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Sample records for human islet isolation

  1. Supravital dithizone staining in the isolation of human and rat pancreatic islets

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    Hansen, W A; Christie, M R; Kahn, R

    1989-01-01

    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...... techniques for the large scale isolation of functionally intact human islets.......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...... 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...

  2. Research-Focused Isolation of Human Islets From Donors With and Without Diabetes at the Alberta Diabetes Institute IsletCore.

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    Lyon, James; Manning Fox, Jocelyn E; Spigelman, Aliya F; Kim, Ryekjang; Smith, Nancy; O'Gorman, Doug; Kin, Tatsuya; Shapiro, A M James; Rajotte, Raymond V; MacDonald, Patrick E

    2016-02-01

    Recent years have seen an increased focus on human islet biology, and exciting findings in the stem cell and genomic arenas highlight the need to define the key features of mature human islets and β-cells. Donor and organ procurement parameters impact human islet yield, although for research purposes islet yield may be secondary in importance to islet function. We examined the feasibility of a research-only human islet isolation, distribution, and biobanking program and whether key criteria such as cold ischemia time (CIT) and metabolic status may be relaxed and still allow successful research-focused isolations, including from donors with type 1 diabetes and type 2 diabetes. Through 142 isolations over approximately 5 years, we confirm that CIT and glycated hemoglobin each have a weak negative impacts on isolation purity and yield, and extending CIT beyond the typical clinical isolation cutoff of 12 hours (to ≥ 18 h) had only a modest impact on islet function. Age and glycated hemoglobin/type 2 diabetes status negatively impacted secretory function; however, these and other biological (sex, body mass index) and procurement/isolation variables (CIT, time in culture) appear to make only a small contribution to the heterogeneity of human islet function. This work demonstrates the feasibility of extending acceptable CIT for research-focused human islet isolation and highlights the biological variation in function of human islets from donors with and without diabetes.

  3. Affinity-purified human interleukin I is cytotoxic to isolated islets of Langerhans

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    Mandrup-Poulsen, T; Bendtzen, K; Nerup, J

    1986-01-01

    . These effects were dose-dependent and reproducible when using three different Interleukin-1 preparations. Highly purified human Interleukin-2, Lymphotoxin, Leucocyte Migration Inhibitory Factor and Macrophage Migration Inhibitory Factor were ineffective. These findings suggest that Interleukin-1 may play......Addition of highly purified human Interleukin-1 to the culture medium of isolated rat islets of Langerhans for 6 days led to 88% inhibition of glucose-induced insulin-release, reduction of islet contents of insulin and glucagon to 31% and 8% respectively, and disintegration of the islets...

  4. Comparison of Modified Celsior Solution and M-Kyoto Solution for Pancreas Preservation in Human Islet Isolation.

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    Noguchi, Hirofumi; Naziruddin, Bashoo; Onaca, Nicholas; Jackson, Andrew; Shimoda, Masayuki; Ikemoto, Tetsuya; Fujita, Yasutaka; Kobayashi, Naoya; Levy, Marlon F; Matsumoto, Shinichi

    2010-06-01

    Since the successful demonstration of the Edmonton protocol, islet transplantation has advanced significantly on several fronts, including improved pancreas preservation systems. In this study, we evaluated two different types of organ preservation solutions for human islet isolation. Modified Celsior (Celsior solution with hydroxyethyl starch and nafamostat mesilate; HNC) solution and modified Kyoto (MK) solution were compared for pancreas preservation prior to islet isolation. Islet yield after purification was significantly higher in the MK group than in the HNC group (MK = 6186 ± 985 IE/g; HNC = 3091 ± 344 IE/g). The HNC group had a longer phase I period (digestion time), a higher volume of undigested tissue, and a higher percentage of embedded islets, suggesting that the solution may inhibit collagenase. However, there was no significant difference in ATP content in the pancreata or in the attainability of posttransplant normoglycemia in diabetic nude mice between the two groups, suggesting that the quality of islets was similar among the two groups. In conclusion, MK solution is better for pancreas preservation before islet isolation than HNC solution due to the higher percentage of islets that can be isolated from the donor pancreas. MK solution should be the solution of choice among the commercially available solutions for pancreatic islet isolation leading to transplantation.

  5. Comparison of modified Celsior solution and M-kyoto solution for pancreas preservation in human islet isolation.

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    Noguchi, Hirofumi; Naziruddin, Bashoo; Onaca, Nicholas; Jackson, Andrew; Shimoda, Masayuki; Ikemoto, Tetsuya; Fujita, Yasutaka; Kobayashi, Naoya; Levy, Marlon F; Matsumoto, Shinichi

    2010-01-01

    Since the successful demonstration of the Edmonton protocol, islet transplantation has advanced significantly on several fronts, including improved pancreas preservation systems. In this study, we evaluated two different types of organ preservation solutions for human islet isolation. Modified Celsior (Celsior solution with hydroxyethyl starch and nafamostat mesilate; HNC) solution and modified Kyoto (MK) solution were compared for pancreas preservation prior to islet isolation. Islet yield after purification was significantly higher in the MK group than in the HNC group (MK = 6186 ± 985 IE/g; HNC = 3091 ± 344 IE/g). The HNC group had a longer phase I period (digestion time), a higher volume of undigested tissue, and a higher percentage of embedded islets, suggesting that the solution may inhibit collagenase. However, there was no significant difference in ATP content in the pancreata or in the attainability of posttransplant normoglycemia in diabetic nude mice between the two groups, suggesting that the quality of islets was similar among the two groups. In conclusion, MK solution is better for pancreas preservation before islet isolation than HNC solution due to the higher percentage of islets that can be isolated from the donor pancreas. MK solution should be the solution of choice among the commercially available solutions for pancreatic islet isolation leading to transplantation.

  6. Human Islet Amyloid Polypeptide

    DEFF Research Database (Denmark)

    Kosicka, Iga

    2014-01-01

    Diabetes mellitus type II is a metabolic disease affecting millions of people worldwide. The disease is associated with occurence of insoluble, fibrillar, protein aggregates in islets of Langerhans in the pancreas - islet amyloid. The main constituent of these protein fibers is the human islet...... of diabetes type II, while revealing the structure(s) of islet amyloid fibrils is necessary for potential design of therapeutic agents....

  7. Assessment of intracellular insulin content during all steps of human islet isolation procedure.

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    Brandhorst, H; Brandhorst, D; Brendel, M D; Hering, B J; Bretzel, R G

    1998-01-01

    This study investigated the recovery of pancreatic insulin content during human islet isolation prior to and after digestion-filtration, continuous Hanks-Ficoll gradient purification (n = 20), and 3-4 day culture at 22 degrees C (n = 6). The native insulin content varied in a wide range from 28.4 U to 360.8 U/pancreas. After digestion the initially measured average insulin content of 115.8 +/- 20.8 U/pancreas (mean +/- SEM) increased to 264.6 +/- 22.8% (p asymetrical distribution of insulin within the pancreas. Sampling of insulin within the pancreatic caput seemed not to be representative for the insulin content of the complete native organ, because the ratio of insulin per gram tissue within the pancreatic cauda compared to the caput (n = 5) was 2.4 +/- 0.4 (p < 0.05). After purification total insulin recovery was 55.3 +/- 4.8% (p < 0.001). Because recovery of islet equivalent number (IEQ) (83.7 +/- 4.4%) exceeded insulin recovery, insulin/IEQ ratio decreased from 656.8 +/- 70.6 microU/IEQ before purification to 436.4 +/- 58.1 microU/IEQ (p < 0.001) after purification. After 22 degrees C culture (n = 6) recovery of insulin and IEQ was 80.1 +/- 8.1% (p < 0.05) and 92.8 +/- 3.5% (p = NS), respectively. Insulin content per IEQ decreased to 85.8 +/- 6.5% (p < 0.05). This study clearly shows that most of islet insulin is lost during purification. This seems to be caused rather by an amplified insulin release than by the loss of islets itself. This release may facilitate the separation of endocrine and exocrine tissue by gradient centrifugation, but may also accelerate islet exhaustion detrimental for long-term insulin independence.

  8. Palmitate activates autophagy in INS-1E β-cells and in isolated rat and human pancreatic islets.

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    Luisa Martino

    Full Text Available We have investigated the in vitro effects of increased levels of glucose and free fatty acids on autophagy activation in pancreatic beta cells. INS-1E cells and isolated rat and human pancreatic islets were incubated for various times (from 2 to 24 h at different concentrations of glucose and/or palmitic acid. Then, cell survival was evaluated and autophagy activation was explored by using various biochemical and morphological techniques. In INS-1E cells as well as in rat and human islets, 0.5 and 1.0 mM palmitate markedly increased autophagic vacuole formation, whereas high glucose was ineffective alone and caused little additional change when combined with palmitate. Furthermore, LC3-II immunofluorescence co-localized with that of cathepsin D, a lysosomal marker, showing that the autophagic flux was not hampered in PA-treated cells. These effects were maintained up to 18-24 h incubation and were associated with a significant decline of cell survival correlated with both palmitate concentration and incubation time. Ultrastructural analysis showed that autophagy activation, as evidenced by the occurrence of many autophagic vacuoles in the cytoplasm of beta cells, was associated with a diffuse and remarkable swelling of the endoplasmic reticulum. Our results indicate that among the metabolic alterations typically associated with type 2 diabetes, high free fatty acids levels could play a role in the activation of autophagy in beta cells, through a mechanism that might involve the induction of endoplasmic reticulum stress.

  9. Experimental studies on islet isolation and islet graft function

    NARCIS (Netherlands)

    Suijlichem, Paul Tjepke Robert van

    1994-01-01

    In the first part of the introduction (Chapter 1) of this thesis an overview of the numerous techniques used in islet isolation procedures is presented. The differing lines of approach for the dissociation of the pancreas which have been applied, and are still being further developed, indicate that

  10. The morphology of islets within the porcine donor pancreas determines the isolation result: successful isolation of pancreatic islets can now be achieved from young market pigs.

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    Krickhahn, Mareike; Bühler, Christoph; Meyer, Thomas; Thiede, Arnulf; Ulrichs, Karin

    2002-01-01

    Clinical islet allotransplantation has become an increasingly efficient "routine" therapy in recent years. Shortage of human donor organs leads to porcine pancreatic islets as a potential source for islet xenotransplantation. Yet it is still very difficult to isolate sufficient numbers of intact porcine islets, particularly from young market pigs. In the following study islets were successfully isolated from retired breeders [4806 +/- 720 islet equivalents per gram organ (IEQ/g); n = 25; 2-3 years old; RB] and also from young hybrid pigs [2868 +/- 260 IEQ/g; n = 65; 4-6 months old; HY] using LiberasePI and a modified version of Ricordi's digestion-filtration technique. As expected, isolations from RB showed significantly better results (p organs from RB (80%) contained mainly large islets (diameter > 200 microm), in contrast to only 35% of all pancreases from HY. Remarkably, the islet size in situ, regardless whether detected in RB or HY, strongly determined the isolation result. A donor organ with predominantly large islets resulted in significantly higher numbers of IEQs compared with a donor organ with predominantly small islets [RB(Large Islets): 5680 +/- 3,318 IEQ/g (n= 20); RB(Small Islets): 1353 +/- 427 IEQ/g (n = 5); p organ prior to the isolation process. Under these conditions highly successful isolations can reliably be performed even from young market pigs.

  11. The Choice of Enzyme for Human Pancreas Digestion Is a Critical Factor for Increasing the Success of Islet Isolation

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    Qi, Meirigeng; Valiente, Luis; McFadden, Brian; Omori, Keiko; Bilbao, Shiela; Juan, Jemily; Rawson, Jeffrey; Scott, Stephen; Ferreri, Kevin; Mullen, Yoko; El-Shahawy, Mohamed; Dafoe, Donald; Kandeel, Fouad; Al-Abdullah, Ismail H.

    2015-01-01

    Background We evaluated 3 commercially available enzymes for pancreatic digestion by comparing key parameters during the islet isolation process, as well as islet quality after isolation. Methods Retrospectively compared and analyzed islet isolations from pancreata using 3 different enzyme groups: liberase HI (n = 63), collagenase NB1/neutral protease (NP) (n = 43), and liberase mammalian tissue-free collagenase/thermolysin (MTF C/T) (n = 115). A standardized islet isolation and purification method was used. Islet quality assessment was carried out using islet count, viability, in vitro glucose-stimulated insulin secretion (GSIS), glucose-stimulated oxygen consumption rate, and in vivo transplantation model in mice. Results Donor characteristics were not significantly different among the 3 enzyme groups used in terms of age, sex, hospital stay duration, cause of death, body mass index, hemoglobin A1c, cold ischemia time, and pancreas weight. Digestion efficacy (percentage of digested tissue by weight) was significantly higher in the liberase MTF C/T group (73.5 ± 1.5 %) when compared to the liberase HI group (63.6 ± 2.3 %) (P < 0.001) and the collagenase NB1/NP group (61.7 ± 2.9%) (P < 0.001). The stimulation index for GSIS was significantly higher in the liberase MTF C/T group (5.3 ± 0.5) as compared to the liberase HI (2.9 ± 0.2) (P < 0.0001) and the collagenase NB1/NP (3.6 ± 2.9) (P = 0.012) groups. Furthermore, the liberase MTF C/T enzymes showed the highest success rate of transplantation in diabetic non-obese diabetic severe combined immunodeficiency mice (65%), which was significantly higher than the liberase HI (42%, P = 0.001) and the collagenase NB1/NP enzymes (41%, P < 0.001). Conclusions Liberase MTF C/T is superior to liberase HI and collagenase NB1/NP in terms of digestion efficacy and GSIS in vitro. Moreover, liberase MTF C/T had a significantly higher success rate of transplantation in diabetic NOD Scid mice compared to liberase HI and

  12. Human umbilical cord matrix-derived stem cells exert trophic effects on β-cell survival in diabetic rats and isolated islets

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    Yunting Zhou

    2015-12-01

    Full Text Available Human umbilical cord matrix-derived stem cells (uMSCs, owing to their cellular and procurement advantages compared with mesenchymal stem cells derived from other tissue sources, are in clinical trials to treat type 1 (T1D and type 2 diabetes (T2D. However, the therapeutic basis remains to be fully understood. The immunomodulatory property of uMSCs could explain the use in treating T1D; however, the mere immune modulation might not be sufficient to support the use in T2D. We thus tested whether uMSCs could exert direct trophic effects on β-cells. Infusion of uMSCs into chemically induced diabetic rats prevented hyperglycemic progression with a parallel preservation of islet size and cellularity, demonstrating the protective effect of uMSCs on β-cells. Mechanistic analyses revealed that uMSCs engrafted long-term in the injured pancreas and the engraftment markedly activated the pancreatic PI3K pathway and its downstream anti-apoptotic machinery. The pro-survival pathway activation was associated with the expression and secretion of β-cell growth factors by uMSCs, among which insulin-like growth factor 1 (IGF1 was highly abundant. To establish the causal relationship between the uMSC-secreted factors and β-cell survival, isolated rat islets were co-cultured with uMSCs in the transwell system. Co-culturing improved the islet viability and insulin secretion. Furthermore, reduction of uMSC-secreted IGF1 via siRNA knockdown diminished the protective effects on islets in the co-culture. Thus, our data support a model whereby uMSCs exert trophic effects on islets by secreting β-cell growth factors such as IGF1. The study reveals a novel therapeutic role of uMSCs and suggests that multiple mechanisms are employed by uMSCs to treat diabetes.

  13. Expression and regulation of nampt in human islets.

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    Karen Kover

    Full Text Available Nicotinamide phosphoribosyltransferase (Nampt is a rate-limiting enzyme in the mammalian NAD+ biosynthesis of a salvage pathway and exists in 2 known forms, intracellular Nampt (iNampt and a secreted form, extracellular Nampt (eNampt. eNampt can generate an intermediate product, nicotinamide mononucleotide (NMN, which has been reported to support insulin secretion in pancreatic islets. Nampt has been reported to be expressed in the pancreas but islet specific expression has not been adequately defined. The aim of this study was to characterize Nampt expression, secretion and regulation by glucose in human islets. Gene and protein expression of Nampt was assessed in human pancreatic tissue and isolated islets by qRT-PCR and immunofluorescence/confocal imaging respectively. Variable amounts of Nampt mRNA were detected in pancreatic tissue and isolated islets. Immunofluorescence staining for Nampt was found in the exocrine and endocrine tissue of fetal pancreas. However, in adulthood, Nampt expression was localized predominantly in beta cells. Isolated human islets secreted increasing amounts of eNampt in response to high glucose (20 mM in a static glucose-stimulated insulin secretion assay (GSIS. In addition to an increase in eNampt secretion, exposure to 20 mM glucose also increased Nampt mRNA levels but not protein content. The secretion of eNampt was attenuated by the addition of membrane depolarization inhibitors, diazoxide and nifedipine. Islet-secreted eNampt showed enzymatic activity in a reaction with increasing production of NAD+/NADH over time. In summary, we show that Nampt is expressed in both exocrine and endocrine tissue early in life but in adulthood expression is localized to endocrine tissue. Enzymatically active eNampt is secreted by human islets, is regulated by glucose and requires membrane depolarization.

  14. Discovery of novel glucose-regulated proteins in isolated human pancreatic islets using LC-MS/MS-based proteomics.

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    Schrimpe-Rutledge, Alexandra C; Fontès, Ghislaine; Gritsenko, Marina A; Norbeck, Angela D; Anderson, David J; Waters, Katrina M; Adkins, Joshua N; Smith, Richard D; Poitout, Vincent; Metz, Thomas O

    2012-07-06

    The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is characterized by insulin resistance and insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent regulator of beta-cell function under physiological conditions, identification of the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of human beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins (∼p < 0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (pleiotropic regulator 1), processing (retinoblastoma binding protein 6), and function (nuclear RNA export factor 1), in addition to neuron navigator 1 and plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and synaptotagmin-17. Up-regulation of dicer 1 and SLC27A2 and down-regulation of phospholipase Cβ4 were confirmed by Western blots. Many proteins found to be differentially abundant after high glucose stimulation are annotated as uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles.

  15. Effect of interleukin-1 on the biosynthesis of proinsulin and insulin in isolated rat pancreatic islets

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    Hansen, Birgit Sehested; Linde, S; Spinas, G A

    1988-01-01

    Insulin dependent diabetes mellitus (IDDM) is often preceded or associated with lymphocytic infiltration in the islets of Langerhans (insulitis). We recently demonstrated that interleukin-1 (IL-1) produced by activated macrophages exerts a bimodal effect on insulin release and biosynthesis...... in isolated rat islets. In the present study we have further analysed the effect of recombinant human interleukin-1 beta (rIL-1) on the biosynthesis and conversion of proinsulin 1 and 2 in rat islets. By RP-HPLC-analysis of islets labelled with [3H]leucine we found that exposure to 6 ng/ml of IL-1 for 24 h...

  16. Discovery of novel glucose-regulated proteins in isolated human pancreatic islets using LC-MS/MS-based proteomics

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    Rutledge, Alexandra C.; Fontes, Ghislaine; Gritsenko, Marina A.; Norbeck, Angela D.; Anderson, David J.; Waters, Katrina M.; Adkins, Joshua N.; Smith, Richard D.; Poitout, Vincent; Metz, Thomas O.

    2012-07-06

    The prevalence of diabetes mellitus is increasing dramatically throughout the world, and the disease has become a major public health issue. The most common form of the disease, type 2 diabetes, is due in part to insufficient insulin production from the pancreatic beta-cell. Since glucose is the most potent and physiologically important regulators of beta-cell function under physiological conditions, understanding the insulin secretory defect underlying type 2 diabetes requires a better understanding of glucose regulation of beta-cell function. To this aim, a bottom-up LC-MS/MS-based proteomics approach was used to profile pooled islets from multiple donors under basal (5 mM) or high (15 mM) glucose conditions. Our analysis discovered 256 differentially abundant proteins ({approx}p < 0.05) after 24 h of high glucose exposure from more than 4500 identified in total. Several novel glucose-regulated proteins were elevated under high glucose conditions, including regulators of mRNA splicing (Pleiotropic regulator 1), processing (Retinoblastoma binding protein 6), and function (Nuclear RNA export factor 1), in addition to Neuron navigator 1 and Plasminogen activator inhibitor 1. Proteins whose abundances markedly decreased during incubation at 15 mM glucose included Bax inhibitor 1 and Synaptotagmin-17. Many proteins found to be differentially abundant after high glucose stimulation were uncharacterized or hypothetical. These findings expand our knowledge of glucose regulation of the human islet proteome and suggest many hitherto unknown responses to glucose that require additional studies to explore novel functional roles.

  17. The Pattern of Neural Elements in the Islets of Normal and Diseased Pancreas and in Isolated Islets

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    Parviz M Pour

    2011-07-01

    Full Text Available Context The association between islet cells and neural elements, the so-called “neuro-insular complex”, has been known for centuries. Objective We examined the expression of beta-III tubulin, in normal pancreases from organ donors, surgical specimens of chronic pancreatitis, surgical specimens of ductal type carcinoma, isolated and purified islets of a 57-year-old male and the pancreases of adult Syrian golden hamsters by immunohistochemistry using a monoclonal antibody to beta-tubulin. Results In the normal pancreas of humans and hamsters, beta-III tubulin was expressed in alpha- and beta-cells, but not in PP cells, neural fibers and gangliae. Occasionally, intra-and peri-insular neural elements were also found. In chronic pancreatitis and pancreatic cancer samples, the number of beta-cells and the immunoreactivity of the beta-III tubulin antibody in islet cells were decreased in most cases. In cultured human islets, devoid of neural elements, no correlation was found between the expression of beta-III tubulin and islet cell hormones. Conclusion Beta-III tubulin is only expressed in the islets derived from the dorsal pancreas and in neural elements. In chronic pancreatitis and pancreatic cancer swelling of intra- and peri-insular nerves occurs, possibly in response to the loss of beta-cells. The secretion of insulin and the expression of beta-tubulin seem to be regulated by nerves.

  18. Tacrolimus inhibits the revascularization of isolated pancreatic islets.

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    Ryuichi Nishimura

    Full Text Available AIMS: Immunosuppressive drugs could be crucial factors for a poor outcome after islet allotransplantation. Unlike rapamycin, the effects of tacrolimus, the current standard immunosuppressant used in islet transplantation, on graft revascularization remain unclear. We examined the effects of tacrolimus on islet revascularization using a highly sensitive imaging system, and analyzed the gene expression in transplanted islets by introducing laser microdissection techniques. METHODS: Islets isolated from C57BL/6-Tg (CAG-EGFP mice were transplanted into the nonmetallic dorsal skinfold chamber on the recipients. Balb/c athymic mice were used as recipients and were divided into two groups: including a control group (n = 9 and tacrolimus-treated group (n = 7. The changes in the newly-formed vessels surrounding the islet grafts were imaged and semi-quantified using multi-photon laser-scanning microscopy and a Volocity system. Gene expression in transplanted islets was analyzed by the BioMark dynamic system. RESULTS: The revascularization process was completed within 14 days after pancreatic islet transplantation at subcutaneous sites. The newly-formed vascular volume surrounding the transplanted islets in the tacrolimus-treated group was significantly less than that in the control group (p<0.05. Although the expression of Vegfa (p<0.05 and Ccnd1 (p<0.05 was significantly upregulated in the tacrolimus-treated group compared with that of the control group, no differences were observed between the groups in terms of other types of gene expression. CONCLUSIONS: The present study demonstrates that tacrolimus inhibits the revascularization of isolated pancreatic islets without affecting the characteristics of the transplanted grafts. Further refinements of this immunosuppressive regimen, especially regarding the revascularization of islet grafts, could improve the outcome of islet allotransplantation.

  19. The influence of porcine pancreas digestion parameters and islet histomorphology on islet isolation outcome.

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    Kinasiewicz, J; Sabat, M; Antosiak-Iwańska, M; Godlewska, E; Sitarek, E; Orłowski, T

    2011-01-01

    Transplantation of the pig islets of Langerhans is considered as the future treatment for patients suffering from type I diabetes mellitus. Despite the adaptation of modified Ricordi method and highly purified collagenase, the results of pancreas digestions are precarious. Selection of proper donor and optimal digestion procedure are fundamental. The aim of this study was to assess the impact of pancreas procuring parameters on pig islets yield. The pancreata were harvested from 69 market sows weighting over 150 kg. After intraductal injection of cold collagenase solution pancreata were transported in UW solution or under conditions of two layer method (TLM). In laboratory pancreata were digested at 37 degrees C according to Ricordi isolation method or stationary in the bottle. The particular parameters of isolation procedure were considered as substantial. Pig weight, volume of infused collagenase solution, TLM application and pancreas dividing before digestion positively affected islet yield. Additionally, the influence of pancreatic islet tissue histomorphology on isolation outcome was studied. Proper donor selection as well as adequate digestion parameters could improve pig islet recovery during islet isolation.

  20. Mechanisms of pancreatic islet cell destruction. Dose-dependent cytotoxic effect of soluble blood mononuclear cell mediators on isolated islets of Langerhans

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    Mandrup-Poulsen, T; Bendtzen, K; Nerup, J

    1986-01-01

    contents of insulin and glucagon in a dose-dependent manner. A maximal effect on islet function was obtained with supernatant concentrations down to 5%. Supernatants of mononuclear cells stimulated with tuberculin were more potent than supernatants produced by lectin stimulation. Culture medium......Supernatants of peripheral blood mononuclear cells from healthy human donors stimulated with recall antigen (purified protein derivative of tuberculin) or lectin (phytohaemagglutinin) markedly inhibited the insulin release from isolated human and rat islets of Langerhans, and decreased rat islet...... reconstituted with tuberculin or phytohaemagglutinin did not impair islet function. Electron microscopy demonstrated that supernatants were cytotoxic to islet cells. The cytotoxic mononuclear cell mediator(s) was non-dialysable, sensitive to heating to 56 degrees C, labile even when stored at -70 degrees C...

  1. A novel high-throughput assay for islet respiration reveals uncoupling of rodent and human islets.

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    Jakob D Wikstrom

    Full Text Available BACKGROUND: The pancreatic beta cell is unique in its response to nutrient by increased fuel oxidation. Recent studies have demonstrated that oxygen consumption rate (OCR may be a valuable predictor of islet quality and long term nutrient responsiveness. To date, high-throughput and user-friendly assays for islet respiration are lacking. The aim of this study was to develop such an assay and to examine bioenergetic efficiency of rodent and human islets. METHODOLOGY/PRINCIPAL FINDINGS: The XF24 respirometer platform was adapted to islets by the development of a 24-well plate specifically designed to confine islets. The islet plate generated data with low inter-well variability and enabled stable measurement of oxygen consumption for hours. The F1F0 ATP synthase blocker oligomycin was used to assess uncoupling while rotenone together with myxothiazol/antimycin was used to measure the level of non-mitochondrial respiration. The use of oligomycin in islets was validated by reversing its effect in the presence of the uncoupler FCCP. Respiratory leak averaged to 59% and 49% of basal OCR in islets from C57Bl6/J and FVB/N mice, respectively. In comparison, respiratory leak of INS-1 cells and C2C12 myotubes was measured to 38% and 23% respectively. Islets from a cohort of human donors showed a respiratory leak of 38%, significantly lower than mouse islets. CONCLUSIONS/SIGNIFICANCE: The assay for islet respiration presented here provides a novel tool that can be used to study islet mitochondrial function in a relatively high-throughput manner. The data obtained in this study shows that rodent islets are less bioenergetically efficient than human islets as well as INS1 cells.

  2. Lung-Derived Microscaffolds Facilitate Diabetes Reversal after Mouse and Human Intraperitoneal Islet Transplantation.

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    Nasser Abualhassan

    Full Text Available There is a need to develop three-dimensional structures that mimic the natural islet tissue microenvironment. Endocrine micro-pancreata (EMPs made up of acellular organ-derived micro-scaffolds seeded with human islets have been shown to express high levels of key beta-cell specific genes and secrete quantities of insulin per cell similar to freshly isolated human islets in a glucose-regulated manner for more than three months in vitro. The aim of this study was to investigate the capacity of EMPs to restore euglycemia in vivo after transplantation of mouse or human islets in chemically diabetic mice. We proposed that the organ-derived EMPs would restore the extracellular components of the islet microenvironment, generating favorable conditions for islet function and survival. EMPs seeded with 500 mouse islets were implanted intraperitoneally into streptozotocin-induced diabetic mice and reverted diabetes in 67% of mice compared to 13% of controls (p = 0.018, n = 9 per group. Histological analysis of the explanted grafts 60 days post-transplantation stained positive for insulin and exhibited increased vascular density in a collagen-rich background. EMPs were also seeded with human islets and transplanted into the peritoneal cavity of immune-deficient diabetic mice at 250 islet equivalents (IEQ, 500 IEQ and 1000 IEQ. Escalating islet dose increased rates of normoglycemia (50% of the 500 IEQ group and 75% of the 1000 IEQ group, n = 3 per group. Human c-peptide levels were detected 90 days post-transplantation in a dose-response relationship. Herein, we report reversal of diabetes in mice by intraperitoneal transplantation of human islet seeded on EMPs with a human islet dose as low as 500 IEQ.

  3. Preservation of beta cell function in adult human pancreatic islets for several months in vitro

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    Brunstedt, J; Andersson, A; Frimodt-Møller, C

    1979-01-01

    Islets of Langerhans were isolated from four human kidney donors, aged 16 to 21 years by the collagenase method described for isolation of rodent islets. So far the human islets have been kept in tissue culture, without attachment, in medium RPMI 1640 supplemented with 10% calf serum for more than...... 9 months, with preservation of the ability to release insulin in response to glucose stimulation. Replacement of calf serum with serum from normal human subjects did not affect B-cell survival, but resulted in elevated insulin values partly due to lower insulin degrading activity. Thus the described...

  4. Interleukin 1 dose-dependently affects the biosynthesis of (pro)insulin in isolated rat islets of Langerhans

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    Spinas, G A; Hansen, B S; Linde, S

    1987-01-01

    Human crude and recombinant interleukin 1 (IL-1) was found to dose- and time-dependently affect the biosynthesis of (pro)insulin in isolated rat islets of Langerhans. Incubation of rat islets with either 0.5 U/ml or 5 U/ml of crude IL-1 for 1 h had no detectable effect on (pro)insulin biosynthesis...

  5. Characterization of the Human Pancreatic Islet Proteome by Two-Dimensional LC/MS/MS

    Energy Technology Data Exchange (ETDEWEB)

    Metz, Thomas O.; Jacobs, Jon M.; Gritsenko, Marina A.; Fontes, Ghislaine; Qian, Weijun; Camp, David G.; Poitout, Vincent J.; Smith, Richard D.

    2006-12-01

    Research to elucidate the pathogenesis of type 1 diabetes mellitus has traditionally focused on the genetic and immunological factors associated with the disease, and, until recently, has not considered the target cell. While there have been reports detailing proteomic analyses of established islet cell lines or isolated rodent islets, the information gained is not always easily extrapolated to humans. Therefore, extensive characterization of the human islet proteome could result in better understanding of islet biology and lead to more effective treatment strategies. We have applied a two-dimensional LC-MS/MS-based analysis to the characterization of the human islet proteome, resulting in the detection of 29,021 unique peptides corresponding to 4,925 proteins. As expected, major islet hormones (insulin, glucagon, somatostatin), beta-cell enriched secretory products (IAPP), ion channels (K-ATP channel), and transcription factors (PDX-1, Nkx 6.1, HNF-1 beta) were detected. In addition, significant proteome coverage of metabolic enzymes and cellular pathways was obtained, including the insulin signaling cascade and the MAP kinase, NF-κβ, and JAK/STAT pathways. This work represents the most extensive characterization of the human islet proteome to date and provides a peptide reference library that may be utilized in future studies of islet biology and type 1 diabetes.

  6. Cytokines cause functional and structural damage to isolated islets of Langerhans

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Bendtzen, K; Bendixen, G

    1985-01-01

    -dependent diabetes mellitus, isolated human or rat islets of Langerhans were incubated for 7 days with cytokine-rich, cell-free supernatants of blood mononuclear cells from healthy human donors stimulated with or without purified protein derivative of tuberculin or phytohaemagglutinin. Glucose stimulated insulin......Cytokines are soluble, antigen non-specific, non-immunoglobulin mediators produced and secreted by blood mononuclear cells interacting in the cellular immune-response. To test the possibility that cytokines participate in the autoimmune destruction of the pancreatic beta-cells leading to insulin......-release, and contents of insulin and glucagon in islets incubated with cytokine-rich supernatants were markedly reduced. This impairment of islet function was due to a cytotoxic effect of cytokine-rich supernatants as judged by disintegration of normal light-microscopic morphology....

  7. Expression of innate immunity genes and damage of primary human pancreatic islets by epidemic strains of Echovirus: implication for post-virus islet autoimmunity.

    Directory of Open Access Journals (Sweden)

    Luis Sarmiento

    Full Text Available Three large-scale Echovirus (E epidemics (E4,E16,E30, each differently associated to the acute development of diabetes related autoantibodies, have been documented in Cuba. The prevalence of islet cell autoantibodies was moderate during the E4 epidemic but high in the E16 and E30 epidemic. The aim of this study was to evaluate the effect of epidemic strains of echovirus on beta-cell lysis, beta-cell function and innate immunity gene expression in primary human pancreatic islets. Human islets from non-diabetic donors (n = 7 were infected with the virus strains E4, E16 and E30, all isolated from patients with aseptic meningitis who seroconverted to islet cell antibody positivity. Viral replication, degree of cytolysis, insulin release in response to high glucose as well as mRNA expression of innate immunity genes (IFN-b, RANTES, RIG-I, MDA5, TLR3 and OAS were measured. The strains of E16 and E30 did replicate well in all islets examined, resulting in marked cytotoxic effects. E4 did not cause any effects on cell lysis, however it was able to replicate in 2 out of 7 islet donors. Beta-cell function was hampered in all infected islets (P<0.05; however the effect of E16 and E30 on insulin secretion appeared to be higher than the strain of E4. TLR3 and IFN-beta mRNA expression increased significantly following infection with E16 and E30 (P<0.033 and P<0.039 respectively. In contrast, the expression of none of the innate immunity genes studied was altered in E4-infected islets. These findings suggest that the extent of the epidemic-associated islet autoimmunity may depend on the ability of the viral strains to damage islet cells and induce pro-inflammatory innate immune responses within the infected islets.

  8. Isolation, characterization, and chromosomal mapping of the human Nkx6.1 gene (NKX6A), a new pancreatic islet homeobox gene

    Energy Technology Data Exchange (ETDEWEB)

    Inoue, Hiroshi; Permutt, M.A.; Veile, R. [Washington Univ. School of Medicine, St. Louis, MO (United States)] [and others

    1997-03-01

    Nkx6.1 (gene symbol NKX6A), a new member of the NK homeobox gene family, was recently identified in rodent pancreatic islet 13-cell lines. The pattern of expression suggested that this gene product might be important for control of islet development and/or regulation of insulin biosynthesis. We now report cloning of human NKX6A, characterization of its genomic structure, and its chromosomal localization. The predicted protein of human NKX6A contained 367 amino acids and had 97% identity to the hamster protein. The highly conserved NK decapeptide and homeodomain regions were identical between human and hamster, suggesting functional importance of these domains. The coding region spanned approximately 4.8 kb and was composed of three exons. The gene was localized to four CEPH {open_quotes}B{close_quotes} yeast artificial chromosome clones (914B4, 951G9, 981D6, and 847133), and a nearby polymorphic marker (D4S1538) on chromosome 4 was identified <1270 kb from the gene. Using fluorescence in situ hybridization, we also determined that NKX6A maps to 4q21.2-q22. 11 refs., 2 figs.

  9. Collagen V Is a Potential Substrate for Clostridial Collagenase G in Pancreatic Islet Isolation

    Directory of Open Access Journals (Sweden)

    Hiroki Shima

    2016-01-01

    Full Text Available The clostridial collagenases, H and G, play key roles in pancreatic islet isolation. Collagenases digest the peptide bond between Yaa and the subsequent Gly in Gly-Xaa-Yaa repeats. To fully understand the pancreatic islet isolation process, identification of the collagenase substrates in the tissue is very important. Although collagen types I and III were reported as possible substrates for collagenase H, the substrate for collagenase G remains unknown. In this study, collagen type V was focused upon as the target for collagenases. In vitro digestion experiments for collagen type V were performed and analyzed by SDS-PAGE and mass spectrometry. Porcine pancreatic tissues were digested in vitro under three conditions and observed during digestion. The results revealed that collagen type V was only digested by collagenase G and that the digestion was initiated from the N-terminal part. Tissue degradation during porcine islet isolation was only observed in the presence of both collagenases H and G. These findings suggest that collagen type V is one of the substrates for collagenase G. The enzymatic activity of collagenase G appears to be more important for pancreatic islet isolation in large mammals such as pigs and humans.

  10. Characterization of the human pancreatic islet proteome by two-dimensional LC/MS/MS.

    Science.gov (United States)

    Metz, Thomas O; Jacobs, Jon M; Gritsenko, Marina A; Fontès, Ghislaine; Qian, Wei-Jun; Camp, David G; Poitout, Vincent; Smith, Richard D

    2006-12-01

    The pancreatic beta-cell plays a central role in the maintenance of glucose homeostasis and in the pathogenesis of both type 1 and type 2 diabetes mellitus. Elucidation of the insulin secretory defects observed in diabetes first requires a better understanding of the complex mechanisms regulating insulin secretion, which are only partly understood. While there have been reports detailing proteomic analyses of islet cell lines or isolated rodent islets, the information gained is not always applicable to humans. Therefore, definition of the human islet proteome could contribute to a better understanding of islet biology and lead to more effective treatment strategies. We have applied a two-dimensional LC-MS/MS-based analysis to the characterization of the human islet proteome, resulting in the confident identification of 29,021 different tryptic peptides covering 3365 proteins (> or =2 unique peptide identifications per protein). As expected, the three major islet hormones (insulin, glucagon, and somatostatin) were detected, as well as various beta-cell enriched secretory products, ion channels, and transcription factors. In addition, significant proteome coverage of metabolic enzymes and cellular pathways was observed, including the integrin signaling cascade and the MAP kinase, NF-kappa beta, and JAK/STAT pathways. The resulting peptide reference library provides a resource for future higher throughput and quantitative studies of islet biology.

  11. Extensive Loss of Islet Mass Beyond the First Day After Intraportal Human Islet Transplantation in a Mouse Model.

    Science.gov (United States)

    Liljebäck, Hanna; Grapensparr, Liza; Olerud, Johan; Carlsson, Per-Ola

    2016-01-01

    Clinical islet transplantation is characterized by a progressive deterioration of islet graft function, which renders many patients once again dependent on exogenous insulin administration within a couple of years. In this study, we aimed to investigate possible engraftment factors limiting the survival and viability of experimentally transplanted human islets beyond the first day after their transplantation to the liver. Human islets were transplanted into the liver of nude mice and characterized 1 or 30 days after transplantation by immunohistochemistry. The factors assessed were endocrine mass, cellular death, hypoxia, vascular density and amyloid formation in the transplanted islets. One day posttransplantation, necrotic cells, as well as apoptotic cells, were commonly observed. In contrast to necrotic death, apoptosis rates remained high 1 month posttransplantation, and the total islet mass was reduced by more than 50% between 1 and 30 days posttransplantation. Islet mass at 30 days posttransplantation correlated negatively to apoptotic death. Vascular density within the transplanted islets remained less than 30% of that in native human islets up to 30 days posttransplantation and was associated with prevailing hypoxia. Amyloid formation was rarely observed in the 1-day-old transplants, but was commonly observed in the 30-day-old islet transplants. We conclude that substantial islet cell death occurs beyond the immediate posttransplantation phase, particularly through apoptotic events. Concomitant low vascularization with prevailing hypoxia and progressive amyloid development was observed in the human islet grafts. Strategies to improve engraftment at the intraportal site or change of implantation site in the clinical setting are needed.

  12. A modified method for isolating mouse islets of an adequate quality, quantity, and purity.

    Science.gov (United States)

    Xu, Jiejie; Peng, Baogang; Zhang, Caiyun; Xu, Jiwei; Ma, Yi; Lu, Xinjun

    2017-08-01

    Mouse islets are widely used in diabetes research. Thus an adequate quality, quantity, and purity of islets are needed for high-quality investigations. We performed a combination of filtration and density gradient separation and optimized many steps in the islet isolation procedure, including perfusion, digestion, and purification. Our results show that an increased quality, quantity, and purity of isolated islets can be achieved using these modifications. Moreover, this method can guarantee maximal recovery and purity of the isolated islets and is easy to perform with practice.

  13. Isolation of viable porcine islets by selective osmotic shock without enzymatic digestion.

    Science.gov (United States)

    Atwater, I; Guajardo, M; Caviedes, P; Jeffs, S; Parrau, D; Valencia, M; Romero, C; Arriagada, C; Caamaño, E; Salas, A; Olguin, F; Atlagich, M; Maas, R; Mears, D; Rojas, E

    2010-01-01

    Islet transplantation is a potential cure for type 1 diabetes, but clinical results have been disappointing. Currently, islet isolation is by enzymatic digestion of the pancreas which has significant pitfalls: warm ischemia exposure, collagenase-induced damage to the islet mass and viability, poor reproducibility, high cost, a relatively low number of islets obtained per whole pancreas, and selection of islets for collagenase resistance rather than for glucose responsiveness. In the present study we performed a series of experiments in a porcine model to demonstrate the feasibility of a new isolation method based on selective osmotic shock (SOS) using very high glucose solutions, doubling or tripling physiological osmotic strength. The SOS method can be carried out at room temperature or in the cold eliminating warm ischemia time which damages the islets. The SOS method does not depend on the texture of the pancreas so all pancreases can be processed identically and the process can be fully automated. The SOS method isolates all the islets of the pancreas regardless of size and shape allowing a greater number of islets to be harvested. The SOS method avoids exposure to toxins in collagenase solutions, is inexpensive and selects for islets with high concentrations of Glut 2 transporters, representing the best glucose responding islets. The SOS method showed a comparable recovery of islets from young pig pancreas and the islets showed improved viability. We conclude that the selective osmotic shock (SOS) method of separating islets from the pancreatic tissue is superior to the collagenase method.

  14. Prevention of core cell damage in isolated islets of Langerhans by low temperature preconditioning

    Institute of Scientific and Technical Information of China (English)

    Yun-Fu Cui; Ming Ma; Gui-Yu Wang; De-En Han; Brigitte Vollmar; Michael D. Menger

    2005-01-01

    AIM: To study the core cell damage in isolated islets of Langerhans and its prevention by low temperature preconditioning (26 ℃).METHODS: Islets were cultured at 37 ℃ for 7-14 d after isolation, and then at 26 ℃ for 2, 4 and 7 d before additional culture at 37 ℃ for another 7 d. Core cell damage in the isolated islets was monitored by video-microscopy and analyzed quantitatively by use of a computer-assisted image analysis system. The analysis included daily measurement of the diameter and the area of the isolated islets and the area of the core cell damage that developed in those islets over time during culture. Histology and TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay were used to characterize the cell damage and to monitor islet function.RESULTS: Microscopic analysis showed that during the 7 to 14 d of culture at 37 ℃, core cell damage occurred in the larger islets with diameters >200 μm, which included both necrotic and apoptotic cell death. Low temperature (26 ℃) culture could prevent core cell damage of isolated islets. The 7-d culture procedure at 26 ℃ could inhibit most of the core cell (excluding diameters>300 μm) damages when the islets were re-warmed at 37 ℃.CONCLUSION: Our results indicate that core cell damage within isolated islets of Langerhans correlates with the size of islets. Low temperature (26 ℃) culture can prevent core cell damage in isolated islets, and successfully precondition these islets for incubation at 37 ℃. These novel findings may help to understand the pathophysiology of early loss of islet tissue after transplantation, and may provide a new strategy to improve graft function in the clinical setting of islet transplantation.

  15. Amount and distribution of collagen in the pancreas have no effect on porcine islet isolation outcome.

    Science.gov (United States)

    Hilling, D; Rijkelijkhuizen, J K R A; Töns, H A M; Terpstra, O T; Bouwman, E

    2009-01-01

    Xenotransplantation of porcine islets of Langerhans is considered to be a possible alternative for clinical islet transplantation. However, porcine islet isolation procedures have been shown to produce highly variable yields between pigs with similar backgrounds. One of the variables that could account for this is the collagen substrate within the pancreas. We determined the amount and distribution of collagen within porcine pancreata as they determined islet isolation outcomes. This study involved the histological examination of 140 porcine pancreata (64 juvenile and 76 adult) and islet isolation from 58 adult organs. To quantify the amount of collagen, tissue samples were stained with Sirius Red. Collagen distribution was determined by assessing the presence of collagen in the endocrine-exocrine interface (the "islet capsule"), in tissue samples double-stained with Sirius Red and anti-insulin. Strong variation in total collagen was observed in both adult and juvenile pigs. The mean collagen content in the juvenile group was significantly lower than that in the adult group. Apparently, the pancreas undergoes a process of fibrosis as pigs age. The vast majority of islets from both adult and juvenile pancreata had no or only a limited collagen capsule. However, islet encapsulation was highly variable between pancreata. We observed no significant correlation between total collagen content or the percentage islet encapsulation and islet yield. Although total collagen content and islet encapsulation show great variability between pancreata, neither the amount nor the distribution of collagen affected porcine islet isolation outcome.

  16. The human insulin gene is part of a large open chromatin domain specific for human islets

    OpenAIRE

    Mutskov, Vesco; Felsenfeld, Gary

    2009-01-01

    Knowledge of how insulin (INS) gene expression is regulated will lead to better understanding of normal and abnormal pancreatic β cell function. We have mapped histone modifications over the INS region, coupled with an expression profile, in freshly isolated islets from multiple human donors. Unlike many other human genes, in which active modifications tend to be concentrated within 1 kb around the transcription start site, these marks are distributed over the entire coding region of INS as w...

  17. Pathogen inactivation of human serum facilitates its clinical use for islet cell culture and subsequent transplantation.

    Science.gov (United States)

    Ståhle, Magnus U; Brandhorst, Daniel; Korsgren, Olle; Knutson, Folke

    2011-01-01

    Serum is regarded as an essential supplement to promote survival and growth of cells during culture. However, the potential risk of transmitting diseases disqualifies the use of serum for clinical cell therapy in most countries. Hence, most clinical cell therapy programs have replaced human serum with human serum albumin, which can result in inferior quality of released cell products. Photochemical treatment of different blood products utilizing Intercept® technology has been shown to inactivate a broad variety of pathogens of RNA and DNA origin. The present study assesses the feasibility of using pathogen-inactivated, blood group-compatible serum for use in human pancreatic islet culture. Isolated human islets were cultured at 37°C for 3-4 days in CMRL 1066 supplemented with 10% of either pathogen-inactivated or nontreated human serum. Islet quality assessment included glucose-stimulated insulin release (perifusion), ADP/ATP ratio, cytokine expression, and posttransplant function in diabetic nude mice. No differences were found between islets cultured in pathogen-inactivated or control serum regarding stimulated insulin release, intracellular insulin content, and ADP/ATP ratio. Whether media was supplemented with treated or nontreated serum, islet expression of IL-6, IL-8, MCP-1, or tissue factor was not affected. The final diabetes-reversal rate of mice receiving islets cultured in pathogen-inactivated or nontreated serum was 78% and 87%, respectively (NS). As reported here, pathogen-inactivated human serum does not affect viability or functional integrity of cultured human islets. The implementation of this technology for RNA- and DNA-based pathogen inactivation should enable reintroduction of human serum for clinical cell therapy.

  18. St. John's wort extract and hyperforin protect rat and human pancreatic islets against cytokine toxicity.

    Science.gov (United States)

    Novelli, Michela; Beffy, Pascale; Menegazzi, Marta; De Tata, Vincenzo; Martino, Luisa; Sgarbossa, Anna; Porozov, Svetlana; Pippa, Anna; Masini, Matilde; Marchetti, Piero; Masiello, Pellegrino

    2014-02-01

    The extract of Hypericum perforatum (St. John's wort, SJW) and its component hyperforin (HPF) were previously shown to inhibit cytokine-induced activation of signal transducer and activator of transcription-1 and nuclear factor κB and prevent apoptosis in a cultured β-cell line. Objective of this study was to assess the protection exerted by SJW and HPF on isolated rat and human islets exposed to cytokines in vitro. Functional, ultrastructural, biomolecular and cell death evaluation studies were performed. In both rat and human islets, SJW and HPF counteracted cytokine-induced functional impairment and down-regulated mRNA expression of pro-inflammatory target genes, such as iNOS, CXCL9, CXCL10, COX2. Cytokine-induced NO production from cultured islets, evaluated by nitrites measurement in the medium, was significantly reduced in the presence of the vegetal compounds. Noteworthy, the increase in apoptosis and necrosis following 48-h exposure to cytokines was fully prevented by SJW and partially by HPF. Ultrastructural morphometric analysis in human islets exposed to cytokines for 20 h showed that SJW or HPF avoided early β-cell damage (e.g., mitochondrial alterations and loss of insulin granules). In conclusion, SJW compounds protect rat and human islets against cytokine effects by counteracting key mechanisms of cytokine-mediated β-cell injury and represent promising pharmacological tools for prevention or limitation of β-cell dysfunction and loss in type 1 diabetes.

  19. Islet Stellate Cells Isolated from Fibrotic Islet of Goto-Kakizaki Rats Affect Biological Behavior of Beta-Cell

    Directory of Open Access Journals (Sweden)

    Feng-Fei Li

    2016-01-01

    Full Text Available We previously isolated islet stellate cells (ISCs from healthy Wistar rat islets. In the present study, we isolated “already primed by diabetic environment” ISCs from islets of Goto-Kakizaki rats, determined the gene profile of these cells, and assessed the effects of these ISCs on beta-cell function and survival. We detected gene expression of ISCs by digital gene expression. INS-1 cell proliferation, apoptosis, and insulin production were measured after being treated with ISCs supernatant (SN. We observed the similar expression pattern of ISCs and PSCs, but 1067 differentially expressed genes. Insulin production in INS-1 cells cultured with ISC-SN was significantly reduced. The 5-ethynyl-2′-deoxyuridine-positive INS-1 cells treated with ISC-SN were decreased. Propidium iodide- (PI- positive INS-1 cells were 2.6-fold higher than those in control groups. Caspase-3 activity was increased. In conclusion, ISCs presented in fibrotic islet of GK rats might be special PSCs, which impaired beta-cell function and proliferation and increased beta-cell apoptosis.

  20. Anakinra and tocilizumab enhance survival and function of human islets during culture: implications for clinical islet transplantation.

    Science.gov (United States)

    Sahraoui, Afaf; Kloster-Jensen, Kristine; Ueland, Thor; Korsgren, Olle; Foss, Aksel; Scholz, Hanne

    2014-01-01

    Pretreatment culture before islet transplantation represents a window of opportunity to ameliorate the proinflammatory profile expressed by human β-cells in duress. Anakinra (IL-1 receptor antagonist) and tocilizumab (monoclonal IL-6 receptor antibody) are two known anti-inflammatory agents successfully used in the treatment of inflammatory states like rheumatoid arthritis. Both compounds have also been shown to reduce blood glucose and glycosylated hemoglobin in diabetic patients. We therefore sought to evaluate the impact of anakinra and tocilizumab on human β-cells. The islets were precultured with or without anakinra or tocilizumab and then transplanted in a marginal mass model using human islets in immunodeficient mice. Islet viability was evaluated in an in vitro model. The pretreatment culture led to a significantly improved engraftment in treated islets compared to the vehicle. Anakinra and tocilizumab are not toxic to human islets and significantly reduce markers of inflammation and cell death. These results strongly support a pretreatment culture with anakinra and tocilizumab prior to human islet transplantation.

  1. Isolation and culture of adult Sertoli cells and their effects on the function of co-cultured allogeneic islets in vitro

    Institute of Scientific and Technical Information of China (English)

    TENG Yan; XUE Wu-jun; DING Xiao-ming; FENG Xin-shun; XIANG He-li; JIANG Ya-zhuo; TIAN Pu-xun

    2005-01-01

    Background Globally, 180 million people suffer from diabetes mellitus. Islet transplantation is believed to be an almost ideal therapy for insulin-dependent patients. How to maintain the viability and the function of isolated human islets is a challenge in clinical practice. Sertoli cells are considered ‘nurse cells'in the seminiferous tubules and have been used in cell graft protocols for neurodegenerative diseases and diabetes in many studies. Many researchers have used immature murine testes as the primarily source of Sertoli cells in islet transplantation because they are easily purified. Mature human Sertoli cells have been seldom investigated. In the present study, we developed a method for the isolation and culture of Sertoli cells derived from adult human testes, and investigated their effects on the function of allogeneic islets when they were cultured together in vitro. Methods Adult Sertoli cells were prepared successfully by two-step enzyme digestion with trypsin, collagenase and hyaluronidase. They were identified by morphological characteristics and their activity was determined by MTT colorimetry over a 28-day culture time in vitro. A glucose-stimulated insulin secretion test was performed to detect the effects of Sertoli cells on allogeneic islets' function when they were co-cultured for 21 days in vitro. Results In cultured cells, mature human Sertoli cells accounted for more than 90% of total cells. The activity of Sertoli cells reached 95% and they remained highly cytoactive for a long time in vitro (P>0.05). Compared with the islets cultured alone, the co-cultured islets with allogeneic Sertoli cells maintained higher sensitivity to glucose stimulation for the duration of the experiment (P<0.01). Conclusions A method of isolation and culture of Sertoli cells from adult testes has been established. Sertoli cells could enhance allogeneic islets' function when they were co-cultured in vitro. They could be a helper cell in islet transplantation.

  2. The human insulin gene is part of a large open chromatin domain specific for human islets.

    Science.gov (United States)

    Mutskov, Vesco; Felsenfeld, Gary

    2009-10-13

    Knowledge of how insulin (INS) gene expression is regulated will lead to better understanding of normal and abnormal pancreatic beta cell function. We have mapped histone modifications over the INS region, coupled with an expression profile, in freshly isolated islets from multiple human donors. Unlike many other human genes, in which active modifications tend to be concentrated within 1 kb around the transcription start site, these marks are distributed over the entire coding region of INS as well. Moreover, a region of approximately 80 kb around the INS gene, which contains the {tyrosine hydroxylase (TH)-(INS)-insulin-like growth factor 2 antisense (IGF2AS)-insulin-like growth factor 2 (IGF2)} gene cluster, unusually is marked by almost uniformly elevated levels of histone acetylation and H3K4 dimethylation, extending both downstream into IGF2 and upstream beyond the TH gene. This is accompanied by islet specific coordinate expression with INS of the neighboring TH and IGF2 genes. The presence of islet specific intergenic transcripts suggests their possible function in the maintenance of this unusual large open chromatin domain.

  3. A 3D map of the islet routes throughout the healthy human pancreas

    Science.gov (United States)

    Ionescu-Tirgoviste, Constantin; Gagniuc, Paul A.; Gubceac, Elvira; Mardare, Liliana; Popescu, Irinel; Dima, Simona; Militaru, Manuella

    2015-01-01

    Islets of Langerhans are fundamental in understanding diabetes. A healthy human pancreas from a donor has been used to asses various islet parameters and their three-dimensional distribution. Here we show that islets are spread gradually from the head up to the tail section of the pancreas in the form of contracted or dilated islet routes. We also report a particular anatomical structure, namely the cluster of islets. Our observations revealed a total of 11 islet clusters which comprise of small islets that surround large blood vessels. Additional observations in the peripancreatic adipose tissue have shown lymphoid-like nodes and blood vessels captured in a local inflammatory process. Our observations are based on regional slice maps of the pancreas, comprising of 5,423 islets. We also devised an index of sphericity which briefly indicates various islet shapes that are dominant throughout the pancreas. PMID:26417671

  4. Neurotransmitters act as paracrine signals to regulate insulin secretion from the human pancreatic islet.

    Science.gov (United States)

    Rodriguez-Diaz, Rayner; Menegaz, Danusa; Caicedo, Alejandro

    2014-08-15

    In this symposium review we discuss the role of neurotransmitters as paracrine signals that regulate pancreatic islet function. A large number of neurotransmitters and their receptors has been identified in the islet, but relatively little is known about their involvement in islet biology. Interestingly, neurotransmitters initially thought to be present in autonomic axons innervating the islet are also present in endocrine cells of the human islet. These neurotransmitters can thus be released as paracrine signals to help control hormone release. Here we propose that the role of neurotransmitters may extend beyond controlling endocrine cell function to work as signals modulating vascular flow and immune responses within the islet.

  5. Generation of pancreatic islet cells from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG DongHui; JIANG Wei; SHI Yan; DENG HongKui

    2009-01-01

    Efficiently obtaining functional pancreaUc islet cells derived from human embryonic stem (hES) cells not only provides great potential to solve the shortage of islets sources for type I diabetes cell therapy,but also benefits the study of the development of the human pancreas and diabetes pathology. In 2001,hES cells were reported to have the capacity to generate insulin-producing cells by spontaneous differentiation in vitro. Since then, many strategies (such as overexpression of key transcription factors,delivery of key proteins for pancreatic development, co-transplantation of differentiated hES cells along with fetal pancreas, stepwise differentiation by mimicking in vivo pancreatic development) have been employed in order to induce the differentiation of pancreatic islet cells from hES cells. Moreover, patient-specific induced pluripotent stem (iPS) cells can be generated by reprogramming somatic cells.iPS cells have characteristics similar to those of ES cells and offer a new cell source for type I diabetes cell therapy that reduces the risk of immunologic rejection. In this review, we summarize the recent progress made in the differentiation of hES and iPS cells into functional pancreatic islet cells and discuss the challenges for their future study.

  6. Generation of pancreatic islet cells from human embryonic stem cells

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Efficiently obtaining functional pancreatic islet cells derived from human embryonic stem(hES) cells not only provides great potential to solve the shortage of islets sources for type I diabetes cell therapy,but also benefits the study of the development of the human pancreas and diabetes pathology.In 2001,hES cells were reported to have the capacity to generate insulin-producing cells by spontaneous differentiation in vitro.Since then,many strategies(such as overexpression of key transcription factors,delivery of key proteins for pancreatic development,co-transplantation of differentiated hES cells along with fetal pancreas,stepwise differentiation by mimicking in vivo pancreatic development) have been employed in order to induce the differentiation of pancreatic islet cells from hES cells.Moreover,patient-specific induced pluripotent stem(iPS) cells can be generated by reprogramming somatic cells.iPS cells have characteristics similar to those of ES cells and offer a new cell source for type I diabetes cell therapy that reduces the risk of immunologic rejection.In this review,we summarize the recent progress made in the differentiation of hES and iPS cells into functional pancreatic islet cells and discuss the challenges for their future study.

  7. Porcine islet isolation outcome is not affected by the amount and distribution of collagen in the pancreas.

    Science.gov (United States)

    Hilling, Denise E; Rijkelijkhuizen, Josephine K R A; Töns, H Annemiek M; Terpstra, Onno T; Bouwman, Eelco

    2010-01-01

    Variable islet yields in porcine islet isolation may be caused by the collagen substrate within the pancreas. The aim of the present study was to determine the total amount and distribution of collagen within porcine pancreata and their relationship to islet isolation outcome. A total of 64 juvenile and 76 adult porcine pancreata of eight purebred breeds were histologically examined. The amount of collagen was quantitatively assessed in tissue samples stained with Sirius Red. Collagen distribution was semi-quantitatively determined by assessing the presence of collagen in the endocrine-exocrine interface and within the islet, in tissue samples stained with Sirius Red and anti-insulin. Islet isolation was performed in 58 pancreata of the adult group. Total collagen content and islet encapsulation ranged widely in both adult and juvenile pigs. However, the majority of islets in adult and juvenile pigs had no or only a limited collagen capsule. The difference in collagen content between adult and juvenile pigs could not be explained by age. Furthermore, no differences between adult and juvenile pigs were found in islet encapsulation or the amount of intra-islet collagen. In adult pigs, no significant relationships were found between obtained islet yield and total collagen content, islet encapsulation or amount of collagen within the islet. Considering the limitations in experimental design (staining method) and study material, isolation outcome does not seem to be affected by the total collagen content or collagen distribution. The influence of other matrix elements and collagen subtypes should be investigated.

  8. Aspects of structural landscape of human islet amyloid polypeptide

    Science.gov (United States)

    He, Jianfeng; Dai, Jin; Li, Jing; Peng, Xubiao; Niemi, Antti J.

    2015-01-01

    The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation.

  9. Aspects of structural landscape of human islet amyloid polypeptide

    Energy Technology Data Exchange (ETDEWEB)

    He, Jianfeng, E-mail: hjf@bit.edu.cn; Dai, Jin, E-mail: daijing491@gmail.com [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Li, Jing, E-mail: jinglichina@139.com [Institute of Biopharmaceutical Research, Yangtze River Pharmaceutical Group Beijing Haiyan Pharmaceutical Co., Ltd, Beijing 102206 (China); Peng, Xubiao, E-mail: xubiaopeng@gmail.com [Department of Physics and Astronomy, Uppsala University, P.O. Box 803, S-75108 Uppsala (Sweden); Niemi, Antti J., E-mail: Antti.Niemi@physics.uu.se [School of Physics, Beijing Institute of Technology, Beijing 100081 (China); Department of Physics and Astronomy, Uppsala University, P.O. Box 803, S-75108 Uppsala (Sweden); Laboratoire de Mathematiques et Physique Theorique CNRS UMR 6083, Fédération Denis Poisson, Université de Tours, Parc de Grandmont, F37200 Tours (France)

    2015-01-28

    The human islet amyloid polypeptide (hIAPP) co-operates with insulin to maintain glycemic balance. It also constitutes the amyloid plaques that aggregate in the pancreas of type-II diabetic patients. We have performed extensive in silico investigations to analyse the structural landscape of monomeric hIAPP, which is presumed to be intrinsically disordered. For this, we construct from first principles a highly predictive energy function that describes a monomeric hIAPP observed in a nuclear magnetic resonance experiment, as a local energy minimum. We subject our theoretical model of hIAPP to repeated heating and cooling simulations, back and forth between a high temperature regime where the conformation resembles a random walker and a low temperature limit where no thermal motions prevail. We find that the final low temperature conformations display a high level of degeneracy, in a manner which is fully in line with the presumed intrinsically disordered character of hIAPP. In particular, we identify an isolated family of α-helical conformations that might cause the transition to amyloidosis, by nucleation.

  10. A review of piscine islet xenotransplantation using wild-type tilapia donors and the production of transgenic tilapia expressing a "humanized" tilapia insulin.

    Science.gov (United States)

    Wright, James R; Yang, Hua; Hyrtsenko, Olga; Xu, Bao-You; Yu, Weiming; Pohajdak, Bill

    2014-01-01

    Most islet xenotransplantation laboratories have focused on porcine islets, which are both costly and difficult to isolate. Teleost (bony) fish, such as tilapia, possess macroscopically visible distinct islet organs called Brockmann bodies which can be inexpensively harvested. When transplanted into diabetic nude mice, tilapia islets maintain long-term normoglycemia and provide human-like glucose tolerance profiles. Like porcine islets, when transplanted into euthymic mice, they are rejected in a CD4 T-cell-dependent manner. However, unlike pigs, tilapia are so phylogenetically primitive that their cells do not express α(1,3)Gal and, because tilapia are highly evolved to live in warm stagnant waters nearly devoid of dissolved oxygen, their islet cells are exceedingly resistant to hypoxia, making them ideal for transplantation within encapsulation devices. Encapsulation, especially when combined with co-stimulatory blockade, markedly prolongs tilapia islet xenograft survival in small animal recipients, and a collaborator has shown function in diabetic cynomolgus monkeys. In anticipation of preclinical xenotransplantation studies, we have extensively characterized tilapia islets (morphology, embryologic development, cell biology, peptides, etc.) and their regulation of glucose homeostasis. Because tilapia insulin differs structurally from human insulin by 17 amino acids, we have produced transgenic tilapia whose islets stably express physiological levels of humanized insulin and have now bred these to homozygosity. These transgenic fish can serve as a platform for further development into a cell therapy product for diabetes.

  11. Human pancreatic islet progenitor cells demonstrate phenotypic plasticity in vitro

    Indian Academy of Sciences (India)

    Maithili P Dalvi; Malati R Umrani; Mugdha V Joglekar; Anandwardhan A Hardikar

    2009-10-01

    Phenotypic plasticity is a phenomenon that describes the occurrence of 2 or more distinct phenotypes under diverse conditions. This article discusses the work carried out over the past few years in understanding the potential of human pancreatic islet-derived progenitors for cell replacement therapy in diabetes. The phenotypic plasticity exhibited by pancreatic progenitors during reversible epithelial-to-mesenchymal transition (EMT) and possible role of microRNAs in regulation of this process is also presented herein.

  12. Islet-like cell aggregates generated from human adipose tissue derived stem cells ameliorate experimental diabetes in mice.

    Directory of Open Access Journals (Sweden)

    Vikash Chandra

    Full Text Available BACKGROUND: Type 1 Diabetes Mellitus is caused by auto immune destruction of insulin producing beta cells in the pancreas. Currently available treatments include transplantation of isolated islets from donor pancreas to the patient. However, this method is limited by inadequate means of immuno-suppression to prevent islet rejection and importantly, limited supply of islets for transplantation. Autologous adult stem cells are now considered for cell replacement therapy in diabetes as it has the potential to generate neo-islets which are genetically part of the treated individual. Adopting methods of islet encapsulation in immuno-isolatory devices would eliminate the need for immuno-suppressants. METHODOLOGY/PRINCIPAL FINDINGS: In the present study we explore the potential of human adipose tissue derived adult stem cells (h-ASCs to differentiate into functional islet like cell aggregates (ICAs. Our stage specific differentiation protocol permit the conversion of mesodermic h-ASCs to definitive endoderm (Hnf3β, TCF2 and Sox17 and to PDX1, Ngn3, NeuroD, Pax4 positive pancreatic endoderm which further matures in vitro to secrete insulin. These ICAs are shown to produce human C-peptide in a glucose dependent manner exhibiting in-vitro functionality. Transplantation of mature ICAs, packed in immuno-isolatory biocompatible capsules to STZ induced diabetic mice restored near normoglycemia within 3-4 weeks. The detection of human C-peptide, 1155±165 pM in blood serum of experimental mice demonstrate the efficacy of our differentiation approach. CONCLUSIONS: h-ASC is an ideal population of personal stem cells for cell replacement therapy, given that they are abundant, easily available and autologous in origin. Our findings present evidence that h-ASCs could be induced to differentiate into physiologically competent functional islet like cell aggregates, which may provide as a source of alternative islets for cell replacement therapy in type 1 diabetes.

  13. Involvement of gene methylation changes in the differentiation of human amniotic epithelial cells into islet-like cell clusters.

    Science.gov (United States)

    Peng, Lin; Wang, Jian; Lu, Guangxiu

    2014-09-01

    Insulin-dependent diabetes results from destruction of the insulin-producing β-cells of the pancreas. Islet cell transplantation is a promising cure for diabetes. Here, we induced human amniotic epithelial cells (hAECs) to differentiate into islet-like cell clusters by nicotinamide plus betacellulin in vitro, and further investigated the DNA methylation status by a Nimble MeDIP microarray before and after cell differentiation to shed light on the molecular mechanisms of this differentiation. In addition, 5-Aza-2'-deoxycytidine was used to investigate whether the differentiation of hAECs into islet-like cells occurred through demethylation. Purified hAECs (CK18(+)/E-cadherin(+)/CD29(+)/CD90(-)/CD34(-)/CD45(-)) were isolated from human amnia. After induction, hAECs were found to be insulin positive and sensitive to glucose, indicating successful induction to islet-like cells. The methylation status of cell cytoskeleton-related genes was down-regulated and that of negative regulation of cell adhesion-related genes was up-regulated. The methylation status of pancreas development-related genes such as HNF1α and DGAT1 was decreased in hAECs after induction. After brief demethylation, INS gene expression was up-regulated in islet-like cell clusters, suggesting that DNA methylation changes were associated with the differentiation of hAECs into islet-like cell clusters.

  14. Limitations in the Use of Fluorescein Diacetate/Propidium Iodide (FDA/PI) and Cell Permeable Nucleic Acid Stains for Viability Measurements of Isolated Islets of Langerhans.

    Science.gov (United States)

    Boyd, Vinc; Cholewa, Olivia Maria; Papas, Klearchos K

    2008-03-01

    BACKGROUND: A review of current literature shows that the combined use of the cell permeable esterase-substrate fluorescein diacetate (FDA) and the cell impermeant nucleic acid stain propidium iodide (PI) to be one of the most common fluorescence-based methods to assess the viability of isolated islets of Langerhans, and it is currently used for islet product release prior to transplantation in humans. However, results from this assay do not correlate with islet viability and function or islet transplantation success in animals or humans (Eckhard et al. 2004; Ricordi et al. 2001). This may be in part attributed to considerable differences as well as discrepancies in the use of these reagents on islets. We critically surveyed the literature and evaluated the impact of a number of variables associated with the use of FDA/PI to determine their reliability in assessing islet cell viability. In addition, we evaluated other fluorescent stains, such as SYTO(R)13, SYTO(R)24 and SYBR(R)14 as possible alternatives to FDA. RESULTS: We found that the stability of stains in storage and stock solutions, the number of islets stained, concentration of stains, staining incubation time, the buffer/media used, and the method of examining islets were significant in the final scoring of viability. For archival file photos, the exposure time and camera/software settings can also impact interpretation of viability. Although our results show that FDA does detect intracellular esterase activity and staining with PI does assess cell membrane integrity, the results obtained from using these stains did not correlate directly with expected islet function and viability per transplantation into diabetic athymic nude mice (Papas et al. 2007). In addition, the use of two nucleic acid stains, such as SYTO(R)13 and PI, for live/dead scoring exhibited staining anomalies which limit their accuracy in assessing islet viability. CONCLUSIONS: From a review of the literature and from our observations on

  15. Induction of human umbilical cord blood-derived stem cells with embryonic stem cell phenotypes into insulin producing islet-like structure.

    Science.gov (United States)

    Sun, Bo; Roh, Kyung-Hwan; Lee, Sae-Rom; Lee, Yong-Soon; Kang, Kyung-Sun

    2007-03-23

    Success in islet-transplantation-based therapies for type I diabetes, coupled with a worldwide shortage of transplant-ready islets, has motivated efforts to develop renewable sources of islet-replacement tissue. Embryonic stem cells (ESCs) have been successfully induced into insulin producing islet-like structure in several studies. However, the source of the ESCs has presented ethical and technical concerns. Here, we isolated a population of stem cells from human cord blood (UCB), which expressed embryo stage specific maker, SSEA-4, and the multi-potential stem cell marker, Oct4. Subsequently, we successfully induced them into insulin-producing islet-like structures, which co-express insulin and C-peptide. These findings might have a significant potential to advance human UCB derived stem-cell-based therapeutics for diabetes.

  16. Human Islet Amyloid Polypeptide Transgenic Mice: In Vivo and Ex Vivo Models for the Role of hIAPP in Type 2 Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    J. W. M. Höppener

    2008-01-01

    Full Text Available Human islet amyloid polypeptide (hIAPP, a pancreatic islet protein of 37 amino acids, is the main component of islet amyloid, seen at autopsy in patients with type 2 diabetes mellitus (DM2. To investigate the roles of hIAPP and islet amyloid in DM2, we generated transgenic mice expressing hIAPP in their islet beta cells. In this study, we found that after a long-term, high-fat diet challenge islet amyloid was observed in only 4 of 19 hIAPP transgenic mice. hIAPP transgenic females exhibited severe glucose intolerance, which was associated with a downregulation of GLUT-2 mRNA expression. In isolated islets from hIAPP males cultured for 3 weeks on high-glucose medium, the percentage of amyloid containing islets increased from 5.5% to 70%. This ex vivo system will allow a more rapid, convenient, and specific study of factors influencing islet amyloidosis as well as of therapeutic strategies to interfere with this pathological process.

