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PubMed1987

Islet amyloid polypeptide-like immunoreactivity in the islet B cells of type 2 (non-insulin-dependent) diabetic and non-diabetic individuals.

A novel peptide, islet amyloid polypeptide (IAPP), with structural resemblance to calcitonin gene-related peptide has recently been purified from amyloid deposits in an insulinoma and from islets of Langerhans. By immunohistochemical methods, using antisera to a synthetic undecapeptide of IAPP and to insulin, we show that freshly fixed islet B cells in man, guinea pig, rat, mouse and hamster exhibit strong IAPP-immunoreactivity while A cells are unreactive. In human autopsy material, all of 11 non-diabetic individuals had IAPP immunoreactivity of the islets. In comparison 8 of the 13 patients with Type 2 (non-insulin-dependent) diabetes had no IAPP immunoreactive cells. The proportion of islet cells having IAPP immunoreactivity exceeded 10% in only 1 of the 5 remaining diabetic patients while in all 13 patients substantially more than 10% of the islet cells contained immunoreactive insulin. IAPP-positive amyloid deposits were found in 20-99% of the islets in 12 of the Type 2 diabetic patients while 6 of 11 non-diabetic subjects had amyloid in 3-11% of their islets. In islets with IAPP-immunoreactive amyloid, very few IAPP-cells were seen despite a strong reaction of the B cells with antiserum to insulin. This study shows that IAPP is a normal islet B cell component and that IAPP immunoreactivity in B cells is diminished in Type 2 diabetes while IAPP is deposited as amyloid fibrils in the islets of Langerhans. Although the function of IAPP is unknown, its occurrence in the islet B cells and its structural relation to calcitonin gene-related peptide makes a hormonal nature probable.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged↗

PubMed2003

IL-1beta and IFN-gamma induce the expression of diverse chemokines and IL-15 in human and rat pancreatic islet cells, and in islets from pre-diabetic NOD mice.

AIMS/HYPOTHESIS: Cytokines and chemokines are important mediators of immune responses due to their ability to recruit and activate leukocytes. Using microarray analysis we observed that rat beta cells exposed to IL-1beta and IFN-gamma have increased mRNA levels of chemokines and IL-15. The aim of this study was to characterize the expression of IP-10, MIP-3alpha, fractalkine and IL-15 in rat beta cells, human pancreatic islets, and in islets isolated from NOD mice, both during the pre-diabetic period and following islet transplantation. METHODS: FACS-purified rat beta cells and human islets were cultured with IL-1beta, IFN-gamma and/or TNF-alpha. Islets were isolated from NOD or BALB/c mice at different ages. For syngeneic islet transplantation, 2- or 3-week-old NOD islets were grafted under the kidney capsule of spontaneously diabetic NOD recipients. Chemokine and IL-15 mRNA expression and protein release were evaluated, respectively, by RT-PCR and ELISA. RESULTS: Human islets and rat beta cells express IP-10, MIP-3alpha, fractalkine and IL-15 mRNAs upon exposure to cytokines. The expression of IL-15, IP-10 and fractalkine is regulated by IFN-gamma, while the expression of MIP-3alpha is IL-1beta-dependent. Moreover, cytokines induced IL-15, IP-10, Mig, I-TAC and MIP-3alpha protein accumulation in culture medium from human islets. In vivo, there was an age-related increase in IL-15, IP-10 and MIP-3alpha expression in islets isolated from NOD mice. Following syngeneic islet transplantation, increased expression of IL-1beta, IFN-gamma, fractalkine, IP-10, MCP-1 and MIP-3alpha mRNAs were observed in the grafts. CONCLUSION/INTERPRETATION: Cytokine-exposed islets or beta cells express chemokines and IL-15. This could contribute to the recruitment and activation of mononuclear cells and development of insulitis in early Type 1 diabetes and during graft destruction.

Adult↗

PubMed1987

Cryopreservation of mouse pancreatic islets: effects of different glucose concentrations in the post-thaw culture medium on islet recovery.

