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D W Gray

Publications and source records attributed to D W Gray.

At least 109 records · Page 6Linked to original sources
PubMed1989

The role of exocrine tissue in pancreatic islet transplantation.

Isolated pancreatic islet transplantation has been proposed as a possible way of treating diabetes, but despite extensive experimental research, successful clinical transplantation remains elusive. A major problem has been the isolation of sufficient viable islet tissue for transplantation, especially from the human pancreas. It is possible to improve the yield of islet tissue by omitting purification steps, and unpurified dispersed pancreas has been successfully transplanted experimentally. However, attempts to apply the same technique clinically have been unsuccessful and have produced unacceptable complications. There is evidence that exocrine contamination may impair the implantation of islet tissue when transplanted to restricted sites, such as the kidney capsule. Yet, complete purification of islet tissue is probably not necessary for safe transplantation with adequate implantation of tissue in sites such as the spleen or liver. Exocrine tissue may be more immunogenic than islet tissue, and complete purification may have advantages for the prevention of rejection.

Animals↗
PubMed1989

Pancreatic islet transplantation.

Successful transplantation of the endocrine pancreas in Type I diabetes with complete correction of the glucose metabolic abnormalities may prevent progression or development of the microangiopathic complications of the disease. This could be achieved by transplantation of the whole pancreas as a vascularized graft (the only available clinical approach at present), transplantation of isolated islets or of fetal pancreas. Since transplantation of the pancreas as a vascularized graft is associated with a significant peri-operative morbidity and requires immunosuppression to prevent rejection it is unlikely to be applicable at a relatively early stage of the disease, before microangiopathic complications become irreversible. For this reason transplantation of pancreatic islets or fetal pancreas is the only approach likely to be acceptable. Transplantation of isolated islets in the rodent does correct experimental diabetes, but recurrence of disease is seen in the spontaneous diabetes of the Biobreeding (BB) rat and the non-obese diabetic (NOD) mouse. Furthermore suppression of rejection of allogenic islets has proved difficult. In large mammals, including man, the lack of a technique to prepare isolated islets in a relatively pure form has been a further obstacle to development. Recently this problem has been overcome, allowing separation of islets in relatively large numbers with adequate purity, bringing closer the time when clinical trials might be considered. However, rejection and disease recurrence are likely to remain major obstacles, although experimental work suggests that both can be overcome.

Animals↗
PubMed1989

Protease activity in pancreatic islet isolation by enzymatic digestion.

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)

Animals↗
PubMed1989

Separation of pancreatic islets by fluorescence-activated sorting.

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.

Animals↗
PubMed1989

Metabolic function of intraportal and intrasplenic islet autografts in cynomolgus monkeys.

Intraportal islet autografting can restore near-normal glucose homeostasis in large diabetic animals, but the long-term failure rate of such grafts remains high. To assess the effect of the site of transplantation, we compared the hormonal responses to glucose (500 mg/kg i.v.) of intraportal (IP) and intrasplenic (IS) islet autografts in the cynomolgus monkey previously rendered diabetic by total pancreatectomy. Intravenous glucose tolerance tests (IVGTTs) 6 wk after IP grafting (n = 10) demonstrated nearly normal plasma glucose changes, with qualitatively normal but quantitatively reduced insulin and glucagon responses; only two animals have maintained these responses for greater than 2 yr. IVGTTs 6 wk after IS grafting (n = 4) demonstrated more abnormal plasma glucose changes, with qualitatively normal but weak insulin responses and glucagon levels that did not fall in response to hyperglycemia; only one animal has maintained fasting normoglycemia for greater than 9 mo. These results suggest that IS transplantation confers no benefit over IP transplantation in this model.

Animals↗
PubMed1989

Normalization of hyperglycemia in diabetic rats by intraportal transplantation of cryopreserved islets from four donors.

Techniques for freezing rat islets have been examined by the intensive use of the supravital stains fluorescein diacetate and ethidium bromide. By the use of a simple scoring system, the effect of the cooling rate, treatment with dimethyl sulfoxide (DMSO), rate of thawing, and postthaw culture were examined. These studies showed the most effective method to be a 24-h culture of islets, followed by partial incubation with 20% DMSO at 0 degrees C, followed by seeding at -8 degrees C in an alcohol bath. The islets were then cooled at a rate of -0.25 degrees C/min to -40 degrees C followed by quenching in liquid nitrogen at -196 degrees C. Rapid thawing at 37 degrees C was then followed by a 24-h culture. Islets from four Lewis rat donors were cryopreserved, counted, and transplanted intraportally into streptozocin-induced diabetic Lewis rats. Corresponding control transplants were performed with islets from four donors only cultured for 48 h. The results showed that reversal of hyperglycemia in severely diabetic rats was obtained at 5, 5, 6, 6, 6, or 8 days with cryopreserved islets from four donors, compared to reversal of diabetes at 1, 4, 5, 6, 7, and 12 days with islets from four donors subjected to culture alone. The new cryopreservation technique has several small modifications over previously described methods and results in a significant improvement in islet survival.

Animals↗
PubMed1988

Exocrine contamination impairs implantation of pancreatic islets transplanted beneath the kidney capsule.