  17. Optimization and Scale-up Isolation and Culture of Neonatal Porcine Islets: Potential for Clinical Application.

    Science.gov (United States)

    Ellis, Cara; Lyon, James G; Korbutt, Gregory S

    2016-01-01

    One challenge that must be overcome to allow transplantation of neonatal porcine islets (NPIs) to become a clinical reality is defining a reproducible and scalable protocol for the efficient preparation of therapeutic quantities of clinical grade NPIs. In our standard protocol, we routinely isolate NPIs from a maximum of four pancreases, requiring tissue culture in 16 Petri dishes (four per pancreas) in Ham's F10 and bovine serum albumin (BSA). We have now developed a scalable and technically simpler protocol that allows us to isolate NPIs from a minimum of 12 pancreases at a time by employing automated tissue chopping, collagenase digestion in a single vessel, and tissue culture/media changes in 75% fewer Petri dishes. For culture, BSA is replaced with human serum albumin and supplemented with Z-VAD-FMK general caspase inhibitor and a protease inhibitor cocktail. The caspase inhibitor was added to the media for only the first 90 min of culture. NPIs isolated using the scalable protocol had significantly more cellular insulin recovered (56.9 ± 1.4 µg) when compared to the standard protocol (15.0 ± 0.5 µg; p < 0.05). Compared to our standard protocol, recovery of β-cells (6.0 × 10(6) ± 0.2 vs. 10.0 × 10(6) ± 0.4; p < 0.05) and islet equivalents (35,135 ± 186 vs. 41,810 ± 226; p < 0.05) was significantly higher using the scalable protocol. During a static glucose stimulation assay, the SI of islets isolated by the standard protocol were significantly lower than the scale-up protocol (4.3 ± 0.2 vs. 5.5 ± 0.1; p < 0.05). Mice transplanted with NPIs using the scalable protocol had significantly lower blood glucose levels than the mice that receiving NPIs from the standard protocol (p < 0.01) and responded significantly better to a glucose tolerance test. Based on the above findings, this improved simpler scalable protocol is a significantly more efficient means for preparing therapeutic quantities

  18. IMPROVEMENT OF HUMAN ISLET FUNCTION BY ADENOVIRUS MEDIATED HO-1 GENE TRANSFER

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective To investigate in vitro heme oxygenase-1 gene (HO-1) delivery to human pancreatic islets by adenovirus vectors. Methods Recombinant adenovirus containing HO-1 or enhanced green fluorescent protein gene(EGFP) was generated by using the AdEasy System. The purified human pancreatic islets were infected with recombinant adenovirus vectors at various multiplicity of infection (MOI). Transduction was confirmed by fluorescence photographs and Western blot. Glucose-stimulated insulin secretion was detected by using Human insulin radioimmunoassay kits and was used to assess the function of human islets infected by recombinant adenovirus.Results Viral titers of Ad-hHO-1 and Ad-EGFP were 1.96×109 and 1.99×109 pfu/mL, respectively. Human pancreatic islets were efficiently infected by recombinant adenovirus vectors in vitro. Transfection of human islets at an MOI of 20 did not inhibit islet function. Recombinant adenovirus mediated HO-1gene transfer significantly improved the islet function of insulin release when simulated by high level glucose. Conclusion Recombinant adenovirus is efficient to deliver exogenous gene into human pancreatic islets in vitro. HO-1 gene transfection can improve human islet function.

  19. Stress increased ghrelin secretion from pancreatic isolated islets in male rats.

    Science.gov (United States)

    Rostamkhani, Fatemeh; Zardooz, Homeira; Goshadrou, Fatemeh; Baveisi, Mahyar; Hedayati, Mehdi

    2016-01-01

    It has been demonstrated that plasma ghrelin is likely affected by stress, but little attention has been paid to the effect of stress on ghrelin release from pancreatic islets. This study investigates the effect of stress on ghrelin secretion from pancreatic islets in rats. Male Wistar rats were divided into control and stressed groups. The stressed group was further divided into foot-shock and psychological stress subgroups. Stress was induced by a communication box. After stress exposure, blood sampling was performed to determine the plasma levels of corticosterone, glucose, and ghrelin. Then the animals' pancreatic islets were isolated to assess their ghrelin output at 5.6, 8.3, and 16.7 mM glucose concentrations. Acute exposure to foot-shock and psychological stress both increased plasma corticosterone concentration. Moreover, plasma glucose concentration increased in the foot-shock stress group. Chronic exposure to foot-shock decreased plasma ghrelin concentration, whereas acute exposure had no significant effect. Acute and chronic exposure to foot-shock and psychological stress increased ghrelin secretion from isolated islets in the presence of different glucose concentrations. The results of the present study suggest that ghrelin secretion from isolated islets is not glucose-dependent. However, ghrelin secretion appears to be intensely responsive to both acute and chronic stress.

  20. Alginate Microencapsulation of Human Islets Does Not Increase Susceptibility to Acute Hypoxia

    Directory of Open Access Journals (Sweden)

    I. K. Hals

    2013-01-01

    Full Text Available Islet transplantation in diabetes is hampered by the need of life-long immunosuppression. Encapsulation provides partial immunoprotection but could possibly limit oxygen supply, a factor that may enhance hypoxia-induced beta cell death in the early posttransplantation period. Here we tested susceptibility of alginate microencapsulated human islets to experimental hypoxia (0.1–0.3% O2 for 8 h, followed by reoxygenation on viability and functional parameters. Hypoxia reduced viability as measured by MTT by 33.8±3.5% in encapsulated and 42.9±5.2% in nonencapsulated islets (P<0.2. Nonencapsulated islets released 37.7% (median more HMGB1 compared to encapsulated islets after hypoxic culture conditions (P<0.001. Glucose-induced insulin release was marginally affected by hypoxia. Basal oxygen consumption was equally reduced in encapsulated and nonencapsulated islets, by 22.0±6.1% versus 24.8±5.7%. Among 27 tested cytokines/chemokines, hypoxia increased the secretion of IL-6 and IL-8/CXCL8 in both groups of islets, whereas an increase of MCP-1/CCL2 was seen only with nonencapsulated islets. Conclusion. Alginate microencapsulation of human islets does not increase susceptibility to acute hypoxia. This is a positive finding in relation to potential use of encapsulation for islet transplantation.

  1. Enzymes for Pancreatic Islet Isolation Impact Chemokine-Production and Polarization of Insulin-Producing β-Cells with Reduced Functional Survival of Immunoisolated Rat Islet-Allografts as a Consequence.

    Science.gov (United States)

    de Vos, Paul; Smink, Alexandra M; Paredes, Genaro; Lakey, Jonathan R T; Kuipers, Jeroen; Giepmans, Ben N G; de Haan, Bart J; Faas, Marijke M

    2016-01-01

    The primary aim of this study was to determine whether normal variations in enzyme-activities of collagenases applied for rat-islet isolation impact longevity of encapsulated islet grafts. Also we studied the functional and immunological properties of rat islets isolated with different enzyme preparations to determine whether this impacts these parameters. Rat-islets were isolated from the pancreas with two different collagenases with commonly accepted collagenase, neutral protease, and clostripain activities. Islets had a similar and acceptable glucose-induced insulin-release profile but a profound statistical significant difference in production of the chemokines IP-10 and Gro-α. The islets were studied with nanotomy which is an EM-based technology for unbiased study of ultrastructural features of islets such as cell-cell contacts, endocrine-cell condition, ER stress, mitochondrial conditions, and cell polarization. The islet-batch with higher chemokine-production had a lower amount of polarized insulin-producing β-cells. All islets had more intercellular spaces and less interconnected areas with tight cell-cell junctions when compared to islets in the pancreas. Islet-graft function was studied by implanting encapsulated and free islet grafts in rat recipients. Alginate-based encapsulated grafts isolated with the enzyme-lot inducing higher chemokine production and lower polarization survived for a two-fold shorter period of time. The lower survival-time of the encapsulated grafts was correlated with a higher influx of inflammatory cells at 7 days after implantation. Islets from the same two batches transplanted as free unencapsulated-graft, did not show any difference in survival or function in vivo. Lack of insight in factors contributing to the current lab-to-lab variation in longevity of encapsulated islet-grafts is considered to be a threat for clinical application. Our data suggest that seemingly minor variations in activity of enzymes applied for islet-isolation

  2. Regulation of the JNK3 signaling pathway during islet isolation: JNK3 and c-fos as new markers of islet quality for transplantation.

    Directory of Open Access Journals (Sweden)

    Saida Abdelli

    Full Text Available Stress conditions generated throughout pancreatic islet processing initiate the activation of pro-inflammatory pathways and beta-cell destruction. Our goal is to identify relevant and preferably beta-specific markers to assess the activation of beta-cell stress and apoptotic mechanisms, and therefore the general quality of the islet preparation prior to transplantation. Protein expression and activation were analyzed by Western blotting and kinase assays. ATP measurements were performed by a luminescence-based assay. Oxygen consumption rate (OCR was measured based on standard protocols using fiber optic sensors. Total RNA was used for gene expression analyzes. Our results indicate that pancreas digestion initiates a potent stress response in the islets by activating two stress kinases, c-Jun N-terminal Kinase (JNK and p38. JNK1 protein levels remained unchanged between different islet preparations and following culture. In contrast, levels of JNK3 increased after islet culture, but varied markedly, with a subset of preparations bearing low JNK3 expression. The observed changes in JNK3 protein content strongly correlated with OCR measurements as determined by the Spearman's rank correlation coefficient rho [Formula: see text] in the matching islet samples, while inversely correlating with c-fos mRNA expression [Formula: see text]. In conclusion, pancreas digestion recruits JNK and p38 kinases that are known to participate to beta-cell apoptosis. Concomitantly, the islet isolation alters JNK3 and c-fos expression, both strongly correlating with OCR. Thus, a comparative analysis of JNK3 and c-fos expression before and after culture may provide for novel markers to assess islet quality prior to transplantation. JNK3 has the advantage over all other proposed markers to be islet-specific, and thus to provide for a marker independent of non-beta cell contamination.

  3. Transplanted human pancreatic islets after long-term insulin independence

    DEFF Research Database (Denmark)

    Muller, Y D; Gupta, Shashank; Morel, P;

    2013-01-01

    independence. Islets were pooled from two donors with respectively one and five HLA mismatches. Insulin-positive islets were found throughout the right and left liver, and absent in the pancreas. Two- and three-dimensional analysis showed that islets lost their initial rounded and compact morphology, had...

  4. Human Islet Oxygen Consumption Rate and DNA Measurements Predict Diabetes Reversal in Nude Mice

    OpenAIRE

    Papas, K.K.; Colton, C. K.; Nelson, R. A.; Rozak, P.R.; Avgoustiniatos, E.S.; Scott, W. E.; Wildey, G. M.; Pisania, A.; Weir, G. C.; Hering, B. J.

    2007-01-01

    There is a need for simple, quantitative and prospective assays for islet quality assessment that are predictive of islet transplantation outcome. The current state-of-the-art athymic nude mouse bioassay is costly, technically challenging and retrospective. In this study, we report on the ability of 2 parameters characterizing human islet quality: (1) oxygen consumption rate (OCR), a measure of viable volume; and (2) OCR/DNA, a measure of fractional viability, to predict diabetes reversal in ...

  5. Regional differences in islet distribution in the human pancreas--preferential beta-cell loss in the head region in patients with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Xiaojun Wang

    Full Text Available While regional heterogeneity in islet distribution has been well studied in rodents, less is known about human pancreatic histology. To fill gaps in our understanding, regional differences in the adult human pancreas were quantitatively analyzed including the pathogenesis of type 2 diabetes (T2D. Cadaveric pancreas specimens were collected from the head, body and tail regions of each donor, including subjects with no history of diabetes or pancreatic diseases (n = 23 as well as patients with T2D (n = 12. The study further included individuals from whom islets were isolated (n = 7 to study islet yield and function in a clinical setting of islet transplantation. The whole pancreatic sections were examined using an innovative large-scale image capture and unbiased detailed quantitative analyses of the characteristics of islets from each individual (architecture, size, shape and distribution. Islet distribution/density is similar between the head and body regions, but is >2-fold higher in the tail region. In contrast to rodents, islet cellular composition and architecture were similar throughout the pancreas and there was no difference in glucose-stimulated insulin secretion in islets isolated from different regions of the pancreas. Further studies revealed preferential loss of large islets in the head region in patients with T2D. The present study has demonstrated distinct characteristics of the human pancreas, which should provide a baseline for the future studies integrating existing research in the field and helping to advance bi-directional research between humans and preclinical models.

  6. Selection of polymers for application in scaffolds applicable for human pancreatic islet transplantation.

    Science.gov (United States)

    Smink, Alexandra M; de Haan, Bart J; Paredes-Juarez, Genaro A; Wolters, Anouk H G; Kuipers, Jeroen; Giepmans, Ben N G; Schwab, Leendert; Engelse, Marten A; van Apeldoorn, Aart A; de Koning, Eelco; Faas, Marijke M; de Vos, Paul

    2016-05-13

    The liver is currently the site for transplantation of islets in humans. This is not optimal for islets, but alternative sites in humans are not available. Polymeric scaffolds in surgically accessible areas are a solution. As human donors are rare, the polymers should not interfere with functional survival of human-islets. We applied a novel platform to test the adequacy of polymers for application in scaffolds for human-islet transplantation. Viability, functionality, and immune parameters were included to test poly(D,L-lactide-co-ε-caprolactone) (PDLLCL), poly(ethylene oxide terephthalate)/polybutylene terephthalate (PEOT/PBT) block copolymer, and polysulfone. The type of polymer influenced the functional survival of human islets. In islets cultured on PDLLCL the glucagon-producing α-cells and insulin-producing β-cells contained more hormone granules than in islets in contact with PEOT/PBT or polysulfone. This was studied with ultrastructural analysis by electron microscopy (nanotomy) during 7 d of culture. PDLLCL was also associated with statistically significant lower release of double-stranded DNA (dsDNA, a so called danger-associate molecular pattern (DAMP)) from islets on PDLLCL when compared to the other polymers. DAMPs support undesired immune responses. Hydrophilicity of the polymers did not influence dsDNA release. Islets on PDLLCL also showed less cellular outgrowth. These outgrowing cells were mainly fibroblast and some β-cells undergoing epithelial to mesenchymal cell transition. None of the polymers influenced the glucose-stimulated insulin secretion. As PDLLCL was associated with less release of DAMPs, it is a promising candidate for creating a scaffold for human islets. Our study demonstrates that for sensitive, rare cadaveric donor tissue such as pancreatic islets it might be necessary to first select materials that do not influence functionality before proposing the biomaterial for in vivo application. Our presented platform may facilitate

  7. Activation of GPR119 Stimulates Human β-Cell Replication and Neogenesis in Humanized Mice with Functional Human Islets

    Science.gov (United States)

    Ansarullah; Free, Colette; Christopherson, Jenica; Chen, Quanhai; Gao, Jie; Liu, Chengyang; Naji, Ali; Rabinovitch, Alex; Guo, Zhiguang

    2016-01-01

    Using humanized mice with functional human islets, we investigated whether activating GPR119 by PSN632408, a small molecular agonist, can stimulate human β-cell regeneration in vivo. Human islets were transplanted under the left kidney capsule of immunodeficient mice with streptozotocin- (STZ-) induced diabetes. The recipient mice were treated with PSN632408 or vehicle and BrdU daily. Human islet graft function in the mice was evaluated by nonfasting glucose levels, oral glucose tolerance, and removal of the grafts. Immunostaining for insulin, glucagon, and BrdU or Ki67 was performed in islet grafts to evaluate α- and β-cell replication. Insulin and CK19 immunostaining was performed to evaluate β-cell neogenesis. Four weeks after human islet transplantation, 71% of PSN632408-treated mice achieved normoglycaemia compared with 24% of vehicle-treated mice. Also, oral glucose tolerance was significantly improved in the PSN632408-treated mice. PSN632408 treatment significantly increased both human α- and β-cell areas in islet grafts and stimulated α- and β-cell replication. In addition, β-cell neogenesis was induced from pancreatic duct cells in the islet grafts. Our results demonstrated that activation of GPR119 increases β-cell mass by stimulating human β-cell replication and neogenesis. Therefore, GPR119 activators may qualify as therapeutic agents to increase human β-cell mass in patients with diabetes. PMID:27413754

  8. Isolation and purification of canine islets%犬胰岛的分离与纯化

    Institute of Scientific and Technical Information of China (English)

    冯金发; 许评; 宋纯; 周毅

    2011-01-01

    目的 探讨影响胰岛分离与纯化结果的相关因素,寻找获得足够数量、高纯度、具有活性的胰岛细胞的方法,为临床胰岛移植应用提供实验基础.方法 寻找自动分离技术连续分离22只犬的胰岛,连续性密度梯度离心法纯化胰岛,观察分离纯化出来的胰岛细胞大体形态、超微结构,用葡萄糖刺激释放实验检验其活性.结果 纯化前胰岛计数平均为( 155 040±310) IEQ/胰腺,纯化后的胰岛平均计数为(74200±185)IEQ/胰腺,纯度约为(89.4±2.6)%,回收率约为(47.8±1.3)%,活率达到(93.0±1.7)%.胰岛分离纯化的结果无论在数量上还是在质量上都随着分离技术的不断更新,操作技术的娴熟而有很大提高.葡萄糖刺激释放实验显示低糖组与高糖组比较P <0.001,提示分离纯化后获得的胰岛功能状态良好.结论 从犬中分离和纯化出足够数量的胰岛细胞,可用于临床研究.%Objective To investigate factors influencing islet isolation and purification in dogs and to explore how to obtain massive purified viable islets from canine pancreas.To provide experimental evidence for pancreatic islet transplantation in clinical practice.Methods Islets were isolated from pancreas of 22 dogs by modified automated techniques,followed by purification using continuous density gradients.The purity of islets was determined by dithizone (DTZ) staining,and the function of islets was evaluated by glucose stimulating insulin release test.Results The crude islets yield was ( 155 040 ±310) IEQ/pancreas while the yield was (74 200 ±185) IEQ/pancreas after purification.The average purity was (89.4 ±2.6) %.The recovery rate was(47.8 ±1.3) %.Islet viability was(93.0 ± 1.7)%.The modified techniques improved both the yield and the quality in islet isolation and purification.The islets were morphologically intact after purification.Glucose stimulating insulin release test showed that the difference of the secreted

  9. Effect of Amorphophallus Konjac oligosaccharides on STZ-induced diabetes model of isolated islets.

    Science.gov (United States)

    Lu, Xiu-Ju; Chen, Xiu-Min; Fu, De-Xian; Cong, Wei; Ouyang, Fan

    2002-12-27

    Three oligosaccharide fractions from the root of Amorphophallus Konjac, which was reported with hypoglycemic effects on diabetes subjects, were isolated and studied using the STZ-treated diabetes model. Among them, one fraction named as KOS-A, was found with nitric oxide (NO(*)) free radical regulation effect, while the other two were not. At concentrations less than 1.5 mM, KOS-A positively decreased STZ-induced NO(*) level of islets, but normal NO(*) release for non-STZ-treated islets was not affected within the range. At 15 mM, KOS-A played a contrary role and increased NO(*) level for islets both with and without STZ-treatment. Islets insulin secretion changed corresponding to NO(*) level in the assay. Increased insulin secretion appeared parallel to the decrease of NO(*), and normal insulin release was not affected by KOS-A less than 1.5 mM. Structure determination of KOS-A shows that it is a tetrasaccharide with Mw of 666 Da and reductive end of alpha-D-mannose. These results indicate that low dosage of KOS-A, with its function on attenuating STZ-induced NO(*) level, doesn't alter normal NO(*) and insulin secretion pathways of isolated islets. The NO(*) attenuation function of KOS-A on the diabetes model is mainly resulted from environmental free radical scavenging by the oligosaccharide. Present results also imply the mechanism of clinical Amorphophallus Konjac hypoglycemic function maybe related with free radical attenuation and lower risks of islets damage from NO(*) radical.

  10. AN ANALYSIS OF THE ROLE OF COLLAGENASE AND PROTEASE IN THE ENZYMATIC DISSOCIATION OF THE RAT PANCREAS FOR ISLET ISOLATION

    NARCIS (Netherlands)

    WOLTERS, GHJ; VOSSCHEPERKEUTER, GH; VANDEIJNEN, JHM; VANSCHILFGAARDE, R

    1992-01-01

    Crude Clostridium histolyticum collagenase is widely used for the enzymatic degradation of pancreatic extracellular matrix in order to isolate the islets of Langerhans. The variable enzymatic composition of crude collagenases is a critical issue which contributes to the poor reproducibility of islet

  11. A rapid, efficient, and economic device and method for the isolation and purification of mouse islet cells.

    Science.gov (United States)

    Zongyi, Yin; Funian, Zou; Hao, Li; Ying, Cheng; Jialin, Zhang; Baifeng, Li

    2017-01-01

    Rapid, efficient, and economic method for the isolation and purification of islets has been pursued by numerous islet-related researchers. In this study, we compared the advantages and disadvantages of our developed patented method with those of commonly used conventional methods (Ficoll-400, 1077, and handpicking methods). Cell viability was assayed using Trypan blue, cell purity and yield were assayed using diphenylthiocarbazone, and islet function was assayed using acridine orange/ethidium bromide staining and enzyme-linked immunosorbent assay-glucose stimulation testing 4 days after cultivation. The results showed that our islet isolation and purification method required 12 ± 3 min, which was significantly shorter than the time required in Ficoll-400, 1077, and HPU groups (34 ± 3, 41 ± 4, and 30 ± 4 min, respectively; P 1000 islets). In summary, the MCT method is a rapid, efficient, and economic method for isolating and purifying murine islet cell clumps. This method overcomes some of the shortcomings of conventional methods, showing a relatively higher quality and yield of islets within a shorter duration at a lower cost. Therefore, the current method provides researchers with an alternative option for islet isolation and should be widely generalized.

  12. Single-Cell Sequencing of Human Pancreatic Islets-New Kids on the Block.

    Science.gov (United States)

    Prasad, Rashmi B; Groop, Leif

    2016-10-11

    RNA sequencing of human pancreatic islets has provided important insights into the islet transcriptome but little information on the specific cells. In this issue, Segerstolpe et al. (2016) and Xin et al. (2016b) report on the transcriptome of single pancreatic cells from non-diabetic and type 2 diabetic donors. Copyright © 2016. Published by Elsevier Inc.

  13. Survival of free and encapsulated human and rat islet xenografts transplanted into the mouse bone marrow.

    Science.gov (United States)

    Meier, Raphael P H; Seebach, Jörg D; Morel, Philippe; Mahou, Redouan; Borot, Sophie; Giovannoni, Laurianne; Parnaud, Geraldine; Montanari, Elisa; Bosco, Domenico; Wandrey, Christine; Berney, Thierry; Bühler, Leo H; Muller, Yannick D

    2014-01-01

    Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow) and 10 days (kidney capsule). Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.

  14. Survival of free and encapsulated human and rat islet xenografts transplanted into the mouse bone marrow.

    Directory of Open Access Journals (Sweden)

    Raphael P H Meier

    Full Text Available Bone marrow was recently proposed as an alternative and potentially immune-privileged site for pancreatic islet transplantation. The aim of the present study was to assess the survival and rejection mechanisms of free and encapsulated xenogeneic islets transplanted into the medullary cavity of the femur, or under the kidney capsule of streptozotocin-induced diabetic C57BL/6 mice. The median survival of free rat islets transplanted into the bone marrow or under the kidney capsule was 9 and 14 days, respectively, whereas that of free human islets was shorter, 7 days (bone marrow and 10 days (kidney capsule. Infiltrating CD8+ T cells and redistributed CD4+ T cells, and macrophages were detected around the transplanted islets in bone sections. Recipient mouse splenocytes proliferated in response to donor rat stimulator cells. One month after transplantation under both kidney capsule or into bone marrow, encapsulated rat islets had induced a similar degree of fibrotic reaction and still contained insulin positive cells. In conclusion, we successfully established a small animal model for xenogeneic islet transplantation into the bone marrow. The rejection of xenogeneic islets was associated with local and systemic T cell responses and macrophage recruitment. Although there was no evidence for immune-privilege, the bone marrow may represent a feasible site for encapsulated xenogeneic islet transplantation.

  15. Inhibitory effect of kisspeptins on insulin secretion from isolated mouse islets.

    Science.gov (United States)

    Vikman, J; Ahrén, B

    2009-11-01

    Islet hormone secretion is regulated by a variety of factors, and many of these signal through G protein-coupled receptors (GPCRs). A novel islet GPCR is GPR54, which couples to the Gq isoform of G proteins, which in turn signal through the phospholipase C pathway. Ligands for GPR54 are kisspeptins, which are peptides encoded in the KISS1 gene and also expressed in islet beta-cells. The KISS1 gene encodes a hydrophobic 145-amino acid protein that is cleaved into a 54-amino acid protein, kisspeptin-54 or KP54. Shorter kisspeptins also exist, such as kisspeptin-10 (KP10) and kisspeptin-13 (KP13). The involvement of GPR54 and kisspeptins in the regulation of islet function is not known. To address this problem, we incubated isolated mouse islets in the presence of KP13 and KP54 for 60 min and measured insulin secretion. We found that both KP13 and KP54 at 10 nM, 100 nM and 1microM inhibited insulin secretion in the presence of 2.8 mM glucose. However, by increasing the glucose concentration, this inhibitory action of the kisspeptins vanished. Thus, at 11.1 mM glucose, KP13 and KP54 inhibited insulin secretion only at high doses, and at 16.7 mM they no longer inhibited insulin secretion in any of the doses. We conclude that kisspeptins inhibit insulin secretion at glucose concentrations below 11.1 mM. This suggests that kisspeptins are regulating insulin secretion at physiological concentrations of glucose. The mechanisms by which kisspeptins regulate islet function and insulin secretion are unknown and will be further investigated.

  16. Human islets and dendritic cells generate post-translationally modified islet auto-antigens

    NARCIS (Netherlands)

    McLaughlin, Rene J; de Haan, Anne; Zaldumbide, Arnaud; de Koning, Eelco J; de Ru, Arnoud H; van Veelen, Peter A; van Lummel, Menno; Roep, Bart O

    2016-01-01

    Initiation of type 1 diabetes (T1D) requires a break in peripheral tolerance. New insights into neo-epitope formation indicate that post-translational modification of islet auto-antigens, for example via deamidation, may be an important component of disease initiation or exacerbation. Indeed, deamid

  17. Proteins differentially expressed in human beta-cells-enriched pancreatic islet cultures and human insulinomas

    DEFF Research Database (Denmark)

    Terra, Letícia F; Teixeira, Priscila C; Wailemann, Rosangela A M

    2013-01-01

    In view of the great demand for human beta-cells for physiological and medical studies, we generated cell lines derived from human insulinomas which secrete insulin, C-peptide and express neuroendocrine and islet markers. In this study, we set out to characterize their proteomes, comparing them t...

  18. Human pancreatic islet preparations release HMGB1: (ir)relevance for graft engraftment.

    Science.gov (United States)

    Nano, Rita; Racanicchi, Leda; Melzi, Raffaella; Mercalli, Alessia; Maffi, Paola; Sordi, Valeria; Ling, Zhidong; Scavini, Marina; Korsgren, Olle; Celona, Barbara; Secchi, Antonio; Piemonti, Lorenzo

    2013-01-01

    High levels of donor-derived high-mobility group box 1 (HMGB1) protein have been associated with poor islet graft outcome in mouse models. The aim of our work was to determine whether HMGB1 released by human islets had independent proinflammatory effects that influence engraftment in humans. Human islet preparations contained and released HMGB1 in different amounts, as determined by Western blot and ELISA (median 17 pg/ml/IEQ/24 h; min-max 0-211, n = 74). HMGB1 release directly correlated with brain death, donor hyperamilasemia, and factors related to the pancreas digestion procedure (collagenase and digestion time). HMGB1 release was significantly positively associated with the release of other cytokines/chemokines, particularly with the highly released "proinflammatory" CXCL8/IL-8, CXCL1/GRO-α, and the IFN-γ-inducible chemokines CXCL10/IP-10 and CXCL9/MIG. HMGB1 release was not modulated by Toll-like receptor 2, 3, 4, 5, and 9 agonists or by exposure to IL-1β. When evaluated after islet transplantation, pretransplant HMGB1 release was weakly associated with the activation of the coagulation cascade (evaluated as serum cross-linked fibrin products), but not with the immediate posttransplant inflammatory response. Concordantly, HMGB1 did not affect short-term human islet function. Our data show that human islet HMGB1 release is a sign of "damaged" islets, although without any independent direct role in graft failure.

  19. Noninvasive imaging of islet grafts using positron-emission tomography

    Science.gov (United States)

    Lu, Yuxin; Dang, Hoa; Middleton, Blake; Zhang, Zesong; Washburn, Lorraine; Stout, David B.; Campbell-Thompson, Martha; Atkinson, Mark A.; Phelps, Michael; Gambhir, Sanjiv Sam; Tian, Jide; Kaufman, Daniel L.

    2006-07-01

    Islet transplantation offers a potential therapy to restore glucose homeostasis in type 1 diabetes patients. However, islet transplantation is not routinely successful because most islet recipients gradually lose graft function. Furthermore, serological markers of islet function are insensitive to islet loss until the latter stages of islet graft rejection. A noninvasive method of monitoring islet grafts would aid in the assessment of islet graft survival and the evaluation of interventions designed to prolong graft survival. Here, we show that recombinant adenovirus can engineer isolated islets to express a positron-emission tomography (PET) reporter gene and that these islets can be repeatedly imaged by using microPET after transplantation into mice. The magnitude of signal from engineered islets implanted into the axillary cavity was directly related to the implanted islet mass. PET signals attenuated over the following weeks because of the transient nature of adenovirus-mediated gene expression. Because the liver is the preferred site for islet implantation in humans, we also tested whether islets could be imaged after transfusion into the mouse liver. Control studies revealed that both intrahepatic islet transplantation and hyperglycemia altered the biodistribution kinetics of the PET probe systemically. Although transplanted islets were dispersed throughout the liver, clear signals from the liver region of mice receiving PET reporter-expressing islets were detectable for several weeks. Viral transduction, PET reporter expression, and repeated microPET imaging had no apparent deleterious effects on islet function after implantation. These studies lay a foundation for noninvasive quantitative assessments of islet graft survival using PET. diabetes | transplantation

  20. Ontogeny of neuro-insular complexes and islets innervation in the human pancreas.

    Directory of Open Access Journals (Sweden)

    Alexandra E. Proshchina

    2014-04-01

    Full Text Available The ontogeny of the neuro-insular complexes (NIC and the islets innervation in human pancreas has not been studied in detail. Our aim was to describe the developmental dynamics and distribution of the nervous system structures in the endocrine part of human pancreas. We used doublestaining with antibodies specific to pan-neural markers (neuron-specific enolase (NSE and S100 protein and to hormones of pancreatic endocrine cells. NSE and S100-positive nerves and ganglia were identified in the human fetal pancreas from gestation week (gw 10 onwards. Later the density of S100 and NSE-positive fibers increased. In adults this network was sparse. The islets innervation started to form from gw 14. NSE-containing endocrine cells were identified from gw 12 onwards. Additionally, S100-positive cells were detected both in the periphery and within some of the islets starting at gw 14. The analysis of islets innervation has shown that the fetal pancreas contained neuro-insular complexes and the number of these complexes was reduced in adults. The highest density of neuro-insular complexes is detected during middle and late fetal periods, when the mosaic islets, typical for adults, form. The close integration between the developing pancreatic islets and the nervous system structures may play an important role not only in the hormone secretion, but also in the islets morphogenesis.

  1. KU-32, a Novel Drug for Diabetic Neuropathy, Is Safe for Human Islets and Improves In Vitro Insulin Secretion and Viability

    Directory of Open Access Journals (Sweden)

    Kevin Farmer

    2012-01-01

    Full Text Available KU-32 is a novel, novobiocin-based Hsp90 inhibitor that protects against neuronal glucotoxicity and reverses multiple clinical indices of diabetic peripheral neuropathy in a rodent model. However, any drug with potential for treating diabetic complications must also have no adverse effects on the function of pancreatic islets. Thus, the goal of the current study was to assess the effect of KU-32 on the in vitro viability and function of human islets. Treating human islets with KU-32 for 24 hours showed no toxicity as assessed using the alamarBlue assay. Confocal microscopy confirmed that with a minimum of 2-day exposure, KU-32 improved cellular viability by blocking apoptosis. Functionally, isolated human islets released more glucose-stimulated insulin when preincubated in KU-32. However, diabetic BKS-db/db mice, a model for type 2 diabetes, administered KU-32 for 10 weeks did not show any significant changes in blood glucose and insulin levels, despite having greater insulin staining/beta cell in the pancreas compared to untreated BKS db/db mice. In summary, KU-32 did not harm isolated human islets and may even be protective. However, the effect does not appear significant enough to alter the in vivo metabolic parameters of diabetic mice.

  2. KU-32, a novel drug for diabetic neuropathy, is safe for human islets and improves in vitro insulin secretion and viability.

    Science.gov (United States)

    Farmer, Kevin; Williams, S Janette; Novikova, Lesya; Ramachandran, Karthik; Rawal, Sonia; Blagg, Brian S J; Dobrowsky, Rick; Stehno-Bittel, Lisa

    2012-01-01

    KU-32 is a novel, novobiocin-based Hsp90 inhibitor that protects against neuronal glucotoxicity and reverses multiple clinical indices of diabetic peripheral neuropathy in a rodent model. However, any drug with potential for treating diabetic complications must also have no adverse effects on the function of pancreatic islets. Thus, the goal of the current study was to assess the effect of KU-32 on the in vitro viability and function of human islets. Treating human islets with KU-32 for 24 hours showed no toxicity as assessed using the alamarBlue assay. Confocal microscopy confirmed that with a minimum of 2-day exposure, KU-32 improved cellular viability by blocking apoptosis. Functionally, isolated human islets released more glucose-stimulated insulin when preincubated in KU-32. However, diabetic BKS-db/db mice, a model for type 2 diabetes, administered KU-32 for 10 weeks did not show any significant changes in blood glucose and insulin levels, despite having greater insulin staining/beta cell in the pancreas compared to untreated BKS db/db mice. In summary, KU-32 did not harm isolated human islets and may even be protective. However, the effect does not appear significant enough to alter the in vivo metabolic parameters of diabetic mice.

  3. Differential sensitivity to beta-cell secretagogues in cultured rat pancreatic islets exposed to human interleukin-1 beta.

    Science.gov (United States)

    Eizirik, D L; Sandler, S; Hallberg, A; Bendtzen, K; Sener, A; Malaisse, W J

    1989-08-01

    The early stages of insulin-dependent diabetes mellitus are characterized by a selective inability to secrete insulin in response to glucose, coupled to a better response to nonnutrient secretagogues. The deficient glucose response may be a result of the autoimmune process directed toward the beta-cells. Interleukin-1 (IL-1) has been suggested to be one possible mediator of immunological damage of the beta-cells. In the present study we characterized the sensitivity of beta-cells to different secretagogues after human recombinant IL-1 beta (rIL-1 beta) exposure. Furthermore, experiments were performed to clarify the biochemical mechanisms behind the defective insulin response observed in these islets. Rat pancreatic islets were isolated and kept in tissue culture (medium RPMI-1640 plus 10% calf serum) for 5 days. The islets were subsequently exposed to 60 pM human recombinant IL-1 beta during 48 h in the same culture conditions as above and examined immediately after IL-1 exposure. The rIL-1 beta-treated islets showed a marked reduction of glucose-stimulated insulin release. Stimulation with arginine plus different glucose concentrations, and leucine plus glutamine partially counteracted the rIL-1 beta-induced reduction of insulin release. The activities of the glycolytic enzymes hexokinase, glucokinase, and glyceraldehyde 3-phosphate dehydrogenase, were similar in control and IL-1-exposed islets. Treatment with IL-1 also did not impair the activities of NADH+- and NADPH+-dependent glutamate dehydrogenase, glutamate-aspartate transaminase, glutamate-alanine transaminase, citrate synthase, and NAD+-linked isocitrate dehydrogenase. The oxidation of D-[6-14C]glucose and L-[U-14C]leucine were decreased by 50% in IL-1-treated islets. Furthermore, there was a significant decrease in the ratios of [2-14C]pyruvate oxidation/[1-14C]pyruvate decarboxylation and L-[U-14C]leucine oxidation/L-[1-14C]leucine decarboxylation, indicating that IL-1 decreases the proportion of

  4. Amyloid Deposition in Transplanted Human Pancreatic Islets: A Conceivable Cause of Their Long-Term Failure

    Directory of Open Access Journals (Sweden)

    Arne Andersson

    2008-01-01

    Full Text Available Following the encouraging report of the Edmonton group, there was a rejuvenation of the islet transplantation field. After that, more pessimistic views spread when long-term results of the clinical outcome were published. A progressive loss of the β-cell function meant that almost all patients were back on insulin therapy after 5 years. More than 10 years ago, we demonstrated that amyloid deposits rapidly formed in human islets and in mouse islets transgenic for human IAPP when grafted into nude mice. It is, therefore, conceivable to consider amyloid formation as one potential candidate for the long-term failure. The present paper reviews attempts in our laboratories to elucidate the dynamics of and mechanisms behind the formation of amyloid in transplanted islets with special emphasis on the impact of long-term hyperglycemia.

  5. Isolation, Culture and Induced Differentiation of Fetal Porcine Islet Derived Pancreatic Stem Cell

    Institute of Scientific and Technical Information of China (English)

    FENG Ruo-peng; ZHANG Hui-ru; WANG Yun; QIAO Hai; ZHAO Ting; SHEN Wen-zheng; DOU Zhong-ying

    2007-01-01

    To isolate and culture the porcine pancreatic stem cells and investigate their function, the fetal porcine pancreatic stem cells were isolated by the method of suspending plus adhering culture. The isolated cells were then identified by irnmunohistochemical staining, and their culture viability measured through the MTT method in vitro. This induced them to differentiate into endocrine cells and detect their function. The isolated IPSCS did not express nestin, but expressed CK-19, a marker of ductal epithelia cells and oc-actin, a smooth muscle marker, demonstrating the growth characteristics of ES-like cells, and strong proliferative ability, after 18 passages. They could excrete insulin, and showed ultrastructure changes after being induced. Porcine pancreatic stem cells can be isolated by this method, induced to form islet-like clusters, and can secret insulin.

  6. The Peri-islet Basement Membrane, a Barrier to Infiltrating Leukocytes in Type 1 Diabetes in Mouse and Human

    DEFF Research Database (Denmark)

    Korpos, Eva; Kadri, Nadir; Kappelhoff, Reinhild

    2013-01-01

    We provide the first comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet basement membrane (BM) and subjacent interstitial matrix (IM), in development of type 1 diabetes in NOD mice and in human type 1 diabetes. Our data...... activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human type 1 diabetic samples and, hence, potentially a novel therapeutic target specifically acting at the islet penetration stage. Interestingly, the peri-islet BM and underlying...... IM are reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies....

  7. Volumetric properties of human islet amyloid polypeptide in liquid water.

    Science.gov (United States)

    Brovchenko, I; Andrews, M N; Oleinikova, A

    2010-04-28

    The volumetric properties of human islet amyloid polypeptide (hIAPP) in water were studied in a wide temperature range by computer simulations. The intrinsic density rho(p) and the intrinsic thermal expansion coefficient alpha(p) of hIAPP were evaluated by taking into account the difference between the volumetric properties of hydration and bulk water. The density of hydration water rho(h) was found to decrease almost linearly with temperature upon heating and its thermal expansion coefficient was found to be notably higher than that of bulk water. The peptide surface exposed to water is more hydrophobic and its rho(h) is smaller in conformation with a larger number of intrapeptide hydrogen bonds. The two hIAPP peptides studied (with and without disulfide bridge) show negative alpha(p), which is close to zero at 250 K and decreases to approximately -1.5 x 10(-3) K(-1) upon heating to 450 K. The analysis of various structural properties of peptides shows a correlation between the intrinsic peptide volumes and the number of intrapeptide hydrogen bonds. The obtained negative values of alpha(p) can be attributed to the shrinkage of the inner voids of the peptides upon heating.

  8. Islet neogenesis potential of human adult stem cells and its applications in cell replacement therapy for diabetes

    Directory of Open Access Journals (Sweden)

    Bhonde RR

    2008-11-01

    Full Text Available In recent years regenerative biology has reached to greater heights due to its therapeutic potential in treating degenerative diseases; as they are not curable by modern medicine. With the advent of research in stem cells and developmental biology the regenerative potential of adult resident stem cells is becoming clearer. The long term objective of regenerative medicine or cell therapy is to treat patients with their own stem cells. These stem cells could be derived from the diseased organs such as skin, liver, pancreas etc. or from reservoirs of multipotent stem cells such as bone marrow or cord blood.Manipulating the ability of tissue resident stem cells as well as from multipotent reservoirs such as bone marrow, umbilical cord and cord blood to give rise to endocrine cells may open new avenues in the treatment of diabetes. A better understanding of stem cell biology would almost certainly allow for the establishment of efficient and reliable cell transplantation experimental programs in the clinic. We show here that multipotent mesenchymal stem cells can be isolated from various sources such as the bone marrow, placenta, umbilical cord. Upon stimulation with specific growth factors they differentiate into islet like clusters (ILCs. When ILCs obtained from the above mentioned sources were transplanted in experimental diabetic mice, restoration of normoglycemia was observed within three weeks of transplantation with concomitant increase in the body weight. These euglycemic mice exhibited normal glucose tolerance test indicating normal utilization of glucose. Allthough the MSCs isolated from all the sources had the same characteristics; they showed significant differences in their islet differentiation potential. ILCs isolated for the human bone marrow did not show any pancreatic hormones in vitro, but upon transplantation they matured into insulin and somatostatin producing hormones. Placental MSCs as well as ILCs showed insulin trascripts

  9. Adaptive changes of human islets to an obesogenic environment in the mouse

    OpenAIRE

    Gargani, Sofia

    2013-01-01

    Introduction: Under normal healthy conditions, organisms maintain a dynamic endocrinecell mass throughout life. Pancreatic beta cell mass are able to maintain plasma glucose levels increasing insulin secretion in conditions as obesity. Beta cell inability to compensate in insulin demand provokes hyperglycemia and Type 2 Diabetes. Clinically, most obese individuals do not develop diabetes because islets compensate for insulin resistance. Direct evidence that human islet mass adapts longitudina...

  10. Efficient gene delivery and silencing of mouse and human pancreatic islets

    Directory of Open Access Journals (Sweden)

    Moerman Ericka

    2010-03-01

    Full Text Available Abstract Background In view of the importance of beta cells in glucose homeostasis and the profound repercussions of beta cell pathology on human health, the acquisition of tools to study pancreatic islet function is essential for the design of alternative novel therapies for diabetes. One promising approach toward this goal involves the modification of gene expression profile of beta cells. Results This study describes a new method of gene and siRNA delivery into human pancreatic islets by microporation technology. We demonstrated that mild islet distention with accutase greatly enhanced the transfection efficiency without compromising in vitro function (secretion, apoptosis and viability. As an example, the recently identified gene involved in type 2 diabetes, ZnT8, can be over-expressed or silenced by RNA interference using this technology. Microporation can also be used on rodent islets. Conclusions Taken together, our results demonstrate that microporation technology can be used to modify gene expression in whole rodent and human islets without altering their in vitro function and will be key to the elucidation of the factors responsible for proper islet function.

  11. Controlled aggregation of primary human pancreatic islet cells leads to glucose-responsive pseudoislets comparable to native islets

    NARCIS (Netherlands)

    Hilderink, Janneke; Spijker, Siebe; Carlotti, Françoise; Lange, Lydia; Engelse, Marten; van Blitterswijk, Clemens; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

    2015-01-01

    Clinical islet transplantation is a promising treatment for patients with type 1 diabetes. However, pancreatic islets vary in size and shape affecting their survival and function after transplantation because of mass transport limitations. To reduce diffusion restrictions and improve islet cell surv

  12. Islet isolation and GMP, ISO 9001:2000: what do we need--a 3-year experience.

    Science.gov (United States)

    Hengster, P; Hermann, M; Pirkebner, D; Draxl, A; Margreiter, R

    2005-10-01

    Pancreatic islet cell isolation and transplantation has been performed for many years at several institutions. Although all institutions aim to produce high-quality islets, applied standards widely deviate from standards in the pharmaceutical industry. The legal situation within the European Union has changed requirements for setting up and running such a laboratory. The process is now clearly defined as a production of a pharmaceutical and therefore must be licensed by federal authorities. Analysis of workload for establishing an islet isolation program that fulfil GMP and ISO 9001 criteria including an estimation of costs and the impact of such a system on the isolation process. The definition of quality parameters and documentation is a central issue of all islet isolation laboratories. Therefore, GMP and ISO 9001:2000 do not add additional work per se. On the other hand, clear guidelines, a clear policy, working place descriptions, forms, checklists, and, particularly standard operating procedures, are instrumental for smooth functioning within the department. Collection of data such as errors, improvement measures, and preventive measures reduces subsequent costs. A clear definition of responsibilities minimizes organizational problems. Steering of inspection devices prevents bias errors and validating the processes clearly points out incorrect assumptions. Documentation helps to prove the correctness of the production at any time and is of use also for scientific evaluations. We strongly feel that GMP criteria are mandatory and together with an ISO 9001:2000 quality management system offers significant advantages for the process of islet isolation and a continuous improvement process.

  13. Cytotoxic T-lymphocyte-mediated killing of human pancreatic islet cells in vitro.

    Science.gov (United States)

    Campbell, Peter D; Estella, Eugene; Dudek, Nadine L; Jhala, Gaurang; Thomas, Helen E; Kay, Thomas W H; Mannering, Stuart I

    2008-09-01

    Cytotoxic T lymphocytes (CTL) are believed to play an essential role in beta-cell destruction leading to development of type 1 diabetes and allogeneic islet graft failure. We aimed to identify the mechanisms used by CTL to kill human beta cells. CTL clones that recognize epitopes from influenza virus and Epstein-Barr virus restricted by human leukocyte antigen (HLA)-A0201 and -B0801, respectively, were used to investigate the susceptibility of human beta cells to CTL. In a short-term (5-hour) assay, CTL killed human islet cells of the appropriate major histocompatibility complex (MHC) class I type that had been pulsed with viral peptides. Killing was increased by pretreating islets with interferon gamma that increases MHC class I on target cells. Killing was abolished by incubation of CTL with the perforin inhibitor concanamycin A. The Fas pathway did not contribute to killing because blocking with neutralizing anti-Fas ligand antibody did not significantly reduce beta-cell killing. In conclusion, we report a novel way of investigating the interaction between CTL and human islets. Human islets were rapidly killed in vitro by MHC class I-restricted CTL predominantly by the granule exocytosis pathway.

  14. Pancreatic islet transplantation

    Directory of Open Access Journals (Sweden)

    Corrêa-Giannella Maria

    2009-09-01

    Full Text Available Abstract Background No formulation of exogenous insulin available to date has yet been able to mimic the physiological nictemeral rhythms of this hormone, and despite all engineering advancements, the theoretical proposal of developing a mechanical replacement for pancreatic β cell still has not been reached. Thus, the replacement of β cells through pancreas and pancreatic islet transplantation are the only concrete alternatives for re-establishing the endogenous insulin secretion in type 1 diabetic patients. Since only 1 to 1.5% of the pancreatic mass corresponds to endocrine tissue, pancreatic islets transplantation arises as a natural alternative. Data from the International Islet Transplant Registry (ITR from 1983 to December 2000 document a total of 493 transplants performed around the world, with progressively worse rates of post-transplant insulin independence. In 2000, the "Edmonton Protocol" introduced several modifications to the transplantation procedure, such as the use of a steroid-free immunosuppression regimen and transplantation of a mean islet mass of 11,000 islet equivalents per kilogram, which significantly improved 1-year outcomes. Although the results of a 5-year follow-up in 65 patients demonstrated improvement in glycemic instability in a significant portion of them, only 7.5% of the patients have reached insulin independence, indicating the need of further advances in the preservation of the function of transplanted islet. In addition to the scarcity of organs available for transplantation, islets transplantation still faces major challenges, specially those related to cell loss during the process of islet isolation and the losses related to the graft site, apoptosis, allorejection, autoimmunity, and immunosuppression. The main strategies to optimize islet transplantation aim at improving all these aspects. Conclusion Human islet transplantation should be regarded as an intervention that can decrease the frequency of

  15. Role and mechanism of rosiglitazone on the impairment of insulin secretion induced by free fatty acids on isolated rat islets

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Background Prolonged exposure of pancreatic β-cells to fatty acids increases basal insulin secretion but inhibits glucose-stimulated insulin secretion. Rosiglitazone is a new antidiabetic agent of the thiazolidinediones. However, the relationship between thiazolidinediones and insulin secretion is highly controversial. The aim of this study is to explore the effect and mechanism of rosiglitazone on insulin secretion of islets under chronic exposure to free fatty acids (FFA).Methods Pancreatic islets were isolated from the pancreata of male Sprague-Dawley rats by the collagenase digestion and by the dextran gradient centrifugation method. The purified islets were cultured in the presence or absence of rosiglitazone and palmitate for 48 hours. The insulin secretion was measured by radioimmunoassay. The mRNA level of peroxisome proliferator-activated receptor (, uncoupling protein 2 (UCP-2) and insulin were determined by real-time polymerase chain reaction (PCR). The cell cytotoxicity assay was measured by cell counting kit-8. Results Islets exposed to elevated palmitate for 48 hours showed an increased basal and a decreased glucose-stimulated insulin secretion (P<0.01). The mRNA level of UCP-2 was increased by 3.7 fold in the 0.5 mmol/L concentration of palmitate. When islets were cultured with palmitate (0.5 mmol/L) in the presence of rosiglitazone (1.0 μmol/L), both basal and glucose-stimulated insulin secretion reversed to a pattern of control islets (P<0.05, P<0.01). The addition of rosiglitazone in the culture medium decreased the mRNA level of UCP-2 by 2.2 fold, having a statistically significant difference (P<0.05) as compared with islets cultured with palmitate alone. The cell viability was not affected. Conclusion The protective effects of rosiglitazone on insulin secretion of isolated pancreatic islets under chronic exposure to palmitate might be mediated through the downregulation of UCP-2 expression.

  16. Islet-like clusters derived from mesenchymal stem cells in Wharton's Jelly of the human umbilical cord for transplantation to control type 1 diabetes.

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    Kuo Ching Chao

    Full Text Available BACKGROUND: There is a widespread interest in developing renewable sources of islet-replacement tissue for type I diabetes mellitus. Human mesenchymal cells isolated from the Wharton's jelly of the umbilical cord (HUMSCs, which can be easily obtained and processed compared with embryonic and bone marrow stem cells, possess stem cell properties. HUMSCs may be a valuable source for the generation of islets. METHODOLOGY AND PRINCIPAL FINDINGS: HUMSCs were induced to transform into islet-like cell clusters in vitro through stepwise culturing in neuron-conditioned medium. To assess the functional stability of the islet-like cell clusters in vivo, these cell clusters were transplanted into the liver of streptozotocin-induced diabetic rats via laparotomy. Glucose tolerance was measured on week 12 after transplantation accompanied with immunohistochemistry and electron microscopy analysis. These islet-like cell clusters were shown to contain human C-peptide and release human insulin in response to physiological glucose levels. Real-time RT-PCR detected the expressions of insulin and other pancreatic beta-cell-related genes (Pdx1, Hlxb9, Nkx2.2, Nkx6.1, and Glut-2 in these islet-like cell clusters. The hyperglycemia and glucose intolerance in streptozotocin-induced diabetic rats was significantly alleviated after xenotransplantation of islet-like cell clusters, without the use of immunosuppressants. In addition to the existence of islet-like cell clusters in the liver, some special fused liver cells were also found, which characterized by human insulin and nuclei-positive staining and possessing secretory granules. CONCLUSIONS AND SIGNIFICANCE: In this study, we successfully differentiate HUMSCs into mature islet-like cell clusters, and these islet-like cell clusters possess insulin-producing ability in vitro and in vivo. HUMSCs in Wharton's Jelly of the umbilical cord seem to be the preferential source of stem cells to convert into insulin

  17. Extracellular matrix components supporting human islet function in alginate-based immunoprotective microcapsules for treatment of diabetes

    NARCIS (Netherlands)

    Llacua Carrasco, Luis; de Haan, Bart J; Smink, Sandra A; de Vos, Paul

    2016-01-01

    In the pancreas, extracellular matrix (ECM) components play an import role in providing mechanical and physiological support, and also contribute to the function of islets. These ECM-connections are damaged during islet-isolation from the pancreas and are not fully recovered after encapsulation and

  18. Candidate genes expressed in human islets and their role in the pathogenesis of type 1 diabetes

    DEFF Research Database (Denmark)

    Storling, Joachim; Brorsson, Caroline Anna

    2013-01-01

    In type 1 diabetes (T1D), the insulin-producing β cells are destroyed by an immune-mediated process leading to complete insulin deficiency. There is a strong genetic component in T1D. Genes located in the human leukocyte antigen (HLA) region are the most important genetic determinants of disease...... exposure to proinflammatory cytokines highlighting that these genes may be involved in the response of β cells to immune attack. In this review, the compiling evidence that many of the candidate genes are expressed in islets and β cells will be presented. Further, we perform the first systematic human...... islet expression analysis of all genes located in 50 T1D-associated GWAS loci using a published RNA sequencing dataset. We find that 336 out of 857 genes are expressed in human islets and that many of these interact in protein networks. Finally, the potential pathogenetic roles of some candidate genes...

  19. Differential expression of glutamic acid decarboxylase in rat and human islets

    DEFF Research Database (Denmark)

    Petersen, J S; Russel, S; Marshall, M O;

    1993-01-01

    The GABA synthesizing enzyme GAD is a prominent islet cell autoantigen in type I diabetes. The two forms of GAD (GAD64 and GAD67) are encoded by different genes in both rats and humans. By in situ hybridization analysis of rat and human pancreases, expression of both genes was detected in rat isl...

  20. Pig islets xenotransplantation: recent progress and current perspectives

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    Haitao eZhu

    2014-03-01

    Full Text Available Islet xenotransplantation is a prospective treatment to bridge the gap between available human cells and needs of patients with diabetes. Pig is the ideal candidate to obtain such available islet cells. However, potential clinical application of pig islet transplantation still faces obstacles such as inadequate yield of high-quality functional islets and xenorejection of the transplants. Adequate amounts of available islets can be obtained based on selection of a suitable pathogen-free source herd and the development of isolation and purification methods. Several studies demonstrated feasibility of successful pre-clinical pig islet xenotransplantation and provided insights and possible mechanisms of xenogeneic immune recognition and rejection. Particularly promising is the achievement of long-term insulin independence in diabetic models by means of distinct islet products and novel immunotherapeutic strategies. Nonetheless, further efforts are needed to obtain much more data on safety and efficacy to translate these findings into clinical practice

  1. Insulin resistance alters islet morphology in nondiabetic humans

    DEFF Research Database (Denmark)

    Mezza, Teresa; Muscogiuri, Giovanna; Sorice, Gian Pio

    2014-01-01

    Type 2 diabetes is characterized by poor glucose uptake in metabolic tissues and manifests when insulin secretion fails to cope with worsening insulin resistance. In addition to its effects on skeletal muscle, liver, and adipose tissue metabolism, it is evident that insulin resistance also affects...... pancreatic β-cells. To directly examine the alterations that occur in islet morphology as part of an adaptive mechanism to insulin resistance, we evaluated pancreas samples obtained during pancreatoduodenectomy from nondiabetic subjects who were insulin-resistant or insulin-sensitive. We also compared...... insulin sensitivity, insulin secretion, and incretin levels between the two groups. We report an increased islet size and an elevated number of β- and α-cells that resulted in an altered β-cell-to-α-cell area in the insulin- resistant group. Our data in this series of studies suggest that neogenesis from...

  2. An Apparent Deficiency of Lymphatic Capillaries in the Islets of Langerhans in the Human Pancreas.

    Science.gov (United States)

    Korsgren, Erik; Korsgren, Olle

    2016-04-01

    The lymphatic system is crucial for efficient immune surveillance and for the maintenance of a physiological pressure in the interstitial space. Even so, almost no information is available concerning the lymph drainage of the islets of Langerhans in the human pancreas. Immunohistochemical staining allowed us to distinguish lymphatic capillaries from blood capillaries. Almost no lymphatic capillaries were found within the islets in pancreatic biopsy specimens from subjects without diabetes or from subjects with type 1 or type 2 diabetes. Lymphatic capillaries were, however, found at the islet-exocrine interface, frequently located along blood capillaries and other fibrotic structures within or close to the islet capsule. Lymphatic capillaries were regularly found in the exocrine pancreas, with small lymphatic vessels located close to and around acini. Larger collecting lymphatic vessels were located in fibrotic septa between the exocrine lobules and adjacent to the ductal system of the pancreas. In summary, we report a pronounced deficiency of lymphatic capillaries in human islets, a finding with implications for immune surveillance and the regulation of interstitial fluid transport in the endocrine pancreas as well as for the pathophysiology of both type 1 and type 2 diabetes. © 2016 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  3. Improvement in The Function of Isolated Rat Pancreatic Islets through Reduction of Oxidative Stress Using Traditional Iranian Medicine

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    Mahban Rahimifard

    2014-06-01

    Full Text Available Objective: Pancreatic islets have fewer antioxidant enzymes than other tissues and thus are vulnerable to oxidative stress. In the present study, the effects of nine specifically selected Iranian medical plants on the mitochondria function and survival of isolated rat islets were examined. Materials and Methods: In this experimental study, following laparotomy, pancreases of rats were removed and the islets isolated and incubated in vitro for 24 hours. Logarithmic doses of plant materials were added to the islets and incubated for an additional 24 hours after which the viability of the cells and production of reactive oxygen species (ROS were measured. Levels of insulin production in relation to static and stimulated glucose concentrations were also determined. Results: The tested compounds markedly increased survival of the islet cells, their mitochondrial activity, and insulin levels at the same time as reducing production of ROS. Greatest effects were observed in the following order: Peganum harmala, Glycyrrhiza glabra, Satureja hortensis, Rosmarinus officinalis, Teucrium scordium, Aloe vera, Zingiber officinale, Silybum marianum, and Hypericum perforatum at doses of 10, 103, 104, 10, 102, 102, 10-1, 10 and 103 μgmL-1, respectively. Conclusion: Based on these results, we suggest that pretreatment with these selected Iranian medical plants can improve the outcomes of pancreas transplants and grafts through the control of oxidative stress damage.

  4. Potential for clinical pancreatic islet xenotransplantation

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    Bottino R

    2014-09-01

    Full Text Available Rita Bottino,1 Santosh Nagaraju,2 Vikas Satyananda,2 Hidetaka Hara,2 Martin Wijkstrom,2 Massimo Trucco,1 David KC Cooper2 1Institute of Cellular Therapeutics, Allegheny Health Network, 2Thomas E Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh, Pittsburgh, PA, USA Abstract: Diabetes mellitus is increasing worldwide. Type 1 diabetes can be treated successfully by islet allotransplantation, the results of which are steadily improving. However, the number of islets that can be obtained from deceased human donors will never be sufficient to cure more than a very small percentage of patients who might benefit from transplantation. Although there are some differences in glucose metabolism between pigs and humans, the use of pigs could provide an unlimited supply of islets, and the insulin produced would undoubtedly control glucose levels. Transplantation of islets into the portal vein results in islets residing in the liver; however, an early inflammatory response and rejection remain problematic, even when the recipient is receiving immunosuppressive therapy. In the long term, immunosuppressive drugs may exhibit toxicities to patients and specifically harm the islet cells. In contrast, encapsulation techniques provide islets with a physical barrier that prevents antibodies binding to the islet graft while still allowing insulin to be released into the recipient's circulation; in theory, patients receiving encapsulated grafts might not require exogenous immunosuppressive therapy. Nonhuman primates with encapsulated pig islet transplants have remained insulin-independent for several weeks, but long-term efficacy remains uncertain. Furthermore, techniques are now available to knock out genes from the pig and/or insert human genes, thus rendering the antigenic structure of pigs closer to that of humans, and providing protection from the human immune response. Islet transplantation from genetically engineered pigs has been

  5. AMOUNT AND DISTRIBUTION OF COLLAGEN IN PANCREATIC TISSUE OF DIFFERENT SPECIES IN THE PERSPECTIVE OF ISLET ISOLATION PROCEDURES

    NARCIS (Netherlands)

    VANSUYLICHEM, PTR; VANDEIJNEN, JEHM; WOLTERS, GHJ; VANSCHILFGAARDE, R

    1995-01-01

    Because collagen is the major target in the enzymatic dissociation of the pancreas for islet isolation, we determined the amount of collagen and its distribution in a comparative study comprising normal pancreata of rat, dog, man, young pig, and adult pig. Collagen content was determined using a

  6. AMOUNT AND DISTRIBUTION OF COLLAGEN IN PANCREATIC TISSUE OF DIFFERENT SPECIES IN THE PERSPECTIVE OF ISLET ISOLATION PROCEDURES

    NARCIS (Netherlands)

    VANSUYLICHEM, PTR; VANDEIJNEN, JEHM; WOLTERS, GHJ; VANSCHILFGAARDE, R

    1995-01-01

    Because collagen is the major target in the enzymatic dissociation of the pancreas for islet isolation, we determined the amount of collagen and its distribution in a comparative study comprising normal pancreata of rat, dog, man, young pig, and adult pig. Collagen content was determined using a col

  7. A novel Gymnema sylvestre extract stimulates insulin secretion from human islets in vivo and in vitro.

    Science.gov (United States)

    Al-Romaiyan, A; Liu, B; Asare-Anane, H; Maity, C R; Chatterjee, S K; Koley, N; Biswas, T; Chatterji, A K; Huang, G-C; Amiel, S A; Persaud, S J; Jones, P M

    2010-09-01

    Many plant-based products have been suggested as potential antidiabetic agents, but few have been shown to be effective in treating the symptoms of Type 2 diabetes mellitus (T2DM) in human studies, and little is known of their mechanisms of action. Extracts of Gymnema sylvestre (GS) have been used for the treatment of T2DM in India for centuries. The effects of a novel high molecular weight GS extract, Om Santal Adivasi, (OSA(R)) on plasma insulin, C-peptide and glucose in a small cohort of patients with T2DM are reported here. Oral administration of OSA(R) (1 g/day, 60 days) induced significant increases in circulating insulin and C-peptide, which were associated with significant reductions in fasting and post-prandial blood glucose. In vitro measurements using isolated human islets of Langerhans demonstrated direct stimulatory effects of OSA(R) on insulin secretion from human ß-cells, consistent with an in vivo mode of action through enhancing insulin secretion. These in vivo and in vitro observations suggest that OSA(R) may provide a potential alternative therapy for the hyperglycemia associated with T2DM.

  8. Direct long-term effects of L-asparaginase on rat and human pancreatic islets

    DEFF Research Database (Denmark)

    Clausen, Niels; Nielsen, Jens Høiriis

    1989-01-01

    L-Asparaginase, an effective agent in the treatment of acute lymphoblastic leukemia, may induce a diabetic state. The pathogenesis of the diabetogenic effect was studied in cultured pancreatic islets. Mean serum concentrations in three children with acute lymphoblastic leukemia were 2.4 U/mL (range...... 1.4-4.5) before and 31.5 U/mL (range 18.6-51.8) immediately after an intravenous injection of 1000 U/kg L-asparaginase. Glucose-induced insulin release from pancreatic islets of rat and man was measured after 3 and 7 days of culture in media with or without clinically relevant concentrations...... of Escherichia coli L-asparaginase (0.01-100 U/mL). After culture, the remaining insulin, glucagon, and DNA in the islets were determined. After 7 days of culture of adult rat or human islets, both the accumulation of insulin in the medium and the content of insulin and glucagon in the islets were significantly...

  9. Functional proteomics screen enables enrichment of distinct cell types from human pancreatic islets.

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    Revital Sharivkin

    Full Text Available The current world-wide epidemic of diabetes has prompted attempts to generate new sources of insulin-producing cells for cell replacement therapy. An inherent challenge in many of these strategies is the lack of cell-surface markers permitting isolation and characterization of specific cell types from differentiating stem cell populations. Here we introduce an iterative proteomics procedure allowing tag-free isolation of cell types based on their function. Our method detects and associates specific cell-surface markers with particular cell functionality by coupling cell capture on antibody arrays with immunofluorescent labeling. Using this approach in an iterative manner, we discovered marker combinations capable of enriching for discrete pancreatic cell subtypes from human islets of Langerhans: insulin-producing beta cells (CD9high/CD56+, glucagon-producing alpha cells (CD9-/CD56+ and trypsin-producing acinar cells (CD9-/CD56-. This strategy may assist future beta cell research and the development of diagnostic tools for diabetes. It can also be applied more generally for function-based purification of desired cell types from other limited and heterogeneous biological samples.

  10. Unstable Expression of Commonly Used Reference Genes in Rat Pancreatic Islets Early after Isolation Affects Results of Gene Expression Studies.

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    Lucie Kosinová

    Full Text Available The use of RT-qPCR provides a powerful tool for gene expression studies; however, the proper interpretation of the obtained data is crucially dependent on accurate normalization based on stable reference genes. Recently, strong evidence has been shown indicating that the expression of many commonly used reference genes may vary significantly due to diverse experimental conditions. The isolation of pancreatic islets is a complicated procedure which creates severe mechanical and metabolic stress leading possibly to cellular damage and alteration of gene expression. Despite of this, freshly isolated islets frequently serve as a control in various gene expression and intervention studies. The aim of our study was to determine expression of 16 candidate reference genes and one gene of interest (F3 in isolated rat pancreatic islets during short-term cultivation in order to find a suitable endogenous control for gene expression studies. We compared the expression stability of the most commonly used reference genes and evaluated the reliability of relative and absolute quantification using RT-qPCR during 0-120 hrs after isolation. In freshly isolated islets, the expression of all tested genes was markedly depressed and it increased several times throughout the first 48 hrs of cultivation. We observed significant variability among samples at 0 and 24 hrs but substantial stabilization from 48 hrs onwards. During the first 48 hrs, relative quantification failed to reflect the real changes in respective mRNA concentrations while in the interval 48-120 hrs, the relative expression generally paralleled the results determined by absolute quantification. Thus, our data call into question the suitability of relative quantification for gene expression analysis in pancreatic islets during the first 48 hrs of cultivation, as the results may be significantly affected by unstable expression of reference genes. However, this method could provide reliable information

  11. Ultrastructural studies of time-course and cellular specificity of interleukin-1 mediated islet cytotoxicity

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Egeberg, J; Nerup, J

    1987-01-01

    Previous electron-microscopic studies of isolated islets of Langerhans exposed to the monokine interleukin-1 for 7 days have indicated that interleukin-1 is cytotoxic to all islet cells. To study the time-course and possible cellular specificity of interleukin-1 cytotoxicity to islets exposed...... to interleukin-1 for short time periods, isolated rat or human islets were incubated with or without 25 U/ml highly purified human interleukin-1 for 24 h. Samples of rat islets were taken after 5 min, 30 min, 1, 2, 4, 6, 8, 10, 12, 16, 20 and 24 h and samples of human islets after 5 min, 30 min and 24 h...... of incubation and examined by electron microscopy in a blinded fashion. Already after 30 min, accumulation of opaque intracytoplasmic bodies without apparent surrounding membranes, and autophagic vacuoles were seen in about 20% of the beta cells examined in rat islets exposed to interleukin-1. After 16 h...

  12. Nitric Oxide Overproduction Reduces Insulin Secretion from Isolated Islets in Fetal Hypothyroid Rats.

    Science.gov (United States)

    Rouintan, Z; Farrokhfall, K; Karbalaei, N; Ghasemi, A

    2016-02-01

    Thyroid hormones have developmental effects during fetal life. Fetal hypothyroidism leads to glucose intolerance and reduced insulin secretion capacity. Activity of nitric oxide synthases follows a heterogeneous pattern in hypothyroidism. Overactivity of constitutive nitric oxide synthase (NOS), inhibits glucose-stimulated insulin release. The aim of this study was to examine if reduction in insulin secretion in fetal hypothyroidism is due to overproduction of nitric oxide. Pregnant Wistar rats were divided into 2 groups; the experimental group consumed water containing 0.02% of 6-propyl-2-thiouracil till delivery, while the control group consumed tap water. After delivery serum thyroid hormones were measured. Intravenous glucose tolerance test was performed in 6-month old offspring (n=8). After 3 weeks recovery, pancreatic islets were isolated and insulin secretion, inducible and constitutive nitric oxide synthase activity were measured (n=4). Compared to controls, during intravenous glucose tolerance test, fetal hypothyroid rats had high plasma glucose concentration (p=0.003) and low plasma insulin levels (p=0.012) at 5-20 min and their insulin secretion from isolated islets at basal glucose concentration and in the presence of l-arginine was lower. The nitric oxide synthase inhibitor, NG-nitro-l-arginine methyl ester significantly improved insulin secretion in fetal hypothyroid rats at basal glucose concentration and in the presence of l-arginine. The results showed higher NOS activities in fetal hypothyroid rats (constitutive 17.60±1.09 vs. 47.34±4.44 and inducible 4.09±0.96 vs. 19.97±1.14 pmol/min/mg proteins, p=0.002). In conclusion, NO overproduction through NOS participates in decreased insulin secretion in fetal hypothyroid rats.

  13. Automated digital image analysis of islet cell mass using Nikon's inverted eclipse Ti microscope and software to improve engraftment may help to advance the therapeutic efficacy and accessibility of islet transplantation across centers.

    Science.gov (United States)

    Gmyr, Valery; Bonner, Caroline; Lukowiak, Bruno; Pawlowski, Valerie; Dellaleau, Nathalie; Belaich, Sandrine; Aluka, Isanga; Moermann, Ericka; Thevenet, Julien; Ezzouaoui, Rimed; Queniat, Gurvan; Pattou, Francois; Kerr-Conte, Julie

    2015-01-01

    Reliable assessment of islet viability, mass, and purity must be met prior to transplanting an islet preparation into patients with type 1 diabetes. The standard method for quantifying human islet preparations is by direct microscopic analysis of dithizone-stained islet samples, but this technique may be susceptible to inter-/intraobserver variability, which may induce false positive/negative islet counts. Here we describe a simple, reliable, automated digital image analysis (ADIA) technique for accurately quantifying islets into total islet number, islet equivalent number (IEQ), and islet purity before islet transplantation. Islets were isolated and purified from n = 42 human pancreata according to the automated method of Ricordi et al. For each preparation, three islet samples were stained with dithizone and expressed as IEQ number. Islets were analyzed manually by microscopy or automatically quantified using Nikon's inverted Eclipse Ti microscope with built-in NIS-Elements Advanced Research (AR) software. The AIDA method significantly enhanced the number of islet preparations eligible for engraftment compared to the standard manual method (p < 0.001). Comparisons of individual methods showed good correlations between mean values of IEQ number (r(2) = 0.91) and total islet number (r(2) = 0.88) and thus increased to r(2) = 0.93 when islet surface area was estimated comparatively with IEQ number. The ADIA method showed very high intraobserver reproducibility compared to the standard manual method (p < 0.001). However, islet purity was routinely estimated as significantly higher with the manual method versus the ADIA method (p < 0.001). The ADIA method also detected small islets between 10 and 50 µm in size. Automated digital image analysis utilizing the Nikon Instruments software is an unbiased, simple, and reliable teaching tool to comprehensively assess the individual size of each islet cell preparation prior to transplantation. Implementation of this

  14. Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices.

    Science.gov (United States)

    Johansson, Ulrika; Ria, Massimiliano; Åvall, Karin; Dekki Shalaly, Nancy; Zaitsev, Sergei V; Berggren, Per-Olof; Hedhammar, My

    2015-01-01

    Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79%) with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year), the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial number of islets

  15. Pancreatic Islet Survival and Engraftment Is Promoted by Culture on Functionalized Spider Silk Matrices.

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    Ulrika Johansson

    Full Text Available Transplantation of pancreatic islets is one approach for treatment of diabetes, however, hampered by the low availability of viable islets. Islet isolation leads to disruption of the environment surrounding the endocrine cells, which contributes to eventual cell death. The reestablishment of this environment is vital, why we herein investigated the possibility of using recombinant spider silk to support islets in vitro after isolation. The spider silk protein 4RepCT was formulated into three different formats; 2D-film, fiber mesh and 3D-foam, in order to provide a matrix that can give the islets physical support in vitro. Moreover, cell-binding motifs from laminin were incorporated into the silk protein in order to create matrices that mimic the natural cell environment. Pancreatic mouse islets were thoroughly analyzed for adherence, necrosis and function after in vitro maintenance on the silk matrices. To investigate their suitability for transplantation, we utilized an eye model which allows in vivo imaging of engraftment. Interestingly, islets that had been maintained on silk foam during in vitro culture showed improved revascularization. This coincided with the observation of preserved islet architecture with endothelial cells present after in vitro culture on silk foam. Selected matrices were further evaluated for long-term preservation of human islets. Matrices with the cell-binding motif RGD improved human islet maintenance (from 36% to 79% with preserved islets architecture and function for over 3 months in vitro. The islets established cell-matrix contacts and formed vessel-like structures along the silk. Moreover, RGD matrices promoted formation of new, insulin-positive islet-like clusters that were connected to the original islets via endothelial cells. On silk matrices with islets from younger donors (<35 year, the amount of newly formed islet-like clusters found after 1 month in culture were almost double compared to the initial

  16. Leptin modulates β cell expression of IL-1 receptor antagonist and release of IL-1β in human islets

    Science.gov (United States)

    Maedler, Kathrin; Sergeev, Pavel; Ehses, Jan A.; Mathe, Zoltan; Bosco, Domenico; Berney, Thierry; Dayer, Jean-Michel; Reinecke, Manfred; Halban, Philippe A.; Donath, Marc Y.

    2004-01-01

    High concentrations of glucose induce β cell production of IL-1β, leading to impaired β cell function and apoptosis in human pancreatic islets. IL-1 receptor antagonist (IL-1Ra) is a naturally occurring antagonist of IL-1β and protects cultured human islets from glucotoxicity. Therefore, the balance of IL-1β and IL-1Ra may play a crucial role in the pathogenesis of diabetes. In the present study, we observed expression of IL-1Ra in human pancreatic β cells of nondiabetic individuals, which was decreased in tissue sections of type 2 diabetic patients. In vitro, chronic exposure of human islets to leptin, a hormone secreted by adipocytes, decreased β cell production of IL-1Ra and induced IL-1β release from the islet preparation, leading to impaired β cell function, caspase-3 activation, and apoptosis. Exogenous addition of IL-1Ra protected cultured human islets from the deleterious effects of leptin. Antagonizing IL-1Ra by introduction of small interfering RNA to IL-1Ra into human islets led to caspase-3 activation, DNA fragmentation, and impaired β cell function. Moreover, siIL-1Ra enhanced glucose-induced β cell apoptosis. These findings demonstrate expression of IL-1Ra in the human β cell, providing localized protection against leptin- and glucose-induced islet IL-1β. PMID:15141093

  17. Human Monoclonal Islet Cell Antibodies From a Patient with Insulin- Dependent Diabetes Mellitus Reveal Glutamate Decarboxylase as the Target Antigen

    Science.gov (United States)

    Richter, Wiltrud; Endl, Josef; Eiermann, Thomas H.; Brandt, Michael; Kientsch-Engel, Rosemarie; Thivolet, Charles; Jungfer, Herbert; Scherbaum, Werner A.