It was the aim of this study to investigate the influence of the glucose concentration of the post-thaw culture medium on islet B-cell survival after cryopreservation by the combined assessments of islet recovery, islet DNA and insulin contents, and insulin release. Collagenase isolated mouse islets were kept in culture for 3 days in the presence of 11.1 mM glucose and then transferred to freezing ampoules containing Hanks' solution supplemented with 10% calf serum and 2 M dimethyl sulfoxide. After a 20-min incubation at 0 degrees C the islets were cooled at a rate of 25 degrees C/min to -70 degrees C and subsequently plunged into liquid nitrogen. After 2 hr the frozen islets were rapidly thawed at 37 degrees C, transferred to culture dishes, and cultured for another 3 days in the presence of 2.8, 5.6, 11.1, 16.7, or 28 mM glucose. Nonfrozen control islets were treated identically after a preceding 3-day culture at 11.1 mM glucose. The percentage recovery of cryopreserved islets was decreased compared to that of nonfrozen islets, but was increased when higher glucose concentrations were used in the post-thaw culture medium. Since the DNA content of the cryopreserved islets was slightly decreased, the overall survival rate of the cryopreserved B-cells, when cultured at the higher glucose concentrations after thawing, was found to be about 75%. The insulin content of the cryopreserved islets was decreased but the glucose-stimulated insulin release was essentially the same as that of the nonfrozen islets.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Mass spectrometric characterization of arachidonate-containing plasmalogens in human pancreatic islets and in rat islet beta-cells and subcellular membranes.

Pancreatic islets, when stimulated with D-glucose, secrete insulin by processes requiring glycolytic metabolism and generation of ATP. Hydrolysis of membrane phospholipids also occurs in glucose-stimulated islets, resulting in accumulation of nonesterified arachidonate, which facilitates Ca2+ entry and the rise in beta-cell [Ca2+] that triggers insulin secretion. Glucose-induced hydrolysis of arachidonate from islet phospholipids is mediated in part by an ATP-stimulated, Ca(2+)-independent (ASCI) phospholipase A2 (PLA2) which prefers plasmenyl over diacyl phospholipid substrates. Here we characterize the endogenous plasmalogen content of islet cells and subcellular membranes. Fast atom bombardment mass spectrometric analyses demonstrated that three of the most abundant molecular species of ethanolamine phospholipids in rat pancreatic islets were plasmalogens with sn-2 arachidonate residues and palmitic, oleic, or stearic aldehyde residues, respectively, in the sn-1 position. Purified populations of beta-cells prepared by fluorescence-activated cell sorting were also found to contain these plasmenylethanolamine molecular species in abundance similar to that in intact islets and greater than that in islet alpha-cells. Both islet plasma membranes (PM) and endoplasmic reticulum (ER) also contained these plasmenylethanolamine species, which accounted for 42% (PM) to 64% (ER) of the ethanolamine phospholipid arachidonate content of these membranes, as measured by stable isotope dilution mass spectrometry. Plasmenylethanolamine species were also abundant constituents of human pancreatic islets (accounting for 58% of their ethanolamine phospholipid arachidonate content) and were hydrolyzed more rapidly than diacyl ethanolamine phospholipid by human islet cytosolic ASCI-PLA2. Both secretagogue-induced eicosanoid release and insulin secretion from human islets were attenuated by an ASCI-PLA2 suicide substrate which sterically resembles plasmalogens. These observations are consistent with the hypotheses that islet beta-cell ASCI-PLA2-catalyzed hydrolysis of arachidonate from endogenous plasmenylethanolamine substrates may occur in membrane compartments which participate in regulation of the beta-cell cytosolic [Ca2+] and that this may be an intermediary biochemical event in the induction of insulin secretion.

Aldehydes↗

PubMed1993

Islet transplantation. The connection of experiment and clinic exemplified by the transplantation of islets of Langerhans.

