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Biomedical subjects

B E Tuch

Publications and source records attributed to B E Tuch.

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

Pig fetal pancreatic monolayers. A model of potential use in transplantation.

Endocrine-rich monolayers of pig fetal pancreas that are free of fibroblasts have been established with the ultimate aim of providing guidelines for the culture of the human equivalent. The immunogenic potential of the monolayers--hence their capacity to be grafted--has also been analyzed. Fetuses ranging from 50 to 90 days were used, and, following digestion with collagenase (4 mg/ml, 15-20 min), the pancreatic suspension was plated onto tissue culture vessels containing RPMI 1640. The fetal calf serum concentration was kept low (5%) initially to inhibit fibroblast proliferation, but subsequently increased to 7%. Monolayers from a typical litter of 8-10 fetal pigs produced 6-8 x 10(8) viable epithelial cells by day 10 of culture, of which 75% were endocrine cells. This represents an 8-fold increase in a two-week period. The ratio of beta:alpha:delta:pancreatic polypeptide cells was 19:33:18:5. These monolayers synthesized both DNA, (pro)insulin and protein, and displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+ and 1.3 microM 12-0-tetradecanoyl-phorbol-13-acetate. Static stimulation with 20 mM glucose however, did not elicit a response in insulin secretion. These cells displayed no reaction to allogeneic lymphocytes in a mixed lymphocyte culture, whereas freshly obtained porcine epithelial cells did. Methods may need to be found to increase the proportion of B cells in this enriched endocrine cell population. In general however, guidelines have been established that may be useful in developing a monolayer of human fetal pancreatic cells with the eventual aim of transplantation. The reduction in immunogenicity of the pig fetal pancreatic cells suggests that they too might be a potential source for transplantation.

Animals↗
PubMed1990

Maturation of insulinogenic response to glucose in human fetal pancreas with retinoic acid.

Although the human fetal pancreas early in the second trimester of pregnancy contains functional beta cells, its ability to release insulin in response to glucose is either poor or lacking. It is tissue of this age which usually has been grafted into diabetic humans, in unsuccessful attempts so far, to reverse the hyperglycemic state. One of the explanations for this failure may be the inability of the transplanted fetal tissue to mature. For this reason, it is important to establish a method of maturing the tissue in vitro before grafting. We have cultured explants of human fetal pancreas for a period of three weeks in the presence of an agent known to induce the differentiation of cells, retinoic acid. This substance, at a concentration of 1 microM, was able to induce both a first and second phase insulinogenic response to 20 mM glucose. No increase in insulin release was observed throughout the period of culture; nor was the insulin content of the treated explants different from the controls. Explants exposed to retinoic acid and then grafted into diabetic nude mice were able to normalize the elevated blood glucose levels of these animals after 54-93 days. Oral glucose tolerance tests were normalized and glucose-responsive human C-peptide was measurable in the plasma. Removal of the grafts, which contained 2.92 +/- 0.78 mU/mg insulin resulted in recurrence of hyperglycemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Streptozotocin is not toxic to the human fetal B cell.

It has been generally assumed that because streptozotocin is toxic to the adult B cell of most species, it should also damage B cells obtained at earlier stages of development. This paper examines whether this is true for human fetal pancreata obtained from the therapeutic termination of pregnancies during the first half of the second trimester. Experiments were carried out both in vivo and in vitro. For the former experiments diced explants of the human fetal pancreas were grafted beneath the renal capsule of nude mice 3 weeks before streptozotocin was administered to make the animals diabetic. The grafts were removed 1 week, 2-4 weeks or 3 months later, and were found to be of similar weight and insulin content to the control grafts. In 2 of the animals with grafts remaining for 3 months the diabetes had even been reversed by the implant, hyperglycaemia recurring when the graft was removed. In contrast, rat fetal pancreata grafted beneath the renal capsule of nude mice, subsequently rendered diabetic, were adversely affected by streptozotocin, the insulin content of the implants being 12% of levels in control grafts. Adequate uptake of streptozotocin by the implanted human fetal pancreas was established by measuring tissue levels of the drug 30 min after its injection. Histological examination of the grafted human fetal pancreas showed no deleterious effect of streptozotocin on the number of granulated B cells one day after injection, although by this time the host was diabetic.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Effect of normoglycemia on the functional maturation of the human fetal beta cell.

Previous studies from this laboratory have shown that human fetal pancreas of gestational age 14-20 weeks was capable not only of growing but also of maturing in its insulinogenic response to an acute static challenge with glucose, once it was removed from its usual physiological environment and passaged for up to 37 weeks in the diabetic nude mouse (absolute age of 58 weeks, where absolute age = gestational age + duration of passage in the mouse). In order to analyze the dynamics of insulin secretion during this process of maturation, these experiments were repeated, the tissue being perifused after removal from the mouse. Normoglycemic nude mice rather than diabetic ones were used because of their hardier nature. Human fetal pancreata of gestational age 14-20 weeks were passaged in these normoglycemic animals, either subcutaneously or beneath the renal capsule, for 11 to 70 weeks (absolute age up to 86 weeks). The tissue was removed and, after a mean of 1 day in organ culture, perifused for 50 min with 20 mmol/L of glucose. While overall there was a slight but significant increase in insulin secretion over basal levels, there was no time-dependent maturation of the response to glucose, and no adult-type response at any stage, as occurs both physiologically and in tissue passaged in the diabetic nude mouse by this period.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Production of insulin-like growth factor-II by human fetal pancreas in culture.

