Monkey Islets Research Articles

The pathological characters of islets aging in old rhesus monkeys

The loss of β cell mass and function in aged population plays a critical role in the prevalence of Type 2 diabetes. However, the causal relations between aging and age-related pancreatic islets degeneration still have not been fully elucidated. Rhesus monkey is one of the most ideal nonhuman primate animal models of a wide range of human diseases, including diabetes and aging-related diseases. In the present study, we observed the overall physiological function, glycolipid metabolism and islet function of middle-age and elderly rhesus monkeys, and compared their gene expression profiles by transcriptome sequencing of isolated islets. Through these analyses, we are aimed to evaluate the pathological characters of islets of old rhesus monkeys in the process of aging, and to provide some tips for the prevention and treatment of diabetes in the elderly population. The results suggested that there was no significant physiological disorder in monkeys of approximately 20 years old, except the glucose metabolism was mildly disturbed. In pancreas tissues and isolated islets of elderly monkeys, we found that the islets sizes were distinctly decreased, and the insulin secretion was compromised. Notably, the islets fibrosis and proportion of insulin/glucagon co-expressing cells increased significantly. Moreover, the β cell identity markers, transcription factors PDX1 and Nkx6.1 were losing with advancing age. Analysis of the RNA sequencing of isolated islets showed the genes related to type 1 diabetes and β cell function changed markedly. In conclusion, we found that in the elderly monkeys around 20 years old, the decreased islets size and compromised insulin secretion may contribute to the disturbed glucose metabolism, and the loss of β cell identity markers is a typical molecular change of islet senescence.

Production of genetically modified pigs expressing human insulin and C-peptide as a source of islets for xenotransplantation.

Islet xenotransplantation is a promising treatment for type I diabetes. Numerous studies of islet xenotransplantation have used pig-to-nonhuman primate transplantation models. Some studies reported long-term survival and successful function of porcine islets in diabetic monkeys. Genetic engineering techniques may improve the survival and function of porcine islets. A recent study reported the generation of transgenic pigs expressing human insulin rather than porcine insulin by changing one amino acid at the end of the β-chain in insulin. However, C-peptide from pigs still existed. In this study, we generated transgenic pigs expressing human proinsulin to express human insulin and C-peptide using fibroblasts from proinsulin knockout pigs as donor cells for somatic cell nuclear transfer. Eleven live piglets were delivered from three surrogates and characterized to confirm the genotype and phenotype of the generated piglets. Genotype analysis of the generated piglets showed that five of the eleven piglets contained the human proinsulin gene. Insulin expression was confirmed in the serum and pancreas in two of the five piglets. C-peptide derived from human proinsulin was also confirmed by liquid chromatography tandem mass spectrometry. Non-fasting blood glucose level was measured to verify the function of the insulin derived from the human proinsulin. Two piglets expressing insulin showed normal glucose levels similar to that in the wild-type control. In conclusion, human insulin- and C-peptide-expressing pigs without porcine insulin and C-peptide were successfully established. These pigs can be used as a source of islets for islet xenotransplantation.

A comparison of the anatomical structure of the pancreas in experimental animals.

As basic knowledge for evaluation of pancreatic toxicity, anatomical structures were compared among experimental animal species, including rats, dogs, monkeys, and minipigs. In terms of gross anatomy, the pancreases of dogs, monkeys, and minipigs are compact and similar to that of humans. The rat pancreas is relatively compact at the splenic segment, but the duodenal segment is dispersed within the mesentery. In terms of histology, the islet of each animal is characterized by a topographic distribution pattern of α- versus β-cells. β-cells occupy the large central part of the rat islet, and α-cells are located in the periphery and occasionally exhibit cuffing. In dog islets, β-cells are distributed in all parts and α-cells are scattered in the center or periphery of the islet (at body and left lobe); whereas β-cells occupy all parts of the islet and no α-cells are present in the islet (at right lobe). Monkey islets show two distinct patterns, that is, α-cell-rich or β-cell-rich islets, and the former represent peripheral β-cells forming an irregular ring. Minipig islets show an irregular outline, and both α- and β-cells are present in all parts of the islet, intermingling with each other. According to morphometry, the endocrine tissue accounts for <2% of the pancreas roughly in rats and minipigs, and that of monkeys accounts for >7% of the pancreas (at tail). The endocrine tissue proportion tends to increase as the position changes from right to left in the pancreas in each species.

