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Sample records for isolated mouse islets

  1. Isolation of Mouse Pancreatic Islets of Langerhans.

    PubMed

    Ramírez-Domínguez, Miriam

    2016-01-01

    The aim of any pancreatic islet isolation is obtaining pure, viable and functional pancreatic islets, either for in vitro or in vivo purposes. The islets of Langerhans are complex microorgans with the important role of regulating glucose homeostasis. Imbalances in glucose homeostasis lead to diabetes, which is defined by the American Diabetes Association as a "group of metabolic diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action or both" (American Diabetes Association 2011). Currently, the rising demand of human islets is provoking a shortage of this tissue, limiting research and clinical practice on this field. In this scenario, it is essential to investigate and improve islet isolation procedures in animal models, while keeping in mind the anatomical and functional differences between species. This chapter discusses the main aspects of mouse islet isolation research, highlighting the critical factors and shortcomings to take into account for the selection and/or optimization of a mouse islet isolation protocol. PMID:27586420

  2. Filtration is a time-efficient option to Histopaque, providing good-quality islets in mouse islet isolation.

    PubMed

    Ramírez-Domínguez, Miriam; Castaño, Luis

    2015-03-01

    Pancreatic islet transplantation is a promising therapy for Type I Diabetes. For many years the method used worldwide for islet purification in both rodent and human islet isolation has been Ficoll-based density gradients, such as Histopaque. However, it is difficult to purify islets in laboratories with staff limitations when large scale isolations are required. We hypothesized that filtration could be a more simple and fast alternative to obtain good quality islets. Four separate islet isolations were performed per method, comparing filtration and Histopaque purification with handpicking as the gold standard method for islet purity. Different parameters of quality were assessed: yield in number of islets per pancreas, purity by dithizone staining, viability by Fluorescein Diacetate/Propidium Iodide vital staining and in vitro functionality assessed by Glucose Stimulated Insulin Secretion. Time efficiency and cost were also analyzed. The overall quality of the islets obtained both by Histopaque and filtration was good. Filtration saved almost 90 % of the time consumed by Histopaque purification, and was also cheaper. However, one-third of the islets were lost. Since human and rodent islets share similar size but different density, filtration appears as a purification method with potential interest in translation to clinic. PMID:24443076

  3. Clinical Islet Isolation.

    PubMed

    Hawthorne, Wayne J; Williams, Lindy; Chew, Yi Vee

    2016-01-01

    The overarching success of islet transplantation relies on the success in the laboratory to isolate the islets. This chapter focuses on the processes of human islet cell isolation and the ways to optimally provide islet cells for transplantation. The major improvements in regards to the choice of enzyme type, way the digested pancreas tissue is handled to best separate islets from the acinar and surrounding tissues, the various methods of purification of the islets, their subsequent culture and quality assurance to improve outcomes to culminate in safe and effective islet transplantation will be discussed. After decades of improvements, islet cell isolation and transplantation now clearly offer a safe, effective and feasible therapeutic treatment option for an increasing number of patients suffering from type 1 diabetes specifically for those with severe hypoglycaemic unawareness. PMID:27586424

  4. Islet Insulin Secretion Measurements in the Mouse.

    PubMed

    Hugill, Alison; Shimomura, Kenju; Cox, Roger D

    2016-01-01

    This article describes detailed protocols for in vitro measurements of insulin function and secretion in isolated mouse islets for the analysis of glucose homeostasis. We specify a method of enzyme digestion and hand picking to isolate and release the greatest number of high quality islets from the pancreas of the mouse. We describe an effective method for generating dynamic measurements of insulin secretion using a perifusion assay including a detailed protocol for constructing a peristaltic pump and tubing assembly. In addition we describe an alternative and simple technique for measuring insulin secretion using static incubation of isolated islets. © 2016 by John Wiley & Sons, Inc. PMID:27584553

  5. Isolation of Pancreatic Islets from Nonhuman Primates.

    PubMed

    Berman, Dora M

    2016-01-01

    Nonhuman primates (NHP) constitute a highly relevant pre-clinical animal model to develop strategies for beta cell replacement. The close phylogenetic and immunologic relationship between NHP and humans results in cross-reactivity of various biological agents with NHP cells, as well as a very similar cytoarchitecture between islets from human and NHP that is strikingly different from that observed in rodent islets. The composition and location of endocrine cells in human or NHP islets, randomly distributed and associated with blood vessels, have functional consequences and a predisposition for paracrine interactions. Furthermore, translation of approaches that proved successful in rodent models to the clinic has been limited. Consequently, data collected from NHP studies can form the basis for an IND submission to the FDA. This chapter describes in detail the key aspects for isolation of islets from NHP, from organ procurement up to assessment of islet function, comparing and emphasizing the similarities between isolation procedures for human and NHP islets. PMID:27586422

  6. Historical Background of Pancreatic Islet Isolation.

    PubMed

    Ramírez-Domínguez, Miriam

    2016-01-01

    Until the discovery of insulin in the twentieth century, diabetes mellitus was a mortal disease with an unclear origin and physiology. Despite the appearance of the concept in an Egyptian papyrus dated c.1550 BC, and the documentation of its study by ancient Chinese, the term "diabetes" was only coined by the Greek Aretaeus in the second century AD. In Europe, the study of diabetes was largely ignored until the seventeenth century, when the characteristic sweet flavor of diabetic urine was first described. However, the link between diabetes and the pancreas was not discovered until 1889 by Minkowski and von Mering, long after the first description of the pancreatic islets by Paul Langerhans in 1869. One of the most significant milestones in the field was the discovery of insulin by Banting and collaborators in 1922, which led to the therapeutic development of insulin administration as a life-saving intervention for type 1 diabetic patients. On the other hand, the isolation of islets was first reported by Bensley in 1911, a critical technical achievement that paved the way for clinical islet transplantation. Here we discuss the history of islet isolation, since the firsts studies of diabetes by ancient civilizations to the birth and parallel evolution of islet isolation and transplantation. PMID:27586418

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

    PubMed Central

    Pawlick, Rena L.; Kahana, Meygal; Pepper, Andrew R.; Bruni, Antonio; Gala-Lopez, Boris; Kin, Tatsuya; Mitrani, Eduardo; Shapiro, A. M. James

    2016-01-01

    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. PMID:27227978

  8. Seven consecutive successful clinical islet isolations with pancreatic ductal injection.

    PubMed

    Matsumoto, Shinichi; Noguichi, Hirofumi; Shimoda, Masayuki; Ikemoto, Tetsuya; Naziruddin, Bashoo; Jackson, Andrew; Tamura, Yoshiko; Olson, Greg; Fujita, Yasutaka; Chujo, Daisuke; Takita, Morihito; Kobayashi, Naoya; Onaca, Nicholas; Levy, Marlon

    2010-01-01

    Inconsistent islet isolation is one of the issues of clinical islet transplantation. In the current study, we applied ductal injection to improve the consistency of islet isolation. Seven islet isolations were performed with the ductal injection of ET-Kyoto solution (DI group) and eight islet isolations were performed without the ductal injection (standard group) using brain-dead donor pancreata. Isolated islets were evaluated based on the Edmonton protocol for transplantation. The DI group had significantly higher islet yields (588,566 +/- 64,319 vs. 354,836 +/- 89,649 IE, p < 0.01) and viability (97.3 +/- 1.2% vs. 92.6 +/- 1.2%, p < 0.02) compared with the standard group. All seven isolated islet preparations in the DI group (100%), versus only three out of eight isolated islet preparations (38%) in the standard group met transplantation criteria. The islets from the DI group were transplanted into three type 1 diabetic patients and all three patients became insulin independent. Ductal injection significantly improved quantity and quality of isolated islets and resulted in high success rate of clinical islet transplantation. This simple modification will reduce the risk of failure of clinical islet isolation.

  9. Expression and function of monoacylglycerol lipase in mouse β-cells and human islets of Langerhans.

    PubMed

    Li, Chen; Vilches-Flores, Alonso; Zhao, Min; Amiel, Stephanie A; Jones, Peter M; Persaud, Shanta J

    2012-01-01

    Elements of the endocannabinoid system (ECS) are expressed by islet endocrine cells and activation of CB1 and CB2 cannabinoid receptors regulates insulin secretion from mouse and human β-cells. The current study aimed to investigate the expression and function, in mouse and human β-cells, of monoacylglycerol lipase (MGL), an enzyme that facilitates degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). We found that MGL mRNA is expressed by MIN6 β-cells, mouse islets, human islets and enriched human islet β-cells, and immunohistochemistry indicated that MGL localisation in human islets is consistent with its expression by some β- and -α-cells. Blockade of MGL activity with the pharmacological inhibitor URB602 led to increased [Ca(2+)](i )and enhanced insulin secretion from MIN6 β-cells, and MGL inhibition also elevated insulin and glucagon secretion from isolated human islets in vitro. These data imply a stimulatory role for endogenous 2-AG in islets that is amplified when its degradation is blocked. PMID:22739267

  10. Anx7 is required for nutritional control of gene expression in mouse pancreatic islets of Langerhans.

    PubMed Central

    Srivastava, Meera; Eidelman, Ofer; Leighton, Ximena; Glasman, Mirta; Goping, Gertrude; Pollard, Harvey B.

    2002-01-01

    BACKGROUND: Gene expression in islets of Langerhans is profoundly sensitive to glucose and other nutrients. Islets of Langerhans in the Anx7(+/-) knockout mouse exhibit a profound reduction in ITPR3 protein expression, defective intracellular calcium signaling, and defective insulin secretion. Additional data presented here also show that mRNA for ITPR3 is virtually undetectable in isolated Anx7(+/-) islets. IP3Receptor type 3 (ITPR3) expression in islets of Langerhans is closely regulated by secretory stimuli, and it has been suggested that the level of the ITPR3 expression controls the ability of the islets to respond to nutritional signals. We report that although control islets respond to glucose in vitro by a transient increment in ITPR3 mRNA, the islets from the Anx7(+/-) mouse remain low. We therefore hypothesized that the Anx7/IP3 Receptor(3)/Ca(2+) signaling pathway plays a role in beta cell responses to glucose, and that in the absence of the Anx7/ITPR3 signaling system, the islets would be unable to discriminate between fed or fasted states in vivo. MATERIALS AND METHODS: To test this hypothesis, we subjected Anx7(+/-) and control mice to either food and water ad libidum or to an overnight fast with access to water only. We then isolated the respective islets and compared nutrient-dependent changes in global gene expression under the four conditions using genome-based microarray technology. RESULTS: Anx7 protein expression in these islets is only about 50% of control levels in normal littermate controls, and IPTR3 message and protein are virtually zero. cDNA microarray analyses show that in control animals gene expression is significantly affected by the fasting state. Many of the affected genes have historical relevance to development and differentiation of islets. These include preproglucagon, APOJ, cadherin2, phosphoglucoisomerase, oncostatin M, PAX6, HGF, and cytokeratin 18. However, there are also many other nutritionally sensitive genes in control

  11. Small Mouse Islets Are Deficient in Glucagon-Producing Alpha Cells but Rich in Somatostatin-Secreting Delta Cells

    PubMed Central

    Grapengiesser, Eva; Hellman, Bo

    2016-01-01

    Small and big mouse islets were compared with special reference to their content of glucagon-producing α-cells and somatostatin-producing δ-cells. Areas stained for glucagon and somatostatin were measured in the largest cross section of small (diameter < 60 μm) and big (diameter > 100 μm) islets. Comparison of the areas indicated proportionally more δ- than α-cells in the small islets. After isolation with collagenase these islets were practically devoid of α-cells. We evaluated the functional importance of the islet size by measuring the Ca2+ signal for insulin release. A majority of the small islets responded to the hyperpolarization action of somatostatin with periodic decrease of cytoplasmic Ca2+ when glucose was elevated after tolbutamide blockade of the KATP channels. PMID:27504459

  12. Experimental studies on islets isolation, purification and function in rats.

    PubMed

    Pang, Xinlu; Xue, Wujun; Feng, Xinshun; Tian, Xiaohui; Teng, Yan; Ding, Xiaoming; Pan, Xiaoming; Guo, Qi; He, Xiaoli

    2015-01-01

    To develop a simple and effective method of islet isolation and purification in rats. Collagenase P was injected into pancreatic duct followed by incubation in water bath to digest the pancreas and isolate islet, then discontinuous gravity gradient purification was used to purify the islet. The purified islets were identified by dithizone staining. The viability of islets was assessed by fluorescence staining of acridine orange (AO) and propidium iodide (PI). The function of purified islets was determined by glucose-stimulated insulin release test and transplantation of rat with streptozocin-induced diabetes. 738±193 islets were recovered after purification. The average purity was 77±13%, the viability of islets was more than 95%. When inspected by glucose stimulation, the secreted insulin concentration was 24.31±5.47 mIU/L when stimulated by low concentration glucose and 37.62±4.29 mIU/L by high concentration glucose. There was significant difference between the two phases (P<0.05). The blood sugar concentration recovered to normal level after two days in the animals with islet transplantation. In conclusion, islets can be procured with good function and shape by using the method of injecting collagenase into pancreatic duct followed by incubation in water bath and purification using discontinuous gravity gradient. PMID:26885021

  13. Experimental studies on islets isolation, purification and function in rats

    PubMed Central

    Pang, Xinlu; Xue, Wujun; Feng, Xinshun; Tian, Xiaohui; Teng, Yan; Ding, Xiaoming; Pan, Xiaoming; Guo, Qi; He, Xiaoli

    2015-01-01

    To develop a simple and effective method of islet isolation and purification in rats. Collagenase P was injected into pancreatic duct followed by incubation in water bath to digest the pancreas and isolate islet, then discontinuous gravity gradient purification was used to purify the islet. The purified islets were identified by dithizone staining. The viability of islets was assessed by fluorescence staining of acridine orange (AO) and propidium iodide (PI). The function of purified islets was determined by glucose-stimulated insulin release test and transplantation of rat with streptozocin-induced diabetes. 738±193 islets were recovered after purification. The average purity was 77±13%, the viability of islets was more than 95%. When inspected by glucose stimulation, the secreted insulin concentration was 24.31±5.47 mIU/L when stimulated by low concentration glucose and 37.62±4.29 mIU/L by high concentration glucose. There was significant difference between the two phases (P<0.05). The blood sugar concentration recovered to normal level after two days in the animals with islet transplantation. In conclusion, islets can be procured with good function and shape by using the method of injecting collagenase into pancreatic duct followed by incubation in water bath and purification using discontinuous gravity gradient. PMID:26885021

  14. Experimental studies on islets isolation, purification and function in rats.

    PubMed

    Pang, Xinlu; Xue, Wujun; Feng, Xinshun; Tian, Xiaohui; Teng, Yan; Ding, Xiaoming; Pan, Xiaoming; Guo, Qi; He, Xiaoli

    2015-01-01

    To develop a simple and effective method of islet isolation and purification in rats. Collagenase P was injected into pancreatic duct followed by incubation in water bath to digest the pancreas and isolate islet, then discontinuous gravity gradient purification was used to purify the islet. The purified islets were identified by dithizone staining. The viability of islets was assessed by fluorescence staining of acridine orange (AO) and propidium iodide (PI). The function of purified islets was determined by glucose-stimulated insulin release test and transplantation of rat with streptozocin-induced diabetes. 738±193 islets were recovered after purification. The average purity was 77±13%, the viability of islets was more than 95%. When inspected by glucose stimulation, the secreted insulin concentration was 24.31±5.47 mIU/L when stimulated by low concentration glucose and 37.62±4.29 mIU/L by high concentration glucose. There was significant difference between the two phases (P<0.05). The blood sugar concentration recovered to normal level after two days in the animals with islet transplantation. In conclusion, islets can be procured with good function and shape by using the method of injecting collagenase into pancreatic duct followed by incubation in water bath and purification using discontinuous gravity gradient.

  15. Pancreatic Islets: Methods for Isolation and Purification of Juvenile and Adult Pig Islets.

    PubMed

    Brandhorst, Heide; Johnson, Paul R V; Brandhorst, Daniel

    2016-01-01

    The current situation of organ transplantation is mainly determined by the disbalance between the number of available organs and the number of patients on the waiting list. This obvious dilemma might be solved by the transplantation of porcine organs into human patients. The metabolic similarities which exist between both species made pancreatic islets of Langerhans to that donor tissue which will be most likely transplanted in human recipients. Nevertheless, the successful isolation of significant yields of viable porcine islets is extremely difficult and requires extensive experiences in the field. This review is focussing on the technical challenges, pitfalls and particularities that are associated with the isolation of islets from juvenile and adult pigs considering donor variables that can affect porcine islet isolation outcome.

  16. Pancreatic Islets: Methods for Isolation and Purification of Juvenile and Adult Pig Islets.

    PubMed

    Brandhorst, Heide; Johnson, Paul R V; Brandhorst, Daniel

    2016-01-01

    The current situation of organ transplantation is mainly determined by the disbalance between the number of available organs and the number of patients on the waiting list. This obvious dilemma might be solved by the transplantation of porcine organs into human patients. The metabolic similarities which exist between both species made pancreatic islets of Langerhans to that donor tissue which will be most likely transplanted in human recipients. Nevertheless, the successful isolation of significant yields of viable porcine islets is extremely difficult and requires extensive experiences in the field. This review is focussing on the technical challenges, pitfalls and particularities that are associated with the isolation of islets from juvenile and adult pigs considering donor variables that can affect porcine islet isolation outcome. PMID:27586421

  17. Cell permeable peptide of JNK inhibitor prevents islet apoptosis immediately after isolation and improves islet graft function.

    PubMed

    Noguchi, Hirofumi; Nakai, Yusuke; Matsumoto, Shinichi; Kawaguchi, Miho; Ueda, Michiko; Okitsu, Teru; Iwanaga, Yasuhiro; Yonekawa, Yukihide; Nagata, Hideo; Minami, Kohtaro; Masui, Yumi; Futaki, Shiroh; Tanaka, Koichi

    2005-08-01

    Although application of the Edmonton protocol has markedly improved outcomes for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has proven to remain low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. Pancreas preservation with the two-layer method (TLM) has proven to improve transplant results by protecting isolated islets against apoptosis through the mitochondrial pathway. However, pancreas storage with TLM cannot protect against activation of c-Jun NH2-terminal kinase (JNK) in isolated islets. This study investigated whether delivery of a JNK inhibitory peptide (JNKI) via the protein transduction system can prevent apoptosis of islet cells immediately after isolation. For efficient delivery of the (JNKI into isolated islets, we synthesized JNKI as a C-terminal fusion peptide with the 11-arginine protein transduction domain (11R-JNKI). 11R efficiently delivered the JNKI into isolated islets and 11R-JNKI prevented islet apoptosis immediately after isolation and improved islet graft function. These findings suggest that peptide drugs could be useful for the prevention of the impairment of islet cells and lead to improvement in the outcomes for pancreatic islet transplantation.

  18. Different responses of mouse islets and MIN6 pseudo-islets to metabolic stimulation: a note of caution.

    PubMed

    Schulze, Torben; Morsi, Mai; Brüning, Dennis; Schumacher, Kirstin; Rustenbeck, Ingo

    2016-03-01

    MIN6 cells and MIN6 pseudo-islets are popular surrogates for the use of primary beta cells and islets. Even though it is generally agreed that the stimulus-secretion coupling may deviate from that of beta cells or islets, direct comparisons are rare. The present side-by-side comparison of insulin secretion, cytosolic Ca(2+) concentration ([Ca(2+)] i ) and oxygen consumption rate (OCR) points out where similarities and differences exist between MIN6 cells and normal mouse beta cells. In mouse islets and MIN6 pseudo-islets depolarization by 40 mM KCl was a more robust insulinotropic stimulus than 30 mM glucose. In MIN6 pseudo-islets, but not in mouse islets, the response to 30 mM glucose was much lower than to 40 mM KCl and could be suppressed by a preceding stimulation with 40 mM KCl. In MIN6 pseudo-islets, glucose was less effective to raise [Ca(2+)] i than in primary islets. In marked contrast to islets, the OCR response of MIN6 pseudo-islets to 30 mM glucose was smaller than to 40 mM KCl and was further diminished by a preceding stimulation with 40 mM KCl. The same pattern was observed when MIN6 pseudo-islets were cultured in 5 mM glucose. As with insulin secretion memory effects on the OCR remained after wash-out of a stimulus. The differences between MIN6 cells and primary beta cells were generally larger in the responses to glucose than to depolarization by KCl. Thus, the use of MIN6 cells in investigations on metabolic signalling requires particular caution.

  19. Pancreatic Ductal Perfusion at Organ Procurement Enhances Islet Yield in Human Islet Isolation

    PubMed Central

    Shimoda, Masayuki; Kanak, Mazhar A.; Shahbazov, Rauf; Kunnathodi, Faisal; Lawrence, Michael C.; Naziruddin, Bashoo; Levy, Marlon F.

    2015-01-01

    Objective Pancreas preservation is a major factor influencing the results of islet cell transplantation. This study evaluated the effects of two different solutions for pancreatic ductal perfusion (PDP) at organ procurement. Methods Eighteen human pancreases were assigned to three groups: non-PDP (control), PDP with ET-Kyoto solution, and PDP with cold storage/purification stock solution. Pancreatic islets were isolated according to the modified Ricordi method. Results No significant differences in donor characteristics, including cold ischemia time, were observed between the three groups. All islet isolations in the PDP groups had >400,000 IEQ in total islet yield post-purification, a significant increase when compared with the control (P = 0.04 and <0.01). The islet quality assessments—including an in vivo diabetic nude mice assay and the response of high-mobility group box protein 1 to cytokine stimulation—also showed no significant differences. The proportion of TUNEL-positive cells showing apoptosis in islets in the PDP groups was significantly lower than in the control group (P < 0.05). Conclusion Both ET-Kyoto solution and cold storage/purification stock solution are suitable for PDP and consistently resulted in isolation success. Further studies with a larger number of pancreas donors should be done to compare the effects of the PDP solutions. PMID:25058879

  20. Kyoto islet isolation method: the optimized one for non-heart-beating donors with highly efficient islet retrieval.

    PubMed

    Okitsu, T; Matsumoto, S; Iwanaga, Y; Noguchi, H; Nagata, H; Yonekawa, Y; Maekawa, T; Tanaka, K

    2005-10-01

    The availability of pancreata for clinical cadaveric islet transplantation is restricted to non-heart-beating donors (NHBDs) in Japan. This forced us to modify the current standard islet isolation protocol that was made up for brain-dead donors and make it suitable for NHBDs. The Kyoto islet isolation method is the one with induction of several steps based on the ideas both already reported literally and invented originally by ourselves. Using this islet isolation method, we isolated islets from 13 human pancreata of NHBDs and transplanted 11 preparations to six type-1 diabetic patients. The rate to meet release criteria of Edmonton protocol was 84.6%. Establishment of this method allowed us to begin a clinical islet transplantation program in Japan and to continue to perform the preparation of islets from NHBDs with high rate to meet the release criteria of the Edmonton protocol.

  1. Characterization of the mouse pancreatic islet proteome and comparative analysis with other mouse tissues

    SciTech Connect

    Petyuk, Vladislav A.; Qian, Weijun; Hinault, Charlotte; Gritsenko, Marina A.; Singhal, Mudita; Monroe, Matthew E.; Camp, David G.; Kulkarni, Rohit N.; Smith, Richard D.

    2008-08-01

    The pancreatic islets of Langerhans and insulin-producing beta cells in particular play a central role in the maintenance of glucose homeostasis and the islet dysfunction is associated with the pathogenesis of both type 1 and type 2 diabetes mellitus. To contribute to the understanding of the biology of the pancreatic islets we applied proteomic techniques based on liquid chromatography coupled with mass spectrometry. Here as an initial step we present the first comprehensive proteomic characterization of pancreas islets of the mouse, the commonly used animal model for diabetes research. Two-dimensional SCX LC/RP LC-MS/MS has been applied to characterize of the mouse islet proteome, resulting in the confident identification of 17,350 different tryptic peptides covering 2,612 proteins with at least two unique peptide identifications per protein. The dataset also allowed identification of a number of post-translational modifications including several modifications relevant to oxidative stress and phosphorylation. While many of the identified phosphorylation sites corroborates with previous known sites, the oxidative modifications observed on cysteinyl residues potentially reveal novel information related to the role of oxidation stress in islet functions. Comparative analysis of the islet proteome database with 15 available proteomic datasets from other mouse tissues and cells revealed a set of 68 proteins uniquely detected only in the pancreatic islets. Besides proteins with known functions, like islet secreted peptide hormones, this unique set contains a number of proteins with yet unknown functions. The resulting peptide and protein database will be available at ncrr.pnl.gov web site of the NCRR proteomic center (ncrr.pnl.gov).

  2. Chaotic electrical activity of living β-cells in the mouse pancreatic islet

    NASA Astrophysics Data System (ADS)

    Kanno, Takahiro; Miyano, Takaya; Tokuda, Isao; Galvanovskis, Juris; Wakui, Makoto

    2007-02-01

    To test for chaotic dynamics of the insulin producing β-cell and explore its biological role, we observed the action potentials with the perforated patch clamp technique, for isolated cells as well as for intact cells of the mouse pancreatic islet. The time series obtained were analyzed using nonlinear diagnostic algorithms associated with the surrogate method. The isolated cells exhibited short-term predictability and visible determinism, in the steady state response to 10 mM glucose, while the intact cells did not. In the latter case, determinism became visible after the application of a gap junction inhibitor. This tendency was enhanced by the stimulation with tolbutamide. Our observations suggest that, thanks to the integration of individual chaotic dynamics via gap junction coupling, the β-cells will lose memory of fluctuations occurring at any instant in their electrical activity more rapidly with time. This is likely to contribute to the functional stability of the islet against uncertain perturbations.

  3. In vitro infection of mouse pancreatic islet cells with coxsackie viruses.

    PubMed

    Bopegamage, S A; Petrovicová, A

    1994-10-01

    We have demonstrated the ability of 4 standard coxsackie viruses (B4, B5, A7, and A9) and one fresh isolate (A7) from a newly diabetic child with homologous serological response, to infect in vitro grown mouse pancreatic islet cells. Up to the 9th day after infection the multiplication of viruses in the cells was proved using virus titration and immunofluorescence test. Isolated pancreatic cells proved to be a suitable model for detailed studies of experimental infection of pancreatic cells with coxsackie viruses.