Since approximately 25 years islet transplantation as a mean for the treatment of diabetes has been developed from early attempts in rodents to first clinical applications in man in recent years. The review describes the results obtained in diabetic rats including metabolic effects as well as prevention of late complications. In the syngeneic system (Lewis-rats) the intraportal transplantation of 1,000-2,000 isolated islets may cure streptozotocin-induced diabetes for the whole lifespan of the animal. In the allogeneic system (across a major histocompatibility barrier), however, isolated islets are highly immunogenic. According to present knowledge, mainly class-II antigen bearing cells are responsible while the role of the endocrine cells seems to be less important. By various in vitro methods (e.g. low temperature culture at 24 degrees C) it was possible to induce immunoalteration of allogeneic islets which was followed by long-term acceptance by the host in rodents. Whether the same technique is effective in larger animals and in man remains open. New technologies for the isolation of high numbers of pure islets from human pancreatic glands have been the key for the use of islet transplantation in man in several centers. The review describes the details regarding institutions as well as results in patients on the basis of the International Islet Transplant Registry which is located at Giessen University. Although longer lasting insulin independency (longest lasting effect 2 1/2 years) has been observed only in a few patients, islet transplantation has been proven to be a very safe procedure for the patient. It is hoped that with increasing experiences regarding the factors which at present limit the function as number and quality of islets, immunosuppressive protocols etc. islet transplantation will develop to a definite alternative to whole-organ transplantation. One of the most important challenges for modern medicine is the fact of a worldwide increase of diabetic long-term complications such as kidney failure, coronary and peripheral vascular disease, blindness and neuropathy. Although intensified insulin treatment in a small percentage of patients may moderate morbidity and mortality, from experimental work there is sufficient evidence suggesting that only the restoration of normoglycemia early in the course of the disease may prevent complications. This, however, can only be achieved by the replacement of the destroyed islets of Langerhans either by transplantation of an intact vascularised pancreas or by isolated islets.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2003

Metallothionein protects islets from hypoxia and extends islet graft survival by scavenging most kinds of reactive oxygen species.

Islet transplantation is a promising therapy for Type 1 diabetes, but many attempts have failed due to early graft hypoxia or immune rejection, which generate reactive oxygen species (ROS). In the current study, we determined that transgenic overexpression of the antioxidant metallothionein (MT) in pancreatic beta cells provided broad resistance to oxidative stress by scavenging most kinds of ROS including H2O2, peroxynitrite radical released from streptozotocin, 3-morpholinosydnonimine (SIN-1), and superoxide radical produced by xanthine/xanthine oxidase. MT also reduced nitric oxide-induced beta cell death. A direct test of hypoxia/reperfusion sensitivity was made by exposing FVB and MT islets to hypoxia (1% O2). MT markedly reduced ROS production and improved islet cell survival. Because MT protected beta cells from a broad spectrum of ROS and from hypoxia, we considered it to be an ideal candidate for improving islet transplantation. We first tested syngeneic transplantation by implanting islets under the kidney capsule of the same strain, FVB mice, thereby eliminating the immune rejection component. Under these conditions, MT islets maintained much greater insulin content than control islets. Allotransplantation was then tested. MT transgenic and normal FVB islets were implanted under the kidney capsule of BALB/c mice that were previously treated with streptozotocin to induce diabetes. We found that MT islets extended the duration of euglycemia 2-fold longer than nontransgenic islets. The benefit of MT was due to protection from ROS since nitrotyrosine staining, an indicator of free radical damage, was much lower in MT grafts than in FVB grafts. The time course of protection suggested that the major mode of MT action may have been protection from hypoxia or hypoxia/reperfusion. These data demonstrate that treatment with a broad spectrum antioxidant protects islets from ROS damage such as that produced during the early phase of islet transplantation.

Animals↗

PubMed2000

Isolated human islets trigger an instant blood mediated inflammatory reaction: implications for intraportal islet transplantation as a treatment for patients with type 1 diabetes.