DPP IV inhibitor suppresses STZ-induced islets injury dependent on activation of the IGFR/Akt/mTOR signaling pathways by GLP-1 in monkeys

BackgroundTo evaluate the protective effect of the DPP IV inhibitor in STZ-induced islet injury and to identify the molecular events that protect islet against apoptosis. Methods4 diabetic monkeys were treated with streptozotocin (70mg/kg) in the presence or absence of the DPP IV inhibitor (Sitagliptin), continuing administered for 4weeks after STZ. The monkeys were evaluated by plasma DPP IV activity, serum active GLP-1 response, blood glucose, insulin and C-P levels, the insulin resistance index (HOMA-IR), and the expression of insulin, caspase-3, IGF receptor (IGFR), p-Akt and p-mTOR in pancreas islets tissues. To test that DPP IV inhibitors might against islets apoptosis via IGFR/Akt/mTOR signaling pathways, the isolated islets from the normal monkeys were pre-treated with or without 10mM STZ for 1h, followed by GLP-1 (10μM) in the presence or absence of NVP-AEW541 or Wortmannin for 24h, to determined islets function and islet apoptosis. ResultsDPP IV inhibitors treatment showed depressing the degradation of GLP-1 and significantly increased serum GLP-1 levels in DM monkeys. Moreover, treatment of diabetic monkeys with the DPP IV inhibitor or treatment of isolated islets with GLP-1 can decrease islet apoptosis, and enhanced islet function and survival, and the expression of IGF receptor, p-Akt and p-mTOR in islets. When the IGFR/Akt/mTOR signaling pathways was blocked by NVP-AEW541 or Wortmannin, the protective effects of GLP1 on STZ-induced islets injury were inhibited in vitro. ConclusionsOur data provides evidence that DPP IV inhibitors confer resistance to STZ-induced islet injury. The protective effects of DPP IV inhibitor on STZ-induced islets injury were dependent on activation of the IGFR/Akt/mTOR signaling pathways by GLP-1 in islets of monkeys.

Amyloidogenic peptide oligomer accumulation in autophagy-deficient β cells induces diabetes.

Islet amyloid accumulation is a hallmark of human type 2 diabetes (T2D). In contrast to human islet amyloid polypeptide (hIAPP), murine islet amyloid polypeptide (mIAPP) does not exhibit amyloidogenic propensity. Because autophagy is important in the clearance of amyloid-like proteins, we studied transgenic mice with β cell-specific expression of hIAPP to evaluate the contribution of autophagy in T2D-associated accumulation of hIAPP. In mice with β cell-specific expression of hIAPP, a deficiency in autophagy resulted in development of overt diabetes, which was not observed in mice expressing hIAPP alone or lacking autophagy alone. Furthermore, lack of autophagy in hIAPP-expressing animals resulted in hIAPP oligomer and amyloid accumulation in pancreatic islets, leading to increased death and decreased mass of β cells. Expression of hIAPP in purified monkey islet cells or a murine β cell line resulted in pro-hIAPP dimer formation, while dimer formation was absent or reduced dramatically in cells expressing either nonamyloidogenic mIAPP or nonfibrillar mutant hIAPP. In autophagy-deficient cells, accumulation of pro-hIAPP dimers increased markedly, and pro-hIAPP trimers were detected in the detergent-insoluble fraction. Enhancement of autophagy improved the metabolic profile of hIAPP-expressing mice fed a high-fat diet. These results suggest that autophagy promotes clearance of amyloidogenic hIAPP, autophagy deficiency exacerbates pathogenesis of human T2D, and autophagy enhancers have therapeutic potential for islet amyloid accumulation-associated human T2D.