  4. Islet isolation from human pancreas with extended cold ischemia time.

    PubMed

    Kühtreiber, W M; Ho, L T; Kamireddy, A; Yacoub, J A W; Scharp, D W

    2010-01-01

    The general consensus among transplant centers is that a cold ischemia time (CIT) beyond 8 hours results in reduced yields and quality of human islets. We sought to optimize the isolation process and enzymes for pancreata with extended CIT. We processed 16 extended CIT pancreata (13.2 +/- 0.7 hours). Donors averaged 50.8 +/- 2.6 (standard error of the mean) years old with a body mass index of 28.6 +/- 1.5. Glands were shipped in cold organ preservation solution without oxygenated perfluorocarbon. Isolations were performed under a protocol optimized for digestion with the new cGMP collagenase from Roche. Purification used continuous Euroficoll/University of Wisconsin gradients. Islets were cultured in two types of Prodo cGMP islet culture media and/or in Miami 1A media. Glucose-stimulated insulin secretion assays were performed after 8 to 16 days of culture. Prepurification yield averaged 415 +/- 41 KIEQ postpurification, 359 +/- 29 KIEQ (purification loss 13.5%); and postculture 317 +/- 27 KIEQ (culture loss 11.7%). Our process liberated an average of 4278 IEQ/g of pancreas (97 +/- 5 g). Most islets were recovered in the purest fraction (purity 79.7% +/- 1.9%). Culture loss in our enhanced culture media was 11.7%. After 2 to 3 days in culture, viability was 92% +/- 1%. Islets exhibited compactness and dithizone staining. Glucose-stimulated insulin secretion assays performed after 3 to 23 days in our PIM(R) media resulted in a stimulation index of 6.8 +/- 1.7 (G50 to G350). We concluded that our human islet isolation process permitted the recovery of large numbers of high-quality human islets from extended CIT pancreata and that our cGMP islet culture media was superior to the current standard CMRL-based media. PMID:20692399

  5. Impact of donor-related variables on islet isolation outcome in dogs.

    PubMed

    van der Burg, M P; Guicherit, O R; Frölich, M; Scherft, J P; Bruijn, J A; Gooszen, H G

    1994-01-01

    Clinical human islet transplantation programmes are considerably hampered by the variability of islet isolation outcome. The effects of the islet content of the pancreas and other donor-related variables on isolation outcome have not been evaluated systematically so far--either in large animals, or in man. We studied the impact of interindividual differences in age, body weight and pancreatic islet content on the outcome of collagenase isolation of islets from the splenic pancreas of beagle dogs (n = 31). The islet volume of the splenic pancreas amounted to a mean (+/- SEM) 15.7 +/- 0.9 microliters per gramme pancreas, and varied three-fold (from 8.4 to 27.3 microliters). Isolated islet yield was 7.6 +/- 0.7 microliters/g and varied nine-fold (1.8-16.3 microliters). Animals also varied in age eight-fold (8-67 months) and body weight two-fold (8.6-18.3 kg). Differences in body weight and age explained 60% of variance in the fractional islet volume of the pancreas and 50% of the variance in islet yield (p < 0.001). Fractional islet volume of the splenic pancreas also explained 50% of the variance in islet yield (p < 0.001). We conclude that the outcome of islet isolation may be predictable after controlling for the variable islet content of pancreases, and other donor-related variables, and suggest that similar studies should be done in man.

  6. Biosynthesis of glucagon in isolated pancreatic islets of guinea pigs

    PubMed Central

    Hellerström, Claes; Howell, Simon L.; Edwards, John C.; Andersson, Arne; Östenson, Claes-Göran

    1974-01-01

    1. The biosynthesis of glucagon in guinea-pig A2 cells was investigated by incubation of isolated islets of Langerhans in the presence of [3H]tryptophan for periods of up to 14 days. Proteins were extracted from islets and incubation media and analysed by gel filtration. 2. In addition to very-high-molecular-weight (100000) proteins, the principal tryptophan-containing biosynthetic product after incubation for up to 17h was a protein of minimum mol.wt. 9000, which co-eluted on gel filtration with a peak of glucagon-like immunoreactivity, but was apparently devoid of biological activity in a fat-cell assay. A discrete peak of labelled glucagon was only recovered after incubation for at least 6 days. Losses of glucagon during the extraction and rapid secretion of newly synthesized glucagon into incubation media were excluded as reasons for the lack of recovery of labelled hormone from islets after shorter incubations. 3. The 9000-mol.wt. protein was localized to A2 cells in experiments using B-cell-depleted islets, and to A2-cell granules by subcellular fractionation and electron-microscopic radioautography. Only glucagon was secreted into the incubation medium. 4. Possible relationships between the 9000-mol.wt. protein and glucagon are discussed in the light of postulated mechanisms of glucagon biosynthesis. PMID:4615708

  7. A Multicenter Study: North American Islet Donor Score in Donor Pancreas Selection for Human Islet Isolation for Transplantation.

    PubMed

    Wang, Ling-Jia; Kin, Tatsuya; O'Gorman, Doug; Shapiro, A M James; Naziruddin, Bashoo; Takita, Morihito; Levy, Marlon F; Posselt, Andrew M; Szot, Gregory L; Savari, Omid; Barbaro, Barbara; McGarrigle, James; Yeh, Chun Chieh; Oberholzer, Jose; Lei, Ji; Chen, Tao; Lian, Moh; Markmann, James F; Alvarez, Alejandro; Linetsky, Elina; Ricordi, Camillo; Balamurugan, A N; Loganathan, Gopalakrishnan; Wilhelm, Joshua J; Hering, Bernhard J; Bottino, Rita; Trucco, Massimo; Liu, Chengyang; Min, Zaw; Li, Yanjing; Naji, Ali; Fernandez, Luis A; Ziemelis, Martynas; Danobeitia, Juan S; Millis, J Michael; Witkowski, Piotr

    2016-01-01

    Selection of an optimal donor pancreas is the first key task for successful islet isolation. We conducted a retrospective multicenter study in 11 centers in North America to develop an islet donor scoring system using donor variables. The data set consisting of 1,056 deceased donors was used for development of a scoring system to predict islet isolation success (defined as postpurification islet yield >400,000 islet equivalents). With the aid of univariate logistic regression analyses, we developed the North American Islet Donor Score (NAIDS) ranging from 0 to 100 points. The c index in the development cohort was 0.73 (95% confidence interval 0.70-0.76). The success rate increased proportionally as the NAIDS increased, from 6.8% success in the NAIDS < 50 points to 53.7% success in the NAIDS ≥ 80 points. We further validated the NAIDS using a separate set of data consisting of 179 islet isolations. A comparable outcome of the NAIDS was observed in the validation cohort. The NAIDS may be a useful tool for donor pancreas selection in clinical practice. Apart from its utility in clinical decision making, the NAIDS may also be used in a research setting as a standardized measurement of pancreas quality. PMID:26922947

  8. 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.

    PubMed

    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 < 0.002). A retrospective histological analysis of almost all donor pancreases showed that the majority of 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 < 0.02]. In addition, isolation results were strongly influenced by the quality of the LiberasePI batch, and therefore single batch testing is invariably required. Purification was performed using Ficoll or OptiPrep density gradient centrifugation manually or in the COBE cell processor. Although islet purity was highest when OptiPrep was used, final islet yields did not differ between the different purification methods. Our study demonstrates that islet size in situ is an extremely critical parameter for highly successful islet isolation; consequently, we are now performing a morphological screening of each donor organ prior to the

  9. Glucose-Stimulated Calcium Dynamics in Islets of Langerhans in Acute Mouse Pancreas Tissue Slices

    PubMed Central

    Stožer, Andraž; Dolenšek, Jurij; Rupnik, Marjan Slak

    2013-01-01

    In endocrine cells within islets of Langerhans calcium ions couple cell stimulation to hormone secretion. Since the advent of modern fluorimetry, numerous in vitro studies employing primarily isolated mouse islets have investigated the effects of various secretagogues on cytoplasmic calcium, predominantly in insulin-secreting beta cells. Due to technical limitations, insights of these studies are inherently limited to a rather small subpopulation of outermost cells. The results also seem to depend on various factors, like culture conditions and duration, and are not always easily reconcilable with findings in vivo. The main controversies regard the types of calcium oscillations, presence of calcium waves, and the level of synchronized activity. Here, we set out to combine the in situ acute mouse pancreas tissue slice preparation with noninvasive fluorescent calcium labeling and subsequent confocal laser scanning microscopy to shed new light on the existing controversies utilizing an innovative approach enabling the characterization of responses in many cells from all layers of islets. Our experiments reproducibly showed stable fast calcium oscillations on a sustained plateau rather than slow oscillations as the predominant type of response in acute tissue slices, and that calcium waves are the mechanistic substrate for synchronization of oscillations. We also found indirect evidence that even a large amplitude calcium signal was not sufficient and that metabolic activation was necessary to ensure cell synchronization upon stimulation with glucose. Our novel method helped resolve existing controversies and showed the potential to help answer important physiological questions, making it one of the methods of choice for the foreseeable future. PMID:23358454

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

    PubMed

    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.

  11. Islet distribution of Peptide YY and its regulatory role in primary mouse islets and immortalised rodent and human beta-cell function and survival.

    PubMed

    Khan, Dawood; Vasu, Srividya; Moffett, R Charlotte; Irwin, Nigel; Flatt, Peter R

    2016-11-15

    Recent evidence suggests that the classic gut peptide, Peptide YY (PYY), could play a fundamental role in endocrine pancreatic function. In the present study expression of PYY and its NPY receptors on mouse islets and immortalised rodent and human beta-cells was examined together with the effects of both major circulating forms of PYY, namely PYY(1-36) and PYY(3-36), on beta-cell function, murine islet adaptions to insulin deficiency/resistance, as well as direct effects on cultured beta-cell proliferation and apoptosis. In vivo administration of PYY(3-36), but not PYY(1-36), markedly (p < 0.05) decreased food intake in overnight fasted mice. Neither form of PYY affected glucose disposal or insulin secretion following an i.p. glucose challenge. However, in vitro, PYY(1-36) and PYY(3-36) inhibited (p < 0.05 to p < 0.001) glucose, alanine and GLP-1 stimulated insulin secretion from immortalised rodent and human beta-cells, as well as isolated mouse islets, by impeding alterations in membrane potential, [Ca(2+)]i and elevations of cAMP. Mice treated with multiple low dose streptozotocin presented with severe (p < 0.01) loss of beta-cell mass accompanied by notable increases (p < 0.001) in alpha and PP cell numbers. In contrast, hydrocortisone-induced insulin resistance increased islet number (p < 0.01) and beta-cell mass (p < 0.001). PYY expression was consistently observed in alpha-, PP- and delta-, but not beta-cells. Streptozotocin decreased islet PYY co-localisation with PP (p < 0.05) and somatostatin (p < 0.001), whilst hydrocortisone increased PYY co-localisation with glucagon (p < 0.05) in mice. More detailed in vitro investigations revealed that both forms of PYY augmented (p < 0.05 to p < 0.01) immortalised human and rodent beta-cell proliferation and protected against streptozotocin-induced cytotoxicity, to a similar or superior extent as the well characterised beta-cell proliferative and anti-apoptotic agent GLP-1. Taken together

  12. Comparison of surface modification chemistries in mouse, porcine, and human islets.

    PubMed

    SoRelle, Jeffrey A; Kanak, Mazhar A; Itoh, Takeshi; Horton, Joshua M; Naziruddin, Bashoo; Kane, Robert R

    2015-03-01

    Beta cell replacement therapy, the transplantation of isolated pancreatic islets by intraportal infusion, offers patients with brittle type 1 diabetes blood glucose regulation with a minimally invasive technique. Chemical modification of islets prior to transplantation, providing a nanothin barrier that potentially includes active protective compounds, has been proposed as a strategy to minimize the inflammatory and immune reactions that often significantly limit graft function and duration. Chemical modification also has the potential to allow the use of alternative sources of islets, such as porcine islets, for transplantation. This investigation compared three orthogonal covalent islet modification techniques across three species (human, porcine, and murine), using multiple measures to determine biocompatibility and effectiveness. All three conjugation chemistries were well tolerated, and the overall efficiency, gross uniformity, and stability of the surface modifications were dependent upon the conjugation chemistry as well as the islet source (human, porcine, or murine). Notably, the reductive modification of surface disulfides was shown to afford intense and long-lasting modification of human islets. This study demonstrates that murine, human, and porcine islets tolerate a variety of covalent modifications, that these modifications are relatively stable, and that the murine islet model may not be predictive for some chemical contexts. PMID:24829144

  13. Mouse and human islets survive and function after coating by biosilicification

    PubMed Central

    Jaroch, David B.; Lu, Jing; Madangopal, Rajtarun; Stull, Natalie D.; Stensberg, Matthew; Shi, Jin; Kahn, Jennifer L.; Herrera-Perez, Ruth; Zeitchek, Michael; Sturgis, Jennifer; Robinson, J. Paul; Yoder, Mervin C.; Porterfield, D. Marshall; Mirmira, Raghavendra G.

    2013-01-01

    Inorganic materials have properties that can be advantageous in bioencapsulation for cell transplantation. Our aim was to engineer a hybrid inorganic/soft tissue construct by inducing pancreatic islets to grow an inorganic shell. We created pancreatic islets surrounded by porous silica, which has potential application in the immunoprotection of islets in transplantation therapies for type 1 diabetes. The new method takes advantage of the islet capsule surface as a template for silica formation. Mouse and human islets were exposed to medium containing saturating silicic acid levels for 9–15 min. The resulting tissue constructs were then cultured for up to 4 wk under normal conditions. Scanning electron microscopy and energy dispersive X-ray spectroscopy was used to monitor the morphology and elemental composition of the material at the islet surface. A cytokine assay was used to assess biocompatibility with macrophages. Islet survival and function were assessed by confocal microscopy, glucose-stimulated insulin release assays, oxygen flux at the islet surface, expression of key genes by RT-PCR, and syngeneic transplant into diabetic mice. PMID:24002572

  14. ISOLATION OF MOUSE NEUTROPHILS

    PubMed Central

    Swamydas, Muthulekha; Luo, Yi; Dorf, Martin E.; Lionakis, Michail S.

    2015-01-01

    Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT 19.6. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments. PMID:26237011

  15. Impact of adverse pancreatic injury at surgical procurement upon islet isolation outcome.

    PubMed

    Andres, Axel; Kin, Tatsuya; O'Gorman, Doug; Bigam, David; Kneteman, Norman; Senior, Peter; Shapiro, Am James

    2014-11-01

    The consequence of a pancreas injury during the procurement for islet isolation purpose is unknown. The goal of this work was to assess the injuries of the pancreata procured for islet isolation, and to determine their effect on the islet yield. Between January 2007 and October 2013, we prospectively documented every injury of the pancreata processed in our centre for islet isolation. Injuries involving the main duct were classified as major, the others as minor. Donors' characteristics and islet yields were compared between the groups of injuries. A pancreas injury was identified in 42 of 452 pancreata received for islet isolation (9.3%). In 15 cases, the injury was major (3.3% of all pancreata). Although a minor injury did not affect the islet yield, a major injury was significantly associated with unfavourable outcomes (postpurification mean islet equivalent of 364 ± 181, 405 ± 190 and 230 ± 115 × 10(3) for absence of injury, minor injury and major injury, respectively). A major injury was significantly more prevalent in lean and short donors. We recommend assessing the quality of the pancreas in the islet isolation centre before starting the isolation procedure. Each centre should determine its own policy based on its financial resources and on the wait list.

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

    PubMed

    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.

  17. Novel Stable Isotope Analyses Demonstrate Significant Rates of Glucose Cycling in Mouse Pancreatic Islets

    PubMed Central

    Pound, Lynley D.; Trenary, Irina; O’Brien, Richard M.

    2015-01-01

    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. PMID:25552595

  18. Enhanced function of immuno-isolated islets in diabetes therapy by co-encapsulation with an anti-inflammatory drug

    PubMed Central

    Dang, Tram T.; Thai, Anh V.; Cohen, Joshua; Slosberg, Jeremy E.; Siniakowicz, Karolina; Doloff, Joshua C.; Ma, Minglin; Hollister-Lock, Jennifer; Tang, Katherine; Gu, Zhen; Cheng, Hao; Weir, Gordon C.; Langer, Robert; Anderson, Daniel G.

    2013-01-01

    Immuno-isolation of islets has the potential to enable the replacement of pancreatic function in diabetic patients. However, host response to the encapsulated islets frequently leads to fibrotic overgrowth with subsequent impairment of the transplanted grafts. Here, we identified and incorporated anti-inflammatory agents into islet-containing microcapsules to address this challenge. In vivo subcutaneous screening of 16 small molecule anti-inflammatory drugs was performed to identify promising compounds that could minimize the formation of fibrotic cell layers. Using parallel non-invasive fluorescent and bioluminescent imaging, we identified dexamethasone and curcumin as the most effective drugs in inhibiting the activities of inflammatory proteases and reactive oxygen species in the host response to subcutaneously injected biomaterials. Next, we demonstrated that co-encapsulating curcumin with pancreatic rat islets in alginate microcapsules reduced fibrotic overgrowth and improved glycemic control in a mouse model of chemically-induced type I diabetes. These results showed that localized administration of anti-inflammatory drug can improve the longevity of encapsulated islets and may facilitate the translation of this technology towards a long-term cure for type I diabetes. PMID:23660251

  19. The Different Faces of the Pancreatic Islet.

    PubMed

    Abdulreda, Midhat H; Rodriguez-Diaz, Rayner; Cabrera, Over; Caicedo, Alejandro; Berggren, Per-Olof

    2016-01-01

    Type 1 diabetes (T1D) patients who receive pancreatic islet transplant experience significant improvement in their quality-of-life. This comes primarily through improved control of blood sugar levels, restored awareness of hypoglycemia, and prevention of serious and potentially life-threatening diabetes-associated complications, such as kidney failure, heart and vascular disease, stroke, nerve damage, and blindness. Therefore, beta cell replacement through transplantation of isolated islets is an important option in the treatment of T1D. However, lasting success of this promising therapy depends on durable survival and efficacy of the transplanted islets, which are directly influenced by the islet isolation procedures. Thus, isolating pancreatic islets with consistent and reliable quality is critical in the clinical application of islet transplantation.Quality of isolated islets is important in pre-clinical studies as well, as efforts to advance and improve clinical outcomes of islet transplant therapy have relied heavily on animal models ranging from rodents, to pigs, to nonhuman primates. As a result, pancreatic islets have been isolated from these and other species and used in a variety of in vitro or in vivo applications for this and other research purposes. Protocols for islet isolation have been somewhat similar across species, especially, in mammals. However, given the increasing evidence about the distinct structural and functional features of human and mouse islets, using similar methods of islet isolation may contribute to inconsistencies in the islet quality, immunogenicity, and experimental outcomes. This may also contribute to the discrepancies commonly observed between pre-clinical findings and clinical outcomes. Therefore, it is prudent to consider the particular features of pancreatic islets from different species when optimizing islet isolation protocols.In this chapter, we explore the structural and functional features of pancreatic islets from

  20. Anti-caspase-3 preconditioning increases proinsulin secretion and deteriorates posttransplant function of isolated human islets.

    PubMed

    Brandhorst, Daniel; Brandhorst, Heide; Maataoui, Vidya; Maataoui, Adel; Johnson, Paul R V

    2013-06-01

    Human islet isolation is associated with adverse conditions inducing apoptosis and necrosis. The aim of the present study was to assess whether antiapoptotic preconditioning can improve in vitro and posttransplant function of isolated human islets. A dose-finding study demonstrated that 200 μmol/L of the caspase-3 inhibitor Ac-DEVD-CMK was most efficient to reduce the expression of activated caspase-3 in isolated human islets exposed to severe heat shock. Ac-DEVD-CMK-pretreated or sham-treated islets were transplanted into immunocompetent or immunodeficient diabetic mice and subjected to static glucose incubation to measure insulin and proinsulin secretion. Antiapoptotic pretreatment significantly deteriorated graft function resulting in elevated nonfasting serum glucose when compared to sham-treated islets transplanted into diabetic nude mice (p < 0.01) and into immunocompetent mice (p < 0.05). Ac-DEVD-CMK pretreatment did not significantly change basal and glucose-stimulated insulin release compared to sham-treated human islets but increased the proinsulin release at high glucose concentrations (20 mM) thus reducing the insulin-to-proinsulin ratio in preconditioned islets (p < 0.05). This study demonstrates that the caspase-3 inhibitor Ac-DEVD-CMK interferes with proinsulin conversion in preconditioned islets reducing their potency to cure diabetic mice. The mechanism behind this phenomenon is unclear so far but may be related to the ketone CMK linked to the Ac-DEVD molecule. Further studies are required to identify biocompatible caspase inhibitors suitable for islet preconditioning.

  1. Tissue dissociation enzymes for isolating human islets for transplantation: factors to consider in setting enzyme acceptance criteria.

    PubMed

    McCarthy, Robert C; Breite, Andrew G; Green, Michael L; Dwulet, Francis E

    2011-01-27

    Tissue dissociation enzymes are critical reagents that affect the yield and quality of human pancreatic islets required for islet transplantation. The United States Food and Drug Administration's oversight of this procedure recommends laboratories to set acceptance criteria for enzymes used in the manufacture of islet products for transplantation. Currently, many laboratories base this selection on personal experience because biochemical analysis is not predictive of success of the islet isolation procedure. This review identifies the challenges of correlating results from enzyme biochemical analysis to their effectiveness in human islet isolation and suggests a path forward to address these challenges to improve control of the islet manufacturing process.

  2. Comparison of Neutral Proteases and Collagenase Class I as Essential Enzymes for Human Islet Isolation

    PubMed Central

    Brandhorst, Heide; Kurfürst, Manfred; Johnson, Paul R.; Korsgren, Olle; Brandhorst, Daniel

    2016-01-01

    Background Efficient islet isolation requires synergistic interaction between collagenase class I (CI) and class II (CII). The CI degradation alters the ratio between CI and CII and is responsible for batch-to-batch variations. This study compares the role of neutral protease (NP) plus clostripain (CP) with CI as essential enzymes for human islet isolation. Methods Human islets were isolated using 4 different enzyme mixtures composed of CII plus either intact (CI-115) or degraded CI (CI-100). Blends were administered either with or without NP/CP. Purified islets were cultured for 3 to 4 days before islet quality assessment. Results Whereas using intact CI-115 without NP/CP did not significantly reduce islet yield (3429 ± 631 vs 3087 ± 970 islet equivalent/g, nonsignificant), administration of degraded CI-100 without NP/CP decreased islet yield from 3501 ± 580 to 1312 ± 244 islet equivalent/g (P < 0.01), doubled the amount of undigested tissue from 11.8 ± 1.6 to 24.4 ± 1.2% (P < 0.01) and triplicated the percentage of trapped islets from 7.7 ± 2.8 to 22.5 ± 3.6% (P < 0.05). Islet yield did not vary between supplemented CI-115 and CI-100, but was increased using CI-115 when NP/CP was omitted (P < 0.05). A trend toward higher viability and increased secretory insulin response was noted in both CI-100 and CI-115 when NP/CP was not added. Conclusions This study suggests that NP/CP can compensate reduced CI activity. Future attempts to optimize enzyme blends should consider the possibility to increase the proportion of collagenase CI to reduce the need for potentially harmful NPs. PMID:27500241

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

    PubMed

    Li, Feng-Fei; Chen, Bi-Jun; Li, Wei; Li, Ling; Zha, Min; Zhou, S; Bachem, M G; Sun, Zi-Lin

    2016-01-01

    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.

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

    PubMed Central

    Li, Feng-Fei; Chen, Bi-Jun; Li, Wei; Li, Ling; Zha, Min; Zhou, S.; Bachem, M. G.; Sun, Zi-Lin

    2016-01-01

    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. PMID:26697502

  5. Characterization of pancreatic islets in two selectively bred mouse lines with different susceptibilities to high-fat diet-induced glucose intolerance.

    PubMed

    Nagao, Mototsugu; Asai, Akira; Inaba, Wataru; Kawahara, Momoyo; Shuto, Yuki; Kobayashi, Shunsuke; Sanoyama, Daisuke; Sugihara, Hitoshi; Yagihashi, Soroku; Oikawa, Shinichi

    2014-01-01

    Hereditary predisposition to diet-induced type 2 diabetes has not yet been fully elucidated. We recently established 2 mouse lines with different susceptibilities (resistant and prone) to high-fat diet (HFD)-induced glucose intolerance by selective breeding (designated selectively bred diet-induced glucose intolerance-resistant [SDG-R] and -prone [SDG-P], respectively). To investigate the predisposition to HFD-induced glucose intolerance in pancreatic islets, we examined the islet morphological features and functions in these novel mouse lines. Male SDG-P and SDG-R mice were fed a HFD for 5 weeks. Before and after HFD feeding, glucose tolerance was evaluated by oral glucose tolerance test (OGTT). Morphometry and functional analyses of the pancreatic islets were also performed before and after the feeding period. Before HFD feeding, SDG-P mice showed modestly higher postchallenge blood glucose levels and lower insulin increments in OGTT than SDG-R mice. Although SDG-P mice showed greater β cell proliferation than SDG-R mice under HFD feeding, SDG-P mice developed overt glucose intolerance, whereas SDG-R mice maintained normal glucose tolerance. Regardless of whether it was before or after HFD feeding, the isolated islets from SDG-P mice showed impaired glucose- and KCl-stimulated insulin secretion relative to those from SDG-R mice; accordingly, the expression levels of the insulin secretion-related genes in SDG-P islets were significantly lower than those in SDG-R islets. These findings suggest that the innate predispositions in pancreatic islets may determine the susceptibility to diet-induced diabetes. SDG-R and SDG-P mice may therefore be useful polygenic animal models to study the gene-environment interactions in the development of type 2 diabetes.