Insulin-like growth factor-II (IGF-II) is a polypeptide hormone thought to be involved in fetal development because of its wide distribution in fetal tissues and its presence in the fetal circulation. We have developed a highly sensitive radioreceptor assay for IGF-II using rat liver microsomal membranes and have used this assay to test for the presence of IGF-II in the human fetal pancreas and the release of IGF-II by the human fetal pancreas in organ culture. IGF-II was present in extracts of pancreatic tissue (0.056 +/- 0.012 pmol/mg tissue, n = 5) and was released in culture at the rate of 0.027-0.134 pmol/mg tissue per day with release being maintained for at least 3 weeks in culture. The rate of release was not affected by the gestational age of the fetus over 14 to 20 weeks but was significantly related to the rate of insulin release (r = 0.712, P less than 0.001, n = 34). Chronic exposure to 12-0-tetradecanoylphorbol-13-acetate (TPA), which inhibits insulin release in the human fetal pancreas, caused an 85% drop in IGF-II production, which was reversed when TPA was removed. These studies demonstrate that IGF-II is produced by the human fetal pancreas in a pattern similar to that of insulin. We suggest that control of IGF-II release, like that of insulin, may involve protein-kinase C and that IGF-II may have a paracrine or autocrine role in the development of fetal pancreatic function.

Fetus↗
PubMed1988

Chronic stimulation of human fetal pancreas with phorbol inhibits insulin secretion.

Acute exposure to agents that activate protein kinase C is known to cause insulin release both from the fetal and adult pancreas. These experiments were designed to test the effect of chronic exposure of the human fetal pancreas to such agents. Nine to twelve days after commencement of culture of this tissue, exposed to 0.165-1.3 microM 12-O-tetradecanoylphorbol-13-acetate, insulin secretion was reduced and remained less than that for controls thereafter. Exchange of the test for the control medium resulted in partial recovery of insulin release. Insulin content of the treated explants was also significantly reduced. The insulinogenic response to an acute challenge of either 20 mM glucose or 10 mM theophylline/2.8 mM glucose at the end of the culture was no different from that for controls.

Enzyme Activation↗
PubMed1988

Immunomodulation of human fetal cells by the fungal metabolite gliotoxin.

Gliotoxin (GT) is a fungal metabolite that reduces the ability of murine macrophages to react immunologically in vitro. It is also capable of modulating the immunogenicity of murine bone marrow cells, so that the onset of graft-versus-host disease in fully allogeneic bone marrow chimeras is delayed. The present study examines the effect of GT on human fetal cells, both in terms of reduction of immunogenicity and toxicity. GT (10 micrograms/ml) significantly decreased the responsiveness in mixed lymphocyte cultures of cells derived from human fetal pancreas, spleen, liver and bone marrow. This concentration of GT was, however, mildly toxic to explants of the pancreas, with a significant reduction in insulin secretion from this tissue during the first day of its organ culture, but not thereafter. GT-treated pancreatic explants were lighter and contained less insulin than the untreated controls 3 months after the tissue had been implanted beneath the renal capsule of nude mice. This difference was not apparent 3 weeks after transplantation into these animals. It is hypothesized that the immunomodulating effect of GT (at a concentration less than 10 micrograms/ml) may be of benefit in treating allografted human fetal pancreas before it is transplanted, as it has for murine adult bone marrow cells.

Bone Marrow↗
PubMed1988

Activators of protein kinase C stimulate insulin secretion from the human fetal pancreas.

The human fetal pancreas, like the adult pancreas, releases insulin in response to agents that increase intracellular levels of calcium and cyclic AMP. It differs from the adult pancreas in that glucose causes either minimal or no insulin secretion from it. Whether activation of protein kinase C will cause release of insulin from this immature cell, as it does from the adult pancreas, is unknown. This was the phenomenon examined in the experiments below. Activators of protein kinase C, 1.3 microM 12-O-tetradecanoylphorbol-13-acetate (TPA) and 25.1 microM 1-oleoyl-2-acetylglycerol, caused significant release of insulin from cultured explants of human fetal pancreas. Lower concentrations of TPA--0.65 microM or less--had no such effect. Agents that are known to inhibit protein kinase C, 100 microM 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, 2 mM polymyxin B, and 1 mM dibucaine, caused abolition of the insulinogenic effect of 1.3 microM TPA. Glucose, at 2.8, 5, and 10 mM, also inhibited the positive effect of TPA. However, when the concentration of glucose was increased to 20 or 30 mM, stimulation of insulin secretion returned to levels achieved with TPA alone. These levels were significantly higher than the minimal response achieved with 20 mM glucose. Such data support the involvement of protein kinase C activation in insulin secretion from the human fetal pancreas. It is postulated that this involvement may occur physiologically.

Enzyme Activation↗