    1992-09-01

    The autoimmune phenomena associated with destruction of the β cell in pancreatic islets and development of type 1 (insulin-dependent) diabetes mellitus (IDDM) include circulating islet cell antibodies. We have immortalized peripheral blood lymphocytes from prediabetic individuals and patients with newly diagnosed IDDM by Epstein-Barr virus transformation. IgG-positive cells were selected by anti-human IgG-coupled magnetic beads and expanded in cell culture. Supernatants were screened for cytoplasmic islet cell antibodies using the conventional indirect immunofluorescence test on cryostat sections of human pancreas. Six islet cell-specific B-cell lines, originating from a patient with newly diagnosed IDDM, could be stabilized on a monoclonal level. All six monoclonal islet cell antibodies (MICA 1-6) were of the IgG class. None of the MICA reacted with human thyroid, adrenal gland, anterior pituitary, liver, lung, stomach, and intestine tissues but all six reacted with pancreatic islets of different mammalian species and, in addition, with neurons of rat cerebellar cortex. MICA 1-6 were shown to recognize four distinct antigenic epitopes in islets. Islet cell antibody-positive diabetic sera but not normal human sera blocked the binding of the monoclonal antibodies to their target epitopes. Immunoprecipitation of 35S-labeled human islet cell extracts revealed that a protein of identical size to the enzyme glutamate decarboxylase (EC 4.1.1.15) was a target of all MICA. Furthermore, antigen immunotrapped by the MICA from brain homogenates showed glutamate decarboxylase enzyme activity. MICA 1-6 therefore reveal glutamate decarboxylase as the predominant target antigen of cytoplasmic islet cell autoantibodies in a patient with newly diagnosed IDDM.

  18. Whole-Genome Bisulfite Sequencing of Human Pancreatic Islets Reveals Novel Differentially Methylated Regions in Type 2 Diabetes Pathogenesis.

    Science.gov (United States)

    Volkov, Petr; Bacos, Karl; Ofori, Jones K; Esguerra, Jonathan Lou S; Eliasson, Lena; Rönn, Tina; Ling, Charlotte

    2017-04-01

    Current knowledge about the role of epigenetics in type 2 diabetes (T2D) remains limited. Only a few studies have investigated DNA methylation of selected candidate genes or a very small fraction of genomic CpG sites in human pancreatic islets, the tissue of primary pathogenic importance for diabetes. Our aim was to characterize the whole-genome DNA methylation landscape in human pancreatic islets, to identify differentially methylated regions (DMRs) in diabetic islets, and to investigate the function of DMRs in islet biology. Here, we performed whole-genome bisulfite sequencing, which is a comprehensive and unbiased method to study DNA methylation throughout the genome at a single nucleotide resolution, in pancreatic islets from donors with T2D and control subjects without diabetes. We identified 25,820 DMRs in islets from individuals with T2D. These DMRs cover loci with known islet function, e.g., PDX1, TCF7L2, and ADCY5 Importantly, binding sites previously identified by ChIP-seq for islet-specific transcription factors, enhancer regions, and different histone marks were enriched in the T2D-associated DMRs. We also identified 457 genes, including NR4A3, PARK2, PID1, SLC2A2, and SOCS2, that had both DMRs and significant expression changes in T2D islets. To mimic the situation in T2D islets, candidate genes were overexpressed or silenced in cultured β-cells. This resulted in impaired insulin secretion, thereby connecting differential methylation to islet dysfunction. We further explored the islet methylome and found a strong link between methylation levels and histone marks. Additionally, DNA methylation in different genomic regions and of different transcript types (i.e., protein coding, noncoding, and pseudogenes) was associated with islet expression levels. Our study provides a comprehensive picture of the islet DNA methylome in individuals with and without diabetes and highlights the importance of epigenetic dysregulation in pancreatic islets and T2D

  19. Robot-assisted pancreatoduodenectomy with preservation of the vascular supply for autologous islet cell isolation and transplantation: a case report

    Directory of Open Access Journals (Sweden)

    Giulianotti Piero

    2012-03-01

    Full Text Available Abstract Introduction For patients with chronic pancreatitis presenting with medically intractable abdominal pain, surgical intervention may be the only treatment option. However, extensive pancreatic resections are typically performed open and are associated with a substantial amount of postoperative pain, wound complications and long recovery time. Minimally invasive surgery offers an avenue to improve results; however, current limitations of laparoscopic surgery render its application in the setting of chronic pancreatitis technically demanding. Additionally, pancreatic resections are associated with a high incidence of diabetes. Transplantation of islets isolated from the resected pancreas portion offers a way to prevent post-surgical diabetes; however, preservation of the vascular supply during pancreatic resection, which determines islet cell viability, is technically difficult using current laparoscopic approaches. With recent advances in the surgical field, robotic surgery now provides a means to overcome these obstacles to achieve the end goals of pain relief and preserved endocrine function. We present the first report of a novel, minimally invasive robotic approach for resection of the pancreatic head that preserves vascular supply and enables the isolation of a high yield of viable islets for transplantation. Case presentation A 35-year old Caucasian woman presented with intractable chronic abdominal pain secondary to chronic pancreatitis, with a stricture of her main pancreatic duct at the level of the ampulla of Vater and distal dilatation. She was offered a robotic-assisted pylorus-preserving pancreatoduodenectomy and subsequent islet transplantation, to both provide pain relief and preserve insulin-secretory reserves. Conclusion We present a novel, minimally invasive robotic approach for resection of the pancreatic head with complete preservation of the vascular supply, minimal warm ischemia time (less than three minutes and

  20. Characterisation of the insulinotropic activity of an aqueous extract of Gymnema sylvestre in mouse beta-cells and human islets of Langerhans.

    Science.gov (United States)

    Liu, Bo; Asare-Anane, Henry; Al-Romaiyan, Altaf; Huang, Guocai; Amiel, Stephanie A; Jones, Peter M; Persaud, Shanta J

    2009-01-01

    Leaves of the Gymnema sylvestre (GS) plant have been used to treat diabetes mellitus for millennia, but the previously documented insulin secretagogue effects of GS extracts in vitro may be non-physiological through damage to the beta-cells. We have now examined the effects of a novel GS extract (termed OSA) on insulin secretion from the MIN6 beta-cell line and isolated human islets of Langerhans. Insulin secretion from MIN6 cells was stimulated by OSA in a concentration-dependent manner, with low concentrations (0.06-0.25 mg/ml) having no deleterious effects on MIN6 cell viability, while higher concentrations (> or = 0.5 mg/ml) caused increased Trypan blue uptake. OSA increased beta-cell Ca2+ levels, an effect that was mediated by Ca2+ influx through voltage-operated calcium channels. OSA also reversibly stimulated insulin secretion from isolated human islets and its insulin secretagogue effects in MIN6 cells and human islets were partially dependent on the presence of extracellular Ca2+. These data indicate that low concentrations of the GS isolate OSA stimulate insulin secretion in vitro, at least in part as a consequence of Ca2+ influx, without compromising beta-cell viability. Identification of the component of the OSA extract that stimulates regulated insulin exocytosis, and further investigation of its mode(s) of action, may provide promising lead targets for Type 2 diabetes therapy.

  1. Microencapsulated 3-dimensional sensor for the measurement of oxygen in single isolated pancreatic islets.

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    Wanyu Chen

    Full Text Available BACKGROUND: Oxygen consumption reflects multiple processes in pancreatic islets including mechanisms contributing to insulin secretion, oxidative stress and viability, providing an important readout in studies of islet function, islet viability and drug testing. Due to the scarcity, heterogeneity, and intrinsic kinetic properties of individual islets, it would be of great benefit to detect oxygen consumption by single islets. We present a novel method we have developed to image oxygen in single islets. METHODOLOGY/PRINCIPAL FINDINGS: Using a microfluidics system, individual islets and a fluorescent oxygen-sensitive dye were encased within a thin alginate polymer layer. Insulin secretion by the encapsulated islets was normal. Fluorescent signal from the encased dye, detected using a standard inverted fluorescence microscope and digital camera, was stable and proportional to the amount of oxygen in the media. When integrated into a perifusion system, the sensing system detected changes in response to metabolic substrates, mitochondrial poisons, and induced-oscillations. Glucose responses averaged 30.1±7.1% of the response to a metabolic inhibitor (cyanide, increases were observed in all cases (n = 6, and the system was able to resolve changes in oxygen consumption that had a period greater than 0.5 minutes. The sensing system operated similarly from 2-48 hours following encapsulation, and viability and function of the islets were not significantly affected by the encapsulation process. CONCLUSIONS/SIGNIFICANCE: An oxygen-dependent dye situated around and within a pancreatic islet encapsulated by a thin layer of alginate was sensitive to changes in oxygen consumption, and was not harmful to the function or viability of islets over the course of two days. The microcapsule-based sensing method is particularly suited to assessing the effects of compounds (dose responses and time courses and chronic changes occurring over the course of days. The

  2. Novel Small Molecule Glucagon-Like Peptide-1 Receptor Agonist Stimulates Insulin Secretion in Rodents and From Human Islets

    Science.gov (United States)

    Sloop, Kyle W.; Willard, Francis S.; Brenner, Martin B.; Ficorilli, James; Valasek, Kathleen; Showalter, Aaron D.; Farb, Thomas B.; Cao, Julia X.C.; Cox, Amy L.; Michael, M. Dodson; Gutierrez Sanfeliciano, Sonia Maria; Tebbe, Mark J.; Coghlan, Michael J.

    2010-01-01

    OBJECTIVE The clinical effectiveness of parenterally-administered glucagon-like peptide-1 (GLP-1) mimetics to improve glucose control in patients suffering from type 2 diabetes strongly supports discovery pursuits aimed at identifying and developing orally active, small molecule GLP-1 receptor agonists. The purpose of these studies was to identify and characterize novel nonpeptide agonists of the GLP-1 receptor. RESEARCH DESIGN AND METHODS Screening using cells expressing the GLP-1 receptor and insulin secretion assays with rodent and human islets were used to identify novel molecules. The intravenous glucose tolerance test (IVGTT) and hyperglycemic clamp characterized the insulinotropic effects of compounds in vivo. RESULTS Novel low molecular weight pyrimidine-based compounds that activate the GLP-1 receptor and stimulate glucose-dependent insulin secretion are described. These molecules induce GLP-1 receptor-mediated cAMP signaling in HEK293 cells expressing the GLP-1 receptor and increase insulin secretion from rodent islets in a dose-dependent manner. The compounds activate GLP-1 receptor signaling, both alone or in an additive fashion when combined with the endogenous GLP-1 peptide; however, these agonists do not compete with radiolabeled GLP-1 in receptor-binding assays. In vivo studies using the IVGTT and the hyperglycemic clamp in Sprague Dawley rats demonstrate increased insulin secretion in compound-treated animals. Further, perifusion assays with human islets isolated from a donor with type 2 diabetes show near-normalization of insulin secretion upon compound treatment. CONCLUSIONS These studies characterize the insulinotropic effects of an early-stage, small molecule GLP-1 receptor agonist and provide compelling evidence to support pharmaceutical optimization. PMID:20823098

  3. Diabetes mellitus is associated with an increased expression of resistin in human pancreatic islet cells.

    Science.gov (United States)

    Al-Salam, Suhail; Rashed, Hameed; Adeghate, Ernest

    2011-01-01

    The pattern of distribution of resistin in the pancreas of diabetic patients was investigated to determine whether diabetes mellitus influences the expression of resistin. Pancreatic tissue samples retrieved, during pancreatectomy for pancreatic cancer, from cancer patients with and without type 2 diabetes were processed for immunohistochemistry. The pancreatic tissue samples were retrieved from non-cancerous and clear margins. An immunofluorescence technique was used to examine the expression of resistin and its co-localization with insulin and glucagon in pancreatic islet cells. Resistin was observed in many cells located in the central region of pancreatic islet. The expression of resistin increased significantly (p diabetic patients compared to control. Resistin co-localized with insulin but not glucagon in pancreatic islet cells of both normal and diabetic patients. However, the degree of co-localization was higher in pancreata of diabetic patients compared to normal. The number of human pancreatic islet cells expressing resistin increased significantly after the onset of type 2 diabetes. In conclusion, resistin may play a role in the regulation of pancreatic β-cell function.

  4. The role of islet neogenesis-associated protein (INGAP) in islet neogenesis.

    Science.gov (United States)

    Lipsett, Mark; Hanley, Stephen; Castellarin, Mauro; Austin, Emily; Suarez-Pinzon, Wilma L; Rabinovitch, Alex; Rosenberg, Lawrence

    2007-01-01

    Islet Neogenesis-Associated Protein (INGAP) is a member of the Reg family of proteins implicated in various settings of endogenous pancreatic regeneration. The expression of INGAP and other RegIII proteins has also been linked temporally and spatially with the induction of islet neogenesis in animal models of disease and regeneration. Furthermore, administration of a peptide fragment of INGAP (INGAP peptide) has been demonstrated to reverse chemically induced diabetes as well as improve glycemic control and survival in an animal model of type 1 diabetes. Cultured human pancreatic tissue has also been shown to be responsive to INGAP peptide, producing islet-like structures with function, architecture and gene expression matching that of freshly isolated islets. Likewise, studies in normoglycemic animals show evidence of islet neogenesis. Finally, recent clinical studies suggest an effect of INGAP peptide to improve insulin production in type 1 diabetes and glycemic control in type 2 diabetes.

  5. Long-term effect of pH on B-cell function in isolated islets of Langerhans in tissue culture

    DEFF Research Database (Denmark)

    Brunstedt, J; Nielsen, Jens Høiriis

    1978-01-01

    Collagenase isolated mouse pancreatic islets were maintained in tissue culture for up to 5 months in a culture medium buffered with Hepes and the pH varying between 6.8 and 7.6. The amount of insulin released into the medium and the insulin response to glucose and glucose plus theophylline were...... measured during the culture period. It was found that islets cultured at pH 7.2 maintained the ability to release insulin into the medium for at least 5 months, which was longer than islets cultured at the other pH values. During the first weeks, the islets cultured at pH 7.6 had a higher response to both...... glucose and glucose plus theophylline than islets cultured at the other pH values, but later they lost their insulin releasing ability....

  6. Uptake of the glycosphingolipid sulfatide in the gastrointestinal tract and pancreas in vivo and in isolated islets of Langerhans

    Directory of Open Access Journals (Sweden)

    Fredman Pam

    2006-10-01

    Full Text Available Abstract Background The glycosphingolipid sulfatide has previously been found in several mammalian tissues, but information on the uptake of exogenously administered sulfatide in different organs in vivo is limited. In pancreatic beta cells, sulfatide has been shown to be involved in insulin processing and secretion in vitro. In this study, we examined the uptake of exogenously administered sulfatide and its distribution to the pancreatic beta cells. This might encourage future studies of the function(s of sulfatide in beta cell physiology in vivo. Radioactive sulfatide was given orally to mice whereafter the uptake of sulfatide in the gastrointestinal tract and subsequent delivery to the pancreas was examined. Sulfatide uptake in pancreas was also studied in vivo by i.p. administration of radioactive sulfatide in mice, and in vitro in isolated rat islets. Isolated tissue/islets were analysed by scintillation counting, autoradiography and thin-layer chromatography-ELISA. Results Sulfatide was taken up in the gastrointestinal tract for degradation or further transport to other organs. A selective uptake of short chain and/or hydroxylated sulfatide fatty acid isoforms was observed in the small intestine. Exogenously administered sulfatide was found in pancreas after i.p, but not after oral administration. The in vitro studies in isolated rat islets support that sulfatide, independently of its fatty acid length, is endocytosed and metabolised by pancreatic islets. Conclusion Our study supports a selective uptake and/or preservation of sulfatide in the gastrointestinal tract after oral administration and with emphasises on pancreatic sulfatide uptake, i.p. administration results in sulfatide at relevant location.

  7. Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes

    DEFF Research Database (Denmark)

    Bacos, Karl; Gillberg, Linn; Volkov, Petr

    2016-01-01

    Aging associates with impaired pancreatic islet function and increased type 2 diabetes (T2D) risk. Here we examine whether age-related epigenetic changes affect human islet function and if blood-based epigenetic biomarkers reflect these changes and associate with future T2D. We analyse DNA...... methylation genome-wide in islets from 87 non-diabetic donors, aged 26-74 years. Aging associates with increased DNA methylation of 241 sites. These sites cover loci previously associated with T2D, for example, KLF14. Blood-based epigenetic biomarkers reflect age-related methylation changes in 83 genes...... demonstrate that blood-based epigenetic biomarkers reflect age-related DNA methylation changes in human islets, and associate with insulin secretion in vivo and T2D....

  8. A systems genetics approach identifies genes and pathways for type 2 diabetes in human islets

    DEFF Research Database (Denmark)

    Taneera, Jalal; Lang, Stefan; Sharma, Amitabh;

    2012-01-01

    Close to 50 genetic loci have been associated with type 2 diabetes (T2D), but they explain only 15% of the heritability. In an attempt to identify additional T2D genes, we analyzed global gene expression in human islets from 63 donors. Using 48 genes located near T2D risk variants, we identified...... gene coexpression and protein-protein interaction networks that were strongly associated with islet insulin secretion and HbA(1c). We integrated our data to form a rank list of putative T2D genes, of which CHL1, LRFN2, RASGRP1, and PPM1K were validated in INS-1 cells to influence insulin secretion...... of genes potentially involved in T2D....

  9. Pancreas procurement from multiorgan donors for islet trasplantation.

    Science.gov (United States)

    Ricordi, C; Mazzeferro, V; Casavilla, A; Scotti, C; Pinna, A; Tzakis, A; Starzl, T E

    1992-01-01

    The outcome of human islet isolation procedures can be significantly effected by the technique used for pancreas procurement. In fact, the final step of islet purification using discontinuous density gradients requires a significant difference between the density of the islets and the density of the non-endocrine component of the gland. Therefore, any procedure during multi-organ procurement that will result in edema or degranulation of the acinar tissue will result in failure of the islet purification step. In this report a technique for combined harvesting of liver and pancreas is presented. The use of this procedure can be of assistance to avoid damage to the pancreas that could result in a compromised islet purification for improper handling of the gland even before it arrives to the isolation facility.

  10. Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats

    Institute of Scientific and Technical Information of China (English)

    XIAO Mei; DOU ZhongYing; AN LiLong; YANG XueYi; GE Xin; QIAO Hai; ZHAO Ting; MA XiaoFei; FAN JingZhua; ZHU MengYang

    2008-01-01

    The major obstacle in using pancreatic islet transplantation to cure type Ⅰ and some type Ⅱ diabetes is the shortage of the donors. One of ways to overcome such obstacle is to isolate and clone pancreatic stem cells as "seed cells" and induce their differentiation into functional islets as an abundant trans-plantation source. In this study, a monoclonal human pancreatic stem cell (mhPSC) line was obtained from abortive fetal pancreatic tissues. Pancreatic tissues were taken from abortive fetus by sterile procedures, and digested into single cells and cell clusters with 0.1% type Ⅳ collagenase. Cultured in modified glucose-low DMEM with 10% fetal bovine serum (FBS), these single cells and cell clusters adhered to culture dishes, and then primary epidermal-like pancreatic stem ceils started to clone. After digesting with 0.25% trypsin and 0.04% EDTA, fibroblasts and other cells were gradually eliminated and epithelioid pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the mhPSCs were obtained. After addition of 10 ng/mL epidermal growth factor (EGF) in cell culture medium, the mhPSCs quickly grew and formed a gravelstone-like monolayer. Continuously proliferated, a mhPSC line, which was derived from a male abortive fetus of 4 months old, has been passed through 50 generations. More than 1×109 mhPSCs were cryo-preserved in liquid nitrogen. Karyotype analysis showed that the chromosome set of the mhPSC line was normal diploid. Immunocytochemistry results demonstrated that the mhPSC line was positive for the pdxl, glucagon, nestin and CK19, and negative for the insulin, CD34, CD44 and CD45 protein expression. RT-PCR revealed further that the mhPSCs expressed transcription factors of the pdx1, glucagon, nestin and CK19. Also, in vitro induced with β-mercaptoethanol, the mhPSCs differentiated into nerve cells that expressed the NF protein. Induced with nicotinamide, the mhPSCs differentiated into functional islet

  11. Establishing a human pancreatic stem cell line and transplanting induced pancreatic islets to reverse experimental diabetes in rats

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The major obstacle in using pancreatic islet transplantation to cure type I and some type II diabetes is the shortage of the donors. One of ways to overcome such obstacle is to isolate and clone pancreatic stem cells as "seed cells" and induce their differentiation into functional islets as an abundant trans-plantation source. In this study, a monoclonal human pancreatic stem cell (mhPSC) line was obtained from abortive fetal pancreatic tissues. Pancreatic tissues were taken from abortive fetus by sterile procedures, and digested into single cells and cell clusters with 0.1% type IV collagenase. Cultured in modified glucose-low DMEM with 10% fetal bovine serum (FBS), these single cells and cell clusters adhered to culture dishes, and then primary epidermal-like pancreatic stem cells started to clone. After digesting with 0.25% trypsin and 0.04% EDTA, fibroblasts and other cells were gradually eliminated and epithelioid pancreatic stem cells were gradually purified during generations. Using clone-ring selection, the mhPSCs were obtained. After addition of 10 ng/mL epidermal growth factor (EGF) in cell culture medium, the mhPSCs quickly grew and formed a gravelstone-like monolayer. Continuously proliferated, a mhPSC line, which was derived from a male abortive fetus of 4 months old, has been passed through 50 generations. More than 1×109 mhPSCs were cryo-preserved in liquid nitrogen. Karyotype analysis showed that the chromosome set of the mhPSC line was normal diploid. Immunocytochemistry results demonstrated that the mhPSC line was positive for the pdx1, glucagon, nestin and CK19, and negative for the insulin, CD34, CD44 and CD45 protein expression. RT-PCR revealed further that the mhPSCs expressed transcription factors of the pdx1, glucagon, nestin and CK19. Also, in vitro induced with β-mercaptoethanol, the mhPSCs differentiated into nerve cells that expressed the NF protein. Induced with nicotinamide, the mhPSCs differentiated into functional islet

  12. [Effects of medium-weight molecules isolated from plasma of healthy and burnt animals on the cellular composition of cultured bone marrow erythroblastic islets].

    Science.gov (United States)

    Volchegorskiĭ, I A; Tishevskaia, N V; Kuznetsov, D A

    2002-01-01

    The so-called median-weight molecular fractions (M, B = 0.5-1.5 kDa) exerting a marked hemopoietic effect in the cultured bone marrow erythroblastic islets were isolated from the plasma deproteinates of healthy dogs and animals with acute inflammation (thermal skin burn) by Sephadex G-15 gel chromatography. Acute inflammation is attended by the occurrence of blood medium-weight molecular components that suppress erythropiesis and by the concurrent high rate of granulocytopoiesis in the cultured bone marrow erythroblastic islets. Medium-weight molecular fractions from the blood of healthy dogs substantially were found to suppress erythropoiesis and to stimulate granulocytopoiesis in the cultured erythroblastic islets.

  13. Leptin Regulated Insulin Secretion via Stimulating IRS2-associated Phosphoinositide 3-kinase Activity in the isolated Rat Pancreatic Islets

    Institute of Scientific and Technical Information of China (English)

    袁莉; 安汉祥; 李卓娅; 邓秀玲

    2003-01-01

    To investigate the molecular mechanism of leptin regulating insulin secretion through determining the regulation of insulin secretion and the insulin receptor substrate (IRS)-2-associated phosphoinositide 3-kinase (PI3K) activity by leptin in the isolated rat pancreatic islets, pancreatic islets were isolated from male SD rats by the collagenase method. The purified islets were incubated with leptin 2 nmol/L for 1 h in the presence of 5.6 mmol/L or 11.1 mmol/L glucose. Insulin release was measured using radioimmunoassay. IRS-2-associated activity of PI3K was determined by immunoprecipitate assay and Western blot. The results showed that in the presence of 5.6 mmol/L glucose, leptin had no significant effect on both insulin secretion and IRS-2-associated PI3K activity, but in the presence of 11.1 mmol/L glucose, insulin release was significantly inhibited after the islets were exposed to leptin for 1 h (P<0. 01). PI3K inhibitor wortmannin blocked the inhibitory regulation of leptin on insulin release (P<0. 05). Western Blot assay revealed that 2 nmol/L leptin could significantly increase the IRS-2-associated activity of PI3K by 51.5 % (P<0. 05) in the presence of 11.1 mmol/L glucose. It was concluded that Leptin could significantly inhibit insulin secretion in the presence of 11.1 mmol/L glucose by stimulating IRS-2-associated activity of PI3K, which might be the molecular mechanism of leptin regulating insulin secretion.

  14. Blood-based biomarkers of age-associated epigenetic changes in human islets associate with insulin secretion and diabetes.

    Science.gov (United States)

    Bacos, Karl; Gillberg, Linn; Volkov, Petr; Olsson, Anders H; Hansen, Torben; Pedersen, Oluf; Gjesing, Anette Prior; Eiberg, Hans; Tuomi, Tiinamaija; Almgren, Peter; Groop, Leif; Eliasson, Lena; Vaag, Allan; Dayeh, Tasnim; Ling, Charlotte

    2016-03-31

    Aging associates with impaired pancreatic islet function and increased type 2 diabetes (T2D) risk. Here we examine whether age-related epigenetic changes affect human islet function and if blood-based epigenetic biomarkers reflect these changes and associate with future T2D. We analyse DNA methylation genome-wide in islets from 87 non-diabetic donors, aged 26-74 years. Aging associates with increased DNA methylation of 241 sites. These sites cover loci previously associated with T2D, for example, KLF14. Blood-based epigenetic biomarkers reflect age-related methylation changes in 83 genes identified in human islets (for example, KLF14, FHL2, ZNF518B and FAM123C) and some associate with insulin secretion and T2D. DNA methylation correlates with islet expression of multiple genes, including FHL2, ZNF518B, GNPNAT1 and HLTF. Silencing these genes in β-cells alter insulin secretion. Together, we demonstrate that blood-based epigenetic biomarkers reflect age-related DNA methylation changes in human islets, and associate with insulin secretion in vivo and T2D.

  15. Autoimmunity against INS-IGF2 protein expressed in human pancreatic islets.

    Science.gov (United States)

    Kanatsuna, Norio; Taneera, Jalal; Vaziri-Sani, Fariba; Wierup, Nils; Larsson, Helena Elding; Delli, Ahmed; Skärstrand, Hanna; Balhuizen, Alexander; Bennet, Hedvig; Steiner, Donald F; Törn, Carina; Fex, Malin; Lernmark, Åke

    2013-10-04

    Insulin is a major autoantigen in islet autoimmunity and progression to type 1 diabetes. It has been suggested that the insulin B-chain may be critical to insulin autoimmunity in type 1 diabetes. INS-IGF2 consists of the preproinsulin signal peptide, the insulin B-chain, and eight amino acids of the C-peptide in addition to 138 amino acids from the IGF2 gene. We aimed to determine the expression of INS-IGF2 in human pancreatic islets and autoantibodies in newly diagnosed children with type 1 diabetes and controls. INS-IGF2, expressed primarily in beta cells, showed higher levels of expression in islets from normal compared with donors with either type 2 diabetes (p = 0.006) or high HbA1c levels (p INS-IGF2 autoantibody levels were increased in newly diagnosed patients with type 1 diabetes (n = 304) compared with healthy controls (n = 355; p INS-IGF2 revealed that more patients than controls had doubly reactive insulin-INS-IGF2 autoantibodies. These data suggest that INS-IGF2, which contains the preproinsulin signal peptide, the B-chain, and eight amino acids of the C-peptide may be an autoantigen in type 1 diabetes. INS-IGF2 and insulin may share autoantibody-binding sites, thus complicating the notion that insulin is the primary autoantigen in type 1 diabetes.

  16. Treatment with Tacrolimus and Sirolimus Reveals No Additional Adverse Effects on Human Islets In Vitro Compared to Each Drug Alone but They Are Reduced by Adding Glucocorticoids

    Directory of Open Access Journals (Sweden)

    Kristine Kloster-Jensen

    2016-01-01

    Full Text Available Tacrolimus and sirolimus are important immunosuppressive drugs used in human islet transplantation; however, they are linked to detrimental effects on islets and reduction of long-term graft function. Few studies investigate the direct effects of these drugs combined in parallel with single drug exposure. Human islets were treated with or without tacrolimus (30 μg/L, sirolimus (30 μg/L, or a combination thereof for 24 hrs. Islet function as well as apoptosis was assessed by glucose-stimulated insulin secretion (GSIS and Cell Death ELISA. Proinflammatory cytokines were analysed by qRT-PCR and Bio-Plex. Islets exposed to the combination of sirolimus and tacrolimus were treated with or without methylprednisolone (1000 μg/L and the expression of the proinflammatory cytokines was investigated. We found the following: (i No additive reduction in function and viability in islets existed when tacrolimus and sirolimus were combined compared to the single drug. (ii Increased expression of proinflammatory cytokines mRNA and protein levels in islets took place. (iii Methylprednisolone significantly decreased the proinflammatory response in islets induced by the drug combination. Although human islets are prone to direct toxic effect of tacrolimus and sirolimus, we found no additive effects of the drug combination. Short-term exposure of glucocorticoids could effectively reduce the proinflammatory response in human islets induced by the combination of tacrolimus and sirolimus.

  17. Distinct differences in the responses of the human pancreatic β-cell line EndoC-βH1 and human islets to proinflammatory cytokines.

    Science.gov (United States)

    Oleson, Bryndon J; McGraw, Jennifer A; Broniowska, Katarzyna A; Annamalai, Mani; Chen, Jing; Bushkofsky, Justin R; Davis, Dawn B; Corbett, John A; Mathews, Clayton E

    2015-09-01

    While insulinoma cells have been developed and proven to be extremely useful in studies focused on mechanisms controlling β-cell function and viability, translating findings to human β-cells has proven difficult because of the limited access to human islets and the absence of suitable insulinoma cell lines of human origin. Recently, a human β-cell line, EndoC-βH1, has been derived from human fetal pancreatic buds. The purpose of this study was to determine whether human EndoC-βH1 cells respond to cytokines in a fashion comparable to human islets. Unlike most rodent-derived insulinoma cell lines that respond to cytokines in a manner consistent with rodent islets, EndoC-βH1 cells fail to respond to a combination of cytokines (IL-1, IFN-γ, and TNF) in a manner consistent with human islets. Nitric oxide, produced following inducible nitric oxide synthase (iNOS) expression, is a major mediator of cytokine-induced human islet cell damage. We show that EndoC-βH1 cells fail to express iNOS or produce nitric oxide in response to this combination of cytokines. Inhibitors of iNOS prevent cytokine-induced loss of human islet cell viability; however, they do not prevent cytokine-induced EndoC-βH1 cell death. Stressed human islets or human islets expressing heat shock protein 70 (HSP70) are resistant to cytokines, and, much like stressed human islets, EndoC-βH1 cells express HSP70 under basal conditions. Elevated basal expression of HSP70 in EndoC-βH1 cells is consistent with the lack of iNOS expression in response to cytokine treatment. While expressing HSP70, EndoC-βH1 cells fail to respond to endoplasmic reticulum stress activators, such as thapsigargin. These findings indicate that EndoC-βH1 cells do not faithfully recapitulate the response of human islets to cytokines. Therefore, caution should be exercised when making conclusions regarding the actions of cytokines on human islets when using this human-derived insulinoma cell line.

  18. Ex vivo expanded human regulatory T cells delay islet allograft rejection via inhibiting islet-derived monocyte chemoattractant protein-1 production in CD34+ stem cells-reconstituted NOD-scid IL2rγnull mice.

    Directory of Open Access Journals (Sweden)

    Fang Xiao

    Full Text Available Type 1 diabetes mellitus (T1DM is an autoimmune disease caused by immune-mediated destruction of insulin-secreting β cells of the pancreas. Near complete dependence on exogenous insulin makes T1DM very difficult to control, with the result that patients are exposed to high blood glucose and risk of diabetic complications and/or intermittent low blood glucose that can cause unconsciousness, fits and even death. Allograft transplantation of pancreatic islets restores normoglycemia with a low risk of surgical complications. However, although successful immediately after transplantation, islets are progressively lost, with most of the patients requiring exogenous insulin within 2 years post-transplant. Therefore, there is an urgent requirement for the development of new strategies to prevent islet rejection. In this study, we explored the importance of human regulatory T cells in the control of islets allograft rejection. We developed a pre-clinical model of human islet transplantation by reconstituting NOD-scid IL2rγnull mice with cord blood-derived human CD34+ stem cells and demonstrated that although the engrafted human immune system mediated the rejection of human islets, their survival was significantly prolonged following adoptive transfer of ex vivo expanded human Tregs. Mechanistically, Tregs inhibited the infiltration of innate immune cells and CD4+ T cells into the graft by down-regulating the islet graft-derived monocyte chemoattractant protein-1. Our findings might contribute to the development of clinical strategies for Treg therapy to control human islet rejection. We also show for the first time that CD34+ cells-reconstituted NOD-scid IL2rγnull mouse model could be beneficial for investigating human innate immunity in vivo.

  19. Islet transplantation and antioxidant management A comprehensive review

    Institute of Scientific and Technical Information of China (English)

    Seyed Sajad Mohseni Salehi Monfared; Bagher Larijani; Mohammad Abdollahi

    2009-01-01

    Islet transplantation as a promising treatment for type 1 diabetes has received widespread attention.Oxidative stress plays an essential role in cell injury during islet isolation and transplantation procedures.Antioxidants have been used in various studies to improve islet transplantation procedures. The present study reviews the role of oxidative stress and the benefits of antioxidants in islet transplantation procedures. The bibliographical databases Pubmed and Scopus were searched up to November 2008.All relevant human and animal in-vivo and in-vitro studies, which investigated antioxidants on islets,were included. Almost all the tested antioxidants used in the in-vitro studies enhanced islet viability and insulin secretion. Better control of blood glucose after transplantation was the major outcome of antioxidant therapy in all in-vivo studies. The data also indicated that antioxidants improved islet transplantation procedures. Although there is still insufficient evidence to draw definitive conclusions about the efficacy of individual supplements, the benefits of antioxidants in islet isolation procedures cannot be ignored.

  20. Pancreatic hormones are expressed on the surfaces of human and rat islet cells through exocytotic sites

    DEFF Research Database (Denmark)

    Larsson, L I; Hutton, J C; Madsen, O D

    1989-01-01

    . Electron microscopy reveals the labeling to occur at sites of exocytotic granule release, involving the surfaces of extruded granule cores. The surfaces of islet cells were labeled both by polyclonal and monoclonal antibodies, excluding that receptor-interacting, anti-idiotypic hormone antibodies were...... responsible for the staining. Human insulin cells were surface-labeled by monoclonal antibodies recognizing the mature secretory products, insulin and C-peptide but not with monoclonal antibodies specific for proinsulin. Thus, routing of unprocessed preproinsulin to the cell surface may not account...... for these results. It is concluded that the staining reflects interactions between the appropriate antibodies and exocytotic sites of hormone release....

  1. Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process.

    Science.gov (United States)

    Ghosal, Abhisek; Sekar, Thillai V; Said, Hamid M

    2014-08-01

    Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na(+)-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na(+)-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS.

  2. TGFβ Pathway Inhibition Redifferentiates Human Pancreatic Islet β Cells Expanded In Vitro.

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    Ginat Toren-Haritan

    Full Text Available In-vitro expansion of insulin-producing cells from adult human pancreatic islets could provide an abundant cell source for diabetes therapy. However, proliferation of β-cell-derived (BCD cells is associated with loss of phenotype and epithelial-mesenchymal transition (EMT. Nevertheless, BCD cells maintain open chromatin structure at β-cell genes, suggesting that they could be readily redifferentiated. The transforming growth factor β (TGFβ pathway has been implicated in EMT in a range of cell types. Here we show that human islet cell expansion in vitro involves upregulation of the TGFβ pathway. Blocking TGFβ pathway activation using short hairpin RNA (shRNA against TGFβ Receptor 1 (TGFBR1, ALK5 transcripts inhibits BCD cell proliferation and dedifferentiation. Treatment of expanded BCD cells with ALK5 shRNA results in their redifferentiation, as judged by expression of β-cell genes and decreased cell proliferation. These effects, which are reproducible in cells from multiple human donors, are mediated, at least in part, by AKT-FOXO1 signaling. ALK5 inhibition synergizes with a soluble factor cocktail to promote BCD cell redifferentiation. The combined treatment may offer a therapeutically applicable way for generating an abundant source of functional insulin-producing cells following ex-vivo expansion.

  3. Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal.

    Science.gov (United States)

    Jalili, Reza B; Moeen Rezakhanlou, Alireza; Hosseini-Tabatabaei, Azadeh; Ao, Ziliang; Warnock, Garth L; Ghahary, Aziz

    2011-07-01

    Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet-extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post-transplantation. FPCM was developed by embedding fibroblasts within type-I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over-growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome.

  4. Differentiation of human multipotent dermal fibroblasts into islet-like cell clusters

    Directory of Open Access Journals (Sweden)

    Liu Wei

    2010-06-01

    Full Text Available Abstract Background We have previously obtained a clonal population of cells from human foreskin that is able to differentiate into mesodermal, ectodermal and endodermal progenies. It is of great interest to know whether these cells could be further differentiated into functional insulin-producing cells. Results Sixty-one single-cell-derived dermal fibroblast clones were established from human foreskin by limiting dilution culture. Of these, two clones could be differentiated into neuron-, adipocyte- or hepatocyte-like cells under certain culture conditions. In addition, those two clones were able to differentiate into islet-like clusters under pancreatic induction. Insulin, glucagon and somatostatin were detectable at the mRNA and protein levels after induction. Moreover, the islet-like clusters could release insulin in response to glucose in vitro. Conclusions This is the first study to demonstrate that dermal fibroblasts can differentiate into insulin-producing cells without genetic manipulation. This may offer a safer cell source for future stem cell-based therapies.

  5. Label-Free Detection of Insulin and Glucagon within Human Islets of Langerhans Using Raman Spectroscopy

    Science.gov (United States)

    Hilderink, Janneke; Otto, Cees; Slump, Cees; Lenferink, Aufried; Engelse, Marten; van Blitterswijk, Clemens; de Koning, Eelco; Karperien, Marcel; van Apeldoorn, Aart

    2013-01-01

    Intrahepatic transplantation of donor islets of Langerhans is a promising therapy for patients with type 1 diabetes. It is of critical importance to accurately monitor islet quality before transplantation, which is currently done by standard histological methods that are performed off-line and require extensive sample preparation. As an alternative, we propose Raman spectroscopy which is a non-destructive and label-free technique that allows continuous real-time monitoring of the tissue to study biological changes as they occur. By performing Raman spectroscopic measurements on purified insulin and glucagon, we showed that the 520 cm-1 band assigned to disulfide bridges in insulin, and the 1552 cm-1 band assigned to tryptophan in glucagon are mutually exclusive and could therefore be used as indirect markers for the label-free distinction between both hormones. High-resolution hyperspectral Raman imaging for these bands showed the distribution of disulfide bridges and tryptophan at sub-micrometer scale, which correlated with the location of insulin and glucagon as revealed by conventional immunohistochemistry. As a measure for this correlation, quantitative analysis was performed comparing the Raman images with the fluorescence images, resulting in Dice coefficients (ranging between 0 and 1) of 0.36 for insulin and 0.19 for glucagon. Although the use of separate microscope systems with different spatial resolution and the use of indirect Raman markers cause some image mismatch, our findings indicate that Raman bands for disulfide bridges and tryptophan can be used as distinctive markers for the label-free detection of insulin and glucagon in human islets of Langerhans. PMID:24167603

  6. The castaway: characteristic islet features affect the ecology of the most isolated European lizard

    Directory of Open Access Journals (Sweden)

    Petros Lymberakis

    2016-12-01

    Full Text Available The ecological importance of islet endemics are in the front line of conservation efforts and thus the good knowledge of their biology is required. Podarcis levendis is a lacertid lizard, endemic to two rocky islets in the Cretan Sea, Greece, that was raised to specific level in 2008 and since then no data on its biology are available. Here we present the first ecological information on the species, focusing on population density, tail autotomy and feeding preferences. We recorded regenerated and damaged tails in the field and estimated population density with the transect method. We also dissected museum specimens and analyzed their stomach content. Regenerated tails were common and reached a considerable 71%. The latter finding could be attributed to the intense intraspecific competition due to high population density but also to the seasonal predation pressure by migratory birds. The diet of P. levendis coincides with that of other insular congenerics, including high percentages of plant material.

  7. MicroRNA expression in alpha and beta cells of human pancreatic islets.

    Directory of Open Access Journals (Sweden)

    Dagmar Klein

    Full Text Available microRNAs (miRNAs play an important role in pancreatic development and adult β-cell physiology. Our hypothesis is based on the assumption that each islet cell type has a specific pattern of miRNA expression. We sought to determine the profile of miRNA expression in α-and β-cells, the main components of pancreatic islets, because this analysis may lead to a better understanding of islet gene regulatory pathways. Highly enriched (>98% subsets of human α-and β-cells were obtained by flow cytometric sorting after intracellular staining with c-peptide and glucagon antibody. The method of sorting based on intracellular staining is possible because miRNAs are stable after fixation. MiRNA expression levels were determined by quantitative high throughput PCR-based miRNA array platform screening. Most of the miRNAs were preferentially expressed in β-cells. From the total of 667 miRNAs screened, the Significant Analysis of Microarray identified 141 miRNAs, of which only 7 were expressed more in α-cells (α-miRNAs and 134 were expressed more in β-cells (β-miRNAs. Bioinformatic analysis identified potential targets of β-miRNAs analyzing the Beta Cell Gene Atlas, described in the T1Dbase, the web platform, supporting the type 1 diabetes (T1D community. cMaf, a transcription factor regulating glucagon expression expressed selectively in α-cells (TFα is targeted by β-miRNAs; miR-200c, miR-125b and miR-182. Min6 cells treated with inhibitors of these miRNAs show an increased expression of cMaf RNA. Conversely, over expression of miR-200c, miR-125b or miR-182 in the mouse alpha cell line αTC6 decreases the level of cMAF mRNA and protein. MiR-200c also inhibits the expression of Zfpm2, a TFα that inhibits the PI3K signaling pathway, at both RNA and protein levels.In conclusion, we identified miRNAs differentially expressed in pancreatic α- and β-cells and their potential transcription factor targets that could add new insights into different

  8. Regulation of leptin on insulin secretion and sulfonulurea receptor 1 transcription level in isolated rats pancreatic islets

    Institute of Scientific and Technical Information of China (English)

    袁莉; 安汉祥; 邓秀玲; 李卓娅

    2003-01-01

    Objective To investigate the regulation of leptin on insulin secretion and expression of ATP-sensitive potassium channel subunit sulfonulurea receptor 1 (SUR1) mRNA, and to determine whether the effects of leptin are mediated through known intracellular signaling transduction. Methods Pancreatic islets were isolated by the collagenase method from male SD rats. The purified islets were incubated with different concentrations of leptin for 2 h in the presence of different concentrations of glucose. Insulin release was measured using radioimmunoassay. Expression of SUR1 mRNA was detected by RT-PCR. Results In the presence of leptin 2 nmol/L, insulin release was significantly inhibited at either 11.1 or 16.7 mmol/L glucose concentration (bothP<0.05), but insulin release was not altered at glucose of 5.6 mmol/L physiological concentration. The dose-response experiment showed that the maximal effect of leptin on insulin secretion achieved at 2 nmol/L. Exposure of islets to 2 nmol/L leptin induced a significant increase of SUR1 transcription evels by 71% (P<0.01) at 11.1 mmol/L glucose and by 56% (P<0.05) at 16.7 mmol/L glucose concentration. Selective phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin significantly prevented the leptin effect on insulin secretion and SUR1 mRNA expression. Conclusions Regulatory effects of leptin on insulin secretion could be biphasic at different concentrations of glucose and leptin. The stimulatory regulation of SUR1 transcription levels may be mediated through activation of PI 3-kinase pathway, which may be a possible mechanism of leptin in regulating insulin secretion.

  9. Islet transplantation: immunological perspectives.

    Science.gov (United States)

    Inverardi, Luca; Kenyon, Norma S; Ricordi, Camillo

    2003-10-01

    Clinical trials of islet transplantation are showing remarkable success, but they require administration of chronic immunosuppression, and are underscoring the large gap that exists between the number of human donors available and the number of patients that could benefit from the procedure. Recent progress has been made in the definition of key immunological mechanisms that are involved in determining islet transplant outcome. Clinical and preclinical studies, and studies in small animal model systems, will all eventually contribute to the definition of efficient and safe protocols for islet transplantation. If the use of xenografts is successful, it might represent a solution to the shortage of human organs.

  10. Single-cell transcriptomes identify human islet cell signatures and reveal cell-type–specific expression changes in type 2 diabetes

    Science.gov (United States)

    Bolisetty, Mohan; Kursawe, Romy; Sun, Lili; Sivakamasundari, V.; Kycia, Ina

    2017-01-01

    Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type–specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis. PMID:27864352

  11. Ultrastructural studies of time-course and cellular specificity of interleukin-1 mediated islet cytotoxicity

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Egeberg, J; Nerup, J

    1987-01-01

    of incubation and examined by electron microscopy in a blinded fashion. Already after 30 min, accumulation of opaque intracytoplasmic bodies without apparent surrounding membranes, and autophagic vacuoles were seen in about 20% of the beta cells examined in rat islets exposed to interleukin-1. After 16 h......Previous electron-microscopic studies of isolated islets of Langerhans exposed to the monokine interleukin-1 for 7 days have indicated that interleukin-1 is cytotoxic to all islet cells. To study the time-course and possible cellular specificity of interleukin-1 cytotoxicity to islets exposed...... of incubation with interleukin-1, more than 80% of rat beta cells showed signs of degeneration. Beta cell specific changes similar to those observed in rat islets exposed to IL-1 for 30 min were seen in human islets exposed to IL-1 for 24 h. The described changes were not observed in alpha cells in interleukin...

  12. Histochemical analysis of the role of class I and class II Clostridium histolyticum collagenase in the degradation of rat pancreatic extracellular matrix for islet isolation

    NARCIS (Netherlands)

    VosScheperkeuter, GH; vanSuylichem, PTR; Wolters, GHJ; vanSchilfgaarde, R

    1997-01-01

    To understand why class II Clostridium histolyticum collagenase is much more effective than class I in the isolation of rat pancreatic islets, we analyzed the role of these collagenases in pancreatic tissue dissociation. Crude collagenase was purified and then fractionated into class I and II with d

  13. Adult Human Pancreatic Islet Beta-Cells Display Limited Turnover and Long Lifespan as Determined by In-Vivo Thymidine Analog Incorporation and Radiocarbon Dating

    Energy Technology Data Exchange (ETDEWEB)

    Perl, S; Kushner, J A; Buchholz, B A; Meeker, A K; Stein, G M; Hsieh, M; Kirby, M; Pechhold, S; Liu, E H; Harlan, D M; Tisdale, J F

    2010-03-15

    Diabetes mellitus results from an absolute or relative deficiency of insulin producing pancreatic beta-cells. The adult human beta-cell's turnover rate remains unknown. We employed novel techniques to examine adult human islet beta-cell turnover and longevity in vivo. Subjects enrolled in NIH clinical trials received thymidine analogues [iododeoxyuridine (IdU) or bromodeoxyuridine (BrdU)] 8-days to 4-years prior to death. Archival autopsy samples from ten patients (aged 17-74 years) were employed to assess beta-cell turnover by scoring nuclear analog labeling within insulin staining cells. Human adult beta-cell longevity was determined by estimating the cells genomic DNA integration of atmospheric carbon-14 ({sup 14}C). DNA was purified from pancreatic islets isolated from cadaveric donors; whole islet prep DNA was obtained from a 15 year old donor, and purified beta-cell DNA was obtained from two donors (age 48 and 80 years). {sup 14}C levels were then determined using accelerator mass spectrometry (AMS). Cellular 'birth date' was determined by comparing the subject's DNA {sup 14}C content relative to a well-established {sup 14}C atmospheric prevalence curve. In the two subjects less than age 20 years, 1-2% of the beta-cell nuclei co-stained for BrdU/IdU. No beta-cell nuclei co-stained in the eight patients more than 30 years old. Consistent with the BrdU/IdU turnover data, beta-cell DNA {sup 14}C content indicated the cells 'birth date' occurred within the subject's first 30 years of life. Under typical circumstances, adult human beta-cells and their cellular precursors are established by young adulthood.

  14. Human fetal islet transplantation in type 1 diabetic patients: comparison of metabolic effects between single and multiple implantation regimens.

    Science.gov (United States)

    Djordjevic, P B; Lalic, N M; Jotic, A; Paunovic, I; Lalic, K; Raketic, N; Nikolic, D; Zamaklar, M; Rajkovic, N; Lukic, L; Dimitrijevic-Sreckovic, V; Dragasevic, M; Nikolic, D; Markovic, I

    2004-11-01

    Previous studies suggest that multiple transplantations might be equally efficient to a single regimen for human adult islets. The aim of this study was to compare metabolic parameters after each of the two regimens of human fetal islet (HFI) transplantation in type 1 diabetics. In group A (single transplant, n = 9), 180 +/- 20 x 1000 HFI equivalents (IEQs) were implanted by a single IM injection; in group B (multiple transplants, n = 8) islets were implanted as three consecutive injections (60 +/- 10 x 1000 IEQs) at 7-day intervals. We analyzed the metabolic parameters on days -1, 30, 60, 90, 120, 150, and 180 after the procedure. Among the metabolic parameters, we evaluated insulin secretion capacity-ISC (C peptide, RIA), metabolic control (HbA1c, chromatography), and insulin daily dose IDD. We found that C peptide levels increased, peaking on day 90 (A: 0.38 +/- 0.15; B: 0.34 +/- 0.19 nmol/L, P = NS) and then rapidly decreasing without differences, the HbA1c levels and IDD decreased in the same manner without differences between the groups. Our results demonstrate that multiple and single islet transplant regimens are equally efficient to temporarily restore a significant ISC with improvement of metabolic and clinical parameters. The results imply that the two regimens have an equal clinical value.

  15. Glucose stimulates human beta cell replication in vivo in islets transplanted into NOD–severe combined immunodeficiency (SCID) mice

    Science.gov (United States)

    Levitt, H. E.; Cyphert, T. J.; Pascoe, J. L.; Hollern, D. A.; Abraham, N.; Lundell, R. J.; Rosa, T.; Romano, L. C.; Zou, B.; O’Donnell, C. P.; Stewart, A. F.; Garcia-Ocaña, A.; Alonso, L. C.

    2011-01-01

    Aims/hypothesis We determined whether hyperglycaemia stimulates human beta cell replication in vivo in an islet transplant model Methods Human islets were transplanted into streptozotocin-induced diabetic NOD–severe combined immunodeficiency mice. Blood glucose was measured serially during a 2 week graft revascularisation period. Engrafted mice were then catheterised in the femoral artery and vein, and infused intravenously with BrdU for 4 days to label replicating beta cells. Mice with restored normoglycaemia were co-infused with either 0.9% (wt/vol.) saline or 50% (wt/vol.) glucose to generate glycaemic differences among grafts from the same donors. During infusions, blood glucose was measured daily. After infusion, human beta cell replication and apoptosis were measured in graft sections using immunofluorescence for insulin, and BrdU or TUNEL. Results Human islet grafts corrected diabetes in the majority of cases. Among grafts from the same donor, human beta cell proliferation doubled in those exposed to higher glucose relative to lower glucose. Across the entire cohort of grafts, higher blood glucose was strongly correlated with increased beta cell replication. Beta cell replication rates were unrelated to circulating human insulin levels or donor age, but tended to correlate with donor BMI. Beta cell TUNEL reactivity was not measurably increased in grafts exposed to elevated blood glucose. Conclusions/interpretation Glucose is a mitogenic stimulus for transplanted human beta cells in vivo. Investigating the underlying pathways may point to mechanisms capable of expanding human beta cell mass in vivo. PMID:20936253

  16. Cathelicidin Antimicrobial Peptide: A Novel Regulator of Islet Function, Islet Regeneration, and Selected Gut Bacteria.

    Science.gov (United States)

    Pound, Lynley D; Patrick, Christopher; Eberhard, Chandra E; Mottawea, Walid; Wang, Gen-Sheng; Abujamel, Turki; Vandenbeek, Roxanne; Stintzi, Alain; Scott, Fraser W

    2015-12-01

    Cathelicidin antimicrobial peptide (CAMP) is a naturally occurring secreted peptide that is expressed in several organs with pleiotropic roles in immunomodulation, wound healing, and cell growth. We previously demonstrated that gut Camp expression is upregulated when type 1 diabetes-prone rats are protected from diabetes development. Unexpectedly, we have also identified novel CAMP expression in the pancreatic β-cells of rats, mice, and humans. CAMP was present even in sterile rat embryo islets, germ-free adult rat islets, and neogenic tubular complexes. Camp gene expression was downregulated in young BBdp rat islets before the onset of insulitis compared with control BBc rats. CAMP treatment of dispersed islets resulted in a significant increase in intracellular calcium mobilization, an effect that was both delayed and blunted in the absence of extracellular calcium. Additionally, CAMP treatment promoted insulin and glucagon secretion from isolated rat islets. Thus, CAMP is a promoter of islet paracrine signaling that enhances islet function and glucoregulation. Finally, daily treatment with the CAMP/LL-37 peptide in vivo in BBdp rats resulted in enhanced β-cell neogenesis and upregulation of potentially beneficial gut microbes. In particular, CAMP/LL-37 treatment shifted the abundance of specific bacterial populations, mitigating the gut dysbiosis observed in the BBdp rat. Taken together, these findings indicate a novel functional role for CAMP/LL-37 in islet biology and modification of gut microbiota. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  17. Islet expression of the DNA repair enzyme 8-oxoguanosine DNA glycosylase (Ogg1 in human type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Yoon Kun-Ho

    2002-04-01

    Full Text Available Abstract Background It has become increasingly clear that β-cell failure plays a critical role in the pathogenesis of type 2 diabetes. Free-radical mediated β-cell damage has been intensively studied in type 1 diabetes, but not in human type 2 diabetes. Therefore, we studied the protein expression of the DNA repair enzyme Ogg1 in pancreases from type 2 diabetics. Ogg1 was studied because it is the major enzyme involved in repairing 7,8-dihydro-8-oxoguanosine DNA adducts, a lesion previously observed in a rat model of type 2 diabetes. Moreover, in a gene expression screen, Ogg1 was over-expressed in islets from a human type 2 diabetic. Methods Immunofluorescent staining of Ogg1 was performed on pancreatic specimens from healthy controls and patients with diabetes for 2–23 years. The intensity and islet area stained for Ogg1 was evaluated by semi-quantitative scoring. Results Both the intensity and the area of islet Ogg1 staining were significantly increased in islets from the type 2 diabetic subjects compared to the healthy controls. A correlation between increased Ogg1 fluorescent staining intensity and duration of diabetes was also found. Most of the staining observed was cytoplasmic, suggesting that mitochondrial Ogg1 accounts primarily for the increased Ogg1 expression. Conclusion We conclude that oxidative stress related DNA damage may be a novel important factor in the pathogenesis of human type 2 diabetes. An increase of Ogg1 in islet cell mitochondria is consistent with a model in which hyperglycemia and consequent increased β-cell oxidative metabolism lead to DNA damage and the induction of Ogg1 expression.

  18. A Novel Efficient Technique of Pancreatic Islet Cell Isolation and Purification%一种高效的小鼠胰岛分离纯化新技术

    Institute of Scientific and Technical Information of China (English)

    李敏; 宋陆军; 高晓东; 常文举; 秦新裕

    2011-01-01

    Objective:To explore the method of obtaining enough mouse islets with high purity in order to set up an animal model for clinical islet transplantation.Methods: The 6~8 weeks male C57BL/6 mice weighing 25~30 g were intraperitoneal anesthetized before the operation of common bile duct ligation.Then we isolated and purified mice islets by adopting collagenV retro perfusion,situ digestion,gradient centrifugation in Ficoll-400 solution and sorted islets using sterile capillary pipettes in the phase contrast microscope.Islets purity was assessed by dithizone staining.Eventually, we cultured islet cells and observed the shape of islets on day 3, 8 and 24 separately.Results:From this way, we could obtain 390± 20 islets in each mouse.The isolated islets were round or mass, 50- 150 μm in diameter, complete and bright.The purity of the islets was more than 85%.Conclusions: We improve the method of isolating islets including retro perfusion,situ digestion and gradient centrifugation in Ficoll-400 solution, which is timesaving and effective.The cultured islet cells had high activity and formed single cells in petri dishes within 24 days, which is suitable for further study.%目的:探讨获得足够数量和较高纯度小鼠胰岛的分离及纯化方法,为进行小鼠的胰岛移植提供实验条件.方法:将6~8周,体质量25~30 g的雄性C57BL/6 小鼠腹腔麻醉后结扎胆总管,采用胶原酶Ⅴ逆行灌注、原位消化、Ficoll-400梯度离心并用无菌的毛细吸管在相差显微镜下观察并分选胰岛,双硫腙(DTZ)染色鉴定胰岛细胞.体外培养胰岛单细胞,并于第3、8、24天观察胰岛形态.结果:采用该法分离纯化后,每只小鼠可得到390±20个胰岛,胰岛呈圆形或团块状,直径50~150 μm,形态完整,折光性好,纯度达85%以上,24 d后在培养皿内铺成单个细胞.结论:本研究所用逆行灌注、原位消化及Ficoll-400梯度离心分离、纯化胰岛细胞的方法省时、高效,培养

  19. Residue specific effects of human islet polypeptide amyloid on self-assembly and on cell toxicity.

    Science.gov (United States)

    Khemtemourian, Lucie; Guillemain, Ghislaine; Foufelle, Fabienne; Killian, J Antoinette

    2017-08-01

    Type 2 diabetes mellitus is characterized histopathologically by the presence of fibrillary amyloid deposits in the pancreatic islets of Langerhans. Human islet amyloid polypeptide (hIAPP), the 37-residue pancreatic hormone, is the major constituent of these amyloid deposits. The propensity of IAPP to form amyloid fibrils is strongly dependent on its primary sequence. An intriguing example is His at residue 18. Although H18 is located outside the amyloidogenic region, it has been suggested that this residue and its charge state play an important role in the kinetics of conformational changes and fibril formation as well as in mediating cell toxicity. To gain more insight into the importance of this residue, we have synthesized four analogues (H18R-IAPP, H18K-IAPP, H18A-IAPP and H18E-IAPP) and we performed a full biophysical study on the properties of these peptides. Kinetic experiments as monitored by thioflavin-T fluorescence, transmission electron microscopy, circular dichroism and cell toxicity assays revealed that all variants are less fibrillogenic and less toxic than native hIAPP both at neutral pH and at low pH. This demonstrates that the effect of H18 in native IAPP is not simply determined by its charge state, but rather that residue 18 is important for specific intra- and intermolecular interactions that occur during fibril formation and that may involve charge, size and hydrophobicity. Furthermore, our results indicate that H18R-IAPP has a strong inhibiting effect on native hIAPP fibril formation. Together these results highlight the large impact of modifying a single residue outside the amyloidogenic domain on fibril formation and cell toxicity induced by IAPP, opening up new avenues for design of inhibitors or modulators of IAPP aggregation. Copyright © 2017. Published by Elsevier B.V.

  20. Identification of the ectonucleotidases expressed in mouse, rat, and human Langerhans islets: potential role of NTPDase3 in insulin secretion.

    Science.gov (United States)

    Lavoie, Elise G; Fausther, Michel; Kauffenstein, Gilles; Kukulski, Filip; Künzli, Beat M; Friess, Helmut; Sévigny, Jean

    2010-10-01

    Extracellular nucleotides and adenosine regulate endocrine pancreatic functions such as insulin secretion by Langerhans islet β-cells via the activation of specific P2 and P1 receptors. Membrane-bound ectonucleotidases regulate the local concentration of these ligands and consequently control the activation of their receptors. The objective of this study was to identify and localize the major ectonucleotidases, namely NTPDases and ecto-5'-nucleotidase, present in the endocrine pancreas. In addition, the potential implication of ecto-ATPase activity on insulin secretion was investigated in the rat β-cell line INS-1 (832/13). The localization of ectonucleotidase activity and protein was carried out in situ by enzyme histochemistry and immunolocalization in mouse, rat, and human pancreas sections. NTPDase1 was localized in all blood vessels and acini, and NTPDase2 was localized in capillaries of Langerhans islets and in peripheral conjunctive tissue, whereas NTPDase3 was detected in all Langerhans islet cell types. Interestingly, among the mammalian species tested, ecto-5'-nucleotidase was present only in rat Langerhans islet cells, where it was coexpressed with NTPDase3. Notably, the inhibition of NTPDase3 activity by BG0136 and NF279 facilitated insulin release from INS-1 (832/13) cells under conditions of low glycemia, probably by affecting P2 receptor activation. NTPDase3 activity also regulated the inhibitory effect of exogenous ATP in the presence of a high glucose concentration most likely by controlling adenosine production. In conclusion, all pancreatic endocrine cells express NTPDase3 that was shown to modulate insulin secretion in rat INS-1 (832/13) β-cells. Ecto-5'-nucleotidase is expressed in rat Langerhans islet cells but absent in human and mouse endocrine cells.

  1. Human Islet Amyloid Polypeptide Fibril Binding to Catalase: A Transmission Electron Microscopy and Microplate Study

    Directory of Open Access Journals (Sweden)

    Nathaniel G. N. Milton

    2010-01-01

    Full Text Available The diabetes-associated human islet amyloid polypeptide (IAPP is a 37-amino-acid peptide that forms fibrils in vitro and in vivo. Human IAPP fibrils are toxic in a similar manner to Alzheimer's amyloid-β (Aβ and prion protein (PrP fibrils. Previous studies have shown that catalase binds to Aβ fibrils and appears to recognize a region containing the Gly-Ala-Ile-Ile sequence that is similar to the Gly-Ala-Ile-Leu sequence found in human IAPP residues 24-27. This study presents a transmission electron microscopy (TEM—based analysis of fibril formation and the binding of human erythrocyte catalase to IAPP fibrils. The results show that human IAPP 1-37, 8-37, and 20-29 peptides form fibrils with diverse and polymorphic structures. All three forms of IAPP bound catalase, and complexes of IAPP 1-37 or 8-37 with catalase were identified by immunoassay. The binding of biotinylated IAPP to catalase was high affinity with a KD of 0.77nM, and could be inhibited by either human or rat IAPP 1-37 and 8-37 forms. Fibrils formed by the PrP 118-135 peptide with a Gly-Ala-Val-Val sequence also bound catalase. These results suggest that catalase recognizes a Gly-Ala-Ile-Leu—like sequence in amyloid fibril-forming peptides. For IAPP 1-37 and 8-37, the catalase binding was primarily directed towards fibrillar rather than ribbon-like structures, suggesting differences in the accessibility of the human IAPP 24-27 Gly-Ala-Ile-Leu region. This suggests that catalase may be able to discriminate between different structural forms of IAPP fibrils. The ability of catalase to bind IAPP, Aβ, and PrP fibrils demonstrates the presence of similar accessible structural motifs that may be targets for antiamyloid therapeutic development.

  2. Trefoil factor 3 stimulates human and rodent pancreatic islet beta-cell replication with retention of function.

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    Fueger, Patrick T; Schisler, Jonathan C; Lu, Danhong; Babu, Daniella A; Mirmira, Raghavendra G; Newgard, Christopher B; Hohmeier, Hans E

    2008-05-01

    Both major forms of diabetes involve a decline in beta-cell mass, mediated by autoimmune destruction of insulin-producing cells in type 1 diabetes and by increased rates of apoptosis secondary to metabolic stress in type 2 diabetes. Methods for controlled expansion of beta-cell mass are currently not available but would have great potential utility for treatment of these diseases. In the current study, we demonstrate that overexpression of trefoil factor 3 (TFF3) in rat pancreatic islets results in a 4- to 5-fold increase in [(3)H]thymidine incorporation, with full retention of glucose-stimulated insulin secretion. This increase was almost exclusively due to stimulation of beta-cell replication, as demonstrated by studies of bromodeoxyuridine incorporation and co-immunofluorescence analysis with anti-bromodeoxyuridine and antiinsulin or antiglucagon antibodies. The proliferative effect of TFF3 required the presence of serum or 0.5 ng/ml epidermal growth factor. The ability of TFF3 overexpression to stimulate proliferation of rat islets in serum was abolished by the addition of epidermal growth factor receptor antagonist AG1478. Furthermore, TFF3-induced increases in [3H]thymidine incorporation in rat islets cultured in serum was blocked by overexpression of a dominant-negative Akt protein or treatment with triciribine, an Akt inhibitor. Finally, overexpression of TFF3 also caused a doubling of [3H]thymidine incorporation in human islets. In summary, our findings reveal a novel TFF3-mediated pathway for stimulation of beta-cell replication that could ultimately be exploited for expansion or preservation of islet beta-cell mass.

  3. Pancreatic islet blood flow and its measurement.

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    Jansson, Leif; Barbu, Andreea; Bodin, Birgitta; Drott, Carl Johan; Espes, Daniel; Gao, Xiang; Grapensparr, Liza; Källskog, Örjan; Lau, Joey; Liljebäck, Hanna; Palm, Fredrik; Quach, My; Sandberg, Monica; Strömberg, Victoria; Ullsten, Sara; Carlsson, Per-Ola

    2016-05-01

    Pancreatic islets are richly vascularized, and islet blood vessels are uniquely adapted to maintain and support the internal milieu of the islets favoring normal endocrine function. Islet blood flow is normally very high compared with that to the exocrine pancreas and is autonomously regulated through complex interactions between the nervous system, metabolites from insulin secreting β-cells, endothelium-derived mediators, and hormones. The islet blood flow is normally coupled to the needs for insulin release and is usually disturbed during glucose intolerance and overt diabetes. The present review provides a brief background on islet vascular function and especially focuses on available techniques to measure islet blood perfusion. The gold standard for islet blood flow measurements in experimental animals is the microsphere technique, and its advantages and disadvantages will be discussed. In humans there are still no methods to measure islet blood flow selectively, but new developments in radiological techniques hold great hopes for the future.

  4. Pig islets for islet xenotransplantation: current status and future perspectives

    Institute of Scientific and Technical Information of China (English)

    Hu Qinghua; Liu Zhongwei; Zhu Haitao

    2014-01-01

    Objective To review the current status and progress on pig islet xenotransplantation.Data sources Data used in this review were mainly from English literature of Pubmed database.The search terms were "pig islet" and "xenotransplantation".Study selection The original articles and critical reviews selected were relevant to this review's theme.Results Pigs are suggested to be an ideal candidate for obtaining available islet cells for transplantation.However,the potential clinical application of pig islet is still facing challenges including inadequate yield of high-quality functional islets and xenorejection of the transplants.The former can be overcome mainly by selection of a suitable pathogen-free source herd and the development of isolation and purification technology.While the feasibility of successful preclinical pig islet xenotranplantation provides insights in the possible mechanisms of xenogeneic immune recognition and rejection to overwhelm the latter.In addition,the achievement of long-term insulin independence in diabetic models by means of distinct islet products and novel immunotherapeutic strategies is promising.Conclusions Pig islet xenotransplantation is one of the prospective treatments to bridge the gap between the needs of transplantation in patients with diabetes and available islet cells.Nonetheless,further studies and efforts are needed to translate obtained findings into tangible applications.

  5. Selective destruction of mouse islet beta cells by human T lymphocytes in a newly-established humanized type 1 diabetic model

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    Zhao, Yong, E-mail: yongzhao@uic.edu [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Guo, Chengshan; Hwang, David; Lin, Brian; Dingeldein, Michael; Mihailescu, Dan; Sam, Susan; Sidhwani, Seema [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Zhang, Yongkang [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Jain, Sumit [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Skidgel, Randal A. [Department of Pharmacology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Prabhakar, Bellur S. [Department of Immunology and Microbiology, University of Illinois at Chicago, Chicago, IL 60612 (United States); Mazzone, Theodore [Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612 (United States); Holterman, Mark J. [Department of Surgery, University of Illinois at Chicago, Chicago, IL 60612 (United States)

    2010-09-03

    Research highlights: {yields} Establish a human immune-mediated type 1 diabetic model in NOD-scid IL2r{gamma}{sup null} mice. {yields} Using the irradiated diabetic NOD mouse spleen mononuclear cells as trigger. {yields} The islet {beta} cells were selectively destroyed by infiltrated human T cells. {yields} The model can facilitate translational research to find a cure for type 1 diabetes. -- Abstract: Type 1 diabetes (T1D) is caused by a T cell-mediated autoimmune response that leads to the loss of insulin-producing {beta} cells. The optimal preclinical testing of promising therapies would be aided by a humanized immune-mediated T1D model. We develop this model in NOD-scid IL2r{gamma}{sup null} mice. The selective destruction of pancreatic islet {beta} cells was mediated by human T lymphocytes after an initial trigger was supplied by the injection of irradiated spleen mononuclear cells (SMC) from diabetic nonobese diabetic (NOD) mice. This resulted in severe insulitis, a marked loss of total {beta}-cell mass, and other related phenotypes of T1D. The migration of human T cells to pancreatic islets was controlled by the {beta} cell-produced highly conserved chemokine stromal cell-derived factor 1 (SDF-1) and its receptor C-X-C chemokine receptor (CXCR) 4, as demonstrated by in vivo blocking experiments using antibody to CXCR4. The specificity of humanized T cell-mediated immune responses against islet {beta} cells was generated by the local inflammatory microenvironment in pancreatic islets including human CD4{sup +} T cell infiltration and clonal expansion, and the mouse islet {beta}-cell-derived CD1d-mediated human iNKT activation. The selective destruction of mouse islet {beta} cells by a human T cell-mediated immune response in this humanized T1D model can mimic those observed in T1D patients. This model can provide a valuable tool for translational research into T1D.

  6. The human pancreatic islet transcriptome: expression of candidate genes for type 1 diabetes and the impact of pro-inflammatory cytokines.

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    Décio L Eizirik

    Full Text Available Type 1 diabetes (T1D is an autoimmune disease in which pancreatic beta cells are killed by infiltrating immune cells and by cytokines released by these cells. Signaling events occurring in the pancreatic beta cells are decisive for their survival or death in diabetes. We have used RNA sequencing (RNA-seq to identify transcripts, including splice variants, expressed in human islets of Langerhans under control conditions or following exposure to the pro-inflammatory cytokines interleukin-1β (IL-1β and interferon-γ (IFN-γ. Based on this unique dataset, we examined whether putative candidate genes for T1D, previously identified by GWAS, are expressed in human islets. A total of 29,776 transcripts were identified as expressed in human islets. Expression of around 20% of these transcripts was modified by pro-inflammatory cytokines, including apoptosis- and inflammation-related genes. Chemokines were among the transcripts most modified by cytokines, a finding confirmed at the protein level by ELISA. Interestingly, 35% of the genes expressed in human islets undergo alternative splicing as annotated in RefSeq, and cytokines caused substantial changes in spliced transcripts. Nova1, previously considered a brain-specific regulator of mRNA splicing, is expressed in islets and its knockdown modified splicing. 25/41 of the candidate genes for T1D are expressed in islets, and cytokines modified expression of several of these transcripts. The present study doubles the number of known genes expressed in human islets and shows that cytokines modify alternative splicing in human islet cells. Importantly, it indicates that more than half of the known T1D candidate genes are expressed in human islets. This, and the production of a large number of chemokines and cytokines by cytokine-exposed islets, reinforces the concept of a dialog between pancreatic islets and the immune system in T1D. This dialog is modulated by candidate genes for the disease at both the

  7. ISLET FORMATION AND REGENERATION

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective To explore the mechanisms of differentiation and development of pancreatic endocrine cells as well as pancreatic regeneration. Methods Human embryonic pancreatic tissue at 7-14 weeks of gestation was collected. Diabetes mellitus rat model was induced with 65 mg/kg of streptozotocin. Insulin, glucagon, somatostatin, nestin, and cytokeratin 19 (CK19)of pancreatic tissues were observed by immunohistochemistry. Results At 9 weeks of gestation, pancreatic epithelial cells began to co-express insulin, glucagon, somatostatin, and CK19 before migration. Islet cells gradually congregated along with the increase of aging, and at 14 weeks of gestation histological examination showed islet formation. At 12 weeks of gestation, nestin-positive cells could be seen in the pancreatic mesenchyme. During early embryogenesis, islet cells of pancreatic ducts co-expressed insulin, glucagon, and somatostatin. During pancreatic regeneration after damage, nestin expression of islet cells increased. Conclusion In the early stage of embryogenesis, islet cells of primary pancreatic ducts can be differentiated to multipotential endocrine cells before migration. During tissue regeneration, pancreatic stem cells may differentiate and proliferate to form pancreatic islet.

  8. Glucose-induced β cell production of IL-1β contributes to glucotoxicity in human pancreatic islets

    Science.gov (United States)

    Maedler, Kathrin; Sergeev, Pavel; Ris, Frédéric; Oberholzer, José; Joller-Jemelka, Helen I.; Spinas, Giatgen A.; Kaiser, Nurit; Halban, Philippe A.; Donath, Marc Y.