  6. Combining Donor Characteristics with Immunohistological Data Improves the Prediction of Islet Isolation Success

    PubMed Central

    Girman, Peter; Zacharovova, Klara; Kriz, Jan; Fabryova, Eva; Leontovyc, Ivan; Koblas, Tomas; Kosinova, Lucie; Neskudla, Tomas; Vavrova, Ema; Habart, David; Loukotova, Sarka; Zahradnicka, Martina; Lipar, Kvetoslav; Voska, Ludek; Skibova, Jelena

    2016-01-01

    Variability of pancreatic donors may significantly impact the success of islet isolation. The aim of this study was to evaluate donor factors associated with isolation failure and to investigate whether immunohistology could contribute to organ selection. Donor characteristics were evaluated for both successful (n = 61) and failed (n = 98) islet isolations. Samples of donor pancreatic tissue (n = 78) were taken for immunohistochemical examination. Islet isolations with 250000 islet equivalents were considered successful. We confirmed that BMI of less than 25 kg/m2 (P < 0.001), cold ischemia time more than 8 hours (P < 0.01), hospitalization longer than 96 hours (P < 0.05), higher catecholamine doses (P < 0.05), and edematous pancreases (P < 0.01) all unfavorably affected isolation outcome. Subsequent immunohistochemical examination of donor pancreases confirmed significant differences in insulin-positive areas (P < 0.001). ROC analyses then established that the insulin-positive area in the pancreas could be used to predict the likely success of islet isolation (P < 0.001). At the optimal cutoff point (>1.02%), sensitivity and specificity were 89% and 76%, respectively. To conclude, while the insulin-positive area, determined preislet isolation, as a single variable, is sufficient to predict isolation outcome and helps to improve the success of this procedure, its combination with the established donor scoring system might further improve organ selection. PMID:27803935

  7. Ca2+ controls slow NAD(P)H oscillations in glucose-stimulated mouse pancreatic islets

    PubMed Central

    Luciani, Dan S; Misler, Stanley; Polonsky, Kenneth S

    2006-01-01

    Exposure of pancreatic islets of Langerhans to physiological concentrations of glucose leads to secretion of insulin in an oscillatory pattern. The oscillations in insulin secretion are associated with oscillations in cytosolic Ca2+ concentration ([Ca2+]c). Evidence suggests that the oscillations in [Ca2+]c and secretion are driven by oscillations in metabolism, but it is unclear whether metabolic oscillations are intrinsic to metabolism or require Ca2+ feedback. To address this question we explored the interaction of Ca2+ concentration and islet metabolism using simultaneous recordings of NAD(P)H autofluorescence and [Ca2+]c, in parallel with measurements of mitochondrial membrane potential (ΔΨm). All three parameters responded to 10 mm glucose with multiphasic dynamics culminating in slow oscillations with a period of ∼5 min. This was observed in ∼90% of islets examined from various mouse strains. NAD(P)H oscillations preceded those of [Ca2+]c, but their upstroke was often accelerated during the increase in [Ca2+]c, and Ca2+ influx was a prerequisite for their generation. Prolonged elevations of [Ca2+]c augmented NAD(P)H autofluorescence of islets in the presence of 3 mm glucose, but often lowered NAD(P)H autofluorescence of islets exposed to 10 mm glucose. Comparable rises in [Ca2+]c depolarized ΔΨm. The NAD(P)H lowering effect of an elevation of [Ca2+]c was reversed during inhibition of mitochondrial electron transport. These findings reveal the existence of slow oscillations in NAD(P)H autofluorescence in intact pancreatic islets, and suggest that they are shaped by Ca2+ concentration in a dynamic balance between activation of NADH-generating mitochondrial dehydrogenases and a Ca2+-induced decrease in NADH. We propose that a component of the latter reflects mitochondrial depolarization by Ca2+, which reduces respiratory control and consequently accelerates oxidation of NADH. PMID:16455690

  8. Efflux of 86Rb from rat and mouse pancreatic islets: the role of membrane depolarization.

    PubMed Central

    Matthews, E. K.; Shotton, P. A.

    1984-01-01

    The efflux of 86Rb from rat or mouse perifused islets preloaded with the isotope has been used as an index of the potassium permeability of the islet beta-cell membrane. Cellular transmembrane potentials were altered by changing [K]O or by direct electrical stimulation and the effects on potassium permeability examined. Omission of KCl from the medium perifusing rat islets induced a biphasic change in 86Rb efflux, a brief decline being superseded by a pronounced increase in efflux. Re-introduction of KCl, 4.7 mM, caused a further increase in 86Rb efflux preceding a return to control values. Increasing [K]O from 4.7 mM to 10 mM, 20 mM or 47 mM caused a phasic increase in 86Rb efflux with the magnitude of both the peak and average rate of efflux being dependent upon the extent of the change in [K]O. The increase in 86Rb efflux produced by [K]O, 47 mM, was attenuated by Co2+, 2.56 mM (51% inhibition) or quinine, 10 microM (47% inhibition), but efflux remained significantly (P less than 0.001) above control values. Electrical stimulation of single microdissected mouse pancreatic islets by currents of 0.1 to 0.5 mA evoked a rapid, phasic increase in 86Rb efflux. The magnitude of the response was unaffected by EGTA, 2 mM, or nupercaine, 100 microM. These observations are discussed in relation to the mechanisms controlling the potassium permeability, membrane potential and insulin secretion of the pancreatic islet beta-cell.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6391599

  9. Use of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells

    PubMed Central

    Xin, Yurong; Kim, Jinrang; Ni, Min; Wei, Yi; Okamoto, Haruka; Lee, Joseph; Adler, Christina; Cavino, Katie; Murphy, Andrew J.; Yancopoulos, George D.; Lin, Hsin Chieh; Gromada, Jesper

    2016-01-01

    This study provides an assessment of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells. The system combines microfluidic technology and nanoliter-scale reactions. We sequenced 622 cells, allowing identification of 341 islet cells with high-quality gene expression profiles. The cells clustered into populations of α-cells (5%), β-cells (92%), δ-cells (1%), and pancreatic polypeptide cells (2%). We identified cell-type–specific transcription factors and pathways primarily involved in nutrient sensing and oxidation and cell signaling. Unexpectedly, 281 cells had to be removed from the analysis due to low viability, low sequencing quality, or contamination resulting in the detection of more than one islet hormone. Collectively, we provide a resource for identification of high-quality gene expression datasets to help expand insights into genes and pathways characterizing islet cell types. We reveal limitations in the C1 Fluidigm cell capture process resulting in contaminated cells with altered gene expression patterns. This calls for caution when interpreting single-cell transcriptomics data using the C1 Fluidigm system. PMID:26951663

  10. Antiapoptotic effects of cerium oxide and yttrium oxide nanoparticles in isolated rat pancreatic islets.

    PubMed

    Hosseini, A; Baeeri, M; Rahimifard, M; Navaei-Nigjeh, M; Mohammadirad, A; Pourkhalili, N; Hassani, S; Kamali, M; Abdollahi, M

    2013-05-01

    Type I diabetes mellitus is a metabolic disease caused by the impairment of pancreatic β-cells mainly mediated through oxidative stress and related apoptosis. Islets transplantation seems a promising treatment for these patients, but during islets transplant, various types of stresses related to the isolation and transplantation procedure compromise the function and viability of islets. We recently hypothesized that the combination of cerium oxide (CeO2) and yttrium oxide (Y2O3) nanoparticles with a potential free radical scavenger behavior should be useful to make isolated islets survive until transplanted. In the present study, oxidative stress-induced apoptosis in isolated rat pancreatic islets exposed to hydrogen peroxide (H2O2) and the protective effects of CeO2 and Y2O3 nanoparticles were investigated. Exposure of islets to H2O2 (50 µm, 2 h) increased intracellular oxidant formation such as reactive oxygen species and subsequently apoptosis and decreased viability, glucose-induced adenosine triphosphate (ATP) production and glucose-stimulated insulin secretion. Pretreatment with CeO2 and/or Y2O3 nanoparticles reduced the oxidant formation and apoptosis and increased viability, glucose-induced ATP production and glucose-stimulated insulin secretion. These results suggest that this combination may protect β-cell apoptosis by improving the oxidative stress-mediated apoptotic pathway.

  11. [Treatment of type 1 diabetics by transplantation of isolated pancreatic islets].

    PubMed

    Ono, Junko

    2006-04-01

    Type 1 diabetes is an autoimmune disease with selective destruction of insulin-producing pancreatic beta cells. Since insulin plays pivotal roles in energy homeostasis by transferring glucose into cells, type 1 diabetic patients can not survive without insulin replacement. Insulin secretion is precisely controlled by ingested glucose as well as hormones and neural factors, therefore it is impossible to reproduce the physiological secretory pattern of insulin via exogenous insulin, even by multiple or continuous delivery by injection. Transplantation of beta cells has long been expected as the fundamental treatment to cure type 1 diabetics, and transplantation of the whole pancreas, both exocrine pancreas and islets, has been applied with success, resulting insulin independence. However, the exocrine pancreas, which releases amylase and trypsin to the digestive tract, is not indispensable for insulin replacement, so the interest in islet transplantation has increased enormously. In the past 20 years, the techniques for isolating large numbers of human islets have been advanced and more potent immunosuppressive agents have also been introduced, permitting newer attempts at islet transplantation. In 2000, insulin independence was first achieved in Canada using the Edmonton protocol. The success rates have increased gradually using this protocol, and 5 institutes in Japan have started to prepare human islet transplantation under the control of the Japan Pancreas and Islet Transplant Society. In 2004, insulin independence by islet transplantation was first achieved at Kyoto University Hospital and the number of islet transplantations has increased, though very slowly. By the end of 2005, approximately 100 patients were on the waiting list for islet transplantation in Japan. Many problems remain unsolved in islet transplantation to meet clinical practice: these are the shortage of insulin-producing cells, further progress in immunosuppressive agents that do not interfere

  12. Validation of Islet Transport From a Geographically Distant Isolation Center Enabling Equitable Access and National Health Service Funding of a Clinical Islet Transplant Program for England.

    PubMed

    Aldibbiat, Ali; Huang, Guo Cai; Zhao, Min; Holliman, Graham N; Ferguson, Linda; Hughes, Stephen; Brigham, Ken; Wardle, Julie; Williams, Rob; Dickinson, Anne; White, Steven A; Johnson, Paul R V; Manas, Derek; Amiel, Stephanie A; Shaw, James A M

    2012-01-01

    Islet transplantation has become established as a successful treatment for type 1 diabetes complicated by recurrent severe hypoglycemia. In the UK access has been limited to a few centrally located units. Our goal was to validate a quality-assured system for safe/effective transport of human islets in the UK and to successfully undertake the first transplants with transported islets. Pancreases were retrieved from deceased donors in the north of England and transported to King's College London using two-layer method (TLM) or University of Wisconsin solution alone. Islets were isolated and transported back to Newcastle in standard blood transfusion or gas-permeable bags with detailed evaluation pre- and posttransport. In the preclinical phase, islets were isolated from 10 pancreases with mean yield of 258,000 islet equivalents. No significant differences were seen between TLM and University of Wisconsin solution organ preservation. A significant loss of integrity was demonstrated in islets shipped in gas-permeable bags, whereas sterility, number, purity, and viability were maintained in blood transfusion bags. Maintenance of secretory granules and glucose-stimulated insulin secretion was confirmed following transport. A Standard Operating Procedure enabling final pretransplant quality control from a simple side-arm sample was validated. Moreover, levels of insulin and cytokines in transport medium were low, enabling transplant without centrifugation/resuspension at the recipient site. Six clinical transplants of transported islets were undertaken in five recipients with 100% primary graft function and resolution of severe hypoglycemia. Safe and clinically effective islet transport has been established facilitating sustainable NHS funding of a clinical islet transplant program for the UK.

  13. The Dominance of Pilus Islet 1 in Pneumococcal Isolates Collected From Patients and Healthy Individuals

    PubMed Central

    Khodaei, Farzaneh; Ahmadi, Ali; Sayahfar, Shirin; Irajian, Gholamreza; Talebi, Malihe

    2016-01-01

    Background Pili in Streptococcus pneumoniae have been shown to be one of the adherence factors for epithelial cells in the human upper respiratory tract. Two types of pilus-like structures (pilus islet-1 and pilus islet-2) have been distinguished in S. pneumoniae. Objectives To investigate the presence of pilus islet-1 (PI-1) in S. pneumoniae and the correlation between our isolates. Materials and Methods In this study, 162 S. pneumoniae isolates were collected from clinical specimens, and normal flora were also examined for the distribution of PI-1 using the presence of the rlrA and rrgC genes as markers for this islet and sipA as an indicator of pilus islet-2 (PI-2). BOX-PCR analyses were performed to determine the genetic relationship between isolates. Results The results confirmed the presence of rlrA and rrgC genes in both clinical (n = 39) and normal flora (n = 26) isolates. The minimal inhibitory concentration results revealed that the rate of resistance of these isolates to the three antibiotics tested ranged from 26% for penicillin to 46% for erythromycin and tetracycline. Furthermore, 12% of the isolates were resistant to all three antibiotics. Strain typing using repetitive element BOX-PCR analysis among the 65 isolates identified 8 different band patterns. Conclusions Our results indicated that the dissemination of PI-1 was widespread in S. pneumoniae isolates, although no PI-2 isolates were detected. Furthermore, the frequency of rlrA and rrgC of clinical isolates was significantly more than that of normal flora isolates. PMID:27540452

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

    PubMed

    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.

  15. 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.

    PubMed

    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

  16. Porcine islet isolation: prospective comparison of automated and manual methods of pancreatic collagenase digestion.

    PubMed

    Toomey, P; Chadwick, D R; Contractor, H; Bell, P R; James, R F; London, N J

    1993-02-01

    A prospective study was undertaken to compare an automated method of porcine pancreatic digestion with a simpler manual procedure. These techniques have not previously been compared directly. After intraductal distension with collagenase, seven porcine pancreata were divided longitudinally; half of each was digested by the automated method and half by the manual technique. Islet yield and purity were measured. Compared with the manual technique, the automated method isolated a significantly greater total volume of islet tissue (median (range) 3.56 (1.39-5.30) versus 1.07 (0.46-1.92) mm3/g, P = 0.022), increased the median (range) number of 105-microns islet equivalents isolated (5875 (2294-8746) versus 1766 (759-3168) per g, P = 0.022) and improved the islet cleavage index (median (range) 92 (89-99) versus 82 (78-92) per cent, P = 0.035). It is concluded that, although the automated method is more complicated to set up, it greatly improves the yield of intact islets from the porcine pancreas. PMID:8443669

  17. Three-dimensional culture of mouse pancreatic islet on a liver-derived perfusion-decellularized bioscaffold for potential clinical application.

    PubMed

    Xu, Tianxin; Zhu, Mingyan; Guo, Yibing; Wu, Di; Huang, Yan; Fan, Xiangjun; Zhu, Shajun; Lin, Changchun; Li, Xiaohong; Lu, Jingjing; Zhu, Hui; Zhou, Pengcheng; Lu, Yuhua; Wang, Zhiwei

    2015-10-01

    The cutting-edge technology of three-dimensional liver decellularized bioscaffold has a potential to provide a microenvironment that is suitable for the resident cells and even develop a new functional organ. Liver decellularized bioscaffold preserved the native extracellular matrix and three-dimensional architecture in support of the cell culture. The goal of this study was to discover if three-dimensional extracellular matrix derived from mouse liver could facilitate the growth and maintenance of physiological functions of mouse isolated islets. We generated a whole organ liver decellularized bioscaffold which could successfully preserve extracellular matrix proteins and the native vascular channels using 1% Triton X-100/0.1% ammonium protocol. To evaluate the potential of decellularized liver as a scaffold for islets transplantation, the liver decellularized bioscaffold was infused with mouse primary pancreatic islets which were obtained through Collagenase P digestion protocol. Its yield, morphology, and quality were estimated by microscopic analysis, dithizone staining, insulin immunofluorescence and glucose stimulation experiments. Comparing the three-dimensional culture in liver decellularized bioscaffold with the orthodoxy two-dimensional plate culture, hematoxylin-eosin staining, immunohistochemistry, and insulin gene expression were tested. Our results demonstrated that the liver decellularized bioscaffold could support cellular culture and maintenance of cell functions. In contrast with the conventional two-dimensional culture, three-dimensional culture system could give rise to an up-regulated insulin gene expression. These findings demonstrated that the liver bioscaffold by a perfusion-decellularized technique could serve as a platform to support the survival and function of the pancreatic islets in vitro. Meanwhile three-dimensional culture system had a superior role in contrast with the two-dimensional culture. This study advanced the field of

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

    NASA Astrophysics Data System (ADS)

    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

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

    PubMed Central

    Shima, Hiroki; Inagaki, Akiko; Imura, Takehiro; Yamagata, Youhei; Watanabe, Kimiko; Igarashi, Kazuhiko; Goto, Masafumi; Murayama, Kazutaka

    2016-01-01

    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. PMID:27195301

  20. Assessment of benzene induced oxidative impairment in rat isolated pancreatic islets and effect on insulin secretion.

    PubMed

    Bahadar, Haji; Maqbool, Faheem; Mostafalou, Sara; Baeeri, Maryam; Rahimifard, Mahban; Navaei-Nigjeh, Mona; Abdollahi, Mohammad

    2015-05-01

    Benzene (C6H6) is an organic compound used in petrochemicals and numerous other industries. It is abundantly released to our environment as a chemical pollutant causing widespread human exposure. This study mainly focused on benzene induced toxicity on rat pancreatic islets with respect to oxidative damage, insulin secretion and glucokinase (GK) activity. Benzene was dissolved in corn oil and administered orally at doses 200, 400 and 800mg/kg/day, for 4 weeks. In rats, benzene significantly raised the concentration of plasma insulin. Also the effect of benzene on the release of glucose-induced insulin was pronounced in isolated islets. Benzene caused oxidative DNA damage and lipid peroxidation, and also reduced the cell viability and total thiols groups, in the islets of exposed rats. In conclusion, the current study revealed that pancreatic glucose metabolism is susceptible to benzene toxicity and the resultant oxidative stress could lead to functional abnormalities in the pancreas.

  1. Clinical islet isolation and transplantation outcomes with deceased cardiac death donors are similar to neurological determination of death donors.

    PubMed

    Andres, Axel; Kin, Tatsuya; O'Gorman, Doug; Livingstone, Scott; Bigam, David; Kneteman, Norman; Senior, Peter; Shapiro, A M James

    2016-01-01

    In islet transplantation, deceased cardiac death (DCD) donation has been identified as a potential extended source. There are currently no studies comparing outcomes between these categories, and our goal was to compare islet isolation success rates and transplantation outcomes between DCD and neurological determination of death (NDD) donors. Islet isolations from 15 DCD and 418 NDD were performed in our centre between September 2008 and September 2014. Donor variables, islet yields, metabolic function of isolated isled and insulin requirements at 1-month post-transplant were compared. Compared to NDD, pancreata from DCD were more often procured locally and donors required less vasopressive support (P < 0.001 and P = 0.023, respectively), but the other variables were similar between groups. Pre- and postpurification islet yields were similar between NDD and DCD (576 vs. 608 × 10(3) islet equivalent, P = 0.628 and 386 vs. 379, P = 0.881, respectively). The metabolic function was similar between NDD and DCD, as well as the mean decrease in insulin requirement at 1-month post-transplantation (NDD: 64.82%; DCD: 60.17% reduction, P = 0.517). These results support the broader use of DCD pancreata for islet isolation. A much larger DCD islet experience will be required to truly determine noninferiority of both short- and long-term outcomes.

  2. Determination of Optimal Sample Size for Quantification of β-Cell Area, Amyloid Area and β-Cell Apoptosis in Isolated Islets.

    PubMed

    Meier, Daniel T; Entrup, Leon; Templin, Andrew T; Hogan, Meghan F; Samarasekera, Thanya; Zraika, Sakeneh; Boyko, Edward J; Kahn, Steven E

    2015-08-01

    Culture of isolated rodent islets is widely used in diabetes research to assess different endpoints, including outcomes requiring histochemical staining. As islet yields during isolation are limited, we determined the number of islets required to obtain reliable data by histology. We found that mean values for insulin-positive β-cell area/islet area, thioflavin S-positive amyloid area/islet area and β-cell apoptosis do not vary markedly when more than 30 islets are examined. Measurement variability declines as more islets are quantified, so that the variability of the coefficient of variation (CV) in human islet amyloid polypeptide (hIAPP) transgenic islets for β-cell area/islet area, amyloid area/islet area and β-cell apoptosis are 13.20% ± 1.52%, 10.03% ± 1.76% and 6.78% ± 1.53%, respectively (non-transgenic: 7.65% ± 1.17% β-cell area/islet area and 8.93% ± 1.56% β-cell apoptosis). Increasing the number of islets beyond 30 had marginal effects on the CV. Using 30 islets, 6 hIAPP-transgenic preparations are required to detect treatment effects of 14% for β-cell area/islet area, 30% for amyloid area/islet area and 23% for β-cell apoptosis (non-transgenic: 9% for β-cell area/islet area and 45% for β-cell apoptosis). This information will be of value in the design of studies using isolated islets to examine β cells and islet amyloid.

  3. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation

    PubMed Central

    Balamurugan, Appakalai N.; Green, Michael L.; Breite, Andrew G.; Loganathan, Gopalakrishnan; Wilhelm, Joshua J.; Tweed, Benjamin; Vargova, Lenka; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G.; Williams, Stuart K.; Hering, Bernhard J.; Dwulet, Francis E.; McCarthy, Robert C.

    2016-01-01

    Background Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. Methods We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Results Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. Conclusions A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival

  4. Effects of methyl mercury on the activity and gene expression of mouse Langerhans islets and glucose metabolism.

    PubMed

    Maqbool, Faheem; Bahadar, Haji; Niaz, Kamal; Baeeri, Maryam; Rahimifard, Mahban; Navaei-Nigjeh, Mona; Ghasemi-Niri, Seyedeh Farnaz; Abdollahi, Mohammad

    2016-07-01

    Mercury (Hg) is a well-known heavy metal and causes various toxic effects. It is abundantly present in fish in the form of methyl mercury (MeHg). Also, various other forms of mercury can enter human body either from environment like inhalation or through dental amalgams. The present study was designed to assess MeHg induced toxicity in mouse plasma and pancreatic islets with respect to insulin secretion, oxidative balance, glucose tolerance, gene expression, caspases 3 and 9 activities. MeHg was dissolved in tap water and administered at doses 2.5, 5 and 10 mg/kg/day, for 4 weeks. In mice, MeHg significantly caused increase in plasma insulin as well as C-peptides. Glucose intolerance, insulin resistance and hyperglycemia are main consequences of our study that correlate with the gene expression changes of glucose homeostasis as well. MeHg caused increase lipid peroxidation in a dose-dependent manner in plasma as well as pancreatic islets. In addition, total thiol molecules and ferrous reducing antioxidant power in MeHg treated group was decreased in plasma as well as pancreatic islets. Caspases 3 and 9 activities of pancreatic islets were upregulated in MeHg exposed animals. Reactive oxygen species were extremely high in pancreatic islets of MeHg treated groups. MeHg disrupted gluconeogenesis/glycogenolysis pathways and insulin secretory functions of islets by targeting GDH, GLUT2 and GCK genes of pancreatic islets. In conclusion, the current study revealed that insulin pathways, oxidative balance and glucose metabolism encoded genetic makeup are susceptible to MeHg toxicity and the subsequent oxidative stress and alternations in gene expression could lead toward functional abnormalities in other organs. PMID:27178136

  5. Multi-Center Analysis of Novel and Established Variables Associated with Successful Human Islet Isolation Outcomes

    PubMed Central

    Kaddis, J.S; Danobeitia, J.S.; Niland, J.C.; Stiller, T.; Fernandez, L.A.

    2010-01-01

    Islet transplantation is a promising therapy used to achieve glycometabolic control in a select subgroup of individuals with type I diabetes. However, features that characterize human islet isolation success prior to transplantation are not standardized and lack validation. We conducted a retrospective analysis of 806 isolation records from 14 pancreas processing laboratories, considering variables from relevant studies in the last 15 years. The outcome was defined as post-purification islet equivalent count, dichotomized into yields ≥ 315,000 or ≤ 220,000. Univariate analysis showed that donor cause of death and use of hormonal medications negatively influenced outcome. Conversely, pancreata from heavier donors and those containing elevated levels of surface fat positively influence outcome, as did heavier pancreata and donors with normal amylase levels. Multivariable logistic regression analysis identified the positive impact on outcome of surgically intact pancreata and donors with normal liver function, and confirmed that younger donors, increased body mass index, shorter cold ischemia times, no administration of fluid/electrolyte medications, absence of organ edema, use of University of Wisconsin preservation solution, and a fatty pancreas improves outcome. In conclusion, this multi-center analysis highlights the importance of carefully reviewing of all donor, pancreas, and processing parameters prior to isolation and transplantation. PMID:20055802

  6. Nonenzymatic cryogenic isolation of therapeutic cells: novel approach for enzyme-free isolation of pancreatic islets using in situ cryopreservation of islets and concurrent selective freeze destruction of acinar tissue.

    PubMed

    Taylor, Michael J; Baicu, Simona C

    2014-01-01

    Cell-based therapies, which all involve processes for procurement and reimplantation of living cells, currently rely upon expensive, inconsistent, and even toxic enzyme digestion processes. A prime example is the preparation of isolated pancreatic islets for the treatment of type 1 diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods, we have conceptualized an alternative approach based on the hypothesis that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ. Pancreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water to distend the acinar; 3) freezing the entire Px solid to < -160°C for storage in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen Px into small fragments; 5) thawing the frozen fragments, filtering, and washing to remove the CPA. Finally, the filtered effluent (cryoisolate) was stained with dithizone for identification of intact islets and with Syto 13/PI for fluorescence viability testing and glucose-stimulated insulin release assessment. As predicted, the cryoisolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact and viable (>90%) embedded islets. Islets were typically larger (range 50-500 µm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7. An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze

  7. Nonenzymatic cryogenic isolation of therapeutic cells: novel approach for enzyme-free isolation of pancreatic islets using in situ cryopreservation of islets and concurrent selective freeze destruction of acinar tissue.

    PubMed

    Taylor, Michael J; Baicu, Simona C

    2014-01-01

    Cell-based therapies, which all involve processes for procurement and reimplantation of living cells, currently rely upon expensive, inconsistent, and even toxic enzyme digestion processes. A prime example is the preparation of isolated pancreatic islets for the treatment of type 1 diabetes by transplantation. To avoid the inherent pitfalls of these enzymatic methods, we have conceptualized an alternative approach based on the hypothesis that cryobiological techniques can be used for differential freeze destruction of the pancreas (Px) to release islets that are selectively cryopreserved in situ. Pancreata were procured from juvenile pigs using approved procedures. The concept of cryoisolation is based on differential processing of the pancreas in five stages: 1) infiltrating islets in situ preferentially with a cryoprotectant (CPA) cocktail via antegrade perfusion of the major arteries; 2) retrograde ductal infusion of water to distend the acinar; 3) freezing the entire Px solid to < -160°C for storage in liquid nitrogen; 4) mechanically crushing and pulverizing the frozen Px into small fragments; 5) thawing the frozen fragments, filtering, and washing to remove the CPA. Finally, the filtered effluent (cryoisolate) was stained with dithizone for identification of intact islets and with Syto 13/PI for fluorescence viability testing and glucose-stimulated insulin release assessment. As predicted, the cryoisolate contained small fragments of residual tissue comprising an amorphous mass of acinar tissue with largely intact and viable (>90%) embedded islets. Islets were typically larger (range 50-500 µm diameter) than their counterparts isolated from juvenile pigs using conventional enzyme digestion techniques. Functionally, the islets from replicate cryoisolates responded to a glucose challenge with a mean stimulation index = 3.3 ± 0.7. An enzyme-free method of islet isolation relying on in situ cryopreservation of islets with simultaneous freeze

  8. Different digestion enzymes used for human pancreatic islet isolation: a mixed treatment comparison (MTC) meta-analysis.