Islet transplantation offers a logical means to treat insulin-dependent diabetes. However, for reasons poorly understood, the clinical results with islet transplantation have been vastly inferior to those obtained with whole organ pancreas transplantation. The conventional technique for transplanting isolated islets is by intraportal injection, with the islets being trapped in the liver. Human islets exposed to human blood trigged an "instant blood mediated inflammatory reaction", IBMIR, characterised by platelet consumption, and activation of the coagulation and complement systems. The islets became surrounded by clots and infiltrated with leukocytes, and there was evidence of islet damage as reflected in insulin dumping. When heparin and a complement inhibitor (SCRI), was added to the system, IBMIR was suppressed and islet damage reduced. After intraportal pig-to-pig islet intraportal allotransplantation similar morphological changes was found, corroborating the in vitro findings. Thus, IBMIR inflicts a significant damage to human islets exposed to human blood and IBMIR will also, most likely, enhance the subsequent specific, cell mediated, rejection. Platelet and complement activation seem to be the most important factors in the pathogenesis of IBMIR. The results presented strongly suggest that IBMIR observed both in vitro and in vivo when isolated islets come in contact with blood could provide an explanation for the unsatisfactory results seen in clinical islet allotransplantation.

Blood Coagulation↗

PubMed2005

Improved human pancreatic islet isolation for a prospective cohort study of islet transplantation vs best medical therapy in type 1 diabetes mellitus.

HYPOTHESIS: A local multiorgan donor pancreas procurement program can provide a source for optimized isolation of purified viable islets for transplantation into patients with type 1 diabetes mellitus receiving best medical therapy. DESIGN: Prospective before-after cohort study. SETTING: Tertiary referral center. PATIENTS: Glycemic control was assessed in 10 patients with diabetes-induced renal dysfunction who were enrolled in a best medical therapy program and then crossed over to islet transplantation. INTERVENTIONS: Thirty human pancreata were retrieved from local multiorgan donors and consecutively processed with intraductal collagenase perfusion, continuous digestion, and density gradient purification (group 1, n = 9) or similarly processed but impure tissue fractions cultured in vitro and then repurified to retrieve additional islets (group 2, n = 21). Islets were implanted by percutaneous portal embolization, providing more than 10 000 islet equivalents (IE) per kilogram of body weight (infusions from 1-3 donors per patient) under cover of antithymocyte globulin, sirolimus, or mycophenolate mofetil and tacrolimus. MAIN OUTCOME MEASURES: Islet yields, purity, and cell viability (caspase 3, terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine 5-triphosphate nick-end labeling stain, and insulin secretion in vitro) were compared. In patients, monitored metabolic parameters were C-peptide secretion, insulin requirements, glycemic excursion, and hemoglobin A(1c) (HbA(1c)). RESULTS: For group 1 vs group 2, no differences were observed in pancreas age (43 vs 44 years), cold storage (5 vs 4 hours), or weight (73 vs 82 g). Group 2 yielded 453 690 IE vs 214 109 IE in group 1 (P = .002). Grafts contained 50% or more endocrine cells in both groups. No difference occurred in cell viability or insulin secretion. Islets from 90% of group 2 pancreata met release criteria for transplantation. C-peptide secretion was detected in all recipients and persisted with a median follow-up to 12 months (range, 6-21 months) after full islet transplantation. Daily insulin dependence was reversed in all patients for at least 3 months. Five patients resumed small insulin doses. Compared with the best care program, all patients had improved metabolic stability. The mean +/- SE HbA(1c) level at entry into the study was 7.8% +/- 0.5%, and this decreased to 6.9% +/- 0.2% after best care (P = .38) and further to 6.2% +/- 0.2% at 6 months after transplantation (P = .002 vs entry; P = .15 vs best care; analysis of variance). CONCLUSIONS: Local pancreas donor retrieval with islet isolation and culture conditioning enabled an offer of islets for transplantation for 90% of consecutively processed pancreata. Isolated islets secreted insulin during prolonged follow-up after implantation into patients, yielding metabolic control comparable with that achieved by best medical therapy.

Adult↗

PubMed2005

Islet isolation for allotransplantation: variables associated with successful islet yield and graft function.

