle to inhibit hepatocyte growth.

Animals↗
PubMed1987

Regulation of DNA synthesis in human fetal hepatocytes by placental lactogen, growth hormone, and insulin-like growth factor I/somatomedin-C.

Hepatocytes were isolated by gentle collagenase digestion of liver fragments from human fetuses of 8-16 weeks gestation obtained following prostaglandin-induced pregnancy terminations. They were maintained on collagen-coated tissue culture dishes in selective arginine-free medium for up to 72 hr, and the action of hormones and growth factors on DNA synthesis was studied by autoradiography following incubation with 3H-thymidine. The labeling index of hepatocytes was consistently enhanced by 25-250 ng/ml human placental lactogen (HPL), 25-250 ng/ml human growth hormone (HGH), 10-50 ng/ml insulin-like growth factor I/somatomedin-C (IGF I/Sm-C), and 10% dialyzed fetal calf serum, reaching a maximum of three- to four-fold greater than in basal medium alone. Under basal conditions, 30% of hepatocytes stained positively for the presence of IGF peptides using a monoclonal antibody raised against purified human IGF I/Sm-C. Although this proportion did not change following treatment with HGH and HPL, IGF I/Sm-C released by cells into culture medium was considerably increased in the presence of both hormones. Incubation with the SmC 1.2 monoclonal antibody abolished the increase in labeling index in response to IGF I/Sm-C and partially blocked the response to both HPL and HGH. These results indicate that both HPL and HGH stimulate DNA synthesis in human fetal hepatocytes and suggest that this effect is at least partly indirect through the release and paracrine action of IGF I/Sm-C.

DNA Replication↗
PubMed1987

Identification of somatomedin/insulin-like growth factor immunoreactive cells in the human fetus.

Somatomedins/insulin-like growth factors (Sm/IGFs) are present in blood and in extracts from multiple tissues of the human fetus and induce the proliferation of cultured human fetal cells. To identify the cellular location of immunoreactive Sm/IGF in human fetal tissues, we have performed immunocytochemistry in tissues from prostaglandin-induced human fetal abortuses of 12 to 20 wk in gestation. Every tissue studied except the cerebral cortex contains Sm/IGF immunoreactive cells. Cells staining positively include hepatocytes, hepatic hemopoietic cells, columnar epithelia of the pulmonary airways, intestine and kidney tubules, adrenal cortical cells, dermal cells, skeletal and cardiac muscle fibers, and pancreatic islet and acinar cells. Immunostaining was specific for Sm/IGFs, but because of the cross-reactivity of the antibodies it was not possible to determine whether the immunoreactivity represented Sm-C/IGF I, IGF II, or both. Liver contained the greatest proportion of immunoreactive cells, while the thymus and spleen had only a few immunostained cells. With the possible exception of dermal and some adrenal cortical cells, the immunoreactive cells do not appear to be the primary sites of Sm/IGF synthesis, because parallel in situ hybridization histochemical studies using Sm/IGF oligodeoxyribonucleotide probes show that Sm/IGF mRNAs are localized predominantly to fibroblasts and mesenchymal cells. Therefore the immunoreactive cells identified in this study may define sites of action of Sm/IGFs.

Adrenal Glands↗
PubMed1987

Effects of human placental lactogen and growth hormone on the production of insulin and somatomedin C/insulin-like growth factor I by human fetal pancreas in tissue culture.

We have investigated the ability of glucose, human GH and human placental lactogen (hPL) to alter the content and release of somatomedin C/insulin-like growth factor I (SM-C/IGF-I), and the biosynthesis, content and release of insulin from cultured human fetal pancreas. Fetal pancreatic explants obtained from glands following prostaglandin-induced abortion between 12 and 21 weeks of gestation were maintained in free-floating culture for 3-5 days before the experiments. The explants were then cultured for 3 days in medium containing either 2.7 or 16.7 mmol glucose/l with or without GH (4.5 or 45.5 nmol/l) or hPL (4.6 or 46.5 nmol/l). Serum-free medium from the final 24 h of culture was collected and SM-C/IGF-I and insulin were measured radioimmunologically in both conditioned medium and tissue explants extracted with acid ethanol. Insulin biosynthesis, determined by immunoprecipitation of [3H]leucine incorporated into insulin, was not significantly altered by any experimental variable. Incubation in the presence of 16.7 mmol glucose/l caused an increase of insulin release from explants, but had no consistent action on insulin content, compared with medium containing 2.7 mmol glucose/l. The pancreatic content and release of SM-C/IGF-I were independent of these glucose concentrations. Neither GH nor hPL altered insulin or SM-C/IGF-I content or release in the presence of the lower glucose concentration. At the higher glucose concentration, 45.5 nmol GH/l did not alter insulin release but caused a significant increase in SM-C/IGF-I content.(ABSTRACT TRUNCATED AT 250 WORDS)

Culture Techniques↗
PubMed1987

Growth hormone regulation of somatomedin C/insulin-like growth factor I production and DNA replication in fetal rat islets in tissue culture.

The regulation of DNA replication by growth hormone and the production of somatomedin C/insulin-like growth factor I (SM-C/IGF-I) and insulin by fetal rat islets in culture has been studied. Islets were cultured for 3 days in medium containing 2.7 or 16.7 mM glucose, various concentrations of fetal calf serum (FCS), and 100-1000 ng/ml human growth hormone (GH). DNA replication was determined by incorporation of [3H]thymidine into islet DNA; SM-C/IGF-I and insulin secreted into the medium were measured by specific radioimmunoassays. Glucose caused a twofold stimulation of islet DNA replication in medium containing greater than or equal to 1% FCS but failed to stimulate DNA replication at lower serum concentrations. In the presence of 16.7 mM glucose, GH (100-1000 ng/ml) stimulated DNA replication at all serum concentrations. In medium containing 2.7 mM glucose, GH was stimulatory only in the presence of 1% FCS. Somatomedin C/IGF-I release into the culture medium could be detected in all experimental groups. Glucose alone did not affect SM-C/IGF-I release, and in serum concentrations less than 0.1% FCS, GH also failed to increase the release of the peptide. In medium containing 1% FCS and 16.7 mM glucose, 100-1000 ng/ml GH caused a 50-100% increase in SM-C/IGF-I release into the medium. Addition of 100 ng/ml exogenous SM-C/IGF-I to medium containing 16.7 mM glucose and 0.1-1.0% FCS caused a twofold stimulation of the islet DNA replication. This effect could be abolished by the addition of an antibody to SM-C/IGF-I.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