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Query: UMLS:C0011849 (diabetes)
 277,896 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ability of the pancreatic beta-cell to repair itself after a cytotoxic injury and reassume its functional activities may be a key issue in affording protection from insulin-dependent diabetes mellitus. The molecular mechanisms behind the functional responses of the beta-cell after cytotoxic damage are still largely unknown. The present study in an attempt to elucidate this issue. Mouse pancreatic islets were isolated with collagenase and, after overnight culture, exposed for 30 min at 37 C to 2.2 mM streptozotocin (SZ) or vehicle alone (controls). The islets were subsequently cultured for 6 days in medium RPMI-1640 plus 10% calf serum. After the culture they were subjected to light microscopical examinations or different functional tests during short term incubations. The SZ-treated islets showed markedly diminished insulin release after stimulation with the beta-cell nutrients glucose and leucine plus glutamine. Compounds known to increase intracellular cAMP [theophylline and (Bu)2-cAMP] were able to partially counteract the SZ-induced reduction of insulin release. Stimulation with arginine could also slightly restore the impaired insulin release. Glucose-stimulated oxygen uptake, proinsulin biosynthesis, and insulin and insulin mRNA contents were also decreased, with values at about 50% of the controls. However, the cellular contents of DNA and RNA and total protein biosynthesis rates were essentially normal. Besides mild degranulation in some islets, the morphological appearance of the SZ-treated islets did not reveal any obvious differences compared to the control islets. The present observations suggest that after a toxic injury there remains a population of partially damaged beta-cells, which are able to maintain most of their basal metabolic functions, but fail to maintain adequate insulin biosynthesis and release.
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PMID:Preferential reduction of insulin production in mouse pancreatic islets maintained in culture after streptozotocin exposure. 245 14

Clinical pancreatic islet transplantation has been impeded by the inability to isolate an adequate mass of functional tissue that will ameliorate diabetes. A simplified method of canine islet isolation was developed that allowed for either intrasplenic or intrahepatic transplantation. Following total pancreatectomy, parenchymal digestion was accomplished by intraductal collagenase perfusion and stationary incubation. The digested tissue was dispersed by filtration through a steel mesh (400 microns), washed, and separated on a discontinuous dextran density gradient. Enhanced islet tissue (2-4 ml) was recovered from the uppermost interface of the gradient and autotransplanted. The islet isolation procedure was tested in two series of dogs undergoing either intrasplenic or intrahepatic engraftment. Immediate and sustained normoglycemia (plasma glucose less than 200 mg%) was obtained in 5 of 8 dogs (63%) in the intrasplenic group and 6 of 8 dogs (75%) in the intrahepatic group. The mean fasting plasma glucose concentration 2 weeks after transplantation was 102.8 +/- 6.4 mg% in the intrasplenic group and 103.3 +/- 8.4 mg% in the intraportal group. The mean IVGTT K-values 2 weeks after transplantation were -1.41 +/- 0.35% and -1.21 +/- 0.13%, respectively. On the basis of insulin content, the islet yield was 33.0 +/- 3.7% of the total pancreas in the intrasplenic group and 33.0 +/- 3.1% in the intrahepatic group. Islet mass was enhanced 10.2 +/- 1.5 and 20.0 +/- 6.2 fold, respectively, on the basis of insulin/amylase ratios. The success rate in this canine model compared favorably with previously published results from other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A simple method of canine pancreatic islet isolation and intrahepatic transplantation. 249 6

Preservation of the Lewis rat pancreas prior to islet isolation was accomplished by initial intraductal distension with the University of Wisconsin (UW) hydroxyethyl starch-lactobionate solution to which collagenase had been added, followed by simple cold storage at 4 degrees C for 0, 3, 12, 24, and 48 hr (n = 16-21 at each interval). The pancreases were then processed by digestion and mechanical dispersion to produce free islets of Langerhans. The mean islet yields (+/- standard errors of the means) were controls = 819 +/- 58 (n = 21), 3 hr = 867 +/- 51 (n = 20), 12 hr = 770 +/- 71 (n = 16), 24 hr = 805 +/- 62 (n = 18), and 48 hr = 722 +/- 55 (n = 16). None of these means differed significantly. The islets from pairs of donor pancreases (mean dose of islets = 1586 +/- 72) were transplanted intraportally into single isogeneic recipients with streptozotocin-induced diabetes (plasma glucose greater than 400). The preservation interval directly influenced the outcome of these islet isografts in the following manner: (i) Rates of functional success (nonfasting glucose less than 200 mg/dl) were 100% after storage times of 0 hr (n = 10), 3 hr (n = 8), and 12 hr (n = 8); 86% with a storage time of 24 hr (n = 7); and 0% after 48 hr (n = 8). (ii) Return of euglycemia was increasingly delayed with increasing preservation intervals.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Experimental pancreatic preservation prior to islet isolation and transplantation. 249 69