    2002-01-01

    In type 2 diabetes, chronic hyperglycemia is suggested to be detrimental to pancreatic β cells, causing impaired insulin secretion. IL-1β is a proinflammatory cytokine acting during the autoimmune process of type 1 diabetes. IL-1β inhibits β cell function and promotes Fas-triggered apoptosis in part by activating the transcription factor NF-κB. Recently, we have shown that increased glucose concentrations also induce Fas expression and β cell apoptosis in human islets. The aim of the present study was to test the hypothesis that IL-1β may mediate the deleterious effects of high glucose on human β cells. In vitro exposure of islets from nondiabetic organ donors to high glucose levels resulted in increased production and release of IL-1β, followed by NF-κB activation, Fas upregulation, DNA fragmentation, and impaired β cell function. The IL-1 receptor antagonist protected cultured human islets from these deleterious effects. β cells themselves were identified as the islet cellular source of glucose-induced IL-1β. In vivo, IL-1β–producing β cells were observed in pancreatic sections of type 2 diabetic patients but not in nondiabetic control subjects. Similarly, IL-1β was induced in β cells of the gerbil Psammomys obesus during development of diabetes. Treatment of the animals with phlorizin normalized plasma glucose and prevented β cell expression of IL-1β. These findings implicate an inflammatory process in the pathogenesis of glucotoxicity in type 2 diabetes and identify the IL-1β/NF-κB pathway as a target to preserve β cell mass and function in this condition. PMID:12235117

  9. A new approach for pancreatic tissue engineering: human endometrial stem cells encapsulated in fibrin gel can differentiate to pancreatic islet beta-cell.

    Science.gov (United States)

    Niknamasl, Azadeh; Ostad, Seyed Nasser; Soleimani, Mansoureh; Azami, Mahmoud; Salmani, Maryam Kabir; Lotfibakhshaiesh, Nasrin; Ebrahimi-Barough, Somayeh; Karimi, Roya; Roozafzoon, Reza; Ai, Jafar

    2014-10-01

    Metabolic diabetes mellitus as the most serious and prevalent metabolic disease in the world has various complications. The most effective treatment of type I diabetes seems to be islet cell transplantation. Shortage of donors and difficult procedures and high rate of rejection have always restricted this approach. Tissue engineering is a novel effective solution to many medical problems such as diabetes. Endometrial mesenchymal stem cells as a lineage which have the potential to differentiate to mesodermal and endodermal tissues seem to be suitable for this purpose. Fibrin hydrogel with a high degree of biocompatibility and specific properties making it similar to normal pancreas seems to be an ideal scaffold. After successfully isolating stem cells (hEnSCs) from human endometrium, a three-step protocol was used to differentiate them into pancreatic beta cells. Fibrin was used as 3D scaffold. After 2 weeks, cells formed clusters like islets cells, and secretion of insulin was measured by chemiluminescence. PDX1, proinsulin, and c-peptide as special markers of β cells were detected by immunofluorescence. Expression of glucagon, PDX1, and insulin genes in mRNA level was detected by Real time PCR and gel electrophoresis. The former showed higher levels of gene expression in 3D cultures. SEM analysis showed good integrity between cells and scaffold. No toxicity was detected with fibrin scaffold by MTT assay.

  10. Islet formation in mice and men: lessons for the generation of functional insulin-producing β-cells from human pluripotent stem cells.

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    Nair, Gopika; Hebrok, Matthias

    2015-06-01

    The Islets of Langerhans are crucial 'micro-organs' embedded in the glandular exocrine pancreas that regulate nutrient metabolism. They not only synthesize, but also secrete endocrine hormones in a modulated fashion in response to physiologic metabolic demand. These highly sophisticated structures with intricate organization of multiple cell types, namely endocrine, vascular, neuronal and mesenchymal cells, have evolved to perform this task to perfection over time. Not surprisingly, islet architecture and function are dissimilar between humans and typically studied model organisms, such as rodents and zebrafish. Further, recent findings also suggest noteworthy differences in human islet development from that in mouse, including delayed appearance and gradual resolution of key differentiation markers, a single-phase of endocrine differentiation, and prenatal association of developing islets with neurovascular milieu. In light of these findings, it is imperative that a systematic study is undertaken to compare islet development between human and mouse. Illuminating inter-species differences in islet development will likely be critical in furthering our pursuit to generate an unlimited supply of truly functional and fully mature β-cells from human pluripotent stem cell (hPSC) sources for therapeutic purposes.

  11. Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

    Science.gov (United States)

    Xu, Zhi-Xue; Zhang, Qiang; Ma, Gong-Li; Chen, Cong-Heng; He, Yan-Ming; Xu, Li-Hui; Zhang, Yuan; Zhou, Guang-Rong; Li, Zhen-Hua

    2016-01-01

    The abnormal fibrillation of human islet amyloid polypeptide (hIAPP) has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−)-epigallocatechin gallate (EGCG) is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III)/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III) and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III) and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III) could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III)/EGCG complex in molar ratio of 1 : 1 as Al(EGCG)(H2O)2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes. PMID:28074190

  12. Influence of Aluminium and EGCG on Fibrillation and Aggregation of Human Islet Amyloid Polypeptide

    Directory of Open Access Journals (Sweden)

    Zhi-Xue Xu

    2016-01-01

    Full Text Available The abnormal fibrillation of human islet amyloid polypeptide (hIAPP has been implicated in the development of type II diabetes. Aluminum is known to trigger the structural transformation of many amyloid proteins and induce the formation of toxic aggregate species. The (−-epigallocatechin gallate (EGCG is considered capable of binding both metal ions and amyloid proteins with inhibitory effect on the fibrillation of amyloid proteins. However, the effect of Al(III/EGCG complex on hIAPP fibrillation is unclear. In the present work, we sought to view insight into the structures and properties of Al(III and EGCG complex by using spectroscopic experiments and quantum chemical calculations and also investigated the influence of Al(III and EGCG on hIAPP fibrillation and aggregation as well as their combined interference on this process. Our studies demonstrated that Al(III could promote fibrillation and aggregation of hIAPP, while EGCG could inhibit the fibrillation of hIAPP and lead to the formation of hIAPP amorphous aggregates instead of the ordered fibrils. Furthermore, we proved that the Al(III/EGCG complex in molar ratio of 1 : 1 as Al(EGCG(H2O2 could inhibit the hIAPP fibrillation more effectively than EGCG alone. The results provide the invaluable reference for the new drug development to treat type II diabetes.

  13. Protection of Human Pancreatic Islets from Lipotoxicity by Modulation of the Translocon.

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    R Cassel

    Full Text Available Type 2 diabetes is characterized by peripheral insulin resistance and pancreatic beta cell dysfunction. Elevated free fatty acids (FFAs may impair beta cell function and mass (lipotoxicity. Altered calcium homeostasis may be involved in defective insulin release. The endoplasmic reticulum (ER is the major intracellular calcium store. Lipotoxicity induces ER stress and in parallel an ER calcium depletion through unknown ER calcium leak channels. The main purposes of this study is first to identify one of these channels and secondly, to check the opportunity to restore beta cells function (i.e., insulin secretion after pharmacological inhibition of ER calcium store depletion. We investigated the functionality of translocon, an ER calcium leak channel and its involvement on FFAs-induced alterations in MIN6B1 cells and in human pancreatic islets. We evidenced that translocon acts as a functional ER calcium leak channel in human beta cells using anisomycin and puromycin (antibiotics, respectively blocker and opener of this channel. Puromycin induced a significant ER calcium release, inhibited by anisomycin pretreatment. Palmitate treatment was used as FFA model to induce a mild lipotoxic effect: ER calcium content was reduced, ER stress but not apoptosis were induced and glucose induced insulin secretion was decreased in our beta cells. Interestingly, translocon inhibition by chronic anisomycin treatment prevented dysfunctions induced by palmitate, avoiding reticular calcium depletion, ER stress and restoring insulin secretion. Our results provide for the first time compelling evidence that translocon actively participates to the palmitate-induced ER calcium leak and insulin secretion decrease in beta cells. Its inhibition reduces these lipotoxic effects. Taken together, our data indicate that TLC may be a new potential target for the treatment of type 2 diabetes.

  14. Identification of a novel human islet amyloid polypeptide beta-sheet domain and factors influencing fibrillogenesis.

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    Jaikaran, E T; Higham, C E; Serpell, L C; Zurdo, J; Gross, M; Clark, A; Fraser, P E

    2001-05-04

    Human islet amyloid polypeptide (hIAPP) accumulates as pancreatic amyloid in type 2 diabetes and readily forms fibrils in vitro. Investigations into the mechanism of hIAPP fibril formation have focused largely on residues 20 to 29, which are considered to comprise a primary amyloidogenic domain. In rodents, proline substitutions within this region and the subsequent beta-sheet disruption, prevents fibril formation. An additional amyloidogenic fragment within the C-terminal sequence, residues 30 to 37, has been identified recently. We have extended these observations by examining a series of overlapping peptide fragments from the human and rodent sequences. Using protein spectroscopy (CD/FTIR), electron microscopy and X-ray diffraction, a previously unrecognised amyloidogenic domain was localised within residues 8 to 20. Synthetic peptides corresponding to this region exhibited a transition from random coil to beta-sheet conformation and assembled into fibrils having a typical amyloid-like morphology. The comparable rat 8-20 sequence, which contains a single His18Arg substitution, was also capable of assembling into amyloid-like fibrils. Examination of peptide fragments corresponding to residues 1 to 13 revealed that the immediate N-terminal region is likely to have only a modulating influence on fibril formation or conformational conversion. The contributions of charged residues as they relate to the amyloid-forming 8-20 sequence were also investigated using IAPP fragments and by assessing the effects of pH and counterions. The identification of these principal amyloidogenic sequences and the effects of associated factors provide details on the IAPP aggregation pathway and structure of the peptide in its fibrillar state. Copyright 2001 Academic Press.

  15. Downregulation of Type II Diabetes Mellitus and Maturity Onset Diabetes of Young Pathways in Human Pancreatic Islets from Hyperglycemic Donors

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    Jalal Taneera

    2014-01-01

    Full Text Available Although several molecular pathways have been linked to type 2 diabetes (T2D pathogenesis, it is uncertain which pathway has the most implication on the disease. Changes in the expression of an entire pathway might be more important for disease pathogenesis than changes in the expression of individual genes. To identify the molecular alterations in T2D, DNA microarrays of human pancreatic islets from donors with hyperglycemia n=20 and normoglycemia n=58 were subjected to Gene Set Enrichment Analysis (GSEA. About 178 KEGG pathways were investigated for gene expression changes between hyperglycemic donors compared to normoglycemic. Pathway enrichment analysis showed that type II diabetes mellitus (T2DM and maturity onset diabetes of the young (MODY pathways are downregulated in hyperglycemic donors, while proteasome and spliceosome pathways are upregulated. The mean centroid of gene expression of T2DM and MODY pathways was shown to be associated positively with insulin secretion and negatively with HbA1c level. To conclude, downregulation of T2DM and MODY pathways is involved in islet function and might be involved in T2D. Also, the study demonstrates that gene expression profiles from pancreatic islets can reveal some of the biological processes related to regulation of glucose hemostats and diabetes pathogenesis.

  16. High-fat diet-induced insulin resistance does not increase plasma anandamide levels or potentiate anandamide insulinotropic effect in isolated canine islets.

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    Orison O Woolcott

    Full Text Available Obesity has been associated with elevated plasma anandamide levels. In addition, anandamide has been shown to stimulate insulin secretion in vitro, suggesting that anandamide might be linked to hyperinsulinemia.To determine whether high-fat diet-induced insulin resistance increases anandamide levels and potentiates the insulinotropic effect of anandamide in isolated pancreatic islets.Dogs were fed a high-fat diet (n = 9 for 22 weeks. Abdominal fat depot was quantified by MRI. Insulin sensitivity was assessed by the euglycemic-hyperinsulinemic clamp. Fasting plasma endocannabinoid levels were analyzed by liquid chromatography-mass spectrometry. All metabolic assessments were performed before and after fat diet regimen. At the end of the study, pancreatic islets were isolated prior to euthanasia to test the in vitro effect of anandamide on islet hormones. mRNA expression of cannabinoid receptors was determined in intact islets. The findings in vitro were compared with those from animals fed a control diet (n = 7.Prolonged fat feeding increased abdominal fat content by 81.3±21.6% (mean±S.E.M, P<0.01. In vivo insulin sensitivity decreased by 31.3±12.1% (P<0.05, concomitant with a decrease in plasma 2-arachidonoyl glycerol (from 39.1±5.2 to 15.7±2.0 nmol/L but not anandamide, oleoyl ethanolamide, linoleoyl ethanolamide, or palmitoyl ethanolamide. In control-diet animals (body weight: 28.8±1.0 kg, islets incubated with anandamide had a higher basal and glucose-stimulated insulin secretion as compared with no treatment. Islets from fat-fed animals (34.5±1.3 kg; P<0.05 versus control did not exhibit further potentiation of anandamide-induced insulin secretion as compared with control-diet animals. Glucagon but not somatostatin secretion in vitro was also increased in response to anandamide, but there was no difference between groups (P = 0.705. No differences in gene expression of CB1R or CB2R between groups were found.In canines, high-fat diet

  17. Angiopoetin-2 Signals Do Not Mediate the Hypervascularization of Islets in Type 2 Diabetes

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    Shah, Payal; Lueschen, Navina; Ardestani, Amin; Oberholzer, Jose; Olerud, Johan; Carlsson, Per-Ola; Maedler, Kathrin

    2016-01-01

    Aims Changes in the islet vasculature have been implicated in the regulation of β-cell survival and function during the progression to type 2 diabetes (T2D). Failure of the β-cell to compensate for the increased insulin demand in obesity eventually leads to diabetes; as a result of the complex interplay of genetic and environmental factors (e.g. ongoing inflammation within the islets) and impaired vascular function. The Angiopoietin/Tie (Ang/Tie) angiogenic system maintains vasculature and is closely related to organ inflammation and angiogenesis. In this study we aimed to identify whether the vessel area within the islets changes in diabetes and whether such changes would be triggered by the Tie-antagonist Ang-2. Methods Immunohistochemical and qPCR analyses to follow islet vascularization and Ang/Tie levels were performed in human pancreatic autopsies and isolated human and mouse islets. The effect of Ang-2 was assessed in β-cell-specific Ang-2 overexpressing mice during high fat diet (HFD) feeding. Results Islet vessel area was increased in autopsy pancreases from patients with T2D. The vessel markers Tie-1, Tie-2 and CD31 were upregulated in mouse islets upon HFD feeding from 8 to 24 weeks. Ang-2 was transiently upregulated in mouse islets at 8 weeks of HFD and under glucolipotoxic conditions (22.2 mM glucose/ 0.5 mM palmitate) in vitro in human and mouse islets, in contrast to its downregulation by cytokines (IL-1β, IFN-ɣ and TNF-α). Ang-1 on the other hand was oppositely regulated, with a significant loss under glucolipotoxic condition, a trend to reduce in islets from patients with T2D and an upregulation by cytokines. Modulation of such changes in Ang-2 by its overexpression or the inhibition of its receptor Tie-2 impaired β-cell function at basal conditions but protected islets from cytokine induced apoptosis. In vivo, β-cell-specific Ang-2 overexpression in mice induced hypervascularization under normal diet but contrastingly led to

  18. Interleukin-1 beta-induced nitric oxide production from isolated rat islets is modulated by D-glucose and 3-isobutyl-1-methyl xanthine

    DEFF Research Database (Denmark)

    Andersen, H U; Mauricio, D; Karlsen, Allan Ertman

    1996-01-01

    Interleukin-1 beta has been proposed to cause selective beta-cell destruction via the induction of nitric oxide synthesis. The cytotoxic effect of interleukin-1 beta is modulated by the concentration of D-glucose in the medium. The aim of this study was to investigate if D......-glucose-mediated modulation of interleukin-1 beta effects on insulin release from isolated rat islets was related to modulation of nitric oxide production. Further, we wished to investigate the effects of agents increasing the intracellular concentration of cAMP on interleukin-1 beta-induced nitrite production. We...... demonstrated that D-glucose potentiated interleukin-1 beta-induced nitrite production in rat islets without affecting the mRNA level of the inducible nitric oxide synthase. This effect was dissociated from interleukin-1 beta action on insulin release, since a relative protection against interleukin-1 beta...

  19. Pancreatic beta cells and islets take up thiamin by a regulated carrier-mediated process: studies using mice and human pancreatic preparations

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    Mee, Lisa; Nabokina, Svetlana M.; Sekar, V. Thillai; Subramanian, Veedamali S.; Maedler, Kathrin; Said, Hamid M.

    2009-01-01

    Thiamin is essential for the normal function of the endocrine pancreas, but very little is known about uptake mechanism(s) and regulation by beta cells. We addressed these issues using mouse-derived pancreatic beta-TC-6 cells, and freshly isolated primary mouse and human pancreatic islets. Results showed that thiamin uptake by beta-TC-6 cells involves a pH (but not Na+)-dependent carrier-mediated process that is saturable at both the nanomolar (apparent Km = 37.17 ± 9.9 nM) and micromolar (apparent Km = 3.26 ± 0.86 μM) ranges, cis-inhibited by thiamin structural analogs, and trans-stimulated by unlabeled thiamin. Involvement of carrier-mediated process was also confirmed in primary mouse and human pancreatic islets. Both THTR-1 and THTR-2 were found to be expressed in these mouse and human pancreatic preparations. Maintaining beta-TC-6 cells in the presence of a high level of thiamin led to a significant (P < 0.01) decrease in thiamin uptake, which was associated with a significant downregulation in level of expression of THTR-1 and THTR-2 at the protein and mRNA levels and a decrease in transcriptional (promoter) activity. Modulators of intracellular Ca2+/calmodulin- and protein-tyrosine kinase-mediated pathways also altered thiamin uptake. Finally, confocal imaging of live beta-TC-6 cells showed that clinical mutants of THTR-1 have mixed expression phenotypes and all led to impairment in thiamin uptake. These studies demonstrate for the first time that thiamin uptake by the endocrine pancreas is carrier mediated and is adaptively regulated by the prevailing vitamin level via transcriptional mechanisms. Furthermore, clinical mutants of THTR-1 impair thiamin uptake via different mechanisms. PMID:19423748

  20. Effects of growth hormone, prolactin, and placental lactogen on insulin content and release, and deoxyribonucleic acid synthesis in cultured pancreatic islets

    DEFF Research Database (Denmark)

    Nielsen, Jens Høiriis

    1982-01-01

    The direct effects of human GH (hGH), ovine pituitary PRL (oPRL), and human chorionic somatomammotropin [placental lactogen (hPL)] on the endocrine pancreas were studied in isolated pancreatic islets maintained in tissue culture. Islets of Langerhans were isolated by collagenase treatment of panc...... and related hormones have a direct stimulatory effect on both the insulin production and DNA synthesis in isolated islets of Langerhans. Whether the effect is directly on the beta-cell or mediated via locally produced growth factors remains to be determined....

  1. Differential transcriptome analysis of diabetes-resistant and -sensitive mouse islets reveals significant overlap with human diabetes susceptibility genes.

    Science.gov (United States)

    Kluth, Oliver; Matzke, Daniela; Schulze, Gunnar; Schwenk, Robert W; Joost, Hans-Georg; Schürmann, Annette

    2014-12-01

    Type 2 diabetes in humans and in obese mice is polygenic. In recent genome-wide association studies, genetic markers explaining a small portion of the genetic contribution to the disease were discovered. However, functional evidence linking these genes with the pathogenesis of diabetes is scarce. We performed RNA sequencing-based transcriptomics of islets from two obese mouse strains, a diabetes-susceptible (NZO) and a diabetes-resistant (B6-ob/ob) mouse, after a short glucose challenge and compared these results with human data. Alignment of 2,328 differentially expressed genes to 106 human diabetes candidate genes revealed an overlap of 20 genes, including TCF7L2, IGFBP2, CDKN2A, CDKN2B, GRB10, and PRC1. The data provide a functional validation of human diabetes candidate genes, including those involved in regulating islet cell recovery and proliferation, and identify additional candidates that could be involved in human β-cell failure. © 2014 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  2. Cytotoxicity of human pI 7 interleukin-1 for pancreatic islets of Langerhans

    DEFF Research Database (Denmark)

    Bendtzen, K; Mandrup-Poulsen, T; Nerup, J

    1986-01-01

    . This cytotoxic activity was eliminated from crude cytokine preparations by adsorption with immobilized, purified antibody to interleukin-1 (IL-1). The islet-inhibitory activity and the IL-1 activity (determined by its comitogenic effect on thymocytes) were recovered by acid wash. Purified natural IL-1...

  3. The antilipolytic agent 3,5-dimethylpyrazole inhibits insulin release in response to both nutrient secretagogues and cyclic adenosine monophosphate agonists in isolated rat islets.

    Science.gov (United States)

    Masiello, P; Novelli, M; Bombara, M; Fierabracci, V; Vittorini, S; Prentki, M; Bergamini, E

    2002-01-01

    This study intended to test the hypothesis that intracellular lipolysis in the pancreatic beta cells is implicated in the regulation of insulin secretion stimulated by nutrient secretagogues or cyclic adenosine monophosphate (cAMP) agonists. Indeed, although lipid signaling molecules were repeatedly reported to influence beta-cell function, the contribution of intracellular triglycerides to the generation of these molecules has remained elusive. Thus, we have studied insulin secretion of isolated rat pancreatic islets in response to various secretagogues in the presence or absence of 3,5-dimethylpyrazole (DMP), a water-soluble and highly effective antilipolytic agent, as previously shown in vivo. In vitro exposure of islets to DMP resulted in an inhibition (by approximately 50%) of the insulin release stimulated not only by high glucose, but also by another nutrient secretagogue, 2-ketoisocaproate, as well as the cAMP agonists 3-isobutyl-1-methylxanthine and glucagon. The inhibitory effect of DMP, which was not due to alteration of islet glucose oxidation, could be reversed upon addition of sn-1,2-dioctanoylglycerol, a synthetic diglyceride, which activates protein kinase C. The results provide direct pharmacologic evidence supporting the concept that endogenous beta-cell lipolysis plays an important role in the generation of lipid signaling molecules involved in the control of insulin secretion in response to both fuel stimuli and cAMP agonists.

  4. PDZ-domain containing-2 (PDZD2) drives the maturity of human fetal pancreatic progenitor-derived islet-like cell clusters with functional responsiveness against membrane depolarization.

    Science.gov (United States)

    Leung, Kwan Keung; Suen, Po Man; Lau, Tse Kin; Ko, Wing Hung; Yao, Kwok Ming; Leung, Po Sing

    2009-09-01

    We recently reported the isolation and characterization of a population of pancreatic progenitor cells (PPCs) from early trimester human fetal pancreata. The PPCs, being the forerunners of adult pancreatic cell lineages, were amenable to growth and differentiation into insulin-secreting islet-like cell clusters (ICCs) upon stimulation by adequate morphogens. Of note, a novel morphogenic factor, PDZ-domain containing-2 (PDZD2) and its secreted form (sPDZD2) were ubiquitously expressed in the PPCs. Our goals for this study were to evaluate the potential role of sPDZD2 in stimulating PPC differentiation and to establish the optimal concentration for such stimulation. We found that 10(-9)M sPDZD2 promoted PPC differentiation, as evidenced by the upregulation of the pancreatic endocrine markers (PDX-1, NGN3, NEURO-D, ISL-1, NKX 2.2, NKX 6.1) and INSULIN mRNA. Inhibited endogenous production of sPDZD2 suppressed expression of these factors. Secreted PDZD2 treatment significantly elevated the C-peptide content of the ICCs and increased the basal rate of insulin secretion. However, they remained unresponsive to glucose stimulation, reflected by a minimal increase in GLUT-2 and GLUCOKINASE mRNA expression. Interestingly, sPDZD2 treatment induced increased expression of the L-type voltage-gated calcium channel (Ca(v)1.2) in the ICCs, triggering calcium ion influx under KCl stimulation and conferring an ability to secrete insulin in response to KCl. Pancreatic progenitor cells from 10- and 13-week fetal pancreata showed peak expression of endogenous sPDZD2, implying that sPDZD2 has a specific role in islet development during the first trimester. In conclusion, our data suggest that sPDZD2 promotes functional maturation of human fetal PPC-derived ICCs, thus enhancing its transplanting potentials.

  5. Reversed-phase high-performance liquid chromatographic analyses of insulin biosynthesis in isolated rat and mouse islets

    DEFF Research Database (Denmark)

    Linde, S; Hansen, Bruno A.; Welinder, B S

    1989-01-01

    Two RP-HPLC systems were developed for the separation of the products of the conversion of proinsulin into insulin in rat and mouse islets, including proinsulin I and II. Peaks were identified by microsequencing and radiosequencing. It was confirmed that mouse C-peptide I has a two amino acid...... deletion compared to rat C-peptide I. A marked species difference in the ratio between insulin I and II was observed, i.e., 2:1 in the rat and 1:2 in the mouse. Pulse-chase experiments in rat islets have demonstrated that the ratio between insulin I and II in newly synthesized insulin is higher than...

  6. Islet Transplantation

    Science.gov (United States)

    ... transplanted islet cells failed. But in recent years, scientists have begun to make rapid advances in transplant technology, and some of the most exciting new research comes to us from researchers at the University of ... Canada. These scientists have used a new procedure called the Edmonton ...

  7. Transplante isogênico de ilhotas de Langerhans no fígado de ratos: metodologia para separação e purificação das ilhotas de Langerhans Isogenic islet transplantation on the rat liver: Method for isolation and purification of the rat islets

    Directory of Open Access Journals (Sweden)

    Eleazar CHAIB

    2000-01-01

    Full Text Available A maior indicação do transplante de pâncreas ou de ilhotas de Langerhans é o diabetes mellitus do tipo I. O processo deve suprir as necessidades de insulina mantendo os níveis glicêmicos dentro da normalidade. Estudou-se o transplante isogênico de ilhotas de Langerhans no fígado de ratos WAG-RT1u. Com o método de separação e purificação das ilhotas de Langerhans obteve-se 2.834 ± 551,64 ilhotas com pureza de 83 ± 2,45%. O transplante de 2.834 ± 551,64 ilhotas de Langerhans no fígado destes animais, normalizou a glicemia que chegou a 35 mmol/L após indução do diabetes pela estreptozotocina, ficando em 9,62 ± 2,65 mmol/L nos primeiros 10 dias após o enxerto e 7,43 ± 0,27 mmol/L nos dias subseqüentes (P The major indication for pancreas or islet transplantation is diabetes mellitus type I. This process has to supply the insulin necessity keeping glucose under control. We have studied isogenic islet transplantation on the rat (WAG-RT1u liver. The method of isolation and purification of the islets obtained 2.834 ± 551,64 islets with purity of 83 ± 2,45%. Diabetes was induced by streptozotocin and seric glucose prior transplantation was 35 mmol/L. The islet transplantation of 2.834 ± 551,64 islets in the rat liver has normalized glucose test from 9,62 ± 2,65 mmol/L 10 days after transplantation to 7,43 ± 0,27 mmol/L later in the follow-up (P <0,05. The median survival time of the islets was 73 days. In conclusion both the method of isolation and purification of the islets and islet transplantation was effective in the control of the diabetes induced by streptozotocin with median survival time of both islet and rat more than 73 days when rats were sacrificied.

  8. The effects of exendin-4 treatment on graft failure: an animal study using a novel re-vascularized minimal human islet transplant model.

    Directory of Open Access Journals (Sweden)

    Afaf Sahraoui

    Full Text Available Islet transplantation has become a viable clinical treatment, but is still compromised by long-term graft failure. Exendin-4, a glucagon-like peptide 1 receptor agonist, has in clinical studies been shown to improve insulin secretion in islet transplanted patients. However, little is known about the effect of exendin-4 on other metabolic parameters. We therefore aimed to determine what influence exendin-4 would have on revascularized minimal human islet grafts in a state of graft failure in terms of glucose metabolism, body weight, lipid levels and graft survival. Introducing the bilateral, subcapsular islet transplantation model, we first transplanted diabetic mice with a murine graft under the left kidney capsule sufficient to restore normoglycemia. After a convalescent period, we performed a second transplantation under the right kidney capsule with a minimal human islet graft and allowed for a second recovery. We then performed a left-sided nephrectomy, and immediately started treatment with exendin-4 with a low (20μg/kg/day or high (200μg/kg/day dose, or saline subcutaneously twice daily for 15 days. Blood was sampled, blood glucose and body weight monitored. The transplanted human islet grafts were collected at study end point and analyzed. We found that exendin-4 exerts its effect on failing human islet grafts in a bell-shaped dose-response curve. Both doses of exendin-4 equally and significantly reduced blood glucose. Glucagon-like peptide 1 (GLP-1, C-peptide and pro-insulin were conversely increased. In the course of the treatment, body weight and cholesterol levels were not affected. However, immunohistochemistry revealed an increase in beta cell nuclei count and reduced TUNEL staining only in the group treated with a low dose of exendin-4 compared to the high dose and control. Collectively, these results suggest that exendin-4 has a potential rescue effect on failing, revascularized human islets in terms of lowering blood glucose

  9. The effects of exendin-4 treatment on graft failure: an animal study using a novel re-vascularized minimal human islet transplant model.

    Science.gov (United States)

    Sahraoui, Afaf; Winzell, Maria Sörhede; Gorman, Tracy; Smith, Dave M; Skrtic, Stanko; Hoeyem, Merete; Abadpour, Shadab; Johansson, Lars; Korsgren, Olle; Foss, Aksel; Scholz, Hanne

    2015-01-01

    Islet transplantation has become a viable clinical treatment, but is still compromised by long-term graft failure. Exendin-4, a glucagon-like peptide 1 receptor agonist, has in clinical studies been shown to improve insulin secretion in islet transplanted patients. However, little is known about the effect of exendin-4 on other metabolic parameters. We therefore aimed to determine what influence exendin-4 would have on revascularized minimal human islet grafts in a state of graft failure in terms of glucose metabolism, body weight, lipid levels and graft survival. Introducing the bilateral, subcapsular islet transplantation model, we first transplanted diabetic mice with a murine graft under the left kidney capsule sufficient to restore normoglycemia. After a convalescent period, we performed a second transplantation under the right kidney capsule with a minimal human islet graft and allowed for a second recovery. We then performed a left-sided nephrectomy, and immediately started treatment with exendin-4 with a low (20μg/kg/day) or high (200μg/kg/day) dose, or saline subcutaneously twice daily for 15 days. Blood was sampled, blood glucose and body weight monitored. The transplanted human islet grafts were collected at study end point and analyzed. We found that exendin-4 exerts its effect on failing human islet grafts in a bell-shaped dose-response curve. Both doses of exendin-4 equally and significantly reduced blood glucose. Glucagon-like peptide 1 (GLP-1), C-peptide and pro-insulin were conversely increased. In the course of the treatment, body weight and cholesterol levels were not affected. However, immunohistochemistry revealed an increase in beta cell nuclei count and reduced TUNEL staining only in the group treated with a low dose of exendin-4 compared to the high dose and control. Collectively, these results suggest that exendin-4 has a potential rescue effect on failing, revascularized human islets in terms of lowering blood glucose, maintaining beta

  10. Lixisenatide accelerates restoration of normoglycemia and improves human beta-cell function and survival in diabetic immunodeficient NOD–scid IL-2rgnull RIP-DTR mice engrafted with human islets

    Directory of Open Access Journals (Sweden)

    Yang C

    2015-08-01

    Full Text Available Chaoxing Yang,1 Matthias Loehn,2 Agata Jurczyk,1 Natalia Przewozniak,1 Linda Leehy,1 Pedro L Herrera,3 Leonard D Shultz,4 Dale L Greiner,1 David M Harlan,5 Rita Bortell1 1Program in Molecular Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, USA; 2Sanofi-Aventis, Diabetes Division, Frankfurt, Germany; 3University of Geneva, Geneva, Switzerland; 4The Jackson Laboratory, Bar Harbor, ME, USA; 5Department of Medicine, Diabetes Center of Excellence, University of Massachusetts Medical School, Worcester, MA, USA Objective: Glucagon-like peptide-1 induces glucose-dependent insulin secretion and, in rodents, increases proliferation and survival of pancreatic beta cells. To investigate the effects on human beta cells, we used immunodeficient mice transplanted with human islets. The goal was to determine whether lixisenatide, a glucagon-like peptide-1 receptor agonist, improves human islet function and survival in vivo. Methods: Five independent transplant studies were conducted with human islets from five individual donors. Diabetic human islet-engrafted immunodeficient mice were treated with lixisenatide (50, 150, and 500 µg/kg or vehicle. Islet function was determined by blood glucose, plasma human insulin/C-peptide, and glucose tolerance tests. Grafts were analyzed for total beta- and alpha-cell number, percent proliferation, and levels of apoptosis. Results: Diabetic mice transplanted with marginal human islet mass and treated with lixisenatide were restored to euglycemia more rapidly than vehicle-treated mice. Glucose tolerance tests, human plasma insulin, and glucose-stimulation indices of lixisenatide-treated mice were significantly improved compared to vehicle-treated mice. The percentages of proliferating or apoptotic beta cells at graft recovery were not different between lixisenatide-treated and vehicle-treated mice. Nevertheless, in one experiment we found a significant twofold to threefold

  11. Human Islet Amyloid Polypeptide N-Terminus Fragment Self-Assembly: Effect of Conserved Disulfide Bond on Aggregation Propensity

    Science.gov (United States)

    Ilitchev, Alexandre I.; Giammona, Maxwell J.; Do, Thanh D.; Wong, Amy G.; Buratto, Steven K.; Shea, Joan-Emma; Raleigh, Daniel P.; Bowers, Michael T.

    2016-06-01

    Amyloid formation by human islet amyloid polypeptide (hIAPP) has long been implicated in the pathogeny of type 2 diabetes mellitus (T2DM) and failure of islet transplants, but the mechanism of IAPP self-assembly is still unclear. Numerous fragments of hIAPP are capable of self-association into oligomeric aggregates, both amyloid and non-amyloid in structure. The N-terminal region of IAPP contains a conserved disulfide bond between cysteines at position 2 and 7, which is important to hIAPP's in vivo function and may play a role in in vitro aggregation. The importance of the disulfide bond in this region was probed using a combination of ion mobility-based mass spectrometry experiments, molecular dynamics simulations, and high-resolution atomic force microscopy imaging on the wildtype 1-8 hIAPP fragment, a reduced fragment with no disulfide bond, and a fragment with both cysteines at positions 2 and 7 mutated to serine. The results indicate the wildtype fragment aggregates by a different pathway than either comparison peptide and that the intact disulfide bond may be protective against aggregation due to a reduction of inter-peptide hydrogen bonding.

  12. The Pancreatic Islet Regulome Browser

    Science.gov (United States)

    Mularoni, Loris; Ramos-Rodríguez, Mireia; Pasquali, Lorenzo

    2017-01-01

    The pancreatic islet is a highly specialized tissue embedded in the exocrine pancreas whose primary function is that of controlling glucose homeostasis. Thus, understanding the transcriptional control of islet-cell may help to puzzle out the pathogenesis of glucose metabolism disorders. Integrative computational analyses of transcriptomic and epigenomic data allows predicting genomic coordinates of putative regulatory elements across the genome and, decipher tissue-specific functions of the non-coding genome. We herein present the Islet Regulome Browser, a tool that allows fast access and exploration of pancreatic islet epigenomic and transcriptomic data produced by different labs worldwide. The Islet Regulome Browser is now accessible on the internet or may be installed locally. It allows uploading custom tracks as well as providing interactive access to a wealth of information including Genome-Wide Association Studies (GWAS) variants, different classes of regulatory elements, together with enhancer clusters, stretch-enhancers and transcription factor binding sites in pancreatic progenitors and adult human pancreatic islets. Integration and visualization of such data may allow a deeper understanding of the regulatory networks driving tissue-specific transcription and guide the identification of regulatory variants. We believe that such tool will facilitate the access to pancreatic islet public genomic datasets providing a major boost to functional genomics studies in glucose metabolism related traits including diabetes. PMID:28261261

  13. Improving Islet Engraftment by Gene Therapy

    Directory of Open Access Journals (Sweden)

    Xiaojie Wang

    2011-01-01

    Full Text Available Islet cell transplantation is currently the only feasible long-term treatment option for patients with type 1 diabetes. However, the majority of transplanted islets experience damage and apoptosis during the isolation process, a blood-mediated inflammatory microenvironment in the portal vein upon islet infusion, hypoxia induced by the low oxygenated milieu, and poor-revascularization-mediated lack of nutrients, and impaired hormone modulation in the local transplanted site. Strategies using genetic modification methods through overexpression or silencing of those proteins involved in promoting new formation of blood vessels or inhibition of apoptosis may overcome these hurdles and improve islet engraftment outcomes.

  14. Munc18b Increases Insulin Granule Fusion, Restoring Deficient Insulin Secretion in Type-2 Diabetes Human and Goto-Kakizaki Rat Islets with Improvement in Glucose Homeostasis

    Directory of Open Access Journals (Sweden)

    Tairan Qin

    2017-02-01

    Infusion of Ad-Munc18b into GK rat pancreas led to sustained improvement in glucose homeostasis. However, Munc18b overexpression in normal islets increased only newcomer SG fusion. Therefore, Munc18b could potentially be deployed in human T2D to rescue the deficient GSIS.

  15. Adult Human Biliary Tree Stem Cells Differentiate to β-Pancreatic Islet Cells by Treatment with a Recombinant Human Pdx1 Peptide.

    Directory of Open Access Journals (Sweden)

    Vincenzo Cardinale

    Full Text Available Generation of β-pancreatic cells represents a major goal in research. The aim of this study was to explore a protein-based strategy to induce differentiation of human biliary tree stem cells (hBTSCs towards β-pancreatic cells. A plasmid containing the sequence of the human pancreatic and duodenal homeobox 1 (PDX1 has been expressed in E. coli. Epithelial-Cell-Adhesion-Molecule positive hBTSCs or mature human hepatocyte cell line, HepG2, were grown in medium to which Pdx1 peptide was added. Differentiation toward pancreatic islet cells were evaluated by the expression of the β-cell transcription factors, Pdx1 and musculoapo-neurotic fibrosarcoma oncogene homolog A, and of the pancreatic hormones, insulin, glucagon, and somatostatin, investigated by real time polymerase chain reaction, western blot, light microscopy and immunofluorescence. C-peptide secretion in response to high glucose was also measured. Results indicated how purified Pdx1 protein corresponding to the primary structure of the human Pdx1 by mass spectroscopy was efficiently produced in bacteria, and transduced into hBTSCs. Pdx1 exposure triggered the expression of both intermediate and mature stage β-cell differentiation markers only in hBTSCs but not in HepG2 cell line. Furthermore, hBTSCs exposed to Pdx1 showed up-regulation of insulin, glucagon and somatostatin genes and formation of 3-dimensional islet-like structures intensely positive for insulin and glucagon. Finally, Pdx1-induced islet-like structures exhibited glucose-regulated C-peptide secretion. In conclusion, the human Pdx1 is highly effective in triggering hBTSC differentiation toward functional β-pancreatic cells.

  16. Acidic pH retards the fibrillization of human islet amyloid polypeptide due to electrostatic repulsion of histidines

    Science.gov (United States)

    Li, Yang; Xu, Weixin; Mu, Yuguang; Zhang, John Z. H.

    2013-08-01

    The human Islet Amyloid Polypeptide (hIAPP) is the major constituent of amyloid deposits in pancreatic islets of type-II diabetes. IAPP is secreted together with insulin from the acidic secretory granules at a low pH of approximately 5.5 to the extracellular environment at a neutral pH. The increased accumulation of extracellular hIAPP in diabetes indicates that changes in pH may promote amyloid formation. To gain insights and underlying mechanisms of the pH effect on hIAPP fibrillogenesis, all-atom molecular dynamics simulations in explicit solvent model were performed to study the structural properties of five hIAPP protofibrillar oligomers, under acidic and neutral pH, respectively. In consistent with experimental findings, simulation results show that acidic pH is not conducive to the structural stability of these oligomers. This provides a direct evidence for a recent experiment [L. Khemtemourian, E. Domenech, J. P. F. Doux, M. C. Koorengevel, and J. A. Killian, J. Am. Chem. Soc. 133, 15598 (2011)], 10.1021/ja205007j, which suggests that acidic pH inhibits the fibril formation of hIAPP. In addition, a complementary coarse-grained simulation shows the repulsive electrostatic interactions among charged His18 residues slow down the dimerization process of hIAPP by twofold. Besides, our all-atom simulations reveal acidic pH mainly affects the local structure around residue His18 by destroying the surrounding hydrogen-bonding network, due to the repulsive interactions between protonated interchain His18 residues at acidic pH. It is also disclosed that the local interactions nearby His18 operating between adjacent β-strands trigger the structural transition, which gives hints to the experimental findings that the rate of hIAPP fibril formation and the morphologies of the fibrillar structures are strongly pH-dependent.

  17. The Mitochondrial Peptidase Pitrilysin Degrades Islet Amyloid Polypeptide in Beta-Cells.

    Directory of Open Access Journals (Sweden)

    Hanjun Guan

    Full Text Available Amyloid formation and mitochondrial dysfunction are characteristics of type 2 diabetes. The major peptide constituent of the amyloid deposits in type 2 diabetes is islet amyloid polypeptide (IAPP. In this study, we found that pitrilysin, a zinc metallopeptidase of the inverzincin family, degrades monomeric, but not oligomeric, islet amyloid polypeptide in vitro. In insulinoma cells when pitrilysin expression was decreased to 5% of normal levels, there was a 60% increase in islet amyloid polypeptide-induced apoptosis. In contrast, overexpression of pitrilysin protects insulinoma cells from human islet amyloid polypeptide-induced apoptosis. Since pitrilysin is a mitochondrial protein, we used immunofluorescence staining of pancreases from human IAPP transgenic mice and Western blot analysis of IAPP in isolated mitochondria from insulinoma cells to provide evidence for a putative intramitochondrial pool of IAPP. These results suggest that pitrilysin regulates islet amyloid polypeptide in beta cells and suggest the presence of an intramitochondrial pool of islet amyloid polypeptide involved in beta-cell apoptosis.

  18. Apelin is a novel islet peptide

    DEFF Research Database (Denmark)

    Ringström, Camilla; Nitert, Marloes Dekker; Bennet, Hedvig;

    2010-01-01

    Apelin, a recently discovered peptide with wide tissue distribution, regulates feeding behavior, improves glucose utilization, and inhibits insulin secretion. We examined whether apelin is expressed in human islets, as well as in normal and type 2 diabetic (T2D) animal islets. Further, we studied...

  19. Secretion of neurotensin from a human pancreatic islet cell carcinoma cell line (QGP-1N).

    Science.gov (United States)

    Tateishi, K; Funakoshi, A; Kitayama, N; Matsuoka, Y

    1993-12-10

    Effects of various secretagogues on secretion of neurotensin from a pancreatic islet cell carcinoma cell line (QGP-1N) were examined. Carbachol stimulated secretion of neurotensin concentration-dependently in the range of 10(-6) - 10(-4) M. The neurotensin secretion stimulated with 10(-5) M carbachol was completely inhibited by atropine at 10(-5) M. Phorbol ester and calcium ionophore (A23187) stimulated secretion of neurotensin. The removal of extracellular Ca2+ suppressed the secretion through the stimulation with 10(-5) M carbachol. Fluoride, an activator of guanine nucleotide-binding (G) protein, stimulated secretion of neurotensin. Neurotensin released into culture medium through stimulation with carbachol coeluted with neurotensin 1-13 on a gel-chromatography. Our results suggest that secretion of neurotensin from QGP-1N cells is mainly regulated by acetylcholine through muscarinic receptors coupled to G protein and that an increase in intracellular Ca2+ and protein kinase C play an important role in stimulus-secretion coupling.

  20. Isolation, identification and differentiation of human embryonic cartilage stem cells.

    Science.gov (United States)

    Fu, Changhao; Yan, Zi; Xu, Hao; Zhang, Chen; Zhang, Qi; Wei, Anhui; Yang, Xi; Wang, Yi

    2015-07-01

    We isolated human embryonic cartilage stem cells (hECSCs), a novel stem cell population, from the articular cartilage of eight-week-old human embryos. These stem cells demonstrated a marker expression pattern and differentiation potential intermediate to those of human embryonic stem cells (hESCs) and human adult stem cells (hASCs). hECSCs expressed markers associated with both hESCs (OCT4, NANOG, SOX2, SSEA-3 and SSEA-4) and human adult stem cells (hASCs) (CD29, CD44, CD90, CD73 and CD10). These cells also differentiated into adipocytes, osteoblasts, chondrocytes, neurons and islet-like cells under specific inducing conditions. We identified N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) as an inducer of chondrogenic differentiation in hECSCs. Similar results using N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) were obtained for two other types of human embryonic tissue-derived stem cells, human embryonic hepatic stem cells (hEHSCs) and human embryonic amniotic fluid stem cells (hEASCs), both of which exhibited a marker expression pattern similar to that of hECSCs. The isolation of hECSCs and the discovery that N(6), 2'-O-dibutyryl cyclic adenosine 3':5'-monophosphate (Bt2cAMP) induces chondrogenic differentiation in different stem cell populations might aid the development of strategies in tissue engineering and cartilage repair.

  1. Islet amyloid polypeptide, islet amyloid, and diabetes mellitus.

    Science.gov (United States)

    Westermark, Per; Andersson, Arne; Westermark, Gunilla T

    2011-07-01

    Islet amyloid polypeptide (IAPP, or amylin) is one of the major secretory products of β-cells of the pancreatic islets of Langerhans. It is a regulatory peptide with putative function both locally in the islets, where it inhibits insulin and glucagon secretion, and at distant targets. It has binding sites in the brain, possibly contributing also to satiety regulation and inhibits gastric emptying. Effects on several other organs have also been described. IAPP was discovered through its ability to aggregate into pancreatic islet amyloid deposits, which are seen particularly in association with type 2 diabetes in humans and with diabetes in a few other mammalian species, especially monkeys and cats. Aggregated IAPP has cytotoxic properties and is believed to be of critical importance for the loss of β-cells in type 2 diabetes and also in pancreatic islets transplanted into individuals with type 1 diabetes. This review deals both with physiological aspects of IAPP and with the pathophysiological role of aggregated forms of IAPP, including mechanisms whereby human IAPP forms toxic aggregates and amyloid fibrils.

  2. Impact of exposure to low concentrations of nitric oxide on protein profile in murine and human pancreatic islet cells.

    Science.gov (United States)

    Tapia-Limonchi, Rafael; Díaz, Irene; Cahuana, Gladys M; Bautista, Mario; Martín, Franz; Soria, Bernat; Tejedo, Juan R; Bedoya, Francisco J

    2014-01-01

    Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human and rodent pancreatic β cells, but the protein profile of this action remains to be determined. We have applied a 2 dimensional LC-MS-MALDI-TOF/TOF-based analysis to study the impact of protective NO in rat insulin-producing RINm5F cell line and in mouse and human pancreatic islets (HPI) exposed to serum deprivation condition. 24 proteins in RINm5F and 22 in HPI were identified to undergo changes in at least one experimental condition. These include stress response mitochondrial proteins (UQCRC2, VDAC1, ATP5C1, ATP5A1) in RINm5F cells and stress response endoplasmic reticulum proteins (HSPA5, PDIA6, VCP, GANAB) in HPI. In addition, metabolic and structural proteins, oxidoreductases and chaperones related with protein metabolism are also regulated by NO treatment. Network analysis of differentially expressed proteins shows their interaction in glucocorticoid receptor and NRF2-mediated oxidative stress response pathways and eNOS signaling. The results indicate that exposure to exogenous NO counteracts the impact of serum deprivation on pancreatic β cell proteome. Species differences in the proteins involved are apparent.

  3. Impact of exposure to low concentrations of nitric oxide on protein profile in murine and human pancreatic islet cells

    Science.gov (United States)

    Tapia-Limonchi, Rafael; Díaz, Irene; Cahuana, Gladys M; Bautista, Mario; Martín, Franz; Soria, Bernat; Tejedo, Juan R; Bedoya, Francisco J

    2014-01-01

    Homeostatic levels of nitric oxide (NO) protect efficiently against apoptotic death in both human and rodent pancreatic β cells, but the protein profile of this action remains to be determined. We have applied a 2 dimensional LC-MS-MALDI-TOF/TOF-based analysis to study the impact of protective NO in rat insulin-producing RINm5F cell line and in mouse and human pancreatic islets (HPI) exposed to serum deprivation condition. 24 proteins in RINm5F and 22 in HPI were identified to undergo changes in at least one experimental condition. These include stress response mitochondrial proteins (UQCRC2, VDAC1, ATP5C1, ATP5A1) in RINm5F cells and stress response endoplasmic reticulum proteins (HSPA5, PDIA6, VCP, GANAB) in HPI. In addition, metabolic and structural proteins, oxidoreductases and chaperones related with protein metabolism are also regulated by NO treatment. Network analysis of differentially expressed proteins shows their interaction in glucocorticoid receptor and NRF2-mediated oxidative stress response pathways and eNOS signaling. The results indicate that exposure to exogenous NO counteracts the impact of serum deprivation on pancreatic β cell proteome. Species differences in the proteins involved are apparent. PMID:25658244

  4. Disturbed α-Cell Function in Mice with β-Cell Specific Overexpression of Human Islet Amyloid Polypeptide

    Directory of Open Access Journals (Sweden)

    Bo Ahrén

    2008-01-01

    Full Text Available Exogenous administration of islet amyloid polypeptide (IAPP has been shown to inhibit both insulin and glucagon secretion. This study examined α-cell function in mice with β-cell specific overexpression of human IAPP (hIAPP after an oral protein gavage (75 mg whey protein/mouse. Baseline glucagon levels were higher in transgenic mice (41±4.0 pg/mL, n=6 than in wildtype animals (19±5.1 pg/mL, n=5, P=.015. In contrast, the glucagon response to protein was impaired in transgenic animals (21±2.7 pg/mL in transgenic mice versus 38±5.7 pg/mL in wildtype mice at 15 minutes; P=.027. Baseline insulin levels did not differ between the groups, while the insulin response, as the glucagon response, was impaired after protein challenge (P=.018. Glucose levels were not different between the groups and did not change significantly after protein gavage. Acetaminophen was given through gavage to the animals (2 mg/mouse to estimate gastric emptying. The plasma acetaminophen profile was similar in the two groups of mice. We conclude that disturbances in glucagon secretion exist in mice with β-cell specific overexpression of human IAPP, which are not secondary to changes in gastric emptying. The reduced glucagon response to protein challenge may reflect a direct inhibitory influence of hIAPP on glucagon secretion.

  5. Reduced early and late phase insulin response to glucose in isolated spiny mouse (Acomys cahirinus) islets: a defective link between glycolysis and adenylate cyclase.

    Science.gov (United States)

    Nesher, R; Abramovitch, E; Cerasi, E

    1989-09-01

    The spiny mouse (Acomys cahirinus) exhibits low insulin responsiveness to glucose with a nearly absent early phase release. The alternative fuel-secretagogue glyceraldehyde (10 mmol/l) produced a maximal early insulin response in rat islets but failed to affect early response in Acomys; however, it potentiated the late insulin response in both species alike. Glucagon (1.5 mumol/l) potentiated the early insulin response to intermediate (8.3 mmol/l) glucose in rat and Acomys islets by two- and four-fold, respectively. Glucose doubled cyclic AMP levels in rat islets but no significant response was noted in Acomys islets. Isobutylmethylxanthine (0.1 mmol/l) and forskolin (25 mumol/l) caused a significant rise in islet cyclic AMP levels in both types of islets; however, neither agent restored the glucose stimulation of cyclic AMP in spiny mouse islets. Forskolin and isobutylmethylxanthine potentiated early and late phase insulin release in both species; however, neither augmented the early response in the Acomys to the degree observed in rat islets. Thus: (1) A deficient link exists in Acomys between glycolysis and subsequent signals. (2) These islets contain a glucose-insensitive adenylate cyclase. (3) The early insulin response may be potentiated by direct activation of adenylate cyclase. (4) The glucose effects on early and late phase insulin release are probably mediated by distinct pathways. (5) In the spiny mouse the signals mediating the early response are deranged to a greater extent than those activating the late phase insulin release.

  6. A somatostatin-secreting cell line established from a human pancreatic islet cell carcinoma (somatostatinoma): release experiment and immunohistochemical study.

    Science.gov (United States)

    Iguchi, H; Hayashi, I; Kono, A

    1990-06-15

    Production and secretion of somatostatin (SRIF) were studied using a carcinoembryonic antigen (CEA)-producing cell line (QGP-1) established from a human pancreatic islet cell carcinoma. High concentrations of SRIF (274 +/- 51 ng/mg of protein, mean +/- SD, n = 5) and CEA (3083 +/- 347 ng/mg of protein, mean +/- SD, n = 5) were present in QGP-1 cells, and the basal secretion rates of SRIF and CEA by the cells (n = 5) were 46.4 +/- 4.8 and 1690 +/- 78 pg/10(5) cells/h, respectively. Immunohistochemical studies revealed the presence of SRIF in xenografts of QGP-1 cells and colocalization of SRIF and CEA. Secretion of SRIF by QGP-1 cells was stimulated in the presence of high K+ (50 mmol) and theophylline (10 mmol), but arginine (10 mmol) and glucose (300 mg/dl) had no effect on the SRIF secretion. The QGP-1 cell line may be useful for studying the regulation mechanism of SRIF secretion.

  7. Mechanism of Inhibition of Human Islet Amyloid Polypeptide-Induced Membrane Damage by a Small Organic Fluorogen

    Science.gov (United States)

    Li, Xiaoxu; Wan, Mingwei; Gao, Lianghui; Fang, Weihai

    2016-02-01

    Human islet amyloid polypeptide (hIAPP) is believed to be responsible for the death of insulin-producing β-cells. However, the mechanism of membrane damage at the molecular level has not been fully elucidated. In this article, we employ coarse- grained dissipative particle dynamics simulations to study the interactions between a lipid bilayer membrane composed of 70% zwitterionic lipids and 30% anionic lipids and hIAPPs with α-helical structures. We demonstrated that the key factor controlling pore formation is the combination of peptide charge-induced electroporation and peptide hydrophobicity-induced lipid disordering and membrane thinning. According to these mechanisms, we suggest that a water-miscible tetraphenylethene BSPOTPE is a potent inhibitor to rescue hIAPP-induced cytotoxicity. Our simulations predict that BSPOTPE molecules can bind directly to the helical regions of hIAPP and form oligomers with separated hydrophobic cores and hydrophilic shells. The micelle-like hIAPP-BSPOTPE clusters tend to be retained in the water/membrane interface and aggregate therein rather than penetrate into the membrane. Electrostatic attraction between BSPOTPE and hIAPP also reduces the extent of hIAPP binding to the anionic lipid bilayer. These two modes work together and efficiently prevent membrane poration.

  8. Identification of novel type 1 diabetes candidate genes by integrating genome-wide association data, protein-protein interactions, and human pancreatic islet gene expression

    DEFF Research Database (Denmark)

    Bergholdt, Regine; Brorsson, Caroline; Palleja, Albert;

    2012-01-01

    Genome-wide association studies (GWAS) have heralded a new era in susceptibility locus discovery in complex diseases. For type 1 diabetes, >40 susceptibility loci have been discovered. However, GWAS do not inevitably lead to identification of the gene or genes in a given locus associated...... with disease, and they do not typically inform the broader context in which the disease genes operate. Here, we integrated type 1 diabetes GWAS data with protein-protein interactions to construct biological networks of relevance for disease. A total of 17 networks were identified. To prioritize...... and substantiate these networks, we performed expressional profiling in human pancreatic islets exposed to proinflammatory cytokines. Three networks were significantly enriched for cytokine-regulated genes and, thus, likely to play an important role for type 1 diabetes in pancreatic islets. Eight of the regulated...

  9. Discovery of molecular pathways mediating 1,25-dihydroxyvitamin D3 protection against cytokine-induced inflammation and damage of human and male mouse islets of Langerhans

    DEFF Research Database (Denmark)

    Wolden-Kirk, Heidi; Rondas, D; Bugliani, M

    2014-01-01

    . The aim of this study was to clarify the molecular mechanisms by which 1,25(OH)2D3 contributes to β-cell protection against cytokine-induced β-cell dysfunction and death. Human and mouse islets were exposed to IL-1β and interferon-γ in the presence or absence of 1,25(OH)2D3. Effects on insulin secretion....../phenotype. In conclusion, these findings demonstrate a direct protective effect of 1,25(OH)2D3 against inflammation-induced β-cell dysfunction and death in human and murine islets, with, in particular, alterations in chemokine production by the islets. These effects may contribute to the beneficial effects of 1,25(OH)2D3......Protection against insulitis and diabetes by active vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), in nonobese diabetic mice has until now mainly been attributed to its immunomodulatory effects, but also protective effects of this hormone on inflammation-induced β-cell death have been reported...

  10. Rebaudioside A potently stimulates insulin secretion from isolated mouse islets: studies on the dose-, glucose-, and calcium-dependency.

    Science.gov (United States)

    Abudula, Reziwanggu; Jeppesen, Per Bendix; Rolfsen, Stig Eric D; Xiao, Jianzhong; Hermansen, Kjeld

    2004-10-01

    Extracts of leaves of the plant Stevia rebaudiana Bertoni (SrB), have been used for many years in traditional treatment of diabetes in South America. Stevia leaves contain diterpene glycosides, stevioside and rebaudioside A being the most abundant. Recently, it was demonstrated that stevioside stimulates the insulin secretion both in vitro and in vivo. Subsequently, we wanted to elucidate the influence of rebaudioside A on the insulin release from mouse islets using static incubations, as well as perifusion experiments. Rebaudioside A (10(-16) to 10(-6) mol/L) dose-dependently stimulated the insulin secretion in the presence of 16.7 mmol/L glucose (P rebaudioside A, and maximal insulin response was obtained at 10(-10) mol/L (P Rebaudioside A stimulates insulin secretion in a glucose-dependent manner (3.3 to 16.7 mmol/L) and only potentiated insulin secretion at glucose > 6.6 mmol/L. The effect of rebaudioside A is critically dependent on the presence of extracellular Ca2+, ie, rebaudioside A-induced insulin stimulation at high glucose disappears in the absence of extracellular Ca2+. In conclusion, rebaudioside A possesses insulinotropic effects and may serve a potential role as treatment in type 2 diabetes mellitus.

  11. Microwell scaffolds for the extrahepatic transplantation of islets of Langerhans.

    Directory of Open Access Journals (Sweden)

    Mijke Buitinga

    Full Text Available Allogeneic islet transplantation into the liver has the potential to restore normoglycemia in patients with type 1 diabetes. However, the suboptimal microenvironment for islets in the liver is likely to be involved in the progressive islet dysfunction that is often observed post-transplantation. This study validates a novel microwell scaffold platform to be used for the extrahepatic transplantation of islet of Langerhans. Scaffolds were fabricated from either a thin polymer film or an electrospun mesh of poly(ethylene oxide terephthalate-poly(butylene terephthalate (PEOT/PBT block copolymer (composition: 4000PEOT30PBT70 and were imprinted with microwells, ∼400 µm in diameter and ∼350 µm in depth. The water contact angle and water uptake were 39±2° and 52.1±4.0 wt%, respectively. The glucose flux through electrospun scaffolds was three times higher than for thin film scaffolds, indicating enhanced nutrient diffusion. Human islets cultured in microwell scaffolds for seven days showed insulin release and insulin content comparable to those of free-floating control islets. Islet morphology and insulin and glucagon expression were maintained during culture in the microwell scaffolds. Our results indicate that the microwell scaffold platform prevents islet aggregation by confinement of individual islets in separate microwells, preserves the islet's native rounded morphology, and provides a protective environment without impairing islet functionality, making it a promising platform for use in extrahepatic islet transplantation.

  12. Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

    Science.gov (United States)

    Lopez, Ana D.; Kayali, Ayse G.; Hayek, Alberto; King, Charles C.

    2014-01-01

    For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells. PMID:24895054

  13. Isolation, culture, and imaging of human fetal pancreatic cell clusters.

    Science.gov (United States)

    Lopez, Ana D; Kayali, Ayse G; Hayek, Alberto; King, Charles C

    2014-05-18

    For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation(1-9). However in vitro, genesis of insulin producing cells from human fetal ICCs is low(10); results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust(11-17). A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro(11-22), far fewer exist for ICCs(10,23,24). Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue(6). Although the examples presented here focus upon the pancreatic endoderm proliferation and β cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

  14. The heterogeneity of islet autoantibodies and the progression of islet failure in type 1 diabetic patients.

    Science.gov (United States)

    Liu, Jin; Bian, Lingling; Ji, Li; Chen, Yang; Chen, Heng; Gu, Yong; Ma, Bingqin; Gu, Wei; Xu, Xinyu; Shi, Yun; Wang, Jian; Zhu, Dalong; Sun, Zilin; Ma, Jianhua; Jin, Hui; Shi, Xing; Miao, Heng; Xin, Bing; Zhu, Yan; Zhang, Zhenwen; Bu, Ruifang; Xu, Lan; Shi, Guangde; Tang, Wei; Li, Wei; Zhou, Dongmei; Liang, Jun; Cheng, Xingbo; Shi, Bimin; Dong, Jixiang; Hu, Ji; Fang, Chen; Zhong, Shao; Yu, Weinan; Lu, Weiping; Wu, Chenguang; Qian, Li; Yu, Jiancheng; Gao, Jialin; Fei, Xiaoqiang; Zhang, Qingqing; Wang, Xueqin; Cui, Shiwei; Cheng, Jinluo; Xu, Ning; Wang, Guofeng; Han, Guoqing; Xu, Chunrong; Xie, Yun; An, Minmin; Zhang, Wei; Wang, Zhixiao; Cai, Yun; Fu, Qi; Fu, Yu; Zheng, Shuai; Yang, Fan; Hu, Qingfang; Dai, Hao; Jin, Yu; Zhang, Zheng; Xu, Kuanfeng; Li, Yifan; Shen, Jie; Zhou, Hongwen; He, Wei; Zheng, Xuqin; Han, Xiao; Yu, Liping; She, Jinxiong; Zhang, Mei; Yang, Tao

    2016-09-01

    Type 1 diabetes mellitus is heterogeneous in many facets. The patients suffered from type 1 diabetes present several levels of islet function as well as variable number and type of islet-specific autoantibodies. This study was to investigate prevalence and heterogeneity of the islet autoantibodies and clinical phenotypes of type 1 diabetes mellitus; and also discussed the process of islet failure and its risk factors in Chinese type 1 diabetic patients. A total of 1,291 type 1 diabetic patients were enrolled in this study. Demographic information was collected. Laboratory tests including mixed-meal tolerance test, human leukocyte antigen alleles, hemoglobinA1c, lipids, thyroid function and islet autoantibodies were conducted. The frequency of islet-specific autoantibody in newly diagnosed T1DM patients (duration shorter than half year) was 73% in East China. According to binary logistic regressions, autoantibody positivity, longer duration and lower Body Mass Index were the risk factors of islet failure. As the disease developed, autoantibodies against glutamic acid decarboxylase declined as well as the other two autoantibodies against zinc transporter 8 and islet antigen 2. The decrease of autoantibodies was positively correlated with aggressive beta cell destruction. Autoantibodies can facilitate the identification of classic T1DM from other subtypes and predict the progression of islet failure. As there were obvious heterogeneity in autoantibodies and clinical manifestation in different phenotypes of the disease, we should take more factors into consideration when identifying type 1 diabetes mellitus.

  15. Differential interleukin-1 receptor antagonism on pancreatic beta and alpha cells. Studies in rodent and human islets and in normal rats

    DEFF Research Database (Denmark)

    Zumsteg, U; Reimers, J I; Pociot, F;

    1993-01-01

    The monokines interleukin-1 alpha and -beta have been implicated as effector molecules in the immune-mediated pancreatic beta-cell destruction leading to insulin-dependent diabetes mellitus. Here we investigated the effects of interleukin-1 receptor antagonism on insulin and glucagon release of rat......, mouse and human islets exposed to recombinant human interleukin-1 beta, and on interleukin-1 beta induced changes in blood glucose, serum insulin and serum glucagon levels in Wistar Kyoto rats. The interleukin-1 receptor antagonist reduced the co-mitogenic effect of interleukin-1 beta on mouse and rat...

  16. Ethanolic Extract of Butea monosperma Leaves Elevate Blood Insulin Level in Type 2 Diabetic Rats, Stimulate Insulin Secretion in Isolated Rat Islets, and Enhance Hepatic Glycogen Formation

    Directory of Open Access Journals (Sweden)

    Mehdi Bin Samad

    2014-01-01

    Full Text Available We measured a vast range of parameters, in an attempt to further elucidate previously claimed antihyperglycemic activity of Butea monosperma. Our study clearly negates the possibility of antidiabetic activity by inhibited gastrointestinal enzyme action or by reduced glucose absorption. Reduction of fasting and postprandial glucose level was reconfirmed (P<0.05. Improved serum lipid profile via reduced low density lipoprotein (LDL, cholesterol, triglycerides (TG, and increased high density lipoprotein (HDL was also reestablished (P<0.05. Significant insulin secretagogue activity of B. monosperma was found in serum insulin assay of B. monosperma treated type 2 diabetic rats (P<0.01. This was further ascertained by our study on insulin secretion on isolated rat islets (P<0.05. Improved sensitivity of glucose was shown by the significant increase in hepatic glycogen deposition (P<0.05. Hence, we concluded that antihyperglycemic activity of B. monosperma was mediated by enhanced insulin secretion and enhanced glycogen formation in the liver.

  17. Coffee components inhibit amyloid formation of human islet amyloid polypeptide in vitro: possible link between coffee consumption and diabetes mellitus.

    Science.gov (United States)

    Cheng, Biao; Liu, Xinran; Gong, Hao; Huang, Lianqi; Chen, Hong; Zhang, Xin; Li, Chuanzhou; Yang, Muyang; Ma, Bingjun; Jiao, Lihua; Zheng, Ling; Huang, Kun

    2011-12-28

    Global epidemic studies have suggested that coffee consumption is reversely correlated with the incidence of type 2 diabetes mellitus (T2DM), a metabolic disease. The misfolding of human islet amyloid polypeptide (hIAPP) is regarded as one of the causative factors of T2DM. Coffee extracts have three major active components: caffeine, caffeic acid (CA), and chlorogenic acid (CGA). In this study, the effects of these major coffee components, as well as dihydrocaffeic acid (DHCA) (a major metabolite of CGA and CA), on the amyloidogenicity of hIAPP were investigated by thioflavin-T based fluorescence emission, transmission electronic microscopy, circular dichroism, light-induced cross-linking, dynamic light scattering, and MTT-based cell viability assays. The results suggest that all components show varied inhibitory effects on the formation of toxic hIAPP amyloids, in which CA shows the highest potency in delaying the conformational transition of the hIAPP molecule with the most prolonged lag time, whereas caffeine shows the lowest potency. At a 5-fold excess molar ratio of compound to hIAPP, all coffee-derived compounds affect the secondary structures of incubated hIAPP as suggested by the circular dichroism spectra and CDPro deconvolution analysis. Further photoinduced cross-linking based oligomerization and dynamic light scattering studies suggested CA and CGA significantly suppressed the formation of hIAPP oligomers, whereas caffeine showed no significant effect on oligomerization. Cell protection effects were also observed for all three compounds, with the protection efficiency being greatest for CA and least for CGA. These findings suggest that the beneficial effects of coffee consumption on T2DM may be partly due to the ability of the major coffee components and metabolites to inhibit the toxic aggregation of hIAPP.

  18. New insights into side effect of solvents on the aggregation of human islet amyloid polypeptide 11-20.

    Science.gov (United States)

    Mao, Yexuan; Yu, Lanlan; Yang, Ran; Ma, Chuanguo; Qu, Ling-bo; Harrington, Peter de B

    2016-02-01

    The formation of highly ordered fibrils for the human islet amyloid polypeptide (hIAPP) is considered as one of the precipitating factors of type 2 diabetes mellitus. In this study, an emerging new approach microscale thermophoresis and conventional ThT fluorescence assay were utilized to investigate the aggregation behavior of hIAPP(11-20), giving a new insight of the solvent effect on the aggregation of hIAPP(11-20). hIAPP(11-20) displayed different aggregation behaviors in various buffers, revealing that hIAPP(11-20) not only self-aggregates but also binds to solvent components. hIAPP(11-20) had a higher binding affinity for Tris than other selected buffers because multiple hydrogen bonds form, resulting in weaker self-aggregation of hIAPP(11-20) at the early stage of aggregation and prolonging the fibril formation process. hIAPP(11-20) displayed similar self-aggregation in both HEPES and pure water. Negatively charged phosphate ions in the PBS solution 'neutralize' the charges carried by hIAPP(11-20) itself to some extent, causing rapid aggregation of hIAPP(11-20), and leading to a shorter fibrillation process of hIAPP(11-20). These results revealed that solvents contribute to the aggregation of hIAPP(11-20) and demonstrated the affect of solvents on the activity of biomolecules. Additionally, as a new technique, microscale thermophoresis offers a powerful and promising approach to study the early stages of aggregation of peptides or proteins.

  19. Saffold Virus, a Human Cardiovirus, and Risk of Persistent Islet Autoantibodies in the Longitudinal Birth Cohort Study MIDIA.

    Directory of Open Access Journals (Sweden)

    German Tapia

    Full Text Available The aim of this study was to describe the frequency and distribution of Saffold virus in longitudinal stool samples from children, and test for association with development of persistent autoantibodies predictive of type 1 diabetes. A cohort of Norwegian children carrying the HLA genotype associated with highest risk of type 1 diabetes ("DR4-DQ8/DR3-DQ2" was followed with monthly stool samples from 3 to 35 months of age. Blood samples were tested for autoantibodies to insulin, glutamic acid decarboxylase65 and Islet Antigen-2. 2077 stool samples from 27 children with ≥ 2 repeatedly positive islet autoantibodies (cases, and 53 matched controls were analysed for Saffold virus genomic RNA by semi-quantitative real-time reverse transcriptase PCR. Saffold virus was found in 53 of 2077 (2.6% samples, with similar proportions between cases (2.5% and controls (2.6%. The probability of being infected by 3 years of age was 28% (95% CI 0.18-0.40. Viral quantities ranged from <1 to almost 105 copies/μl. Estimated odds ratio between islet autoimmunity and infection episodes prior to seroconversion was 1.98 (95% CI: 0.57-6.91, p = 0.29. Saffold virus had no statistically significant association with islet autoimmunity.

  20. 不同热缺血时间对猪胰岛获取及功能的影响%Effect of different warm-ischemia periods on islet isolation and function in pigs

    Institute of Scientific and Technical Information of China (English)

    张明; 杜成友

    2011-01-01

    , fragmentation of islets increased gradually, more obvious when more than 30 minutes, it was difficult to obtain Integrity islets at 45 minutes. Survival rate of islets was more than 80% at 0, 10, 20 minutes, survival rate of islets was more than 60% at 30 minutes, and survival rate of islets was less than 40% at 45 minutes. The amount of islets was 30; there were significant differences in the amount of islets between 30, 45 minutes and 0 minute (P < 0.05). There were significant differences in glucose-stimulated insulin release test between 30, 45 minutes and 0 minute (P < 0.05). The results suggested that there was no significant difference in morphology, yield, activity, and function of islets after isolation and purification when WIT was less than 20 minutes. Moreover, the function of islets was good; the morphology yield , and function of islets significantly change, and hypofunction when WIT was more than 30 minutes.

  1. Effects of mature Sertoli cells on allogeneic islets cocultured in vitro

    Institute of Scientific and Technical Information of China (English)

    Heli Xiang; Wujun Xue; Yan Teng; Xinshun Feng; Puxun Tian; Xiaoming Ding

    2006-01-01

    Objective: To set up a method for isolation and culture of mature Sertoli cells and to estimate their effects on allogeneic islets cocultured in vitro. Methods: Adult SD rat testicular Sertoli cells were prepared successfully by three-step enzyme digestion. Then they were cocultured respectively with allogeneic islets and activated Wistar rat splenocytes. 24-hour cumulative insulin release and glucose-stimulated insulin secretion test were performed to detect islet function between pure islets culture group and coculture group. Splenocyte proliferation activity was determined by MTT colorimetry assay to observe the inhibition effect of Sertoli cells in different densities. Result: Firstly, in pure islet culture group, the 24-hour cumulative insulin release was gradually decreased in 21-day culture time. Compared to day 3, this change was significant on day 7 (P < 0.05) and on day 10,14,21 (P < 0.01). In contrast, in coculture group, compared to day 3, the 24-hour cumulative insulin release was increased significantly on day 7 (P < 0.01 ), and then gradually decreased on day 10 and 14, but still higher than that of day 3. It was on day 21 that it began to decrease compared to day 3 (P < 0.05). During the culture time in vitro, the 24-hour cumulative insulin release of islet coculture group was significantly higher than that of pure islets culture group (P < 0.01). In the case of stimulation index(SI), there was a similar tendency as insulin release in the two groups. Secondly, mature Sertoli cells(1×106/mL)pretreated by 15 grays irradiation could decrease proliferation activity of activated splenocytes compared to that of control group (P < 0.01 ). This inhibition effect was dose-dependent. Conclusion: Mature Sertoli cells can improve the function and prolong the survival of islet cells cultured in vitro. They can also provide an immune protection to islet cells. The approach described above might be applicable to human islet transplantation as soon as

  2. Isolation, cultivation and transfection of human keratinocytes.

    Science.gov (United States)

    Zare, Sona; Zarei, Mohammad Ali; Ghadimi, Tayyeb; Fathi, Fardin; Jalili, Ali; Hakhamaneshi, Mohammad Saeed

    2014-04-01

    Human keratinocytes could be used in the repair of damaged skin, in tissue engineering applications, gene therapy and recently, the generation of iPS cells. We isolated human keratinocytes from foreskin and subsequently cultured them on fibronectin, collagen type I, gelatin and laminin-coated dishes that contained three different types of serum-free medium (epilife, KSM or CnT). We developed improved conditions for efficient transfection of these human keratinocytes by testing three common transfection methods and a GFP plasmid vector. The isolated cells showed typical keratinocyte morphology and expressed the epithelial cell specific antigen, cytokeratin 14. Collagen type 1, epilife medium and lipofectamin 2000 gave the best results for isolation and transfection of human keratinocytes. Our protocol can be used as a reproducible, simple and efficient method for isolation, cultivation and genetic manipulation of human keratinocytes, which may be useful in cell and gene therapy applications.