AIMS/HYPOTHESIS: Efficient islet isolation is an important prerequisite for successful clinical islet transplantation. Although progressively improved, islet yield and quality are, however, unpredictable and variable and require standardisation. METHODS: Since 1989 we have processed 437 pancreases using the automated method. The donor characteristics, pancreas procurement, and digestion and purification procedures including a wide enzyme characterisation of these pancreases were analysed and correlated with islet yield and transplant outcome. RESULTS: By univariate analysis, islet yield was significantly associated with donor age (r=0.16; p=0.0009), BMI (r=0.19; p=0.0004), good pancreas condition (p=0.0031) and weight (r=0.15; p=0.0056), total collagenase activity (r=0.22; p=0.0001), adjusted collagenase activity/mg (r=0.18; p=0.0002), collagenase activity/solution volume (r=0.18; p=0.0002) and neutral protease activity/solution volume (r=0.14; p=0.0029). A statistically significant contribution to the variability of islet yield in a multivariate analysis performed on donor variables was found for donor BMI (p=0.0008). In a multivariate analysis performed on pancreas variables a contribution was found for pancreas weight (p=0.0064), and for a multivariate analysis performed on digestion variables we found a contribution for digestion time (p=0.0048) and total collagenase activity (p=0.0001). Twenty-four patients with type 1 diabetes received single islet preparations from single donors. In these patients, multivariate analyses showed that the reduction in insulin requirement was significantly associated with morphological aspects of islets (p=0.0010) and that 1-month C-peptide values were associated with islet purity (p=0.0071). CONCLUSIONS/INTERPRETATION: These data provide baseline donor, digestion and purification selection criteria for islet isolation using the automated method and indicate that the morphological aspect may be a clinically relevant measure of islets on which the decision for transplant can be based.

Adolescent↗

PubMed1998

Leptin regulation of islet amyloid polypeptide secretion from mouse pancreatic islets.

Leptin receptors are expressed in pancreatic beta-cells. However, leptin's role in islet hormone secretion is essentially unknown. In the present study, we aimed to elucidate leptin's effect on isolated pancreatic NMRI mouse islets by examining islet amyloid polypeptide (IAPP) and insulin secretion in acute experiments and after 48-hr exposure to leptin (1-100 nM). It was also examined whether a putative effect of leptin was affected by the glucose concentration. Islets were cultured in medium RPMI 1640 + 10% fetal calf serum, and the effects of leptin on islet cell replication, glucose metabolism, and hormone content were subsequently examined. Glucose-stimulated IAPP secretion was reduced both acutely and after 48-hr exposure to leptin, whereas only minor effects were found on insulin release, i.e. an inhibition in islets cultured with 1 nM leptin. An acute inhibitory effect by 10 nM leptin was observed on the ratio of IAPP/insulin release at 5.6-11.1 mM glucose, but this was overcome by 16.7 mM glucose. The islet glucose oxidation rate was enhanced by 1 nM leptin, but decreased at higher concentrations of leptin in acute experiments. In contrast, glucose metabolism was not affected in long-term experiments. Moreover, leptin did not influence islet (pro)insulin synthesis or the cell replication rate after culture. In conclusion, we show that islet IAPP release seems to be more sensitive to leptin than is insulin release. The effect of leptin on islet hormone secretion is dependent on the glucose concentration. The regulation of hormone secretion seems to be dissociated from glucose metabolism, an effect previously described in islets after exposure to certain cytokines. Our data necessarily suggest that a previously proposed negative feedback loop between leptin and insulin can be counteracted by IAPP.

Amyloid↗

PubMed2003

Postnatally disturbed pancreatic islet cell distribution in human islet amyloid polypeptide transgenic mice.