Prevention of Nonimmunologic Loss of Transplanted Islets in Monkeys

The nonimmunologic loss of islets in the pre-, peri-, and early post-islet transplant periods is profound. To determine the potential role that transplantation of only a marginal mass of functioning beta cells may play in triggering late nonimmunologic graft loss, we studied the effect of treatment with alpha-1-antitrypsin (AAT) in the autologous cynomolgus islet transplant model. A marginal mass of autologous islets, that is islets prepared from 70% to 80% of the pancreas, was transplanted at 1600-4100 IEQ/kg into subtotal pancreatectomized, streptozotocin-treated and insulin-deficient diabetic hosts. In this marginal mass islet transplant model, islet function is insidiously lost over time and diabetes recurs in all untreated monkeys by 180 days posttransplantation. Short-term treatment with AAT, an acute phase reactant, in the peri-transplant period serves to terminate inflammation through effects upon expression of TGFβ, NFκB and AKT and favorably altering expression of cell death and survival pathways, as detected by a system biology approach and histology. These effects enabled functional expansion of the islet mass in transplanted hosts such that graft function improves rather than deteriorating over time.

Implementation of a Simplified Method of Islet Isolation for Allogeneic Islet Transplantation in Cynomolgus Monkeys

The present study describes a simple and cost-effective islet isolation procedure. Using this method, allogeneic islets reverse diabetes in cynomolgus monkeys. Pancreatic tissue from 11 cynomolgus monkeys were digested, collected, and purified using a simplified method. Islet quantification, purity, viability, and glucose static incubation were conducted immediately after isolation. Five streptozotocin-induced monkeys with diabetes were transplanted intrahepatically, and liver biopsies from 3 of these monkeys were taken at different time points for histologic study. The mean (SD) of viability, purity, and static glucose incubation stimulation index were 94.4% (2.3%), 91.8% (3.4%), and 2.6 (1.7), respectively. Monkeys who received a mean (SD) dose of 19,968 (2273) islet equivalent per kilogram (n = 4) from 2 to 3 donors who achieved prolonged normoglycemia (57-232 days), whereas the single monkey who received an islet dose of 8000 islet equivalent per kilogram did not experience diabetes reversal. Immunohistochemical assessment of the liver biopsies taken from the monkeys with normoglycemia revealed an insulin- and glucagon-positive islet graft for up to 6 months with minimal peri-islet inflammatory infiltration. This study demonstrates that cynomolgus monkey islets can be successfully and efficiently harvested using a simple isolation method, and these islets can restore normoglycemia in monkeys with diabetes.

New perspectives of vesicular monoamine transporter 2 chemical characteristics in mammals and its constant expression in type 1 diabetes rat models

Vesicular monoamine transporter 2 (VMAT2) has been exploited as a biomarker of β-cell mass in human islets. However, a current report suggested no immunoreactivity of VMAT2 in the β cells of rat islets. To investigate the cellular localization of VMAT2 in islets further, the pancreatic tissues from monkeys and humans were compared with those of rats and mice. The study was performed using among-species comparisons and a type 1 diabetes model (T1DM) for rats by Western blotting, double-label immunofluorescence, and confocal laser scanning microscopy. We found that VMAT2-immunoreactivity (IR) was distributed peripherally in the islets of rodents, but was widely scattered throughout the islets of primates. Consistent with rodent islets, VMAT2-IR did not exist in insulin (INS)-IR cells but was abundantly present in glucagon (GLU)-IR and pancreatic polypeptide (PP)-IR cells in monkey and human islets. VMAT2-IR had no colocalization with INS-IR in any part of the rat pancreas (head, body, and tail). INS-IR cells were reduced dramatically in T1DM rat islets, but no significant alteration in the proportion of VMAT2-IR cells and GLU-IR cells was observed. Furthermore, a strong colocalization of VMAT2-IR with GLU-IR was distributed in the peripheral regions of diabetic islets. For the first time, the current study demonstrates the presence of VMAT2 in α cells and PP cells but not in β cells in the islets of monkeys and humans. This study provides convinced morphologic evidence that VMAT2 is not present in β cells. There needs to be studies for new markers for β cell mass.