    PubMed

    Rheinheimer, Jakeline; Ziegelmann, Patrícia Klarmann; Carlessi, Rodrigo; Reck, Luciana Ross; Bauer, Andrea Carla; Leitão, Cristiane Bauermann; Crispim, Daisy

    2014-01-01

    Collagenases are critical reagents determining yield and quality of isolated human pancreatic islets and may affect islet transplantation outcome. Some islet transplantation centers have compared 2 or more collagenase blends; however, the results regarding differences in quantity and quality of islets are conflicting. Thus, for the first time, a mixed treatment comparison (MTC) meta-analysis was carried out to compile data about the effect of different collagenases used for human pancreas digestion on islet yield, purity, viability and stimulation index (SI). Pubmed, Embase and Cochrane libraries were searched. Of 755 articles retrieved, a total of 15 articles fulfilled the eligibility criteria and were included in the MTC meta-analysis. Our results revealed that Vitacyte and Liberase MTF were associated with a small increase in islet yield (islet equivalent number/g pancreas) when compared with Sevac enzyme [standardized mean difference (95% credible interval - CrI) = -2.19 (-4.25 to -0.21) and -2.28 (-4.49 to -0.23), respectively]. However, all other enzyme comparisons did not show any significant difference regarding islet yield. Purity and viability percentages were not significantly different among any of the analyzed digestion enzymes. Interestingly, Vitacyte and Serva NB1 were associated with increased SI when compared with Liberase MTF enzyme [unstandardized weighted mean difference (95% CrI) = -1.69 (-2.87 to -0.51) and -1.07 (-1.79 to -0.39), respectively]. In conclusion, our MTC meta-analysis suggests that the digestion enzymes currently being used for islet isolation works with similar efficiency regarding islet yield, purity and viability; however, Vitacyte and Serva NB1 enzymes seem to be associated with an improved SI as compared with Liberase MTF. PMID:25437379

  9. Exercise at anaerobic threshold intensity and insulin secretion by isolated pancreatic islets of rats

    PubMed Central

    de Oliveira, Camila Aparecida Machado; Paiva, Mauricio Ferreira; Mota, Clécia Alencar Soares; Ribeiro, Carla; de Almeida Leme, José Alexandre Curiacos; Luciano, Eliete

    2010-01-01

    To evaluate the effect of acute exercise and exercise training at the anaerobic threshold (AT) intensity on aerobic conditioning and insulin secretion by pancreatic islets, adult male Wistar rats were submitted to the lactate minimum test (LMT) for AT determination. Half of the animals were submitted to swimming exercise training (trained), 1 h/day, 5 days/week during 8 weeks, with an overload equivalent to the AT. The other half was kept sedentary. At the end of the experimental period, the rats were submitted to an oral glucose tolerance test and to another LMT. Then, the animals were sacrificed at rest or immediately after 20 minutes of swimming exercise at the AT intensity for pancreatic islets isolation. At the end of the experiment mean workload (% bw) at AT was higher and blood lactate concentration (mmol/L) was lower in the trained than in the control group. Rats trained at the AT intensity showed no alteration in the areas under blood glucose and insulin during OGTT test. Islet insulin content of trained rats was higher than in the sedentary rats while islet glucose uptake did not differ among the groups. The static insulin secretion in response to the high glucose concentration (16.7 mM) of the sedentary group at rest was lower than the sedentary group submitted to the acute exercise and the inverse was observed in relation to the trained groups. Physical training at the AT intensity improved the aerobic condition and altered insulin secretory pattern by pancreatic islets. PMID:21099318

  10. Circadian variation of the pancreatic islet transcriptome.

    PubMed

    Rakshit, Kuntol; Qian, Jingyi; Ernst, Jason; Matveyenko, Aleksey V

    2016-09-01

    Pancreatic islet failure is a characteristic feature of impaired glucose control in diabetes mellitus. Circadian control of islet function is essential for maintaining proper glucose homeostasis. Circadian variations in transcriptional pathways have been described in diverse cell types and shown to be critical for optimization of cellular function in vivo. In the current study, we utilized Short Time Series Expression Miner (STEM) analysis to identify diurnally expressed transcripts and biological pathways from mouse islets isolated at 4 h intervals throughout the 24 h light-dark cycle. STEM analysis identified 19 distinct chronological model profiles, and genes belonging to each profile were subsequently annotated to significantly enriched Kyoto Encyclopedia of Genes and Genomes biological pathways. Several transcriptional pathways essential for proper islet function (e.g., insulin secretion, oxidative phosphorylation), cell survival (e.g., insulin signaling, apoptosis) and cell proliferation (DNA replication, homologous recombination) demonstrated significant time-dependent variations. Notably, KEGG pathway analysis revealed "protein processing in endoplasmic reticulum - mmu04141" as one of the most enriched time-dependent pathways in islets. This study provides unique data set on time-dependent diurnal profiles of islet gene expression and biological pathways, and suggests that diurnal variation of the islet transcriptome is an important feature of islet homeostasis, which should be taken into consideration for optimal experimental design and interpretation of future islet studies. PMID:27495157

  11. Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

    SciTech Connect

    Chow, S.A.; Falany, J.L.; Fischer, L.J. )

    1989-06-01

    The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic beta-cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of 3H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.

  12. 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

    PubMed Central

    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

  13. Islet Culture/Preservation Before Islet Transplantation.

    PubMed

    Noguchi, Hirofumi; Miyagi-Shiohira, Chika; Kurima, Kiyoto; Kobayashi, Naoya; Saitoh, Issei; Watanabe, Masami; Noguchi, Yasufumi; Matsushita, Masayuki

    2015-12-17

    Although islet culture prior to transplantation provides flexibility for the evaluation of isolated islets and the pretreatment of patients, it is well known that isolated islets deteriorate rapidly in culture. Human serum albumin (HSA) is used for medium supplementation instead of fetal bovine serum (FBS), which is typically used for islet culture research, to avoid the introduction of xenogeneic materials. However, FBS contains several factors that are beneficial to islet viability and which also neutralize the endogenous pancreatic enzymes or exogenous enzymes left over from the isolation process. Several groups have reported the comparison of cultures at 22°C and 37°C. Recent studies have demonstrated the superiority of 4°C preservation to 22°C and 37°C cultures. We herein review the current research on islet culture/preservation for clinical islet transplantation. PMID:26858905

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

    PubMed

    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.

  15. Function and expression of sulfonylurea, adrenergic, and glucagon-like peptide 1 receptors in isolated porcine islets.

    PubMed

    Kelly, Amy C; Steyn, Leah V; Kitzmann, Jenna P; Anderson, Miranda J; Mueller, Kate R; Hart, Nathaniel J; Lynch, Ronald M; Papas, Klearchos K; Limesand, Sean W

    2014-01-01

    The scarcity of human cadaveric pancreata limits large-scale application of islet transplantation for patients with diabetes. Islets isolated from pathogen-free pigs provide an economical and abundant alternative source assuming immunologic barriers are appropriate. Membrane receptors involved in insulin secretion that also have potential as imaging targets were investigated in isolated porcine islets. Quantitative (q)PCR revealed that porcine islets express mRNA transcripts for sulfonylurea receptor 1 (Sur1), inward rectifying potassium channel (Kir6.2, associated with Sur1), glucagon-like peptide 1 receptor (GLP1R), and adrenergic receptor alpha 2A (ADRα2A). Receptor function was assessed in static incubations with stimulatory glucose concentrations, and in the presence of receptor agonists. Glibenclamide, an anti-diabetic sulfonylurea, and exendin-4, a GLP-1 mimetic, potentiated glucose-stimulated insulin secretion >2-fold. Conversely, epinephrine maximally reduced insulin secretion 72 ± 9% (P < 0.05) and had a half maximal inhibitory concentration of 60 nm in porcine islets (95% confidence interval of 45-830 nm). The epinephrine action was inhibited by the ADRα2A antagonist yohimbine. Our findings demonstrate that porcine islets express and are responsive to both stimulatory and inhibitory membrane localized receptors, which can be used as imaging targets after transplantation or to modify insulin secretion.

  16. Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

    PubMed Central

    Grant, P. T.; Reid, K. B. M.

    1968-01-01

    1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11·5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain. PMID:4866431

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

    PubMed Central

    Johansson, Ulrika; 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

  18. Improved quality and yield of islets isolated from human pancreata using a two-step digestion method.

    PubMed

    Kenmochi, T; Miyamoto, M; Une, S; Nakagawa, Y; Moldovan, S; Navarro, R A; Benhamou, P Y; Brunicardi, F C; Mullen, Y

    2000-03-01

    A new approach, involving a two-step digestion process and Los Angeles preservation solution #1 (LAP-1), a cold storage solution, was developed for isolation of high-quality islets from human pancreata for transplantation. This approach markedly improves the islet yield, purity and viability, and the isolation success rate. In this method, the pancreas was digested first in warm collagenase solution for up to 20 minutes. After decanting the enzyme solution, partially digested tissue was dissociated by gentle agitation in cold LAP-1 solution without additional collagenase. The digested tissues were stored in cold LAP-1 solution until islet purification on Euro-Ficoll. Forty-six islet isolations were performed consecutively by the new method (group 1). These results were compared to those obtained earlier with 46 consecutive isolations, using our previous method that had been used before development of the new method (group 2). Our old method was a modification of Ricordi's method involving only warm collagenase digestion and the storage of digested tissues in cold Hanks balanced salt solution. All pancreata were partial, containing the body and tail. There were no significant differences in both groups with regard to the donor age, cold ischemic time, harvesting conditions, and pancreatic weight. Pancreas digestion was completed in approximately 1 hour in both groups. The isolation success rate as determined by viable islets after 2 days in culture was 93.5% (43 of 46 cases) in group 1, and 56.5% (26 of the 46) in group 2. Immediately after isolation, the new method yielded a total of 335,739 +/- 36,244 islets equivalent to 150 microm (IEQ) and 6,233 +/- 681 IEQ/g of pancreas with 83 +/- 2.5% purity, whereas the old method yielded a total of 195,587 +/- 25,242 IEQ and 3,763 +/- 5,509 IEQ/g with 69.2 +/- 4.7% purity. Isolated islets in group 1 maintained a good three-dimensional structure, displayed normal insulin release to high glucose stimulation in vitro, and

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

    PubMed Central

    Mahroui, Neda; Mirzaei, Sanaz; Siahpoosh, Zahra; D.4, Pharm.; Nili-Ahmadabadi, Amir; Mohammadirad, Azadeh; Baeeri, Maryam; Hajiaghaie, Reza; Abdollahi, Mohammad

    2014-01-01

    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 concen- trations were also determined. Results The tested compounds markedly increased survival of the islet cells, their mi- tochondrial 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 103μgmL-1, respectively. Conclusion Based on these results, we suggest that pretreatment with these select- ed Iranian medical plants can improve the outcomes of pancreas transplants and grafts through the control of oxidative stress damage. PMID:24567945

  20. Evidence for the presence of glucose cycling in pancreatic islets of the ob/ob mouse

    SciTech Connect

    Khan, A.; Chandramouli, V.; Ostenson, C.G.; Ahren, B.; Schumann, W.C.; Loew, H.L.; Landau, B.R.; Efendic, S.

    1989-06-15

    Pancreatic islets from ob/ob mice incubated with /sub 3/H/sub 2/O and 5.5 mM glucose formed /sup 3/H-labeled glucose, 74 picoatoms incorporated/islet/h. Sixty-three percent of the 3H was bound to carbon 2 of the glucose. The amount of glucose-6-P dephosphorylated to glucose, determined from this incorporation, was 48 pmol/islet/h. Glucose utilization, measured by the formation of /sup 3/H/sub 2/O from (5-/sup 3/H)glucose, was 72 pmol/islet/h. The amount of glucose dephosphorylated was then about 40% of that phosphorylated. Thus, glucose-6-P is dephosphorylated to glucose to a significant extent by intact islets in vitro and presumably by the beta cells of the islets. The extent of this glucose cycling, i.e. glucose----glucose-6-P----glucose, may play a role in determining the extent of glucose-induced insulin secretion.

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

    PubMed Central

    Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

    2016-01-01

    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 from 48 hrs

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

    PubMed

    Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

    2016-01-01

    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 from 48 hrs onwards.

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

    PubMed

    Kosinová, Lucie; Cahová, Monika; Fábryová, Eva; Týcová, Irena; Koblas, Tomáš; Leontovyč, Ivan; Saudek, František; Kříž, Jan

    2016-01-01

    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 from 48 hrs onwards

  4. Human Endothelial Protein C Receptor Overexpression Protects Intraportal Islet Grafts in Mice.

    PubMed

    Gock, H; Lee, K F E; Murray-Segal, L; Mysore, T B; d'Apice, A J F; Salvaris, E J; Cowan, P J

    2016-01-01

    Islet transplantation can potentially cure type 1 diabetes mellitus, but it is limited by a shortage of human donors as well as by islet graft destruction by inflammatory and thrombotic mechanisms. A possible solution to these problems is to use genetically modified pig islets. Endothelial protein C receptor (EPCR) enhances protein C activation and regulates coagulation, inflammation, and apoptosis. We hypothesized that human EPCR (hEPCR) expression on donor islets would improve graft survival and function. Islets from an hEPCR transgenic mouse line strongly expressed the transgene, and hEPCR expression was maintained after islet isolation. Islets were transplanted from hEPCR mice and wild-type (WT) littermates into diabetic mice in a marginal-dose syngeneic intraportal islet transplantation model. The blood glucose level normalized within 5 days in 5 of 7 recipients of hEPCR islets, compared with only 2 of 7 recipients of WT islets (P < .05). Transplanted hEPCR islets had better preserved morphology and more intense insulin staining than WT grafts, and they retained transgene expression. The improved engraftment compared with WT islets suggests that inflammation and coagulation associated with the transplant process can be reduced by hEPCR expression on donor tissue. PMID:27569971

  5. Characterization of proinsulin- and proglucagon-converting activities in isolated islet secretory granules.

    PubMed

    Fletcher, D J; Quigley, J P; Bauer, G E; Noe, B D

    1981-08-01

    The conversion of proglucagon and proinsulin by secretory granules isolated from both prelabeled and unlabeled anglerfish islets was investigated. Either granules isolated from tissue labeled with [3H]tryptophan and [14C]isoleucine or [35S]cysteine, or lysed granules from unlabeled tissue to which exogenously labeled prohormones had been added were incubated under various conditions. Acetic acid extracts of these granule preparations were analyzed for prohormone and hormone content by gel filtration. Both prelabeled and lysed, unlabeled secretory granules converted radiolabeled precursor peptides (Mr 8,000-15,000) to labeled insulin and glucagon. The accuracy of the cleavage process was established by demonstrating comigration of products obtained from in vitro cleavage with insulin and glucagon extracted from intact islets using electrophoresis and high-pressure liquid chromatography (HPLC). The pH optimum for granule-mediated conversion was found to be in the range of pH 4.5-5.5. Conversion of both proglucagon and proinsulin by secretory granules was significantly inhibited in the presence of antipain, leupeptin, p-chloromercuribenzoate (PCMB) or dithiodipyridine (DDP) but not chloroquine, diisopropyl fluorophosphate, EDTA, p-nitrophenyl guanidinobenzoate, soybean trypsin inhibitor, or N-p-tosyl-L-lysine chloromethyl ketone HCl. The inhibitory action of PCMB and DDP was reversed in the presence of dithiothreitol. Both membranous and soluble components of the secretory granules possessed significant converting activity. HPLC and electrophoretic analysis of cleavage products demonstrated that the converting activities of the membranous and soluble components were indistinguishable. The amount of inhibition of proinsulin and proglucagon conversion caused by 600 micrograms/ml porcine proinsulin was significantly lower than that caused by the same concentration of unlabeled anglerfish precursor peptides. These results indicate that the proinsulin and proglucagon

  6. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations

    PubMed Central

    Delalat, Bahman; Rojas-Canales, Darling M.; Rasi Ghaemi, Soraya; Waibel, Michaela; Harding, Frances J.; Penko, Daniella; Drogemuller, Christopher J.; Loudovaris, Thomas; Coates, Patrick T. H.; Voelcker, Nicolas H.

    2016-01-01

    Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response. PMID:27600088

  7. A Combinatorial Protein Microarray for Probing Materials Interaction with Pancreatic Islet Cell Populations.

    PubMed

    Delalat, Bahman; Rojas-Canales, Darling M; Rasi Ghaemi, Soraya; Waibel, Michaela; Harding, Frances J; Penko, Daniella; Drogemuller, Christopher J; Loudovaris, Thomas; Coates, Patrick T H; Voelcker, Nicolas H

    2016-01-01

    Pancreatic islet transplantation has become a recognized therapy for insulin-dependent diabetes mellitus. During isolation from pancreatic tissue, the islet microenvironment is disrupted. The extracellular matrix (ECM) within this space not only provides structural support, but also actively signals to regulate islet survival and function. In addition, the ECM is responsible for growth factor presentation and sequestration. By designing biomaterials that recapture elements of the native islet environment, losses in islet function and number can potentially be reduced. Cell microarrays are a high throughput screening tool able to recreate a multitude of cellular niches on a single chip. Here, we present a screening methodology for identifying components that might promote islet survival. Automated fluorescence microscopy is used to rapidly identify islet derived cell interaction with ECM proteins and immobilized growth factors printed on arrays. MIN6 mouse insulinoma cells, mouse islets and, finally, human islets are progressively screened. We demonstrate the capability of the platform to identify ECM and growth factor protein candidates that support islet viability and function and reveal synergies in cell response. PMID:27600088

  8. Effect of C-peptide Alone or in Combination with Nicotinamide on Insulin Levels from Pancreatic Islets in Mouse

    PubMed Central

    Ahangarpour, Akram; Ali Akbari, Fatemeh Ramezani; Moghadam, Hadi Fathi

    2016-01-01

    Background Both c-peptide and nicotinamide are known to increase blood insulin in diabetes. In the present study, we examined the effect of c-peptide alone or in combination with nicotinamide on insulin levels in pancreatic islets in mice. Methods This study was conducted with 60 adult male Naval Medical Research Institute (NMARI) mice weighing 25 to 30 g. Pancreatic islets from normal mice were isolated by the collagenase digestion method. Mice were divided into ten groups of six (n = 6): control, glyburide (1 and 10 μM), C-peptide (50 and 100 nM), nicotinamide (10, 25, and 100 mM), nicotinamide + C-peptide (100 mM and 100 nM), and buffer in different glucose concentrations (2.8, 5.6, and 16.7 mM). Insulin secretion was measured using insulin radioimmunoassay method. Results Insulin secretion significantly increased at 16.7 mM glucose concentration compared with 2.8 and 5.6 mM glucose concentrations. Incubation of islets at 2.8 and 5.6 mM glucose concentrations and nicotinamide + C-peptide, nicotinamide 25 and 100 mM, and C-peptide 100 nM significantly increased insulin secretion compared with the control group. In addition, incubation of islets at 16.7 mM glucose with nicotinamide + C-peptide significantly increased insulin secretion. Glyburide at 10 μM concentration was more effective than nicotinamide at 10 and 100 mM, C-peptide 50 and 100 nM in the presence of 16.7 mM glucose concentration. However, the combination of nicotinamide + C-peptide was more effective than glyburide at a concentration of 10 μM in the presence of a 16.7 mM glucose concentration. Conclusions This paper suggests that c-peptide, nicotinamide, and the combination of c-peptide and nicotinamide in-creases insulin secretion from pancreatic islets. PMID:27540321

  9. Specific glucagon-related peptides isolated from anglerfish islets are metabolic cleavage products of (pre)proglucagon-II.

    PubMed

    Noe, B D; Andrews, P C

    1986-01-01

    Sequence analyses of cDNAs prepared from anglerfish islet mRNA have demonstrated the presence of mRNAs coding for two different preproglucagons, aPPG-I and aPPG-II. Each of these precursors was predicted to contain 29 residue and 34 residue glucagon-related peptides as potential cleavage products. Recently, several glucagon-related peptides found in extracts of anglerfish islets have been isolated and characterized. In order to determine whether any of these peptides could be identified as metabolic cleavage products in anglerfish islets, differentially radiolabeled Mr 2,500-8,000 peptides from islet extracts were subjected to reverse phase HPLC under varying conditions. The potential cleavage products aPPG-II[52-80] and aPPG-II[89-122] could be readily identified among the extract peptides. Both peptides became labeled appropriately (as predicted from their sequences) with 13 different amino acids and demonstrated glucagon-like immunoreactivity in a radioimmunoassay. Conversely, a third peptide (aPPG-II[89-119]) could be found among the labeled products in small amounts only. These results demonstrate that glucagon-II[52-80] and aGLP-II[89-112] are primary cleavage products of aPPG-II and suggest that aGLP-IIc[89-119] may be a peptide generated more slowly by post-translational modification of aGLP-II. PMID:3526301

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

    PubMed Central

    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

  11. A comparative evaluation of culture conditions for short-term maintenance (<24 hr) of human islets isolated using the Edmonton protocol.

    PubMed

    Matsumoto, Shinichi; Goel, Shilpa; Qualley, Sabrina; Strong, D. Michael; Reems, Jo Anna

    2003-01-01

    Once human islets are isolated, they are typically transplanted into type 1 diabetic recipients within 2 h of isolation. This time restriction makes it difficult for patients to travel from distant locations to receive an islet transplant and it also makes it difficult to complete pre-release quality control assessments (i.e., endotoxin and gram stain) before the expiration of the islet product. Therefore, there were two goals for this study. The first was to measure the stability of islets after a 24 h culture period using CMRL media 1066 (CMRL) supplemented with either fetal bovine serum (FBS); albumin or insulin transferrin and selenium (ITS). The second was to determine the impact of cell concentration and media depth on islet stability. The results of the study indicated that culture recoveries at 37 degrees C with CMRL + ITS (also known as Memphis media) were higher (64.1 +/- 8.3%) than with CMRL supplemented with FBS (38.7 +/- 9.7%) or albumin (47.6 +/- 8.2%) and that post-culture islet viabilities, post-culture purities and stimulation indexes (SIs) were comparable. In the second series of experiments, the results showed that islets recoveries and SIs in cultures with low islet concentrations (300 IE/ml) were significantly better than cultures at high islet concentrations (1500 IE/ml). Additionally, at a shallow media depth (1.4 vs. 7 mm of media) the SI of the islets improved, and this effect was independent of the additive (i.e., FBS, albumin and ITS).

  12. Glucocorticoids in vivo induce both insulin hypersecretion and enhanced glucose sensitivity of stimulus-secretion coupling in isolated rat islets.

    PubMed

    Rafacho, Alex; Marroquí, Laura; Taboga, Sebastião R; Abrantes, Júlia L F; Silveira, Leonardo R; Boschero, Antonio C; Carneiro, Everardo M; Bosqueiro, José R; Nadal, Angel; Quesada, Ivan

    2010-01-01

    Although glucocorticoids are widely used as antiinflammatory agents in clinical therapies, they may cause serious side effects that include insulin resistance and hyperinsulinemia. To study the potential functional adaptations of the islet of Langerhans to in vivo glucocorticoid treatment, adult Wistar rats received dexamethasone (DEX) for 5 consecutive days, whereas controls (CTL) received only saline. The analysis of insulin release in freshly isolated islets showed an enhanced secretion in response to glucose in DEX-treated rats. The study of Ca(2+) signals by fluorescence microscopy also demonstrated a higher response to glucose in islets from DEX-treated animals. However, no differences in Ca(2+) signals were found between both groups with tolbutamide or KCl, indicating that the alterations were probably related to metabolism. Thus, mitochondrial function was explored by monitoring oxidation of nicotinamide dinucleotide phosphate autofluorescence and mitochondrial membrane potential. Both parameters revealed a higher response to glucose in islets from DEX-treated rats. The mRNA and protein content of glucose transporter-2, glucokinase, and pyruvate kinase was similar in both groups, indicating that changes in these proteins were probably not involved in the increased mitochondrial function. Additionally, we explored the status of Ca(2+)-dependent signaling kinases. Unlike calmodulin kinase II, we found an augmented phosphorylation level of protein kinase C alpha as well as an increased response of the phospholipase C/inositol 1,4,5-triphosphate pathway in DEX-treated rats. Finally, an increased number of docked secretory granules were observed in the beta-cells of DEX animals using transmission electron microscopy. Thus, these results demonstrate that islets from glucocorticoid-treated rats develop several adaptations that lead to an enhanced stimulus-secretion coupling and secretory capacity.

  13. Isolation and analysis of mouse microglial cells.

    PubMed

    Garcia, Jenny A; Cardona, Sandra M; Cardona, Astrid E

    2014-01-01

    Microglia are mononuclear phagocytes that make up about 10% of the central nervous system (CNS). They are known for their surveillant behavior, which involves continuous monitoring of neural tissue by extending and retracting their processes. Microglial cells are derived from myeloid progenitor cells and play important roles in homeostasis as well as inflammatory and immune responses in the brain. This unit describes several microglial cell isolation protocols that can be easily adapted for projects requiring a rapid and efficient analysis of mouse microglial cells by flow cytometry. Methods for visualizing microglial cells using in situ immunohistochemistry and immunochemistry in free-floating sections are also included.

  14. In vivo synchronous membrane potential oscillations in mouse pancreatic beta-cells: lack of co-ordination between islets.