OBJECTIVE: Islet amyloid polypeptide (IAPP)/amylin is produced by the pancreatic islet beta-cells, which also produce insulin. To study potential functions of IAPP, we have generated transgenic mice overexpressing human IAPP (hIAPP) in the beta-cells. These mice show a diabetic phenotype when challenged with an oral glucose load. In this study, we examined the islet cytoarchitecture in the hIAPP mice by examining islet cell distribution in the neonatal period, as well as 1, 3 and 6 months after birth. RESULTS: Neonatal transgenic mice exhibited normal islet cell distribution with beta-cells constituting the central islet portion, whereas glucagon and somatostatin-producing cells constituted the peripheral zone. In contrast, in hIAPP transgenic mice at the age of 1 month, the glucagon-immunoreactive (IR) cells were dispersed throughout the islets. Furthermore, at the age of 3 and 6 months, the islet organisation was similarly severely disturbed as at 1 month. Expression of both endogenous mouse IAPP and transgenic hIAPP was clearly higher in 6-month-old mice as compared to newborns, as revealed by mRNA in situ hybridisation. CONCLUSIONS: Mice transgenic for hIAPP have islets with disrupted islet cytoarchitecture in the postnatal period, particularly affecting the distribution of glucagon-IR cells. This islet cellular phenotype of hIAPP transgenic mice is similar to that of other mouse models of experimental diabetes and might contribute to the impaired glucose homeostasis.

Amyloid↗

PubMed1995

Hyperproliferative state of liver acini and pancreatic islets after intraportal transplantation of a small mass of islets of Langerhans in streptozotocininduced diabetic rats.

After the portal-embolic transplantation of a low number of isologous islets of Langerhans into streptozotocin-diabetic rats altered liver acini emerge down-stream to the islet grafts. The hepatocytes of these altered liver acini store high amounts of glycogen and/or fat. The proliferation of the hepatocytes of such altered acini and simultaneously their apoptotic elimination were increased strongly, compared with the surrounding unaltered liver acini. After transplantation of a high number of islets, altered liver acini were not observed. The reason for these focal phenomena was supposed to be the persistent systemic hyperglycemic diabetic state which stimulates the beta cells to produce insulin and to secrete it in maximal amounts. This hypothesis has been supported by electron-microscopical examination of the transplanted islets in a former publication, but the proliferative activity of the transplanted islet epithelial cells has not so far been examined. Furthermore, the period of observation has been only three weeks, and it seemed of interest to determine whether the locally increased hepatocytic proliferation and the stimulation of the beta-cells would persist for a longer time. 450-500 (group I, n = 14) or 1500-2000 (group II, n = 8) isologous pancreatic islets were transplanted to adult male Lewis-rats made diabetic by streptozotocin. Group I animals stayed hyperglycemic after transplantation, group II animals became normoglycemic. 5-bromo-2'-desoxyuridine (BrdU) was applied by osmotic minipumps for 6 days before death (6, 13, 21 and 67-77 days after islet transplantation). In group I animals the mitotic index (number of mitotic figures per 1000 hepatocytic nuclei), the apoptotic index (number of apoptotic bodies per 1000 hepatocytic nuclei) and the immunohistochemical-labeling index for BrdU (BrdU-labeled hepatocytic nuclei per 100 hepatocytic nuclei) of these altered acini (A) were compared with the adjacent non-altered acini (NA). The mitotic index in A was 40-fold to 100-fold increased compared with NA (for the whole period of observation). The apoptotic index in A was 6-fold to 20-fold increased compared with NA. The BrdU-LI in A was 20-fold to 80-fold increased compared with NA. In group II animals altered liver acini did not emerge. Furthermore, BrdU-LI of epithelial cells of group I islets were significantly higher compared with group II islets (28.4 vs. 6.3 at day 6, 28.8 vs. 4.2 at day 13, 23.2 vs. 2.9 at day 21, 15.3 vs. 0.1 at days 67 to 77, respectively). It is supposed that two different types of proliferative stimuli were active in this model of low number islet transplantation: 1) The chronic hyperglycemia induces endocrine epithelial cell proliferation in the grafts. 2) The local chronic hyperinsulinism induces glycogen and fat storage, proliferation and apoptotic elimination of the hepatocytes in the liver acini down-stream to the stimulated islets.