Using a model of streptozotocin-induced, ketosis-prone, insulin-dependent diabetes mellitus (IDDM) in the cynomolgus monkey, we performed 11 intraportal transplants of collagenase-digested, Ficoll-purified pancreatic islets (9 ABO-compatible allografts and 2 concordant baboon xenografts). Islets were pretreated with ultraviolet-B irradiation and recipients received cyclosporine A immunosuppression. Two grafts never functioned, five grafts showed evidence of partial function, and four grafts (three allografts and one xenograft) showed evidence of good function, with the animals independent of exogenous insulin with morning fasting blood glucose levels less than 200 mg/dl. Because two grafts functioned only after CsA was either tapered or discontinued, we performed a related study that showed that therapeutic doses of CsA (morning trough serum level 150-250 ng/ml) impaired intravenous glucose tolerance tests (IVGTT) of normal monkeys and may contributed to graft dysfunction in our islet transplantation model. The results show that there is a decrease in release of serum insulin during an IVGTT leading to impairment of glucose utilization, while serum glucagon remains unaffected. After cessation of CsA, the IVGTT did not return to normal for 28 days. Oral glucose tolerance tests were unaffected in CsA-treated monkeys. These initial studies show that the streptozotocin-diabetic monkey is a valuable model to study IDDM and islet transplantation in nonhuman primates. We also confirm studies in rodents, dogs, and sheep by showing that CsA partially inhibits beta cell function in normal monkeys.
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PMID:Pancreatic islet transplantation in cynomolgus monkeys. Initial studies and evidence that cyclosporine impairs glucose tolerance in normal monkeys. 251 1

Human islets of Langerhans were isolated with the principles of collagenase perfusion via the pancreatic duct and gentle dissociation of tissue. The number of islets released was 161 x 10(3), distributed as 76 x 10(3) large (greater than 100-micron) and 85 x 10(3) small (less than 100-micron) islets. Recovery after Ficoll-gradient purification was 61% for the large islets and 42% for the small islets. The final islet volume was 240 microliter, with purity of 70-90% (large islets) and 20-40% (small islets). Perifusion with glucose elicited a biphasic release of insulin, with the response rising sixfold from basal secretion. Implantation of pure islets under the kidney capsule of normal or streptozocin-induced diabetic nude mice resulted in human C-peptide secretion and partial or complete reversal of hyperglycemia, confirmed by histological recovery. The data show that these methods provide large quantities of viable purified human islets.
Diabetes 1989 Jan
PMID:Viable purified islets of Langerhans from collagenase-perfused human pancreas. 253 89

Three methods for the preparation of islets from human fetal pancreas (17.4 +/- 1.2 wk gestational age) were compared. In each method, the pancreases were minced and followed by 1) no collagenase digestion, 2) 5 min of collagenase digestion, or 3) 14 min of collagenase digestion. The culture conditions prevented adherence of the fragments. Culture for 6-7 wk of minced fetal pancreas without collagenase digestion resulted in fragments that were a mixture of cells positive for insulin or glucagon, ducts, necrotic debris, and other unidentified cells with complete degeneration of the acinar cells. Culture of minced pancreas digested for 5 min with collagenase resulted in fragments that superficially appeared to be islets but did not have the size characteristics of human fetal islets and contained fibrous and duct elements not seen in islets. Culture of minced pancreas digested for 14 min with collagenase resulted in islets that were released into the medium and harvested by picking. These islets were morphologically similar to islets of the intact human fetal pancreas and isolated islets from rat neonatal pancreas. These islets and fragments were viable for at least 7-8 wk in culture.
Diabetes 1989 Jul
PMID:Development of a method for isolation of islets from human fetal pancreas. 254 73

Dithizone, a zinc chelating agent, is known to selectively stain the islets of Langerhans in the pancreas. In the present study, we have used this stain to aid the identification of islets in material obtained by collagenase digestion of human pancreas. Islets were shown to rapidly and reversibly stain red on incubation with dithizone solution. Tissue selected on the basis of dithizone staining was shown to contain insulin-positive cells and to accumulate insulin in the medium during a subsequent period in tissue culture. Experiments with rat islets indicated that the dithizone treatment had no effect on insulin release in tissue culture, on acute responses to stimulatory glucose concentrations or on the insulin content of cells. These results suggest that dithizone staining can assist in the identification of islets from the human pancreas and may prove to be a useful tool in developing techniques for the large scale isolation of functionally intact human islets.
Diabetes Res 1989 Feb
PMID:Supravital dithizone staining in the isolation of human and rat pancreatic islets. 254 5