    PubMed Central

    Valdeolmillos, M; Gomis, A; Sánchez-Andrés, J V

    1996-01-01

    1. The properties of the oscillations in electrical activity of different beta-cells within the same islet of Langerhans, and of different islets within the same pancreas, recorded in vivo, are described. 2. Simultaneous recordings of two cells within the same islet showed that the oscillations were synchronous. A rapid increase in blood glucose led to the simultaneous appearance of a transitory phase of continuous electrical activity in both cells. These results indicate that under physiological conditions, the islets operate as a functional syncytium. 3. Simultaneous recordings of cells from two different islets within the same pancreas showed that the oscillations in the electrical activity were not synchronous, which suggests that each islet is a functionally independent unit. Rapid changes in blood glucose led to the appearance of a transitory phase of increased electrical activity in both islets, although of different duration. These results suggest that the endocrine pancreas lacks a pacemaker driving the electrical activity of all the islets. 4. The comparison of the degree of activation of different islets, simultaneously recorded at different glucose concentrations, indicated that all the islets had a similar sensitivity to glucose. Furthermore, when the glucose concentration was increased, the electrical activity in both islets increased in parallel, suggesting that the amount of insulin released due to the increase in glycaemia was produced by the simultaneous response of all the islets and not by the recruitment of islets with different sensitivities to glucose. 5. Our results predict that the synchronous electrical activity of all the cells within an islet will result in widespread intracellular calcium oscillations and pulsatile insulin secretion. The periodicity of the pulses of insulin secretion in different islets is suggested to be of slightly different length and asynchronous. PMID:8735691

  15. The isolated pancreatic islet as a micro-organ and its transplantation to cure diabetes

    PubMed Central

    2010-01-01

    Over the past three decades the pancreatic islet of Langerhans has taken center stage as an endocrine microorgan whose glucoregulatory function is highly explicable on the basis of the increasingly well understood activities of three highly interactive secretory cells. Islet dysfunction underlies both type 1 and type 2 diabetes mellitus (DM); its protection from immune attack and gluco-and lipo-toxicity may prevent the development of DM; and its replacement by non-surgical transplantation may be curative of DM. During a career marked by vision, focus and tenacity, Paul Lacy contributed substantially to the development of each of these concepts. In this review we focus on Lacy's contribution to the development of the concept of the islet as a micro-organ, how this foreshadowed our current detailed understanding of single cell function and cell-cell interactions and how this led to a reduced model of islet function encouraging islet transplantation. Next, we examine how clinical allotransplantation, first undertaken by Lacy, has contributed to a more complex view of the interaction of islet endocrine cells with its circulation and neighboring tissues, both “in situ” and after transplantation. Lastly, we consider recent developments in some alternative approaches to treatment of DM that Lacy could glimpse on the horizon but did not have the chance to participate in. PMID:21099316

  16. Extracellular Matrix Protein-Coated Scaffolds Promote the Reversal of Diabetes After Extrahepatic Islet Transplantation

    PubMed Central

    Salvay, David M.; Rives, Christopher B.; Zhang, Xiaomin; Chen, Fei; Kaufman, Dixon B.; Lowe, William L.; Shea, Lonnie D.

    2008-01-01

    Background The survival and function of transplanted pancreatic islets is limited, owing in part to disruption of islet-matrix attachments during the isolation procedure. Using polymer scaffolds as a platform for islet transplantation, we investigated the hypothesis that replacement of key extracellular matrix components known to surround islets in vivo would improve graft function at an extrahepatic implantation site. Methods Microporous polymer scaffolds fabricated from copolymers of lactide and glycolide were adsorbed with collagen IV, fibronectin, laminin-332 or serum proteins before seeding with 125 mouse islets. Islet-seeded scaffolds were then implanted onto the epididymal fat pad of syngeneic mice with streptozotocin-induced diabetes. Nonfasting glucose levels, weight gain, response to glucose challenges, and histology were used to assess graft function for 10 months after transplantation. Results Mice transplanted with islets seeded onto scaffolds adsorbed with collagen IV achieved euglycemia fastest and their response to glucose challenge was similar to normal mice. Fibronectin and laminin similarly promoted euglycemia, yet required more time than collagen IV and less time than serum. Histopathological assessment of retrieved grafts demonstrated that coating scaffolds with specific extracellular matrix proteins increased total islet area in the sections and vessel density within the transplanted islets, relative to controls. Conclusions Extracellular matrix proteins adsorbed to microporous scaffolds can enhance the function of transplanted islets, with collagen IV maximizing graft function relative to the other proteins tested. These scaffolds enable the creation of well-defined microenvironments that promote graft efficacy at extrahepatic sites. PMID:18497687

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

    SciTech Connect

    Zhao, Yong; Guo, Chengshan; Hwang, David; Lin, Brian; Dingeldein, Michael; Mihailescu, Dan; Sam, Susan; Sidhwani, Seema; Zhang, Yongkang; Jain, Sumit; Skidgel, Randal A.; Prabhakar, Bellur S.; Mazzone, Theodore; Holterman, Mark J.

    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.

  18. Effects of glass ionomers and dental resin composites on viability of beta-cells and insulin release in isolated islets of Langerhans.

    PubMed

    Persson-Sjögren, Solveig; Sjögren, Göran

    2003-09-01

    Information on the biocompatibility of glass ionomers and resin composites is sparse. To extend the scale of biological testing we evaluated the influence of those materials on insulin secretion at whole organ level in vitro. The effects on insulin secretion of three glass ionomers and two resin composites, aged for 1 week, were studied in isolated mouse islets of Langerhans at basal (5.5mM) and at stimulatory (11.1mM) D-glucose concentrations. In addition, viability of single mouse beta-cells was evaluated. The effect of glass ionomer specimens aged for 1 and 4 months on insulin secretion at 11.1mM D-glucose was also studied. None of the materials affected the viability of the beta-cells. At 5.5mM D-glucose none of the materials affected the insulin secretion. At 11.1mM D-glucose, the glass ionomers only decreased the secretion and glass ionomers aged for 1 month still decreased insulin release whereas after 4 months ageing only one of the glass ionomers affected the release. The result shows a dynamic effect on insulin release of the elements and/or compounds released from the specimens.

  19. Isolation and Physiological Analysis of Mouse Cardiomyocytes

    PubMed Central

    Roth, Gretchen M.; Bader, David M.; Pfaltzgraff, Elise R.

    2014-01-01

    Cardiomyocytes, the workhorse cell of the heart, contain exquisitely organized cytoskeletal and contractile elements that generate the contractile force used to pump blood. Individual cardiomyocytes were first isolated over 40 years ago in order to better study the physiology and structure of heart muscle. Techniques have rapidly improved to include enzymatic digestion via coronary perfusion. More recently, analyzing the contractility and calcium flux of isolated myocytes has provided a vital tool in the cellular and sub-cellular analysis of heart failure. Echocardiography and EKGs provide information about the heart at an organ level only. Cardiomyocyte cell culture systems exist, but cells lack physiologically essential structures such as organized sarcomeres and t-tubules required for myocyte function within the heart. In the protocol presented here, cardiomyocytes are isolated via Langendorff perfusion. The heart is removed from the mouse, mounted via the aorta to a cannula, perfused with digestion enzymes, and cells are introduced to increasing calcium concentrations. Edge and sarcomere detection software is used to analyze contractility, and a calcium binding fluorescent dye is used to visualize calcium transients of electrically paced cardiomyocytes; increasing understanding of the role cellular changes play in heart dysfunction. Traditionally used to test drug effects on cardiomyocytes, we employ this system to compare myocytes from WT mice and mice with a mutation that causes dilated cardiomyopathy. This protocol is unique in its comparison of live cells from mice with known heart function and known genetics. Many experimental conditions are reliably compared, including genetic or environmental manipulation, infection, drug treatment, and more. Beyond physiologic data, isolated cardiomyocytes are easily fixed and stained for cytoskeletal elements. Isolating cardiomyocytes via perfusion is an extremely versatile method, useful in studying cellular changes

  20. Overexpression of IRS2 in isolated pancreatic islets causes proliferation and protects human {beta}-cells from hyperglycemia-induced apoptosis

    SciTech Connect

    Mohanty, S.; Spinas, G.A.; Maedler, K.; Zuellig, R.A.; Lehmann, R.; Donath, M.Y.; Trueb, T.; Niessen, M. . E-mail: markus.niessen@usz.ch

    2005-02-01

    Studies in vivo indicate that IRS2 plays an important role in maintaining functional {beta}-cell mass. To investigate if IRS2 autonomously affects {beta}-cells, we have studied proliferation, apoptosis, and {beta}-cell function in isolated rat and human islets after overexpression of IRS2 or IRS1. We found that {beta}-cell proliferation was significantly increased in rat islets overexpressing IRS2 while IRS1 was less effective. Moreover, proliferation of a {beta}-cell line, INS-1, was decreased after repression of Irs2 expression using RNA oligonucleotides. Overexpression of IRS2 in human islets significantly decreased apoptosis of {beta}-cells, induced by 33.3 mM D-glucose. However, IRS2 did not protect cultured rat islets against apoptosis in the presence of 0.5 mM palmitic acid. Overexpression of IRS2 in isolated rat islets significantly increased basal and D-glucose-stimulated insulin secretion as determined in perifusion experiments. Therefore, IRS2 is sufficient to induce proliferation in rat islets and to protect human {beta}-cells from D-glucose-induced apoptosis. In addition, IRS2 can improve {beta}-cell function. Our results indicate that IRS2 acts autonomously in {beta}-cells in maintenance and expansion of functional {beta}-cell mass in vivo.

  1. Isolation and Analysis of Mouse Microglial Cells

    PubMed Central

    Garcia, Jenny A.; Cardona, Sandra M.

    2014-01-01

    Microglia are mononuclear phagocytes that make up about 10% of the central nervous system (CNS). They are known for their surveillant behavior which comprises continuously monitoring neural tissue by extending and retracting their processes. Microglial cells are derived from myeloid progenitor cells and play important roles in homeostasis, inflammatory and immune responses in the brain. This Unit describes several microglial cell isolation protocols (Basic Protocol 1, Alternate Protocol, and Basic Protocol 2) that can be easily adapted for projects requiring a rapid and efficient analysis of mouse microglial cells by flow cytometry (Support Protocol 1). Methods for visualizing microglial cells using in situ immunohistochemistry (Basic Protocol 3) and immunochemistry in free-floating sections (Basic Protocol 4) are also included. PMID:24510618

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

    PubMed Central

    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

  3. Small human islets comprised of more β-cells with higher insulin content than large islets

    PubMed Central

    Farhat, Bilal; Almelkar, Akshay; Ramachandran, Karthik; Williams, S. Janette; Huang, Han-Hung; Zamierowksi, David; Novikova, Lesya; Stehno-Bittel, Lisa

    2013-01-01

    For the past 30 y, data have suggested that unique islet populations exist, based on morphology and glucose sensitivity. Yet little has been done to determine the mechanism of these functional differences. The purpose of this study was to determine whether human islets were comprised functionally unique populations, and to elucidate a possible mechanism. Islets or pancreatic sections from 29 human donors were analyzed. Islets were isolated and measured for insulin secretion, cell composition and organization, insulin and glucagon granule density and insulin content. Insulin secretion was significantly greater in small compared with large islets. In sectioned human pancreata, β-cells comprised a higher proportion of the total endocrine cells in small islets (63%) than large islets (39%). A higher percentage of β-cells in small islets contacted blood vessels (44%) compared with large islets (31%). Total insulin content of isolated human islets was significantly greater in the small (1323 ± 512 μIU/IE) compared with large islets (126 ± 48 μIU/IE). There was less immunostaining for insulin in the large islets from human pancreatic sections, especially in the core of the islet, compared with small islets. The results suggest that differences in insulin secretion between large and small islets may be due to a higher percentage of β-cells in small islets with more β-cells in contact with blood vessels and a higher concentration of insulin/β-cell in small islets. PMID:23648896

  4. Isolation of mouse cell proteoglycan mutants

    SciTech Connect

    Keller, K.M.; Keller, J.M.

    1986-05-01

    The sulfated proteoglycans on the surface of cultured mammalian cells have been implicated in a variety of phenomena. To obtain more direct evidence for the role of these molecules in specific cellular functions, they are isolating mutants that produce altered sulfated proteoglycans from a cloned line of Swiss mouse 3T3 cells. This cell type was selected because it exhibits contact inhibition of growth and there is extensive information on its' cell surface and extracellular proteoglycans and other glycoproteins. Cells were chemically mutagenized and subjected to one or more cycles of radiation suicide in the presence of /sup 35/S-sulfate. By replica plating, 150 clones, which appear to incorporate abnormal amounts of /sup 35/S-sulfate, have been selected. After recloning three times via the replica plating technique, the proteoglycans of 29 clones have thus far been analyzed. They have identified four clones which appear to make altered amounts of either cell surface heparan sulfate or chondroitin sulfate. The biochemical bases for the altered levels of the proteoglycans are under study. Of particular interest, however, is the fact that in this limited collection of mutants the chemical alterations correlate with specific altered cellular morphologies.

  5. The optimization of large-scale density gradient isolation of human islets.

    PubMed

    Robertson, G S; Chadwick, D R; Contractor, H; James, R F; London, N J

    1993-01-01

    The use of the COBE 2991 cell processor (COBE Laboratories, Colorado) for large-scale islet purification using discontinuous density gradients has been widely adopted. It minimizes many of the problems such as wall effects, normally encountered during centrifugation, and avoids the vortexing at interfaces that occurs during acceleration and deceleration by allowing the gradient to be formed and the islet-containing interface to be collected while continuing to spin. We have produced cross-sectional profiles of the 2991 bag during spinning which allow the area of interfaces in such step gradients to be calculated. This allows the volumes of the gradient media layers loaded on the machine to be adjusted in order to maximize the area of the gradient interfaces. However, even using the maximal areas possible (144.5 cm2), clogging of tissue at such interfaces limits the volume of digest which can be separated on one gradient to 15 ml. We have shown that a linear continuous density gradient can be produced within the 2991 bag, that allows as much as 40 ml of digest to be successfully purified. Such a system combines the intrinsic advantages of the 2991 with those of continuous density gradients and provides the optimal method for density-dependent islet purification. PMID:8219265

  6. Remodelling sympathetic innervation in rat pancreatic islets ontogeny

    PubMed Central

    Cabrera-Vásquez, Siraam; Navarro-Tableros, Víctor; Sánchez-Soto, Carmen; Gutiérrez-Ospina, Gabriel; Hiriart, Marcia

    2009-01-01

    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. PMID:19534767

  7. In Vivo Islet Protection by a Nuclear Import Inhibitor in a Mouse Model of Type 1 Diabetes

    PubMed Central

    Moore, Daniel J.; Zienkiewicz, Jozef; Kendall, Peggy L.; Liu, Danya; Liu, Xueyan; Veach, Ruth Ann; Collins, Robert D.; Hawiger, Jacek

    2010-01-01

    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. PMID:20949090

  8. Islet cell transplantation.

    PubMed

    Srinivasan, P; Huang, G C; Amiel, S A; Heaton, N D

    2007-04-01

    People with type 1 diabetes have normal exocrine pancreatic function, making islet cell rather than whole organ transplantation an attractive option. Achieving insulin independence in type 1 diabetes was the perceived goal of islet cell transplantation. The success of the Edmonton group in achieving this in a selected group of type 1 patients has led to renewed optimism that this treatment could eventually replace whole organ pancreas transplantation. However the long-term results of this treatment indicate that insulin independence is lost with time in a significant proportion of patients, although they may retain glycaemic stability. In this context, the indications for islet cell transplantation, which have evolved over the last 5 years, indicate that the patients who benefit most are those who experience severe hypoglycaemic reactions despite optimal insulin therapy. This review will summarise the history of islet cell transplantation, islet isolation techniques, the transplant procedure, immunosuppressive therapy, indications for islet cell transplantation, current clinical trials, the early UK islet cell transplant experience using the Edmonton protocol, and some of the challenges that lie ahead. PMID:17403947

  9. Generation of islet-like cells from mouse gall bladder by direct ex vivo reprogramming

    PubMed Central

    Hickey, Raymond D; Galivo, Feorillo; Schug, Jonathan; Brehm, Michael A; Haft, Annelise; Wang, Yuhan; Benedetti, Eric; Gu, Guoqiang; Magnuson, Mark A; Shultz, Leonard D; Lagasse, Eric; Greiner, Dale L; Kaestner, Klaus H; Grompe, Markus

    2013-01-01

    Cell replacement is an emerging therapy for type 1 diabetes. Pluripotent stem cells have received a lot of attention as a potential source of transplantable β-cells, but their ability to form teratomas poses significant risks. Here, we evaluated the potential of primary mouse gall bladder epithelial cells (GBCs) as targets for ex vivo genetic reprogramming to the β-cell fate. Conditions for robust expansion and genetic transduction of primary GBCs by adenoviral vectors were developed. Using a GFP reporter for insulin, conditions for reprogramming were then optimized. Global expression analysis by RNA-sequencing was used to quantitatively compare reprogrammed GBCs (rGBCs) to true β-cells, revealing both similarities and differences. Adenoviral-mediated expression of NEUROG3, Pdx1, and MafA in GBCs resulted in robust induction of pancreatic endocrine genes, including Ins1, Ins2, Neurod1, Nkx2-2 and Isl1. Furthermore, expression of GBC-specific genes was repressed, including Sox17 and Hes1. Reprogramming was also enhanced by addition of retinoic acid and inhibition of Notch signaling. Importantly, rGBCs were able to engraft long term in vivo and remained insulin-positive for 15 weeks. We conclude that GBCs are a viable source for autologous cell replacement in diabetes, but that complete reprogramming will require further manipulations. PMID:23562832

  10. Enalapril and ASS inhibit tumor growth in a transgenic mouse model of islet cell tumors.

    PubMed

    Fendrich, V; Lopez, C L; Manoharan, J; Maschuw, K; Wichmann, S; Baier, A; Holler, J P; Ramaswamy, A; Bartsch, D K; Waldmann, J

    2014-10-01

    Accumulating evidence suggests a role for angiotensin-converting enzymes involving the angiotensin II-receptor 1 (AT1-R) and the cyclooxygenase pathway in carcinogenesis. The effects of ASS and enalapril were assessed in vitro and in a transgenic mouse model of pancreatic neuroendocrine neoplasms (pNENs). The effects of enalapril and ASS on proliferation and expression of the AGTR1A and its target gene vascular endothelial growth factor (Vegfa) were assessed in the neuroendocrine cell line BON1. Rip1-Tag2 mice were treated daily with either 0.6 mg/kg bodyweight of enalapril i.p., 20 mg/kg bodyweight of ASS i.p., or a vehicle in a prevention (weeks 5-12) and a survival group (week 5 till death). Tumor surface, weight of pancreatic glands, immunostaining for AT1-R and nuclear factor kappa beta (NFKB), and mice survival were analyzed. In addition, sections from human specimens of 20 insulinomas, ten gastrinomas, and 12 non-functional pNENs were evaluated for AT1-R and NFKB (NFKB1) expression and grouped according to the current WHO classification. Proliferation was significantly inhibited by enalapril and ASS in BON1 cells, with the combination being the most effective. Treatment with enalapril and ASS led to significant downregulation of known target genes Vegf and Rela at RNA level. Tumor growth was significantly inhibited by enalapril and ASS in the prevention group displayed by a reduction of tumor size (84%/67%) and number (30%/45%). Furthermore, daily treatment with enalapril and ASS prolonged the overall median survival compared with vehicle-treated Rip1-Tag2 (107 days) mice by 9 and 17 days (P=0.016 and P=0.013). The AT1-R and the inflammatory transcription factor NFKB were abolished completely upon enalapril and ASS treatment. AT1-R and NFKB expressions were observed in 80% of human pNENs. Enalapril and ASS may provide an approach for chemoprevention and treatment of pNENs.

  11. Imaging of the islet neural network.

    PubMed

    Tang, S-C; Peng, S-J; Chien, H-J

    2014-09-01

    The islets of Langerhans receive signals from the circulation and nerves to modulate hormone secretion in response to physiological cues. Although the rich islet innervation has been documented in the literature dating as far back as Paul Langerhans' discovery of islets in the pancreas, it remains a challenging task for researchers to acquire detailed islet innervation patterns in health and disease due to the dispersed nature of the islet neurovascular network. In this article, we discuss the recent development of 3-dimensional (3D) islet neurohistology, in which transparent pancreatic specimens were prepared by optical clearing to visualize the islet microstructure, vasculature and innervation with deep-tissue microscopy. Mouse islets were used as an example to illustrate how to apply this 3D imaging approach to characterize (i) the islet parasympathetic innervation, (ii) the islet sympathetic innervation and its reinnervation after transplantation under the kidney capsule and (iii) the reactive cellular response of the Schwann cell network in islet injury. While presenting and characterizing the innervation patterns, we also discuss how to apply the signals derived from transmitted light microscopy, vessel painting and immunostaining of neural markers to verify the location and source of tissue information. In summary, the systematic development of tissue labelling, clearing and imaging methods to reveal the islet neuroanatomy offers insights to help study the neural-islet regulatory mechanisms and the role of neural tissue remodelling in the development of diabetes.

  12. PALMITATE INHIBITS INSULIN GENE EXPRESSION BY ALTERING PDX-1 NUCLEAR LOCALIZATION AND REDUCING MAFA EXPRESSION IN ISOLATED RAT ISLETS OF LANGERHANS*

    PubMed Central

    Hagman, Derek K.; Hays, Lori B.; Parazzoli, Susan D.; Poitout, Vincent

    2005-01-01

    Abnormalities in lipid metabolism have been proposed as contributing factors to both defective insulin secretion from the pancreatic beta cell and peripheral insulin resistance in type 2 diabetes. Previously, we have shown that prolonged exposure of isolated rat islets of Langerhans to excessive fatty acid levels impairs insulin gene transcription. This study was designed to assess whether palmitate alters the expression and binding activity of the key regulatory factors pancreas-duodenum homeobox-1 (PDX-1), MafA, and Beta2, which respectively bind to the A3, C1, and E1 elements in the proximal region of the insulin promoter. Nuclear extracts of isolated rat islets cultured with 0.5 mM palmitate exhibited reduced binding activity to the A3 and C1 elements, but not the E1 element. Palmitate did not affect the overall expression of PDX-1, but reduced its nuclear localization. In contrast, palmitate blocked the stimulation of MafA mRNA and protein expression by glucose. Combined, adenovirus-mediated, over-expression of PDX-1 and MafA in islets completely prevented the inhibition of insulin gene expression by palmitate. These results demonstrate that prolonged exposure of islets to palmitate inhibits insulin gene transcription by impairing nuclear localization of PDX-1 and cellular expression of MafA. PMID:15944145

  13. Expression of Receptors for Tetanus Toxin and Monoclonal Antibody A2B5 by Pancreatic Islet Cells

    NASA Astrophysics Data System (ADS)

    Eisenbarth, G. S.; Shimizu, K.; Bowring, M. A.; Wells, S.

    1982-08-01

    Studies of the reaction of antibody A2B5 and tetanus toxin with pancreatic islet cells, islet cell tumors, and other human amine precursor uptake and decarboxylation (APUD) tumors are described. By indirect immunofluorescence, antibody A2B5 and tetanus toxin were shown to specifically bind to the plasma membrane of human, rat, chicken, and mouse islet cells. The binding of antibody A2B5 to the cell surface of living islet cells has allowed isolation of these cells from a suspension of pancreatic cells by using a fluorescence-activated cell sorter. In studies designed to determine whether tetanus toxin and antibody A2B5 bound to the same surface antigen, A2B5 and tetanus toxin did not compete for binding to normal islet cells, a human islet cell tumor, or a rat islet cell tumor. In addition to binding to islet cell tumors, antibody A2B5 reacts with frozen sections, isolated cells, and cell lines of neural, neural crest, and APUD origin.

  14. Cooperation by Fibroblasts and Bone Marrow-Mesenchymal Stem Cells to Improve Pancreatic Rat-to-Mouse Islet Xenotransplantation

    PubMed Central

    Meana, Alvaro; Otero, Jesus; Esteban, Manuel M.

    2013-01-01

    Experimental and clinical experiences highlight the need to review some aspects of islet transplantation, especially with regard to site of grafting and control of the immune response. The subcutaneous space could be a good alternative to liver but its sparse vasculature is its main limitation. Induction of graft tolerance by using cells with immunoregulatory properties is a promising approach to avoid graft rejection. Both Fibroblasts and Mesenchymal Stem Cells (MSCs) have shown pro-angiogenic and immunomodulatory properties. Transplantation of islets into the subcutaneous space using plasma as scaffold and supplemented with fibroblasts and/or Bone Marrow-MSCs could be a promising strategy to achieve a functional extra-hepatic islet graft, without using immunosuppressive drugs. Xenogenic rat islets, autologous fibroblasts and/or allogenic BM-MSCs, were mixed with plasma, and coagulation was induced to constitute a Plasma-based Scaffold containing Islets (PSI), which was transplanted subcutaneously both in immunodeficient and immunocompetent diabetic mice. In immunodeficient diabetic mice, PSI itself allowed hyperglycemia reversion temporarily, but the presence of pro-angiogenic cells (fibroblasts or BM-MSCs) within PSI was necessary to improve graft re-vascularization and, thus, consistently maintain normoglycemia. In immunocompetent diabetic mice, only PSI containing BM-MSCs, but not those containing fibroblasts, normalized glycemia lasting up to one week after transplantation. Interestingly, when PSI contained both fibroblasts and BM-MSCs, the normoglycemia period showed an increase of 4-times with a physiological-like response in functional tests. Histology of immunocompetent mice showed an attenuation of the immune response in those grafts with BM-MSCs, which was improved by co-transplantation with fibroblasts, since they increased BM-MSC survival. In summary, fibroblasts and BM-MSCs showed similar pro-angiogenic properties in this model of islet

  15. Pancreatic islet transplantation for treating diabetes.

    PubMed

    Matsumoto, Shinichi; Noguchi, Hirofumi; Yonekawa, Yukihide; Okitsu, Teru; Iwanaga, Yasuhiro; Liu, Xiaoling; Nagata, Hideo; Kobayashi, Naoya; Ricordi, Camillo

    2006-01-01

    Pancreatic islet transplantation is one of the options for treating diabetes and has been shown to improve the quality of life of severe diabetic patients. Since the Edmonton protocol was announced, islet transplantation have advanced considerably, including islet after kidney transplantation, utilisation of non-heart-beating donors, single-donor islet transplantation and living-donor islet transplantation. These advances were based on revised immunosuppression protocols, improved pancreas procurement and islet isolation methods, and enhanced islet engraftment. Further improvements are necessary to make islet transplantation a routine clinical treatment. To synergise efforts towards a cure for type 1 diabetes, a Diabetes Research Institute (DRI) Federation is currently being established to include leading diabetes research centres worldwide, including DRIs in Miami, Edmonton and Kyoto among others.

  16. Human islet isolation for autologous transplantation: comparison of yield and function using SERVA/Nordmark versus Roche enzymes.

    PubMed

    Anazawa, T; Balamurugan, A N; Bellin, M; Zhang, H J; Matsumoto, S; Yonekawa, Y; Tanaka, T; Loganathan, G; Papas, K K; Beilman, G J; Hering, B J; Sutherland, D E R

    2009-10-01

    Islet autotransplantation (IAT) is used to preserve as much insulin-secretory capacity as possible in patients undergoing total pancreatectomy for painful chronic pancreatitis. The enzyme used to dissociate the pancreas is a critical determinant of islet yield, which is correlated with posttransplant function. Here, we present our experience with IAT procedures to compare islet product data using the new enzyme SERVA/Nordmark (SN group; n = 46) with the standard enzyme Liberase-HI (LH group; n = 40). Total islet yields (mean +/- standard deviation; 216,417 +/- 79,278 islet equivalent [IEQ] in the LH group; 227,958 +/- 58,544 IEQ in the SN group; p = 0.67) were similar. However, the percentage of embedded islets is higher in the SN group compared to the LH group. Significant differences were found in pancreas digestion time, dilution time, and digested pancreas weight between the two groups. Multivariate linear regression analysis showed the two groups differed in portal venous pressure changes. The incidence of graft function and insulin independence was not different between the two groups. The SN and LH enzymes are associated with similar outcomes for IAT. Further optimization of the collagenase/neutral protease ratio is necessary to reduce the number of embedded islets obtained when using the SN enzyme.

