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Biomedical subjects

B E Tuch

Publications and source records attributed to B E Tuch.

At least 55 records · Page 3Linked to original sources
PubMed1993

Resistance of the human fetal beta-cell to the toxic effect of multiple low-dose streptozotocin.

The human fetal beta-cell is different from the adult beta-cell not only in that it is unable to release significant amounts of insulin during an acute glucose challenge, but also in that it is resistant to the toxic effects of a bolus of streptozotocin (STZ) and chronic exposure to cytokines tumour necrosis factor-alpha and interferon-gamma. To examine further the differences between the immature and mature beta-cell, these cells were exposed to multiple low doses of STZ (MLS) both in vivo and in vitro, the regimen used being classically toxic to rodent adult beta-cells. The in vivo experiments were conducted after the tissue had been grafted into the athymic mouse. There was no adverse effect of MLS on the insulin content of engrafted human fetal pancreatic explants, either 1, 4, or 15 weeks after the injection of 70 mg/kg/i.p. STZ daily for 5 days. Diabetes, induced in the mice by MLS or a bolus of STZ, was reversed in five of seven MLS-treated mice at 59 +/- 8 days and four of seven STZ-treated animals at 75 +/- 10 days (no significant difference). The growth rate of the implanted tissues also was not adversely affected by the MLS, nor was the percentage of beta-cells 2 weeks after completion of the MLS. In contrast, mouse fetal beta-cells transplanted into the athymic mouse were adversely affected, their insulin content 2 weeks after MLS being only 0.7% that of controls.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1993

Chronic hyperglycemia and the human fetal beta cell.

It is well known that the ability of the immature rodent fetal beta cell to release insulin in response to a glucose challenge can be enhanced by chronic exposure to a high concentration of glucose in vitro. It might be thought that the human fetal beta cell would mature similarly in vitro, because neonates born of diabetic mothers release insulin in a more mature manner than normal infants. Using an organ culture of human fetal pancreatic explants, we have examined this possibility by exposing the tissue to 0-30 mM glucose. Six weeks of exposure of pancreatic explants to as high a concentration of glucose as 30 mM did not cause significant enhancement of the insulinogenic response to an acute challenge with 20 mM glucose. In contrast, chronic insulin release was enhanced, although culture medium containing 2.8 mM glucose was equally as efficacious as that with 30 mM glucose. Just as with insulin, proinsulin levels in the culture media containing no glucose also were suppressed. Degranulation of the beta cell exposed to high concentrations of glucose did not occur, the insulin content of the explants at the end of culture being enhanced in those maintained in 5.6-30 mM but not 2.8 mM glucose. Desensitization to the acute stimulatory effect of 10 mM theophylline did not eventuate, even in explants exposed to 30 mM glucose. In contrast to the human explants, rat fetal pancreatic explants did mature when exposed to 11.2 mM glucose for 1 week.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Release of proinsulin from the human fetal beta cell.

beta cells in the human fetal pancreas are immature in that they release little or no insulin in response to nutrients, such as glucose. The aim of this study was to examine further the immaturity of these cells, specifically regarding the storage and release of the precursor of insulin, proinsulin. Explants of human fetal pancreas were cultured in vitro for 3 weeks. Levels of proinsulin remained relatively constant throughout at 0.04 +/- 0.002 (S.E.M.) pmol/mg per day with a molar ratio of proinsulin to insulin of 2.2 +/- 0.11%. This low ratio was slightly greater than that observed in culture medium conditioned by adult human islets (0.3 +/- 0.1%), but similar to that found in acid-ethanol extracts of cultured explants (1.4 +/- 0.3%). Passaging of human fetal pancreas for 3 months in diabetic nude mice, which should have caused some maturation of the fetal beta cell, did not change the proportion of proinsulin present. Culture of explants in the presence of 12-O-tetra-decanoylphorbol-13-acetate resulted in some inhibition of proinsulin release, but much less than that for insulin, so that the molar ratio increased to 15.4 +/- 1.6% from the control 3.5 +/- 0.3%. Static stimulation of cultured explants with 10 mmol Ca2+/l, 10 mmol theophylline/l, and these two agents together caused 15-, 4- and 10-fold enhancement respectively of proinsulin release; glucose, leucine, arginine and KCl had no effect. In contrast, all these agents caused significant insulin release, the last four to a much smaller extent (less than or equal to three fold) than the first three (10-, 19- and 65-fold respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Reversal of diabetes by human fetal pancreas. Optimization of requirements in the hyperglycemic nude mouse.