The immunobiology of pig‐to‐nonhuman primate islet xenotransplantation: insights, innovation, and impact

Previous studies of pig‐to‐non‐human primate (NHP) islet xenotransplantation have provided important insights into the immune recognition and effector pathways operative in this relevant preclinical model. The specifics of the xenograft product, microenvironment at the implantation site, and the immunosuppressive regimen significantly influence the mechanisms underlying the rejection of xenogeneic islets.Our current understanding of the immunological barriers to survival of pig islets in NHPs is largely based on studies on intraportal islet xenografts and on comparisons with islet allografts. The demonstration of cell‐mediated rejection of intraportal porcine islet xenografts at about 1 month posttransplant in monkeys immunosuppressed with the same protocols that prevent monkey islet allograft rejection indicates that islet xenograft rejection involves cellular mechanisms that are not present in acute islet allograft rejection. While these mechanisms remain poorly defined the demonstration of long‐term diabetes reversal after intraportal islet xenotransplantation in non‐human primates immunosuppressed with anti‐CD40L but not with anti‐CD40 antibody‐based protocols suggests that the therapeutic efficacy of anti‐CD40L in this transplantation setting likely involves the depletion of donor‐reactive, activated T cells besides CD40:CD40L costimulation blockade.Rejection of intraportal islet xenografts in NHPs immunosuppressed with CTLA4‐Ig and rapamycin was mediated largely by IL‐15‐primed, CXCR3+CD8+ memory T cells recruited by IP‐10 (CXCL10) positive pig islets and macrophages that showed staining for IL‐12 and iNOS. Adding basiliximab induction and tacrolimus maintenance therapy to this protocol prevented rejection in 24 of 26 recipients followed for up to 275 days. Comparison of both groups suggests, though by no means conclusive, that prolongation of graft survival in this large cohort was associated with reduced direct T cell responses to xenoantigens, reduced proportion of intrahepatic (intragraft) B cells and IFN‐γ+ and IL‐17+ CD4 and CD8 T cells, and increased local production of immunoregulatory molecules linked with Tregs, including TGF‐β, Foxp3, HO‐1, and IL‐10. Anti‐pig non‐Gal IgG antibody elicitation was suppressed in both groups.We are currently exploring the concept of negative vaccination to markedly minimize the need for immunosuppression in islet xenotransplantation. Peritransplant administration of donor apoptotic cells extended pig‐to‐mouse islet xenograft survival to &gt;250 days when combined with peritransplant B cell‐depletion and rapamycin. This costimulation blockade‐sparing, antigen‐specific immunotherapy is expected to cause rapid pretransplant clonal deletion of indirect and anergy of direct xenospecific T cells while inducing regulatory T cells. As anti‐CD40L antibodies, B cell depleting antibodies are expected to interfere with indirect antigen presentation, costimulation, and cytokine production required for optimal T cell proliferation, memory formation, and intragraft CD8+ effector function. It is conceivable that additional strategies must be employed in NHPs and eventually in diabetic patients to achieve – as previously with anti‐CD40L antibodies – more complete, yet selective depletion of donor‐reactive, activated T‐cells for the purpose of stable xenograft acceptance.

Porcine Islet Adaptation to Metabolic Need of the Monkeys in Pig to Monkey Intraportal Islet Xenotransplantation

To evaluate successful islet engraftment, specific metabolic functional tests are required in pig-to- monkey islet xenotransplantation model. Among several tests, we conducted intravenous glucose tolerance test (IVGTT) at the regular interval after islet transplantation, and measured porcine c-peptide level in recipient monkeys. Following 2 months after transplantation, glucose response upon stimulation via IVGTT showed normoglycemic pattern. Interestingly, however, c-peptide level revealed gradual adaptation in response to metabolic need of the monkeys. Initial c-peptide response to glucose stimulation was more augmented during long-term follow-up period than that in early period of islet transplantation. The area under the curve (AUC) of c-peptide level for 15 minutes after glucose challenge was significantly increased from 26.8 to 36.8 (p=0.0132). Histopathologic finding also identified that the host endothelial cells were well vascularized around porcine islets in the liver. In conclusion, we demonstrated that porcine islets completely adapted to the liver of recipient monkeys as revealed by functional and morphological evaluation and this result may predict long-term graft survival in pig-to-monkey islet xenotransplantation.