The effect of exocrine contamination on islets implanted under the kidney capsule has been studied by histological examination of pure or exocrine-contamination human, monkey, or rat islets transplanted to the kidney capsule of the nude rat, monkey, or rat, respectively. Exocrine contamination resulted in an appearance suggestive of impaired islet implantation, due to tissue necrosis and subsequent fibrosis. The effect of exocrine contamination was examined quantitatively in a rat islet isograft model in which handpicked DA rat islets were transplanted under the kidney capsule of normal DA rats. The islets were either pure or deliberately recontaminated with exocrine tissue (50 or 90% contamination). Four hundred pure islets were placed under one kidney capsule and 400 islets (of similar size and from the same islet preparation) were contaminated and then placed under the contralateral kidney capsule. After 2 weeks the kidneys were removed and extracted for insulin content. The insulin content of kidneys bearing islets contaminated by either 50 or 90% exocrine tissue was significantly reduced when compared to the contralateral kidney bearing pure islets. These findings support the view that exocrine contamination of islets resulted in impaired islet implantation when transplanted to a confined site such as the kidney subcapsule.

Animals↗
PubMed1988

Is abdominal wall tenderness a useful sign in the diagnosis of non-specific abdominal pain?

Pain arising from the abdominal wall has been implicated as a cause of non-specific abdominal pain (NSAP), and the presence of abdominal wall tenderness (AWT) has been proposed as an accurate diagnostic test for NSAP. One hundred and fifty eight patients admitted to hospital with abdominal pain were tested for the presence of positive AWT. In 53 patients the final diagnosis was appendicitis and positive AWT was found in five. Thirty eight patients were found to have a variety of other recognised pathological diagnoses, none of whom had a positive AWT. In 67 patients a diagnosis of NSAP was made in the absence of other pathological diagnosis, 19 of whom had positive AWT, which was significantly different from the other diagnostic groups. This study confirms the presence of AWT in up to 28% of patients with NSAP, and suggests that testing for AWT is of value in patients with abdominal pain, although a positive AWT is not as accurate a predictor of NSAP as previously reported.

Abdomen↗
PubMed1987

Regulation and specificity of glucose-stimulated insulin gene expression in human islets of Langerhans.

The insulin response of cultured human islets of Langerhans was measured at both mRNA and polypeptide levels in response to natural and pharmacological stimuli. We report a dosage dependent stimulation of both mRNA levels and insulin secretion by extracellular glucose, and present evidence that islet responsiveness can be divided into two temporal phases: an early response, apparently under post-transcriptional control, and a late phase in which insulin messenger accumulates. Although glucose effects in man are similar to rodents, there are important differences, especially with respect to modulation of glucose stimulation by activators of beta-cell protein kinases.

Cells, Cultured↗
PubMed1987

Survival of cryopreserved isolated adult human pancreatic islets of Langerhans.

Human islets of Langerhans were isolated from the pancreata of 13 adult organ donors, cultured for 24 h, cryopreserved, stored in liquid nitrogen at -196 degrees C for 6-88 days, thawed, and then cultured again. The number of islets recovered after this procedure was 80% of that present at the beginning. The viability of cultured/cryopreserved islets was then compared with that of islets from the same donor cultured for 24 hr, and was assessed by supravital staining, insulin response to glucose, and survival after implantation under the kidney capsule of nude rats. Supravital staining showed more nonviable cells in cryopreserved islets than in their cultured counterparts. Significant response to glucose was seen before and after cryopreservation in 2 of 4 sets of islets. Xenografts of 200 cultured islets (from 13 donors) were implanted in 15 nude rats under the kidney capsule, and a further 15 rats had cryopreserved islets (from the same 13 donors) similarly implanted beneath the kidney capsule. Two weeks later tissue was visible at the site of implantation in all 30 rats. Histological examination of both groups showed the tissue to have the morphology of islets, confirmed by immunohistochemical chemical localization of insulin. The insulin content of kidneys bearing 200 cultured islets was 7.88 +/- 1.6 mU (n = 13) versus 6.84 +/- 1.43 mU (n = 13) for kidneys bearing cryopreserved islets. Thus this technique for cryopreservation of isolated adult human islets enables a high recovery of endocrine tissue that survives after transplantation to nude rats, but some evidence of damage was apparent from insulin secretion studies and electron-microscopic studies.

Animals↗
PubMed1987

The use of fluorescein diacetate and ethidium bromide as a viability stain for isolated islets of Langerhans.

There is a need for a simple, rapid, sensitive method for assessing the viability of isolated islets of Langerhans. In this study the fluorescent dyes fluorescein diacetate (FDA) and ethidium bromide (EB) have been used to provide a viability assay for isolated rat islets. Discrimination of living from dead islets is efficient; in a blind sorting experiment using freshly isolated islets and islets killed by either heat or alcohol, viability determined by insulin secretion in response to glucose stimulation correlated well with viability as determined by FDA/EB staining. Furthermore, it is possible to discriminate degrees of viability, and a scoring system is described for this purpose which is shown to correlate with another index of viability, the ATP content. A reliable viability stain should not itself be toxic; FDA/EB stained islets remain viable after staining, showing normal response to glucose stimulation and normal function after transplantation.

Animals↗