It has been established previously that the grafted human fetal pancreas takes 2-3 months to reverse diabetes in a recipient hyperglycemic immunoincompetent mouse. The factors responsible for this have never been clarified. It was the aim of these experiments to investigate the effect of grafting various amounts of human fetal pancreas of gestational age 14-20 weeks into these animals, to determine if the time required for normalization of blood glucose levels could be altered. The amount of tissue grafted ranged from 2.6-49 mg (median 12.6 mg), which is the equivalent of 8-100% (median 31%) of a complete pancreas. The 25 diabetic nude mice became normoglycemic 66 days (range 28-147) after administration of streptozotocin, with normal or better-than-normal responses in an oral glucose tolerance test, and detectable human C-peptide levels, which fluctuated in parallel with the glucose levels. The minimal time required for correction of diabetes was 28 days, this occurring when 49 mg of tissue was grafted. Blood glucose levels rose from 2.9 +/- 0.3 mmol/L to 21.3 +/- 1.9 mmol/L after removal of the implants. Analysis of the results by stepwise multiple-linear-regression analysis revealed that the time required for normalization of blood glucose levels was dependent on three unrelated factors: the amount of tissue grafted (P = 0.002); the age of the fetal pancreas, as determined by the heel-toe length (P = 0.042); and the rate of growth of the implant (P = 0.031). These data demonstrate that it is advantageous to graft as much human fetal pancreas as possible, ideally from fetuses as old as possible, in order to achieve the most rapid reversal of diabetes in athymic mice. The clinical relevance of this information remains to be established.

Animals↗
PubMed1991

Regulation of blood glucose to human levels by human fetal pancreatic xenografts.

Previous experiments xenografting human fetal pancreas into athymic mice made diabetic with streptozotocin have demonstrated that normoglycemia can be achieved 1-3 months after implantation, but that the blood glucose levels obtained were significantly lower than those of control mice. These lower levels could be due either to genetic regulation by the grafted human beta cell--human neonatal blood glucose levels are lower than those of athymic mice--or to excess release of insulin from the increasing number of beta cells in the implant. In order to address the latter possibility, human fetal pancreas was grafted beneath the renal capsule of athymic mice, the pancreas of which was intact, and they and their ungrafted litter mates monitored during their life span--71 +/- 17 weeks after surgery for the 4 successfully grafted mice. The random blood glucose level in all mice was the same initially and remained so until a significant lowering 20 weeks later--2.0 +/- 0.2 vs. 3.4 +/- 0.4; this difference was maintained thereafter. Data obtained from the oral glucose tolerance tests conducted at 8 weekly intervals paralleled these results. Human C-peptide was detectable in blood by 26 weeks, with a rise demonstrable during the oral glucose tolerance test. Peak levels did not change during the ensuing year. At no stage did any of the mice develop clinical hypoglycemia, despite an annual 23 +/- 6-fold increase in size of the graft and the presence of 0.3-9.2 U insulin therein. The insulin content of the mouse pancreas (0.08 +/- 0.03 U) was unaffected by the presence of the human fetal beta cells. These data show that the xenografted human fetal pancreas grows and releases insulin in a controlled but not excessive manner, with the blood glucose levels probably being determined by genetic information stored in the grafted beta cell.

Animals↗
PubMed1991

Role of tumor necrosis factor-alpha and interferon-gamma as growth factors to the human fetal beta-cell.

The effects of the cytokines tumor necrosis factor-alpha and interferon-gamma on the adult beta-cell have been well described: a reduction of insulin secretion and content and death of the cell. For this reason and because these cytokines may be released from activated lymphocytes and macrophages that infiltrate islets in insulin-dependent diabetes, they have been implicated in the pathophysiology of this form of diabetes. As to whether the human fetal beta-cell, which differs from the adult beta-cell in not releasing insulin in response to the nutrient glucose and not being adversely affected by the toxin streptozotocin, is similarly affected is unknown. To examine this question we cultured monolayers of a single cell suspension of human fetal pancreas in the presence or absence of 1000 U/mL of these cytokines for 7 days. Chronic insulin release was enhanced for the first 2 days of culture, but unchanged thereafter. Acute insulin release in response to the secretagogue theophylline (10 mM) was enhanced on day 7, but not earlier. There was an increase in the insulin content of the cells by the fourth day, probably due to an increase in the number of beta-cells present (45 +/- 5% vs. 22 +/- 3%). Microscopically, non-beta-cells also seemed to increase in number; there was an increase in both DNA and cell number by the seventh day. In contrast to these beneficial effects on the human fetal beta-cell, treatment of adult rat insulinoma cells, represented by RIN-m5F cells, resulted in inhibition of insulin secretion during the first day of culture and subsequent death of 86% of the cells by the sixth day of culture. It is hypothesized that the functional immaturity and lack of normal (adult) metabolic activity of the human fetal beta-cell somehow confers protection on these cells from the cytotoxic effects of tumor necrosis factor-alpha and interferon-gamma. Indeed, our findings suggest that these cytokines may be trophic for the developing beta-cell.