Porcine Islet Adaptation to Metabolic Need of the Monkeys in Pig to Monkey Intraportal Islet Xenotransplantation

To evaluate successful islet engraftment, specific metabolic functional tests are required in pig-to- monkey islet xenotransplantation model. Among several tests, we conducted intravenous glucose tolerance test (IVGTT) at the regular interval after islet transplantation, and measured porcine c-peptide level in recipient monkeys. Following 2 months after transplantation, glucose response upon stimulation via IVGTT showed normoglycemic pattern. Interestingly, however, c-peptide level revealed gradual adaptation in response to metabolic need of the monkeys. Initial c-peptide response to glucose stimulation was more augmented during long-term follow-up period than that in early period of islet transplantation. The area under the curve (AUC) of c-peptide level for 15 minutes after glucose challenge was significantly increased from 26.8 to 36.8 (p=0.0132). Histopathologic finding also identified that the host endothelial cells were well vascularized around porcine islets in the liver. In conclusion, we demonstrated that porcine islets completely adapted to the liver of recipient monkeys as revealed by functional and morphological evaluation and this result may predict long-term graft survival in pig-to-monkey islet xenotransplantation.

Multifunctional Capsule‐in‐Capsules for Immunoprotection and Trimodal Imaging

Type I diabetes mellitus (T1DM) is a T-cell-mediated autoimmune disease that results in destruction of insulin-producing β cells and subsequent hyperglycemia.[1,2] The current way of treating T1DM is insulin replacement therapy through repetitive injections of recombinant insulin. In more serious cases, a cadaveric pancreas[3] or purified pancreatic islets[4,5] can be transplanted to restore proper glucose regulation. However, the risks of surgery and the accompanying life-long immunosuppression outweigh the disadvantages of continued administration of insulin. The immunoisolation of islets by alginate microencapsulation is an emerging and promising solution to circumvent immune rejection and so overcome this limitation.[6] While the semipermeable alginate membrane blocks penetration of immune cells and antibodies, it allows the unhindered passage of nutrients, metabolites, and insulin that are produced by encapsulated islet cells.[7] Intraperitoneal administration of microencapsulated islets in monkeys and humans has showed considerable promise for the treatment of T1DM.[8,9]

Islet architecture: A comparative study

Emerging reports on the organization of the different hormone-secreting cell types (alpha, glucagon; beta, insulin; and delta, somatostatin) in human islets have emphasized the distinct differences between human and mouse islets, raising questions about the relevance of studies of mouse islets to human islet physiology. Here, we examine the differences and similarities between the architecture of human and mouse islets. We studied islets from various mouse models including ob/ob and db/db and pregnant mice. We also examined the islets of monkeys, pigs, rabbits and birds for further comparisons. Despite differences in overall body and pancreas size as well as total beta-cell mass among these species, the distribution of their islet sizes closely overlaps, except in the bird pancreas in which the delta-cell population predominates (both in singlets and clusters) along with a small number of islets. Markedly large islets (>10,000 µm2) were observed in human and monkey islets as well as in islets from ob/ob and pregnant mice. The fraction of α-, β-, and δ-cells within an islet varied between islets in all the species examined. Furthermore, there was variability in the distribution of alpha- and delta-cells with the same species. In summary, human and mouse islets share common architectural features that may reflect demand for insulin. Comparative studies of islet architecture may lead to a better understanding of islet development and function.

Islet amyloid in transgenic mouse and monkey islets; formation and degradation in vivo and in vitro

Islet amyloid in transgenic mouse and monkey islets; formation and degradation <i>in vivo</i> and <i>in vitro</i>

Effect of various immunosuppressive monotherapies on survival and histopathology of monkey islet xenografts in rats

The isolation and testing of monkey islets after transplantation in small animal models provides basic information about their functional capacity. We describe the effect of cyclosporine A (CsA), tacrolimus (FK506) or prednisolone monotherapy on preventing monkey islet graft rejection after xenoTx in a rat model. Histopathological aspects are reported. Indian bonnet monkey (Macaca radiata radiata) islets were isolated by a simple stationary digestion technique using collagenase. The islets were purified with dextran density gradients and were transplanted under the renal capsule of normal or diabetic rats. The rats received a daily dose of CsA, or FK506, or prednisolone, and the grafts were removed at different intervals to determine islet survival. The effect of discontinuation of CsA on islet graft survival was also monitored. Histological examination of islets transplanted into normal or streptozotocin-induced diabetic rats was carried out. In diabetic rats, islet survival was determined by the graft's ability to achieve euglycemia. Reversal of diabetes was achieved in all transplanted diabetic rats, demonstrating the efficacy of the isolated monkey islets. Histological examination indicated that monkey islets survived in the presence of continuous high-dose immunosuppressive monotherapy in rats. Various types of infiltrating cells were observed in the grafted area at varying times after transplantation, depending on the immunosuppressive treatment. After discontinuation of CsA, the grafts were protected for a short period. This study provided evidence for monkey islet survival after transplantation into rats receiving immunosuppressive monotherapy. Basic information on infiltrating cell types may be important in the study of xenograft rejection.