  3. Possible modulatory effect of endogenous islet catecholamines on insulin secretion

    Directory of Open Access Journals (Sweden)

    Gagliardino Juan J

    2001-10-01

    Full Text Available Abstract Background The possible participation of endogenous islet catecholamines (CAs in the control of insulin secretion was tested. Methods Glucose-induced insulin secretion was measured in the presence of 3-Iodo-L-Tyrosine (MIT, a specific inhibitor of tyrosine-hydroxylase activity, in fresh and precultured islets isolated from normal rats. Incubated islets were also used to measure CAs release in the presence of low and high glucose, and the effect of α2-(yohimbine [Y] and idazoxan [I] and α1-adrenergic antagonists (prazosin [P] and terazosin [T] upon insulin secretion elicited by high glucose. Results Fresh islets incubated with 16.7 mM glucose released significantly more insulin in the presence of 1 μM MIT (6.66 ± 0.39 vs 5.01 ± 0.43 ng/islet/h, p Conclusion Our results suggest that islet-originated CAs directly modulate insulin release in a paracrine manner.

  4. Characterization of a novel functional protein in the pancreatic islet: islet homeostasis protein regulation of glucagon synthesis in α cells.

    Science.gov (United States)

    Oh, Seh-Hoon; Darwiche, Houda; Cho, Jae-Hyoung; Shupe, Thomas; Petersen, Bryon E

    2012-01-01

    We have identified a novel protein in bone marrow-derived insulin-producing cells. Here we characterize this protein, hereby named islet homeostasis protein (IHoP), in the pancreatic islet. Detection of IHoP mRNA and protein was performed using reverse transcriptase-polymerase chain reaction, immunocytochemistry, and in situ hybridization. Islet homeostasis protein functions were utilizing proliferation, insulin secretion by in vitro assays, and following small interfering RNA protocols for suppression of IHoP. We found that IHoP did not homolog with known pancreatic hormones. Islet homeostasis protein expression was seen in both bone marrow-derived insulin-producing cells and isolated pancreatic islets. Immunohistochemistry on pancreatic islet revealed that IHoP localized to the glucagon-synthesizing α cells. Inhibition of IHoP by small interfering RNA resulted in the loss of glucagon expression, which induced low blood glucose levels (63-85 mg/dL). Subsequently, cellular apoptosis was observed throughout the islet, including the insulin-producing β cells. Islets of preonset diabetic patients showed normal expression of IHoP and glucagon; however, IHoP was lost upon onset of the disease. These data suggest that IHoP could be a new functional protein in the islet and may play a role in islet homeostasis.

  5. Pancreatic islet insulin secretion and metabolism in adult rats malnourished during neonatal life

    DEFF Research Database (Denmark)

    Barbosa, Francisco B; Capito, Kirsten; Kofod, Hans;

    2002-01-01

    Pancreatic islets were isolated from rats that had been nursed by dams fed with a control or an 8.7% protein diet during the first 12 d of the lactation period. Glucose-induced insulin secretion from islets in the 8.7% protein group was reduced 50%. The islet insulin and DNA content were similar,...

  6. Saffold Virus, a Human Cardiovirus, and Risk of Persistent Islet Autoantibodies in the Longitudinal Birth Cohort Study MIDIA.

    Science.gov (United States)

    Tapia, German; Bøås, Håkon; de Muinck, Eric J; Cinek, Ondrej; Stene, Lars C; Torjesen, Peter A; Rasmussen, Trond; Rønningen, Kjersti S

    2015-01-01

    The aim of this study was to describe the frequency and distribution of Saffold virus in longitudinal stool samples from children, and test for association with development of persistent autoantibodies predictive of type 1 diabetes. A cohort of Norwegian children carrying the HLA genotype associated with highest risk of type 1 diabetes ("DR4-DQ8/DR3-DQ2") was followed with monthly stool samples from 3 to 35 months of age. Blood samples were tested for autoantibodies to insulin, glutamic acid decarboxylase65 and Islet Antigen-2. 2077 stool samples from 27 children with ≥ 2 repeatedly positive islet autoantibodies (cases), and 53 matched controls were analysed for Saffold virus genomic RNA by semi-quantitative real-time reverse transcriptase PCR. Saffold virus was found in 53 of 2077 (2.6%) samples, with similar proportions between cases (2.5%) and controls (2.6%). The probability of being infected by 3 years of age was 28% (95% CI 0.18-0.40). Viral quantities ranged from Saffold virus had no statistically significant association with islet autoimmunity.

  7. Genetic differences between avian and human isolates of Candida dubliniensis.

    LENUS (Irish Health Repository)

    McManus, Brenda A

    2009-09-01

    When Candida dubliniensis isolates obtained from seabird excrement and from humans in Ireland were compared by using multilocus sequence typing, 13 of 14 avian isolates were genetically distinct from human isolates. The remaining avian isolate was indistinguishable from a human isolate, suggesting that transmission may occur between humans and birds.

  8. Differentiation of fetal pancreatic stem cells into neuron-like and islet-like cells in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiufeng Hua; Yanwei Wang; Peiwen Lian; Shouxin Zhang; Jianyuan Li; Haiyan Wang; Shulin Chen; Wei Gao

    2012-01-01

    Pancreatic stem cells were isolated and cultured from aborted human fetal pancreases of gestational age 14-20 weeks.They were seeded at a density of 1 × 104 in serum-free media for differentiation into neuron-like cells, expressing β-tubulin III and glial fibrillary acidic protein.These neuron-like cells displayed a synapse-like morphology and appeared to form a neuronal network.Pancreatic stem cells were also seeded at a density of 1 × 105 for differentiation into islet-like cells, expressing insulin and glucagon, with an islet-like morphology.These cells had glucose-stimulated secretion of human insulin and C-peptide.Results suggest that pancreatic stem cells can be differentiated into neuron-like and islet-like cells.

  9. Tat-biliverdin reductase A protects INS-1 cells from human islet amyloid polypeptide-induced cytotoxicity by alleviating oxidative stress and ER stress.

    Science.gov (United States)

    Lee, Su Jin; Kang, Hyung Kyung; Eum, Won Sik; Park, Jinseu; Choi, Soo Young; Kwon, Hyeok Yil

    2017-02-15

    Human islet amyloid polypeptide (hIAPP), a major constituent of islet amyloid deposits, induces pancreatic β-cell apoptosis and eventually contributes to β-cell deficit in patients with type 2 diabetes mellitus (T2DM). In this study, Tat-mediated transduction of biliverdin reductase A (BLVRA) was investigated in INS-1 cells to examine whether exogenous supplementation of BLVRA prevented hIAPP-induced apoptosis and dysfunction in insulin secretion in β-cells. Tat-BLVRA fusion protein was efficiently delivered into INS-1 cells in a time- and dose-dependent manner. Exposure of cells to hIAPP induced apoptotic cell death, which was dose-dependently inhibited by pre-treatment with Tat-BLVRA for 1 h. Transduced Tat-BLVRA reduced hIAPP-evoked generation of reactive oxygen species, a crucial mediator of β-cell destruction. Immunoblot analysis showed that Tat-BLVRA suppressed hIAPP-induced increase in the levels of proteins involved in endoplasmic reticulum (ER) stress and apoptosis signaling. Transduced Tat-BLVRA also recovered hIAPP-induced dysfunction in basal and glucose-stimulated insulin secretions. These results suggested that transduced Tat-BLVRA enhanced the tolerance of β-cells against IAPP-induced cytotoxicity by alleviating oxidative stress and ER stress. Therefore, Tat-mediated transduction of BLVRA may provide a potential tool to ameliorate β-cell deficit in pancreas with T2DM.

  10. Correction of diabetic pattern of insulin release from islets of the spiny mouse (Acomys cahirinus) by glucose priming in vitro.

    Science.gov (United States)

    Nesher, R; Abramovitch, E; Cerasi, E

    1985-04-01

    Insulin release kinetics were studied in perifused islets of Langerhans, isolated from mildly hyperglycaemic and from normoglycaemic spiny mice (Acomys cahirinus), a rodent predisposed to develop spontaneously non-ketotic diabetes. In both groups, insulin response to glucose (16.7 mmol/l) was delayed in comparison with that of rat islets, the release kinetics being analogous to that of human Type 2 (non-insulin-dependent) diabetes. Thirty min priming of the isolated Acomys islets with glucose (16.7 mmol/l) resulted in potentiation of the insulin release to a second stimulation. The degree of potentiation decreased exponentially with the time interval between stimulations, showing a t1/2 of 18 min. Induction of potentiation by glucose was time-dependent, giving a maximal effect after 20 min of priming. In addition to overall amplification of the insulin response, priming with glucose accelerated markedly the initial release rates, correcting the dynamics of the response. We conclude that: (1) decreased and delayed insulin secretion is found in Acomys cahirinus before the development of hyperglycaemia; (2) induction of time-dependent potentiation in the islet by priming with glucose corrects the diabetic-type dynamics of insulin release; (3) therefore the deficient insulin release of Acomys is of a functional nature, the mechanism of potentiation bypassing the defect; (4) since insulin release in Acomys resembles that in prediabetic and diabetic man, similar conclusions might apply to the islet dysfunction in Type 2 diabetes.

  11. Total excitation of the isolated human heart

    NARCIS (Netherlands)

    Durrer, D.; Dam, R.Th. van; Freud, G.E.; Janse, M.J.; Meijler, F.L.; Arzbaecher, R.C.

    1970-01-01

    To obtain information conceming the time course and instantaneous distribution of the excitatory process of the normal human healt, studies were made on isolated human hearts from seven individuals who died from various cerebral conditions, but who had no history of cardiac disease. Measurements wer

  12. Immature transformed rat islet beta-cells differentially express C-peptides derived from the genes coding for insulin I and II as well as a transfected human insulin gene

    DEFF Research Database (Denmark)

    Blume, N; Petersen, J S; Andersen, L C;

    1992-01-01

    Synthetic peptides representing unique sequences in rat proinsulin C-peptide I and II were used to generate highly specific antisera, which, when applied on sections of normal rat pancreas, confirm a homogeneous coexpression of the two C-peptides in all islet beta-cells. Insulin gene expression...... is induced in the transformed heterogeneous rat islet cell clone, NHI-6F, by transient in vivo passage. During this process a transfected human insulin gene is coactivated with the endogenous nonallelic rat insulin I and II genes. Newly established cultures from NHI-6F insulinomas having a high frequency...

  13. Differential interleukin-1 receptor antagonism on pancreatic beta and alpha cells. Studies in rodent and human islets and in normal rats

    DEFF Research Database (Denmark)

    Zumsteg, U; Reimers, J I; Pociot, F

    1993-01-01

    The monokines interleukin-1 alpha and -beta have been implicated as effector molecules in the immune-mediated pancreatic beta-cell destruction leading to insulin-dependent diabetes mellitus. Here we investigated the effects of interleukin-1 receptor antagonism on insulin and glucagon release of rat......, mouse and human islets exposed to recombinant human interleukin-1 beta, and on interleukin-1 beta induced changes in blood glucose, serum insulin and serum glucagon levels in Wistar Kyoto rats. The interleukin-1 receptor antagonist reduced the co-mitogenic effect of interleukin-1 beta on mouse and rat...... thymocytes with a 50% inhibitory concentration of 10- and 100-fold molar excess, respectively. Complete inhibition was obtained with a 100-1,000-fold molar excess. However, at a 100-fold molar excess the interleukin-1 receptor antagonist did not antagonise the potentiating effect of interleukin-1 beta on rat...

  14. Altered islet morphology but normal islet secretory function in vitro in a mouse model with microvascular alterations in the pancreas.

    Directory of Open Access Journals (Sweden)

    Elena Kostromina

    Full Text Available BACKGROUND: Our previous studies have shown that signal transducer and activator of transcription 3 (STAT3 signaling is important for the development of pancreatic microvasculature via its regulation of vascular endothelial growth factor-A (VEGF-A. Pancreas-specific STAT3-KO mice exhibit glucose intolerance and impaired insulin secretion in vivo, along with microvascular alterations in the pancreas. However, the specific role of STAT3 signaling in the regulation of pancreatic islet development and function is not entirely understood. METHODOLOGY/PRINCIPAL FINDINGS: To investigate the role of STAT3 signaling in the formation and maintenance of pancreatic islets, we studied pancreas-specific STAT3-KO mice. Histological analysis showed that STAT3 deficiency affected pancreatic islet morphology. We found an increased proportion of small-sized islets and a reduced fraction of medium-sized islets, indicating abnormal islet development in STAT3-KO mice. Interestingly, the islet area relative to the whole pancreas area in transgenic and control mice was not significantly different. Immunohistochemical analysis on pancreatic cryosections revealed abnormalities in islet architecture in STAT3-KO mice: the pattern of peripheral distribution of glucagon-positive α-cells was altered. At the same time, islets belonging to different size categories isolated from STAT3-KO mice exhibited normal glucose-stimulated insulin secretion in perifusion experiments in vitro when compared to control mice. CONCLUSIONS: Our data demonstrate that STAT3 signaling in the pancreas is required for normal islet formation and/or maintenance. Altered islet size distribution in the KO mice does not result in an impaired islet secretory function in vitro. Therefore, our current study supports that the glucose intolerance and in vivo insulin secretion defect in pancreas-specific STAT3-KO mice is due to altered microvasculature in the pancreas, and not intrinsic beta-cell function.

  15. Teucrium polium complex with molybdate enhance cultured islets secretory function.

    Science.gov (United States)

    Mohseni Salehi Monfared, Seyed Sajad; Pournourmohammadi, Shirin

    2010-02-01

    Islet transplantation has become a promising treatment in the therapy of type 1 diabetes. Its function improvement, after isolation and before transplantation, is crucial because of their loss both in number and function of islets after isolation procedures. Trace elements sodium orthovanadate (SOV) and sodium molybdate (SM), as well as medicinal plant Teucrium polium L. (TP), showed and possessed high beneficial antioxidative potential and even hypoglycemic properties via their effect on islets. We evaluated the effect of these components in combination on cultured islet function in order to improve pancreatic islet transplantation. Rat pancreatic islets were cultured for 24 h then incubated with different concentrations of TP (0.01 and 0.1 mg/mL) alone and in combination with SOV (1 mM) or SM (1 mM). Insulin concentration in buffer media was measured as islet secretory function. Administration of TP (0.01 mg/mL), SM, and SOV alone or in combination with each other significantly increased insulin secretion at high glucose concentration (16.7 mM); insulin secretion was significantly greater in the group containing both TP and SM than other treated groups (p molybdate with TP could improve islet cells function before transplantation.

  16. Towards engineering a novel transplantation site for pancreatic islets

    NARCIS (Netherlands)

    Smink, Alexandra Maria

    2016-01-01

    Intraportal pancreatic islet transplantation is a promising therapy for type 1 diabetes, but the liver is not an optimal site as it is associated with massive cell-death in the graft. Several alternative sites were investigated, but the human body does not contain an adequate islet transplantation s

  17. The voltage-gated proton channel Hv1 is expressed in pancreatic islet β-cells and regulates insulin secretion

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Qing [Department of Biophysics, School of Physics Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071 (China); Che, Yongzhe [School of Medicine, Nankai University, Tianjin 300071 (China); Li, Qiang; Zhang, Shangrong [Department of Biophysics, School of Physics Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071 (China); Gao, Ying-Tang [Key Laboratory of Artificial Cell, Third Central Clinical College of Tianjin Medical University, Tianjin 300170 (China); Wang, Yifan; Wang, Xudong; Xi, Wang; Zuo, Weiyan [Department of Biophysics, School of Physics Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071 (China); Li, Shu Jie, E-mail: shujieli@nankai.edu.cn [Department of Biophysics, School of Physics Science, The Key Laboratory of Bioactive Materials, Ministry of Education, Nankai University, Tianjin 300071 (China)

    2015-12-25

    The voltage-gated proton channel Hv1 is a potent acid extruder that participates in the extrusion of the intracellular acid. Here, we showed for the first time, Hv1 is highly expressed in mouse and human pancreatic islet β-cells, as well as β-cell lines. Imaging studies demonstrated that Hv1 resides in insulin-containing granules in β-cells. Knockdown of Hv1 with RNA interference significantly reduces glucose- and K{sup +}-induced insulin secretion in isolated islets and INS-1 (832/13) β-cells and has an impairment on glucose- and K{sup +}-induced intracellular Ca{sup 2+} homeostasis. Our data demonstrated that the expression of Hv1 in pancreatic islet β-cells regulates insulin secretion through regulating Ca{sup 2+} homeostasis.

  18. Transcriptional profiling of type 1 diabetes genes on chromosome 21 in a rat beta-cell line and human pancreatic islets

    DEFF Research Database (Denmark)

    Bergholdt, R.; Karlsen, A.E.; Hagedorn, Peter;

    2007-01-01

    We recently finemapped a type 1 diabetes (T1D)-linked region on chromosome 21, indicating that one or more T1D-linked genes exist in this region with 33 annotated genes. In the current study, we have taken a novel approach using transcriptional profiling in predicting and prioritizing the most...... likely candidate genes influencing beta-cell function in this region. Two array-based approaches were used, a rat insulinoma cell line (INS-1alphabeta) overexpressing pancreatic duodenum homeobox 1 (pdx-1) and treated with interleukin 1beta (IL-1beta) as well as human pancreatic islets stimulated...... with a mixture of cytokines. Several candidate genes with likely functional significance in T1D were identified. Genes showing differential expression in the two approaches were highly similar, supporting the role of these specific gene products in cytokine-induced beta-cell damage. These were genes involved...

  19. 简便快捷的小鼠胰岛分离纯化方法研究%A Convenient and Efficient Method of Mouse Pancreatic Islet Isolation and Purification

    Institute of Scientific and Technical Information of China (English)

    合湫; 苏恒; 李超; 沈涛; 严新民; 薛元明

    2012-01-01

    目的 探讨小鼠胰岛分离与纯化的方法.方法 采用多点注射灌注胶原酶法消化胰腺,不连续密度梯度离心联合人工挑取的方法分离、纯化胰岛.双硫腙特异性染色后计算胰岛产量及纯度.葡萄糖刺激后测定培养上清液胰岛素水平检测胰岛功能.结果 (1)每只小鼠采用上述分离、纯化法平均得到(114±15)个胰岛,平均纯度为(77.12±3.23)%,胰岛细胞存活率>90%; (2)分离、纯化的胰岛培养上清液中胰岛素水平在无糖、低糖(2.8 mmol/L)和高糖(22.2 mmol/L)刺激下分别为(23.80±3.52) mIU/L、 (67.57 ±4.04) mIU/L和(164.32±10.75) mIU/L,各组间比较,差异有统计学意义(P<0.05).两两比较,低糖组的胰岛素水平为无糖组的2.84倍(P< 0.05),高糖组的胰岛素水平为低糖组的2.43倍(P<0.05),高糖组的胰岛素水平为无糖组的6.90倍(P<0.05).结论 采用多点注射灌注胶原酶法消化胰腺,不连续密度梯度离心联合人工挑取的方法分离、纯化的小鼠胰岛产量及纯度较高,形态完整,不同浓度葡萄糖刺激后胰岛素分泌反应良好,是一种简便、快捷的小鼠胰岛分离方法.%Objective To investigate a convenient and efficient method of mouse pancreatic islets isolation and purification. Methods The isolation of mouse pancreatic islets was carried out by stationary digestion after multi-point injection of collagenase IV solution into the pancreas. A discontinuous Histopaque solution (1119, 1110, 1080 and 1060) was applied for the purification of the islets. Islet cell purity and viability were determined by dithizone ( DTZ) and Trypan blue staining. The islets were cultured overnight and stimulated with different concentration of glucose (0, 2.8 and 22.2mmol/L) for 1 hour. Supernatants were collected for insulin assays by an insulin RIA kit. Results (1) By this optimized islets isolation and purification protocol, each mouse pancreas could yield (114 ± 15) islets, with an

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

    Science.gov (United States)

    Gray, D W; Sutton, R; McShane, P; Peters, M; Morris, P J

    1988-11-01

    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.

  1. Proghrelin-derived peptides influence the secretion of insulin, glucagon, pancreatic polypeptide and somatostatin: a study on isolated islets from mouse and rat pancreas

    DEFF Research Database (Denmark)

    Qader, S.S.; Hakanson, R.; Lundquist, I.;

    2008-01-01

    , acyl ghrelin inhibited insulin secretion at low doses and stimulated at high. In rat islets, acyl ghrelin inhibited insulin secretion in a dose-dependent manner but the IC(50) for the acyl ghrelin-induced inhibition of insulin release was 7.5 x 10(-8) M, while the EC(50) and IC(50) values, with respect...... to stimulation of glucagon release and to inhibition of PP and somatostatin release, were in the 3 x 10(-12)-15 x 10(-12) M range. The corresponding EC(50) and IC(50) values for obestatin ranged from 5 x 10(-12) to 20 x 10(-12) M. Desacyl ghrelin per se did not affect islet hormone secretion. However, at a ten...

  2. Influence and mechanism of ghrelin on insulin release in isolated rat islets%Ghrelin对离体大鼠胰岛分泌胰岛素的影响及其机制

    Institute of Scientific and Technical Information of China (English)

    周晓磊; 薛承锐

    2011-01-01

    Objective To investigate the influence of ghrelin administration on the insulin secretion, and the expression of Kir6. 2 channels in islets. Methods Ghrelin was intraperitoneally administrated in Wistar rats at the doses 10 nmol/kg every day for 2 weeks. Islets were isolated for insulin release experiments. Single β cells were isolated for electrophysiological experiments. Meanwhile, the expression levels of Kir6. 2 mRNA and protein in islets were detected. Results At 11. 1 and 16. 7 mmol/L glucose,the levels of insulin release in control group were 22. 5 and 43.5 uIU/islet per h, and those in ghrelin-treated group were 15. 2 and 30. 1 uIU/islet per h (P <0. 05 ). In control group, the resting membrane potential reached ( - 73.2 ± 24. 8 ) mV, but in ghrelin-treated group, it was hyperpolarized to ( - 95.4 ± 33.7 )mV ( P < 0. 05 ). The Kir6. 2 expression levels were significantly up-regulated by ghrelin ( P < 0. 05 ).Conclusion Ghrelin via pancreatic GHSR up-regulates the Kir6. 2 expression in islets, hyperpolarizing the resting membrane potential, and resulting in the inhibition of insulin release.%目的 观察外源性Ghrelin对胰岛素分泌和胰岛内向整流钾通道(Kir6.2)表达的影响.方法 Wistar大鼠按10 nmoL/kg的剂量予Ghrelin腹腔注射2周后,分离胰岛进行胰岛素释放实验,分离β细胞检测静息膜电位;检测胰岛Kir6.2 mRNA和蛋白表达变化.结果 在11.1、16.7mmol/L葡萄糖刺激下,对照组的胰岛素释放分别为22.5、43.5 uIU/胰岛/h,Ghrelin组为15.2、30.1uIU/胰岛/h,较对照组显著降低(P<0.05).对照组β细胞的静息膜电位为(-73.2±24.8)mV,Ghrelin组为(-95.4±33.7)mV,较对照组显著降低(P<0.05).而Ghrelin组Kir6.2表达显著高于对照组(P<0.05).结论 Ghrelin与其受体结合后抑制胰岛素分泌,其机制可能与上调Kir6.2表达,使β细胞兴奋性降低有关.

  3. Overexpression of thioredoxin in islets transduced by a lentiviral vector prolongs graft survival in autoimmune diabetic NOD mice

    Directory of Open Access Journals (Sweden)

    Sytwu Huey-Kang

    2009-08-01

    Full Text Available Abstract Pancreatic islet transplantation is considered an appropriate treatment to achieve insulin independence in type I diabetic patients. However, islet isolation and transplantation-induced oxidative stress and autoimmune-mediated destruction are still the major obstacles to the long-term survival of graft islets in this potential therapy. To protect islet grafts from inflammatory damage and prolong their survival, we transduced islets with an antioxidative gene thioredoxin (TRX using a lentiviral vector before transplantation. We hypothesized that the overexpression of TRX in islets would prolong islet graft survival when transplanted into diabetic non-obese diabetic (NOD mice. Methods Islets were isolated from NOD mice and transduced with lentivirus carrying TRX (Lt-TRX or enhanced green fluorescence protein (Lt-eGFP, respectively. Transduced islets were transplanted under the left kidney capsule of female diabetic NOD mice, and blood glucose concentration was monitored daily after transplantation. The histology of the islet graft was assessed at the end of the study. The protective effect of TRX on islets was investigated. Results The lentiviral vector effectively transduced islets without altering the glucose-stimulating insulin-secretory function of islets. Overexpression of TRX in islets reduced hydrogen peroxide-induced cytotoxicity in vitro. After transplantation into diabetic NOD mice, euglycemia was maintained for significantly longer in Lt-TRX-transduced islets than in Lt-eGFP-transduced islets; the mean graft survival was 18 vs. 6.5 days (n = 9 and 10, respectively, p Conclusion We successfully transduced the TRX gene into islets and demonstrated that these genetically modified grafts are resistant to inflammatory insult and survived longer in diabetic recipients. Our results further support the concept that the reactive oxygen species (ROS scavenger and antiapoptotic functions of TRX are critical to islet survival after

  4. Islet β-cell ghrelin signaling for inhibition of insulin secretion.

    Science.gov (United States)

    Dezaki, Katsuya; Yada, Toshihiko

    2012-01-01

    Ghrelin, an acylated 28-amino acid peptide, was isolated from the stomach, where circulating ghrelin is produced predominantly. In addition to its unique role in regulating growth-hormone release, mealtime hunger, lipid metabolism, and the cardiovascular system, ghrelin is involved in the regulation of glucose metabolism. Ghrelin is expressed in pancreatic islets and released into pancreatic microcirculations. Ghrelin inhibits insulin release in mice, rats, and humans. Pharmacological and genetic blockades of islet-derived ghrelin markedly augment glucose-induced insulin release. The signal transduction mechanisms of ghrelin in islet β-cells are very unique, being distinct from those utilized for growth-hormone release. Ghrelin attenuates the glucose-induced cAMP production and PKA activation, which drives activation of Kv channels and suppression of the glucose-induced [Ca(2+)](i) increase and insulin release in β-cells. Insulinostatic function of the ghrelin-GHS-R system in islets is a potential therapeutic target for type 2 diabetes. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. [Effects of chemical constituents of Crossostephium chinense on insulin secretion in rat islets in vitro].

    Science.gov (United States)

    Zou, Lei; Wu, Qi; Yang, Xiuwei; Fu, Dexian

    2009-06-01

    To investigate the effects of the chemical constituents of the whole herbs of Crossostephium chinense on insulin secretion in rat islets. Islets were isolated from rat pancreata, cultured in vitro, and measured by color signals of dithizone stained digestion solution for detection of pancreatic islets. The morphological observation of islets was carried out by inverted microscope. The effects of test compounds, scopoletin (1), scopolin (2), tanacetin (3), quercetagetin-3,6,7-trimethylether (4) and 5-O-methyl-myo-inositol (5) isolated from the whole herbs of C. chinense, on the insulin secreting level from islets were compared with those of glybenclamide as a positive control substances, and the difference in insulin secreting level from islets between the presence and absence of test compounds was assayed. There was no difference in basal insulin secretion before and after 2 h incubation period of rat islets. The islets treated with quercetagetin-3,6,7-trimethylether have about 2-fold higher insulin secreting level (P < 0.01) compared a normal control group. The islets treated with 5-O-methyl-myo-inositol have about 1.5-fold higher insulin secreting level (P < 0.05) compared to a normal control group. Whereas the islets treated with scopoletin show about 1.9-fold lower basal insulin secreting level (P < 0.05) than a normal control group. In this paper the developed cultivation method of isolated pancreatic islets from rat can be used as a kind of islet-based drug screening model for diabetes mellitus in vitro. Quercetagetin-3,6,7-trimethylether and 5-O-methyl-myo-inositol could enhance rat islet insulin secretion and further in vivo studies are needed to clarify the nature of such an observation. However, scopletin suppress rat islet insulin secretion.

  6. Islet cytotoxicity of interleukin 1. Influence of culture conditions and islet donor characteristics

    DEFF Research Database (Denmark)

    Mandrup-Poulsen, T; Spinas, G A; Prowse, S J

    1987-01-01

    We recently demonstrated that the macrophage product interleukin 1 (IL-1) is cytotoxic to isolated pancreatic islets and hypothesized that IL-1 is responsible for beta-cell destruction in insulin-dependent diabetes mellitus (IDDM). We studied whether the variation in IDDM preponderance with age...

  7. Retention of gene expression in porcine islets after agarose encapsulation and long-term culture

    Energy Technology Data Exchange (ETDEWEB)

    Dumpala, Pradeep R., E-mail: pdumpala@rixd.org [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Holdcraft, Robert W.; Martis, Prithy C.; Laramore, Melissa A. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States); Parker, Thomas S.; Levine, Daniel M. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); Smith, Barry H. [The Rogosin Institute, 505 East 70th Street, New York, NY 10021 (United States); NewYork-Presbyterian Hospital, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021 (United States); Gazda, Lawrence S. [The Rogosin Institute – Xenia Division, 740 Birch Road, Xenia, OH 45385 (United States)

    2016-08-05

    Agarose encapsulation of porcine islets allows extended in vitro culture, providing ample time to determine the functional capacity of the islets and conduct comprehensive microbiological safety testing prior to implantation as a treatment for type 1 diabetes mellitus. However, the effect that agarose encapsulation and long-term culture may have on porcine islet gene expression is unknown. The aim of the present study was to compare the transcriptome of encapsulated porcine islets following long-term in vitro culture against free islets cultured overnight. Global gene expression analysis revealed no significant change in the expression of 98.47% of genes. This indicates that the gene expression profile of free islets is highly conserved following encapsulation and long-term culture. Importantly, the expression levels of genes that code for critical hormones secreted by islets (insulin, glucagon, and somatostatin) as well as transcripts encoding proteins involved in their packaging and secretion are unchanged. While a small number of genes known to play roles in the insulin secretion and insulin signaling pathways are differentially expressed, our results show that overall gene expression is retained following islet isolation, agarose encapsulation, and long-term culture. - Highlights: • Effect of agarose encapsulation and 8 week culture on porcine islets was analyzed. • Transcriptome analysis revealed no significant change in a majority (98%) of genes. • Agarose encapsulation allows for long-term culture of porcine islets. • Islet culture allows for functional and microbial testing prior to clinical use.

  8. Mild exposure of RIN-5F β-cells to human islet amyloid polypeptide aggregates upregulates antioxidant enzymes via NADPH oxidase-RAGE: An hormetic stimulus

    Directory of Open Access Journals (Sweden)

    Elisabetta Borchi

    2014-01-01

    Full Text Available The presence of amyloid aggregates of human islet amyloid polypeptide (hIAPP, a hallmark of type 2 diabetes, contributes to pancreatic β-cell impairment, where oxidative stress plays a key role. A contribution of NADPH oxidase to reactive oxygen species (ROS generation after cell exposure to micromolar concentrations of hIAPP aggregates has been suggested. However, little is known about β-cells exposure to lower amounts of hIAPP aggregates, similar to those found in human pancreas. Thus, we aimed to investigate the events resulting from RIN-5F cells exposure to nanomolar concentrations of toxic hIAPP aggregates. We found an early and transient rise of NADPH oxidase activity resulting from increased Nox1 expression following the engagement of receptor for advanced glycation end-products (RAGE by hIAPP aggregates. Unexpectedly, NADPH oxidase activation was not accompanied by a significant ROS increase and the lipoperoxidation level was significantly reduced. Indeed, cell exposure to hIAPP aggregates affected the antioxidant defences, inducing a significant increase of the expression and activity of catalase and glutathione peroxidase. We conclude that exposure of pancreatic β-cells to nanomolar concentrations of hIAPP aggregates for a short time induces an hormetic response via the RAGE-Nox1 axis; the latter stimulates the enzymatic antioxidant defences that preserve the cells against oxidative stress damage.

  9. Glucose metabolism, islet architecture, and genetic homogeneity in imprinting of [Ca2+](i and insulin rhythms in mouse islets.

    Directory of Open Access Journals (Sweden)

    Craig S Nunemaker

    Full Text Available We reported previously that islets isolated from individual, outbred Swiss-Webster mice displayed oscillations in intracellular calcium ([Ca2+](i that varied little between islets of a single mouse but considerably between mice, a phenomenon we termed "islet imprinting." We have now confirmed and extended these findings in several respects. First, imprinting occurs in both inbred (C57BL/6J as well as outbred mouse strains (Swiss-Webster; CD1. Second, imprinting was observed in NAD(PH oscillations, indicating a metabolic component. Further, short-term exposure to a glucose-free solution, which transiently silenced [Ca2+](i oscillations, reset the oscillatory patterns to a higher frequency. This suggests a key role for glucose metabolism in maintaining imprinting, as transiently suppressing the oscillations with diazoxide, a K(ATP-channel opener that blocks [Ca2+](i influx downstream of glucose metabolism, did not change the imprinted patterns. Third, imprinting was not as readily observed at the level of single beta cells, as the [Ca2+](i oscillations of single cells isolated from imprinted islets exhibited highly variable, and typically slower [Ca2+](i oscillations. Lastly, to test whether the imprinted [Ca2+](i patterns were of functional significance, a novel microchip platform was used to monitor insulin release from multiple islets in real time. Insulin release patterns correlated closely with [Ca2+](i oscillations and showed significant mouse-to-mouse differences, indicating imprinting. These results indicate that islet imprinting is a general feature of islets and is likely to be of physiological significance. While islet imprinting did not depend on the genetic background of the mice, glucose metabolism and intact islet architecture may be important for the imprinting phenomenon.

  10. Small intestinal submucosa improves islet survival and function during in vitro culture

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hui Tian; Wu-Jun Xue; Xiao-Ming Ding; Xin-Lu Pang; Yan Teng; Pu-Xun Tian; Xin-Shun Feng

    2005-01-01

    AIM: To evaluate the recovery and function of isolated rat pancreatic islets during in vitro culture with small intestinal submucosa (SIS).METHODS: Pancreatic islets were isolated from Wistar rats by standard surgical procurement followed by intraductal collagenase distension, mechanical dissociation and Euroficoll purification. Purified islets were cultured in plates coated with multilayer SIS (SIS-treated group) or without multilayer SIS (standard cultured group) for 7 and 14 d in standard islet culture media of RPMI 1640. After isolation and culture, islets from both experimental groups were stained with dithizone and counted. Recovery of islets was determined by the ratio of counts after the culture to the yield of islets immediately following islet isolation. Viability of islets after the culture was assessed by the glucose challenge test with low (2.7 mmol/L) and high glucose (16.7 mmol/L)solution supplemented with 50 mmol/L 3-isobutyl-1-methylxanthine (IBMX) solution. Apoptosis of islet cells after the culture was measured by relative quantification of histone-complexed DNA fragments using ELISA.RESULTS: After 7 or 14 d of in vitro tissue culture, the recovery of islets in SIS-treated group was significantly higher than that cultured in plates without SIS coating. The recovery of islets in SIS-treated group was about twice more than that of in the control group. In SIS-treated group, there was no significant difference in the recovery of islets between short- and long-term periods of culture (95.8±1.0% vs 90.8±1.5%, P>0.05). When incubated with high glucose (16.7 mmol/L) solution,insulin secretion in SIS-treated group showed a higher increase than that in control group after 14 d of culture (20.7±1.1 mU/L vs11.8±1.1 mU/L, P0.05).Much less apoptosis of islet cells occurred in SIS-treated group than in control group after the culture.CONCLUSION: Co-culture of isolated rat islets with native sheet-like SIS might build an extracellular matrix for islets and

  11. Altered Expression of Somatostatin Receptors in Pancreatic Islets from NOD Mice Cultured at Different Glucose Concentrations In Vitro and in Islets Transplanted to Diabetic NOD Mice In Vivo

    Directory of Open Access Journals (Sweden)

    Eva Ludvigsen

    2011-01-01

    Full Text Available Somatostatin acts via five receptors (sst1-5. We investigated if the changes in pancreatic islet sst expression in diabetic NOD mice compared to normoglycemic mice are a consequence of hyperglycemia or the ongoing immune reaction in the pancreas. Pancreatic islets were isolated from NOD mice precultured for 5 days and further cultured for 3 days at high or low glucose before examined. Islets were also isolated from NOD mice and transplanted to normal or diabetic mice in a number not sufficient to cure hyperglycemia. After three days, the transplants were removed and stained for sst1-5 and islet hormones. Overall, changes in sst islet cell expression were more common in islets cultured in high glucose concentration in vitro as compared to the islet transplantation in vivo to diabetic mice. The beta and PP cells exhibited more frequent changes in sst expression, while the alpha and delta cells were relatively unaffected by the high glucose condition. Our findings suggest that the glucose level may alter sst expressed in islets cells; however, immune mechanisms may counteract such changes in islet sst expression.

  12. Bisphenol A accelerates toxic amyloid formation of human islet amyloid polypeptide: a possible link between bisphenol A exposure and type 2 diabetes.

    Science.gov (United States)

    Gong, Hao; Zhang, Xin; Cheng, Biao; Sun, Yue; Li, Chuanzhou; Li, Ting; Zheng, Ling; Huang, Kun

    2013-01-01

    Bisphenol A (BPA) is a chemical compound widely used in manufacturing plastic products. Recent epidemiological studies suggest BPA exposure is positively associated with the incidence of type 2 diabetes mellitus (T2DM), however the mechanisms underlying this link remain unclear. Human islet amyloid polypeptide (hIAPP) is a hormone synthesized and secreted by the pancreatic β-cells. Misfolding of hIAPP into toxic oligomers and mature fibrils can disrupt cell membrane and lead to β-cell death, which is regarded as one of the causative factors of T2DM. To test whether there are any connections between BPA exposure and hIAPP misfolding, we investigated the effects of BPA on hIAPP aggregation using thioflavin-T based fluorescence, transmission electronic microscopy, circular dichroism, dynamic light scattering, size-exclusion chromatography, fluorescence-dye leakage assay in an artificial micelle system and the generation of reactive oxygen species in INS-1 cells. We demonstrated that BPA not only dose-dependently promotes the aggregation of hIAPP and enhances the membrane disruption effects of hIAPP, but also promotes the extent of hIAPP aggregation related oxidative stress. Taken together, our results suggest that BPA exposure increased T2DM risk may involve the exacerbated toxic aggregation of hIAPP.

  13. Targeted Mass Spectrometry Approach Enabled Discovery of O-Glycosylated Insulin and Related Signaling Peptides in Mouse and Human Pancreatic Islets.

    Science.gov (United States)

    Yu, Qing; Canales, Alejandra; Glover, Matthew S; Das, Rahul; Shi, Xudong; Liu, Yang; Keller, Mark P; Attie, Alan D; Li, Lingjun

    2017-08-07

    O-Linked glycosylation often involves the covalent attachment of sugar moieties to the hydroxyl group of serine or threonine on proteins/peptides. Despite growing interest in glycoproteins, little attention has been directed to glycosylated signaling peptides, largely due to lack of enabling analytical tools. Here we explore the occurrence of naturally O-linked glycosylation on the signaling peptides extracted from mouse and human pancreatic islets using mass spectrometry (MS). A novel targeted MS-based method is developed to increase the likelihood of capturing these modified signaling peptides and to provide improved sequence coverage and accurate glycosite localization, enabling the first large-scale discovery of O-glycosylation on signaling peptides. Several glycosylated signaling peptides with multiple glycoforms are identified, including the first report of glycosylated insulin-B chain and insulin-C peptide and BigLEN. This discovery may reveal potential novel functions as glycosylation could influence their conformation and biostability. Given the importance of insulin and its related peptide hormones and previous studies of glycosylated insulin analogues, this natural glycosylation may provide important insights into diabetes research and therapeutic treatments.

  14. Effects of cholesterol on pore formation in lipid bilayers induced by human islet amyloid polypeptide fragments: A coarse-grained molecular dynamics study

    Science.gov (United States)

    Xu, Weixin; Wei, Guanghong; Su, Haibin; Nordenskiöld, Lars; Mu, Yuguang

    2011-11-01

    Disruption of the cellular membrane by the amyloidogenic peptide, islet amyloid polypeptide (IAPP), has been considered as one of the mechanisms of β-cell death during type 2 diabetes. The N-terminal region (residues 1-19) of the human version of IAPP is suggested to be primarily responsible for the membrane-disrupting effect of the full-length hIAPP peptide. However, the detailed assembly mode of hIAPP1-19 with membrane remains unclear. To gain insight into the interactions of hIAPP1-19 oligomer with the model membrane, we have employed coarse-grained molecular dynamics self-assembly simulations to study the aggregation of hIAPP1-19 fragments in the binary lipid made of zwitterionic dipalmitoylphosphatidylcholine (DPPC) and anionic dipalmitoylphosphatidylserine (DPPS) in the presence and absence of different levels of cholesterol content. The membrane-destabilizing effect of hIAPP1-19 is found to be modulated by the presence of cholesterol. In the absence of cholesterol, hIAPP1-19 aggregates prefer to locate inside the bilayer, forming pore-like assemblies. While in the presence of cholesterol molecules, the lipid bilayer becomes more ordered and stiff, and the hIAPP1-19 aggregates are dominantly positioned at the bilayer-water interface. The action of cholesterol may suggest a possible way to maintain the membrane integrity by small molecule interference.

  15. Nestin-positive progenitor cells isolated from human fetal pancreas have phenotypic markers identical to mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Ling Zhang; Tian-Pei Hong; Jiang Hu; Yi-Nan Liu; Yong-Hua Wu; Ling-Song Li

    2005-01-01

    AIM: To isolate nestin-positive progenitor cells from human fetal pancreas and to detect their surface markers and their capability of proliferation and differentiation into pancreatic islet endocrine cells in vitro.METHODS: Islet-like cell clusters (ICCs) were isolated from human fetal pancreas by using collagenase digestion. The free-floating ICCs were handpicked and cultured in a new dish. After the ICCs developed into monolayer epithelium-like cells, they were passaged and induced for differentiation. Reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence stain, fluorescenceactivated cell sorting (FACS) and radioimmunoassay (RIA)were used to detect the expression of cell markers. RESULTS: (1) The monolayer epithelium-like cells had highly proliferative potential and could be passaged more than 16 timesin vitro; (2) RT-PCR analysis and immunofluorescence stain showed that these cells expressed both nestin and ABCG2, two of stem cellmarkers; (3) FACS analysis revealed that CD44, CD90and CD147 were positive, whereas CD34, CD38, CD45, CD71, CD117, CD133 and HLA-DR were negative on the nestin-positive cells; (4) RT-PCR analysis showed that the mRNA expression of insulin, glucagon and pancreaticduodenal homeobox gene-1 was detected, whereas the expression of nestin and neurogenin 3 disappeared in these cells treated with serum-free media supplemented with the cocktail of growth factors. Furthermore, the intracellular insulin content was detected by RIA after the induction culture.CONCLUSION: Nestin-positive cells isolated from human fetal pancreas possess the characteristics of pancreatic progenitor cells since they have highly proliferative potential and the capability of differentiation into insulinproducing cells in vitro. Interestingly, the nestin-positive pancreatic progenitor cells share many phenotypic markers with mesenchymal stem cells derived from bone marrow.

  16. Remodelling sympathetic innervation in rat pancreatic islets ontogeny

    Directory of Open Access Journals (Sweden)

    Hiriart Marcia

    2009-06-01

    Full Text Available Abstract Background Pancreatic islets are not fully developed at birth and it is not clear how they are vascularised and innervated. Nerve Growth Factor (NGF is required to guide sympathetic neurons that innervate peripheral organs and also in cardiovascular system and ovary angiogenesis. Pancreatic beta cells of a transgenic mouse that over-expressed NGF in attracts sympathetic hyper-innervation towards them. Moreover, we have previously demonstrated that adult beta cells synthesize and secrete NGF; however, we do not know how is NGF secreted during development, nor if it might be trophic for sympathetic innervation and survival in the pancreas. We analyzed sympathetic innervation and vasculature development in rat pancreatic islets at different developmental stages; foetal (F19, early postnatal (P1, weaning period (P20 and adults. We temporarily correlated these events to NGF secretion by islet cells. Results Sympathetic fibres reached pancreatic islets in the early postnatal period, apparently following blood vessels. The maximal number of sympathetic fibres (TH immunopositive in the periphery of the islets was observed at P20, and then fibres entered the islets and reached the core where beta cells are mainly located. The number of fibres decreased from that stage to adulthood. At all stages studied, islet cells secreted NGF and also expressed the high affinity receptor TrkA. Foetal and neonatal isolated islet cells secreted more NGF than adults. TrkA receptors were expressed at all stages in pancreatic sympathetic fibres and blood vessels. These last structures were NGF–immunoreactive only at early stages (foetal and P0. Conclusion The results suggest that NGF signalling play an important role in the guidance of blood vessels and sympathetic fibres toward the islets during foetal and neonatal stages and could also preserve innervation at later stages of life.

  17. Microencapsulation of pancreatic islets for use in a bioartificial pancreas.

    Science.gov (United States)

    Opara, Emmanuel C; McQuilling, John P; Farney, Alan C

    2013-01-01

    Islet transplantation is the most exciting treatment option for individuals afflicted with Type 1 diabetes. However, the severe shortage of human pancreas and the need to use risky immunosuppressive drugs to prevent transplant rejection remain two major obstacles for the routine use of islet transplantation in diabetic patients. Successful development of a bioartificial pancreas using the approach of microencapsulation with perm-selective coating of islets with biopolymers for graft immunoisolation holds tremendous promise for diabetic patients because it has great potential to overcome these two barriers. In this chapter, we provide a detailed description of the microencapsulation process.

  18. Effect of micro- and macroencapsulation on oxygen consumption by pancreatic islets.

    Science.gov (United States)

    Cornolti, Roberta; Figliuzzi, Marina; Remuzzi, Andrea

    2009-01-01

    Immunoisolation of pancreatic islets is extensively investigated for glycemic control in diabetic experimental animals. We previously reported that subcutaneous xenotransplantation of bovine islets protected by a selective polysulfone membrane successfully controlled glycemia in diabetic rats for up to 20 days. We then wondered whether immunoisolated islets have adequate oxygen supply in this device, where only diffusive transport allows cell function and survival. Here we set up an experimental technique to measure oxygen consumption rate (OCR) using a Clark's electrode inserted in a glass thermostated chamber connected to a data recorder and acquisition system. Bovine islets were isolated from 6-month-old calves, encapsulated in sodium alginate microcapsules or inserted in polysulfone hollow fibers. After 1 and 2 days in culture a series of measurements was performed using free islets (at normal or high-glucose concentration), islets encapsulated in microcapsules, or in hollow fibers. In free islets OCR averaged from 2.0 +/- 0.8 pmol/IEQ/min at low-glucose concentration and from 2.5 +/- 1.0 pmol/IEQ/min at high-glucose concentration (p hollow fibers was comparable, and not significantly different from that measured in free islets. Two days after isolation OCR averaged 2.3 +/- 0.6 in free islets, 2.3 +/- 0.9 in alginate microcapsules, and 2.2 +/- 0.7 pmol/IEQ/min in hollow fibers. These results show that OCR by bovine islets is comparable to that previously reported for other species. OCR increases in islets stimulated with high glucose and may be considered as a functional index. Moreover, islet encapsulation in alginate microcapsule, as well as in hollow fiber membranes, did not significantly affect in vitro OCR, suggesting adequate islet oxygenation in these conditions.

  19. [Updated inventory of mosquitoes (Dipbra: Culkidae) from the French islets of Europa, Juan-de-Nova and Grande-Glorieuse (Mozambique channel, Indian Ocean)].

    Science.gov (United States)

    Girod, R; Le Goff, G

    2006-05-01

    The islets of Europa, Juan-de-Nova and Grande-Glorieuse are French territories isolated in the Mozambique Channel (Indian Ocean) which have remained relatively preserved from anthropization all along their history These three islets have been classified entire nature reserves from 1975 and are today inhabited only by a permanent military detachment of about fifteen men even if they occasionally greet technical and scientific staff. Sanitary and environmental issues brought about assessment of the present culicid fauna. The authors propose a synthesis of the culicid knowledge from the islets and make an inventory of new species. The role played by humans in importation of culicids is discussed as well as sanitary consequences of their adaptation to environment

  20. Conservation of the sequence of islet amyloid polypeptide in five mammals is consistent with its putative role as an islet hormone.

    OpenAIRE

    Nishi, M.; Chan, S J; Nagamatsu, S; Bell, G I; Steiner, D. F.

    1989-01-01

    Islet amyloid polypeptide (IAPP) is a 37-amino acid peptide found in the pancreatic amyloid deposits of type II (non-insulin-dependent) diabetic patients and insulinomas. We previously reported the nucleotide sequence of a human cDNA, which indicated that IAPP is a C-terminally amidated peptide derived by proteolytic processing of an 89-amino acid precursor. We now report the isolation of cDNA clones coding for cat, rat, mouse, and guinea pig IAPP precursors, obtained using the combination of...

  1. Effects of thioacetamide on pancreatic islet B-cell function

    NARCIS (Netherlands)

    Malaisse, WJ; Lebrun, P; Sener, A; Wolters, GHJ; Ravazzola, M

    2004-01-01

    Thioacetamide (0.01-1.3 mM) fails to exert any significant immediate effect upon insulin release from rat isolated islets. However, when administered (4 mumol/g body wt) intraperitoneally 24 h before sacrifice, it reduced food intake and body weight and affected the secretory response of isolated is

  2. Mitis group streptococci express variable pilus islet 2 pili.

    Directory of Open Access Journals (Sweden)

    Dorothea Zähner

    Full Text Available BACKGROUND: Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguinis are members of the Mitis group of streptococci and agents of oral biofilm, dental plaque and infective endocarditis, disease processes that involve bacteria-bacteria and bacteria-host interactions. Their close relative, the human pathogen S. pneumoniae uses pilus-islet 2 (PI-2-encoded pili to facilitate adhesion to eukaryotic cells. METHODOLOGY/PRINCIPAL FINDINGS: PI-2 pilus-encoding genetic islets were identified in S. oralis, S. mitis, and S. sanguinis, but were absent from other isolates of these species. The PI-2 islets resembled the genetic organization of the PI-2 islet of S. pneumoniae, but differed in the genes encoding the structural pilus proteins PitA and PitB. Two and three variants of pitA (a pseudogene in S. pneumoniae and pitB, respectively, were identified that showed ≈20% difference in nucleotide as well as corresponding protein sequence. Species-independent combinations of pitA and pitB variants indicated prior intra- and interspecies horizontal gene transfer events. Polyclonal antisera developed against PitA and PitB of S. oralis type strain ATCC35037 revealed that PI-2 pili in oral streptococci were composed of PitA and PitB. Electronmicrographs showed pilus structures radiating >700 nm from the bacterial surface in the wild type strain, but not in an isogenic PI-2 deletion mutant. Anti-PitB-antiserum only reacted with pili containing the same PitB variant, whereas anti-PitA antiserum was cross-reactive with the other PitA variant. Electronic multilocus sequence analysis revealed that all PI-2-encoding oral streptococci were closely-related and cluster with non-PI-2-encoding S. oralis strains. CONCLUSIONS/SIGNIFICANCE: This is the first identification of PI-2 pili in Mitis group oral streptococci. The findings provide a striking example of intra- and interspecies horizontal gene transfer. The PI-2 pilus diversity provides a possible key to

  3. Islet transplantation in rodents: do encapsulated islets really work?

    Directory of Open Access Journals (Sweden)

    Yngrid Ellyn Dias Maciel de Souza

    2011-06-01

    Full Text Available CONTEXT: Diabetes mellitus type I affects around 240 million people in the world and only in the USA 7.8% of the population. It has been estimated that the costs of its complications account for 5% to 10% of the total healthcare spending around the world. According to World Health Organization, 300 million people are expected to develop diabetes mellitus by the year 2025. The pancreatic islet transplantation is expected to be less invasive than a pancreas transplant, which is currently the most commonly used approach. OBJECTIVES: To compare the encapsulated and free islet transplantation in rodents looking at sites of islet implantation, number of injected islets, viability and immunosuppression. METHODS: A literature search was conducted using MEDLINE/PUBMED and SCIELO with terms about islet transplantation in the rodent from 2000 to 2010. We found 2,636 articles but only 56 articles from 2000 to 2010 were selected. RESULTS: In these 56 articles used, 34% were encapsulated and 66% were nonencapsulated islets. Analyzing both types of islets transplantation, the majority of the encapsulated islets were implanted into the peritoneal cavity and the nonencapsulated islets into the liver, through the portal vein. In addition, the great advantage of the peritoneal cavity as the site of islet transplantation is its blood supply. Both vascular endothelial cells and vascular endothelial growth factor were used to stimulate angiogenesis of the islet grafts, increasing the vascularization rapidly after implantation. It also has been proven that there is influence of the capsules, since the larger the capsule more chances there are of central necrosis. In some articles, the use of immunosuppression demonstrated to increase the life expectancy of the graft. CONCLUSION: While significant progress has been made in the islets transplantation field, many obstacles remain to be overcome. Microencapsulation provides a means to transplant islets without

  4. Increased expression of miR-187 in human islets from individuals with type 2 diabetes is associated with reduced glucose-stimulated insulin secretion.

    Science.gov (United States)

    Locke, J M; da Silva Xavier, G; Dawe, H R; Rutter, G A; Harries, L W

    2014-01-01

    Type 2 diabetes is characterised by progressive beta cell dysfunction, with changes in gene expression playing a crucial role in its development. MicroRNAs (miRNAs) are post-transcriptional regulators of gene expression and therefore alterations in miRNA levels may be involved in the deterioration of beta cell function. Global TaqMan arrays and individual TaqMan assays were used to measure islet miRNA expression in discovery (n = 20) and replication (n = 20) cohorts from individuals with and without type 2 diabetes. The role of specific dysregulated miRNAs in regulating insulin secretion, content and apoptosis was subsequently investigated in primary rat islets and INS-1 cells. Identification of miRNA targets was assessed using luciferase assays and by measuring mRNA levels. In the discovery and replication cohorts miR-187 expression was found to be significantly increased in islets from individuals with type 2 diabetes compared with matched controls. An inverse correlation between miR-187 levels and glucose-stimulated insulin secretion (GSIS) was observed in islets from normoglycaemic donors. This correlation paralleled findings in primary rat islets and INS-1 cells where overexpression of miR-187 markedly decreased GSIS without affecting insulin content or apoptotic index. Finally, the gene encoding homeodomain-interacting protein kinase-3 (HIPK3), a known regulator of insulin secretion, was identified as a direct target of miR-187 and displayed reduced expression in islets from individuals with type 2 diabetes. Our findings suggest a role for miR-187 in the blunting of insulin secretion, potentially involving regulation of HIPK3, which occurs during the pathogenesis of type 2 diabetes.

  5. Interleukin-1 beta-induced nitric oxide production from isolated rat islets is modulated by D-glucose and 3-isobutyl-1-methyl xanthine

    DEFF Research Database (Denmark)

    Andersen, H U; Mauricio, D; Karlsen, Allan Ertman

    1996-01-01

    Interleukin-1 beta has been proposed to cause selective beta-cell destruction via the induction of nitric oxide synthesis. The cytotoxic effect of interleukin-1 beta is modulated by the concentration of D-glucose in the medium. The aim of this study was to investigate if D-glucose-mediated modula......Interleukin-1 beta has been proposed to cause selective beta-cell destruction via the induction of nitric oxide synthesis. The cytotoxic effect of interleukin-1 beta is modulated by the concentration of D-glucose in the medium. The aim of this study was to investigate if D...... effects on acute insulin release was found at high (28 mmol/l) concentrations of D-glucose, and blocking nitrite production by the L-arginine analog aminoguanidine, which selectively inhibits the cytokine-inducible nitric oxide synthase, did not result in protection against the inhibitory action...... that could be reproduced by the cAMP analog dibutyryl cAMP. Addition of 3-isobutyl-1-methyl xanthine resulted in a threefold reduction in the mRNA level of interleukin-1 beta-induced inducible nitric oxide synthase. We conclude that interleukin-1 beta-induced islet nitric oxide synthesis is augmented by D...

  6. Intraportal islet transplantation: the impact of the liver microenvironment.

    Science.gov (United States)

    Delaune, Vaihere; Berney, Thierry; Lacotte, Stéphanie; Toso, Christian

    2017-03-01

    The portal vein remains the preferred site for pancreatic islet transplantation due to its easy access and low morbidity. However, despite great progress in isolation and transplantation protocols over the past few years, it is still associated with the early loss of some 50-70% of transplanted islets. The complex liver microenvironment itself presumably plays an important role in this loss. The present review focuses on the specifics of the liver microenvironment, notably the localized hepatic ischemia/reperfusion injury following transplantation, the low oxygenation of the portal vein, the instant blood-mediated inflammatory reaction, the endogenous liver immune system, and the gut-liver axis, and how they can each have an impact on the transplanted islets. It identifies the potential, or already applied, clinical interventions for improving intraportal islet survival, and pinpoints those promising areas still lacking preclinical research. Future interventions on clinical intraportal islet transplantation need to take into account the global context of the liver microenvironment, with multi-point interventions being most likely to improve early islet survival and engraftment. © 2017 The Authors. Transplant International published by John Wiley & Sons Ltd on behalf of Steunstichting ESOT.

  7. Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets in in vitro culture

    Institute of Scientific and Technical Information of China (English)

    Xiao-Bo Chen; Yong-Xiang Li; Yang Jiao; Wei-Ping Dong; Ge Li; Jing Chen; Jian-Ming Tan

    2007-01-01

    AIM:To ifivestigate the influence of heme oxygenase-1(HO-1)gene transfer on the viability and function of cultured rat islets in vitro.METHODS:Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion,and purified by discontinuous Ficoll density gradient centrifugation.Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene(Ad-HO-1)or enhanced green fluorescent protein gene(Ad-EGFP),and then cultured for seven days.Transfection was confirmed by fluorescence microscopy and Western blot.Islet viability was evaluated by acridine orange/propidium iodide fluorescent staining.Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets.Stimulation index (SI)was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation.RESULTS:After seven days culture,the viability of cultured rat islets decreased significantly(92% ± 6% vs 52% ± 13%,P < 0.05),and glucose-stimulated insulin release also decreased significantly(6.47 ± 0.55 mIU/L/30IEQ vs 4.57 ± 0.40 mIU/L/30IEQ,14.93 ± 1.17mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ,P < 0.05).Transfection of rat Islets with adenoviral vectors at an MOI of 20 was efficient,and did not impair islet function.At 7 d post-transfection,the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% ± 15% vs 52% ± 13%,P < 0.05).There was no significant difference in insulin release upon low glucose stimulation(2.8 mmol/L)among Ad-HO-1 transfected group,Ad-EGFP transfected group,and control group(P > 0.05),while when stimulated by high glucose(16.7 mmol/L)solution,insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group,respectively(12.50 ± 2.17 mIU/L/30IEQ vs 8.87 ± 0.65 mIU/L/30IEQ;12.50 ± 2.17 mIU/L/30IEQ vs 9.63 ± 0.71 mIU/L/30IEQ

  8. Embryonic and foetal Islet-1 positive cells in human hearts are also positive to c-Kit

    Directory of Open Access Journals (Sweden)

    C. Serradifalco

    2011-12-01

    Full Text Available During embryogenesis, the mammalian heart develops from a primitive heart tube originating from two bilateral primary heart fields located in the lateral plate mesoderm. Cells belongings to the pre-cardiac mesoderm will differentiate into early cardiac progenitors, which express early transcription factors which are also common to the Isl-1 positive cardiac progenitor cells isolated from the developing pharyngeal mesoderm and the foetal and post-natal mice hearts. A second population of cardiac progenitor cells positive to c-Kit has been abundantly isolated from adult hearts. Until now, these two populations have been considered two different sets of progenitor cells present in the heart in different stages of an individual life. In the present study we collected embryonic, foetal and infant hearts, and we tested the hypotheses that c-Kit positive cells, usually isolated from the adult heart, are also present in the intra-uterine life and persist in the adult heart after birth, and that foetal Isl-1 positive cells are also positive to c-Kit. Using immunohistochemistry we studied the temporal distribution of Isl-1 positive and c-Kit/CD105 double positive cells, and by immunofluorescence and confocal analysis we studied the co-localization of c-Kit and Isl-1 positive cells. The results indicated that cardiomyocytes and interstitial cells were positive for c-Kit from the 9th to the 19th gestational week, that cells positive for both c-Kit and CD105 appeared in the interstitium at the 17th gestational week and persisted in the postnatal age, and that the Isl-1 positive cells were a subset of the c-Kit positive population.

  9. MLVA genotyping of human Brucella isolates from Peru

    NARCIS (Netherlands)

    H.L. Smits; B. Espinosa; R. Castillo; E. Hall; A. Guillen; M. Zevaleta; R.H. Gilman; P. Melendez; C. Guerra; A. Draeger; A. Broglia; K. Nöckler

    2009-01-01

    Recent human Brucella melitensis isolates from Peru were genotyped by multiple locus variable number repeat analysis. All 24 isolates originated from hospitalized patients living in the central part of Peru and consisted of six genomic groups comprising two to four isolates and nine unique genotypes

  10. Islet expression of the DNA repair enzyme 8-oxoguanosine DNA glycosylase (Ogg1) in human type 2 diabetes

    OpenAIRE

    Yoon Kun-Ho; Wang-Rodriguez Jessica; Dib Sergio A.; Anachkov Kamen A; Tyrberg Björn; Levine Fred

    2002-01-01

    Abstract Background It has become increasingly clear that β-cell failure plays a critical role in the pathogenesis of type 2 diabetes. Free-radical mediated β-cell damage has been intensively studied in type 1 diabetes, but not in human type 2 diabetes. Therefore, we studied the protein expression of the DNA repair enzyme Ogg1 in pancreases from type 2 diabetics. Ogg1 was studied because it is the major enzyme involved in repairing 7,8-dihydro-8-oxoguanosine DNA adducts, a lesion previously o...

  11. IL-12p40 is not required for islet allograft rejection

    Institute of Scientific and Technical Information of China (English)

    En-guang BI; Wei SHI; Jia ZOU; Zhen-hua HAO; Zhen-hu LI; Duan CAI; Hua-qun ZHANG; Bing SUN

    2006-01-01

    Aim: To investigate whether IL-12p40 plays a crucial role in regulating islet allograft rejection in a streptozotocin (STZ)-induced diabetes mouse model. Methods: C57BL/6 and IL-12p40 gene knockout mice were selected as recipient mice, to which the diabetes was induced with a treatment of STZ (150-200 mg/kg) by a single ip injection. BALB/c mice were selected as donor mice and islet cells were isolated from the mice. The 500 islets were transplanted into recipient mice beneath the capsule of the left kidney. Following the islet transplantation the glucose from the mice sera was monitored and the rejection rate of islets was analyzed. Results: STZ could induce diabetes in the recipient mice within 1 week. After transplantation of allograft islets, the increased glucose in wild-type (WT) mice returned to normal level and was maintained for 10 d. Unexpectedly, the rejection rate of islet allograft between IL-12p40-deficient mice and WT mice was similar. Conclusion: The results suggested that, although islet allograft rejection is believed to be Th1-cell predominant, the Th1 response inducer, IL-12 and IL-23 are not essential to induce islet allograft rejection.

  12. Therapeutic Strategies to Increase Human β-Cell Growth and Proliferation by Regulating mTOR and GSK-3/β-Catenin Pathways.

    Science.gov (United States)

    Rohatgi, Nidhi; Remedi, Maria S; Kwon, Guim; Pappan, Kirk L; Marshall, Connie A; McDaniel, Michael L

    2010-01-01

    This perspective delineates approaches to develop therapeutic strategies to stimulate the proliferative potential of adult human β-cells in vitro. Previous findings demonstrated that nutrients, through regulation of mTOR signaling, promote regenerative processes including DNA synthesis, cell cycle progression and β-cell proliferation in rodent islets but rarely in human islets. Recently, we discovered that regulation of the Wnt/GSK-3/β-catenin pathway by directly inhibiting GSK-3 with pharmacologic agents, in combination with nutrient activation of mTOR, was required to increase growth and proliferation in human islets. Studies also revealed that nuclear translocation of β-catenin in response to GSK-3 inhibition regulated these processes and was rapamycin sensitive, indicating a role for mTOR. Human islets displayed a high level of insulin resistance consistent with the inability of exogenous insulin to activate Akt and engage the Wnt pathway by GSK-3 inhibition. This insulin resistance in human islets is not present in rodent islets and may explain the differential requirement in human islets to inhibit GSK-3 to enhance these regenerative processes. Human islets exhibited normal insulin secretion but a loss of insulin content, which was independent of all treatment conditions. The loss of insulin content may be related to insulin resistance, the isolation process or culture conditions. In this perspective, we provide strategies to enhance the proliferative capacity of adult human β-cells and highlight important differences between human and rodent islets: the lack of a nutrient response, requirement for direct GSK-3 inhibition, insulin resistance and loss of insulin content that emphasize the physiological significance of conducting studies in human islets.

  13. Adaptation of pancreatic islet cyto-architecture during development

    Science.gov (United States)

    Striegel, Deborah A.; Hara, Manami; Periwal, Vipul

    2016-04-01

    Plasma glucose in mammals is regulated by hormones secreted by the islets of Langerhans embedded in the exocrine pancreas. Islets consist of endocrine cells, primarily α, β, and δ cells, which secrete glucagon, insulin, and somatostatin, respectively. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Varying demands and available nutrients during development produce changes in the local connectivity of β cells in an islet. We showed in earlier work that graph theory provides a framework for the quantification of the seemingly stochastic cyto-architecture of β cells in an islet. To quantify the dynamics of endocrine connectivity during development requires a framework for characterizing changes in the probability distribution on the space of possible graphs, essentially a Fokker-Planck formalism on graphs. With large-scale imaging data for hundreds of thousands of islets containing millions of cells from human specimens, we show that this dynamics can be determined quantitatively. Requiring that rearrangement and cell addition processes match the observed dynamic developmental changes in quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that there is a transient shift in preferred connectivity for β cells between 1-35 weeks and 12-24 months.

  14. Islet cell development.

    Science.gov (United States)

    Rojas, Anabel; Khoo, Adrian; Tejedo, Juan R; Bedoya, Francisco J; Soria, Bernat; Martín, Franz

    2010-01-01

    Over the last years, there has been great success in driving stem cells toward insulin-expressing cells. However, the protocols developed to date have some limitations, such as low reliability and low insulin production. The most successful protocols used for generation of insulin-producing cells from stem cells mimic in vitro pancreatic organogenesis by directing the stem cells through stages that resemble several pancreatic developmental stages. Islet cell fate is coordinated by a complex network of inductive signals and regulatory transcription factors that, in a combinatorial way, determine pancreatic organ specification, differentiation, growth, and lineage. Together, these signals and factors direct the progression from multipotent progenitor cells to mature pancreatic cells. Later in development and adult life, several of these factors also contribute to maintain the differentiated phenotype of islet cells. A detailed understanding of the processes that operate in the pancreas during embryogenesis will help us to develop a suitable source of cells for diabetes therapy. In this chapter, we will discuss the main transcription factors involved in pancreas specification and beta-cell formation.

  15. Protection of rat islet viability following heme oxygenase-1 gene transfection via adenoviral vector in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiaobo Chen; Yongxiang Li; Weiping Dong; Yang Jiao; Jianming Tan

    2007-01-01

    Objective: To investigate the effect of Heme oxygenase-1 (HO-1) gene transfection on the viability of cultured rat islets, and to explore the potential value of HO-1 gene in islet transplantation. Methods:Recombinant adenovirus vector containing human HO-1 gene(Ad-HO-1 ) or enhanced green fluorescent protein gene(Ad-EGFP) was generated by using AdEasy system respectively.The rat islets were transfected with Ad-HO-1, Ad-EGFP or blank vector and then cultured for 7 days. Transfection was confirmed by expression of EGFP and human HO-1 protein detected by fluorescence photographs and western blot, respectively. The insulin release upon different concentration of glucose stimulation was detected using insulin radioimmunoassay kit, and stimulation index (SI) was calculated. Glucose-stimulated insulin release was usedto assess islet viability. Results:Adenovirus vector successfully transferred HO-1 gene to rat islet cells in vitro, and the insulin release upon high level of glucose stimulation and stimulation index(SI) of Ad-HO-1-infected islets were significantly higher than those of Ad-EGFP-infected islets and control islets(P < 0.05).Conclusion: Adenovirus-mediated HO-1 gene transfection is a feasible strategy to confer cytoprotection and therefore protect the viability of cultured rat islets.

  16. Macro-or microencapsulation of pig islets to cure type 1 diabetes

    Institute of Scientific and Technical Information of China (English)

    Denis Dufrane; Pierre Gianello

    2012-01-01

    Although allogeneic islet transplantation can successfully cure type 1 diabetes,it has limited applicability.For example,organs are in short supply; several human pancreas donors are often needed to treat one diabetic recipient; the intrahepatic site may not be the most appropriate site for islet implantation; and immunosuppressive regimens,which are associated with side effects,are often required to prolong survival of the islet graft.An altemative source of insulinproducing cells would therefore be of major interest.Pigs represent a possible alternative source of beta cells.Grafting of pig islets may appear difficult because of the immunologic species barrier,but pig islets have been shown to function in primates for at least 6 mo with clinically incompatible immunosuppression.Therefore,a bioartificial pancreas made of encapsulated pig islets may resolve issues associated with islet allotransplantation.Although several groups have shown that encapsulated pig islets are functional in small-animal models,less is known about the use of bioartificial pancreases in large-animal models.In this review,we summarize current knowledge of encapsulated pig islets,to determine obstacles to implantation in humans and possible solutions to overcome these obstacles.

  17. Nucleotide sequencing, cloning, and expression of Capra hircus Heme Oxygenase-1 in caprine islets to promote insulin secretion in vitro.

    Science.gov (United States)

    Vakhshiteh, Faezeh; Allaudin, Zeenathul Nazariah; Lila, Mohd Azmi B Mohd; Abbasiliasi, Sahar; Ajdari, Zahra

    2015-01-01

    Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.

  18. New Insight in Copper-Ion Binding to Human Islet Amyloid: The Contribution of Metal-Complex Speciation To Reveal the Polypeptide Toxicity.

    Science.gov (United States)

    Magrì, Antonio; La Mendola, Diego; Nicoletti, Vincenzo Giuseppe; Pappalardo, Giuseppe; Rizzarelli, Enrico

    2016-09-05

    Type-2 diabetes (T2D) is considered to be a potential threat on a global level. Recently, T2D has been listed as a misfolding disease, such as Alzheimer's and Parkinson's diseases. Human islet amyloid polypeptide (hIAPP) is a molecule cosecreted in pancreatic β cells and represents the main constituent of an aggregated amyloid found in individuals affected by T2D. The trace-element serum level is significantly influenced during the development of diabetes. In particular, the dys-homeostasis of Cu(2+) ions may adversely affect the course of the disease. Conflicting results have been reported on the protective role played by complex species formed by Cu(2+) ions with hIAPP or its peptide fragments in vitro. The histidine (His) residue at position 18 represents the main binding site for the metal ion, but contrasting results have been reported on other residues involved in metal-ion coordination, in particular those toward the N or C terminus. Sequences that encompass regions 17-29 and 14-22 were used to discriminate between the two models of the hIAPP coordination mode. Due to poor solubility in water, poly(ethylene glycol) (PEG) derivatives were synthesized. A peptide fragment that encompasses the 17-29 region of rat amylin (rIAPP) in which the arginine residue at position 18 was substituted by a histidine residue was also obtained to assess that the PEG moiety does not alter the peptide secondary structure. The complex species formed by Cu(2+) ions with Ac-PEG-hIAPP(17-29)-NH2 , Ac-rIAPP(17-29)R18H-NH2 , and Ac-PEG-hIAPP(14-22)-NH2 were studied by using potentiometric titrations coupled with spectroscopic methods (UV/Vis, circular dichroism, and EPR). The combined thermodynamic and spectroscopic approach allowed us to demonstrate that hIAPP is able to bind Cu(2+) ions starting from the His18 imidazole nitrogen atom toward the N-terminus domain. The stability constants of copper(II) complexes with Ac-PEG-hIAPP(14-22)-NH2 were used to simulate the different

  19. Isolation and characterization of human spermatogonial stem cells

    Directory of Open Access Journals (Sweden)

    Liu Shixue

    2011-10-01

    Full Text Available Abstract Background To isolate and characterization of human spermatogonial stem cells from stem spermatogonium. Methods The disassociation of spermatogonial stem cells (SSCs were performed using enzymatic digestion of type I collagenase and trypsin. The SSCs were isolated by using Percoll density gradient centrifugation, followed by differential surface-attachment method. Octamer-4(OCT4-positive SSC cells were further identified using immunofluorescence staining and flow cytometry technques. The purity of the human SSCs was also determined, and a co-culture system for SSCs and Sertoli cells was established. Results The cell viability was 91.07% for the suspension of human spermatogonial stem cells dissociated using a two-step enzymatic digestion process. The cells isolated from Percoll density gradient coupled with differential surface-attachement purification were OCT4 positive, indicating the cells were human spermatogonial stem cells. The purity of isolated human spermatogonial stem cells was 86.7% as assessed by flow cytometry. The isolated SSCs were shown to form stable human spermatogonial stem cell colonies on the feeder layer of the Sertoli cells. Conclusions The two-step enzyme digestion (by type I collagenase and trypsin process is an economical, simple and reproducible technique for isolating human spermatogonial stem cells. With little contamination and less cell damage, this method facilitates isolated human spermatogonial stem cells to form a stable cell colony on the supporting cell layer.