  17. RNA isolation from mouse pancreas: a ribonuclease-rich tissue.

    PubMed

    Azevedo-Pouly, Ana Clara P; Elgamal, Ola A; Schmittgen, Thomas D

    2014-08-02

    Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

  18. AMYLOID FORMATION IN HUMAN IAPP TRANSGENIC MOUSE ISLETS AND PANCREAS AND HUMAN PANCREAS IS NOT ASSOCIATED WITH ENDOPLASMIC RETICULUM STRESS

    PubMed Central

    Hull, R.L.; Zraika, S.; Udayasankar, J.; Aston-Mourney, K.; Subramanian, S.L.; Kahn, S.E.

    2009-01-01

    Hypothesis Supraphysiological levels of the amyloidogenic peptide human islet amyloid polypeptide (hIAPP) have been associated with beta cell endoplasmic reticulum (ER) stress. However, in human type 2 diabetes, levels of hIAPP are equivalent or decreased relative to matched controls. Thus, we sought to investigate whether ER stress is induced during amyloidogenesis at physiological levels of hIAPP. Methods Islets from hIAPP transgenic mice that develop amyloid, and non-transgenic mice that do not, were cultured for up to seven days in 11.1, 16.7 and 33.3 mmol/l glucose. Pancreata from hIAPP transgenic and non-transgenic mice and human subjects with or without type 2 diabetes were also evaluated. Amyloid formation was determined histologically. ER stress was determined in islets by quantifying mRNA levels of BiP, Atf4 and Chop, and alternate splicing of XBP-1 mRNA or in pancreata by immunostaining for BiP, CHOP and XBP-1. Results Amyloid formation in hIAPP transgenic islets was associated with reduced beta-cell area in a glucose- and time-dependent manner. However, amyloid formation was not associated with significant increases in expression of ER stress markers under any culture condition. Thapsigargin treatment, a positive control, did result in significant ER stress. Amyloid formation in vivo in pancreas samples from hIAPP transgenic mice or humans was not associated with upregulation of ER stress markers. Conclusions/interpretation Our data suggest that ER stress is not an obligatory pathway mediating the toxic effects of amyloid formation at physiological levels of hIAPP. PMID:19352619

  19. Blockade of both B7-H4 and CTLA-4 co-signaling pathways enhances mouse islet allograft survival

    PubMed Central

    Wang, Xiaojie; Hao, Jianqiang; Metzger, Daniel L.; Mui, Alice; Lee, I-Fang; Akhoundsadegh, Noushin; Chen, C. Lieping; Ou, Dawei; Ao, Ziliang; Verchere, C. Bruce; Warnock, Garth L.

    2012-01-01

    Costimulation blockade is an effective way to prevent allograft rejection. In this study, we tested the efficacy of two negative co-signaling molecules in protecting islet allograft function. We used local expression of B7-H4 by adenoviral transduction of islets (Ad-B7-H4) and systemic administration of CTLA-4.Ig to investigate the outcomes of allograft survival. Five groups of streptozotocin-induced diabetic C57BL/6 mice received 400 islets each from BALB/c donors. The groups consisted of control (G1); CTLA-4.Ig (G2); Ad-LacZ (G3); Ad-B7-H4 (G4); and Ad-B7-H4 and CTLA-4.Ig combined (G5). G1 and G3 developed graft failure on average of two weeks. G2, G4 and G5 survived for 43.8 ± 34.8, 54.7 ± 31.2 and 77.8 ± 21.5 d, respectively. Activated T and B cells in the lymph nodes were significantly controlled by CTLA-4.Ig treatment. Significantly reduced infiltrates were also detected in the allografts of G2 compared with G1. By contrast, B7-H4 significantly inhibited Th1-associated IFN-gamma secretion in the early stage and increased Foxp3+ T cells in the long-term surviving allografts. Our study suggests that CTLA-4 and B7-H4 inhibit alloimmune responses through distinct mechanisms, and that combination therapy which activates two negative co-signaling pathways can further enhance islet allograft survival. PMID:22878670

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

    PubMed

    Zhou, Yunting; Hu, Qi; Chen, Fuyi; Zhang, Juan; Guo, Jincheng; Wang, Hongwu; Gu, Jiang; Ma, Lian; Ho, Guyu

    2015-12-01

    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.

  1. Vascular endothelial growth factor coordinates islet innervation via vascular scaffolding

    PubMed Central

    Reinert, Rachel B.; Cai, Qing; Hong, Ji-Young; Plank, Jennifer L.; Aamodt, Kristie; Prasad, Nripesh; Aramandla, Radhika; Dai, Chunhua; Levy, Shawn E.; Pozzi, Ambra; Labosky, Patricia A.; Wright, Christopher V. E.; Brissova, Marcela; Powers, Alvin C.

    2014-01-01

    Neurovascular alignment is a common anatomical feature of organs, but the mechanisms leading to this arrangement are incompletely understood. Here, we show that vascular endothelial growth factor (VEGF) signaling profoundly affects both vascularization and innervation of the pancreatic islet. In mature islets, nerves are closely associated with capillaries, but the islet vascularization process during embryonic organogenesis significantly precedes islet innervation. Although a simple neuronal meshwork interconnects the developing islet clusters as they begin to form at E14.5, the substantial ingrowth of nerve fibers into islets occurs postnatally, when islet vascularization is already complete. Using genetic mouse models, we demonstrate that VEGF regulates islet innervation indirectly through its effects on intra-islet endothelial cells. Our data indicate that formation of a VEGF-directed, intra-islet vascular plexus is required for development of islet innervation, and that VEGF-induced islet hypervascularization leads to increased nerve fiber ingrowth. Transcriptome analysis of hypervascularized islets revealed an increased expression of extracellular matrix components and axon guidance molecules, with these transcripts being enriched in the islet-derived endothelial cell population. We propose a mechanism for coordinated neurovascular development within pancreatic islets, in which endocrine cell-derived VEGF directs the patterning of intra-islet capillaries during embryogenesis, forming a scaffold for the postnatal ingrowth of essential autonomic nerve fibers. PMID:24574008

  2. Combination strategy of multi-layered surface camouflage using hyperbranched polyethylene glycol and immunosuppressive drugs for the prevention of immune reactions against transplanted porcine islets.

    PubMed

    Haque, Muhammad R; Jeong, Jee-Heon; Byun, Youngro

    2016-04-01

    This study suggests a novel method of stabilizing fragile porcine islets to prevent the dissociation after isolation and reducing immune cell invasion in a combination therapy of 'surface camouflaging' and immunosuppressive drugs (FK506, Rapamycin, MR-1, anti-CD19 mAb, and Clodrosome(®)) to effectively alleviate overall immune reactions against xenotransplanted porcine islets. The surface camouflage of pancreatic islets using biocompatible materials improved stabilization of pancreatic islet and prevented the infiltration of immune cells. Firstly, the surface of porcine islets was camouflaged by SH-6-arm-PEG-lipid and gelatin-catechol (artificial extracellular matrix) in order to stabilize the fragile isolated islets. Secondly, three different PEG layers (6-arm-PEG-SH, 6-arm-PEG-catechol, and linear PEG-SH) were chemically conjugated onto the surface of the stabilized porcine islets. Both artificial extracellular matrix (artificial ECM) and PEGylation effectively covered the surface of porcine islets without increasing the size of the whole islet. In addition, the viability and functionality of the islets were not affected by this multi-layer surface modification. The multi-layer modification significantly reduced the attachment of human serum albumin, fibronectin, and immunoglobulin G in comparison to the control collagen surface. The combination effect of multi-layer PEGylation and cocktailed immunosuppressive drugs on the survival time of the transplanted islets was assessed in a xenogeneic porcine-to-mouse model. The median survival time (MST) of 'artificial ECM + PEGylation' group was 4-fold increased compared to that of control group. In addition, the MST of 'artificial ECM + PEGylation + drug' group was 2.16-fold increased, compared to the 'control + drug' group. In conclusion, we proposed a novel porcine islet transplantation protocol using surface multi-layer modification and cocktailed immunosuppressive drugs, for stabilization and

  3. High-Fat Diet-Induced Insulin Resistance Does Not Increase Plasma Anandamide Levels or Potentiate Anandamide Insulinotropic Effect in Isolated Canine Islets

    PubMed Central

    Woolcott, Orison O.; Richey, Joyce M.; Kabir, Morvarid; Chow, Robert H.; Iyer, Malini S.; Kirkman, Erlinda L.; Stefanovski, Darko; Lottati, Maya; Kim, Stella P.; Harrison, L. Nicole; Ionut, Viorica; Zheng, Dan; Hsu, Isabel R.; Catalano, Karyn J.; Chiu, Jenny D.; Bradshaw, Heather; Wu, Qiang; Bergman, Richard N.

    2015-01-01

    Background 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. Objective To determine whether high-fat diet-induced insulin resistance increases anandamide levels and potentiates the insulinotropic effect of anandamide in isolated pancreatic islets. Design and Methods 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). Results 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

  4. Isolation of the mouse homologue of BRCA1 and genetic mapping to mouse chromosome 11

    SciTech Connect

    Bennett, L.M.; Haugen-Strano, A.; Cochran, C.

    1995-10-10

    The BRCA1 gene is in large part responsible for hereditary human breast and ovarian cancer. Here we report the isolation of the murine Brca1 homologue cDNA clones. In addition, we identified genomic P1 clones that contain most, if not all, of the mouse Brca1 locus. DNA sequence analysis revealed that the mouse and human coding regions are 75% identical at the nucleotide level while the predicted amino acid identity is only 58%. A DNA sequence variant in the Brcal locus was identified and used to map this gene on a (Mus m. musculus Czech II x C57BL/KsJ)F1 x C57BL/KsJ intersubspecific backcross to distal mouse chromosome 11. The mapping of this gene to a region highly syntenic with human chromosome 17, coupled with Southern and Northern analyses, confirms that we isolated the murine Brcal homologue rather than a related RING finger gene. The isolation of the mouse Brca1 homologue will facilitate the creation of mouse models for germline BRCA1 defects. 12 refs., 3 figs.

  5. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells. PMID:17460896

  6. [Isolation, purification and identification of epithelial cells derived from fetal islet-like cell clusters].

    PubMed

    Qiao, Hai; Zhao, Ting; Wang, Yun; Yang, Chun-Rong; Xiao, Mei; Dou, Zhong-Ying

    2007-03-01

    The aim of this article is to provide methods for the isolation and identification of pancreatic stem cells and cell source for research and therapy of diabetes. ICCs were isolated by collagenase IV digesting and then cultured; epithelial cells were purified from monolayer cultured ICCs. The growth curve of the epithelial cells was measured by MTT. The expression of molecular markers in the cells was identified by immunohistochemical staining. The surface markers in the epithelial cells were analyzed by FACS. Epithelial cells were purified from isolated human fetal ICCs and passaged 40 times, and 10(6) - 10(8) cells were cryopreservated per passage. The growth curve demonstrated that the epithelial cells proliferated rapidly. The epithelial cells expressed PDX-1, PCNA, CK-7, CK-19, Nestin, Glut2, and Vimentin, but Insulin was undetected. The cells expressed CD29, CD44, and CD166, but did not express CD11a, CD14, CD34, CD45, CD90, CD105, and CD117. Taken together, these results indicate that self-renewable epithelial cells can be isolated and purified from human fetal pancreas. These also show that the epithelial cells originate from ducts and have the characteristics of pancreatic stem cells.

  7. Caprine pancreatic islet xenotransplantation into diabetic immunosuppressed BALB/c mice

    PubMed Central

    Hani, Homayoun; Allaudin, Zeenathul N; Mohd-Lila, Mohd-Azmi; Ibrahim, Tengku A Tengku; Othman, Abas M

    2014-01-01

    Background Type 1 diabetes mellitus is a devastating disease for which there is currently no cure, but only lifetime management. Islet xenotransplantation is a promising technique for the restoration of blood glucose control in patients with diabetes mellitus. The purpose of this study was to explore the potential use of caprine (goat) islet cells as xenogeneic grafts in the treatment for diabetes in a mouse model. Methods Caprine pancreases were harvested and transported to the laboratory under conditions optimized to prevent ischemia. Islets were isolated, purified, and tested for functionality. Caprine islets (2000 islet equivalent) were transplanted beneath the kidney capsules of diabetic BALB/c mice under thalidomide-induced immunosuppression. Blood glucose and insulin levels of grafted mice were evaluated by glucometer and enzyme-linked immunosorbent assay kit, respectively. The functionality and quality of caprine pancreatic islet grafts were assessed by intraperitoneal glucose tolerance tests. Results The viability of purified islet cells exceeded 90%. Recipient mice exhibited normoglycemia (<11 mm glucose) for 30 days. In addition, weight gain negatively correlated with blood glucose level. The findings verified diabetes reversal in caprine islet recipient mice. A significant drop in non-fasting blood glucose level (from 23.3 ± 5.4 to 8.04 ± 0.44 mm) and simultaneous increase in serum insulin level (from 0.01 ± 0.001 to 0.56 ± 0.17 μg/l) and body weights (from 23.64 ± 0.31 to 25.85 ± 0.34 g) were observed (P < 0.05). Immunohistochemical analysis verified insulin production in the transplanted islets. Conclusions Purified caprine islets were demonstrated to successfully sustain viability and functionality for controlling blood glucose levels in an immunosuppressed mouse model of diabetes. These results suggest the use of caprine islets as an addition to the supply of xenogeneic islets for diabetes research. PMID:24645790

  8. Structural characterization of peptides derived from prosomatostatins I and II isolated from the pancreatic islets of two species of teleostean fish: the daddy sculpin and the flounder.

    PubMed

    Conlon, J M; Davis, M S; Falkmer, S; Thim, L

    1987-11-01

    The primary structures of three peptides from extracts from the pancreatic islets of the daddy sculpin (Cottus scorpius) and three analogous peptides from the islets of the flounder (Platichthys flesus), two species of teleostean fish, have been determined by automated Edman degradation. The structures of the flounder peptides were confirmed by fast-atom bombardment mass spectrometry. The peptides show strong homology to residues (49-60), (63-96) and (98-125) of the predicted sequence of preprosomatostatin II from the anglerfish (Lophius americanus). The amino acid sequences of the peptides suggest that, in the sculpin, prosomatostatin II is cleaved at a dibasic amino acid residue processing site (corresponding to Lys61-Arg62 in anglerfish preprosomatostatin II). The resulting fragments are further cleaved at monobasic residue processing sites (corresponding to Arg48 and Arg97 in anglerfish preprosomatostatin II). In the flounder the same dibasic residue processing site is utilised but cleavage at different monobasic sites takes place (corresponding to Arg50 and Arg97 in anglerfish preprosomatostatin II). A peptide identical to mammalian somatostatin-14 was also isolated from the islets of both species and is presumed to represent a cleavage product of prosomatostatin I. PMID:2889597

  9. [Impaired insulin secretion in isolated islets of Goto-Kakizaki rats, an animal model of non obese type 2 diabetes, is a primary event].

    PubMed

    Seiça, Raquel M; Suzuki, K I; Santos, Rosa M; Do Rosário, Luis M

    2004-01-01

    The development of type 2 diabetes is associated with the impairment of insulin secretion. To evaluate the evolution of the secretory response, a chronological study comparing normal Wistar (W) vs Goto-Kakizaki (GK) rats, an animal model of non obese type 2 diabetes, was done. Glucose and arginine were tested in collagenase isolated islets of Langerhans with perfusion and ELISA immunoassay techniques. Fasting glycaemia and insulinemia and glucose tolerance were also evaluated. We have seen, in W rats, a mild glucose intolerance in the first two weeks of age. GK rats were always glucose intolerant with hyperglycaemia and hyperinsulinemia at fasten after one month old. Wistar islets had a characteristic biphasic response to glucose after the first two weeks of age. GK islets were always glucose irresponsive. Arginine induced an increase in insulin secretion in both animal models, independent of age, although GK rats had always a smaller response when compared to W rats. We concluded that 1) in W rats, a biphasic insulin secretion in response to glucose is observed after the first two weeks of age, simultaneously with glycaemia stabilization 2) in GK rats, both first and second phases of glucose-induced insulin release are significantly reduced and a smaller reduction in response to arginine is observed. This beta-cell disfunction is a primary event in this model of type 2 diabetes, preceding fasting hyperglycaemia and hyperinsulinemia.

  10. Isolating Primary Melanocyte-like Cells from the Mouse Heart

    PubMed Central

    Hwang, Hayoung; Liu, Fang; Levin, Mark D.; Patel, Vickas V.

    2014-01-01

    We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes. PMID:25285608

  11. Isolating primary melanocyte-like cells from the mouse heart.

    PubMed

    Hwang, Hayoung; Liu, Fang; Levin, Mark D; Patel, Vickas V

    2014-09-29

    We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

  12. Pancreas preservation for pancreas and islet transplantation

    PubMed Central

    Iwanaga, Yasuhiro; Sutherland, David E.R.; Harmon, James V.; Papas, Klearchos K.

    2010-01-01

    Purpose of review To summarize advances and limitations in pancreas procurement and preservation for pancreas and islet transplantation, and review advances in islet protection and preservation. Recent findings Pancreases procured after cardiac death, with in-situ regional organ cooling, have been successfully used for islet transplantation. Colloid-free Celsior and histidine-tryptophan-ketoglutarate preservation solutions are comparable to University of Wisconsin solution when used for cold storage before pancreas transplantation. Colloid-free preservation solutions are inferior to University of Wisconsin solution for pancreas preservation prior to islet isolation and transplantation. Clinical reports on pancreas and islet transplants suggest that the two-layer method may not offer significant benefits over cold storage with the University of Wisconsin solution: improved oxygenation may depend on the graft size; benefits in experimental models may not translate to human organs. Improvements in islet yield and quality occurred from pancreases treated with inhibitors of stress-induced apoptosis during procurement, storage, isolation or culture. Pancreas perfusion may be desirable before islet isolation and transplantation and may improve islet yields and quality. Methods for real-time, noninvasive assessment of pancreas quality during preservation have been implemented and objective islet potency assays have been developed and validated. These innovations should contribute to objective evaluation and establishment of improved pancreas preservation and islet isolation strategies. Summary Cold storage may be adequate for preservation before pancreas transplants, but insufficient when pancreases are processed for islets or when expanded donors are used. Supplementation of cold storage solutions with cytoprotective agents and perfusion may improve pancreas and islet transplant outcomes. PMID:18685343

  13. Human islet isolation--a prospective randomized comparison of pancreatic vascular perfusion with hyperosmolar citrate or University of Wisconsin solution.

    PubMed

    Robertson, G S; Chadwick, D; Thirdborough, S; Swift, S; Davies, J; James, R; Bell, P R; London, N J

    1993-09-01

    University of Wisconsin solution has become the most commonly used vascular perfusate during multiorgan donation world-wide. In the UK however, hyperosmolar citrate remains in common use. The purpose of this prospective randomized study was to compare the effect of systemic perfusion with UW or HOC on subsequent islet yield and purification for pancreata with short cold ischemic times. Seven pancreata were randomized to each group, with the donor age, pancreas weight, and period of cold ischemia being similar in both. Perfusion with UW was shown to inhibit collagenase digestion, and a higher concentration of this enzyme was needed to achieve comparable numbers of islets with good separation of exocrine and islet tissue after a similar period of digestion. There were no differences in the number, size, purity, or viability of islets between the two groups. In conclusion, UW solution offers no benefits over HOC for pancreata with short cold ischemic times, and because of its expense and need to use greater amounts of collagenase enzyme, we continue to use HOC.

  14. Human islet isolation--a prospective randomized comparison of pancreatic vascular perfusion with hyperosmolar citrate or University of Wisconsin solution.

    PubMed

    Robertson, G S; Chadwick, D; Thirdborough, S; Swift, S; Davies, J; James, R; Bell, P R; London, N J

    1993-09-01

    University of Wisconsin solution has become the most commonly used vascular perfusate during multiorgan donation world-wide. In the UK however, hyperosmolar citrate remains in common use. The purpose of this prospective randomized study was to compare the effect of systemic perfusion with UW or HOC on subsequent islet yield and purification for pancreata with short cold ischemic times. Seven pancreata were randomized to each group, with the donor age, pancreas weight, and period of cold ischemia being similar in both. Perfusion with UW was shown to inhibit collagenase digestion, and a higher concentration of this enzyme was needed to achieve comparable numbers of islets with good separation of exocrine and islet tissue after a similar period of digestion. There were no differences in the number, size, purity, or viability of islets between the two groups. In conclusion, UW solution offers no benefits over HOC for pancreata with short cold ischemic times, and because of its expense and need to use greater amounts of collagenase enzyme, we continue to use HOC. PMID:8212148

  15. Optical mapping system for visualizing arrhythmias in isolated mouse atria.

    PubMed

    Schmidt, Robyn; Nygren, Anders

    2006-01-01

    Optical mapping has become an important technique in the study of cardiac electrophysiology, especially in terms of investigating the mechanisms of cardiac arrhythmias. The increasing availability of transgenic mice as models for cardiovascular disease is driving the need for instrumentation suitable for the study of electrical activity in the mouse heart. In this paper we evaluate our optical mapping system's ability to clearly record induced arrhythmic activity in an isolated mouse atrial preparation. Preliminary results indicate that the signal quality is high enough that individual optically recorded action potentials can be discerned in many pixels, even without post-processing for noise removal. The optical mapping video is clear enough for general observations regarding the patterns of electrical propagation during arrhythmic behaviour. The induced arrhythmias appear to have a regular pattern of activity, and are likely best classified as atrial tachycardias.

  16. Isolation of Mouse and Human Tumor-Associated Macrophages.

    PubMed

    Cassetta, Luca; Noy, Roy; Swierczak, Agnieszka; Sugano, Gaël; Smith, Harriet; Wiechmann, Lisa; Pollard, Jeffrey W

    2016-01-01

    The tumor microenvironment is a complex network of cells that support tumor progression and malignancy. It has been demonstrated that tumor cells can educate the immune system to promote a tumor-friendly environment. Among all these immune cells, tumor-associated macrophages (TAMs) are well represented and their presence in mouse models has been shown to promote tumor progression and metastasis. These effects are through the stimulation of angiogenesis, enhancement of tumor cell invasion and intravasation, immunosuppression, and at the metastatic site tumor cell extravasation and growth. However, the precise mechanisms are not fully understood. Furthermore there is limited information on TAMs derived from human cancers. For this reason it is important to be able to extract TAMs from tumors in order to compare their phenotypes, functions, and transcriptomes with normal resident tissue macrophages. Isolation of these cells is challenging due to the lack of markers and standardized protocols. Here we show an optimized protocol for the efficient isolation and extraction of resident macrophages and TAMs from human and mouse tissues by using multicolor flow cytometry. These protocols allow for the extraction of thousands of macrophages in less than 5 h from tissues as small as half a gram. The isolated macrophages can then be used for both "omics" and in vitro studies. PMID:27325269

  17. Isolation of Mouse and Human Tumor-Associated Macrophages

    PubMed Central

    Cassetta, Luca; Noy, Roy; Swierczak, Agnieszka; Sugano, Gaël; Smith, Harriet; Wiechmann, Lisa; Pollard, Jeffrey W.

    2016-01-01

    The tumor microenvironment is a complex network of cells that support tumor progression and malignancy. It has been demonstrated that tumor cells can educate the immune system to promote a tumor-friendly environment. Among all these immune cells, tumor-associated macrophages (TAMs) are well represented and their presence in mouse models has been shown to promote tumor progression and metastasis. These effects are through the stimulation of angiogenesis, enhancement of tumor cell invasion and intravasation, immunosuppression, and at the metastatic site tumor cell extravasation and growth. However, the precise mechanisms are not fully understood. Furthermore there is limited information on TAMs derived from human cancers. For this reason it is important to be able to extract TAMs from tumors in order to compare their phenotypes, functions, and transcriptomes with normal resident tissue macrophages. Isolation of these cells is challenging due to the lack of markers and standardized protocols. Here we show an optimized protocol for the efficient isolation and extraction of resident macrophages and TAMs from human and mouse tissues by using multicolor flow cytometry. These protocols allow for the extraction of thousands of macrophages in less than 5 h from tissues as small as half a gram. The isolated macrophages can then be used for both “omics” and in vitro studies. PMID:27325269

  18. Glucose intolerance and reduced islet blood flow in transgenic mice expressing the FRK tyrosine kinase under the control of the rat insulin promoter.

    PubMed

    Annerén, Cecilia; Welsh, Michael; Jansson, Leif

    2007-04-01

    The FRK tyrosine kinase has previously been shown to transduce beta-cell cytotoxic signals in response to cytokines and streptozotocin and to promote beta-cell proliferation and an increased beta-cell mass. We therefore aimed to further evaluate the effects of overexpression of FRK tyrosine kinase in beta-cells. A transgenic mouse expressing kinase-active FRK under control of the insulin promoter (RIP-FRK) was studied with regard to islet endocrine function and vascular morphology. Mild glucose intolerance develops in RIP-FRK male mice of at least 4 mo of age. This effect is accompanied by reduced glucose-stimulated insulin secretion in vivo and reduced second-phase insulin secretion in response to glucose and arginine upon pancreas perfusion. Islets isolated from the FRK transgenic mice display a glucose-induced insulin secretory response in vitro similar to that of control islets. However, islet blood flow per islet volume is decreased in the FRK transgenic mice. These mice also exhibit a reduced islet capillary lumen diameter as shown by electron microscopy. Total body weight and pancreas weight are not significantly affected, but the beta-cell mass is increased. The data suggest that long-term expression of active FRK in beta-cells causes an in vivo insulin-secretory defect, which may be the consequence of islet vascular abnormalities that yield a decreased islet blood flow.