Animals↗
PubMed1991

Characterization of endocrine-rich monolayers of human fetal pancreas that display reduced immunogenicity.

Fibroblast-free insulin-secreting monolayers of human fetal pancreas (14-20 wk of gestation) were formed by plating isletlike cell clusters (ICCs) obtained from partially digested pancreases on plates coated with bovine corneal matrix. Human fetal pancreatic cells, freshly digested with collagen, displayed a 17-fold response to human peripheral blood lymphocytes (HPBLs) in mixed-lymphocyte culture. After 14 days in culture, monolayers derived from ICCs exhibited a smaller, twofold response to HPBLs. By comparison, in monolayers produced from single-cell suspensions, fibroblast overgrowth remained a problem. The endocrine component of the monolayers was 65 +/- 13 and 43 +/- 8%, respectively, with the number of beta-cells being 51 and 9%. Cells from both monolayers displayed increased insulin release when exposed to 10 mM theophylline, 10 mM Ca2+, and 0.6-1.3 microM 12-O-tetradecanoylphorbol-13-acetate but not to 20 mM glucose. Monolayers derived from ICCs synthesized DNA, proinsulin, and protein. This study showed that it is possible to establish an endocrine-rich monolayer of human fetal pancreas that has greatly reduced immunogenicity. The existence of residual activity to HPBLs suggests some additional form of immunosuppression is required to prevent rejection of this tissue when grafted into diabetic patients. Subculturing and cryopreservation may also be needed to achieve adequate numbers of beta-cells for clinical transplantation.

Calcium↗
PubMed1990

The role of calcium in insulin release from the human fetal pancreas.

Previous experiments have established that the human fetal pancreas is relatively unresponsive to glucose as regards insulin release, but will secrete this hormone when exposed to agents which increase levels of cAMP or which activate protein kinase C. The current experiments were designed to establish which role another major stimulus, calcium, had in the release of insulin from this organ. For this purpose, cultured explants of human fetal pancreas were exposed to stimuli either in static or dynamic stimulation. The data show that insulin release is enhanced in the presence of 10 mM Ca2+, as well as the calcium ionophores A23187 and ionomycin, the latter agent being effective only if extracellular Ca2+ was present. A biphasic response was seen for Ca2+ but only a second phase response for A23187. Voltage-dependent calcium channels were shown to be present by the ability of the calcium channel blocker, verapamil, to inhibit insulin release caused by an agent that depolarizes membranes, potassium. The essential role of extracellular calcium in the insulinogenic effect of agents which increase cAMP levels--theophylline--and which activate protein kinase C--12-O-tetradecanoylphorbol-13-acetate--was demonstrated by showing (a) partial inhibition of insulin secretion by calcium channel blockers, (b) no enhancement of insulin release in the absence of extracellular calcium and (c) greater enhancement of insulin release in the presence of the calcium channel activator BAY-K-8644, which caused no stimulation by itself. These data put into better perspective our understanding of the mechanisms involved in insulin release from the human fetal pancreas.(ABSTRACT TRUNCATED AT 250 WORDS)

Biological Transport, Active↗
PubMed1990

Grafting of fetal pancreata into neonatal rat brain.

Undigested fetal pancreatic tissue has been previously shown to have immunogenic properties, even after transplantation into the adult rat brain, a relatively immunoprivileged site. In the present study, iso-, allo-, and xenografts of fetal pancreas were placed into neonatal rat brain parenchyma and ventricles in order to determine the extent and quality of its survival in this environment. Adult recipients of the same tissue types were used as controls. Selective survival of insulin-staining beta cells was observed in neonates (n = 33) over the 6-week period of the experiment. Ducts and acini were gradually destroyed in allo- and xenografts, disappearing completely by the 42nd day, while there were no such changes in the isografts. The absence of an acute inflammatory reaction was noted, but there were varying degrees of lymphocytic infiltration, though small (20 +/- 4 lymphocytes per average graft area of 0.16 mm2) in all but one graft. This infiltrate was greatest in allografts, with a significant increase observed after 14 days, corresponding to the time when the ducts started to disappear. Other structures present included fibroblasts and blood vessels. The latter increased significantly with time after transplantation. Unlike isografts placed in the parenchyma of adult rats, allo- and xenografts were rejected from the earliest time observed, 7 days postoperatively. In summary, these data show that beta cells in rat fetal pancreas will survive when grafted across major allogeneic and xenogeneic barriers for up to 6 weeks, without utilization of any form of immunosuppression, provided the recipients are neonates.

Age Factors↗