  20. Advances in pancreatic islet transplantation for the treatment of diabetes%胰岛移植治疗糖尿病的现状和进展

    Institute of Scientific and Technical Information of China (English)

    彭丹凤; 贾伟平

    2012-01-01

    胰岛移植是治疗糖尿病尤其是1型糖尿病的一种简单有效的方法,相较与胰腺移植,它较为简单和方便,但存在组织来源匮乏和免疫移植排斥等障碍.新的胰岛分离纯化方法提高了供移植的胰岛的纯度和活性.成体干细胞研究、异种移植研究,有望解决移植的供源问题.Edmonton方案在胰岛移植的临床应用中具有里程碑意义.新型的免疫抑制剂和免疫诱导剂的研究可以提高临床胰岛移植的成功率.%Objective Pancreatic islet transplantation is effective in treating diabetes, especially in type 1 diabetes. It can provide diabetes management with good glycemic control and insulin independence. Compared to pancreas transplantation, islet transplantation is technically much simplier and safer. However, currently its clinical use is highly restricted by a series of influence factors, including lack of sufficient donor organs and the side effects of immunosuppressive therapy. With recent advances in methods of islet isolation and purification, we can get better donor organs. Deriving islet cells from other sources such as pigs, human pancreatic duct cells, fetal pancreatic stem cells, and embryonic stem cells will overcome shortage of donor organs. The use of the Edmonton protocol has been proved to be the key procedure of clinical islet transplantation. And study of new immunosuppressive drugs and immunomodulators can provide higher rate of success for clinical islet transplantation.

  1. Acceleration of Functional Maturation and Differentiation of Neonatal Porcine Islet Cell Monolayers Shortly In Vitro Cocultured with Microencapsulated Sertoli Cells

    Directory of Open Access Journals (Sweden)

    Francesca Mancuso

    2010-01-01

    Full Text Available The limited availability of cadaveric human donor pancreata as well as the incomplete success of the Edmonton protocol for human islet allografts fasten search for new sources of insulin the producing cells for substitution cell therapy of insulin-dependent diabetes mellitus (T1DM. Starting from isolated neonatal porcine pancreatic islets (NPIs, we have obtained cell monolayers that were exposed to microencapsulated monolayered Sertoli cells (ESCs for different time periods (7, 14, 21 days. To assess the development of the cocultured cell monolayers, we have studied either endocrine cell phenotype differentiation markers or c-kit, a hematopoietic stem cell marker, has recently been involved with growth and differentiation of β-cell subpopulations in human as well as rodent animal models. ESC which were found to either accelerate maturation and differentiation of the NPIs β-cell phenotype or identify an islet cell subpopulation that was marked positively for c-kit. The insulin/c-kit positive cells might represent a new, still unknown functionally immature β-cell like element in the porcine pancreas. Acceleration of maturation and differentiation of our NPI cell monolayers might generate a potential new opportunity to develop insulin-producing cells that may suite experimental trials for cell therapy of T1DM.

  2. Islet Xenotransplantation and Xeno-antigenicity: studies in a preclinical model

    NARCIS (Netherlands)

    P.P.M. Rood (Pleunie)

    2008-01-01

    textabstractShortage of human donor organs is the major limiting factor for clinical islet allotransplantation. Xenotransplantation, using the pig as the source of islets is considered a potential solution to this problem. Since the development of pigs homozygous for α1,3-galactosyltransfer

  3. The role of the disulfide bond in the interaction of islet amyloid polypeptide with membranes

    NARCIS (Netherlands)

    Khemtemourian, L.P.; Engel, M.F.M.; Kruijtzer, J.A.W.; Hoppener, J.W.M.; Liskamp, R.M.J.; Killian, J.A.

    2010-01-01

    Human islet amyloid polypeptide (hIAPP) forms amyloid fibrils in pancreatic islets of patients with type 2 diabetes mellitus. It has been suggested that the N-terminal part, which contains a conserved intramolecular disulfide bond between residues 2 and 7, interacts with membranes, ultimately

  4. An imidazoline compound completely counteracts interleukin-1[beta] toxic effects to rat pancreatic islet [beta] cells

    DEFF Research Database (Denmark)

    Papaccio, Gianpaolo; Nicoletti, Ferdinando; Pisanti, Francesco A

    2002-01-01

    In vitro studies have demonstrated that interleukin (IL)-1beta decreases insulin and DNA contents in pancreatic islet beta cells, causing structural damage, that it is toxic to cultured human islet beta cells and that it is able to induce apoptosis in these cells....

  5. Recent insights in islet amyloid polypeptide-induced membrane disruption and its role in β-cell death in type II diabetes mellitus

    NARCIS (Netherlands)

    Khemtémourian, L.P.|info:eu-repo/dai/nl/304824372; Killian, J.A.|info:eu-repo/dai/nl/071792317; Höppener, J.W.M.; Engel, M.F.M.|info:eu-repo/dai/nl/263614190

    2008-01-01

    The presence of fibrillar protein deposits (amyloid) of human islet amyloid polypeptide (hIAPP) in the pancreatic islets of Langerhans is thought to be related to death of the insulin-producing islet β-cells in type 2 diabetes mellitus (DM2). The mechanism of hIAPP-induced β-cell death is not

  6. Immunosuppression for islet transplantation.

    Directory of Open Access Journals (Sweden)

    Noguchi,Hirofumi

    2006-04-01

    Full Text Available The development by the Edmonton group of a sirolimus-based, steroid-free, low-tacrolimus regimen is a significant breakthrough that allows the rate of insulin independence after islet transplantation to increase from 13% to 80% at 1 year ; however, the rate is reduced to 50% at 3 years, attributed to prolonged tacrolimus exposure. Recently, immunosuppression agents such as cyclosporine, mycophenolate mofetil, and the novel agent FTY 720 have been used instead of tacrolimus. Lymphocytedepleting antibodies such as anti-thymocyte globulin, alemtuzumab, and hOKT3gamma 1 (ala, ala have been launched, and a costimulatory blockade of anti-CD40 monoclonal antibodies and CTLA4-Ig will be attempted in the near future. Moreover, the potential of a novel immunosuppressing peptide could now be realized using new technology called the protein transduction system. In this review, we show some of the most recent contributions to the advancement of knowledge in this field.

  7. Thermolysin in human cultured keratinocyte isolation

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    A. Gragnani

    Full Text Available BACKGROUND: When treating extensively burned patients using cultured epidermal sheets, the main problem is the time required for its production. Conventional keratinocyte isolation is usually done using Trypsin. We used a modification of the conventional isolation method in order to improve this process and increase the number of colonies from the isolated epidermal cell population. PURPOSE: To compare the action of trypsin and thermolysin in the keratinocyte isolation using newborn foreskin. METHODS: This method used thermolysin as it selectively digests the dermo-epidermal junction. After dermis separation, the epidermis was digested by trypsin in order to obtain a cell suspension. RESULTS: Compared to the conventional procedure, these experiments demonstrated that in the thermolysin group, the epidermis was easily detached from the dermis, there was no fibroblast contamination and there were a larger number of keratinocyte colonies which had a significant statistical difference. CONCLUSION: The number of colonies in the thermolysin group was significantly greater than in the trypsin group.

  8. Electrofusion of mesenchymal stem cells and islet cells for diabetes therapy: a rat model.

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    Goichi Yanai

    Full Text Available Islet transplantation is a minimally invasive treatment for severe diabetes. However, it often requires multiple donors to accomplish insulin-independence and the long-term results are not yet satisfying. Therefore, novel ways to overcome these problems have been explored. Isolated islets are fragile and susceptible to pro-apoptotic factors and poorly proliferative. In contrast, mesenchymal stem cells (MSCs are highly proliferative, anti-apoptotic and pluripotent to differentiate toward various cell types, promote angiogenesis and modulate inflammation, thereby studied as an enhancer of islet function and engraftment. Electrofusion is an efficient method of cell fusion and nuclear reprogramming occurs in hybrid cells between different cell types. Therefore, we hypothesized that electrofusion between MSC and islet cells may yield robust islet cells for diabetes therapy. We establish a method of electrofusion between dispersed islet cells and MSCs in rats. The fusion cells maintained glucose-responsive insulin release for 20 days in vitro. Renal subcapsular transplantation of fusion cells prepared from suboptimal islet mass (1,000 islets that did not correct hyperglycemia even if co-transplanted with MSCs, caused slow but consistent lowering of blood glucose with significant weight gain within the observation period in streptozotocin-induced diabetic rats. In the fusion cells between rat islet cells and mouse MSCs, RT-PCR showed new expression of both rat MSC-related genes and mouse β-cell-related genes, indicating bidirectional reprogramming of both β-cell and MSCs nuclei. Moreover, decreased caspase3 expression and new expression of Ki-67 in the islet cell nuclei suggested alleviated apoptosis and gain of proliferative capability, respectively. These results show that electrofusion between MSCs and islet cells yield special cells with β-cell function and robustness of MSCs and seems feasible for novel therapeutic strategy for diabetes

  9. 1,25-Dihydroxyvitamin D/sub 3/ target cells in immature pancreatic islets

    Energy Technology Data Exchange (ETDEWEB)

    Clark, S.A.; Stumpf, W.E.; Sar, M.; DeLuca, H.F.

    1987-07-01

    Target cells of 1,25-dihydroxyvitamin D/sub 3/ were identified by autoradiography in islets from rats of different ages. Nuclei of pancreatic islet cells selectively concentrated 1,25-(/sup 3/H)dihydroxyvitamin D/sub 3/ but not 25-(/sup 3/H)hydroxyvitamin D/sub 3/ or 24,25-(/sup 3/H)dihydroxyvitamin D/sub 3/. Developmental studies of pancreatic islets indicated that target cells, as revealed by significant nuclear concentration of 1,25-(/sup 3/H)dihydroxyvitamin D/sub 3/, are present in islet cells of fetal rats. The percentage of islet cells that concentrated 1,25-(/sup 3/H)dihydroxyvitamin D/sub 3/ increased from 10 to 15% in the fetus to 60% at 1 day of age. Immunocytochemical staining indicated that insulin-containing cells but not glucagon or somatostatin cells concentrated 1,25-(/sup 3/H)dihydroxyvitamin D/sub 3/. Peak uptake of 1,25(/sup 3/H) dihydroxyvitamin D/sub 3/ was calculated to be 400 pmol/mg DNA, with no significant difference in nuclear accumulation between islets cells from neonatal and adult rats or between islets in vivo and isolated islets in vitro. The results of these studies indicate that (1) 1,25-(/sup 3/H)dihydroxyvitamin D/sub 3/ target cells are present in islets before pancreatic ..beta..-cells are morphologically or functionally mature; (2) islet ..beta..-cells concentrate 1,25-dihydroxyvitamin D/sub 3/, but not 25-hydroxyvitamin D/sub 3/ or 24,25-dihydroxyvitamin D/sub 3/. The authors conclude that only the 1,25-dihydroxyvitamin D/sub 3/ metabolite of vitamin D is accumulated by nuclei of developing and mature ..beta..-cells and suggest that 1,25-dihydroxyvitamin D/sub 3/ plays a role in the maturation of islet ..beta..-cells.

  10. Distinct cell clusters touching islet cells induce islet cell replication in association with over-expression of Regenerating Gene (REG protein in fulminant type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    Kaoru Aida

    Full Text Available BACKGROUND: Pancreatic islet endocrine cell-supporting architectures, including islet encapsulating basement membranes (BMs, extracellular matrix (ECM, and possible cell clusters, are unclear. PROCEDURES: The architectures around islet cell clusters, including BMs, ECM, and pancreatic acinar-like cell clusters, were studied in the non-diabetic state and in the inflamed milieu of fulminant type 1 diabetes in humans. RESULT: Immunohistochemical and electron microscopy analyses demonstrated that human islet cell clusters and acinar-like cell clusters adhere directly to each other with desmosomal structures and coated-pit-like structures between the two cell clusters. The two cell-clusters are encapsulated by a continuous capsule composed of common BMs/ECM. The acinar-like cell clusters have vesicles containing regenerating (REG Iα protein. The vesicles containing REG Iα protein are directly secreted to islet cells. In the inflamed milieu of fulminant type 1 diabetes, the acinar-like cell clusters over-expressed REG Iα protein. Islet endocrine cells, including beta-cells and non-beta cells, which were packed with the acinar-like cell clusters, show self-replication with a markedly increased number of Ki67-positive cells. CONCLUSION: The acinar-like cell clusters touching islet endocrine cells are distinct, because the cell clusters are packed with pancreatic islet clusters and surrounded by common BMs/ECM. Furthermore, the acinar-like cell clusters express REG Iα protein and secrete directly to neighboring islet endocrine cells in the non-diabetic state, and the cell clusters over-express REG Iα in the inflamed milieu of fulminant type 1 diabetes with marked self-replication of islet cells.

  11. Isolation of Campylobacter from human stool samples

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    S M Salim

    2014-01-01

    Full Text Available Context: Campylobacter is an undetected cause of diarrhoea especially under 5 years of age in most of the countries. Isolation of this organism is difficult, expensive and cumbersome. Aims: Our objective of this study was to isolate this pathogen from the stool specimens on routinely available blood containing laboratory media using the candle jar for creating the microaerophilic atmosphere in our setup. Settings and Designs: A descriptive study. Materials and Methods: A total of 50 stool samples were inoculated onto selective and non-selective media with and without filtration using a 0.45 μm membrane. The inoculated media were simultaneously incubated in microaerophilic conditions using the Anoxomat as well as in candle jars at temperatures 37°C and 42°C. The culture isolates were confirmed by standard phenotypic tests. A simplex polymerase chain reaction (PCR targeting the 16S ribosomal deoxyribonucleic acid of Campylobacter was performed on the deoxyribonucleic acid (DNA of the culture isolates as well as on the DNA extracted from the stool filtrates. Statistical Analysis: Data was expressed as a proportion. Results: Campylobacter could be isolated in 5 out of 50 stool samples using both the Anoxomat as well as the candle jar. Furthermore, we did not find any difference between the isolation using the selective and blood containing media as well as the different incubation temperatures. All the five were confirmed phenotypically and genotypically to be Campylobacter jejuni. The PCR results corroborated with that of the culture. Conclusions: Isolation by culture was as sensitive as that of the PCR.

  12. Amiloride derivatives enhance insulin release in pancreatic islets from diabetic mice

    Directory of Open Access Journals (Sweden)

    Head W Steven

    2005-12-01

    Full Text Available Abstract Background Amiloride derivatives, commonly used for their diuretic and antihypertensive properties, can also cause a sustained but reversible decrease of intracellular pH (pHi. Using dimethyl amiloride (DMA on normal rodent pancreatic islets, we previously demonstrated the critical influence of islet pHi on insulin secretion. Nutrient-stimulated insulin secretion (NSIS requires a specific pHi-range, and is dramatically enhanced by forced intracellular acidification with DMA. Furthermore, DMA can enable certain non-secretagogues to stimulate insulin secretion, and induce time-dependent potentiation (TDP of insulin release in mouse islets where this function is normally absent. The present study was performed to determine whether pHi-manipulation could correct the secretory defect in islets isolated from mice with type 2 diabetes. Methods Using two mouse models of type 2 diabetes, we compared a pHi-regulation, and b NSIS with and without treatment with amiloride derivatives, in islets isolated from diabetic mice and wild type mice. Results A majority of the islets from the diabetic mice showed a slightly elevated basal pHi and/or poor recovery from acid/base load. DMA treatment produced a significant increase of NSIS in islets from the diabetic models. DMA also enabled glucose to induce TDP in the islets from diabetic mice, albeit to a lesser degree than in normal islets. Conclusion Islets from diabetic mice show some mis-regulation of intracellular pH, and their secretory capacity is consistently enhanced by DMA/amiloride. Thus, amiloride derivatives show promise as potential therapeutic agents for type 2 diabetes.

  13. Short-term glucosamine infusion increases islet blood flow in anesthetized rats.

    Science.gov (United States)

    Gao, Xiang; Jansson, Leif; Persson, A Erik G; Sandberg, Monica

    2013-01-01

    Impaired glucose tolerance and type 2 diabetes in rodents are associated with increased islet blood flow. If this is important for modulation of the endocrine function is at present unknown. We evaluated if glucosamine infusion, which induces peripheral insulin resistance and glucose intolerance, could be used to acutely increase islet blood flow. We infused anaesthetized Sprague-Dawley rats for 2 h with glucosamine (6 mg/kg body weight), in some cases followed by glucose administration. The former induced a 2-fold increase in serum insulin concentrations while plasma glucose remained unchanged. In vitro an augmented insulin response to hyperglycemia and decreased insulin content in batch type islet incubations with glucosamine for 24 h were seen. After 2 h glucosamine exposure in vitro, insulin release was decreased. In vivo glucosamine infusion increased islet blood flow, without affecting other regional blood flow values. Glucose increased islet blood flow to the same extent in control and glucosamine-infused rats. When exposed to 10 mmol/L glucosamine arterioles of isolated perfused islets showed a 10% dilation of their vascular smooth muscle. Thus, application of this model leads to acute hyperinsulinemia in vivo but a decreased insulin release in vitro, which suggests that effects not located to β cells are responsible for the effects seen in vivo. An increased islet blood flow in previously healthy animals was also seen after glucose administration, which can be used to further dissect the importance of blood flow changes in islet function.

  14. Anaerococcus nagyae sp. nov., isolated from human clinical specimens

    NARCIS (Netherlands)

    Veloo, A C M; Vries , de E. D.; Jean-Pierre, H; van Winkelhoff, A J

    We describe a new Anaerococcus species isolated from human clinical specimens. Analyses of 16S rRNA gene sequences of three strains showed <98% similarity with its closest relative Anaerococcus octavius. Phylogenetically the isolated strains form a cluster and can be differentiated from other

  15. Anaerococcus degenerii sp nov., isolated from human clinical specimens

    NARCIS (Netherlands)

    Veloo, A. C. M.; Elgersma, P. E.; van Winkelhoff, A. J.

    Four clinical isolates of gram-positive strict anaerobic cocci were isolated from four different human mixed anaerobic infections. The taxonomical status of the four strains could not be established using standard identification techniques. The biochemical features of the strains were established

  16. Quantitative Assessment of Islets of Langerhans Encapsulated in Alginate

    Science.gov (United States)

    Johnson, Amy S.; O'Sullivan, Esther; D'Aoust, Laura N.; Omer, Abdulkadir; Bonner-Weir, Susan; Fisher, Robert J.; Weir, Gordon C.

    2011-01-01

    Improved methods have recently been developed for assessing islet viability and quantity in human islet preparations for transplantation, and these measurements have proven useful for predicting transplantation outcome. The objectives of this study were to adapt these methods for use with microencapsulated islets, to verify that they provide meaningful quantitative measurements, and to test them with two model systems: (1) barium alginate and (2) barium alginate containing a 70% (w/v) perfluorocarbon (PFC) emulsion, which presents challenges to use of these assays and is of interest in its own right as a means for reducing oxygen supply limitations to encapsulated tissue. Mitochondrial function was assessed by oxygen consumption rate measurements, and the analysis of data was modified to account for the increased solubility of oxygen in the PFC-alginate capsules. Capsules were dissolved and tissue recovered for nuclei counting to measure the number of cells. Capsule volume was determined from alginate or PFC content and used to normalize measurements. After low oxygen culture for 2 days, islets in normal alginate lost substantial viable tissue and displayed necrotic cores, whereas most of the original oxygen consumption rate was recovered with PFC alginate, and little necrosis was observed. All nuclei were recovered with normal alginate, but some nuclei from nonrespiring cells were lost with PFC alginate. Biocompatibility tests revealed toxicity at the islet periphery associated with the lipid emulsion used to provide surfactants during the emulsification process. We conclude that these new assay methods can be applied to islets encapsulated in materials as complex as PFC-alginate. Measurements made with these materials revealed that enhancement of oxygen permeability of the encapsulating material with a concentrated PFC emulsion improves survival of encapsulated islets under hypoxic conditions, but reformulation of the PFC emulsion is needed to reduce toxicity

  17. Unraveling pancreatic islet biology by quantitative proteomics

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, Jianying; Dann, Geoffrey P.; Liew, Chong W.; Smith, Richard D.; Kulkarni, Rohit N.; Qian, Weijun

    2011-08-01

    The pancreatic islets of Langerhans play a critical role in maintaining blood glucose homeostasis by secreting insulin and several other important peptide hormones. Impaired insulin secretion due to islet dysfunction is linked to the pathogenesis underlying both Type 1 and Type 2 diabetes. Over the past 5 years, emerging proteomic technologies have been applied to dissect the signaling pathways that regulate islet functions and gain an understanding of the mechanisms of islet dysfunction relevant to diabetes. Herein, we briefly review some of the recent quantitative proteomic studies involving pancreatic islets geared towards gaining a better understanding of islet biology relevant to metabolic diseases.

  18. That which does not kill us makes us stronger--does Nietzsche's quote apply to islets? A re-evaluation of the passenger leukocyte theory, free radicals, and glucose toxicity in islet cell transplantation.

    Science.gov (United States)

    Wright, J R; Xu, B-Y

    2014-07-01

    In clinical islet transplantation, isolated islets are embolized into the liver via the portal vein (PV); however, up to 70% of the islets are lost in the first few days after transplantation (i.e., too quickly to be mediated by the adaptive immune system). Part of early loss is due to instant blood-mediated inflammatory reaction, an immune/thrombotic process caused by islets interacting with complement. We have shown that glucose toxicity (GT) also plays a critical role based upon the observation that islets embolized into the PVs of diabetic athymic mice are rapidly lost but, if recipients are not diabetic, the islet grafts persist. Using donor islets resistant to the β-cell toxin streptozotocin, we have shown that intraportal islets engrafted in non-diabetic athymic mice for as little as 3 days will maintain normoglycemia when streptozotocin is administered destroying the recipient's native pancreas β-cells. What is the mechanism of GT in β-cells? Chronic exposure to hyperglycemia over-exerts β-cells and their electron transport chains leak superoxide radicals during aerobic metabolism. Here we reinterpret old data and present some compelling new data supporting a new model of early intraportal islet graft loss. We hypothesize that diabetes stimulates overproduction of superoxide in both the β-cells of the islet grafts and the endothelial cells lining the intraportal microvasculature adjacent to where the embolized islets become lodged. This double dose of oxidant damage stresses both the islets, which are highly susceptible to free radicals because of inherent low levels of scavenging enzymes, and the adjacent hepatic endothelial cells. This, superimposed upon localized endothelial damage caused by embolization, precipitates inflammation and coagulation which further damages islet grafts. Based upon this model, we predict that pre-exposing islets to sub-lethal hyperoxia should up-regulate islet free radical scavenging enzyme levels and promote initial

  19. B7-H4 as a protective shield for pancreatic islet beta cells

    Institute of Scientific and Technical Information of China (English)

    Annika; C; Sun; Dawei; Ou; Dan; S; Luciani; Garth; L; Warnock

    2014-01-01

    Auto- and alloreactive T cells are major culprits that damage β-cells in type 1 diabetes(T1D) and islet transplantation. Current immunosuppressive drugs can alleviate immune-mediated attacks on islets. T cell co-stimulation blockade has shown great promise in autoimmunity and transplantation as it solely targets activated T cells, and therefore avoids toxicity of current immunosuppressive drugs. An attractive approach is offered by the newly-identified negative T cell cosignaling molecule B7-H4 which is expressed in normal human islets, and its expression co-localizes with insulin. A concomitant decrease in B7-H4/insulin colocalization is observed in human type 1 diabetic islets. B7-H4 may play protective roles in the pancreatic islets, preserving their function and survival. In this review we outline the protective effect of B7-H4 in the contexts of T1 D, islet cell transplantation, and potentially type 2 diabetes. Current evidence offers encouraging data regarding the role of B7-H4 in reversal of autoimmune diabetes and donor-specific islet allograft tolerance. Additionally, unique expression of B7-H4 may serve as a potential biomarker for the development of T1 D. Futurestudies should continue to focus on the islet-specific effects of B7-H4 with emphasis on mechanistic pathways in order to promote B7-H4 as a potential therapy and cure for T1 D.

  20. First isolation of Streptococcus downei from human dental plaques.

    Science.gov (United States)

    Yoo, So Young; Kim, Kwan-Joong; Lim, Seong-Hoon; Kim, Kwang-Won; Hwang, Ho-Keel; Min, Byung-Moo; Choe, Son-Jin; Kook, Joong-Ki

    2005-08-15

    In this study, we isolated four bacterial strains grown on mitis-salivarius sucrose bacitracin agar. The strains had similar biochemical characteristics to biotypes I or II of mutans streptococci. The four isolates were identified as Streptococcus downei by 16S rDNA and dextranase gene (dex) sequencing as well as polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) targeting dex. To our knowledge, this is the first report of the isolation and identification of S. downei from dental plaque in humans. The results suggest that S. downei can inhabit the human oral cavity.

  1. Comparison of Staphylococcus aureus isolates associated with food intoxication with isolates from human nasal carriers and human infections.

    Science.gov (United States)

    Wattinger, L; Stephan, R; Layer, F; Johler, S

    2012-04-01

    Staphylococcus aureus represents an organism of striking versatility. While asymptomatic nasal colonization is widespread, it can also cause serious infections, toxinoses and life-threatening illnesses in humans and animals. Staphylococcal food poisoning (SFP), one of the most prevalent causes of foodborne intoxication worldwide, results from oral intake of staphylococcal enterotoxins leading to violent vomiting, diarrhea and cramps shortly upon ingestion. The aim of the present study was to compare isolates associated with SFP to isolates collected from cases of human nasal colonization and clinical infections in order to investigate the role of S. aureus colonizing and infecting humans as a possible source of SFP. Spa typing and DNA microarray profiling were used to characterize a total of 120 isolates, comprising 50 isolates collected from the anterior nares of healthy donors, 50 isolates obtained from cases of clinical infections in humans and 20 isolates related to outbreaks of staphylococcal food poisoning. Several common spa types were found among isolates of all three sources (t015, t018, t056, t084). DNA microarray results showed highly similar virulence gene profiles for isolates from all tested sources. These results suggest contamination of foodstuff with S. aureus colonizing and infecting food handlers to represent a source of SFP.

  2. Fetal endocannabinoids orchestrate the organization of pancreatic islet microarchitecture.

    Science.gov (United States)

    Malenczyk, Katarzyna; Keimpema, Erik; Piscitelli, Fabiana; Calvigioni, Daniela; Björklund, Peyman; Mackie, Kenneth; Di Marzo, Vincenzo; Hökfelt, Tomas G M; Dobrzyn, Agnieszka; Harkany, Tibor

    2015-11-10

    Endocannabinoids are implicated in the control of glucose utilization and energy homeostasis by orchestrating pancreatic hormone release. Moreover, in some cell niches, endocannabinoids regulate cell proliferation, fate determination, and migration. Nevertheless, endocannabinoid contributions to the development of the endocrine pancreas remain unknown. Here, we show that α cells produce the endocannabinoid 2-arachidonoylglycerol (2-AG) in mouse fetuses and human pancreatic islets, which primes the recruitment of β cells by CB1 cannabinoid receptor (CB1R) engagement. Using subtractive pharmacology, we extend these findings to anandamide, a promiscuous endocannabinoid/endovanilloid ligand, which impacts both the determination of islet size by cell proliferation and α/β cell sorting by differential activation of transient receptor potential cation channel subfamily V member 1 (TRPV1) and CB1Rs. Accordingly, genetic disruption of TRPV1 channels increases islet size whereas CB1R knockout augments cellular heterogeneity and favors insulin over glucagon release. Dietary enrichment in ω-3 fatty acids during pregnancy and lactation in mice, which permanently reduces endocannabinoid levels in the offspring, phenocopies CB1R(-/-) islet microstructure and improves coordinated hormone secretion. Overall, our data mechanistically link endocannabinoids to cell proliferation and sorting during pancreatic islet formation, as well as to life-long programming of hormonal determinants of glucose homeostasis.

  3. Application of Rotating Wall Vessel (RWV) Cell Culture for Pancreas Islet Cell Transplantation

    Science.gov (United States)

    Rutzky, Lynne P.

    1998-01-01

    Type I insulin-dependent diabetes mellitus (IDDM) remains a major cause of morbidity and mortality in both pediatric and adult populations, despite significant advances in medical management. While insulin therapy treats symptoms of acute diabetes, it fails to prevent chronic complications such as microvascular disease, blindness, neuropathy, and chronic renal failure. Strict control of blood glucose concentrations delays but does not prevent the onset and progression of secondary complications. Although, whole pancreas transplantation restores physiological blood glucose levels, a continuous process of allograft rejection causes vascular and exocrine-related complications. Recent advances in methods for isolation and purification of pancreatic islets make transplantation of islet allografts an attractive alternative to whole pancreas transplantation. However, immunosuppressive drugs are necessary to prevent rejection of islet allografts and many of these drugs are known to be toxic to the islets. Since auto-transplants of isolated islets following total pancreatectomy survive and function in vivo, it is apparent that a major obstacle to successful clinical islet transplantation is the immunogenicity of the islet allografts.

  4. Romboutsia timonensis, a new species isolated from human gut

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    D. Ricaboni

    2016-07-01

    Full Text Available The exploration of the human microbiome was recently revolutionized by microbial culturomics and taxonogenomics. Thanks to this approach, we report here the main characteristics of Romboutsia timonensis strain Marseille-P326, a new bacterium isolated from the right human colon by colonoscopy in a 63-year-old French man with severe anaemia with melaena.

  5. Isolation and characterization of human rhinovirus antigenic variants

    Energy Technology Data Exchange (ETDEWEB)

    Watson, D.G.

    1985-01-01

    Isolation of antigenic variants of human rhinovirus types 2, 14, and 17 was attempted by plaquing untreated virus (P-isolates), selecting variants in the presence of homologous antiserum (C-isolates), and by selecting variants in the presence of antibody following 5-fluorouracil mutagenesis (M-isolates). All viruses were triple-plaque purified and purity neutralization tested prior to isolate selection. Based on a fourfold reduction in neutralizing antibody titer to homologous antiserum, no antigenic variation was found in P-isolates from the three serotypes examined. Antigenic variants of all three serotypes could be isolated by the antiserum selection method (C-isolates). However, antigenic variants of RV17 were isolated at a much higher frequency and showed a larger degree of variation than those of RV2 and RV14. At least two of the variants selected, RV17 (C301) and RV2 (M803), failed to be neutralized by the known 89 rhinovirus antiserum. SDS-polyacrylamide gel electrophoresis of (/sup 35/S) methionine-labelled virion polypeptides revealed that each serotype had a characteristic pattern and that selected RV2 and RV17 isolates had patterns identical to those of the prototype strains. By isoelectric focusing an antigenic variant of RV2 was shown to contain altered virion polypeptides VP1 and VP2 whereas two RV17 antigenic variants demonstrated alterations only in the VP1 polypeptide.

  6. Pancreatic islet isolation by mechanical-enzymatic separation, stationary collagenase digestion and dextran discontinuous density gradient purification: experimental study in dogs Isolamento das ilhotas pancreáticas pela separação mecânica-enzimática digestão estacionária com colagenase e purificação com gradiente de densidade descontínua de dextran: estudo experimental em cães

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    Jaques Waisberg

    2002-04-01

    Full Text Available The prospects for allotransplantation of pancreatic islets in man depend on the development of methods that provide sufficient quantities of pancreatic islets from a single donor, which are capable, when transplanted, of achieve the normalization of carbohydrate metabolism. Objective: Evaluate the efficacy of the isolation of Langerhans islets from dogs, by means of mechanical-enzymatic separation technique with stationary digestion using collagenase, and purification with a discontinuous dextran density gradient. Methods: The counting of islet numbers and evaluation of their sizes was accomplished by staining with diphenylthiocarbazone and using stereoscopic microscopes equipped with eyepiece reticule for the measurement of average diameters of stained islets. Results: The results disclosed that the average number of islets isolated was 81032.20 ± 24736.79 and the average number of islets isolated per kg of body weight was 6938.70 ± 1392.43. The average number of islets isolated per kg of body weight showed significant correlation with body weight and weight of the pancreas resected. Conclusion: The number of islets isolated, of a single donor, by mechanical-enzymatic separation, stationary collagenase digestion and discontinuous dextran density gradient purification can be sufficient to success of pancreatic islets transplant in dogs.A perspectiva do alotransplante de ilhotas pancreáticas no homem está na dependência do desenvolvimento de métodos que propiciem quantidades suficientes de ilhotas pancreáticas, originadas de doador único, capazes de, quando transplantadas, levarem à normalização do metabolismo dos hidratos de carbono. Objetivo: Avaliar, em cães, a eficácia do isolamento das ilhotas de Langerhans por meio da técnica de separação mecânica-enzimática, digestão estacionária com colagenase e purificação pelo gradiente de densidade descontínua de dextran. Métodos: A contagem do número e avaliação do tamanho

  7. Isolation of human apolipoprotein E by chromatofocusing.

    Science.gov (United States)

    Weisweiler, P; Schwandt, P

    1982-09-01

    Human prolipoprotein E is implicated in the transport of serum cholesterol and the binding of lipoproteins to cell receptors. Further investigations on this apolipoprotein would be facilitated by improved purification methods. We prepared human apo E by the combination of high performance gel filtration and chromatofocusing from serum very low density lipoproteins. Chromatofocusing was performed with a pH gradient from 7 to 4. Apo E contained all isoforms, but was homogeneous in SDS-polyacrylamide gel electrophoresis and in double immunodiffusion against a monospecific antiserum. The reported purification method allows a rapid and simple preparation of large amounts of apo E.

  8. [Xenogeneic cell therapeutics: Treatment of type 1 diabetes using porcine pancreatic islets and islet cells].

    Science.gov (United States)

    Godehardt, Antonia W; Schilling-Leiß, Dagmar; Sanzenbacher, Ralf; Tönjes, Ralf R

    2015-11-01

    In view of the existing shortage of human donor organs and tissues, xenogeneic cell therapeutics (xCT) offer an alternative for adequate treatment. In particular, porcine pancreatic islets and islet cells have already entered the field of experimental therapy for type-1 diabetes mellitus (T1DM) patients. Thereby, xCT depict challenging products with a glance on medical, ethical, and regulatory questions. With cross-species transplantation (xenotransplantation), the risk of immunological graft rejection as well as the risk of infectious transmission of microbial and viral pathogens must be considered. This includes the bidirectional transmission of microorganisms from graft to host as well as from host to graft. Crossing the border of species requires a critical risk-benefit evaluation as well as a thorough longtime surveillance of transplant recipients after treatment. The international legal and regulatory requirements for xCT are inter alia based on the World Health Organization criteria summarized in the Changsha Communiqué (2008). In the European Union, they were reflected by the European Medicines Agency (EMA) Guideline on Xenogeneic Cell-based Medicinal Products following the implementation of the Regulation on Advanced Therapies (ATMP). On the basis of this regulation, the first non-clinical and clinical experiences were obtained for porcine islets. The results suggest that supportive treatment of T1DM risk patients with xCT may be an alternative to established allogeneic organ transplantation in the future.

  9. Hawthorn extract inhibits human isolated neutrophil functions.

    Science.gov (United States)

    Dalli, Ernesto; Milara, Javier; Cortijo, Julio; Morcillo, Esteban J; Cosín-Sales, Juan; Sotillo, José Francisco

    2008-06-01

    Hawthorn extract is a popular herbal medicine given as adjunctive treatment for chronic heart failure. In contrast to the cardiac properties of hawthorn extract, its anti-inflammatory effect has been scarcely investigated. This study examines the effects of a dry extract of leaves and flowers of Crataegus laevigata on various functional outputs of human neutrophils in vitro. Incubation of human neutrophils obtained from peripheral blood of healthy donors with C. laevigata extract (0.75-250 microg/ml) inhibited N-formyl-Met-Leu-Phe (FMLP)-induced superoxide anion generation, elastase release and chemotactic migration with potency values of 43.6, 21.9, and 31.6 microg/ml, respectively. By contrast, serum-opsonized zymosan-induced phagocytosis was unaltered by plant extract. C. laevigata extract (125 microg/ml) reduced FMLP-induced leukotriene B(4) production and lipopolysaccharide-induced generation of tumour necrosis factor-alpha and interleukin-8. Extract inhibited FMLP-induced intracellular calcium signal with potency of 17.4 microg/ml. Extract also markedly inhibited the extracellular calcium entry into calcium-depleted neutrophils, and the thapsigargin-induced intracellular calcium response. In conclusion, C. laevigata extract inhibited various functional outputs of activated human neutrophils which may be relevant to the pathophysiology of cardiac failure.

  10. Rfx6 is an Ngn3-dependent winged helix transcription factor required for pancreatic islet cell development.

    Science.gov (United States)

    Soyer, Josselin; Flasse, Lydie; Raffelsberger, Wolfgang; Beucher, Anthony; Orvain, Christophe; Peers, Bernard; Ravassard, Philippe; Vermot, Julien; Voz, Marianne L; Mellitzer, Georg; Gradwohl, Gérard

    2010-01-01

    The transcription factor neurogenin 3 (Neurog3 or Ngn3) controls islet cell fate specification in multipotent pancreatic progenitor cells in the mouse embryo. However, our knowledge of the genetic programs implemented by Ngn3, which control generic and islet subtype-specific properties, is still fragmentary. Gene expression profiling in isolated Ngn3-positive progenitor cells resulted in the identification of the uncharacterized winged helix transcription factor Rfx6. Rfx6 is initially expressed broadly in the gut endoderm, notably in Pdx1-positive cells in the developing pancreatic buds, and then becomes progressively restricted to the endocrine lineage, suggesting a dual function in both endoderm development and islet cell differentiation. Rfx6 is found in postmitotic islet progenitor cells in the embryo and is maintained in all developing and adult islet cell types. Rfx6 is dependent on Ngn3 and acts upstream of or in parallel with NeuroD, Pax4 and Arx transcription factors during islet cell differentiation. In zebrafish, the Rfx6 ortholog is similarly found in progenitors and hormone expressing cells of the islet lineage. Loss-of-function studies in zebrafish revealed that rfx6 is required for the differentiation of glucagon-, ghrelin- and somatostatin-expressing cells, which, in the absence of rfx6, are blocked at the progenitor stage. By contrast, beta cells, whose number is only slightly reduced, were no longer clustered in a compact islet. These data unveil Rfx6 as a novel regulator of islet cell development.

  11. Anaerococcus nagyae sp. nov., isolated from human clinical specimens.

    Science.gov (United States)

    Veloo, A C M; de Vries, E D; Jean-Pierre, H; van Winkelhoff, A J

    2016-04-01

    We describe a new Anaerococcus species isolated from human clinical specimens. Analyses of 16S rRNA gene sequences of three strains showed octavius. Phylogenetically the isolated strains form a cluster and can be differentiated from other species of the genus Anaerococcus based on its phenotypic characteristics and its MALDI-TOF MS profile. We propose the name Anaerococcus nagyae, with A. nagyae DSM101193 (accession number KU043522) as the type strain. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. Distinct replicative and cytopathic characteristics of human immunodeficiency virus isolates.

    Science.gov (United States)

    Fenyö, E M; Morfeldt-Månson, L; Chiodi, F; Lind, B; von Gegerfelt, A; Albert, J; Olausson, E; Asjö, B

    1988-01-01

    According to their capacity to replicate in vitro, human immunodeficiency virus (HIV) isolates can be divided into two major groups, rapid/high and slow/low. Rapid/high viruses can easily be transmitted to a variety of cell lines of T-lymphoid (CEM, H9, and Jurkat) and monocytoid (U937) origin. In contrast, slow/low viruses replicate transiently, if at all, in these cell lines. Except for a few isolates, the great majority of slow/low viruses replicate in peripheral blood mononuclear cells and Jurkat-tatIII cells constitutively expressing the tatIII gene of HIV-1. The viruses able to replicate efficiently cause syncytium formation and are regularly isolated from immunodeficient patients. Poorly replicating HIV isolates, often obtained from individuals with no or mild disease, show syncytium formation and single-cell killing simultaneously or, with some isolates, cell killing only. Images PMID:2459416

  13. Phylogenetic analysis of Escherichia coli strains isolated from human samples

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    Abdollah Derakhshandeh

    2013-12-01

    Full Text Available Escherichia coli (E. coli is a normal inhabitant of the gastrointestinal tract of vertebrates, including humans. Phylogenetic analysis has shown that E. coli is composed of four main phylogenetic groups (A, B1, B2 and D. Group A and B1 are generally associated with commensals, whereas group B2 is associated with extra-intestinal pathotypes. Most enteropathogenic isolates, however, are assigned to group D. In the present study, a total of 102 E. coli strains, isolated from human samples, were used. Phylogenetic grouping was done based on the Clermont triplex PCR method using primers targeted at three genetic markers, chuA, yjaA and TspE4.C2. Group A contained the majority of the collected isolates (69 isolates, 67.64%, followed by group B2 (18 isolates, 17.64% and D (15 isolates, 14.7% and no strains were found to belong to group B1. The distribution of phylogenetic groups in our study suggests that although the majority of strains were commensals, the prevalence of enteropathogenic and extra-intestinal pathotypes was noteworthy. Therefore, the role of E. coli in human infections including diarrhea, urinary tract infections and meningitis should be considered.

  14. Blastocystis phylogeny among various isolates from humans to insects.

    Science.gov (United States)

    Yoshikawa, Hisao; Koyama, Yukiko; Tsuchiya, Erika; Takami, Kazutoshi

    2016-12-01

    Blastocystis is a common unicellular eukaryotic parasite found not only in humans, but also in various kinds of animal species worldwide. Since Blastocystis isolates are morphologically indistinguishable, many molecular biological approaches have been applied to classify these isolates. The complete or partial sequences of the small subunit rRNA gene (SSU rDNA) are mainly used for comparisons and phylogenetic analyses among Blastocystis isolates. However, various lengths of the partial SSU rDNA sequence have been used for phylogenetic inference among genetically different isolates. Based on the complete SSU rDNA sequences, consensus terminology of nine subtypes (STs) of Blastocystis sp. that were supported by phylogenetically monophyletic nine clades was proposed in 2007. Thereafter, eight additional kinds of STs comprising non-human mammalian Blastocystis isolates have been reported based on the phylogeny of SSU rDNA sequences, while STs 11 and 12 were only proposed on the base of partial sequences. Although many sequence data from mammalian and avian Blastocystis are registered in GenBank, only limited data on SSU rDNA are available for poikilotherm-derived Blastocystis isolates. Therefore, the phylogenetic positions of the reptilian/amphibian Blastocystis clades are unstable. The phylogenetic inference of various STs comprising mammalian and/or avian Blastocystis isolates was verified herein based on comparisons between partial and complete SSU rDNA sequences, and the phylogenetic positions of reptilian and amphibian Blastocystis isolates were also investigated using 14 new Blastocystis isolates from reptiles with all known isolates from other reptilians, amphibians, and insects registered in GenBank. Copyright © 2016. Published by Elsevier Ireland Ltd.

  15. Functional Improvement in Rats' Pancreatic Islets Using Magnesium Oxide Nanoparticles Through Antiapoptotic and Antioxidant Pathways.

    Science.gov (United States)

    Moeini-Nodeh, Shermineh; Rahimifard, Mahban; Baeeri, Maryam; Abdollahi, Mohammad

    2017-01-01

    According to undiscovered toxicity and safety of magnesium oxide nanoparticles (MgO NPs) in isolated pancreatic islet cells, this study was designed to examine the effects of its various concentrations on a time-course basis on the oxidative stress, viability, and function of isolated islets of rat's pancreas. Pancreatic islets were isolated and exposed to different MgO NP (oxidative stress biomarkers were investigated and the best exposure time was selected. Then, safety of MgO NPs was investigated by flow cytometry and fluorescent staining, and levels of insulin secretion and caspase activity were measured. The results illustrated a considerable decrease in oxidative stress markers such as reactive oxygen species (ROS) and lipid peroxidation (LPO) levels of pancreatic islets which were treated by MgO NPs for 24 h. Also, in that time of exposure, cell apoptosis investigation by flow cytometry and insulin test showed that MgO NPs, in a concentration of 100 μg/ml, decreased the rate of apoptotic cells via inhibiting caspase-9 activity and made a significant increase in the level of insulin secretion. Data of function and apoptosis biomarkers correlated with each other. It is concluded that the use of MgO NPs in concentration of as low as 100 μg/ml can induce antiapoptotic, antioxidative, and antidiabetic effects in rat pancreatic islets, which support its possible benefit in islet transplantation procedures.

  16. Anti-inflammatory thalidomide improves islet grafts survival and functions in a xenogenic environment.

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    Chunguang Chen

    Full Text Available Thalidomide possesses both anti-inflammatory and anti-angiogenic properties. This study investigates its potential application in islet transplantation with a xenogenic transplantation model. Transplantation was performed using C57Bl/6 mice and NMRI nu/nu mice as recipients of porcine islets. Moreover, islet graft vasculature and inflammation were investigated to identify the mechanisms of thalidomide action. In the immunocompetent environment of C57Bl/6 mice, a fast graft rejection was observed. The group treated with thalidomide 200 mg/kg BW per day achieved and maintained euglycemia in the complete observation period for 42 days. The treated mice had more functional islet graft mass with less leukocyte infiltration. The pro-inflammatory TNF-alpha and VEGF content in islet grafted kidneys was significantly lowered by the treatment. By comparison, thalidomide was not effective in improving graft survival in immunocompromised nude mice. It strongly inhibited the VEGF and TNF-alpha-induced endothelial proliferation of isolated pig islets in a dose dependent manner. The magnitude of thalidomide's inhibitory effect was nearly identical to the effect of VEGF- receptor 2 inhibitor SU416 and anti-TNF-receptor 1 neutralizing antibody, and was reversed by sphingosine-1-phosphate. In conclusion, the anti-inflammatory effect of thalidomide improved islet graft survival and function in a transplantation model with a maximum immune barrier.

  17. Classification of microscopy images of Langerhans islets

    Science.gov (United States)

    Å vihlík, Jan; Kybic, Jan; Habart, David; Berková, Zuzana; Girman, Peter; Kříž, Jan; Zacharovová, Klára

    2014-03-01

    Evaluation of images of Langerhans islets is a crucial procedure for planning an islet transplantation, which is a promising diabetes treatment. This paper deals with segmentation of microscopy images of Langerhans islets and evaluation of islet parameters such as area, diameter, or volume (IE). For all the available images, the ground truth and the islet parameters were independently evaluated by four medical experts. We use a pixelwise linear classifier (perceptron algorithm) and SVM (support vector machine) for image segmentation. The volume is estimated based on circle or ellipse fitting to individual islets. The segmentations were compared with the corresponding ground truth. Quantitative islet parameters were also evaluated and compared with parameters given by medical experts. We can conclude that accuracy of the presented fully automatic algorithm is fully comparable with medical experts.

  18. [Human social activity under conditions of relative social isolation].

    Science.gov (United States)

    Prokhvatilov, A Iu

    1992-01-01

    The differences in using a "social isolation" concept in the psychological literature are presented. The term of "relative social isolation" is clarified. A relationship between human adaptation to the relative social isolation environments and the development of his social qualities and social activities is presented. The "social context", dictating motivation attitudes of a man to the isolation situation, emotional experiences, self-appraisal of activity is of crucial importance for evaluating the real environments of relative social isolations. Social activity of a personality is studied as the relations of a man with the conditions of his activity. The results of studying the dynamics of the psychic state of a man during individual and group isolation are compared. It is concluded that social activity of man and his functional state are interrelated. The particular manifestations and direction of the changes in the social activity of the subject depend on the duration of isolation and are determined first of all by social significance and meaningful and balanced work for a person as well as by the amount and frequency of direct and mediated social contacts under specific conditions of relative social isolation.

  19. The presence of the pilus locus is a clonal property among pneumococcal invasive isolates

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    Melo-Cristino José

    2008-02-01

    Full Text Available Abstract Background Pili were recently recognized in Streptococcus pneumoniae and implicated in the virulence of this bacterium, which led to the proposal of using these antigens in a future pneumococcal vaccine. However, pili were found to be encoded by the rlrA islet that was not universally distributed in the species. We examined the distribution of the pilus islet, using the presence of the rlrA gene as a marker for the locus, among a collection of invasive isolates recovered in Portugal and analyzed its association with capsular serotypes, clusters defined by the pulsed-field gel electrophoretic profiles (PFGE and multilocus sequence types. Results Only a minority of the isolates were positive for the presence of the rlrA gene (27%. There was a high correspondence between the serotype and the presence or absence of rlrA (Wallace coefficient, W = 0.778. In particular, there was an association between the presence of rlrA and the vaccine serotypes 4, 6B, 9V and 14 whereas the gene was significantly absent from other serotypes, namely 1, 7F, 8, 12B and 23F, a group that included a vaccine serotype (23F and serotype 1 associated with enhanced invasiveness. Even within serotypes, there was variation in the presence of the pilus islet between PFGE clones and a higher Wallace coefficient (W = 0.939 indicates that carriage of the islet is a clonal property of pneumococci. Analysis of rlrA negative isolates revealed heterogeneity in the genomic region downstream of the rfl gene, the region where the islet is found in other isolates, compatible with recent loss of the islet in some lineages. Conclusion The pilus islet is present in a minority of pneumococcal isolates recovered from human invasive infections and is therefore not an essential virulence factor in these infections. Carriage of the pilus islet is a clonal property of pneumococci that may vary between isolates expressing the same serotype and loss and acquisition of the islet may be

  20. Two distinct mechanisms mediate the involvement of bone marrow cells in islet remodeling: neogenesis of insulin-producing cells and support of islet recovery.

    Science.gov (United States)

    Iskovich, Svetlana; Goldenberg-Cohen, Nitza; Sadikov, Tamila; Yaniv, Isaac; Stein, Jerry; Askenasy, Nadir

    2015-01-01

    We have recently reported that small-sized bone marrow cells (BMCs) isolated by counterflow centrifugal elutriation and depleted of lineage markers (Fr25lin(-)) have the capacity to differentiate and contribute to regeneration of injured islets. In this study, we assess some of the characteristics of these cells compared to elutriated hematopoietic progenitors (R/O) and whole BMCs in a murine model of streptozotocin-induced chemical diabetes. The GFP(bright)CD45(+) progeny of whole BMCs and R/O progenitors progressively infiltrate the pancreas with evolution of donor chimerism; are found at islet perimeter, vascular, and ductal walls; and have a modest impact on islet recovery from injury. In contrast, Fr25lin(-) cells incorporate in the islets, convert to GFP(dim)CD45(-)PDX-1(+) phenotypes, produce proinsulin, and secrete insulin with significant contribution to stabilization of glucose homeostasis. The elutriated Fr25lin(-) cells express low levels of CD45 and are negative for SCA-1 and c-kit, as removal of cells expressing these markers did not impair conversion to produce insulin. BMCs mediate two synergistic mechanisms that contribute to islet recovery from injury: support of islet remodeling by hematopoietic cells and neogenesis of insulin-producing cells from stem cells.

  1. Culturing pancreatic islets in microfluidic flow enhances morphology of the associated endothelial cells.

    Directory of Open Access Journals (Sweden)

    Krishana S Sankar

    Full Text Available Pancreatic islets are heavily vascularized in vivo with each insulin secreting beta-cell associated with at least one endothelial cell (EC. This structure is maintained immediately post-isolation; however, in culture the ECs slowly deteriorate, losing density and branched morphology. We postulate that this deterioration occurs in the absence of blood flow due to limited diffusion of media inside the tissue. To improve exchange of media inside the tissue, we created a microfluidic device to culture islets in a range of flow-rates. Culturing the islets from C57BL6 mice in this device with media flowing between 1 and 7 ml/24 hr resulted in twice the EC-density and -connected length compared to classically cultured islets. Media containing fluorescent dextran reached the center of islets in the device in a flow-rate-dependant manner consistent with improved penetration. We also observed deterioration of EC morphology using serum free media that was rescued by addition of bovine serum albumin, a known anti-apoptotic signal with limited diffusion in tissue. We further examined the effect of flow on beta-cells showing dampened glucose-stimulated Ca(2+-response from cells at the periphery of the islet where fluid shear-stress is greatest. However, we observed normal two-photon NAD(PH response and insulin secretion from the remainder of the islet. These data reveal the deterioration of islet EC-morphology is in part due to restricted diffusion of serum albumin within the tissue. These data further reveal microfluidic devices as unique platforms to optimize islet culture by introducing intercellular flow to overcome the restricted diffusion of media components.

  2. Human Streptococcus agalactiae isolate in Nile tilapia (Oreochromis niloticus)

    Science.gov (United States)

    Streptococcus agalactiae, the Lancefield group B Streptococcus (GBS), long recognized as a mammalian pathogen, is an emerging pathogen to fish. We show that a GBS serotype Ia, multilocus sequence type ST-7 isolate from a human neonatal meningitis clinical case causes disease signs and mortality in N...

  3. Human Streptococcus agalactiae Isolate in Nile Tilapia (Oreochromis niloticus)

    OpenAIRE

    Evans, Joyce J.; Phillip H. Klesius; Pasnik, David J.; Bohnsack, John F.

    2009-01-01

    Streptococcus agalactiae, the Lancefield group B streptococcus (GBS) long recognized as a mammalian pathogen, is an emerging concern with regard to fish. We show that a GBS serotype Ia multilocus sequence type ST-7 isolate from a clinical case of human neonatal meningitis caused disease and death in Nile tilapia (Oreochromis niloticus).

  4. The Beta Cell in Its Cluster: Stochastic Graphs of Beta Cell Connectivity in the Islets of Langerhans.

    Directory of Open Access Journals (Sweden)

    Deborah A Striegel

    2015-08-01

    Full Text Available Pancreatic islets of Langerhans consist of endocrine cells, primarily α, β and δ cells, which secrete glucagon, insulin, and somatostatin, respectively, to regulate plasma glucose. β cells form irregular locally connected clusters within islets that act in concert to secrete insulin upon glucose stimulation. Due to the central functional significance of this local connectivity in the placement of β cells in an islet, it is important to characterize it quantitatively. However, quantification of the seemingly stochastic cytoarchitecture of β cells in an islet requires mathematical methods that can capture topological connectivity in the entire β-cell population in an islet. Graph theory provides such a framework. Using large-scale imaging data for thousands of islets containing hundreds of thousands of cells in human organ donor pancreata, we show that quantitative graph characteristics differ between control and type 2 diabetic islets. Further insight into the processes that shape and maintain this architecture is obtained by formulating a stochastic theory of β-cell rearrangement in whole islets, just as the normal equilibrium distribution of the Ornstein-Uhlenbeck process can be viewed as the result of the interplay between a random walk and a linear restoring force. Requiring that rearrangements maintain the observed quantitative topological graph characteristics strongly constrained possible processes. Our results suggest that β-cell rearrangement is dependent on its connectivity in order to maintain an optimal cluster size in both normal and T2D islets.

  5. Interleukin-1beta induced changes in the protein expression of rat islets: a computerized database

    DEFF Research Database (Denmark)

    Andersen, H U; Fey, S J; Larsen, Peter Mose

    1997-01-01

    Insulin-dependent diabetes mellitus is caused by an autoimmune destruction of the beta-cells in the islets of Langerhans. The cytokine interleukin 1 inhibits insulin release and is selectively cytotoxic to beta-cells in isolated pancreatic rat islets. The antigen(s) triggering the immune response...... as well as the intracellular mechanisms of action of interleukin 1-mediated beta-cell cytotoxicity are unknown. However, previous studies have found an association of beta-cell destruction with alterations in protein synthesis. Thus, two-dimensional (2-D) gel electrophoresis of pancreatic islet proteins......% of %IOD was 45.7% in the NEPHGE gels. Addition of interleukin-1beta (IL-1beta) to the cultures resulted in statistically significant modulation or de novo synthesis of 105 proteins in the 10% gels. In conclusion, we present the first 10% and 15% acrylamide 2-D gel protein databases of neonatal rat islets...

  6. Islet cryopreservation: improved recovery following taurine pretreatment.

    Science.gov (United States)

    Hardikar, A A; Risbud, M V; Remacle, C; Reusens, B; Hoet, J J; Bhonde, R R

    2001-01-01

    Simple and efficient freezing methods with maximal postthawing recovery form the basis of ideal cryopreservation. Taurine (2-amino ethanesulfonic acid), an end-product of sulphur amino acid metabolism, is one of the most abundant free amino acids in the body. The membrane stabilizing, free radical scavenging, and osmoregulatory roles of taurine have been well documented. We studied the effect of physiological and supra-physiological concentrations (0.3 and 3.0 mM) of taurine on islet cryopreservation. Islet viability on cryopreservation was significantly improved in both the taurine-treated groups (91.9 +/- 2.3% in 0.3 mM and 94.6 +/- 1.58% in 3.0 mM group, p taurine group, as examined under phase contrast and quantified by islet morphometric analysis (p Taurine-treated islets showed significant reduction in lipid peroxidation (0.905 and 0.848 nM MDA/microg protein for 0.3 and 3.0 mM taurine, respectively, p 200 mg/dl) following removal of the graft. Suboptimal islet transplantation using 250 IE suggests that the grafted islet mass was inadequate for diabetes reversal. In addition, no significant differences were observed in the islet insulin content between the three groups following cryopreservation of the islets at -196 degrees C. Our studies indicate that taurine pretreatment and its continued presence during islet cryopreservation improves the postthawing viable recovery of islets.

  7. Progress in Clinical Encapsulated Islet Xenotransplantation.

    Science.gov (United States)

    Cooper, David K C; Matsumoto, Shinichi; Abalovich, Adrian; Itoh, Takeshi; Mourad, Nizar I; Gianello, Pierre R; Wolf, Eckhard; Cozzi, Emanuele

    2016-11-01

    At the 2015 combined congress of the Cell Transplant Society, International Pancreas and Islet Transplant Association, and International Xenotransplantation Association, a symposium was held to discuss recent progress in pig islet xenotransplantation. The presentations focused on 5 major topics - (1) the results of 2 recent clinical trials of encapsulated pig islet transplantation, (2) the inflammatory response to encapsulated pig islets, (3) methods to improve the secretion of insulin by pig islets, (4) genetic modifications to the islet-source pigs aimed to protect the islets from the primate immune and/or inflammatory responses, and (5) regulatory aspects of clinical pig islet xenotransplantation. Trials of microencapsulated porcine islet transplantation to treat unstable type 1 diabetic patients have been associated with encouraging preliminary results. Further advances to improve efficacy may include (1) transplantation into a site other than the peritoneal cavity, which might result in better access to blood, oxygen, and nutrients; (2) the development of a more biocompatible capsule and/or the minimization of a foreign body reaction; (3) pig genetic modification to induce a greater secretion of insulin by the islets, and/or to reduce the immune response to islets released from damaged capsules; and (4) reduction of the inflammatory response to the capsules/islets by improvements in the structure of the capsules and/or in genetic engineering of the pigs and/or in some form of drug therapy. Ethical and regulatory frameworks for islet xenotransplantation are already available in several countries, and there is now a wider international perception of the importance of developing an internationally harmonized ethical and regulatory framework.

  8. IFN-{gamma} gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice

    Energy Technology Data Exchange (ETDEWEB)

    Rabinovitch, A.; Suarez-Pinzon, W.L.; Sorensen, O. [Univ. of Alberta, Edmonton (Canada)] [and others

    1995-05-01

    Insulin-dependent diabetes mellitus in nonobese diabetic (NOD) mice results from selective destruction of pancreatic islet {beta}-cells following islet filtration by mononuclear leukocytes. Cytokines produced by islet-infiltrating mononuclear cells may be involved in {beta}-cell destruction. Therefore, we analyzed cytokine mRNA expression, by reverse-transcriptase PCR (RT-PCR) assay, in mononuclear leukocytes isolated from pancreatic islets of four groups of mice: diabetes-prone female NOD mice; female NOD mice protected from diabetes by injection of CFA at an early age; male NOD mice with a low diabetes incidence; and female BALB/c mice that do not develop diabetes. We found that mRNA levels of IL-1{beta}, IL-2, IL-4, IL-10, and IFN-{gamma} in mononuclear cells from islets of diabetes-prone female NOD mice increased progressively as these cells infiltrated the islets from age 5 wk to diabetes onset (>13 wk). However, only IFN-{gamma} mRNA levels were significantly higher in islet mononuclear cells from 12-wk-old diabetes-prone female NOD mice than from less diabetes-prone NOD mice (CFA-treated females, and males) and normal mice (BALB/c). In contrast, IL-4 mRNA levels were lower in islet mononuclear cells from diabetes-prone female NOD mice than from NOD mice with low diabetes incidence (CFA-treated females and males). Splenic cell mRNA levels of IFN-{gamma} and IL-4 were not different in the four groups of mice. These results suggest that islet {beta}-cell destruction and diabetes in female NOD mice are dependent upon intra-islet IFN-{gamma} production by mononuclear cells, and that CFA-treated female NOD mice and male NOD mice may be protected from diabetes development by down-regulation of IFN-{gamma} production in the islets. 56 refs., 4 figs., 3 tabs.

  9. Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

    Science.gov (United States)

    Vidal, Samuel J.; Quinn, S. Aidan; de la Iglesia-Vicente, Janis; Bonal, Dennis M.; Rodriguez-Bravo, Veronica; Firpo-Betancourt, Adolfo; Cordon-Cardo, Carlos; Domingo-Domenech, Josep

    2014-01-01

    The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice. PMID:24686446

  10. A novel redox-active metalloporphyrin reduces reactive oxygen species and inflammatory markers but does not improve marginal mass engraftment in a murine donation after circulatory death islet transplantation model.

    Science.gov (United States)

    Bruni, Antonio; Pepper, Andrew R; Gala-Lopez, Boris; Pawlick, Rena; Abualhassan, Nasser; Crapo, James D; Piganelli, Jon D; Shapiro, A M James

    2016-07-03

    Islet transplantation is a highly effective treatment for stabilizing glycemic control for select patients with type-1 diabetes. Despite improvements to clinical transplantation, single-donor transplant success has been hard to achieve routinely, necessitating increasing demands on viable organ availability. Donation after circulatory death (DCD) may be an alternative option to increase organ availability however, these organs tend to be more compromised. The use of metalloporphyrin anti-inflammatory and antioxidant (MnP) compounds previously demonstrated improved in vivo islet function in preclinical islet transplantation. However, the administration of MnP (BMX-001) in a DCD islet isolation and transplantation model has yet to be established. In this study, murine donors were subjected to a 15-min warm ischemic (WI) period prior to isolation and culture with or without MnP. Subsequent to one-hour culture, islets were assessed for in vitro viability and in vivo function. A 15-minute WI period significantly reduced islet yield, regardless of MnP-treatment relative to yields from standard isolation. MnP-treated islets did not improve islet viability compared to DCD islets alone. MnP-treatment did significantly reduce the presence of extracellular reactive oxygen species (ROS) (p MnP-treatment. DCD islet grafts harvested 7 d post-transplant exhibited sustained TNF-α and IL-10, while MnP-treated islet-bearing grafts demonstrated reduced IL-10 levels. Taken together, 15-minute WI in murine islet isolation significantly impairs islet yield. DCD islets do indeed demonstrate in vivo function, though MnP therapy was unable to improve viability and engraftment outcomes.

  11. Islet transplantation in type 1 diabetes: ongoing challenges, refined procedures, and long-term outcome.

    Science.gov (United States)

    Shapiro, A M James

    2012-01-01

    Remarkable progress has been made in islet transplantation over a span of 40 years. Once just an experimental curiosity in mice, this therapy has moved forward, and can now provide robust therapy for highly selected patients with type 1 diabetes (T1D), refractory to stabilization by other means. This progress could not have occurred without extensive dynamic international collaboration. Currently, 1,085 patients have undergone islet transplantation at 40 international sites since the Edmonton Protocol was reported in 2000 (752 allografts, 333 autografts), according to the Collaborative Islet Transplant Registry. The long-term results of islet transplantation in selected centers now match registry data of pancreas-alone transplantation, with 6 sites reporting five-year insulin independence rates ≥50%. Islet transplantation has been criticized for the use of multiple donor pancreas organs, but progress has also occurred in single-donor success, with 10 sites reporting increased single-donor engraftment. The next wave of innovative clinical trial interventions will address instant blood-mediated inflammatory reaction (IBMIR), apoptosis, and inflammation, and will translate into further marked improvements in single-donor success. Effective control of auto- and alloimmunity is the key to long-term islet function, and high-resolution cellular and antibody-based assays will add considerable precision to this process. Advances in immunosuppression, with new antibody-based targeting of costimulatory blockade and other T-B cellular signaling, will have further profound impact on the safety record of immunotherapy. Clinical trials will move forward shortly to test out new human stem cell derived islets, and in parallel trials will move forward, testing pig islets for compatibility in patients. Induction of immunological tolerance to self-islet antigens and to allografts is a difficult challenge, but potentially within our grasp.

  12. Genetic regulatory signatures underlying islet gene expression and type 2 diabetes

    Science.gov (United States)

    Varshney, Arushi; Scott, Laura J.; Welch, Ryan P.; Erdos, Michael R.; Chines, Peter S.; Narisu, Narisu; Albanus, Ricardo D’O.; Orchard, Peter; Wolford, Brooke N.; Kursawe, Romy; Vadlamudi, Swarooparani; Cannon, Maren E.; Didion, John P.; Hensley, John; Kirilusha, Anthony; Bonnycastle, Lori L.; Taylor, D. Leland; Watanabe, Richard; Mohlke, Karen L.; Boehnke, Michael; Collins, Francis S.; Parker, Stephen C. J.; Stitzel, Michael L.

    2017-01-01

    Genome-wide association studies (GWAS) have identified >100 independent SNPs that modulate the risk of type 2 diabetes (T2D) and related traits. However, the pathogenic mechanisms of most of these SNPs remain elusive. Here, we examined genomic, epigenomic, and transcriptomic profiles in human pancreatic islets to understand the links between genetic variation, chromatin landscape, and gene expression in the context of T2D. We first integrated genome and transcriptome variation across 112 islet samples to produce dense cis-expression quantitative trait loci (cis-eQTL) maps. Additional integration with chromatin-state maps for islets and other diverse tissue types revealed that cis-eQTLs for islet-specific genes are specifically and significantly enriched in islet stretch enhancers. High-resolution chromatin accessibility profiling using assay for transposase-accessible chromatin sequencing (ATAC-seq) in two islet samples enabled us to identify specific transcription factor (TF) footprints embedded in active regulatory elements, which are highly enriched for islet cis-eQTL. Aggregate allelic bias signatures in TF footprints enabled us de novo to reconstruct TF binding affinities genetically, which support the high-quality nature of the TF footprint predictions. Interestingly, we found that T2D GWAS loci were strikingly and specifically enriched in islet Regulatory Factor X (RFX) footprints. Remarkably, within and across independent loci, T2D risk alleles that overlap with RFX footprints uniformly disrupt the RFX motifs at high-information content positions. Together, these results suggest that common regulatory variations have shaped islet TF footprints and the transcriptome and that a confluent RFX regulatory grammar plays a significant role in the genetic component of T2D predisposition. PMID:28193859

  13. Francisella tularensis subsp. novicida isolated from a human in Arizona

    Directory of Open Access Journals (Sweden)

    Birdsell Dawn N

    2009-11-01

    Full Text Available Abstract Background Francisella tularensis is the etiologic agent of tularemia and is classified as a select agent by the Centers for Disease Control and Prevention. Currently four known subspecies of F. tularensis that differ in virulence and geographical distribution are recognized:tularensis (type A, holarctica (type B, mediasiatica, and novicida. Because of the Select Agent status and differences in virulence and geographical location, the molecular analysis of any clinical case of tularemia is of particular interest. We analyzed an unusual Francisella clinical isolate from a human infection in Arizona using multiple DNA-based approaches. Findings We report that the isolate is F. tularensis subsp. novicida, a subspecies that is rarely isolated. Conclusion The rarity of this novicida subspecies in clinical settings makes each case study important for our understanding of its role in disease and its genetic relationship with other F. tularensis subspecies.

  14. Cholesterol reduction ameliorates glucose-induced calcium handling and insulin secretion in islets from low-density lipoprotein receptor knockout mice.

    Science.gov (United States)

    Souza, J C; Vanzela, E C; Ribeiro, R A; Rezende, L F; de Oliveira, C A; Carneiro, E M; Oliveira, H C F; Boschero, A C

    2013-04-01

    Changes in cellular cholesterol level may contribute to beta cell dysfunction. Islets from low density lipoprotein receptor knockout (LDLR(-/-)) mice have higher cholesterol content and secrete less insulin than wild-type (WT) mice. Here, we investigated the association between cholesterol content, insulin secretion and Ca(2+) handling in these islets. Isolated islets from both LDLR(-/-) and WT mice were used for measurements of insulin secretion (radioimmunoassay), cholesterol content (fluorimetric assay), cytosolic Ca(2+) level (fura-2AM) and SNARE protein expression (VAMP-2, SNAP-25 and syntaxin-1A). Cholesterol was depleted by incubating the islets with increasing concentrations (0-10mmol/l) of methyl-beta-cyclodextrin (MβCD). The first and second phases of glucose-stimulated insulin secretion (GSIS) were lower in LDLR(-/-) than in WT islets, paralleled by an impairment of Ca(2+) handling in the former. SNAP-25 and VAMP-2, but not syntaxin-1A, were reduced in LDLR(-/-) compared with WT islets. Removal of excess cholesterol from LDLR(-/-) islets normalized glucose- and tolbutamide-induced insulin release. Glucose-stimulated Ca(2+) handling was also normalized in cholesterol-depleted LDLR(-/-) islets. Cholesterol removal from WT islets by 0.1 and 1.0mmol/l MβCD impaired both GSIS and Ca(2+) handling. In addition, at 10mmol/l MβCD WT islet showed a loss of membrane integrity and higher DNA fragmentation. Abnormally high (LDLR(-/-) islets) or low cholesterol content (WT islets treated with MβCD) alters both GSIS and Ca(2+) handling. Normalization of cholesterol improves Ca(2+) handling and insulin secretion in LDLR(-/-) islets. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Isolation of human liver angiotensin-converting enzyme by chromatofocusing.

    Science.gov (United States)

    Sakharov IYu; Danilov, S M; Sukhova, N V

    1987-10-01

    Angiotensin-converting enzyme (EC 3.4.15.1) has been isolated from human liver by chromatofocusing. The isolation procedure permitted us to obtain a 9000-fold purified enzyme with a 22% yield. Specific activity of the angiotensin-converting enzyme was 10 units/mg of protein. The molecular mass of enzyme determined by polyacrylamide gel electrophoresis under denaturing conditions was 150,000. The isoelectric point (4.2-4.3) was also determined by chromatofocusing. The Km values of the enzyme for hippuryl-L-histidyl-L-leucine and N-benzyloxycarbonyl-L-phenylalanyl-L-histidyl-L-leucine are 5000 and 125 microM, respectively. The human liver angiotensin-converting enzyme is inhibited by bradykinin-potentiating factor SQ 20881 (IC50 = 18 nM).

  16. Fibrillar dimer formation of islet amyloid polypeptides

    Energy Technology Data Exchange (ETDEWEB)

    Chiu, Chi-cheng [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States); de Pablo, Juan J. [Univ. of Chicago, IL (United States); Argonne National Lab. (ANL), Argonne, IL (United States)

    2015-05-08

    Amyloid deposits of human islet amyloid polypeptide (hIAPP), a 37-residue hormone co-produced with insulin, have been implicated in the development of type 2 diabetes. Residues 20 – 29 of hIAPP have been proposed to constitute the amyloidogenic core for the aggregation process, yet the segment is mostly unstructured in the mature fibril, according to solid-state NMR data. Here we use molecular simulations combined with bias-exchange metadynamics to characterize the conformational free energies of hIAPP fibrillar dimer and its derivative, pramlintide. We show that residues 20 – 29 are involved in an intermediate that exhibits transient β-sheets, consistent with recent experimental and simulation results. By comparing the aggregation of hIAPP and pramlintide, we illustrate the effects of proline residues on inhibition of the dimerization of IAPP. The mechanistic insights presented here could be useful for development of therapeutic inhibitors of hIAPP amyloid formation.