Animals↗

PubMed1997

Transgenic overproduction of islet amyloid polypeptide (amylin) is not sufficient for islet amyloid formation.

Islet amyloid polypeptide forms islet amyloid deposits in non-insulin-dependent diabetes mellitus. We have generated transgenic mice which express human islet amyloid polypeptide in their pancreatic beta cells yet do not develop islet amyloid deposits despite producing levels of the amyloidogenic human peptide 2 - 3 fold higher than the native (mouse) peptide. To determine whether marked overproduction of islet amyloid polypeptide is a potential cause of islet amyloid formation, we increased expression of this transgene by producing homozygous transgenic animals and by making heterozygous mice experimentally insulin resistant with nicotinic acid. Pancreatic content of islet amyloid polypeptide-like immunoreactivity in homozygous and nicotinic acid-treated mice was 2-fold (25 +/- 7 fmol/microg; n = 6) and 3.5-fold (47 +/- 20 fmol/microg; n = 3) higher, respectively, than that of untreated heterozygous animals (13+/-2 fmol/microg; n = 11; both p < 0.05). Despite this marked increase in production of islet amyloid polypeptide, neither group of mice developed gross islet amyloid deposits even after 16 months of age. We conclude that overproduction of islet amyloid polypeptide, even as produced by extreme insulin resistance, is not in itself sufficient for islet amyloid formation.

Amyloid↗

PubMed1987

Low-temperature culture of human islets or in vivo treatment with L3T4 antibody produces a marked prolongation of islet human-to-mouse xenograft survival.

In previous studies we have shown that rejection of islet xenografts transplanted between closely related species (rat to mouse) can be prevented by destruction or alteration of antigen-presenting cells in the donor islets and temporary immunosuppression of the recipients. Relatively few studies have been reported on the survival of islet xenografts transplanted between widely discordant species. In the present study, isolated human islets were transplanted beneath the renal capsule of B6 mice made diabetic by the injection of streptozotocin. The effect of culturing the human islets at 24 degrees C for 7 days with or without the administration of anti-L3T4 for 7 days after transplantation was determined. A marked prolongation of the mean survival time was obtained with low-temperature culture alone (greater than 40.2 +/- 9.9 days), with anti-L3T4 alone (greater than 45.2 +/- 6.3 days), and with the combination of these regimens (greater than 51.9 +/- 5.1 days) as compared to controls (7.5 +/- 1.1 days). The surprising finding was the marked effect of low-temperature culture alone on prolonging human islet xenograft survival because this treatment of the donor islets had no effect on the survival of rat islet xenografts. Intact human islets were present in approximately equal to 80% of the recipients after returning to a diabetic state, whereas xenografts of rat islets were completely destroyed. The findings indicate that complete rejection of islets across this widely discordant species barrier is slower than across a closely related barrier and may be occurring by a different rejection process.

Animals↗

PubMed1996

Islet amyloid formation associated with hyperglycemia in transgenic mice with pancreatic beta cell expression of human islet amyloid polypeptide.

Pancreatic islet amyloid deposits are a characteristic pathologic feature of non-insulin-dependent diabetes mellitus and contain islet amyloid polypeptide (IAPP; amylin). We used transgenic mice that express human IAPP in pancreatic beta cells to explore the potential role of islet amyloid in the pathogenesis of non-insulin-dependent diabetes mellitus. Extensive amyloid deposits were observed in the pancreatic islets of approximately 80% of male transgenic mice > 13 months of age. Islet amyloid deposits were rarely observed in female transgenic mice (11%) and were never seen in nontransgenic animals. Ultrastructural analysis revealed that these deposits were composed of human IAPP-immunoreactive fibrils that accumulated between beta cells and islet capillaries. Strikingly, approximately half of the mice with islet amyloid deposits were hyperglycemic (plasma glucose > 11 mM). In younger (6- to 9-month-old) male transgenic mice, islet amyloid deposits were less commonly observed but were always associated with severe hyperglycemia (plasma glucose > 22 mM). These data indicate that expression of human IAPP in beta cells predisposes male mice to the development of islet amyloid and hyperglycemia. The frequent concordance of islet amyloid with hyperglycemia in these mice suggests an interdependence of these two conditions and supports the hypothesis that islet amyloid may play a role in the development of hyperglycemia.