RETRACTION

OBJECTIVES Transplantation of pancreatic islets is considered a potential curative treatment of type 1 diabetes. Electron microscopy plays a major role in the evaluation of pancreatic islets. The aim of this study was to study the reversal of diabetes by xenotransplantation of monkey pancreatic islets into diabetic recipient by functional and structural findings. METHODS Islets of Langerhans were isolated from monkeys by collagenase digestion method. Two days after the induction of diabetes in rats with streptozotocin, diabetes was confirmed. Freshly isolated islets were transplanted under the renal capsule of the diabetic rats. The recipients received cyclosporin A (30 mg/kg) every day. Fasting plasma glucose was estimated on days 3, 7, and 14 after transplantation. The presence of glucose and ketone in urine was checked. After 14 days, the grafts were removed and processed for light and electron microscopic study. RESULTS After the induction of diabetes, the mean fasting plasma glucose was 347.20 mg/dL. On day 3 after transplantation, the mean fasting plasma glucose value was 100 mg/dL. The mean fasting plasma glucose was 94.6 mg/dL on day 7 and 94.8 mg/dL on day 14. Histology of the monkey islet grafts after 14 days showed the survival of pancreatic islets. Ultrastructure of the same grafts showed the presence of alpha, beta, and delta granules similar to those of native pancreatic islets with the other cellular organelles. CONCLUSION The diabetic state of rats can be reversed by xenotransplantation of isolated monkey islets. Ultrastructural study confirms the normal synthesis and release of islet hormones. The released insulin from transplanted monkey islets had lowered the plasma glucose level of recipient rats.

Validation of Large Particle Flow Cytometry for the Analysis and Sorting of Intact Pancreatic Islets

Accurate quantification of total islet yield is an essential step prior to transplantation and for research. The standard method of manually determining an islet equivalent (IEQ) count is subjective and prone to error. We evaluated Complex Object Parametric Analyzer and Sorter (COPAS) large particle flow cytometry for the determination of islet equivalent counts and purities of islet preparations. Initial validation of the sensitivity and accuracy of the COPAS flow cytometer was performed by analysis and sorting of uniform polystyrene microspheres with sizes similar to islets. Human and Rhesus monkey islets were stained with the zinc-specific fluorescent dye Newport Green to discriminate islet from nonislet tissue. Islet sizes were extrapolated from standard curves obtained using microspheres from which individual islet volumes were calculated. IEQ counts on six islet preparations were performed by the standard manual method and compared with results obtained by automated COPAS flow cytometry. The COPAS flow cytometer was highly accurate in the detection and measurement of both polystyrene microspheres and islets. IEQ counts determined by COPAS flow cytometry were consistent with manual counts although subject to error when assessing preparations with significant numbers of islets embedded within acinar tissue. Size-specific islet sorting with retention of morphology and dithizone staining was also shown using the COPAS flow cytometer. COPAS large particle flow cytometry provides a novel automated approach for quantification of intact islets and determination of islet equivalent yield. In addition, the ability to analyze and sort islets based upon user defined criterion opens unique avenues for experimentation.