  17. Calpain-10 expression is elevated in pancreatic islets from patients with type 2 diabetes.

    Directory of Open Access Journals (Sweden)

    Charlotte Ling

    Full Text Available BACKGROUND: Calpain-10 was the first gene to be identified influencing the risk of type 2 diabetes (T2D by positioning cloning. Studies in beta-cell lines and rodent islets suggest that calpain-10 may act as a regulator of insulin secretion. However, its role in human pancreatic islets remains unclear. The aim of this study was to examine if calpain-10 expression is altered in islets from patients with T2D and if the transcript level correlates with insulin release. We also tested if polymorphisms in the CAPN10 gene are associated with gene expression and insulin secretion in vitro. METHODOLOGY/PRINCIPAL FINDINGS: Calpain-10 mRNA expression was analysed in human pancreatic islets from 34 non-diabetic and 10 T2D multi-organ donors. CAPN10 SNP-43 and SNP-44 were genotyped and related to gene expression and insulin release in response to glucose, arginine and glibenclamide. The mRNA level of calpain-10 was elevated by 64% in pancreatic islets from patients with T2D compared with non-diabetic donors (P = 0.01. Moreover, the calpain-10 expression correlated positively with arginine-stimulated insulin release in islets from non-diabetic donors (r = 0.45, P = 0.015. However, this correlation was lost in islets from patients with T2D (r = 0.09; P = 0.8. The G/G variant of SNP-43 was associated with reduced insulin release in response to glucose (Phuman islets, this correlation is lost in T2D suggesting that a stimulatory effect of calpain-10 could be lost in patients with T2D.

  18. Color-Removal by Microorganisms Isolated from Human Hands

    Directory of Open Access Journals (Sweden)

    Tsukasa Ito

    2013-08-01

    Full Text Available Microorganisms are essential for human life. Microorganisms decompose the carbon compounds in dead animals and plants and convert them into carbon dioxide. Intestinal bacteria assist in food digestion. Some vitamins are produced by bacteria that live in the intestines. Sewage and industrial wastewater are treated by activated sludge composed of microbial communities. All of these are due to the ability of microbes to produce many enzymes that can degrade chemicals. How do teachers make students understand that microorganisms are always associated with humans, and that microorganisms have the ability to degrade chemicals? The presence of microorganisms on humans can be shown by incubating agar plates after they are touched by the hands of students. The ability of microorganisms to degrade chemicals can be shown by an analytical measurement of the degradation of chemicals. When the chemicals are dyes (colorants in water, microbial activity on degradation of dyes can be demonstrated by observing a decreasing degree of color as a result of the enzymatic activity (e.g., azoreductase. Dyes are widely used in the textile, food, and cosmetic industries. They are generally resistant to conventional biological wastewater treatment systems such as the activated sludge process (4. The discharge of wastewater containing dye pollutes surface water. The ability of microorganisms to decolorize and degrade dyes has been widely investigated to use for bioremediation purposes (5. The goal of this tip is to understand the presence of bacteria on human skin and the ability of bacteria to degrade colorant chemicals (decolorization. In this tip, students first cultivate and isolate bacteria on their hands, and then examine potential decolorization activity of each bacterium by observing the degree of color of the liquid in tubes in which bacteria isolated from students’ hands were inoculated. Decolorization activity of bacterial isolates from human skin has been

  19. Isolation and Quantification of Glycosaminoglycans from Human Hair Shaft

    Science.gov (United States)

    Bonovas, Stefanos; Sitaras, Nikolaos

    2016-01-01

    Background There is evidence that glycosaminoglycans (GAGs) are present in the hair shaft within the follicle but there are no studies regarding GAGs isolation and measurement in the human hair shaft over the scalp surface, it means, in the free hair shaft. Objective The purpose of our research was to isolate and measure the total GAGs from human free hair shaft. Methods Seventy-five healthy individuals participated in the study, 58 adults, men and women over the age of 50 and 17 children (aged 4~9). GAGs in hair samples, received from the parietal and the occipital areas, were isolated with 4 M guanidine HCl and measured by the uronic acid-carbazole reaction assay. Results GAGs concentration was significantly higher in the occipital area than in the parietal area, in all study groups. GAG levels from both areas were significantly higher in children than in adults. GAG levels were not associated with gender, hair color or type. Conclusion We report the presence of GAGs in the human free hair shaft and the correlation of hair GAG levels with the scalp area and participants' age. PMID:27746630

  20. Isolated deficiency of IgE in humans: update

    Directory of Open Access Journals (Sweden)

    D.V. Maltsev

    2017-04-01

    Full Text Available Isolated deficiency IgE is one of the most common primary immunodeficiency diseases in human with prevalence of 1 case per 30 people of total population. Genetic basis of this immunodeficiency are polymorphisms of 5923A/G and 7888C/T in AICDA gene of B-lymphocytes. Recently several new studies have been conducted that expand the current understanding of the nature of an isolated IgE deficiency in human. Several recent epidemiological studies specified immunodeficiency frequency in different cohorts of patients and confirmed the relationship of immunological and clinical phenotypes, including recurrent infections, autoimmunity and oncology. The results of a number of clinical cases have expanded the knowledge of the heterogeneity of clinical symptoms of the di­sease. Several studies investigated the complications of an isolated deficiency IgE, including chronic gastritis and peptic stomach ulcer associated with H. pylori, and atherosclerosis and related vascular accidents. The results of comparative clinical studies demonstrate high efficiency of base immunotherapy with normal human immunoglobulin preparations for the intramuscular and intravenous usage.

  1. Islet-selectivity of G-protein coupled receptor ligands evaluated for PET imaging of pancreatic {beta}-cell mass

    Energy Technology Data Exchange (ETDEWEB)

    Cline, Gary W., E-mail: gary.cline@yale.edu [Yale University School of Medicine (United States); Zhao, Xiaojian [Yale University School of Medicine (United States); Jakowski, Amy B.; Soeller, Walter C.; Treadway, Judith L. [Pfizer Global Research and Development, Pfizer Inc., Groton CT (United States)

    2011-09-02

    Highlights: {yields} We screened G-protein coupled receptors for imaging pancreatic. {yields} Database mining and immunohistochemistry identified GPCRs enriched in {beta}-cells. {yields} In vitro and in vivo assays were used to determine exocrine vs endocrine specificity. {yields} GPCR candidates for imaging of {beta}-cell mass are Prokineticin-1R, mGluR5, and GLP-1R. -- Abstract: A critical unmet need exists for methods to quantitatively measure endogenous pancreatic {beta}-cell mass (BCM) for the clinical evaluation of therapies to prevent or reverse loss of BCM and diabetes progression. Our objective was to identify G-protein coupled receptors (GPCRs) that are expressed with a high degree of specificity to islet {beta}-cells for receptor-targeted imaging of BCM. GPCRs enriched in pancreatic islets relative to pancreas acinar and hepatic tissue were identified using a database screen. Islet-specific expression was confirmed by human pancreas immunohistochemistry (IHC). In vitro selectivity assessment was determined from the binding and uptake of radiolabeled ligands to the rat insulinoma INS-1 832/13 cell line and isolated rat islets relative to the exocrine pancreas cell-type, PANC-1. Tail-vein injections of radioligands into rats were used to determine favorable image criteria of in vivo biodistribution to the pancreas relative to other internal organs (i.e., liver, spleen, stomach, and lungs). Database and IHC screening identified four candidate receptors for further in vitro and in vivo evaluation for PET imaging of BCM: prokineticin-1 receptor (PK-1R), metabotropic glutamate receptor type-5 (mGluR5), neuropeptide Y-2 receptor (NPY-2R), and glucagon-like peptide 1 receptor (GLP-1R). In vitro specificity ratios gave the following receptor rank order: PK-1R > GLP-1R > NPY-2R > mGluR5. The biodistribution rank order of selectivity to the pancreas was found to be PK-1R > VMAT2 {approx} GLP-1R > mGluR5. Favorable islet selectivity and biodistribution

  2. A health-economic analysis of porcine islet xenotransplantation.

    Science.gov (United States)

    Beckwith, Jessica; Nyman, John A; Flanagan, Brian; Schrover, Rudolf; Schuurman, Henk-Jan

    2010-01-01

    Islet cell transplantation is a promising treatment for type 1 diabetes. To overcome the shortage of deceased human pancreas donors, porcine islet cell xenotransplantation is being developed as an alternative to allotransplantation. The objective of this study was to perform a cost-effectiveness analysis of porcine islet transplantation in comparison with standard insulin therapy. The patient population for this study was young adults, ages 20 to 40, for whom standard medical care is inadequate in controlling blood glucose levels (hypoglycemia unawareness). Since trial data were lacking, estimates used extrapolations from data found in the literature and ongoing trials in clinical allotransplantation. Cost estimates were based on the data available in the USA. Markov modeling and Monte Carlo simulations using software specifically developed for health-economic evaluations were used. Outcomes data for ongoing clinical islet allotransplantation from the University of Minnesota were used, along with probabilities of complications from the Diabetes Control and Complications Trial. Quality-adjusted life years (QALYs) were the effectiveness measure. The upper limit of being cost-effective is $100,000 per QALY. Cost data from the literature were used and adjusted to 2007 US dollars using the medical care portion of the Consumer Price Index. In both Markov modeling and Monte Carlo simulations, porcine islet xenotransplantation was both more effective and less costly over the course of the 20-yr model. For standard insulin therapy, cumulative cost per patient was $661,000, while cumulative effectiveness was 9.4 QALYs, for a cost of $71,100 per QALY. Transplantation had a cumulative cost of $659 000 per patient, a cumulative effectiveness of 10.9 QALYs, and a cost per QALY of $60,700. Islet transplantation became cost-effective at 4 yr after transplantation, and was more cost-effective than standard insulin treatment at 14 yr. These findings are related to relative high

  3. A protective effect of endomorphins on the oxidative injury of islet.

    Science.gov (United States)

    Tian, L M; Liu, J; Sun, X L; Gao, C X; Fan, Y; Guo, Q

    2010-08-01

    The antioxidative capacity of endomorphins (EMs), endogenous μ-opioid receptor agonists, has been demonstrated by IN VIVO assays. In this study, we attempt to evaluate the effects of endomorphin 1 (EM1) and endomorphin 2 (EM2) on pancreatic islet injuries induced by streptozotocin (STZ), alloxan (ALX) and H(2)O(2), respectively. Wistar rats' islets were isolated and purified. The function of the islet cells, the insulin response to glucose stimulation was examined by insulin Radio Immuno Assay and the cell viability was measured by MTT assay. DNA fragments were performed to evaluate the apoptosis, while the cell cycle distribution was analyzed by PI staining flow cytometric analysis. Furthermore, the islet were treated with EM1, EM2 or ALX for 24 h, and the expression of p53 and p21 protein were determined by Western blot. The results showed that STZ, ALX, and H(2)O(2) displayed clear concentration-dependent inhibitory effects on the pancreatic islet cells. While EMs improved the viability of islet induced by STZ, ALX or H(2)O(2), and EMs enhanced insulin accumulation of the cell supernatant after ALX and STZ stimulation. Our data also showed both that EMs inhibited cell apoptosis and cell cycle G1 arrest induced by STZ and ALX through down-regulaing p53 and p21 expression. Taken together, these results demonstrate that EMs can protect islet cells from STZ, ALX and H(2)O(2) induced injuries. Our observations imply that the endomorphins may have protective effects on islet cells oxidative injury.

  4. Epidemiological relationship of human and swine Streptococcus suis isolates.

    Science.gov (United States)

    Tarradas, C; Luque, I; de Andrés, D; Abdel-Aziz Shahein, Y E; Pons, P; González, F; Borge, C; Perea, A

    2001-06-01

    Two cases of meningitis due to Streptococcus suis in humans are reported here. A butcher and an abattoir worker were referred to a health centre in Castellón (Spain) with fever and symptoms of meningitis. After adequate treatment, a slight hipoacusia persisted as sequelae in both cases. Colonies of S. suis group R, serotype 2 and phenotype MRP+EF+ were isolated from cerebroespinal fluid. Epidemiological studies showed that both workers had in common the handling of pork meat of slaughtered healthy pigs from three closed farms. A study of the tonsils from apparently healthy, slaughtered pigs was carried out. A total of 234 tonsillar samples were obtained and 81 strains of S. suis were isolated from them. Serotype 2 appeared to be the most frequent (50.6%), and the analysis for phenotype showed a high percentage of tonsillar strains with the phenotype MRP+EF+ (35.9%). The humans and 28 tonsillar swine strains showed a similar profile (S. suis group R, serotype 2 and phenotype MRP+EF+). A total of 26 of the swine isolates were analysed by ribotyping using EcoRI. The human strains showed the same six-band hybridization pattern that shared five bands with the pattern most frequently shown by most of the tonsillar N. suis group R, serotype 2 and phenotype MRP+EF+ strains, differing only in the lightest, faintest band which was slightly less anodical in human (> or = 1.8 kb) than in swine (approximately 1.8 kb). From these results, both groups of strains, humans and porcine, showed differences; how can these differences in the pattern of ribotyping be explained if they should have the same origin? Is it possible that they have undergone an adaptation to the new host or perhaps the modification is due to other unknown causes? Further studies in this area are required in order to answer these questions.

  5. Sterols of Pneumocystis carinii hominis Organisms Isolated from Human Lungs

    Science.gov (United States)

    Kaneshiro, Edna S.; Amit, Zunika; Chandra, Jyotsna; Baughman, Robert P.; Contini, Carlo; Lundgren, Bettina

    1999-01-01

    The opportunistic pathogen Pneumocystis carinii causes pneumonia (P. carinii pneumonia, or PCP) in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii carinii organisms have distinct sterols which are not synthesized by mammals and not found in other microbes infecting mammalian lungs. The dominant sterol present in the organism is cholesterol (which is believed to be scavenged from the host), but other sterols in P. carinii carinii have an alkyl group at C-24 of the sterol side chain (C28 and C29 24-alkylsterols) and a double bond at C-7 of the nucleus. Recently, pneumocysterol (C32), which is essentially lanosterol with a C-24 ethylidene group, was detected in lipids extracted from a formalin-fixed human P. carinii-infected lung, and its structures were elucidated by gas-liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectrometry in conjunction with analyses of chemically synthesized authentic standards. The sterol composition of isolated P. carinii hominis organisms has yet to be reported. If P. carinii from animal models is to be used for identifying potential drug targets and for developing chemotherapeutic approaches to clear human infections, it is important to determine whether the 24-alkylsterols of organisms found in rats are also present in organisms in humans. In the present study, sterol analyses of P. carinii hominis organisms isolated from cryopreserved human P. carinii-infected lungs and from bronchoalveolar lavage fluid were performed. Several of the same distinct sterols (e.g., fungisterol and methylcholest-7-ene-3β-ol) previously identified in P. carinii carinii were also present in organisms isolated from human specimens. Pneumocysterol was detected in only some of the samples. PMID:10548595

  6. Innate immune functions of microglia isolated from human glioma patients

    Directory of Open Access Journals (Sweden)

    Grimm Elizabeth

    2006-03-01

    Full Text Available Abstract Background Innate immunity is considered the first line of host defense and microglia presumably play a critical role in mediating potent innate immune responses to traumatic and infectious challenges in the human brain. Fundamental impairments of the adaptive immune system in glioma patients have been investigated; however, it is unknown whether microglia are capable of innate immunity and subsequent adaptive anti-tumor immune responses within the immunosuppressive tumor micro-environment of human glioma patients. We therefore undertook a novel characterization of the innate immune phenotype and function of freshly isolated human glioma-infiltrating microglia (GIM. Methods GIM were isolated by sequential Percoll purification from patient tumors immediately after surgical resection. Flow cytometry, phagocytosis and tumor cytotoxicity assays were used to analyze the phenotype and function of these cells. Results GIM expressed significant levels of Toll-like receptors (TLRs, however they do not secrete any of the cytokines (IL-1β, IL-6, TNF-α critical in developing effective innate immune responses. Similar to innate macrophage functions, GIM can mediate phagocytosis and non-MHC restricted cytotoxicity. However, they were statistically less able to mediate tumor cytotoxicity compared to microglia isolated from normal brain. In addition, the expression of Fas ligand (FasL was low to absent, indicating that apoptosis of the incoming lymphocyte population may not be a predominant mode of immunosuppression by microglia. Conclusion We show for the first time that despite the immunosuppressive environment of human gliomas, GIM are capable of innate immune responses such as phagocytosis, cytotoxicity and TLR expression but yet are not competent in secreting key cytokines. Further understanding of these innate immune functions could play a critical role in understanding and developing effective immunotherapies to malignant human gliomas.

  7. Long-Term Glycemic Control in Adult Patients Undergoing Remote vs. Local Total Pancreatectomy With Islet Autotransplantation.

    Science.gov (United States)

    Kesseli, Samuel J; Wagar, Matthew; Jung, Min K; Smith, Kerrington D; Lin, Yu Kuei; Walsh, R Matthew; Hatipoglu, Betul; Freeman, Martin L; Pruett, Timothy L; Beilman, Gregory J; Sutherland, David E R; Dunn, Ty B; Axelrod, David A; Chaidarun, Sushela S; Stevens, Tyler K; Bellin, Melena; Gardner, Timothy B

    2017-04-01

    Total pancreatectomy with islet autotransplantation (TPIAT) is increasingly performed with remote islet cell processing and preparation, i.e., with islet cell isolation performed remotely from the primary surgical site at an appropriately equipped islet isolation facility. We aimed to determine whether TPIAT using remote islet isolation results in comparable long-term glycemic outcomes compared with TPIAT performed with standard local isolation. We performed a retrospective cohort study of adult patients who underwent TPIAT at three tertiary care centers from 2010 to 2013. Two centers performed remote isolation and one performed local isolation. Explanted pancreata in the remote cohort were transported ∼130 miles to and from islet isolation facilities. The primary outcome was insulin independence 1 year following transplant. Baseline characteristics were similar between groups except the remote cohort had higher preoperative hemoglobin A1c (HbA1c; 5.43 vs. 5.25, P=0.02) and there were more females in the local cohort (58% vs. 76%, P=0.049). At 1 year, 27% of remote and 32% of local patients were insulin independent (P=0.48). Remote patients experienced a greater drop in fasting c-peptide (-1.66 vs. -0.64, P=0.006) and a greater rise in HbA1c (1.65 vs. 0.99, P=0.014) at 1-year follow-up. A preoperative c-peptide >2.7 (odds ratio (OR) 4.4, 95% confidence interval (CI) 1.6-14.3) and >3,000 islet equivalents/kg (OR 11.0, 95% CI 3.2-37.3) were associated with one-year insulin independence in the local group. At 1 year after TPIAT, patients undergoing remote surgery have equivalent rates of long-term insulin independence compared with patients undergoing TPIAT locally, but metabolic control is superior with local isolation.

  8. Control of islet intercellular adhesion molecule-1 expression by interferon-alpha and hypoxia.

    Science.gov (United States)

    Chakrabarti, D; Huang, X; Beck, J; Henrich, J; McFarland, N; James, R F; Stewart, T A

    1996-10-01

    The ability of interferon-alpha (IFN-alpha) to induce the adhesion molecules that characterize the islets of patients with type I diabetes has been investigated. We have found that all tested recombinant IFN-as will induce major histocompatibility complex (MHC) class I on arterial endothelial cells. Some but not all IFN-as will induce intercellular adhesion molecule-1 (ICAM-1). However, there is only a transient and modest increase in VCAM on arterial endothelial cells. IFN-alpha has very little effect on endothelial MHC class II expression but will induce these proteins on monocytes. Thus, there is a close concordance between the biological actions of IFN-alpha and the appearance of those adhesion molecules induced in the islets of patients with type I diabetes. IFN-alpha is also produced in normal human islets during short-term cultures, probably as a result of the ischemia present at the center of the islet. This induction of IFN-alpha by hypoxia may explain the previously reported spontaneous induction of ICAM-1 in human islets and may also be a contributing factor to the failure of islet grafts.

  9. A stereological study of effects of aqueous extract of Tamarindus indica seeds on pancreatic islets in streptozotocin-induced diabetic rats.

    Science.gov (United States)

    Hamidreza, Hamidreza; Heidari, Zahra; Shahraki, Mohammadreza; Moudi, Bita

    2010-10-01

    Tamarindus indica Linn was used as a traditional medicine for the management of diabetes mellitus in human and experimental animals. This study investigated effects of aqueous extract of Tamarindus indica seeds (AETIS) against STZ-induced damages in pancreatic islands by means of stereological methods. sixty matured normoglycemic male Wistar rats, weighing 200-250 gr, were selected and randomly divided into 6 groups (n=10). Control, STZ-induced diabetic; by intraperitoneal injection of 55 mg/Kg streptozotocin, Treated control group (TC); received AETIS at a dose of 200mg/kg/day, and AETIS treated diabetic groups (TD1-3); received respectively AETIS at the dose of 50, 100,and 200 mg/kg/day by gavage from one week after induction of diabetes by STZ. After 8 weeks of experiment, stereological estimation of volume density and total volume of islets and beta cells, volume weighted mean islets volume, mass of beta cells, islets, and pancreas and total number of islets were done. Volume density and total volume of islets, volume weighted mean islets volume, volume density islets/pancreas, volume density beta cells/islet, mass of islets and pancreas of treated diabetic groups (TD1-3) were significantly higher than untreated diabetic group (P0.05). Total number of islets, pancreas wet weight and volume did not show any significant changes between control and experimental groups (P>0.05). Results suggested that AETIS partially restores pancreatic beta cells and repairs STZ-induced damages in rats.

  10. Mold-casted non-degradable, islet macro-encapsulating hydrogel devices for restoration of normoglycemia in diabetic mice.

    Science.gov (United States)

    Rios, Peter Daniel; Zhang, Xiaomin; Luo, Xunrong; Shea, Lonnie D

    2016-11-01

    Islet transplantation is a potential cure for diabetic patients, however this procedure is not widely adopted due to the high rate of graft failure. Islet encapsulation within hydrogels is employed to provide a three-dimensional microenvironment conducive to survival of transplanted islets to extend graft function. Herein, we present a novel macroencapsulation device, composed of PEG hydrogel, that combines encapsulation with lithography techniques to generate polydimethylsiloxane (PDMS) molds. PEG solutions are mixed with islets, which are then cast into PDMS molds for subsequent crosslinking. The molds can also be employed to provide complex architectures, such as microchannels that may allow vascular ingrowth through pre-defined regions of the hydrogel. PDMS molds allowed for the formation of stable gels with encapsulation of islets, and in complex architectures. Hydrogel devices with a thickness of 600 μm containing 500 islets promoted normoglycemia within 12 days following transplantation into the epididymal fat pad, which was sustained over the two-month period of study until removal of the device. The inclusion of microchannels, which had a similar minimum distance between islets and the hydrogel surface, similarly promoted normoglycemia. A glucose challenge test indicated hydrogel devices achieved normoglycemia 90 min post-dextrose injections, similar to control mice with native pancreata. Histochemical staining revealed that transplanted islets, identified as insulin positive, were viable and isolated from host tissue at 8 weeks post-transplantation, yet devices with microchannels had tissue and vascular ingrowth within the channels. Taken together, these results demonstrate a system for creating non-degradable hydrogels with complex geometries for encapsulating islets capable of restoring normoglycemia, which may expand islet transplantation as a treatment option for diabetic patients. Biotechnol. Bioeng. 2016;113: 2485-2495. © 2016 Wiley

  11. Rac1 regulates pancreatic islet morphogenesis.

    OpenAIRE

    2009-01-01

    Abstract Background Pancreatic islets of Langerhans originate from endocrine progenitors within the pancreatic ductal epithelium. Concomitant with differentiation of these progenitors into hormone-producing cells such cells delaminate, aggregate and migrate away from the ductal epithelium. The cellular and molecular mechanisms regulating islet cell delamination and cell migration are poorly understood. Extensive biochemical and cell biological studies using cultured cells demonstrated that Ra...

  12. Isolation and characterization of the human MRE11 homologue

    Energy Technology Data Exchange (ETDEWEB)

    Petrini, J.H.J.; Walsh, M.E.; DiMare, C. [Harvard Medical School, Boston, MA (United States)] [and others

    1995-09-01

    Mutation of the Saccharomyces cerevisiae RAD52 epistasis group gene, MRE11, blocks meiotic recombination, confers profound sensitivity to double-strand break damage, and has a hyperrecombinational phenotype in mitotic cells. We isolated a highly conserved human MRE11 homologue using a two-hybrid screen for DNA ligase I-interacting proteins. Human MRE11 shares approximately 50% identity with its yeast counterpart over the N-terminal half of the protein. MRE11 is expressed at the highest levels in proliferating tissues, but is also observed in other tissues. The MRE11 locus maps to human chromosome 11q21 in a region frequently associated with cancer-related chromosomal abnormalities. A MRE11-related locus was found on chromosome 7q11.2-q11.3. 60 refs., 4 figs.

  13. Genotyping of Giardia lamblia isolates from human in southern Iran.

    Science.gov (United States)

    Sarkari, B; Ashrafmansori, A; Hatam, G R; Motazedian, M H; Asgari, Q; Mohammadpour, I

    2012-09-01

    Giardia lamblia cysts isolated from human faeces in South of Iran were analyzed with PCR-restriction fragment length polymorphism (RFLP) assay, based on the detection of glutamate dehydrogenase (gdh) genes. Among 205 faecal samples from microscopically diagnosed giardiasis patients, the gdh gene was amplified from 172 cases with a semi nested PCR assay and typed by RFLP analysis. Of the 172 positive samples, 128 (74.41%) were typed as assemblage AII, 30 (17.44%) assemblage BIII, 6 (3.49%) assemblage BIV and in 8 (4.66%) isolates, mixed assemblages AII and BIV were detected. Clinical features were available for 52 successfully typed cases and the possible correlation of Giardia assemblages and clinical symptoms was evaluated. Both assemblages caused similar illness, but assemblage AII was significantly more frequently associated with abdominal pain, nausea and vomiting. Since these isolates, A and B, are of human origin, anthroponotic transmission of Giardia can be suggested for the route of infection in this region.

  14. Canine islets in an ultrafiltered environment.

    Science.gov (United States)

    Merrell, R C; Basadonna, G

    1985-11-01

    Molecular sieve membranes can protect pancreatic islets against immune recognition in diabetic patients treated by endocrine tissue replacement. These biocompatible membranes permit the passage of small peptides such as insulin, and preclude the diffusion of immunoglobulins and immunogenic molecules. However, the tissue must function indefinitely in an ultrafiltered environment determined by the sequestering membranes. The chronic perifusion of canine islet tissue was compared in ultrafiltered and microfiltered chambers. The biphasic pattern of insulin release by similar numbers of islets from the same pancrease preparation was not significantly different when tissue was cultured in a micro- or an ultrafiltered environment. The cumulative insulin output of the two systems was quite similar over 3 days of culture. Canine islet tissue can be sustained in an ultrafiltered environment with maintenance of insulin release to glucose stimulation, which is quantitatively similar to islet tissue maintained in chronic perifusion without ultrafiltration.

  15. Fentanyl inhibits glucose-stimulated insulin release from β-cells in rat pancreatic islets

    Institute of Scientific and Technical Information of China (English)

    Tao-Lai Qian; Xin-Hua Wang; Sheng Liu; Liang Ma; Ying Lu

    2009-01-01

    AIM:To explore the effects of fentanyl on insulin release from freshly isolated rat pancreatic islets in static culture.METHODS: Islets were isolated from the pancreas of mature Sprague Dawley rats by common bile duct intraductal collagenase V digestion and were purified by discontinuous Ficoll density gradient centrifugation.The islets were divided into four groups according to the fentanyl concentration: control group (0 ng/mL),group Ⅰ (0.3 ng/mL), group Ⅱ (3.0 ng/mL), and group Ⅲ (30 ng/mL). In each group, the islets were co-cultured for 48 h with drugs under static conditions with fentanyl alone, fentanyl + 0.1 μg/mL naloxone or fentanyl + 1.0 μg/mL naloxone. Cell viability was assessed by the MTT assay. Insulin release in response to low and high concentrations (2.8 mmol/L and 16.7 mmol/L,respectively) of glucose was investigated and electron microscopy morphological assessment was performed.RESULTS: Low- and high-glucose-stimulated insulin release in the control group was significantly higher than in groups Ⅱ and Ⅲ (62.33 ±9.67 μIU vs 47.75 ±96.17 ± 14.17 μIU, 75.17 ± 13.57 μIU, respectively, P <0.01) and was lowest in group Ⅲ ( P < 0.01). After adding 1 μg/mL naloxone, insulin release in groups Ⅱ and Ⅲ was not different from the control group. Electron microscopy studies showed that the islets were damaged by 30 ng/mL fentanyl.CONCLUSION: Fentanyl inhibited glucose-stimulated insulin release from rat islets, which could be prevented by naloxone. Higher concentrations of fentanyl significantly damaged β-cells of rat islets.howed that the isl

  16. Exogenous and endogenous ghrelin counteracts GLP-1 action to stimulate cAMP signaling and insulin secretion in islet β-cells.

    Science.gov (United States)

    Damdindorj, Boldbaatar; Dezaki, Katsuya; Kurashina, Tomoyuki; Sone, Hideyuki; Rita, Rauza; Kakei, Masafumi; Yada, Toshihiko

    2012-07-30

    We studied interactive effects of insulinotropic GLP-1 and insulinostatic ghrelin on rat pancreatic islets. GLP-1 potentiated glucose-induced insulin release and cAMP production in isolated islets and [Ca(2+)](i) increases in single β-cells, and these potentiations were attenuated by ghrelin. Ghrelin suppressed [Ca(2+)](i) responses to an adenylate cyclase activator forskolin. Moreover, GLP-1-induced insulin release and cAMP production were markedly enhanced by [D-lys(3)]-GHRP-6, a ghrelin receptor antagonist, in isolated islets. These results indicate that both exogenous and endogenous islet-derived ghrelin counteracts glucose-dependent GLP-1 action to increase cAMP production, [Ca(2+)](i) and insulin release in islet β-cells, positioning ghrelin as a modulator of insulinotropic GLP-1.

  17. Rac1 regulates pancreatic islet morphogenesis

    Directory of Open Access Journals (Sweden)

    Ståhlberg Anders

    2009-01-01

    Full Text Available Abstract Background Pancreatic islets of Langerhans originate from endocrine progenitors within the pancreatic ductal epithelium. Concomitant with differentiation of these progenitors into hormone-producing cells such cells delaminate, aggregate and migrate away from the ductal epithelium. The cellular and molecular mechanisms regulating islet cell delamination and cell migration are poorly understood. Extensive biochemical and cell biological studies using cultured cells demonstrated that Rac1, a member of the Rho family of small GTPases, acts as a key regulator of cell migration. Results To address the functional role of Rac1 in islet morphogenesis, we generated transgenic mice expressing dominant negative Rac1 under regulation of the Rat Insulin Promoter. Blocking Rac1 function in beta cells inhibited their migration away from the ductal epithelium in vivo. Consistently, transgenic islet cell spreading was compromised in vitro. We also show that the EGF-receptor ligand betacellulin induced actin remodelling and cell spreading in wild-type islets, but not in transgenic islets. Finally, we demonstrate that cell-cell contact E-cadherin increased as a consequence of blocking Rac1 activity. Conclusion Our data support a model where Rac1 signalling controls islet cell migration by modulating E-cadherin-mediated cell-cell adhesion. Furthermore, in vitro experiments show that betacellulin stimulated islet cell spreading and actin remodelling is compromised in transgenic islets, suggesting that betacellulin may act as a regulator of Rac1 activity and islet migration in vivo. Our results further emphasize Rac1 as a key regulator of cell migration and cell adhesion during tissue and organ morphogenesis.

  18. Expression of stem cell markers CK-19 and PDX-1 mRNA in pancreatic islet samples of different purity from rats

    Institute of Scientific and Technical Information of China (English)

    Chuang Yang; Ji-Ming Wang; Cheng-You Du; Dong Xue

    2007-01-01

    BACKGROUND:Islet stem cells are more or less retained in the procedure of islet isolation and puriifcation, and are transplanted together with islet grafts. Keratoprotein (CK-19) and pancreatic duodenal hox gene 1 (PDX-1) are markers of stem cells. This study was undertaken to examine the expression of these markers in pancreatic islet samples of different purity from rats. METHODS: A total of 30 male Sprague-Dawley rats were randomly assigned to 3 groups to undergo perfusion with V-type collagenase via the pancreatic duct, then the pancreas was excised, diced, shaken, digested and centrifuged to obtain islet sediments. The sediment from group A was not puriifed, while that from group B was puriifed with 25% Ficoll-400 and that from group C with 25% and 11% Ficoll-400. RNA was extracted from the different islet samples for reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of the pancreatic stem cell markers CK-19 and PDX-1 was assessed. RESULTS:The purity of islets in samples was (43.6±6.29)%in group A;(65.3±4.40)%in group B;and (77.6±6.36)%in group C (P CONCLUSION:The expression of CK-19 and PDX-1 mRNA in islet samples of different purity suggests the presence of stem cells in all islet samples.

  19. Long-term function of islets encapsulated in a re-designed alginate microcapsule construct in omentum pouches of immune-competent diabetic rats

    Science.gov (United States)

    Pareta, Rajesh; McQuilling, John P; Sittadjody, Sivanandane; Jenkins, Randy; Bowden, Stephen; Orlando, Giuseppe; Farney, Alan C; Brey, Eric M; Opara, Emmanuel C

    2014-01-01

    Objectives Our study aim was to determine encapsulated islet graft viability in an omentum pouch and the effect of FGF-1 released from our redesigned alginate microcapsules on the function of the graft. Methods Isolated rat islets were encapsulated in an inner core made with 1.5% low-viscosity high-mannuronic acid (LVM) alginate followed by an external layer made with 1.25% low-viscosity high-guluronic acid (LVG) alginate with or without FGF-1, in microcapsules measuring 300 – 400 μm in diameter. The two alginate layers were separated by a perm-selective membrane made with 0.1 % Poly-L-Ornithine (PLO), and the inner LVM core was partially chelated using 55 mM sodium citrate for 2 min. Results A marginal mass of encapsulated islet allografts (~2000 islets/kg) in Streptozotocin-diabetic Lewis rats caused significant reduction in blood glucose levels similar to the effect observed with encapsulated islet isografts. Transplantation of allo-islets co-encapsulated with FGF-1 did not result in better glycemic control, but induced greater body weight maintenance in transplant recipients compared to those that received only allo-islets. Histological examination of the retrieved tissue demonstrated morphologically and functionally intact islets in the microcapsules, with no signs of fibrosis. Conclusion We conclude that the omentum is a viable site for encapsulated islet transplantation. PMID:24681880

  20. Sustained NF-κB activation and inhibition in β-cells have minimal effects on function and islet transplant outcomes.

    Directory of Open Access Journals (Sweden)

    Aileen J F King

    Full Text Available The activation of the transcription factor NF-κB leads to changes in expression of many genes in pancreatic β-cells. However, the role of NF-κB activation in islet transplantation has not been fully elucidated. The aim of the present study was to investigate whether the state of NF-κB activation would influence the outcome of islet transplantation. Transgenic mice expressing a dominant active IKKβ (constitutively active or a non-degradable form of IκBα (constitutive inhibition under control of the rat insulin promoter were generated. Islets from these mice were transplanted into streptozotocin diabetic mice in suboptimal numbers. Further, the effects of salicylate (an inhibitor of NF-κB treatment of normal islets prior to transplantation, and the effects of salicylate administration to mice prior to and after islet implantation were evaluated. Transplantation outcomes were not affected using islets expressing a non-degradable form of IκBα when compared to wild type controls. However, the transplantation outcomes using islets isolated from mice expressing a constitutively active mutant of NF-κB were marginally worse, although no aberrations of islet function in vitro could be detected. Salicylate treatment of normal islets or mice had no effect on transplantation outcome. The current study draws attention to the complexities of NF-κB in pancreatic beta cells by suggesting that they can adapt with normal or near normal function to both chronic activation and inhibition of this important transcription factor.

  1. Control of insulin secretion by cytochrome C and calcium signaling in islets with impaired metabolism.

    Science.gov (United States)

    Rountree, Austin M; Neal, Adam S; Lisowski, Mark; Rizzo, Norma; Radtke, Jared; White, Sarah; Luciani, Dan S; Kim, Francis; Hampe, Christiane S; Sweet, Ian R

    2014-07-01

    The aim of the study was to assess the relative control of insulin secretion rate (ISR) by calcium influx and signaling from cytochrome c in islets where, as in diabetes, the metabolic pathways are impaired. This was achieved either by culturing isolated islets at low (3 mm) glucose or by fasting rats prior to the isolation of the islets. Culture in low glucose greatly reduced the glucose response of cytochrome c reduction and translocation and ISR, but did not affect the response to the mitochondrial fuel α-ketoisocaproate. Unexpectedly, glucose-stimulated calcium influx was only slightly reduced in low glucose-cultured islets and was not responsible for the impairment in glucose-stimulated ISR. A glucokinase activator acutely restored cytochrome c reduction and translocation and ISR, independent of effects on calcium influx. Islets from fasted rats had reduced ISR and cytochrome c reduction in response to both glucose and α-ketoisocaproate despite normal responses of calcium. Our data are consistent with the scenario where cytochrome c reduction and translocation are essential signals in the stimulation of ISR, the loss of which can result in impaired ISR even when calcium response is normal.

  2. Postnatal development of numbers and mean sizes of pancreatic islets and beta-cells in healthy mice and GIPR(dn transgenic diabetic mice.

    Directory of Open Access Journals (Sweden)

    Nadja Herbach

    Full Text Available The aim of this study was to examine postnatal islet and beta-cell expansion in healthy female control mice and its disturbances in diabetic GIPR(dn transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPR(dn transgenic mice, aged 10, 45, 90 and 180 days were examined, using state-of-the-art quantitative-stereological methods. Total islet and beta-cell volumes, as well as their absolute numbers increased significantly until 90 days in control mice, and remained stable thereafter. The mean islet volumes of controls also increased slightly but significantly between 10 and 45 days of age, and then remained stable until 180 days. The total volume of isolated beta-cells, an indicator of islet neogenesis, and the number of proliferating (BrdU-positive islet cells were highest in 10-day-old controls and declined significantly between 10 and 45 days. In GIPR(dn transgenic mice, the numbers of islets and beta-cells were significantly reduced from 10 days of age onwards vs. controls, and no postnatal expansion of total islet and beta-cell volumes occurred due to a reduction in islet neogenesis whereas early islet-cell proliferation and apoptosis were unchanged as compared to control mice. Insulin secretion in response to pharmacological doses of GIP was preserved in GIPR(dn transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPR(dn transgenic mice vs. controls. We could show that the increase in islet number is mainly responsible for expansion of islet and beta-cell mass in healthy control mice. GIPR(dn transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis.

  3. Postnatal development of numbers and mean sizes of pancreatic islets and beta-cells in healthy mice and GIPR(dn) transgenic diabetic mice.

    Science.gov (United States)

    Herbach, Nadja; Bergmayr, Martina; Göke, Burkhard; Wolf, Eckhard; Wanke, Ruediger

    2011-01-01

    The aim of this study was to examine postnatal islet and beta-cell expansion in healthy female control mice and its disturbances in diabetic GIPR(dn) transgenic mice, which exhibit an early reduction of beta-cell mass. Pancreata of female control and GIPR(dn) transgenic mice, aged 10, 45, 90 and 180 days were examined, using state-of-the-art quantitative-stereological methods. Total islet and beta-cell volumes, as well as their absolute numbers increased significantly until 90 days in control mice, and remained stable thereafter. The mean islet volumes of controls also increased slightly but significantly between 10 and 45 days of age, and then remained stable until 180 days. The total volume of isolated beta-cells, an indicator of islet neogenesis, and the number of proliferating (BrdU-positive) islet cells were highest in 10-day-old controls and declined significantly between 10 and 45 days. In GIPR(dn) transgenic mice, the numbers of islets and beta-cells were significantly reduced from 10 days of age onwards vs. controls, and no postnatal expansion of total islet and beta-cell volumes occurred due to a reduction in islet neogenesis whereas early islet-cell proliferation and apoptosis were unchanged as compared to control mice. Insulin secretion in response to pharmacological doses of GIP was preserved in GIPR(dn) transgenic mice, and serum insulin to pancreatic insulin content in response to GLP-1 and arginine was significantly higher in GIPR(dn) transgenic mice vs. controls. We could show that the increase in islet number is mainly responsible for expansion of islet and beta-cell mass in healthy control mice. GIPR(dn) transgenic mice show a disturbed expansion of the endocrine pancreas, due to perturbed islet neogenesis.

  4. Pancreatic islet renin angiotensin system: its novel roles in islet function and in diabetes mellitus.

    Science.gov (United States)

    Leung, Po Sing; Carlsson, Per-Ola

    2005-05-01

    Several regulatory systems are implicated in the regulation of islet function and beta cell mass. Of great interest in this context are some endocrine, paracrine/autocrine, and intracrine regulators. These include, to name but a few, the gut peptides, growth factors, prostaglandins, and some vasoactive mediators such as nitric oxide, bradykinins, endothelins, and angiotensins. Apart from its potent vasoconstrictor actions, the renin-angiotensin system (RAS) that generates angiotensin II has several novel functions-stimulation and inhibition of cell proliferation; induction of apoptosis; generation of reactive oxygen species; regulation of hormone secretion; and proinflammatory and profibrogenic actions. In the pancreas, recent evidence supports the presence of an islet RAS, which is subject to activation by islet transplantation and diabetes. Such a local islet RAS, if activated, may drive islet fibrosis and reduce islet blood flow, oxygen tension, and insulin biosynthesis. Moreover, activation of an islet RAS may drive the synthesis of reactive oxygen species, cause oxidative stress-induced beta cell dysfunction and apoptosis, and thus contribute to the islet dysfunction seen in type 2 diabetes and after islet transplantation. Blockade of the RAS could contribute to the development of novel therapeutic strategies in the prevention and treatment of patients with diabetes and in islet transplantation.

  5. Small Islets Transplantation Superiority to Large Ones: Implications from Islet Microcirculation and Revascularization

    Directory of Open Access Journals (Sweden)

    Wenjuan Li

    2014-01-01

    Full Text Available Pancreatic islet transplantation is a promising therapy to regain glycemic control in diabetic patients. The selection of ideal grafts is the basis to guarantee short-term effectivity and longevity of the transplanted islets. Contradictory to the traditional notion, recent findings implied the superiority of small islets for better transplantation outcomes rather than the large and intact ones. However, the mechanisms remain to be elucidated. Recent evidences emphasized the major impact of microcirculation on islet β-cell mass and function. And potentials in islet graft revascularization are crucial for their survival and preserved function in the recipient. In this study, we verified the distinct histological phenotype and functionality of small islets versus large ones both in vitro and in vivo. With efforts to exploring the differences in microcirculation and revascularization of islet grafts, we further evaluated local expressions of angiotensin and vascular endothelial growth factor A (VEGF-A at different levels. Our findings reveal that, apart from the higher density of insulin-producing β-cells, small islets express less angiotensin and more angiotrophic VEGF-A. We therefore hypothesized a logical explanation of the small islet superiority for transplantation outcome from the aspects of facilitated microcirculation and revascularization intrinsically in small islets.

  6. Identification of Donor Origin and Condition of Transplanted Islets In Situ in the Liver of a Type 1 Diabetic Recipient.

    Science.gov (United States)

    van der Torren, Cornelis R; Suwandi, Jessica S; Lee, DaHae; Van't Wout, Ernst-Jan T; Duinkerken, Gaby; Swings, Godelieve; Mulder, Arend; Claas, Frans H J; Ling, Zhidong; Gillard, Pieter; Keymeulen, Bart; In't Veld, Peter; Roep, Bart O

    2017-01-24

    Transplantation of islet allografts into type 1 diabetic recipients usually requires multiple pancreas donors to achieve insulin independence. This adds to the challenges of immunological monitoring of islet transplantation currently relying on surrogate immune markers in peripheral blood. We investigated donor origin and infiltration of islets transplanted in the liver of a T1D patient who died of hemorrhagic stroke 4 months after successful transplantation with two intraportal islet grafts combining six donors. Immunohistological staining for donor HLA using a unique panel of human monoclonal HLA-specific alloantibodies was performed on liver cryosections after validation on cryopreserved kidney, liver, and pancreas and compared with auto- and alloreactive T-cell immunity in peripheral blood. HLA-specific staining intensity and signal-to-noise ratio varied between tissues from very strong on kidney glomeruli, less in liver, kidney tubuli, and endocrine pancreas to least in exocrine pancreas, complicating the staining of inflamed islets in an HLA-disparate liver. Nonetheless, five islets from different liver lobes could be attributed to donors 1, 2, and 5 by staining patterns with multiple HLA types. All islets showed infiltration with CD8+ cytotoxic T cells that was mirrored by progressive alloreactive responses in peripheral blood mononuclear cells (PBMCs) to donors 1, 2, and 5 after transplantation. Stably low rates of peripheral islet autoreactive T-cell responses after islet infusion fit with a complete HLA mismatch between grafts and recipient and exclude the possibility that the islet-infiltrating CD8 T cells were autoreactive. HLA-specific immunohistochemistry can identify donor origin in situ and differentiate graft dysfunction and immunological destruction.

  7. Clinical use of fructosamine in islet transplantation.

    Science.gov (United States)

    Tharavanij, Thipaporn; Froud, Tatiana; Leitao, Cristiane B; Baidal, David A; Paz-Pabon, Charlotte N; Shari, Messinger; Cure, Pablo; Bernetti, Karina; Ricordi, Camillo; Alejandro, Rodolfo

    2009-01-01

    Many islet transplant recipients have medical conditions that could interfere with the accuracy of HbA1c measurements (e.g., anemia/dapsone use). Fructosamine is less prone to have clinical interferences and reflects glucose control in a shorter period of time than HbA1c. This study aimed to validate fructosamine use in islet transplant subjects and to evaluate its effectiveness as a predictor for islet graft dysfunction. Thirty-three islet transplant recipients who had concomitant fructosamine and HbA1c data available were retrospectively analyzed. HbA1c, fructosamine, mean capillary blood glucose, and islet graft function (fasting C-peptide/glucose ratio) were assessed. There was a significant and positive association between fructosamine and HbA1c (p 6% was predictive of this outcome 1 month in advance (OR 2.95, p = 0.003). However, although significantly associated with graft dysfunction, use of this cutoff as a predictor of dysfunction has poor sensitivity (50%) and specificity (77.6%). Fructosamine above the normal range (>270 mumol/L Quest Diagnostics) was also predictive of ensuing dysfunction (OR 2.47, p = 0.03); however, it had similarly poor sensitivity (62%) and specificity (64%). Fructosamine can be used as an alternative to HbA1c for glycemic assessment in islet transplant recipients in situations with HbA1c assay interference. Neither HbA1c nor fructosamine are good predictors of islet graft dysfunction.

  8. Isolation and functional characterization of the human 90K promoter

    DEFF Research Database (Denmark)

    Brakebusch, C; Sures, I; Jallal, B;

    1999-01-01

    90K is a secreted protein thought to be involved in the body's defense against pathogens and cancer. To elucidate its transcriptional regulation, the promoter of human 90K (HGMW-approved symbol LGAL S3BP) was isolated and characterized. Analysis of the 3. 3-kb 5'-flanking region revealed that it ......90K is a secreted protein thought to be involved in the body's defense against pathogens and cancer. To elucidate its transcriptional regulation, the promoter of human 90K (HGMW-approved symbol LGAL S3BP) was isolated and characterized. Analysis of the 3. 3-kb 5'-flanking region revealed...... that it is a TATA-less promoter, but neither GC-rich nor dependent on SP1 sites. RNase protection assays detected one major transcription start site (+1) and several minor transcription start sites upstream and downstream. Deletion studies defined a minimal promoter (-103 --> -49) and indirectly suggested positive...... synergism between different elements within it. Consistent with the proposed function of 90K, its promoter activity could be stimulated by poly(I). poly(C), mimicking viral infection. Two regions mediating induction by poly(I). poly(C) (-171 --> -112, -32 --> 46) were identified by deletion mutants. A small...

  9. Neuron enriched nuclear proteome isolated from human brain.

    Science.gov (United States)

    Dammer, Eric B; Duong, Duc M; Diner, Ian; Gearing, Marla; Feng, Yue; Lah, James J; Levey, Allan I; Seyfried, Nicholas T

    2013-07-05

    The brain consists of diverse cell types including neurons, astrocytes, oligodendrocytes, and microglia. The isolation of nuclei from these distinct cell populations provides an opportunity to identify cell-type-specific nuclear proteins, histone modifications, and regulation networks that are altered with normal brain aging or neurodegenerative disease. In this study, we used a method by which intact neuronal and non-neuronal nuclei were purified from human post-mortem brain employing a modification of fluorescence activated cell sorting (FACS) termed fluorescence activated nuclei sorting (FANS). An antibody against NeuN, a neuron specific splicing factor, was used to isolate neuronal nuclei. Utilizing mass spectrometry (MS) based label-free quantitative proteomics, we identified 1755 proteins from sorted NeuN-positive and negative nuclear extracts. Approximately 20% of these proteins were significantly enriched or depleted in neuronal versus non-neuronal populations. Immunoblots of primary cultured rat neuron, astrocyte, and oligodendrocyte extracts confirmed that distinct members of the major nucleocytoplasmic structural linkage complex (LINC), nesprin-1 and nesprin-3, were differentially enriched in neurons and astrocytes, respectively. These comparative proteomic data sets also reveal a number of transcription and splicing factors that are selectively enriched in a cell-type-specific manner in human brain.

  10. Human dental pulp mesenchymal stem cells isolation and osteoblast differentiation

    Directory of Open Access Journals (Sweden)

    Moustafa Alkhalil

    2015-02-01

    Full Text Available Aim This study was focused on the isolation and characterization of mesenchymal stem cells (MSCs from human dental pulp (DPSC. Methods The study was performed in the Department for Oral and Cranio-Maxillo- Facial Surgey Hamad Medical Corporation, Doha, Qatar and Weill Cornell Medical Colleague Doha, Qatar, in period 2010-2011. Dental pulp was extracted from premolars and third molars of 19 healthy patients. The pulp was digested in a solution of 3 mg/mL collagenase type I and 4 mg/mL dispase for 1 hour at 37C. After filtration, cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM Low Glucoses with 20% Fetal Bovine Serum (FBS, 2mM L-glutamine and antibiotics (100 U/mL penicillin, 100 ug/mL streptomycin at 37 °C under 5% CO2. Cultures were treated with osteoinductive medium for differentiation MSC in to the osteoblast cell line. Staining with Alizarin red were used for the detection of the osteoblast production and calcification new formed tissue. Results On the total of three out of 19 patients it was possible to isolate DPMSCs after 2 to 3 weeks: in one patient it was not possible to expand MSCs because of infection, and in other two patients positive Alizarin red staining reaction showed osteogenic differentiation capability and strong mineralization in vitro. Conclusion The main advantage of using DPSC is absence of morbidity. MSCs could be isolated noninvasively from teeth, routinely extracted in the clinic and discarded as medical waste. Standardization of clinical and laboratory protocols for DPMSCs isolation and team work coordination could lead to significantly improved result.

  11. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand

    Science.gov (United States)

    Nawtaisong, Pruksa; Tanganuchitcharnchai, Ampai; Smith, Derek J.; Day, Nicholas P. J.; Paris, Daniel H.

    2016-01-01

    Background Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development. Methodology/Principal Findings This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera. Conclusions/Significance Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes. PMID:27248711

  12. Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand.

    Directory of Open Access Journals (Sweden)

    Sarah L James

    2016-06-01

    Full Text Available Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development.This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation, in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera.Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes.

  13. Isolation of biologically-active exosomes from human plasma.

    Science.gov (United States)

    Muller, Laurent; Hong, Chang-Sook; Stolz, Donna B; Watkins, Simon C; Whiteside, Theresa L

    2014-09-01

    Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in μg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies.

  14. Plasticity of adult human pancreatic duct cells by neurogenin3-mediated reprogramming

    DEFF Research Database (Denmark)

    Swales, Nathalie; Martens, Geert A; Bonné, Stefan

    2012-01-01

    Duct cells isolated from adult human pancreas can be reprogrammed to express islet beta cell genes by adenoviral transduction of the developmental transcription factor neurogenin3 (Ngn3). In this study we aimed to fully characterize the extent of this reprogramming and intended to improve it....

  15. L-leucine methyl ester stimulates insulin secretion and islet glutamate dehydrogenase

    DEFF Research Database (Denmark)

    Knudsen, P; Kofod, Hans; Lernmark, A

    1983-01-01

    Column perifusion of collagenase-isolated mouse pancreatic islets was used to study the dynamics of insulin release in experiments lasting for several hours. The methyl esters of L-leucine and L-arginine were synthesized. Whereas L-arginine methyl ester (L-arginine OMe) had no effect, L-leucine OMe...

  16. ARGININE STIMULATED GLUCAGON AND INSULIN-SECRETION BY ISLETS OF LANGERHANS OF PREGNANT AND LACTATING RATS

    NARCIS (Netherlands)

    MOES, H; SCHUILING, GA; KOITER, TR

    Glucagon secretion by isolated pancreatic rat islets was not affected by an increase of the glucose concentration from 2.5 to 5.0 mM, but was stimulated by 25 mM arginine. This stimulation was only slightly increased by pregnancy and lactation. Insulin secretion increased, when the glucose

  17. ARGININE STIMULATED GLUCAGON AND INSULIN-SECRETION BY ISLETS OF LANGERHANS OF PREGNANT AND LACTATING RATS

    NARCIS (Netherlands)

    MOES, H; SCHUILING, GA; KOITER, TR

    1993-01-01

    Glucagon secretion by isolated pancreatic rat islets was not affected by an increase of the glucose concentration from 2.5 to 5.0 mM, but was stimulated by 25 mM arginine. This stimulation was only slightly increased by pregnancy and lactation. Insulin secretion increased, when the glucose concentra

  18. Characterization of Staphylococcus caprae Clinical Isolates Involved in Human Bone and Joint Infections, Compared with Goat Mastitis Isolates.

    Science.gov (United States)

    d'Ersu, J; Aubin, G G; Mercier, P; Nicollet, P; Bémer, P; Corvec, S

    2016-01-01

    Staphylococcus caprae is an emerging microorganism in human bone and joint infections (BJI). The aim of this study is to describe the features of S. caprae isolates involved in BJI (H for human) compared with those of isolates recovered in goat mastitis (A for animal). Fourteen isolates of each origin were included. Identifications were performed using a Vitek 2 GP ID card, tuf gene sequencing, and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) Vitek MS. Molecular typing was carried out using pulsed-field gel electrophoresis (PFGE) and DiversiLab technology. The crystal violet method was used to determine biofilm-forming ability. Virulence factors were searched by PCR. Vitek MS technology provides an accurate identification for the two types of isolates compared to that of gold-standard sequencing (sensitivity, 96.4%), whereas the Vitek 2 GP ID card was more effective for H isolates. Molecular typing methods revealed two distinct lineages corresponding to the origin despite few overlaps: H and A. In our experimental conditions, no significant difference was observed in biofilm production ability between H and A isolates. Nine isolates (5 H isolates and 4 A isolates) behaved as weak producers while one A isolate was a strong producer. Concerning virulence factors, the autolysin atlC and the serine aspartate adhesin (sdrZ) genes were detected in 24 isolates (86%), whereas the lipase gene was always detected, except in one H isolate (96%). The ica operon was present in 23 isolates (82%). Fibrinogen-binding (fbe) or collagen-binding (cna) genes were not detected by using primers designed for Staphylococcus aureus or Staphylococcus epidermidis, even in low stringency conditions. Although S. caprae probably remains underestimated in human infections, further studies are needed to better understand the evolution and the adaptation of this species to its host.

  19. Phycocyanin protects INS-1E pancreatic beta cells against human islet amyloid polypeptide-induced apoptosis through attenuating oxidative stress and modulating JNK and p38 mitogen-activated protein kinase pathways.

    Science.gov (United States)

    Li, Xiao-Ling; Xu, Gang; Chen, Tianfeng; Wong, Yum-Shing; Zhao, Hai-Lu; Fan, Rong-Rong; Gu, Xue-Mei; Tong, Peter C Y; Chan, Juliana C N

    2009-07-01

    It is widely accepted that human islet amyloid polypeptide (hIAPP) aggregation plays an important role in the loss of insulin-producing pancreatic beta cells. hIAPP-induced cytotoxicity is mediated by generation of reactive oxygen species (ROS). Phycocyanin (PC) is a natural compound from blue-green algae that is widely used as food supplement. Currently, little is known about the effects of PC on beta cells with the presence of hIAPP. The aim of this study was to investigate the in vitro protective effects of PC on INS-1E rat insulinoma beta cells against hIAPP-induced cell death, as well as the underlying mechanisms. Our results showed that hIAPP-induced cell death with apoptotic characteristics including growth inhibition, chromatin condensation and DNA fragmentation. However, cytotoxicity of hIAPP was significantly attenuated by co-incubation of the cells with PC. The results of Western blotting showed that activation of caspase-3 and cleavage of poly (ADP-ribose) polymerase (PARP) in hIAPP-treated cells was blocked by PC. Moreover, PC significantly prevented the hIAPP-induced overproduction of intracellular ROS and malondialdehyde (MDA), as well as changes in activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) enzymes. Furthermore, hIAPP triggered the activation of mitogen-activated protein kinases (MAPKs), and these effects were effectively suppressed by PC. Taken together, our results suggest that PC protects INS-1E pancreatic beta cells against hIAPP-induced apoptotic cell death through attenuating oxidative stress and modulating c-Jun N-terminal kinase (JNK) and p38 pathways.

  20. Selenium-enriched Spirulina protects INS-1E pancreatic beta cells from human islet amyloid polypeptide-induced apoptosis through suppression of ROS-mediated mitochondrial dysfunction and PI3/AKT pathway.

    Science.gov (United States)

    Li, Xiao-Ling; Wong, Yum-Shing; Xu, Gang; Chan, Juliana C N

    2015-06-01

    Human islet amyloid polypeptide (hIAPP) aggregation is linked to loss of pancreatic beta cells in type 2 diabetes, in part due to oxidative stress. Currently, little is known about the effects of selenium-enriched Spirulina on beta cells with the presence of hIAPP. In this study, INS-1E rat insulinoma cells were used as a model to evaluate in vitro protective effects of Se-enriched Spirulina extract (Se-SE) against hIAPP-induced cell death, as well as the underlying mechanisms. Flow cytometric analysis was used to evaluate cell apoptosis, mitochondrial membrane potential (ΔΨm) and ROS generation. Caspase activity was measured using a fluorometric method. Western blotting was applied to detect protein expression. Our results showed that exposure of INS-1E cells to hIAPP resulted in cell viability loss, LDH release and appearance of sub-G peak. However, cytotoxicity of hIAPP was significantly attenuated by co-treatment with Se-SE. Se-SE also inhibited hIAPP-induced activation of caspase-3, -8 and -9. Additionally, hIAPP-induced accumulation of ROS and superoxide was suppressed by co-treatment with Se-SE. Moreover, Se-SE was able to prevent hIAPP-induced depletion of ΔΨm and intracellular ATP, reduction in mitochondrial mass, changes in the expression of Bcl-2 family members, release of mitochondrial apoptogenic factors. Furthermore, hIAPP-mediated AKT inhibition was restored by co-treatment with Se-SE. Our results showed that Se-SE protects INS-1E cells from hIAPP-induced cell death through preventing ROS overproduction, mitochondrial dysfunction and modulating PI3K/AKT pathway.

  1. Suppression of islet homeostasis protein thwarts diabetes mellitus progression.

    Science.gov (United States)

    Oh, Seh-Hoon; Jorgensen, Marda L; Wasserfall, Clive H; Gjymishka, Altin; Petersen, Bryon E

    2017-05-01

    During progression to type 1 diabetes, insulin-producing β-cells are lost through an autoimmune attack resulting in unrestrained glucagon expression and secretion, activation of glycogenolysis, and escalating hyperglycemia. We recently identified a protein, designated islet homeostasis protein (IHoP), which specifically co-localizes within glucagon-positive α-cells and is overexpressed in the islets of both post-onset non-obese diabetic (NOD) mice and type 1 diabetes patients. Here we report that in the αTC1.9 mouse α-cell line, IHoP was released in response to high-glucose challenge and was found to regulate secretion of glucagon. We also show that in NOD mice with diabetes, major histocompatibility complex class II was upregulated in islets. In addition hyperglycemia was modulated in NOD mice via suppression of IHoP utilizing small interfering RNA (IHoP-siRNA) constructs/approaches. Suppression of IHoP in the pre-diabetes setting maintained normoglycemia, glyconeolysis, and fostered β-cell restoration in NOD mice 35 weeks post treatment. Furthermore, we performed adoptive transfer experiments using splenocytes from IHoP-siRNA-treated NOD/ShiLtJ mice, which thwarted the development of hyperglycemia and the extent of insulitis seen in recipient mice. Last, IHoP can be detected in the serum of human type 1 diabetes patients and could potentially serve as an early novel biomarker for type 1 diabetes in patients.

  2. Novel stable isotope analyses demonstrate significant rates of glucose cycling in mouse pancreatic islets.

    Science.gov (United States)

    Wall, Martha L; Pound, Lynley D; Trenary, Irina; O'Brien, Richard M; Young, Jamey D

    2015-06-01

    A polymorphism located in the G6PC2 gene, which encodes an islet-specific glucose-6-phosphatase catalytic subunit, is the most important common determinant of variations in fasting blood glucose (FBG) levels in humans. Studies of G6pc2 knockout (KO) mice suggest that G6pc2 represents a negative regulator of basal glucose-stimulated insulin secretion (GSIS) that acts by hydrolyzing glucose-6-phosphate (G6P), thereby reducing glycolytic flux. However, this conclusion conflicts with the very low estimates for the rate of glucose cycling in pancreatic islets, as assessed using radioisotopes. We have reassessed the rate of glucose cycling in pancreatic islets using a novel stable isotope method. The data show much higher levels of glucose cycling than previously reported. In 5 mmol/L glucose, islets from C57BL/6J chow-fed mice cycled ∼16% of net glucose uptake. The cycling rate was further increased at 11 mmol/L glucose. Similar cycling rates were observed using islets from high fat-fed mice. Importantly, glucose cycling was abolished in G6pc2 KO mouse islets, confirming that G6pc2 opposes the action of the glucose sensor glucokinase by hydrolyzing G6P. The demonstration of high rates of glucose cycling in pancreatic islets explains why G6pc2 deletion enhances GSIS and why variants in G6PC2 affect FBG in humans. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

  3. Stem cell sources for clinical islet transplantation in type 1 diabetes: embryonic and adult stem cells.

    Science.gov (United States)

    Miszta-Lane, Helena; Mirbolooki, Mohammadreza; James Shapiro, A M; Lakey, Jonathan R T

    2006-01-01

    Lifelong immunosuppressive therapy and inadequate sources of transplantable islets have led the islet transplantation benefits to less than 0.5% of type 1 diabetics. Whereas the potential risk of infection by animal endogenous viruses limits the uses of islet xeno-transplantation, deriving islets from stem cells seems to be able to overcome the current problems of islet shortages and immune compatibility. Both embryonic (derived from the inner cell mass of blastocysts) and adult stem cells (derived from adult tissues) have shown controversial results in secreting insulin in vitro and normalizing hyperglycemia in vivo. ESCs research is thought to have much greater developmental potential than adult stem cells; however it is still in the basic research phase. Existing ESC lines are not believed to be identical or ideal for generating islets or beta-cells and additional ESC lines have to be established. Research with ESCs derived from humans is controversial because it requires the destruction of a human embryo and/or therapeutic cloning, which some believe is a slippery slope to reproductive cloning. On the other hand, adult stem cells are already in some degree specialized, recipients may receive their own stem cells. They are flexible but they have shown mixed degree of availability. Adult stem cells are not pluripotent. They may not exist for all organs. They are difficult to purify and they cannot be maintained well outside the body. In order to draw the future avenues in this field, existent discrepancies between the results need to be clarified. In this study, we will review the different aspects and challenges of using embryonic or adult stem cells in clinical islet transplantation for the treatment of type 1 diabetes.

  4. Insulin release from islets of Langerhans entrapped in a poly(N-isopropylacrylamide-co-acrylic acid) polymer gel.

    Science.gov (United States)

    Vernon, B; Kim, S W; Bae, Y H

    1999-01-01

    A copolymer of N-isopropylacrylamide (98 mol% in feed) and acrylic acid, poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), was prepared by free radical polymerization for development of a thermally reversible polymer to entrap islets of Langerhans for a refillable biohybrid artificial pancreas. A 5 wt% solution of the polymer in Hanks' balanced salt solution forms a gel at 37 degrees C that exhibits no syneresis. Diffusion of fluorescein isothiocyanate (FITC) dextrans having molecular weights of 4400 and 70000 were used to evaluate mass transport in the gel at 37 degrees C. Insulin secretion from islets in the polymer gel was also investigated in both static and dynamic systems. The polymer gel exhibited excellent diffusion of FITC dextran 4400 and FITC dextran 70000 with diffusion ratios, D/D0 (ratio of diffusion in the gel to diffusion in water), of 0.20+/-0.04 and 0.35+/-0.17, respectively. Human islets entrapped in the polymer gel showed prolonged insulin secretion in response to basal (5.5 mM) glucose concentration compared to free human islets. Rat islets showed prolonged insulin secretion in response to high (16.5 mM) glucose concentrations compared to free rat islets. Rat islets in the polymer gel maintained insulin secretion in response to the higher glucose concentration for over 26 days. Rat islets entrapped by the polymer also released higher quantities of insulin more rapidly in response to changes in concentrations of glucose and other stimulants than rat islets entrapped in an alginate control. These results suggest that this material would provide adequate diffusion for rapid insulin release in an application as a synthetic extracellular matrix for a biohybrid artificial pancreas.

  5. Extracellular matrix gel is necessary for in vitro cultivation of insulin producing cells from human umbilical cord blood derived mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    GAO Feng; WU De-quan; HU Yan-hua; JIN Guang-xin

    2008-01-01

    Background Pancreatic islet cell transplantation is an effective approach to treat type 1 diabetes. However, this therapy is not widely used because of the severe shortage of transplantable donor islets. This study investigated whether mesenchymal stem cells (MSCs) derived from human umbilical cord blood (UCB) could be transdifferentiated into insulin producing cells in vitro and the role of extracellular matrix (ECM) gel in this procedure.Methods Human UCB samples were collected and MSCs were isolated. MSCs specific marker proteins were analyzed by a flow cytometer. The capacities of osteoblast and adipocyte to differentiate were tested. Differentiation into islet like cell was induced by a 15-day protocol with or without ECM gel. Pancreatic characteristics were evaluated with immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR) and flow cytometry. Insulin content and release in response to glucose stimulation were detected with chemiluminescent immunoassay system.Results Sixteen MSCs were isolated from 42 term human UCB units (38%). Human UCB-MSCs expressed MSCs specific markers and could be induced in vitro into osteoblast and adipocyte. Islet like cell clusters appeared about 9 days after pancreatic differentiation in the inducing system with ECM gel. The insulin positive cells accounted for (25.2±3.4)% of the induced cells. The induced cells expressed islet related genes and hormones, but were not very responsive to glucose challenge. When MSCs were induced without ECM gel, clusters formation and secretion of functional islet proteins could not be observed.Conclusions Human UCB-MSCs can differentiate into islet like cells in vitro and ECM gel plays an important role in pancreatic endocrine cell maturation and formation of three dimensional structures.

  6. IDDM: an islet or an immune disease?

    Science.gov (United States)

    Boitard, C; Larger, E; Timsit, J; Sempe, P; Bach, J F

    1994-09-01

    Insulin-dependent diabetes develops as a consequence of the selective destruction of insulin-producing cells by an autoimmune reaction. However, the precise series of events which trigger anti-islet autoreactive T cells is still being investigated. Major issues will need to be raised before a comprehensive view of the anti-islet autoimmune reaction can be delineated. These include defining the primary site of activation of autoreactive lymphocytes and exploring hypotheses to explain the chronicity of the diabetes process. These issues all relate with the more general dilemma of the actual role of the islets of Langerhans in breaking self tolerance to beta-cell antigens. By studying non-obese diabetic mice deprived of beta cells following a single injection of a high dose of alloxan at 3 weeks of age, we recently obtained evidence that the activation of autoreactive T cells requires the presence of target islet cells in order to develop.

  7. Oxidation of [1,12-14C]dodecanedioic acid by rat pancreatic islets.

    Science.gov (United States)

    Malaisse, W J; Greco, A V; Mingrone, G

    2000-10-01

    Several aliphatic dioic acids were recently reported to stimulate insulin release in isolated rat pancreatic islets incubated at close-to-physiological D-glucose concentrations. In order to gain insight into the mode of action of these acids in pancreatic islet B-cells, the oxidation of [1,12-14C]dodecanedioic acid (5.0 mM) was now measured in rat islets. Expressed as pmol of [1, 12-14C]dodecanedioic acid equivalent, the production of 14CO2 was close to 1.0 pmol/islet per 120 min, representing about 8% of that attributable to the oxidation of D-[U-14C]-glucose (8.3 mM). The dioic acid and the hexose failed to exert any significant reciprocal effect upon their respective oxidation rate. These findings support the view that the insulinotropic action of dodecanedioic acid, and presumably other aliphatic dioic acids, is causally linked to their capacity to act as nutrients in pancreatic islet cells.

  8. Silicon nanopore membrane (SNM) for islet encapsulation and immunoisolation under convective transport

    Science.gov (United States)

    Song, Shang; Faleo, Gaetano; Yeung, Raymond; Kant, Rishi; Posselt, Andrew M.; Desai, Tejal A.; Tang, Qizhi; Roy, Shuvo

    2016-03-01

    Problems associated with islet transplantation for Type 1 Diabetes (T1D) such as shortage of donor cells, use of immunosuppressive drugs remain as major challenges. Immune isolation using encapsulation may circumvent the use of immunosuppressants and prolong the longevity of transplanted islets. The encapsulating membrane must block the passage of host’s immune components while providing sufficient exchange of glucose, insulin and other small molecules. We report the development and characterization of a new generation of semipermeable ultrafiltration membrane, the silicon nanopore membrane (SNM), designed with approximately 7 nm-wide slit-pores to provide middle molecule selectivity by limiting passage of pro-inflammatory cytokines. Moreover, the use of convective transport with a pressure differential across the SNM overcomes the mass transfer limitations associated with diffusion through nanometer-scale pores. The SNM exhibited a hydraulic permeability of 130 ml/hr/m2/mmHg, which is more than 3 fold greater than existing polymer membranes. Analysis of sieving coefficients revealed 80% reduction in cytokines passage through SNM under convective transport. SNM protected encapsulated islets from infiltrating cytokines and retained islet viability over 6 hours and remained responsive to changes in glucose levels unlike non-encapsulated controls. Together, these data demonstrate the novel membrane exhibiting unprecedented hydraulic permeability and immune-protection for islet transplantation therapy.

  9. Candida nivariensis isolated from an Indonesian human immunodeficiency virus-infected patient suffering from oropharyngeal candidiasis

    NARCIS (Netherlands)

    Wahyuningsih, Retno; SahBandar, Ivo N.; Theelen, Bart; Hagen, Ferry; Poot, Ge; Meis, Jacques F.; Rozalyani, Anna; Sjam, Ridhawati; Widodo, Djoko; Djauzi, Samsuridjal; Boekhout, Teun

    2008-01-01

    Candida nivariensis was isolated from an Indonesian human immunodeficiency virus-infected patient who suffered from oropharyngeal candidiasis and was identified with molecular tools. Our isolate demonstrated low MICs to amphotericin B, flucytosine, posaconazole, caspofungin, and isavueonazole and wa

  10. Isolation of Mesenchymal Stem Cells from Human Deciduous Teeth Pulp

    Directory of Open Access Journals (Sweden)

    Aileen I. Tsai

    2017-01-01

    Full Text Available This study aimed to identify predictors of success rate of mesenchymal stem cell (MSC isolation from human deciduous teeth pulp. A total of 161 deciduous teeth were extracted at the dental clinic of Chang Gung Memorial Hospital. The MSCs were isolated from dental pulps using a standard protocol. In total, 128 colonies of MSCs were obtained and the success rate was 79.5%. Compared to teeth not yielding MSCs successfully, those successfully yielding MSCs were found to have less severe dental caries (no/mild-to-moderate/severe: 63.3/24.2/12.5% versus 12.5/42.4/42.4%, P<0.001 and less frequent pulpitis (no/yes: 95.3/4.7% versus 51.5/48.5%, P<0.001. In a multivariate regression model, it was confirmed that the absence of dental caries (OR = 4.741, 95% CI = 1.564–14.371, P=0.006 and pulpitis (OR = 9.111, 95% CI = 2.921–28.420, P<0.001 was significant determinants of the successful procurement of MSCs. MSCs derived from pulps with pulpitis expressed longer colony doubling time than pulps without pulpitis. Furthermore, there were higher expressions of proinflammatory cytokines, interleukin- (IL- 6 and monocyte chemoattractant protein- (MCP- 1, P<0.01, and innate immune response [toll-like receptor 1 (TLR1 and TLR8, P<0.05; TLR2, TLR3, and TLR6, P<0.01] in the inflamed than noninflamed pulps. Therefore, a carious deciduous tooth or tooth with pulpitis was relatively unsuitable for MSC processing and isolation.