Amyloid↗

PubMed2002

Islet filtration: a simple and rapid new purification procedure that avoids ficoll and improves islet mass and function.

Islet isolation is a time-consuming process. Islet yields vary, and previous in vitro studies suggest that Ficoll may be an islet toxin. Here, we describe an alternative, Ficoll-free method to purify murine islets by filtration through a cell strainer. Collagenase digestion of pancreata was carried out using standard procedures. The pancreatic digest was divided into aliquots and purified either by Ficoll or by filtration. Following filtration, islets were intact and separated from nondigested tissue. Purity was similar to that achieved using Ficoll. However, purification by filtration was faster, increased islet yield, and resulted in higher insulin secretion in vitro. Moreover, when syngeneic diabetic hosts were transplanted with a marginal islet mass, islets purified by filtration restored normoglycemia significantly faster than those isolated by Ficoll. This suggests that Ficoll exposure negatively impacts islet function. In conclusion, islet filtration is a simple and rapid procedure for purification of islets that demonstrate improved functional mass.

Animals↗

PubMed2004

The development of new density gradient media for purifying human islets and islet-quality assessments.

Successful islet transplantation is dependent on the quality and quantity of islets infused. Islets are purified on density gradients, but procedures currently used have limited capacity for pancreatic digests, islet yield, and viability. We aimed to improve islet purification with a modified gradient medium. Biocoll was diluted in University of Wisconsin solution to create linear density gradients of 1.065 to 1.095 g/mL. Properties of islets purified from 22 human pancreas digests with modified medium were compared with 15 preparations using standard medium. The modification increased the capacity of gradients for pancreatic digests from 20 to 60 mL, islet yield increased from 218,000 to 435,318 per isolation, and viability increased from 65.4% to 92.1%. Islet fractions contained greater than 95% of recovered insulin. Islets showed good physiologic responses to secretagogues and restored normoglycemia in streptozotocin-induced diabetic severe combined immunodeficiency disease mice. The new medium enhances yield, purity, and viability of human islet preparations for clinical islet transplantation.

Adenosine↗

PubMed2004

Efficacy of human islet isolation from the tail section of the pancreas for the possibility of living donor islet transplantation.

BACKGROUND: Islet transplantation is on the rise for the treatment of type 1 diabetes. Apparent donor shortages could be alleviated through use of living donor pancreata. A critical issue for using a section of pancreas from living donors is whether islet yields would be sufficient for transplantation. METHODS: After obtaining human pancreata, islets were isolated from the head section (n=20, head group), tail section (n=23, tail group) or whole pancreas (n=24, whole group). Islets were isolated by enzymatic digestion followed by purification, then assessed for yields, purity, morphology, functionality, and insulin content. RESULTS: Fifteen of twenty cases (75%) in the head group, all cases (100%) in the tail group, and 23 of 24 cases (96%) in the whole group were successfully completed for islet isolation. Islet yield per gram pancreas was significantly higher in the tail group compared with both the head and whole groups (head, 1,472+/-326 IE/g; tail, 4,256+/-574 IE/g; whole, 2,424+/-506 IE/g). Total islet yield from the head group was significantly lower compared with both tail and whole groups (head, 75,016+/-18,933 IE; tail, 197,469+/-28,236 IE; whole, 208,207+/-43,414 IE), and the tail group showed similar islet yield to the whole group. The whole group showed significantly lower purities and the head group showed significantly lower morphologic scores. There were no significant differences in viability, function, and insulin content among the three groups. CONCLUSIONS: The tail section of the human pancreas is suitable for islet isolation. The living donor islet transplantation may be feasible using only this section of the pancreas for the first transplantation to reduce hypoglycemic unawareness for small recipients, which might be followed by the second islet transplantation from cadaveric donor.

Adult↗