  19. Lectin-based Isolation and Culture of Mouse Embryonic Motoneurons

    PubMed Central

    Conrad, Rebecca; Jablonka, Sibylle; Sczepan, Teresa; Sendtner, Michael; Wiese, Stefan; Klausmeyer, Alice

    2011-01-01

    Spinal motoneurons develop towards postmitotic stages through early embryonic nervous system development and subsequently grow out dendrites and axons. Neuroepithelial cells of the neural tube that express Nkx6.1 are the unique precursor cells for spinal motoneurons1. Though postmitotic motoneurons move towards their final position and organize themselves into columns along the spinal tract2,3. More than 90% of all these differentiated and positioned motoneurons express the transcription factors Islet 1/2. They innervate the muscles of the limbs as well as those of the body and the inner organs. Among others, motoneurons typically express the high affinity receptors for brain derived neurotrophic factor (BDNF) and Neurotrophin-3 (NT-3), the tropomyosin-related kinase B and C (TrkB, TrkC). They do not express the tropomyosin-related kinase A (TrkA)4. Beside the two high affinity receptors, motoneurons do express the low affinity neurotrophin receptor p75NTR. The p75NTR can bind all neurotrophins with similar but lower affinity to all neurotrophins than the high affinity receptors would bind the mature neurotrophins. Within the embryonic spinal cord, the p75NTR is exclusively expressed by the spinal motoneurons5. This has been used to develop motoneuron isolation techniques to purify the cells from the vast majority of surrounding cells6. Isolating motoneurons with the help of specific antibodies (panning) against the extracellular domains of p75NTR has turned out to be an expensive method as the amount of antibody used for a single experiment is high due to the size of the plate used for panning. A much more economical alternative is the use of lectin. Lectin has been shown to specifically bind to p75NTR as well7. The following method describes an alternative technique using wheat germ agglutinin for a preplating procedure instead of the p75NTR antibody. The lectin is an extremely inexpensive alternative to the p75NTR antibody and the purification grades using

  20. Isolation of Non-parenchymal Cells from the Mouse Liver.

    PubMed

    Mohar, Isaac; Brempelis, Katherine J; Murray, Sara A; Ebrahimkhani, Mohammad R; Crispe, I Nicholas

    2015-01-01

    Hepatocytes comprise the majority of liver mass and cell number. However, in order to understand liver biology, the non-parenchymal cells (NPCs) must be considered. Herein, a relatively rapid and efficient method for isolating liver NPCs from a mouse is described. Using this method, liver sinusoidal endothelial cells, Kupffer cells, natural killer (NK) and NK-T cells, dendritic cells, CD4+ and CD8+ T cells, and quiescent hepatic stellate cells can be purified. This protocol permits the collection of peripheral blood, intact liver tissue, and hepatocytes, in addition to NPCs. In situ perfusion via the portal vein leads to efficient liver digestion. NPCs are enriched from the resulting single-cell suspension by differential and gradient centrifugation. The NPCs can by analyzed or sorted into highly enriched populations using flow cytometry. The isolated cells are suitable for flow cytometry, protein, and mRNA analyses as well as primary culture.

  1. Pancreas donation for islet transplantation.

    PubMed

    Frutos, M A; Ruiz, P; Mansilla, J J

    2005-04-01

    Islet transplantation, though still in the experimental phase, is a therapeutic option that has opened new expectations for the control of diabetes mellitus. Initial results are encouraging for the significant advantages compared with whole pancreas transplantation for selected patients with type 1 diabetes mellitus, with or without kidney failure. However, the success of transplantation, both at centers with more experience and others with less, is limited by the difficulty in obtaining a suitable number of donors and by laboratory isolation techniques. Significant advances require changes in donor selection, perfusion, oxygenation, and transfer of the pancreas, and in the process of isolation, purification, and culture in the laboratory. Of the 32 pancreases sent to the islet isolation laboratory from different hospitals in Andalusia, a viable percentage of islets was finally available in 19. However, in only 4 (18%) procedures were the preparations considered optimal for implantation in 2 recipients. PMID:15866673

  2. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells.

    PubMed

    Reichert, Maximilian; Takano, Shigetsugu; Heeg, Steffen; Bakir, Basil; Botta, Gregory P; Rustgi, Anil K

    2013-01-01

    The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). PDAC resembles duct cells morphologically and, to some extent, at a molecular level. Recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and ductlike cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. This approach is fast (~4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas. PMID:23787893

  3. Isolation, culture and genetic manipulation of mouse pancreatic ductal cells

    PubMed Central

    Reichert, Maximilian; Takano, Shigetsugu; Heeg, Steffen; Bakir, Basil; Botta, Gregory P; Rustgi, Anil K

    2014-01-01

    The most common subtype of pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC). pDac resembles duct cells morphologically and, to some extent, at a molecular level. recently, genetic-lineage labeling has become popular in the field of tumor biology in order to study cell-fate decisions or to trace cancer cells in the mouse. However, certain biological questions require a nongenetic labeling approach to purify a distinct cell population in the pancreas. Here we describe a protocol for isolating mouse pancreatic ductal epithelial cells and duct-like cells directly in vivo using ductal-specific Dolichos biflorus agglutinin (DBA) lectin labeling followed by magnetic bead separation. Isolated cells can be cultured (in two or three dimensions), manipulated by lentiviral transduction to modulate gene expression and directly used for molecular studies. this approach is fast (∼4 h), affordable, results in cells with high viability, can be performed on the bench and is applicable to virtually all genetic and nongenetic disease models of the pancreas. PMID:23787893

  4. Partial regeneration of beta-cells in the islets of Langerhans by Nymphayol a sterol isolated from Nymphaea stellata (Willd.) flowers.

    PubMed

    Subash-Babu, P; Ignacimuthu, S; Agastian, P; Varghese, Babu

    2009-04-01

    Reduction of the beta-cell mass is critical in the pathogenesis of diabetes mellitus. The discovery of agents which induce regeneration of pancreatic beta-cells would be useful to develop new therapeutic approaches to treat diabetes. The present study was aimed at identifying a new agent for the control of diabetes through regeneration of pancreatic beta cells and insulin secretory potential. Nymphaea stellata flower chloroform extract (NSFCExt) showed significant plasma glucose lowering effect. Further NSFCExt was utilized to isolate and identify the lead compound based on bioassay guided fractionation; we found Nymphayol (25,26-dinorcholest-5-en-3beta-ol) a new crystal [space group P2(1) (No. 4), a=9.618(5), b=7.518(5), c=37.491(5)]. It was purified by repeat column. The structure was determined on the basis of X-ray crystallography and spectral data. Oral administration of Nymphayol for 45 days significantly (p<0.05) lowered the blood glucose level and more importantly it effectively increased the insulin content in diabetic rats. In addition, Nymphayol increased the number of beta cell mass enormously. Islet-like cell clusters in the islets of Langerhans were clearly observed based on histochemical and immunohistochemical study. PMID:19272781

  5. Human Pancreatic Islets and Diabetes Research

    PubMed Central

    Kaddis, John S.; Olack, Barbara J.; Sowinski, Janice; Cravens, James; Contreras, Juan L.; Niland, Joyce C.

    2013-01-01

    Human islet research is crucial to understanding the cellular biology of the pancreas in developing therapeutic options for diabetes patients and in attempting to prevent the development of this disease. The national Islet Cell Resource Center Consortium provides human pancreatic islets for diabetes research while simultaneously addressing the need to improve islet isolation and transplantation technologies. Since its inception in 2001, the consortium has supplied 297.6 million islet equivalents to 151 national and international scientists for use in clinical and laboratory projects. Data on the volume, quality, and frequency of shipments substantiate the importance of human islets for diabetes research, as do the number of funded grants for beta-cell projects and publications produced as a direct result of islets supplied by this resource. Limitations in using human islets are discussed, along with the future of islet distribution centers. The information presented here is instructive to clinicians, basic science investigators, and policy makers who determine the availability of funding for such work. Organ procurement coordinators also may find the information useful in explaining to donor families why research consent is so valuable. PMID:19366778

  6. Improvement in Isolation and Identification of Mouse Oogonial Stem Cells

    PubMed Central

    Lu, Zhiyong; Wu, Meng; Zhang, Jinjin; Xiong, Jiaqiang; Cheng, Jing; Shen, Wei; Luo, Aiyue; Fang, Li; Wang, Shixuan

    2016-01-01

    Female germline stem cells (FGSCs) or oogonial stem cells (OSCs) have the capacity to generate newborn oocytes and thus open a new door to fight ovarian aging and female infertility. However, the production and identification of OSCs are difficult for investigators. Rare amount of these cells in the ovary results in the failure of the acquisition of OSCs. Furthermore, the oocyte formation by OSCs in vivo was usually confirmed using tissue sections by immunofluorescence or immunohistochemistry in previous studies. STO or MEF feeder cells are derived from mouse, not human. In our study, we modified the protocol. The cells were digested from ovaries and cultured for 2-3 days and then were purified by magnetic-activated cell sorting (MACS). The ovaries and fetus of mice injected with EGFP-positive OSCs were prepared and put on the slides to directly visualize oocyte and progeny formation under microscope. Additionally, the human umbilical cord mesenchymal stem cells (hUC-MSCs) were also used as feeder cells to support the proliferation of OSCs. The results showed that all the modified procedures can significantly improve and facilitate the generation and characterization of OSCs, and hUC-MSCs as feeder will be useful for isolation and proliferation of human OSCs avoiding contamination from mouse. PMID:26635882

  7. Neuronal control of heart rate in isolated mouse atria.

    PubMed

    Choate, J K; Feldman, R

    2003-09-01

    A novel mouse isolated atrial preparation with intact postganglionic autonomic innervation was used to investigate the neuronal control of heart rate. To establish whether autonomic activation was likely to alter heart rate by modulating the hyperpolarization-activated current (If), the L-type Ca2+ current (ICa,L), or the ACh-activated K+ current (IK,ACh), the effects of nerve stimulation (right stellate ganglion or right vagus, 1-30 Hz) and autonomic agonists (0.1 microM norepinephrine or 0.3 microM carbachol) on heart rate were investigated in the presence of inhibitors of these currents, cesium chloride (Cs+, 1 mM), nifedipine (200 nM), and barium chloride (Ba2+, 0.1 mM), respectively. The positive chronotropic response to stellate ganglion stimulation was reduced by approximately 20% with Cs+ and nifedipine (P < 0.05), whereas the heart rate response to norepinephrine was only reduced with Cs+ (P < 0.05). Ba2+ attenuated the decrease in heart rate with vagal stimulation and carbachol by approximately 60% (P < 0.05). These results are consistent with the idea that sympathetic nerve stimulation modulates If to increase heart rate in the mouse. Activation of ICa,L also appears to contribute to the sympathetic heart rate response. However, the decrease in heart rate with vagal stimulation or carbachol is likely to result primarily from the activation of IK,ACh.

  8. Mouse lysozyme M gene: isolation, characterization, and expression studies.

    PubMed Central

    Cross, M; Mangelsdorf, I; Wedel, A; Renkawitz, R

    1988-01-01

    We have isolated and characterized both cDNA and genomic DNA of the mouse lysozyme M gene. Derivation of the amino acid sequence from the nucleotide sequences revealed six positions in the carboxyl terminus that differ from partial sequences previously published. The differential detection of specific mRNAs from the closely related lysozyme M and P genes has revealed different but overlapping tissue specificities of expression. The M gene is expressed weakly in myeloblasts, moderately in immature macrophages, and strongly in both mature macrophages and macrophage-rich tissues, while high levels of P transcripts are present only in small intestine. Sites of protein accumulation, rather than gene expression, have been identified by comparative quantitation of mRNA and enzyme levels. Images PMID:3413093

  9. Enumeration of islets by nuclei counting and light microscopic analysis.

    PubMed

    Pisania, Anna; Papas, Klearchos K; Powers, Daryl E; Rappel, Michael J; Omer, Abdulkadir; Bonner-Weir, Susan; Weir, Gordon C; Colton, Clark K

    2010-11-01

    Islet enumeration in impure preparations by conventional dithizone staining and visual counting is inaccurate and operator dependent. We examined nuclei counting for measuring the total number of cells in islet preparations, and we combined it with morphological analysis by light microscopy (LM) for estimating the volume fraction of islets in impure preparations. Cells and islets were disrupted with lysis solution and shear, and accuracy of counting successively diluted nuclei suspensions was verified with (1) visual counting in a hemocytometer after staining with crystal violet, and automatic counting by (2) aperture electrical resistance measurement and (3) flow cytometer measurement after staining with 7-aminoactinomycin-D. DNA content averaged 6.5 and 6.9 pg of DNA per cell for rat and human islets, respectively, in agreement with literature estimates. With pure rat islet preparations, precision improved with increasing counts, and samples with about ≥160 islets provided a coefficient of variation of about 6%. Aliquots of human islet preparations were processed for LM analysis by stereological point counting. Total nuclei counts and islet volume fraction from LM analysis were combined to obtain the number of islet equivalents (IEs). Total number of IE by the standard method of dithizone staining/manual counting was overestimated by about 90% compared with LM/nuclei counting for 12 freshly isolated human islet research preparations. Nuclei counting combined with islet volume fraction measurements from LM is a novel method for achieving accurate islet enumeration. PMID:20697375

  10. [Isolation and purification of primary Kupffer cells from mouse liver].

    PubMed

    Sun, Chao; Luo, Qingbo; Lu, Xiuxian; Zheng, Daofeng; He, Diao; Wu, Zhongjun

    2016-08-01

    Objective To isolate and purify Kupffer cells (KCs) from BALB/c mice by an efficient method of low-speed centrifugation and rapid adherence. Methods The mouse liver tissue was perfused in situ and digested with 0.5 g/L collagenase type IV in vitro by water bath. Then, through the low-speed centrifugation, KCs were separated from the mixed hepatocytes, and purified by rapid adherent characteristics. Finally, the production and activity of KCs obtained by this modified method were compared with those isolated by Percoll density gradient centrifugation. We used F4/80 antibody immunofluorescence technique to observe morphological features of KCs, flow cytometry (FCM) to detect the expression of F4/80 antibody and the ink uptake test to observe the phagocytic activity. Moreover, using FCM, we evaluated the expressions of molecules associated with antigen presentation, including major histocompatibility complex class II (MHC II), CD40, CD86 and CD68 on the surface of KCs subjected to hypoxia/reoxygenation (H/R) modeling. And, ELISA was conducted to measure tumor necrosis factor-α (TNF-α) production of the cultured KCs following H/R. Results The yield of KCs was (5.83±0.54)×10(6) per mouse liver and the survival rate of KCs was up to 92% by low-speed centrifugation and rapid adherent method. Compared with Percoll density gradient centrifugation [the yield of KCs was (2.19±0.43)×10(6) per liver], this new method significantly improved the yield of KCs. F4/80 immunofluorescence showed typical morphologic features of KCs such as spindle or polygon shapes and FCM identified nearly 90% F4/80 positive cells. The phagocytic assay showed that lots of ink particles were phagocytosed into the isolated cells. KC H/R models expressed more MHC II, CD40 and CD86 and produced more TNF-α participating in inflammation. Conclusion The efficient method to isolate and purify KCs from BALB /c mice has been successfully established. PMID:27412929

  11. [Isolation and purification of primary Kupffer cells from mouse liver].

    PubMed

    Sun, Chao; Luo, Qingbo; Lu, Xiuxian; Zheng, Daofeng; He, Diao; Wu, Zhongjun

    2016-08-01

    Objective To isolate and purify Kupffer cells (KCs) from BALB/c mice by an efficient method of low-speed centrifugation and rapid adherence. Methods The mouse liver tissue was perfused in situ and digested with 0.5 g/L collagenase type IV in vitro by water bath. Then, through the low-speed centrifugation, KCs were separated from the mixed hepatocytes, and purified by rapid adherent characteristics. Finally, the production and activity of KCs obtained by this modified method were compared with those isolated by Percoll density gradient centrifugation. We used F4/80 antibody immunofluorescence technique to observe morphological features of KCs, flow cytometry (FCM) to detect the expression of F4/80 antibody and the ink uptake test to observe the phagocytic activity. Moreover, using FCM, we evaluated the expressions of molecules associated with antigen presentation, including major histocompatibility complex class II (MHC II), CD40, CD86 and CD68 on the surface of KCs subjected to hypoxia/reoxygenation (H/R) modeling. And, ELISA was conducted to measure tumor necrosis factor-α (TNF-α) production of the cultured KCs following H/R. Results The yield of KCs was (5.83±0.54)×10(6) per mouse liver and the survival rate of KCs was up to 92% by low-speed centrifugation and rapid adherent method. Compared with Percoll density gradient centrifugation [the yield of KCs was (2.19±0.43)×10(6) per liver], this new method significantly improved the yield of KCs. F4/80 immunofluorescence showed typical morphologic features of KCs such as spindle or polygon shapes and FCM identified nearly 90% F4/80 positive cells. The phagocytic assay showed that lots of ink particles were phagocytosed into the isolated cells. KC H/R models expressed more MHC II, CD40 and CD86 and produced more TNF-α participating in inflammation. Conclusion The efficient method to isolate and purify KCs from BALB /c mice has been successfully established.

  12. 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

    PubMed Central

    Samad, Mehdi Bin; Kabir, Ashraf Ul; Ahmed, Arif; Jahan, Mohammad Rajib; Hannan, J. M. A.

    2014-01-01

    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. PMID:24860609

  13. Human Serum Versus Human Serum Albumin Supplementation in Human Islet Pretransplantation Culture: In Vitro and In Vivo Assessment.

    PubMed

    Nacher, Montserrat; Estil Les, Elisabet; Garcia, Ainhoa; Nadal, Belen; Pairó, Mar; Garcia, Cristofer; Secanella, Lluís; Novials, Anna; Montanya, Eduard

    2016-01-01

    There is conflicting evidence favoring both the use of human serum (HS) and of human serum albumin (HSA) in human islet culture. We evaluated the effects of HS versus HSA supplementation on 1) in vitro β-cell viability and function and 2) in vivo islet graft revascularization, islet viability, β-cell death, and metabolic outcome after transplantation. Islets isolated from 14 cadaveric organ donors were cultured for 3 days in CMRL 1066 medium supplemented with HS or HSA. After 3 days in culture, β-cell apoptosis was lower in HS group (1.41 ± 0.27 vs. 2.38 ± 0.39%, p = 0.029), and the recovery of islets was 77 ± 11% and 54 ± 1% in HS- and HSA-cultured groups, respectively. Glucose-stimulated insulin secretion (GSIS) was higher in HS group (29.4, range 10.4-99.9, vs. 22.3, range 8.7-70.6, p = 0.031). In vivo viability and revascularization was determined in HS- and HSA-cultured islets transplanted into the anterior chamber of the eye of Balb/c mice (n = 14), and β-cell apoptosis in paraffin-embedded mouse eyes. Islet viability and β-cell apoptosis were similar in both groups. Revascularization was observed in one graft (HS group) on day 10 after transplantation. Islet function was determined in streptozotocin (STZ)-diabetic nude mice (n = 33) transplanted with 2,000 IEQs cultured with HS or HSA that showed similar blood glucose levels and percentage of normoglycemic animals over time. In conclusion, human islets cultured in medium supplemented with HS showed higher survival in vitro, as well as islet viability and function. The higher in vitro survival increased the number of islets available for transplantation. However, the beneficial effect on viability and function did not translate into an improved metabolic evolution when a similar number of HSA- and HS-cultured islets was transplanted. PMID:25955150

  14. Islet1 regulates establishment of the posterior hindlimb field upstream of the Hand2-Shh morphoregulatory gene network in mouse embryos.

    PubMed

    Itou, Junji; Kawakami, Hiroko; Quach, Thu; Osterwalder, Marco; Evans, Sylvia M; Zeller, Rolf; Kawakami, Yasuhiko

    2012-05-01

    How divergent genetic systems regulate a common pathway during the development of two serial structures, forelimbs and hindlimbs, is not well understood. Specifically, HAND2 has been shown to regulate Shh directly to initiate its expression in the posterior margin of the limb mesenchyme. Although the Hand2-Shh morphoregulatory system operates in both the forelimb and hindlimb bud, a recent analysis suggested that its upstream regulation is different in the forelimb and hindlimb bud. A combination of all four Hox9 genes is required for Hand2 expression in the forelimb-forming region; however, it remains elusive what genetic system regulates the Hand2-Shh pathway in the hindlimb-forming region. By conditional inactivation of Islet1 in the hindlimb-forming region using the Hoxb6Cre transgene, we show that Islet1 is required for establishing the posterior hindlimb field, but not the forelimb field, upstream of the Hand2-Shh pathway. Inactivation of Islet1 caused the loss of posterior structures in the distal and proximal regions, specifically in the hindlimb. We found that Hand2 expression was downregulated in the hindlimb field and that Shh expression was severely impaired in the hindlimb bud. In the Hoxb6Cre; Islet1 mutant pelvis, the proximal element that is formed in a Shh-independent manner, displayed complementary defects in comparison with Pitx1(-/-) hindlimbs. This suggests that Islet1 and Pitx1 function in parallel during girdle development in hindlimbs, which is in contrast with the known requirement for Tbx5 in girdle development in forelimbs. Our studies have identified a role for Islet1 in hindlimb-specific development and have revealed Islet1 functions in two distinct processes: regulation upstream of the Hand2-Shh pathway and contributions to girdle development.

  15. Magnesium deficiency improves glucose homeostasis in the rat: studies in vivo and in isolated islets in vitro.

    PubMed

    Reis, M A; Latorraca, M Q; Carneiro, E M; Boschero, A C; Saad, M J; Velloso, L A; Reyes, F G

    2001-05-01

    The serum mineral levels, glucose disappearance rate (kg), total area under the glucose (DeltaG) and insulin (DeltaI) curves, and static insulin secretion were compared among rats fed a Mg-deficient diet for 6 (DF-6) or 11 (DF-11) weeks, and rats fed a control diet for the same periods (CO-6 and CO-11 groups). No change in glucose homeostasis was observed among DF-6, CO-6 and CO-11 rats. DF-11 rats showed an elevated kg and a reduced DeltaG and DeltaI. For evaluating the effect of supplementation, rats fed a control or Mg-deficient diet for 6 weeks were then fed a Mg- supplemented diet for 5 weeks (SCO and SDF groups respectively). The serum Mg levels in SDF rats were similar to those in CO-11 and SCO rats, but higher than in the DF-11 group. SDF rats showed similar kg, DeltaG and DeltaI compared with the CO-11 and SCO groups. However, a significantly lower kg and higher DeltaG and DeltaI were observed in SDF compared with DF-11 rats. Basal and 8.3 mmol glucose/l-stimulated insulin secretion by islets from DF-11 rats were higher than by islets from CO-11 rats. These results indicate that moderate Mg depletion for a long period may increase the secretion and sensitivity to insulin, while Mg supplementation in formerly Mg-deficient rats may prevent the increase in sensitivity and secretion of insulin. PMID:11348569

  16. DISCOVERY OF NOVEL GLUCOSE-REGULATED PROTEINS IN ISOLATED HUMAN PANCREATIC ISLETS USING LC-MS/MS-BASED PROTEOMICS

    PubMed Central

    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.

    2012-01-01

    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. PMID:22578083

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

    SciTech Connect

    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.

  18. Secretagogue-induced diacylglycerol accumulation in isolated pancreatic islets. Mass spectrometric characterization of the fatty acyl content indicates multiple mechanisms of generation

    SciTech Connect

    Wolf, B.A.; Easom, R.A.; Hughes, J.H.; McDaniel, M.L.; Turk, J. )

    1989-05-16

    Diacylglycerol accumulation has been examined in secretagogue-stimulated pancreatic islets with a newly developed negative ion chemical ionization mass spectrometric method. The muscarinic agonist carbachol induces islet accumulation of diacylglycerol rich in arachidonate and stearate, and a parallel accumulation of {sup 3}H-labeled diacylglycerol occurs in carbachol-stimulated islets that had been prelabeled with ({sup 3}H)glycerol. Islets so labeled do not accumulate {sup 3}H-labeled diacylglycerol in response to D-glucose, but D-glucose does induce islet accumulation of diacylglycerol by mass. This material is rich in palmitate and oleate and contains much smaller amounts of arachidonate. Neither secretagogue influences triacylglycerol labeling, and neither induces release of ({sup 3}H)choline or ({sup 3}H)phosphocholine from islets prelabeled with ({sup 3}H)choline. These observations indicate that the diacylglycerol that accumulates in islets in response to carbachol arises from hydrolysis of glycerolipids, probably including phosphoinositides. The bulk of the diacylglycerol which accumulates in response to glucose does not arise from glycerolipid hydrolysis and must therefore reflect de novo synthesis. The endogenous diacylglycerol which accumulates in secretagogue-stimulated islets may participate in insulin secretion because exogenous diacylglycerol induces insulin secretion from islets, and an inhibitor of diacylglycerol metabolism to phosphatidic acid augments glucose-induced insulin secretion.

  19. Efficient culture of CD8+ T cells from the islets of NOD mice and their use for the study of autoreactive specificities

    PubMed Central

    Jarchum, Irene; Takaki, Toshiyuki; DiLorenzo, Teresa P.