Reversal of Diabetes by Xenotransplantation of Monkey Pancreatic Islets in Rats

Transplantation of pancreatic islets is considered a potential curative treatment of type 1 diabetes. Electron microscopy plays a major role in the evaluation of pancreatic islets. The aim of this study was to study the reversal of diabetes by xenotransplantation of monkey pancreatic islets into diabetic recipient by functional and structural findings. Islets of Langerhans were isolated from monkeys by collagenase digestion method. Two days after the induction of diabetes in rats with streptozotocin, diabetes was confirmed. Freshly isolated islets were transplanted under the renal capsule of the diabetic rats. The recipients received cyclosporin A (30 mg/kg) every day. Fasting plasma glucose was estimated on days 3, 7, and 14 after transplantation. The presence of glucose and ketone in urine was checked. After 14 days, the grafts were removed and processed for light and electron microscopic study. After the induction of diabetes, the mean fasting plasma glucose was 347.20 mg/dL. On day 3 after transplantation, the mean fasting plasma glucose value was 100 mg/dL. The mean fasting plasma glucose was 94.6 mg/dL on day 7 and 94.8 mg/dL on day 14. Histology of the monkey islet grafts after 14 days showed the survival of pancreatic islets. Ultrastructure of the same grafts showed the presence of alpha, beta, and delta granules similar to those of native pancreatic islets with the other cellular organelles. The diabetic state of rats can be reversed by xenotransplantation of isolated monkey islets. Ultrastructural study confirms the normal synthesis and release of islet hormones. The released insulin from transplanted monkey islets had lowered the plasma glucose level of recipient rats.

Insulin secretory characteristics of monkey pancreatic islets: a simple method of islet isolation and the effect of various density gradients on separation

We describe a simple stationary digestion method of islet isolation and separation by various density gradients from monkey pancreas ( Macaca radiata radiata). Effective method, different types and concentrations of collagenase were standardized. Sigma type XI collagenase yielded >1000 islets/gram pancreas at the concentration of 4 mg/ml and 3 ml Hank’s/gram pancreas. Slow digestion with less concentration of collagenase was suitable for monkey islet isolation. Discontinuous density gradients of bovine serum albumin (BSA) and dextran were compared with standard Ficoll for separation of islets. Islet yield (1038±81), insulin secretory response (stimulation index, S.I.11) and histological examination revealed dextran gradients were more appropriate for monkey islets when compared to BSA and Ficoll. Insulin secretory characteristics of monkey islets were studied by exposing them to low and high concentrations glucose (S.I.11.5), arginine (S.I.4.2), leucine (S.I.2.3) and tolbutamide (S.I.1.7). The results indicated that the magnitude of glucose induced insulin secretion of monkey islet is about half as that of rat and mouse islets. However, it is higher than that of porcine and bovine islets. In conclusion, the knowledge of insulin secretory ability of Indian bonnet monkey islets together with the techniques of isolation and separation are useful tool for diabetic research especially islet transplantation.

A simple and cost-effective method for the isolation of islets from nonhuman primates.

Recent advances in islet cell transplantation have led to insulin independence in a majority of islet transplant recipients. However, there exists a need to overcome the shortage of donor tissue and the necessity for life-long immunosuppression. Preclinical studies in large animal models are necessary to evaluate the safety and efficacy of alternative approaches for clinical islet transplantation. The nonhuman primate serves as an appropriate animal model for such investigations; however, a major impediment in performing such preclinical research has been the difficulty in isolating islets of sufficient quantity and quality. The current study describes a simple and cost-effective method to isolate nonhuman primate islets to support preclinical islet transplantation research. The results of islet isolations from 54 cynomolgus monkeys and 4 baboons are reported. The pancreas was infused with Liberase HI and subjected to static digestion. The digested tissue was shaken, filtered through a mesh screen, applied to a discontinuous gradient, and centrifuged in much the same manner as with conventional rodent islet isolations. Islets were collected from the two interfaces, washed, and transplanted. Following purification, cynomolgus monkey islet isolation yields were 50,100 +/- 3120 IE total or 8760 +/- 420 IE/g pancreas with the percent purity and viability of 90.8 +/- 0.9 and 90.7 +/- 0.7, respectively. Total insulin content of the isolated islets was 405 +/- 53 microg insulin with DNA content being and 976 +/- 117 microg DNA, corresponding to a ratio of 0.57 microg insulin/microg DNA. STZ-induced diabetes was reversed in both mouse and nonhuman primate recipients, which possessed significant levels of c-peptide following transplantation and well-granulated islet grafts. The technique yields sufficient numbers of pure and viable islets to support preclinical research to develop improved strategies to prevent the immune destruction of the transplanted islet graft.