  11. Isolation of Mesenchymal Stem Cells from Human Deciduous Teeth Pulp

    Science.gov (United States)

    Tsai, Aileen I.; Hong, Hsiang-Hsi; Fu, Jen-Fen; Chang, Chih-Chun; Wang, I-Kuan; Huang, Wen-Hung; Weng, Cheng-Hao; Hsu, Ching-Wei

    2017-01-01

    This study aimed to identify predictors of success rate of mesenchymal stem cell (MSC) isolation from human deciduous teeth pulp. A total of 161 deciduous teeth were extracted at the dental clinic of Chang Gung Memorial Hospital. The MSCs were isolated from dental pulps using a standard protocol. In total, 128 colonies of MSCs were obtained and the success rate was 79.5%. Compared to teeth not yielding MSCs successfully, those successfully yielding MSCs were found to have less severe dental caries (no/mild-to-moderate/severe: 63.3/24.2/12.5% versus 12.5/42.4/42.4%, P < 0.001) and less frequent pulpitis (no/yes: 95.3/4.7% versus 51.5/48.5%, P < 0.001). In a multivariate regression model, it was confirmed that the absence of dental caries (OR = 4.741, 95% CI = 1.564–14.371, P = 0.006) and pulpitis (OR = 9.111, 95% CI = 2.921–28.420, P < 0.001) was significant determinants of the successful procurement of MSCs. MSCs derived from pulps with pulpitis expressed longer colony doubling time than pulps without pulpitis. Furthermore, there were higher expressions of proinflammatory cytokines, interleukin- (IL-) 6 and monocyte chemoattractant protein- (MCP-) 1, P < 0.01, and innate immune response [toll-like receptor 1 (TLR1) and TLR8, P < 0.05; TLR2, TLR3, and TLR6, P < 0.01] in the inflamed than noninflamed pulps. Therefore, a carious deciduous tooth or tooth with pulpitis was relatively unsuitable for MSC processing and isolation. PMID:28377925

  12. Trichophyton onychocola sp. nov. isolated from human nail.

    Science.gov (United States)

    Hubka, Vit; Cmokova, Adela; Skorepova, Magdalena; Mikula, Peter; Kolarik, Miroslav

    2014-04-01

    A previously undescribed Trichophyton species was isolated from the nail of a 33-year-old man with a history of probable distal lateral subungual onychomycosis (without confirmation by mycological examination). The infection occurred for the first time five years earlier (in 2006) and affected the right great toenail, with complete clinical remission after treatment with ciclopirox olamine. This undescribed species was isolated during probable relapse in 2011, but its etiological significance was not confirmed, that is, direct microscopy was negative and additional clinical samples were not collected. The species is probably geophilic based on phylogenetic analysis (internal transcribed spacer [ITS] rDNA) and is most closely related to the anamorphic T. thuringiense, homothallic Arthroderma ciferrii (anamorph T. georgiae), and heterothallic A. melis. The new species is characterized by yellowish colonies, red reverse on several media, positive urease test, negative hair-perforation test, absence of growth at 34°C, absence of macroconidia, formation of one-celled clavate microconidia, and spiral hyphae. The species grows well on sterilized human hairs placed on agar medium without any additional nutrients and forms gymnothecium-like structures covered by peridial hyphae. The combination of unique micro- and macromorphological features and physiological and sequence data from four unlinked loci (ITS, benA, RPB2, and act1 gene) justified the proposal of a new species T. onychocola sp. nov.

  13. Streptococcus rubneri sp. nov., isolated from the human throat.

    Science.gov (United States)

    Huch, Melanie; De Bruyne, Katrien; Cleenwerck, Ilse; Bub, Achim; Cho, Gyu-Sung; Watzl, Bernhard; Snauwaert, Isabel; Franz, Charles M A P; Vandamme, Peter

    2013-11-01

    The novel, Gram-stain-positive, ovoid, lactic acid bacterial isolates LMG 27205, LMG 27206, LMG 27207(T) and MRI-F 18 were obtained from throat samples of healthy humans. 16S rRNA gene sequence analyses indicated that these isolates belong to the genus Streptococcus, specifically the Streptococcus mitis group, with Streptococcus australis and Streptococcus mitis as the nearest neighbours (99.45 and 98.56 % 16S rRNA gene sequence similarity to the respective type strains). Genotypic fingerprinting by fluorescent amplified fragment length polymorphism (FAFLP) and pulsed-field gel electrophoresis (PFGE), DNA-DNA hybridizations, comparative sequence analysis of pheS, rpoA and atpA and physiological and biochemical tests revealed that these bacteria formed a taxon well separated from its nearest neighbours and other species of the genus Streptococcus with validly published names and, therefore, represent a novel species, for which the name Streptococcus rubneri sp. nov. is proposed, with LMG 27207(T) ( = DSM 26920(T)) as the type strain.

  14. Biofilm formation in Hafnia alvei HUMV-5920, a human isolate

    Directory of Open Access Journals (Sweden)

    Itziar Chapartegui-González

    2016-11-01

    Full Text Available Hafnia alvei is a Gram-negative, rodshaped, facultative anaerobic bacterium of the family Enterobacteriaceae that has been isolated from various mammals, fish, insects and birds. In humans, case reports of Hafnia-associated enteric infections have been chiefly reported in Spain. Although H. alvei shares some virulence mechanisms with other Gram-negative enteropathogens little is known about the factors that contribute to its pathogenesis or virulence factors and regulatory circuits that may enhance the establishment and survival of H. alvei in the environment. The goal of the present study was to analyze the capacity of a H. alvei clinical isolate (strain HUMV-5920 to form biofilms. Biofilm formation by this strain increases during growth at 28 °C compared to 37 °C. Investigation of multicellular behavior by confocal microscopy, crystal violet and calcofluor staining in this strain showed biofilm formation associated with the production of cellulose. Importantly, several genes related to cellulose production including bcsABZC and yhjQ are present in the H. alvei HUMV-5920 chromosome. The ability of H. alvei to adhere to abiotic surfaces and to form biofilms likely contributes to its persistence in the hospital environment or food processing environments, increasing the probability of causing infections. Therefore, a better understanding of the adherence properties of this species will provide greater insights into the diseases it causes.

  15. Reversal of hyperglycemia in diabetic rats by portal vein transplantation of islet-like cells generated from bone marrow mesenchymal stem cells

    Institute of Scientific and Technical Information of China (English)

    Xiao-Hong Wu; Chao Liu; Cui-Ping Liu; Kuan-Feng Xu; Xiao-Dong Mao; Jian Zhu; Jing-Jing Jiang; Dai Cui; Mei Zhang; Yu Xu

    2007-01-01

    AIM: To study the capacity of bone marrow mesenchymal stem cells (BM-MSCs) trans-differentiating into islet-like cells and to observe the effect of portal vein transplantation of islet-like cells in the treatment of streptozotocin-induced diabetic rat.METHODS: BM-MSCs were isolated from SD rats and induced to differentiate into islet-like cells under defined conditions. Differentiation was evaluated with electron microscopy, RT-PCR, immunofluorescence and flow cytometry. Insulin release after glucose challenge was tested with ELISA. Then allogeneic islet-like cells were transplanted into diabetic rats via portal vein. Blood glucose levels were monitored and islet hormones were detected in the liver and pancreas of the recipient by immunohistochemistry.RESULTS: BM-MSCs were spheroid adherent monolayers with high CD90, CD29 and very low CD45 expression.Typical islet-like cells clusters were formed after induction. Electron microscopy revealed that secretory granules were densely packed within the cytoplasm of the differentiated cells. The spheroid cells expressed islet related genes and hormones. The insulin-positive cells accounted for 19.8% and mean fluorescence intensity increased by 2.6 fold after induction. The cells secreted a small amount of insulin that was increased 1.5 fold after glucose challenge. After transplantation, islet-like cells could locate in the liver expressing islet hormones and lower the glucose levels of diabetic rats during d 6 to d 20.CONCLUSION: Rat BM-MSCs could be transdifferentiated into islet-like cells in vitro. Portal vein transplantation of islet-like cells could alleviate the hyperglycemia of diabetic rats.

  16. Sterols of Pneumocystis carinii hominis organisms isolated from human lungs

    DEFF Research Database (Denmark)

    Kaneshiro, E S; Amit, Z; Chandra, Jan Suresh

    1999-01-01

    The opportunistic pathogen Pneumocystis carinii causes pneumonia (P. carinii pneumonia, or PCP) in immunocompromised individuals such as AIDS patients. Rat-derived P. carinii carinii organisms have distinct sterols which are not synthesized by mammals and not found in other microbes infecting...... mammalian lungs. The dominant sterol present in the organism is cholesterol (which is believed to be scavenged from the host), but other sterols in P. carinii carinii have an alkyl group at C-24 of the sterol side chain (C(28) and C(29) 24-alkylsterols) and a double bond at C-7 of the nucleus. Recently...... in conjunction with analyses of chemically synthesized authentic standards. The sterol composition of isolated P. carinii hominis organisms has yet to be reported. If P. carinii from animal models is to be used for identifying potential drug targets and for developing chemotherapeutic approaches to clear human...

  17. [Isolation and physico-chemical characteristics of human cancerocerebral antigen].

    Science.gov (United States)

    Prokopenko, P G; Borisenko, S A; Tatarinov, Iu S

    1984-01-01

    During gel filtration on Sephadex G-200 human cancerocerebral antigen (CCA) was eluted as two protein fractions with molecular mass of 135,000 and 270.000 daltons. Only one band of protein with molecular mass of about 15,000 daltons was noted after electrophoresis in 10% polyacrylamide gel containing SDS. As characteristic properties of CCA were recognized an electrophoretic polymorphism and a distinct trend to polymerization and isomeria. The antigen was not stained with dyes designed for staining base proteins, lipo-,glyco- and ferroproteins; CCA was thermostable (5 min at 80 degrees), it was inactivated by trypsin and protease but was resistant to pronase, hexokinase, alpha-amylase and beta-glucuronidase. A procedure was developed for isolation of CCA from brain, including fractionation with ammonium sulfate, ion exchange chromatography on DEAE-Sephadex A-50. The procedure enabled to obtain the CCA preparations suitable for radioimmunological, immunobiological assays and amino acid analyses.

  18. PIXE analysis of human spermatozoa isolated from seminal plasma

    Science.gov (United States)

    Maeda, K.; Sasa, Y.; Kusuyama, H.; Yoshida, K.; Uda, M.

    1990-04-01

    PIXE has been applied to the multielemental and microanalysis of human spermatozoa. This is the first attempt to determine the chemical compositions of the motile spermatozoa free from contaminations of seminal plasma without loss of component elements during washing. The spermatozoa were isolated from semen by letting them swim into a kind of physiological saline, Tyrode's solution. Relative concentrations of P, K, Ca, Ti, Fe, Zn and Br in motile spermatozoa were determined by the use of the chlorine K X-ray peak intensity for evaluating the amount of Tyrode's solution contained in the sample targets. The concentrations of calcium and iron in spermatozoa were considerably higher than in seminal plasma. The concentrations of P, K, Zn and Br in spermatozoa were not so different from those in seminal plasma.

  19. Percutaneous transhepatic portal catheterization guided by ultrasound technology for islet transplantation in rhesus monkey

    Institute of Scientific and Technical Information of China (English)

    FengGao; Shao-DongAi; ShengLiu; Wen-BinZeng; WeiWang

    2012-01-01

    BACKGROUND: Pig islet xenotransplantation has the potential to overcome the shortage of donated human islets for islet cell transplantation in type 1 diabetes. Testing in non-human primate models is necessary before clinical application in humans. Intraportal islet transplantation in monkeys is usually performed by surgical infusion during laparotomy or laparoscopy. In this paper, we describe a new method of percutaneous transhepatic portal catheterization (PTPC) as an alternative to current methods of islet transplantation in rhesus monkeys. METHODS: We performed ultrasound-guided PTPC in five adult rhesus monkeys weighing 7-8 kg, with portal vein catheterization confirmed by digital subtraction angiography. We monitored for complications in the thoracic and abdominal cavity. To evaluate the safety of ultrasound-guided PTPC, we recorded the changes in portal pressure throughout the microbead transplantation procedure. RESULTS:  Ultrasound-guided PTPC and infusion of 16 000 microbeads/kg body weight into the portal vein was successful in all five monkeys. Differences in the hepatobiliary anatomy of rhesus monkeys compared to humans led to a higher initial complication rate. The first monkey died of abdominal hemorrhage 10 hours post-transplantation. The second suffered from a mild pneumothorax but recovered fully after taking only conservative measures. After gaining experience with the first two monkeys, we decreased both the hepatic puncture time and the number of puncture attempts required, with the remaining three monkeys experiencing no complications. Portal pressures initially increased proportional to the number of transplanted microbeads but returned to pre-infusion levels at 30 minutes post-transplantation. The changes in portal pressures occurring during the procedure were not significantly different. CONCLUSIONS: Ultrasound-guided PTPC is an effective, convenient, and minimally invasive method suitable for use in non-human primate models of

  20. Isolation and characterization of DNA probes for human chromosome 21.

    Science.gov (United States)

    Watkins, P C

    1990-01-01

    A coordinated effort to map and sequence the human genome has recently become a national priority. Chromosome 21, the smallest human chromosome accounting for less than 2% of the human genome, is an attractive model system for developing and evaluating genome mapping technology. Several strategies are currently being explored including the development of chromosome 21 libraries from somatic cell hybrids as reported here, the cloning of chromosome 21 in yeast artificial chromosomes (McCormick et al., 1989b), and the construction of chromosome 21 libraries using chromosome flow-sorting techniques (Fuscoe et al., 1989). This report describes the approaches used to identify DNA probes that are useful for mapping chromosome 21. Probes were successfully isolated from both phage and cosmid libraries made from two somatic cell hybrids that contain human chromosome 21 as the only human chromosome. The 15 cosmid clones from the WA17 library, reduced to cloned DNA sequences of an average size of 3 kb, total 525 kb of DNA which is approximately 1% of chromosome 21. From these clones, a set of polymorphic DNA markers that span the length of the long arm of chromosome 21 has been generated. All of the probes thus far analyzed from the WA17 libraries have been mapped to chromosome 21 both by physical and genetic mapping methods. It is therefore likely that the WA17 hybrid cell line contains human chromosome 21 as the only human component, in agreement with cytogenetic observation. The 153E7b cosmid libraries will provide an alternative source of cloned chromosome 21 DNA. Library screening techniques can be employed to obtain cloned DNA sequences from the same genetic loci of the two different chromosome 21s. Comparative analysis will allow direct estimation of DNA sequence variation for different regions of chromosome 21. Mapped DNA probes make possible the molecular analysis of chromosome 21 at a level of resolution not achievable by classical cytogenetic techniques (Graw et al

  1. Justifying clinical trials for porcine islet xenotransplantation.

    Science.gov (United States)

    Ellis, Cara E; Korbutt, Gregory S

    2015-01-01

    The development of the Edmonton Protocol encouraged a great deal of optimism that a cell-based cure for type I diabetes could be achieved. However, donor organ shortages prevent islet transplantation from being a widespread solution as the supply cannot possibly equal the demand. Porcine islet xenotransplantation has the potential to address these shortages, and recent preclinical and clinical trials show promising scientific support. Consequently, it is important to consider whether the current science meets the ethical requirements for moving toward clinical trials. Despite the potential risks and the scientific unknowns that remain to be investigated, there is optimism regarding the xenotransplantation of some types of tissue, and enough evidence has been gathered to ethically justify clinical trials for the most safe and advanced area of research, porcine islet transplantation. Researchers must make a concerted effort to maintain a positive image for xenotransplantation, as a few well-publicized failed trials could irrevocably damage public perception of xenotransplantation. Because all of society carries the burden of risk, it is important that the public be involved in the decision to proceed. As new information from preclinical and clinical trials develops, policy decisions should be frequently updated. If at any point evidence shows that islet xenotransplantation is unsafe, then clinical trials will no longer be justified and they should be halted. However, as of now, the expected benefit of an unlimited supply of islets, combined with adequate informed consent, justifies clinical trials for islet xenotransplantation.

  2. 诱导人脐带MSCs分化为胰岛样细胞团的促成熟方案及机制%Maturity-promoting protocols and mechanisms directing differentiation of human umbilical cord-derived MSCs towards islet-like clusters

    Institute of Scientific and Technical Information of China (English)

    苏仲春; 陈家劲; 王圳; 陈志明; 王跃春

    2013-01-01

    AIM: To determine the maturity-promoting protocols for directing the differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs)towards islet-like clusters (ILCs) expressing prohormone conver-tase (PC) 1 and PC2 through different combinations of biological products. METHODS: Primary hUC-MSCs were isolated from the whole human umbilical cord by digestion (with collagenase Ⅱ) , filtration and centrifugation, and then were purified by incomplete digestion. The stem cell-specific markers were detected by the methods of flow cytometry, RT-PCR and immunocytofluorescence. The protocols were adopted for inducing the differentiation of hUC-MSCs towards ILCs. Protocol A consisted of IMDM culture medium containing 10 μg/L basic fibroblast growth factor (bFGF) , 10 μg/L epidermal growth factor ( EGF) , 10 mg/L Ginkgo biloba extract ( GBE) and 2% fetal calf serum ( FCS) . Protocol B was based on the protocol A plus 10 μg/L nicotinamide. Protocol C was based on the protocol B plus 10 μg/L hepatocyte growth factor ( HGF). Before and after induction, the morphological changes of hUC-MSCs were observed under inverted microscope. The islet-related mRNA and proteins especially PCI and PC2 were measured by RT-PCR, qPCR and Western blotting. RESULTS: The cell morphology, surface antigen and stem cell-specific markers indicated that hUC-MSCs were successfully isolated and purified from human umbilical cord. hUC-MSCs expressed nestin broadly and Isll to a less degree. ILCs induced by protocol A expressed Glut-2 and MafA mRNA. ILCs induced by protocol B expressed Glut-2, MafA, Nkx6. 1 and PC2 mRNA. ILCs induced by protocol C expressed Glut-2, MafA, Nkx6. 1, PC2, Ngn3, Pdxl, PCI and insulin mRNA. The expression of PCI mRNA was only observed in ILCs induced by protocol C. ILCs induced by protocol B and protocol C both expressed PC2 mRNA and the expression in the cells induced by protocol C was significantly higher than that in the cells induced by protocol B

  3. Prevalence of Salmonella serotypes isolated in Spain from human and non human sources (1983-1987).

    Science.gov (United States)

    Echeita, M A; Usera, M A

    1989-09-01

    Salmonella serotypes over a five year period were studied in order to know their prevalence in Spain. The Salmonella Reference Centre received a total of 17,612 strains from 1983-1987. The majority (16,133) were of human origin and only 1,479 strains were isolated from non-human sources. The serotyping yielded 100 different serotypes, Salmonella enterica serotype Enteritidis (8) being the commonest in both groups, 61.18% of human origin and 31.91% of non-human origin. Salmonella enterica serotype Typhimurium the commonest serotype in many countries, occupies second place in our results with the following percentages 11.87% and 9.67% respectively. Among the strains of human origin Salmonella enterica serotype Typhi occupies fourth place (3.24%). This is very low compared with the high number of clinically diagnosed typhoid fever cases declared in the country: over 5,000 cases per year.

  4. Rhodococcus equi human clinical isolates enter and survive within human alveolar epithelial cells.

    Science.gov (United States)

    Ramos-Vivas, J; Pilares-Ortega, L; Remuzgo-Martínez, S; Padilla, D; Gutiérrez-Díaz, J L; Navas-Méndez, J

    2011-05-01

    Rhodococcus equi is an emerging opportunistic human pathogen associated with immunosuppressed people, especially those infected with the human immunodeficiency virus (HIV). This pathogen resides primarily within lung macrophages of infected patients, which may explain in part its ability to escape normal pulmonary defense mechanisms. Despite numerous studies as a pulmonary pathogen in foals, where a plasmid seems to play an important role in virulence, information on the pathogenesis of this pathogen in humans is still scarce. In this study, fluorescence microscopy and vancomycin protection assays were used to investigate the ability of R. equi human isolates to adhere to and to invade the human alveolar epithelial cell line A549. Our findings indicate that some R. equi clinical strains are capable of adhering, entering and surviving within the alveolar cell line, which may contribute to the pathogen persistence in lung tissues. Copyright © 2011 Institut Pasteur. Published by Elsevier SAS. All rights reserved.

  5. Isolation & molecular characterization of human parainfluenza virus in Chennai, India

    Directory of Open Access Journals (Sweden)

    C P Indumathi

    2015-01-01

    Full Text Available Background & objectives: Human parainfluenza virus (HPIV accounts for a significant proportion of lower respiratory tract infections in children as well as adults. This study was done to detect the presence of different subtypes of HPIV from patients having influenza like illness (ILI. Methods: Throat and nasal swabs from 232 patients with ILI who were negative for influenza viruses were tested by multiplex reverse transcription polymerase chain reaction(mRT-PCR for the detection of human parainfluenza virus. All samples were inoculated in rhesus monkey kidney (LLC-MK2 cell line. Results: Of the 232 samples, 26(11.2% were positive by mRT-PCR and nine (34.6% showed cytopathic effect with syncytium formation for HPIV and all were HPIV-3 serotype, other serotypes like 1,2,4 were negative. The HPIV-3 strains (HN gene were sequenced and analysed. Two novel mutations were identified at amino acid residues 295 and 297. Interpretation & conclusions: The mRT-PCR assay offers a rapid, sensitive and accurate diagnostic method for detection of HPIV which enables early detection and control. In our study there was a predominance of HPIV among 1-5 yr age group and the school going age group was less affected. Further studies need to be done to characterize HPIV isolated from different parts of the country.

  6. Histomorphology of the bottlenose dolphin (Tursiops truncatus) pancreas and association of increasing islet β-cell size with chronic hypercholesterolemia.

    Science.gov (United States)

    Colegrove, Kathleen M; Venn-Watson, Stephanie

    2015-04-01

    Bottlenose dolphins (Tursiops truncatus) can develop metabolic states mimicking prediabetes, including hyperinsulinemia, hyperlipidemia, elevated glucose, and fatty liver disease. Little is known, however, about dolphin pancreatic histomorphology. Distribution and area of islets, α, β, and δ cells were evaluated in pancreatic tissue from 22 dolphins (mean age 25.7years, range 0-51). Associations of these measurements were evaluated by sex, age, percent high glucose and lipids during the last year of life, and presence or absence of fatty liver disease and islet cell vacuolation. The most common pancreatic lesions identified were exocrine pancreas fibrosis (63.6%) and mild islet cell vacuolation (47.4%); there was no evidence of insulitis or amyloid deposition, changes commonly associated with type 2 diabetes. Dolphin islet architecture appears to be most similar to the pig, where α and β cells are localized to the central or periphery of the islet, respectively, or are well dispersed throughout the islet. Unlike pigs, large islets (greater than 10,000μm(2)) were common in dolphins, similar to that found in humans. A positive linear association was identified between dolphin age and islet area average, supporting a compensatory response similar to other species. The strongest finding in this study was a positive linear association between islet size, specifically β-cells, and percent blood samples with high cholesterol (greater than 280mg/dl, R(2)=0.57). This study is the most comprehensive assessment of the dolphin pancreas to date and may help direct future studies, including associations between chronic hypercholesterolemia and β-cell size.

  7. In vivo islet protection by a nuclear import inhibitor in a mouse model of type 1 diabetes.

    Directory of Open Access Journals (Sweden)

    Daniel J Moore

    Full Text Available BACKGROUND: Insulin-dependent Type 1 diabetes (T1D is a devastating autoimmune disease that destroys beta cells within the pancreatic islets and afflicts over 10 million people worldwide. These patients face life-long risks for blindness, cardiovascular and renal diseases, and complications of insulin treatment. New therapies that protect islets from autoimmune destruction and allow continuing insulin production are needed. Increasing evidence regarding the pathomechanism of T1D indicates that islets are destroyed by the relentless attack by autoreactive immune cells evolving from an aberrant action of the innate, in addition to adaptive, immune system that produces islet-toxic cytokines, chemokines, and other effectors of islet inflammation. We tested the hypothesis that targeting nuclear import of stress-responsive transcription factors evoked by agonist-stimulated innate and adaptive immunity receptors would protect islets from autoimmune destruction. PRINCIPAL FINDINGS: Here we show that a first-in-class inhibitor of nuclear import, cSN50 peptide, affords in vivo islet protection following a 2-day course of intense treatment in NOD mice, which resulted in a diabetes-free state for one year without apparent toxicity. This nuclear import inhibitor precipitously reduces the accumulation of islet-destructive autoreactive lymphocytes while enhancing activation-induced cell death of T and B lymphocytes derived from autoimmune diabetes-prone, non-obese diabetic (NOD mice that develop T1D. Moreover, in this widely used model of human T1D we noted attenuation of pro-inflammatory cytokine and chemokine production in immune cells. CONCLUSIONS: These results indicate that a novel form of immunotherapy that targets nuclear import can arrest inflammation-driven destruction of insulin-producing beta cells at the site of autoimmune attack within pancreatic islets during the progression of T1D.

  8. Activin B regulates islet composition and islet mass but not whole body glucose homeostasis or insulin sensitivity

    Science.gov (United States)

    Bonomi, Lara; Brown, Melissa; Ungerleider, Nathan; Muse, Meghan; Matzuk, Martin M.

    2012-01-01

    Based on the phenotype of the activin-like kinase-7 (ALK7)-null mouse, activins A and B have been proposed to play distinct roles in regulating pancreatic islet function and glucose homeostasis, with activin A acting to enhance islet function and insulin release while activin B antagonizes these actions. We therefore hypothesized that islets from activin B-null (BBKO) mice would have enhanced glucose-stimulated insulin secretion. In addition, we hypothesized that this enhanced islet function would translate into increased whole body glucose tolerance. We tested these hypotheses by analyzing glucose homeostasis, insulin secretion, and islet function in BBKO mice. No differences were observed in fasting glucose or insulin levels, glucose tolerance, or insulin sensitivity compared with weight-matched young or older males. Similarly, there were no significant differences in insulin secretion comparing islets from WT or BBKO males at either age. However, BBKO islets were more sensitive to activin A, myostatin (MSTN), and follistatin (FST) treatments, so that activin A and FST inhibited and MSTN enhanced glucose stimulated insulin secretion. While mean islet area and the distribution of islet areas were not different between the genotypes, islet mass, islet number, and the proportion of α-cells/islet were significantly reduced in BBKO islets. These results indicate that activin B does not antagonize activin A to influence whole body glucose homeostasis or β-cell function but does influence islet mass and proportion of α-cells/islet. Therefore, loss of activin B signaling alone does not account for the ALK7-null phenotype, but activin B may have important roles in modulating islet mass, islet number, and the cellular composition of islets. PMID:22739106

  9. Islet Oxygen Consumption Rate (OCR Dose Predicts Insulin Independence in Clinical Islet Autotransplantation.

    Directory of Open Access Journals (Sweden)

    Klearchos K Papas

    Full Text Available Reliable in vitro islet quality assessment assays that can be performed routinely, prospectively, and are able to predict clinical transplant outcomes are needed. In this paper we present data on the utility of an assay based on cellular oxygen consumption rate (OCR in predicting clinical islet autotransplant (IAT insulin independence (II. IAT is an attractive model for evaluating characterization assays regarding their utility in predicting II due to an absence of confounding factors such as immune rejection and immunosuppressant toxicity.Membrane integrity staining (FDA/PI, OCR normalized to DNA (OCR/DNA, islet equivalent (IE and OCR (viable IE normalized to recipient body weight (IE dose and OCR dose, and OCR/DNA normalized to islet size index (ISI were used to characterize autoislet preparations (n = 35. Correlation between pre-IAT islet product characteristics and II was determined using receiver operating characteristic analysis.Preparations that resulted in II had significantly higher OCR dose and IE dose (p<0.001. These islet characterization methods were highly correlated with II at 6-12 months post-IAT (area-under-the-curve (AUC = 0.94 for IE dose and 0.96 for OCR dose. FDA/PI (AUC = 0.49 and OCR/DNA (AUC = 0.58 did not correlate with II. OCR/DNA/ISI may have some utility in predicting outcome (AUC = 0.72.Commonly used assays to determine whether a clinical islet preparation is of high quality prior to transplantation are greatly lacking in sensitivity and specificity. While IE dose is highly predictive, it does not take into account islet cell quality. OCR dose, which takes into consideration both islet cell quality and quantity, may enable a more accurate and prospective evaluation of clinical islet preparations.

  10. Assessment of Toxicological Perturbations and Variants of Pancreatic Islet Development in the Zebrafish Model

    Directory of Open Access Journals (Sweden)

    Karilyn E. Sant

    2016-09-01

    Full Text Available The pancreatic islets, largely comprised of insulin-producing beta cells, play a critical role in endocrine signaling and glucose homeostasis. Because they have low levels of antioxidant defenses and a high perfusion rate, the endocrine islets may be a highly susceptible target tissue of chemical exposures. However, this endpoint, as well as the integrity of the surrounding exocrine pancreas, is often overlooked in studies of developmental toxicology. Disruption of development by toxicants can alter cell fate and migration, resulting in structural alterations that are difficult to detect in mammalian embryo systems, but that are easily observed in the zebrafish embryo model (Danio rerio. Using endogenously expressed fluorescent protein markers for developing zebrafish beta cells and exocrine pancreas tissue, we documented differences in islet area and incidence rates of islet morphological variants in zebrafish embryos between 48 and 96 h post fertilization (hpf, raised under control conditions commonly used in embryotoxicity assays. We identified critical windows for chemical exposures during which increased incidences of endocrine pancreas abnormalities were observed following exposure to cyclopamine (2–12 hpf, Mono-2-ethylhexyl phthalate (MEHP (3–48 hpf, and Perfluorooctanesulfonic acid (PFOS (3–48 hpf. Both islet area and length of the exocrine pancreas were sensitive to oxidative stress from exposure to the oxidant tert-butyl hydroperoxide during a highly proliferative critical window (72 hpf. Finally, pancreatic dysmorphogenesis following developmental exposures is discussed with respect to human disease.

  11. Potentiation of glucose-induced insulin release in islets by desHis1[Glu9]glucagon amide

    DEFF Research Database (Denmark)

    Kofod, Hans; Unson, C G; Merrifield, R B

    1988-01-01

    Glucagon and secretin and some of their hybrid analogs potentiate glucose-induced release of insulin from isolated mouse pancreatic islets. It was recently shown that the synthetic glucagon analog, desHis1[Glu9]glucagon amide, does not stimulate the formation of cyclic adenosine monophosphate...... in the rat hepatocyte membrane, but binds well to the glucagon receptor and is a good competitive antagonist of glucagon. In the present study the effect of this analog on isolated islets was examined. desHis1-[Glu9]glucagon amide at 3 x 10(-7) M, in the presence of 0.01 M D-glucose, increased the release...

  12. Protein phosphatase 1 (PP-1)-dependent inhibition of insulin secretion by leptin in INS-1 pancreatic β-cells and human pancreatic islets.

    Science.gov (United States)

    Kuehnen, Peter; Laubner, Katharina; Raile, Klemens; Schöfl, Christof; Jakob, Franz; Pilz, Ingo; Päth, Günter; Seufert, Jochen

    2011-05-01

    Leptin inhibits insulin secretion from pancreatic β-cells, and in turn, insulin stimulates leptin biosynthesis and secretion from adipose tissue. Dysfunction of this adipoinsular feedback loop has been proposed to be involved in the development of hyperinsulinemia and type 2 diabetes mellitus. At the molecular level, leptin acts through various pathways, which in combination confer inhibitory effects on insulin biosynthesis and secretion. The aim of this study was to identify molecular mechanisms of leptin action on insulin secretion in pancreatic β-cells. To identify novel leptin-regulated genes, we performed subtraction PCR in INS-1 β-cells. Regulated expression of identified genes was confirmed by RT-PCR and Northern and Western blotting. Furthermore, functional impact on β-cell function was characterized by insulin-secretion assays, intracellular Ca²(+) concentration measurements, and enzyme activity assays. PP-1α, the catalytic subunit of protein phosphatase 1 (PP-1), was identified as a novel gene down-regulated by leptin in INS-1 pancreatic β-cells. Expression of PP-1α was verified in human pancreatic sections. PP-1α mRNA and protein expression is down-regulated by leptin, which culminates in reduction of PP-1 enzyme activity in β-cells. In addition, glucose-induced insulin secretion was inhibited by nuclear inhibitor of PP-1 and calyculin A, which was in part mediated by a reduction of PP-1-dependent calcium influx into INS-1 β-cells. These results identify a novel molecular pathway by which leptin confers inhibitory action on insulin secretion, and impaired PP-1 inhibition by leptin may be involved in dysfunction of the adipoinsular axis during the development of hyperinsulinemia and type 2 diabetes mellitus.

  13. Preoperative Computerized Tomography and Magnetic Resonance Imaging of the Pancreas Predicts Pancreatic Mass and Functional Outcomes After Total Pancreatectomy and Islet Autotransplant.

    Science.gov (United States)

    Young, Michael C; Theis, Jake R; Hodges, James S; Dunn, Ty B; Pruett, Timothy L; Chinnakotla, Srinath; Walker, Sidney P; Freeman, Martin L; Trikudanathan, Guru; Arain, Mustafa; Robertson, Paul R; Wilhelm, Joshua J; Schwarzenberg, Sarah J; Bland, Barbara; Beilman, Gregory J; Bellin, Melena D

    2016-08-01

    Approximately two thirds of patients will remain on insulin therapy after total pancreatectomy with islet autotransplant (TPIAT) for chronic pancreatitis. We investigated the relationship between measured pancreas volume on computerized tomography or magnetic resonance imaging and features of chronic pancreatitis on imaging, with subsequent islet isolation and diabetes outcomes. Computerized tomography or magnetic resonance imaging was reviewed for pancreas volume (Vitrea software) and presence or absence of calcifications, atrophy, and dilated pancreatic duct in 97 patients undergoing TPIAT. Relationship between these features and (1) islet mass isolated and (2) diabetes status at 1-year post-TPIAT were evaluated. Pancreas volume correlated with islet mass measured as total islet equivalents (r = 0.50, P pancreas volume and 3 imaging features strongly associated with 1-year insulin use (P = 0.07), islet graft failure (P = 0.003), hemoglobin A1c (P = 0.0004), fasting glucose (P = 0.027), and fasting C-peptide level (P = 0.008). Measures of pancreatic parenchymal destruction on imaging, including smaller pancreas volume and calcifications, associate strongly with impaired islet mass and 1-year diabetes outcomes.

  14. Establishing a cGMP pancreatic islet processing facility: the first experience in Iran.

    Science.gov (United States)

    Larijani, Bagher; Arjmand, Babak; Amoli, Mahsa M; Ao, Ziliang; Jafarian, Ali; Mahdavi-Mazdah, Mitra; Ghanaati, Hossein; Baradar-Jalili, Reza; Sharghi, Sasan; Norouzi-Javidan, Abbas; Aghayan, Hamid Reza

    2012-12-01

    It has been predicted that one of the greatest increase in prevalence of diabetes will happen in the Middle East bear in the next decades. The aim of standard therapeutic strategies for diabetes is better control of complications. In contrast, some new strategies like cell and gene therapy have aimed to cure the disease. In recent years, significant progress has occurred in beta-cell replacement therapies with a progressive improvement of short-term and long term outcomes. In year 2005, considering the impact of the disease in Iran and the promising results of the Edmonton protocol, the funding for establishing a current Good Manufacturing Practice (cGMP) islet processing facility by Endocrinology and Metabolism Research Center was approved by Tehran University of Medical Sciences. Several islet isolations were performed following establishment of cGMP facility and recruitment of all required equipments for process validation and experimental purpose. Finally the first successful clinical islet isolation and transplantation was performed in September 2010. In spite of a high cost of the procedure it is considered beneficial and may prevent long term complications and the costs associated with secondary cares. In this article we will briefly describe our experience in setting up a cGMP islet processing facility which can provide valuable information for regional countries interested to establish similar facilities.

  15. Epidemiological relationships of Campylobacter jejuni strains isolated from humans and chickens in South Korea.

    Science.gov (United States)

    Oh, Jae-Young; Kwon, Yong-Kuk; Wei, Bai; Jang, Hyung-Kwan; Lim, Suk-Kyung; Kim, Cheon-Hyeon; Jung, Suk-Chan; Kang, Min-Su

    2017-01-01

    Thirty-nine human isolates of Campylobacter jejuni obtained from a national university hospital during 2007-2010 and 38 chicken isolates of C. jejuni were collected from poultry farms during 2009-2010 in South Korea were used in this study. Campylobacter genomic species and virulence-associated genes were identified by PCR. Pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to compare their genetic relationships. All isolates were highly resistant to ciprofloxacin, nalidixic acid, and tetracycline. Of all isolates tested, over 94% contained seven virulence associated genes (flaA, cadF, racR, dnaJ, cdtA, cdtB, and cdtC). All isolates were classified into 39 types by PFGE clustering with 90% similarity. Some chicken isolates were incorporated into some PFGE types of human isolates. MLST analysis for the 39 human isolates and 38 chicken isolates resulted in 14 and 23 sequence types (STs), respectively, of which 10 STs were new. STs overlapped in both chicken and human isolates included ST-21, ST-48, ST-50, ST-51, and ST-354, of which ST-21 was the predominant ST in both human and chicken isolates. Through combined analysis of PFGE types and STs, three chicken isolates were clonally related to the three human isolates associated with food poisoning (VII-ST-48, XXII-ST-354, and XXVIII-ST-51). They were derived from geographically same or distinct districts. Remarkably, clonal spread of food poisoning pathogens between animals and humans was confirmed by population genetic analysis. Consequently, contamination of campylobacters with quinolone resistance and potential virulence genes in poultry production and consumption may increase the risk of infections in humans.

  16. Islet Amyloid Polypeptide: Structure, Function, and Pathophysiology

    Directory of Open Access Journals (Sweden)

    Rehana Akter

    2016-01-01

    Full Text Available The hormone islet amyloid polypeptide (IAPP, or amylin plays a role in glucose homeostasis but aggregates to form islet amyloid in type-2 diabetes. Islet amyloid formation contributes to β-cell dysfunction and death in the disease and to the failure of islet transplants. Recent work suggests a role for IAPP aggregation in cardiovascular complications of type-2 diabetes and hints at a possible role in type-1 diabetes. The mechanisms of IAPP amyloid formation in vivo or in vitro are not understood and the mechanisms of IAPP induced β-cell death are not fully defined. Activation of the inflammasome, defects in autophagy, ER stress, generation of reactive oxygen species, membrane disruption, and receptor mediated mechanisms have all been proposed to play a role. Open questions in the field include the relative importance of the various mechanisms of β-cell death, the relevance of reductionist biophysical studies to the situation in vivo, the molecular mechanism of amyloid formation in vitro and in vivo, the factors which trigger amyloid formation in type-2 diabetes, the potential role of IAPP in type-1 diabetes, the development of clinically relevant inhibitors of islet amyloidosis toxicity, and the design of soluble, bioactive variants of IAPP for use as adjuncts to insulin therapy.

  17. Prevotella colorans sp. nov., isolated from a human wound.

    Science.gov (United States)

    Buhl, Michael; Willmann, Matthias; Liese, Jan; Autenrieth, Ingo B; Marschal, Matthias

    2016-08-01

    A strain of obligately anaerobic, Gram-stain-negative and non-spore-forming rod-shaped bacterium was isolated from a human wound and characterized both phenotypically and genotypically. The strain was moderately saccharolytic and proteolytic. Phylogenetic analysis was based on full-length 16S rRNA gene sequence analysis and revealed the strain to represent a member of the genus Prevotella, but to be different from the described species, with the closest relationship to Prevotella bergensis and Prevotella multisaccharivorax. The genomic DNA G+C content was 43.2 mol%. The most abundant cellular long-chain fatty acids were 3-OH iso-C17 : 0, anteiso-C15 : 0 and iso-C15 : 0. In view of phenotypical and biochemical characteristics as well as gene sequencing, strain A1336T is considered to represent a novel species within the genus Prevotella, for which the name Prevotella colorans sp. nov. is proposed. The type strain is A1336T (=DSM 100333T =CCUG 67421T =CCOS 902T).

  18. Human attribute concepts: relative ubiquity across twelve mutually isolated languages.

    Science.gov (United States)

    Saucier, Gerard; Thalmayer, Amber Gayle; Bel-Bahar, Tarik S

    2014-07-01

    It has been unclear which human-attribute concepts are most universal across languages. To identify common-denominator concepts, we used dictionaries for 12 mutually isolated languages-Maasai, Supyire Senoufo, Khoekhoe, Afar, Mara Chin, Hmong, Wik-Mungkan, Enga, Fijian, Inuktitut, Hopi, and Kuna-representing diverse cultural characteristics and language families, from multiple continents. A composite list of every person-descriptive term in each lexicon was closely examined to determine the content (in terms of English translation) most ubiquitous across languages. Study 1 identified 28 single-word concepts used to describe persons in all 12 languages, as well as 41 additional terms found in 11 of 12. Results indicated that attribute concepts related to morality and competence appear to be as cross-culturally ubiquitous as basic-emotion concepts. Formulations of universal-attribute concepts from Osgood and Wierzbicka were well-supported. Study 2 compared lexically based personality models on the relative ubiquity of key associated terms, finding that 1- and 2-dimensional models draw on markedly more ubiquitous terms than do 5- or 6-factor models. We suggest that ubiquitous attributes reflect common cultural as well as common biological processes.

  19. The Use of Isolated Human Lymphocytes in Mycotoxin Cytotoxicity Testing

    Directory of Open Access Journals (Sweden)

    Mike F. Dutton

    2008-08-01

    Full Text Available The cytotoxicity of selected mycotoxins against isolated human lymphocytes was investigated, as a means of detecting mycotoxins in extracts derived from cereal samples. The methodology was based on the ability of viable cells to reduce methyl tetrazolium bromide to a purple formazan dye that could be quantitated by spectrophometric means and hence give a measure of the cytotoxicity of added substances. The results showed that there was good correlation with the occurrence of identified mycotoxins with only a minimum of false positives. For example, of the 13 samples of barley or barley derivatives that were positive for the mycotoxins, fumonisin B1 (FB1 deoxynivalenol (DON and ochratoxin A (OTA, all gave positive cytotoxicity responses. Two samples negative for mycotoxins gave no cytotoxicity responses. There was little variation between the results for lymphocytes drawn from the same healthy volunteer on three different occasions. Furthermore, for two of the mycotoxins tested (FB1 and DON it was possible to correlate general levels of mycotoxins present to the cytotoxic response of the lymphocytes but not for OTA, where it was concluded that interfering substances prevented direct correlation. It was concluded that this method was suited for general application as it could handle relatively high number of samples in a short period of time.

  20. Mesenchymal stem cells as feeder cells for pancreatic islet transplants.

    OpenAIRE

    2010-01-01

    Allogeneic islet transplantation serves as a source of insulin-secreting beta-cells for the maintenance of normal glucose levels and treatment of diabetes. However, limited availability of islets, high rates of islet graft failure, and the need for life-long non-specific immunosuppressive therapy are major obstacles to the widespread application of this therapeutic approach. To overcome these problems, pancreatic islet transplantation was recently suggested as a potential target of the "thera...

  1. Description of Campylobacter fetus subsp. testudinum subsp. nov., isolated from humans and reptiles

    Science.gov (United States)

    A polyphasic study was undertaken to determine the taxonomic position of 13 Campylobacter fetus-like isolates from humans (n=8) and reptiles (n=5). Phenotypic characterization, Genusgenus-specific and sap insertion-PCR initially identified all human isolates as type A Campylobacter fetus. Phylogenet...

  2. Comparison of virulence factors and capsular types of Streptococcus agalactiae isolated from human and bovine infections.

    Science.gov (United States)

    Emaneini, Mohammad; Khoramian, Babak; Jabalameli, Fereshteh; Abani, Samira; Dabiri, Hossein; Beigverdi, Reza

    2016-02-01

    Streptococcus agalactiae is a leading cause of human and bovine infections. A total of 194 S. agalactiae isolates, 55 isolates from bovines and 139 from humans, were analyzed for capsular types, virulence genes (scpB, hly, rib, bca and bac) and mobile genetic elements (IS1548 and GBSi1) using polymerase chain reaction (PCR) and multiplex PCR. Capsular type III was predominant (61%), followed by types V, II, Ib, and IV. The scpB, hly, bca and bac virulence genes were only found among human isolates. Twelve and 2 distinct virulence gene profiles were identified among human and bovine isolates respectively. The virulence gene profiles scpB- hly- IS1548- rib-bca (51%) and scpB- hly- IS1548- bca (19%) were only predominant among human isolates. The rib gene was the most common virulence gene in both human and bovine isolates. The study showed a high prevalence of virulence genes in S. agalactiae strains isolated from human infections, these result can support the idea that S. agalactiae isolated from humans and bovines are generally unrelated and probably belonged to separate populations.

  3. Multilocus sequence types of Finnish bovine Campylobacter jejuni isolates and their attribution to human infections

    Directory of Open Access Journals (Sweden)

    Corander Jukka

    2010-07-01

    Full Text Available Abstract Background Campylobacter jejuni is the most common bacterial cause of human gastroenteritis worldwide. Due to the sporadic nature of infection, sources often remain unknown. Multilocus sequence typing (MLST has been successfully applied to population genetics of Campylobacter jejuni and mathematical modelling can be applied to the sequence data. Here, we analysed the population structure of a total of 250 Finnish C. jejuni isolates from bovines, poultry meat and humans collected in 2003 using a combination of Bayesian clustering (BAPS software and phylogenetic analysis. Results In the first phase we analysed sequence types (STs of 102 Finnish bovine C. jejuni isolates by MLST and found a high diversity totalling 50 STs of which nearly half were novel. In the second phase we included MLST data from domestic human isolates as well as poultry C. jejuni isolates from the same time period. Between the human and bovine isolates we found an overlap of 72.2%, while 69% of the human isolates were overlapping with the chicken isolates. In the BAPS analysis 44.3% of the human isolates were found in bovine-associated BAPS clusters and 45.4% of the human isolates were found in the poultry-associated BAPS cluster. BAPS reflected the phylogeny of our data very well. Conclusions These findings suggest that bovines and poultry were equally important as reservoirs for human C. jejuni infections in Finland in 2003. Our results differ from those obtained in other countries where poultry has been identified as the most important source for human infections. The low prevalence of C. jejuni in poultry flocks in Finland could explain the lower attribution of human infection to poultry. Of the human isolates 10.3% were found in clusters not associated with any host which warrants further investigation, with particular focus on waterborne transmission routes and companion animals.

  4. Renal adenocarcinoma, hepatocellular carcinoma, and pancreatic islet cell carcinoma in a binturong (Arctictis binturong).

    Science.gov (United States)

    Klaphake, Eric; Shoieb, Ahmed; Ramsay, Ed; Schumacher, Juergen; Craig, Linden

    2005-03-01

    A 19-yr-old binturong (Arctictis binturong) with acute upper respiratory disease was euthanized. Postmortem findings included hepatocellular carcinoma, pancreatic islet cell carcinoma, and renal adenocarcinoma with metastasis to the spleen, pleura, and pericardium. A link between primary hepatic and renal neoplasms has been noted in older humans.

  5. Exploration of α1-antitrypsin treatment protocol for islet transplantation: dosing plan and route of administration.

    Science.gov (United States)

    Baranovski, Boris M; Ozeri, Eyal; Shahaf, Galit; Ochayon, David E; Schuster, Ronen; Bahar, Nofar; Kalay, Noa; Cal, Pablo; Mizrahi, Mark I; Nisim, Omer; Strauss, Pnina; Schenker, Eran; Lewis, Eli C

    2016-11-07

    Life-long weekly infusions of human α1-antitrypsin (hAAT) are currently administered as augmentation therapy for patients with genetic AAT deficiency (AATD). Several recent clinical trials attempt to extend hAAT therapy to conditions outside AATD, including type 1 diabetes. Since the endpoint for AATD is primarily the reduction of risk for pulmonary emphysema, the present study explores hAAT dose protocols and routes of administration in attempt to optimize hAAT therapy for islet-related injury. Islet-grafted mice were treated with hAAT (Glassia™; i.p. or s.c.) under an array of clinically relevant dosing plans. Serum hAAT and immunocyte cell membrane association were examined, as well as parameters of islet survival. Results indicate that dividing the commonly prescribed 60 mg/kg i.p. dose to three 20 mg/kg injections is superior in affording islet graft survival; in addition, a short dynamic descending dose protocol (240→120→60→60 mg/kg i.p.) is comparable in outcomes to indefinite 60 mg/kg injections. While hAAT pharmacokinetics after i.p. administration in mice resembles exogenous hAAT treatment in humans, s.c. administration better imitated the physiological progressive rise of hAAT during acute phase responses; nonetheless, only the 60 mg/kg dose depicted an advantage using the s.c. route. Taken together, this study provides a platform for extrapolating an islet-relevant clinical protocol from animal models that use hAAT to protect islets. In addition, the study places emphasis on outcome-oriented analyses of drug efficacy, particularly important when considering that hAAT is presently at an era of drug-repurposing towards an extended list of clinical indications outside genetic AATD.

  6. Detection of CC17 Enterococcus faecium in dogs and a comparison with human isolates.

    Science.gov (United States)

    Kwon, K H; Moon, B Y; Hwang, S Y; Park, Y H

    2012-09-01

    Enterococcus faecium strains of clonal complex (CC) 17 were isolated from domestic dogs. The strains were more prevalent in infectious isolates than in colonized isolates, suggesting that strains of the CC17 lineage may have an advantage in causing infections in dogs. The pulsed field gel electrophoresis patterns of some dog and human isolates were over 90% similar. However, antimicrobial resistance patterns and virulence factors were not identical, which might reflect different use of antimicrobials in veterinary medicine or in host specificity.

  7. Antimicrobial drug resistance of Salmonella isolates from meat and humans, Denmark

    DEFF Research Database (Denmark)

    Skov, Marianne Nielsine; Andersen, Jens Strodl; Aabo, Søren

    2007-01-01

    We compared 8,144 Salmonella isolates collected from meat imported to or produced in Denmark, as well as from Danish patients. Isolates from imported meat showed a higher rate of antimicrobial drug resistance, including multidrug resistance, than did isolates from domestic meat. Isolates from...... humans showed resistance rates lower than those found in imported meat but higher than in domestic meat. These findings indicate that programs for controlling resistant Salmonella spp. are a global issue...

  8. Microwell scaffolds for the extrahepatic transplantation of islets of langerhans

    NARCIS (Netherlands)

    Buitinga, M.; Truckenmuller, R.; Engelse, M.A.; Moroni, L.; Ten Hoopen, H.W.; van Blitterswijk, C.A.; de Koning, E.J.; van Apeldoorn, A.A.; Karperien, M.

    2013-01-01

    Allogeneic islet transplantation into the liver has the potential to restore normoglycemia in patients with type 1 diabetes. However, the suboptimal microenvironment for islets in the liver is likely to be involved in the progressive islet dysfunction that is often observed post-transplantation. Thi

  9. Effects of Acute Cytomegalovirus Infection on Rat Islet Allograft Survival

    NARCIS (Netherlands)

    Smelt, M. J.; Faas, M. M.; Melgert, B. N.; de Vos, P.; de Haan, Bart; de Haan, Aalzen

    2011-01-01

    Transplantation of pancreatic islets is a promising therapy for the treatment of type 1 diabetes mellitus. However, long-term islet graft survival rates are still unsatisfactory low. In this study we investigated the role of cytomegalovirus (CMV) in islet allograft failure. STZ-diabetic rats receive

  10. Pancreatic islet-cell viability, functionality and oxidative status remain unaffected at pharmacological concentrations of commonly used antibiotics in vitro

    Indian Academy of Sciences (India)

    Yogita Shewade; Suraj Tirth; R R Bhonde

    2001-09-01

    Environmental factors such as diet, physical activity, drugs, pollution and life style play an important role in the progression and/or precipitation of diseases like diabetes, hypertension, obesity and cardiovascular disorders. Indiscriminate use of antibiotics to combat infectious diseases is one of the commonest forms of misuse of drugs. Antibiotics seem to have a correlation with diabetes and pancreatic function. There are controversial reports about the effect of antibiotics on the pancreatic islets; some suggesting their harmless action, some depicting a beneficial role and others indicating deleterious effect. Moreover, use of antibiotics is mandatory during islet isolation and cultivation to reduce incidences of microbial contamination. It is likely that antibiotic treatment may adversely affect islet viability and its functioning leading to failure of islet transplantation. The present in vitro study was undertaken to examine the effect of commonly used antibiotics such as gentamycin, penicillin, streptomycin, tetracycline, neomycin, erythromycin and chloramphenicol on islet viability, its functioning and induction of oxidative stress if any. The viability and insulin production data showed that none of the antibiotics used in the present study affect the viability and the functioning of the islets at their pharmacological concentrations. Free radical levels measured in terms of melonyldialdehyde (MDA), nitric oxide (NO) and reduced glutathione (GSH) reveal that except for a marginal increase in lipid peroxidation with tetracycline and slight increase in NO levels with streptomycin, none of these antibiotics affect the oxidative status of the cells. Antioxidant enzymes such as superoxide dismutase and catalase remain unaffected after this treatment. Our results reveal the innocuous nature of the antibiotics used at pharmacological concentrations, suggesting their safety whenever prescribed to combat infections and also during islet isolation procedures.

  11. Decreased 11β-Hydroxysteroid Dehydrogenase 1 Level and Activity in Murine Pancreatic Islets Caused by Insulin-Like Growth Factor I Overexpression.

    Directory of Open Access Journals (Sweden)

    Subrata Chowdhury

    Full Text Available We have reported a high expression of IGF-I in pancreatic islet β-cells of transgenic mice under the metallothionein promoter. cDNA microarray analysis of the islets revealed that the expression of 82 genes was significantly altered compared to wild-type mice. Of these, 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1, which is responsible for the conversion of inert cortisone (11-dehydrocorticosterone, DHC in rodents to active cortisol (corticosterone in the liver and adipose tissues, has not been identified previously as an IGF-I target in pancreatic islets. We characterized the changes in its protein level, enzyme activity and glucose-stimulated insulin secretion. In freshly isolated islets, the level of 11β-HSD1 protein was significantly lower in MT-IGF mice. Using dual-labeled immunofluorescence, 11β-HSD1 was observed exclusively in glucagon-producing, islet α-cells but at a lower level in transgenic vs. wild-type animals. MT-IGF islets also exhibited reduced enzymatic activities. Dexamethasone (DEX and DHC inhibited glucose-stimulated insulin secretion from freshly isolated islets of wild-type mice. In the islets of MT-IGF mice, 48-h pre-incubation of DEX caused a significant decrease in insulin release, while the effect of DHC was largely blunted consistent with diminished 11β-HSD1 activity. In order to establish the function of intracrine glucocorticoids, we overexpressed 11β-HSD1 cDNA in MIN6 insulinoma cells, which together with DHC caused apoptosis and a significant decrease in proliferation. Both effects were abolished with the treatment of an 11β-HSD1 inhibitor. Our results demonstrate an inhibitory effect of IGF-I on 11β-HSD1 expression and activity within the pancreatic islets, which may mediate part of the IGF-I effects on cell proliferation, survival and insulin secretion.

  12. Pancreatic islet function in omega-3 fatty acid-depleted rats : Alteration of calcium fluxes and calcium-dependent insulin release

    OpenAIRE

    Zhang, Ying; Oguzhan, Berrin; Louchami, Karim; Chardigny, Jean-Michel; Portois, Laurence; Carpentier, Yvon; Malaisse, Willy-Jean; Herchuelz, André; Sener, Abdullah

    2006-01-01

    Considering the insufficient supply of long-chain polyunsaturated omega-3 fatty acids often prevailing in Western populations, this report deals mainly with alterations of Ca2+ fluxes and Ca2+-dependent insulin secretory events in isolated pancreatic islets from omega-3-depleted rats. In terms of Ca-45(2+) handling, the islets from omega-3-depleted rats, compared with those from normal animals, displayed an unaltered responsiveness to an increase in extracellular K+ concentration, a lower inf...

  13. Islet Brain 1 Protects Insulin Producing Cells against Lipotoxicity.

    Science.gov (United States)

    Brajkovic, Saška; Ferdaoussi, Mourad; Pawlowski, Valérie; Ezanno, Hélène; Plaisance, Valérie; Zmuda, Erik; Hai, Tsonwin; Annicotte, Jean-Sébastien; Waeber, Gérard; Abderrahmani, Amar

    2016-01-01

    Chronic intake of saturated free fatty acids is associated with diabetes and may contribute to the impairment of functional beta cell mass. Mitogen activated protein kinase 8 interacting protein 1 also called islet brain 1 (IB1) is a candidate gene for diabetes that is required for beta cell survival and glucose-induced insulin secretion (GSIS). In this study we investigated whether IB1 expression is required for preserving beta cell survival and function in response to palmitate. Chronic exposure of MIN6 and isolated rat islets cells to palmitate led to reduction of the IB1 mRNA and protein content. Diminution of IB1 mRNA and protein level relied on the inducible cAMP early repressor activity and proteasome-mediated degradation, respectively. Suppression of IB1 level mimicked the harmful effects of palmitate on the beta cell survival and GSIS. Conversely, ectopic expression of IB1 counteracted the deleterious effects of palmitate on the beta cell survival and insulin secretion. These findings highlight the importance in preserving the IB1 content for protecting beta cell against lipotoxicity in diabetes.

  14. The Study of Non-Viral Nanoscale Delivery Systems for Islet Transplantation

    Science.gov (United States)

    Gutierrez, Diana

    Due to safety concerns associated with using viral systems clinically to expand islet cells and make them available to many more patients, significant emphasis has been placed on producing a safe and effective non-viral delivery system for biological research and gene therapy. To obtain this goal, we propose the use of an innovative technology that utilizes gold nanoparticles (AuNPs) as a non-viral method of delivery. Our laboratory was one of the first to describe the use of AuNPs in human islets and observe AuNPs can penetrate into the core of islets to deliver a gene to the vast majority of the cells, without damaging the cell. Gold nanoparticles proved to be a biocompatible delivery system both in vitro and in vivo. Thus far, gene therapy and molecular biology have focused primarily on delivering DNA of a specific gene into cells. The risk of this approach is that the DNA can be permanently incorporated into the genome and lead to damages in the cell that could result in overexpression of cancerous tumor cells. This risk does not exist with the use of mRNA. Many researchers believe mRNA is too unstable to be used as a molecular tool to overexpress specific proteins. With advances in nanotechnology, and better understanding of the translation process, methods have been developed that allow for expression of specific proteins by intracellular delivery of protein-encoding mRNA. We used AuNPs conjugated to mCherry mRNA to establish a proof of concept of the feasibility of using AuNP-mRNA to achieve increased expression of a specific protein within cells. To do this, we conjugated mCherry mRNA to AuNPs and tested the feasibility for increasing delivery efficacy and preserve functionality of human pancreatic islets. We believe that with this novel technology we can create AuNPs that allow specific mRNA to enter islets and lead to the production of a specific protein within the cell, with the aim to induce beta cell proliferation. In a previous experiment with single

  15. Xenotransplantation of piscine islets into hyperglycemic rats

    Energy Technology Data Exchange (ETDEWEB)

    Weber, C.; Weil, R. III; McIntosh, R.; Hogle, H.; Warden, G.; Reemtsma, K.

    1975-02-01

    Xenotransplantation of piscine islets into hyperglycemic rats usually lowers the blood sugar level of the recipient. The duration of this effect is prolonged by irradiation of the host or by enclosing donor tissue in synthetic envelopes. This prolongation appears to be related to interference with the host's ability to reject the graft; the duration of the prolongation may be limited by the host tissue reaction surrounding the envelope. The availability of anatomically separate piscine islet tissue makes it potentially useful for xenotransplantation into mammals.

  16. Myeloperoxidase impairs the contractile function in isolated human cardiomyocytes.

    Science.gov (United States)

    Kalász, Judit; Pásztor, Enikő T; Fagyas, Miklós; Balogh, Ágnes; Tóth, Attila; Csató, Viktória; Édes, István; Papp, Zoltán; Borbély, Attila

    2015-07-01

    We set out to characterize the mechanical effects of myeloperoxidase (MPO) in isolated left-ventricular human cardiomyocytes. Oxidative myofilament protein modifications (sulfhydryl (SH)-group oxidation and carbonylation) induced by the peroxidase and chlorinating activities of MPO were additionally identified. The specificity of the MPO-evoked functional alterations was tested with an MPO inhibitor (MPO-I) and the antioxidant amino acid Met. The combined application of MPO and its substrate, hydrogen peroxide (H2O2), largely reduced the active force (Factive), increased the passive force (Fpassive), and decreased the Ca(2+) sensitivity of force production (pCa50) in permeabilized cardiomyocytes. H2O2 alone had significantly smaller effects on Factive and Fpassive and did not alter pCa50. The MPO-I blocked both the peroxidase and the chlorinating activities, whereas Met selectively inhibited the chlorinating activity of MPO. All of the MPO-induced functional effects could be prevented by the MPO-I and Met. Both H2O2 alone and MPO + H2O2 reduced the SH content of actin and increased the carbonylation of actin and myosin-binding protein C to the same extent. Neither the SH oxidation nor the carbonylation of the giant sarcomeric protein titin was affected by these treatments. MPO activation induces a cardiomyocyte dysfunction by affecting Ca(2+)-regulated active and Ca(2+)-independent passive force production and myofilament Ca(2+) sensitivity, independent of protein SH oxidation and carbonylation. The MPO-induced deleterious functional alterations can be prevented by the MPO-I and Met. Inhibition of MPO may be a promising therapeutic target to limit myocardial contractile dysfunction during inflammation.

  17. Isolating nasal olfactory stem cells from rodents or humans.

    Science.gov (United States)

    Girard, Stéphane D; Devéze, Arnaud; Nivet, Emmanuel; Gepner, Bruno; Roman, François S; Féron, François

    2011-08-22

    The olfactory mucosa, located in the nasal cavity, is in charge of detecting odours. It is also the only nervous tissue that is exposed to the external environment and easily accessible in every living individual. As a result, this tissue is unique for anyone aiming to identify molecular anomalies in the pathological brain or isolate adult stem cells for cell therapy. Molecular abnormalities in brain diseases are often studied using nervous tissue samples collected post-mortem. However, this material has numerous limitations. In contrast, the olfactory mucosa is readily accessible and can be biopsied safely without any loss of sense of smell(1). Accordingly, the olfactory mucosa provides an "open window" in the adult human through which one can study developmental (e.g. autism, schizophrenia)(2-4) or neurodegenerative (e.g. Parkinson, Alzheimer) diseases(4,5). Olfactory mucosa can be used for either comparative molecular studies(4,6) or in vitro experiments on neurogenesis(3,7). The olfactory epithelium is also a nervous tissue that produces new neurons every day to replace those that are damaged by pollution, bacterial of viral infections. This permanent neurogenesis is sustained by progenitors but also stem cells residing within both compartments of the mucosa, namely the neuroepithelium and the underlying lamina propria(8-10). We recently developed a method to purify the adult stem cells located in the lamina propria and, after having demonstrated that they are closely related to bone marrow mesenchymal stem cells (BM-MSC), we named them olfactory ecto-mesenchymal stem cells (OE-MSC)(11). Interestingly, when compared to BM-MSCs, OE-MSCs display a high proliferation rate, an elevated clonogenicity and an inclination to differentiate into neural cells. We took advantage of these characteristics to perform studies dedicated to unveil new candidate genes in schizophrenia and Parkinson's disease(4). We and others have also shown that OE-MSCs are promising candidates

  18. Molecular typing of Brucella species isolates from livestock and human.

    Science.gov (United States)

    Nagalingam, Mohandoss; Shome, Rajeswari; Balamurugan, Vinayagamurthy; Shome, Bibek Ranjan; NarayanaRao, Krishnamsetty; Vivekananda; Isloor, Shrikrishna; Prabhudas, Krishnamsetty

    2012-01-01

    Although host specificity has been observed in different species of Brucella, crossing the animal host boundary is likely to occur at any time. In this study, Bruce ladder PCR and abortus-melitensis-ovis-suis (AMOS) PCR assays were used to characterize 47 Brucella isolates from Indian origin in order to know exact species for understanding epidemiology of brucellosis. Out of them, 28, 14, and 5 isolates were found to be Brucella abortus, Brucella melitensis, and Brucella suis, respectively. Further analysis by AMOS PCR has identified that all the B. abortus isolates belong to any one of the biovar 1, 2, or 4; of the five B. suis isolates, three belong to biovar 1 and two belong to any one of the biovar 2, 3, 4, or 5. Although this multiplex Bruce ladder PCR is useful in differentiating all Brucella species, elaborate study is required to further characterize the isolates at exact biovar level.

  19. Inhibition of Insulin-Degrading Enzyme Does Not Increase Islet Amyloid Deposition in Vitro.

    Science.gov (United States)

    Hogan, Meghan F; Meier, Daniel T; Zraika, Sakeneh; Templin, Andrew T; Mellati, Mahnaz; Hull, Rebecca L; Leissring, Malcolm A; Kahn, Steven E

    2016-09-01

    Islet amyloid deposition in human type 2 diabetes results in β-cell loss. These amyloid deposits contain the unique amyloidogenic peptide human islet amyloid polypeptide (hIAPP), which is also a known substrate of the protease insulin-degrading enzyme (IDE). Whereas IDE inhibition has recently been demonstrated to improve glucose metabolism in mice, inhibiting it has also been shown to increase cell death when synthetic hIAPP is applied exogenously to a β-cell line. Thus, we wanted to determine whether a similar deleterious effect is observed when hIAPP is endogenously produced and secreted from islets. To address this issue, we cultured hIAPP transgenic mouse islets that have the propensity to form amyloid for 48 and 144 hours in 16.7 mM glucose in the presence and absence of the IDE inhibitor 1. At neither time interval did IDE inhibition increase amyloid formation or β-cell loss. Thus, the inhibition of IDE may represent an approach to improve glucose metabolism in human type 2 diabetes, without inducing amyloid deposition and its deleterious effects.

  20. Islet-intrinsic effects of CFTR mutation.

    Science.gov (United States)

    Koivula, Fiona N Manderson; McClenaghan, Neville H; Harper, Alan G S; Kelly, Catriona

    2016-07-01

    Cystic fibrosis-related diabetes (CFRD) is the most significant extra-pulmonary comorbidity in cystic fibrosis (CF) patients, and accelerates lung decline. In addition to the traditional view that CFRD is a consequence of fibrotic destruction of the pancreas as a whole, emerging evidence may implicate a role for cystic fibrosis transmembrane-conductance regulator (CFTR) in the regulation of insulin secretion from the pancreatic islet. Impaired first-phase insulin responses and glucose homeostasis have also been reported in CF patients. CFTR expression in both human and mouse beta cells has been confirmed, and recent studies have shown differences in endocrine pancreatic morphology from birth in CF. Recent experimental evidence suggests that functional CFTR channels are required for insulin exocytosis and the regulation of membrane potential in the pancreatic beta cell, which may account for the impairments in insulin secretion observed in many CF patients. These novel insights suggest that the pathogenesis of CFRD is more complicated than originally thought, with implications for diabetes treatment and screening in the CF population. This review summarises recent emerging evidence in support of a primary role for endocrine pancreatic dysfunction in the development of CFRD. Summary • CF is an autosomal recessive disorder caused by mutations in the CFTR gene • The vast majority of morbidity and mortality in CF results from lung disease. However CFRD is the largest extra-pulmonary co-morbidity and rapidly accelerates lung decline • Recent experimental evidence shows that functional CFTR channels are required for normal patterns of first phase insulin secretion from the pancreatic beta cell • Current clinical recommendations suggest that insulin is more effective than oral glucose-lowering drugs for the treatment of CFRD. However, the emergence of CFTR corrector and potentiator drugs may offer a personalised approach to treating diabetes in the CF population.

  1. Characterization of clinical and environmental Mycobacterium avium spp. isolates and their interaction with human macrophages.

    Directory of Open Access Journals (Sweden)

    Evelyn Guirado

    Full Text Available Members of the Mycobacterium avium complex (MAC are naturally occurring bacteria in the environment. A link has been suggested between M. avium strains in drinking water and clinical isolates from infected individuals. There is a need to develop new screening methodologies that can identify specific virulence properties of M. avium isolates found in water that predict a level of risk to exposed individuals. In this work we have characterized 15 clinical and environmental M. avium spp. isolates provided by the US Environmental Protection Agency (EPA to improve our understanding of the key processes involved in the binding, uptake and survival of these isolates in primary human macrophages. M. avium serovar 8 was predominant among the isolates studied. Different amounts and exposure of mannose-capped lipoarabinomannan (ManLAM and glycopeptidolipids (GPLs, both major mycobacterial virulence factors, were found among the isolates studied. Reference clinical isolate 104 serovar 1 and clinical isolates 11 and 14 serovar 8 showed an increased association with macrophages. Serum opsonization increased the cell association and survival at 2 h post infection for all isolates. However, only the clinical isolates 104 and 3 among those tested showed an increased growth in primary human macrophages. The other isolates varied in their survival in these cells. Thus we conclude that the amounts of cell envelope ManLAM and GPL, as well as GPL serovar specificity are not the only important bacterial factors for dictating the early interactions of M. avium with human macrophages.

  2. Isolation of single-base genome-edited human iPS cells without antibiotic selection.

    Science.gov (United States)

    Miyaoka, Yuichiro; Chan, Amanda H; Judge, Luke M; Yoo, Jennie; Huang, Miller; Nguyen, Trieu D; Lizarraga, Paweena P; So, Po-Lin; Conklin, Bruce R

    2014-03-01

    Precise editing of human genomes in pluripotent stem cells by homology-driven repair of targeted nuclease-induced cleavage has been hindered by the difficulty of isolating rare clones. We developed an efficient method to capture rare mutational events, enabling isolation of mutant lines with single-base substitutions without antibiotic selection. This method facilitates efficient induction or reversion of mutations associated with human disease in isogenic human induced pluripotent stem cells.

  3. Isolation of tumor suppressor genes from MEN-1 related neoplasms

    Energy Technology Data Exchange (ETDEWEB)

    Yavari, R.; Kinder, B.; Bale, A.E. [Yale Univ. School of Medicine, New Haven, CT (United States)

    1994-09-01

    Multiple Endocrine Neoplasia type 1 (MEN 1) is a cancer predisposition syndrome marked by the development of tumors in specific endocrine tissues such as the pituitary, parathyroid and pancreatic islets. Genetic linkage studies have mapped the MEN 1 gene to 11q13, and allelic loss in related tumors suggests that the gene is a tumor suppressor. Because inactivation of tumor suppressors may be accompanied by underexpression, subtractive hybridization was used to isolate potential candidate genes underexpressed in MEN 1 tumors. cDNA was synthesized from tumor and normal parathyroid tissue by RT-PCR. Biotinylated tumor cDNA was used as a driver and normal cDNA as a tester in subtractive hybridization. Following annealing of the driver and tester amplicons, the biotinylated strands were removed with streptavidin. The subtracted material was then used as a probe to isolate clones from a normal pancreatic islet library. Screening 2 x 10{sup 5} plaques yielded 14 positive clones. Of 6 clones analyzed, 3 were confirmed to be underexpressed in parathyroid tumors. Sequence analysis identified 2 clones as human ribosomal protein S10 (RPS10, chromosome 6) and 1 as the islet amyloid polypeptide (1AP, chromosome 12). The precise function of human RPS10 is not known but the related RPS6 functions as a tumor suppressor in Drosophila. 1AP has been implicated in modulation of G protein activity. The remaining positive clones will be mapped to determine if any fall on chromosome 11q13, and additional subtractions with parathyroid and pancreatic islet neoplasms are underway.

  4. Cold ischemia time and blood compatibility associated with activity of transplanted islet cells%冷缺血时间及血液相容性与移植胰岛细胞活性的关系

    Institute of Scientific and Technical Information of China (English)

    高宏君; 梁泰生; 杨欢; 罗向东; 吴佩钟; 谭臻; 梁芳芳

    2007-01-01

    都发生明显的消耗;加入肝素后血细胞计数与对照组比较差异明显(P<0.05),反应明显减轻.HLA匹配组和HLA错配组胰岛细胞体外培养24 h活性胰岛细胞数量较,差异明显(P<0.05).结论:在冷缺血时间<5 h的情况下获取的胰腺可以用于临床胰岛细胞的移植;血液相容性好能够明显提高胰岛细胞移植的成功率.%BACKGROUND: The quantity and bioactivity of isolated islet are vital to islet transplantation; while, the cold ischemia time and human leucocyte antigen (HLA) typing are key factors to islet quantity and bioactivity which inflect islet transplantation.OBJECTIVE: To observe the effects of cold ischemia time and blood compatibility on quantity and bioactivity of islet cells.DESIGN: Observational study.SETTING: Ruikang Affiliated Hospital of Guangxi College of Traditional Chinese Medicine.MATERIALS: Organs from voluntary donors died of irreversible coma were adopted whose blood-type and HLA typing had been known, pancreases acquisition was carried on after other organs ablation or in the meantime. The blood of identical ABO and HLA matching conformity, or HLA cross-matching hypersensitization (missmatching over 3 Iocuses),or panel reaction antibody (PRA) > 50%, or lymphocyte cytotoxin crossmatching test positive. The isolated and purified islet suspension was filtered by 70 μm filter, which result in preparing 1.2×105/L islet suspention.METHODS: Hypertonic citrate adenine solution was perfused into aorta, and kidney-pancreases and kidney-pancreases-liver were cut together or kidney-pancreases-liver was cut separately. Islet activity was judged by diphenylthiocarbazone (DTZ) dyeing and acridine orange (AO) dyeing; meanwhile, twelve pancreases far from contamination were aquired, mean ablation time was 15 minutes; cold ischemia time ranged from 2.5 to 8 hou