Commercial Collagenase* prepared from Clostridium histolyticum is widely used in isolation of pancreatic islets. It is known that the enzyme is very impure and that there are substantial variations in effectiveness between batches. Our studies suggest that one of the impurities of importance in islet isolation is a protease that has not been very well characterized. Comparison of two batches of enzyme, one of which was known to give good yields of islets and the other poor yields, showed that they had very similar activity against collagen (measured by digestion of insoluble collagen followed by assay of soluble products with ninhydrin) but substantially different activities against azocasein as measured by optical density increase (measured by release of dye). Eighteen batches of Collagenase were examined for efficiency in islet isolation, and the yields obtained correlated with manufacturer's data of activity against casein. The data show that low caseinase activity is associated with performance in islet isolation (r = .5 after adjusting for collagenase activity). The effect of supplementing a batch of collagenase, known to be poor in isolating islets, with proteolytic enzymes was investigated. Trypsin and papain had apparently no effect, but dispase significantly increased yield. Dispase alone failed to digest pancreas. Size-exclusion high-performance liquid chromatography identified a peak associated with high protease activity and efficiency in islet isolation, having an Mr of approximately 30,000, compared to 78,000 for collagenase. The protease, like collagenase, is inhibited by EDTA. Increased Ca2+ and Mg2+ (up to 10 mM) did not affect activity. Both the protease and collagenase are stable under normal use but are inactivated by heating at 56 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Diabetes 1989 Jan
PMID:Protease activity in pancreatic islet isolation by enzymatic digestion. 264 34

More efficient methods of islet isolation must be developed for islet transplantation to become clinically routine. During collagenase dispersal of human pancreas, an amorphous, viscous, gellike material often develops and entraps large numbers of islets, thereby reducing the yield. When donor human pancreas is minced and treated with collagenase, the gel forms most abundantly if the digestion temperature is less than 35 degrees C and if pH falls below 7.2 +/- 0.2. Gel formation appears to be proportional to warm- or cold-ischemia time and may be related to tissue trauma during collection. Once gel has formed, trapped islets cannot be released by filtration, dilution, DNase, incubation temperature, or pH adjustment. These characteristics suggest that the material is gelatin derived from collagen released enzymatically from pancreatic stroma. We demonstrate that gelation is greatly reduced or eliminated when 1) the incubation medium includes glycerol--a common gelatin solvent--at 5% (vol/vol), 2) the minced tissue-to-total incubation volume ratio is greater than or equal to 1:10, 3) free-islet exposure to pancreatic digestion products is minimized by frequent separation of islets, and 4) collagenase concentration is optimized by titration. Gelation is also minimized by maintaining 5) incubation temperature at 38 +/- 1 degree C and 6) pH in the range 7.7-7.9. Variations in these physical and chemical conditions were analyzed by determining islet yields (stereoscopic microscope counts of serially diluted samples) and by insulin radioimmunoassay of acid alcohol extracts of isolated islets after separation through discontinuous Ficoll gradients. When isolation conditions are optimized as stated, we typically recover 3.3 +/- 1.0 x 10(4) islets/g pancreas, corresponding to greater than 10(6) islets per donor.
Diabetes 1989 Jan
PMID:Factors influencing isolation of islets of Langerhans. 264 35

To allow clinical pancreatic islet transplantation, the yield and purity of islets must be improved. Intravital staining of islets with neutral red is a specific, nontoxic technique for labeling islets of various species. Using neutral red-stained rat islets, we investigated the known fluorescence absorbance and emission spectra in comparison with unstained exocrine tissue and have shown that stimulation with light of wavelength between 500 and 560 nm produces detectable emission greater than 610 nm, which is absent from unstained exocrine tissue. The PARTEC cell sorter is an inexpensive alternative to currently available fluorescence-activated cell sorters and has a sorting mechanism based on a piezoelectric valve. We made extensive modifications to this machine to allow passage of particles up to 300 micron diam. Using rat pancreas stained intravitally with neutral red and dispersed by intraductal collagenase technique, we have shown that islets can be accurately identified in a high-speed flow system and sorted to a purity of greater than 90% islet tissue. The islets remain intact and viable as determined by supravital staining and isogeneic transplantation to the kidney capsule site. These studies prove the feasibility of separating intact islets by fluorescence-activated sorting.
Diabetes 1989 Jan
PMID:Separation of pancreatic islets by fluorescence-activated sorting. 264 37


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