    2008-01-01

    During progression to type 1 diabetes (T1D), the pancreatic islets of humans and of the widely studied mouse model of T1D, the nonobese diabetic (NOD) mouse, are infiltrated by cells of the immune system. Here we report that infiltrated mouse islets (“translucent islets”) can be identified visually. We demonstrate the use of an efficient method for the isolation and culture of the islet-infiltrating cells of NOD mice, which results in a high percentage of CD8+ T cells after seven days, with minimal manipulation. We show that islet-infiltrating cells exit the islets soon after they are placed in culture and can be used in flow cytometric experiments several hours later. Importantly, we demonstrate that the cultured cells are antigen-responsive and that specificities present at the beginning of the culture are generally still present on day seven. In addition, some reactivities are undetected without culture, supporting the validity of the seven-day expansion period. This technique greatly facilitates investigations of the CD8+ T cell reactivities that play a pivotal role in the demise of pancreatic beta cells leading to the development of T1D. PMID:18782577

  20. Pancreatic tissue resident mesenchymal stromal cell (MSC)-like cells as a source of in vitro islet neogenesis.

    PubMed

    Gopurappilly, Renjitha; Bhat, Vijay; Bhonde, Ramesh

    2013-10-01

    Insufficient β-cell mass is a common denominator for both type 1 and type 2 diabetes. In vitro generation of β-cells from islet precursor cells, exocrine cells or ductal epithelia provide an alternative source of insulin-producing cells. However the presence of multipotent precursor cells within the pancreas is also deliberated. In this study we isolated mesenchymal stromal cell (MSC)-like cells from adult mouse pancreas by collagenase digestion. We used Knockout DMEM for our isolation procedure and the floating islets and acini were removed after 48 h. This strategy permitted the adhesion of stromal cells with typical mesenchymal morphology. These cells not only expressed MSC-specific markers like Sca-1, CD90.2, CD73, and CD44 but also generated osteocytes, adipocytes, and neurons when induced with specific growth media. Upon exposure to islet differentiation serum-free cocktail a significant upregulation of pancreatic markers like Nkx2.2, Nkx6.1, Pdx1, insulin, and somatostatin was seen. The differentiated islet-like cell aggregates (ICAs) secreted insulin which increased over the days in culture in presence of basal glucose levels. Taken together, our data strongly indicate that there is a tissue-resident precursor population within the pancreas that can be exploited for islet neogenesis in vitro. PMID:23606308

  1. Pancreatic Islet Cell Development and Regeneration

    PubMed Central

    Romer, Anthony I.; Sussel, Lori

    2015-01-01

    Purpose This review will discuss recent advances in understanding mouse and human pancreatic islet cell development, novel concepts related to β cell dysfunction and improved approaches for replenishing β cells to treat diabetes. Recent Findings Considerable knowledge about pancreatic islet development and function has been gained using model systems with subsequent validation in human tissues. Recently, several rodent studies have revealed that differentiated adult islet cells retain remarkable plasticity and can be converted to other islet cell types by perturbing their transcription factor profiles. Furthermore, significant advances have been made in the generation of β-like cells from stem cell populations. Therefore, the generation of functionally mature β cells by the in situ conversion of non-β cell populations or by the directed differentiation of human pluripotent stem cells could represent novel mechanisms for replenishing β cells in diabetic patients. Summary The overall conservation between mouse and human pancreatic development, islet physiology and etiology of diabetes encourages the translation of novel β cell replacement therapies to humans. Further deciphering the molecular mechanisms that direct islet cell regeneration, plasticity and function could improve and expand the β cell replacement strategies for treating diabetes. PMID:26087337

  2. Osteogenic cell fractions isolated from mouse tongue muscle

    PubMed Central

    HARADA, KOJI; HARADA, TOYOKO; FERDOUS, TARANNUM; TAKENAWA, TAKANORI; UEYAMA, YOSHIYA

    2015-01-01

    The use of stem cells represents a promising approach for the treatment of bone defects. However, successful treatments rely upon the availability of cells that are easily obtained and that appropriately differentiate into osteoblasts. The tongue potentially represents a source of autologous cells for such purposes. In the present study, the ability of stem cell antigen-1 (Sca-1) positive cells derived from tongue muscle to differentiate into osteoblasts was investigated. The tongue muscles were excised from Jcl-ICR mice and tongue muscle-derived Sca-1-positive cells (TDSCs) were isolated from the tongue muscle using a magnetic cell separation system with microbeads. TDSCs were cultured in plastic dishes or gelatin sponges of β-tricalcium phosphate (β-TCP) with bone differentiation-inducing medium. The expression of osteogenic markers (Runx2, osterix, alkaline phosphatase, fibronectin, osteocalcin, osteonectin and osteopontin) was investigated in cultured TDSCs by western blot analysis. The formation of mineralized matrices was examined using alizarin red S and Von Kossa staining. Bone formation was investigated in cultured TDSCs by hematoxylin-eosin staining and immunohistochemstry. In the present study, the expression of Sca-1 in mouse tongue muscle was demonstrated and TDSCs were isolated at high purity. TDSCs differentiated into cells of osteoblast lineage, as demonstrated by the upregulation of osteoblastic marker expression. The formation of mineralized matrices was confirmed by alizarin red S or Von Kossa staining in vitro. Bone formation was observed in the gelatin sponges of β-TCP, which were subsequently implanted under the skin of the backs of nude mice. These results suggested that TDSCs retain their osteogenic differentiation potential and therefore the tongue muscle may be used as a source of stem cells for bone regeneration. PMID:25684092

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

    PubMed

    Zhao, Qing; Che, Yongzhe; Li, Qiang; Zhang, Shangrong; Gao, Ying-Tang; Wang, Yifan; Wang, Xudong; Xi, Wang; Zuo, Weiyan; Li, Shu Jie

    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(+)-induced insulin secretion in isolated islets and INS-1 (832/13) β-cells and has an impairment on glucose- and K(+)-induced intracellular Ca(2+) homeostasis. Our data demonstrated that the expression of Hv1 in pancreatic islet β-cells regulates insulin secretion through regulating Ca(2+) homeostasis. PMID:26559003

  4. Trimeprazine increases IRS2 in human islets and promotes pancreatic β cell growth and function in mice

    PubMed Central

    Kuznetsova, Alexandra; Yu, Yue; Hollister-Lock, Jennifer; Opare-Addo, Lynn; Rozzo, Aldo; Sadagurski, Marianna; Norquay, Lisa; Reed, Jessica E.; El Khattabi, Ilham; Bonner-Weir, Susan; Weir, Gordon C.; Sharma, Arun

    2016-01-01

    The capacity of pancreatic β cells to maintain glucose homeostasis during chronic physiologic and immunologic stress is important for cellular and metabolic homeostasis. Insulin receptor substrate 2 (IRS2) is a regulated adapter protein that links the insulin and IGF1 receptors to downstream signaling cascades. Since strategies to maintain or increase IRS2 expression can promote β cell growth, function, and survival, we conducted a screen to find small molecules that can increase IRS2 mRNA in isolated human pancreatic islets. We identified 77 compounds, including 15 that contained a tricyclic core. To establish the efficacy of our approach, one of the tricyclic compounds, trimeprazine tartrate, was investigated in isolated human islets and in mouse models. Trimeprazine is a first-generation antihistamine that acts as a partial agonist against the histamine H1 receptor (H1R) and other GPCRs, some of which are expressed on human islets. Trimeprazine promoted CREB phosphorylation and increased the concentration of IRS2 in islets. IRS2 was required for trimeprazine to increase nuclear Pdx1, islet mass, β cell replication and function, and glucose tolerance in mice. Moreover, trimeprazine synergized with anti-CD3 Abs to reduce the progression of diabetes in NOD mice. Finally, it increased the function of human islet transplants in streptozotocin-induced (STZ-induced) diabetic mice. Thus, trimeprazine, its analogs, or possibly other compounds that increase IRS2 in islets and β cells without adverse systemic effects might provide mechanism-based strategies to prevent the progression of diabetes. PMID:27152363

  5. Necessities for a Clinical Islet Program.

    PubMed

    Hawthorne, Wayne J

    2016-01-01

    For more than two decades we have been refining advances in islet cell transplantation as a clinical therapy for patients suffering from type 1 diabetes. A great deal of effort has gone to making this a viable therapy for a broader range of patients with type 1 diabetes. Clinical results have progressively improved, demonstrating clinical outcomes on par with other organ transplants, specifically in terms of insulin independence, graft and patient survival. We are now at the point where islet cell transplantation, in the form of allotransplantation, has become accepted as a clinical therapy in adult patients affected by type 1 diabetes, in particular those suffering from severe hypoglycaemic unawareness. This chapter provides an overview on how this has been undertaken over the years to provide outcomes on par with other organ transplantation results. In particular this chapter focuses on the processes and facilities that are required to establish a clinical islet isolation and transplantation program. It also outlines the very important underpinning processes of selection of the organ donor for islet isolation, the processes of organ donor operation and preservation of the pancreas by various means and the ideal ways to best improve outcomes for human islet cell isolation. Providing these more optimal conditions we can underpin the isolation processes to provide islets for transplantation and as such a safe, effective and feasible therapeutic option for an increasing number of patients suffering from type 1 diabetes with severe hypoglycaemic unawareness. PMID:27586423

  6. Islet Cell Transplantation

    MedlinePlus

    ... the body use glucose for energy. Islet cell transplantation transfers cells from an organ donor into the ... to make and release insulin. Researchers hope islet transplantation will help people with type 1 diabetes live ...

  7. Pancreatic Islet Transplantation

    MedlinePlus

    ... allo-transplantation?" For each pancreatic islet allo-transplant infusion, researchers use specialized enzymes to remove islets from ... in a lab. Transplant patients typically receive two infusions with an average of 400,000 to 500, ...

  8. The effect of time and temperature on viability and performance of Langerhans islets separated from Balb/c mouse after death

    PubMed Central

    Ghorbani, Rostam; Jalili, Cyrus; Salahshoor, Mohammad Reza; Shiasi, Maryam

    2015-01-01

    Background: Tissue transplantation plays a pivotal role in the treatment of diseases. Pancreatic beta cell transplantation is the best way to obtain normal blood glucose in patients with diabetes type 1. However, it is not clear how long endocrine pancreas cells can be used for transplantation after the donor's death. The present study was conducted to analyze the performance and viability of pancreatic islet cells after death. Materials and Methods: Pancreas was separated from Balb/c mice at different times (0, 1, 4, 6, 12, and 24 h after death) at temperatures of 4°C and 23°C, and was cultured in Roswell_Park_Memorial_Institute (RPMI) 1640. Insulin shock, MTT assay, aldehyde fuchsin staining, dithizone staining, and florescence microscopy methods were applied to analyze the performance of beta cells, cell viability, islets’ diagnosis, islet cells’ diagnosis, and viable and necrotic cells diagnosis, respectively. Results: Islets of Langerhans and beta cells were diagnosed. By increasing the temperature and time, the viability and performance of beta cells decreased significantly (P < 0.05). Conclusion: The best condition for keeping the islets of Langerhans in terms of viability and performance is 4 h after death at temperature of 4°C. PMID:26015919

  9. Pancreatic islet cell transplantation using non-heart-beating donors (NHBDs).

    PubMed

    Matsumoto, Shinichi; Tanaka, Koichi

    2005-01-01

    Recent dramatic improvements in clinical islet cell transplantation demonstrated by the Edmonton group have increased the demand for this treatment, and donor shortage could become a major problem. Utilization of marginal donors could alleviate the donor shortage, and non-heart-beating donors (NHBDs) might be good resources. The University of Pennsylvania group demonstrated that it was possible to isolate islets from NHBDs, and the group actually transplanted islets from NHBDs, for the first time. The patient became insulin-independent; however, there had been no more cases using NHBDs until our group initiated islet transplantations from NHBDs in Japan. In order to utilize NHBDs effectively, we modified the standard islet isolation method. These modifications included minimizing the warm ischemic time, the use of trypsin inhibition during isolation, carrying out density measurement before purification and the use of a less toxic islet purification solution. With these modifications we were able to transplant nine of ten islet preparations from ten NHBDs (90%), into five type-1 diabetic patients. The first transplantation was performed on April 7, 2004 (the first time in Japan), and this patient became insulin-independent after the second islet transplantation (first time in Japan). All patients showed improved glycemic control and reduced insulin requirements, without hypoglycemic events. We also performed living-donor islet transplantation, with our modified islet isolation protocol, on January 19, 2005. The improved islet isolation protocol enabled us to perform effective islet transplantations from NHBDs, and it also enabled us to perform the living-donor islet transplantation.

  10. Characterization of isolated mouse cerebellar cell populations in vitro.

    PubMed

    Schnitzer, J; Schachner, M

    1981-12-01

    Cells from early postnatal mouse cerebellar cortex were isolated by discontinuous BSA gradient centrifugation. Three cellular fractions were obtained and called A (interface at 0-10% BSA), B ( 10-15%) and C (15-25%). These fractions were characterized after maintenance in vitro for 3 days by indirect immunofluorescence labeling with several cell type-specific probes: Tetanus toxin was used as a neuronal marker.Under the described culture conditions Thy-1.2 antibodies served as additional markers for mature neurons and NS-4 antiserum for neurons and oligodendroglial cells. Glial fibrillary acidic (GFA) protein was used as a marker for differentiated astroglia, and fibronectin as a marker for fibroblasts. Monoclonal antibodies to 04 antigen and antiserum to corpus callosum served to distinguish oligodendroglia. Fraction C contains most of the cellular debris and cells with large cell bodies (about 20 micrometers in diameter) which are positive for Thy-1, NS-4, and tetanus toxin. By birthdate labeling with [3H]thymidine these cells can be identified as Purkinje cells and/or Golgi type II cells. Fraction B is relatively heterogeneous. It contains predominantly GFA protien-positive astroglial cells (about 50% of all cells) which can be classified into 3 morphologically distinct cell types, flat epithelioid cells and star-shaped cells with thick or very thin cellular processes. Fraction B is enriched also in 04 antigen-positive oligodendrocytes, fibronectin-positive fibroblasts and Thy-1 negative, but NS-4 and tetanus toxin positive cells with small cell bodies and many fine processes. These small neurons, putative stellate and basket cells, have many fine processes and are morphologically different from th bipolar putative granule cells, some of which are also present in this fraction. Fraction C contains predominantly small neurons, mostly putative granule cell (more than 0% of all cells) which are positive for NS-4 and tetanus toxin, but negative for Thy-1.

  11. Involvement of a proapoptotic gene (BBC3) in islet injury mediated by cold preservation and rewarming.

    PubMed

    Omori, Keiko; Kobayashi, Eiji; Komatsu, Hirotake; Rawson, Jeffrey; Agrawal, Garima; Parimi, Mounika; Oancea, Alina R; Valiente, Luis; Ferreri, Kevin; Al-Abdullah, Ismail H; Kandeel, Fouad; Takahashi, Masafumi; Mullen, Yoko

    2016-06-01

    Long-term pancreatic cold ischemia contributes to decreased islet number and viability after isolation and culture, leading to poor islet transplantation outcome in patients with type 1 diabetes. In this study, we examined mechanisms of pancreatic cold preservation and rewarming-induced injury by interrogating the proapoptotic gene BBC3/Bbc3, also known as Puma (p53 upregulated modulator of apoptosis), using three experimental models: 1) bioluminescence imaging of isolated luciferase-transgenic ("Firefly") Lewis rat islets, 2) cold preservation of en bloc-harvested pancreata from Bbc3-knockout (KO) mice, and 3) cold preservation and rewarming of human pancreata and isolated islets. Cold preservation-mediated islet injury occurred during rewarming in "Firefly" islets. Silencing Bbc3 by transfecting Bbc3 siRNA into islets in vitro prior to cold preservation improved postpreservation mitochondrial viability. Cold preservation resulted in decreased postisolation islet yield in both wild-type and Bbc3 KO pancreata. However, after culture, the islet viability was significantly higher in Bbc3-KO islets, suggesting that different mechanisms are involved in islet damage/loss during isolation and culture. Furthermore, Bbc3-KO islets from cold-preserved pancreata showed reduced HMGB1 (high-mobility group box 1 protein) expression and decreased levels of 4-hydroxynonenal (4-HNE) protein adducts, which was indicative of reduced oxidative stress. During human islet isolation, BBC3 protein was upregulated in digested tissue from cold-preserved pancreata. Hypoxia in cold preservation increased BBC3 mRNA and protein in isolated human islets after rewarming in culture and reduced islet viability. These results demonstrated the involvement of BBC3/Bbc3 in cold preservation/rewarming-mediated islet injury, possibly through modulating HMGB1- and oxidative stress-mediated injury to islets. PMID:27117005

  12. Protein targeting via the "constitutive-like" secretory pathway in isolated pancreatic islets: passive sorting in the immature granule compartment

    PubMed Central

    1992-01-01

    We have suggested the existence of a novel "constitutive-like" secretory pathway in pancreatic islets, which preferentially conveys a fraction of newly synthesized C-peptide, insulin, and proinsulin, and is related to the presence of immature secretory granules (IGs). Regulated exocytosis of IGs results in an equimolar secretion of C- peptide and insulin; however an assay of the constitutive-like secretory pathway recently demonstrated that this route conveys newly synthesized C-peptide in molar excess of insulin (Arvan, P., R. Kuliawat, D. Prabakaran, A.-M. Zavacki, D. Elahi, S. Wang, and D. Pilkey. J. Biol. Chem. 266:14171-14174). We now use this assay to examine the kinetics of constitutive-like secretion. Though its duration is much shorter than the life of mature granules under physiologic conditions, constitutive-like secretion appears comparatively slow (t1/2 approximately equal to 1.5 h) compared with the rate of proinsulin traffic through the ER and Golgi stacks. We have examined whether this slow rate is coupled to the rate of IG exit from the trans-Golgi network (TGN). Escape from the 20 degrees C temperature block reveals a t1/2 less than or equal to 12 min from TGN exit to stimulated release of IGs; the time required for IG formation is too rapid to be rate limiting for constitutive-like secretion. Further, conditions are described in which constitutive-like secretion is blocked yet regulated discharge of IGs remains completely intact. Thus, constitutive-like secretion appears to represent an independent secretory pathway that is kinetically restricted to a specific granule maturation period. The data support a model in which passive sorting due to insulin crystallization results in enrichment of C-peptide in membrane vesicles that bud from IGs to initiate the constitutive-like secretory pathway. PMID:1639842

  13. Islet transplantation at the Diabetes Research Institute Japan.

    PubMed

    Noguchi, Hirofumi; Matsumoto, Shinichi

    2008-01-01

    Since the Edmonton Protocol was announced, more than 600 patients with type 1 diabetes at more than 50 institutions have received islet transplantation to treat their disease. We recently established a new islet isolation protocol, called the Kyoto Islet Isolation Method, based on the Ricordi method. It includes an in-situ cooling system for pancreas procurement, pancreatic ductal protection, a modified two-layer (M-Kyoto /perfluorochemical [PFC]) method of pancreas preservation, and a new islet purification solution (Iodixanol-based solution). Using this islet isolation method, we isolated islets from 19 human pancreata of non-heart-beating donors and transplanted 16 preparations into seven patients with type 1 diabetes between April 7, 2004 and November 18, 2005. The percentage of those meeting the release criteria of the Edmonton Protocol was more than 80%. We also performed living-donor transplantation of islets for unstable diabetes on January 19, 2005. Establishment of this method enables us to make diabetic patients insulin-independent, using islets not only from two or three pancreata of non-heart-beating donors but also using islets from half a pancreas from a living donor.

  14. Quadrupole Magnetic Sorting of Porcine Islets of Langerhans

    PubMed Central

    Shenkman, Rustin M.; Chalmers, Jeffrey J.; Hering, Bernhard J.; Kirchhof, Nicole

    2009-01-01

    Islet transplantation is emerging as a treatment option for selected patients with type 1 diabetes. Inconsistent isolation, purification, and recovery of large numbers of high-quality islets remain substantial impediments to progress in the field. Removing islets as soon as they are liberated from the pancreas during digestion and circumventing the need for density gradient purification is likely to result in substantially increased viable islet yields by minimizing exposure to proteolytic enzymes, reactive oxygen intermediates, and mechanical stress associated with centrifugation. This study capitalized on the hypervascularity of islets compared with acinar tissue to explore their preferential enrichment with magnetic beads to enable immediate separation in a magnetic field utilizing a quadrupole magnetic sorting. The results demonstrate that (1) preferential enrichment of porcine islets is achievable, but homogeneous bead distribution within the pancreas is difficult to achieve with current protocols; (2) greater than 70% of islets in the dissociated pancreatic tissue were recovered by quadrupole magnetic sorting, but their purity was low; and (3) infused islets purified by density gradients and subsequently passed through quadrupole magnetic sorting had similar potency as uninfused islets. These results demonstrate proof of concept and define the steps for implementation of this technology in pig and human islet isolation. PMID:19505179

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

    PubMed

    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.

  16. JANEX-1, a JAK3 inhibitor, protects pancreatic islets from cytokine toxicity through downregulation of NF-{kappa}B activation and the JAK/STAT pathway

    SciTech Connect

    Lv, Na; Kim, Eun-Kyung; Song, Mi-Young; Choi, Ha-Na; Moon, Woo Sung; Park, Sung-Joo; Park, Jin-Woo; Kwon, Kang-Beom; Park, Byung-Hyun

    2009-07-15

    JANEX-1/WHI-P131, a selective Janus kinase 3 (JAK3) inhibitor, has been shown to delay the onset of diabetes in the NOD mouse model. However, the molecular mechanism by which JANEX-1 protects pancreatic {beta}-cells is unknown. In the current study, we investigated the role of JANEX-1 on interleukin (IL)-1{beta} and interferon (IFN)-{gamma}-induced {beta}-cell damage using isolated islets. JANEX-1-pretreated islets showed resistance to cytokine toxicity, namely suppressed nitric oxide (NO) production, reduced inducible form of NO synthase (iNOS) expression, and decreased islet destruction. The molecular mechanism by which JANEX-1 inhibits iNOS expression was mediated through suppression of the nuclear factor {kappa}B (NF-{kappa}B) and JAK/signal transducer and activator of transcription (STAT) pathways. Islets treated with the cytokines downregulated the protein levels of suppressor of cytokine signaling (SOCS)-1 and SOCS-3, but pretreatment with JANEX-1 attenuated these decreases. Additionally, islets from JAK3{sup -/-} mice were more resistant to cytokine toxicity than islets from control mice. These results demonstrate that JANEX-1 protects {beta}-cells from cytokine toxicity through suppression of the NF-{kappa}B and JAK/STAT pathways and upregulation of SOCS proteins, suggesting that JANEX-1 may be used to preserve functional {beta}-cell mass.

  17. Improved physiological properties of gravity-enforced reassembled rat and human pancreatic pseudo-islets.

    PubMed

    Zuellig, R A; Cavallari, G; Gerber, P; Tschopp, O; Spinas, G A; Moritz, W; Lehmann, R

    2014-04-16

    Previously we demonstrated the superiority of small islets vs large islets in terms of function and survival after transplantation, and we generated reaggregated rat islets (pseudo-islets) of standardized small dimensions by the hanging-drop culture method (HDCM). The aim of this study was to generate human pseudo-islets by HDCM and to evaluate and compare the physiological properties of rat and human pseudo-islets. Isolated rat and human islets were dissociated into single cells and incubated for 6-14 days by HDCM. Newly formed pseudo-islets were analysed for dimensions, morphology, glucose-stimulated insulin secretion (GSIS) and total insulin content. The morphology of reaggregated human islets was similar to that of native islets, while rat pseudo-islets had a reduced content of α and δ cells. GSIS of small rat and human pseudo-islets (250 cells) was increased up to 4.0-fold (p < 0.01) and 2.5-fold (p < 0.001), respectively, when compared to their native counterparts. Human pseudo-islets showed a more pronounced first-phase insulin secretion as compared to intact islets. GSIS was inversely correlated to islet size, and small islets (250 cells) contained up to six-fold more insulin/cell than large islets (1500 cells). Tissue loss with this new technology could be reduced to 49.2 ± 1.5% in rat islets, as compared to the starting amount. With HDCM, pseudo-islets of standardized size with similar cellular composition and improved biological function can be generated, which compensates for tissue loss during production. Transplantation of small pseudo-islets may represent an attractive strategy to improve graft survival and function, due to better oxygen and nutrient supply during the phase of revascularization. Copyright © 2014 John Wiley & Sons, Ltd.

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

    PubMed

    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. PMID:26370175

  19. Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters.

    PubMed

    Beamish, Christine A; Strutt, Brenda J; Arany, Edith J; Hill, David J

    2016-04-18

    Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive, glucose-transporter-2-low (Ins(+)Glut2(LO)) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future.

  20. Insulin-positive, Glut2-low cells present within mouse pancreas exhibit lineage plasticity and are enriched within extra-islet endocrine cell clusters.

    PubMed

    Beamish, Christine A; Strutt, Brenda J; Arany, Edith J; Hill, David J

    2016-04-18

    Regeneration of insulin-producing β-cells from resident pancreas progenitors requires an understanding of both progenitor identity and lineage plasticity. One model suggested that a rare β-cell sub-population within islets demonstrated multi-lineage plasticity. We hypothesized that β-cells from young mice (postnatal day 7, P7) exhibit such plasticity and used a model of islet dedifferentiation toward a ductal epithelial-cell phenotype to test this theory. RIPCre;Z/AP(+/+) mice were used to lineage trace the fate of β-cells during dedifferentiation culture by a human placental alkaline phosphatase (HPAP) reporter. There was a significant loss of HPAP-expressing β-cells in culture, but remaining HPAP(+) cells lost insulin expression while gaining expression of the epithelial duct cell marker cytokeratin-19 (Ck19). Flow cytometry and recovery of β-cell subpopulations from whole pancreas vs. islets suggest that the HPAP(+)Ck19(+) cells had derived from insulin-positive, glucose-transporter-2-low (Ins(+)Glut2(LO)) cells, representing 3.5% of all insulin-expressing cells. The majority of these cells were found outside of islets within clusters of <5 β-cells. These insulin(+)Glut2(LO) cells demonstrated a greater proliferation rate in vivo and in vitro as compared to insulin(+)Glut2(+) cells at P7, were retained into adulthood, and a subset differentiated into endocrine, ductal, and neural lineages, illustrating substantial plasticity. Results were confirmed using RIPCre;ROSA- eYFP mice. Quantitative PCR data indicated these cells possess an immature β-cell phenotype. These Ins(+)Glut2(LO) cells may represent a resident population of cells capable of forming new, functional β-cells, and which may be potentially exploited for regenerative therapies in the future. PMID:27010375

  1. Molecular and antimicrobial analyses of non-classical Bordetella isolated from a laboratory mouse

    PubMed Central

    LOONG, Shih Keng; MAHFODZ, Nur Hidayana; WALI, Haryanti Azura Mohamad; TALIB, Siti Aisyah A.; NASRAH, Siti Noraisah Ahmad; WONG, Pooi Fong; ABUBAKAR, Sazaly

    2016-01-01

    Accurate identification and separation of non-classical Bordetella species is very difficult. These species have been implicated in animal infections. B. hinzii, a non-classical Bordetella, has been isolated from mice in experimental facilities recently. We isolated and characterized one non-classical Bordetella isolate from the trachea and lung of an ICR mouse. Isolate BH370 was initially identified as B. hinzii by 16S ribosomal DNA and ompA sequencing. Additionally, isolate BH370 also displayed antimicrobial sensitivity profiles similar to B. hinzii. However, analyses of nrdA sequences determined its identity as Bordetella genogroup 16. The isolation of BH370 from a healthy mouse suggests the possibility of it being a commensal. The nrdA gene was demonstrated to possess greater phylogenetic resolution as compared with 16S ribosomal DNA and ompA for the discrimination of non-classical Bordetella species. PMID:26782013

  2. A model utilizing adult murine stem cells for creation of personalized islets for transplantation.

    PubMed

    Wang, J; Song, L J; Gerber, D A; Fair, J H; Rice, L; LaPaglia, M; Andreoni, K A

    2004-05-01

    Clinical islet cell transplantation has demonstrated great promise for diabetes treatment. Two major obstacles are the organ donor shortage and the immunoresponse. The purpose of this study was to create a model using the patient's own adult stem cell sources, possibly in combination with non-self cells, such as pancreatic, hepatic, or embryonic stem cells, to create "personalized" islets. We hypothesize that the reconstructed islets have the normal capability to produce insulin and glucagon with reduced immunoresponses after transplantation. Stem cells are a proliferating population of master cells that have the ability for self-renewal and multilineage differentiation. The recently developed photolithograph-based, biologic, microelectromechanic system (BioMEMS) technique supplies a useful tool for biomedical applications. Our lab has developed a novel method that integrates the adult stem cell and BioMEMS to reconstruct personalized islets. We selected islet-derived progenitor cells (IPC) for repairing and reconstructing STZ-diabetic islets. A6(+)/PYY(+) or A6(+)/ngn3(+) cells were selected to manipulate the neoislets. After 3 to 4 weeks in culture, the reconstructed cells formed islet-like clusters containing insulin or glucagon producing cells. The pilot results showed the ability of these reconstructed islets to correct hyperglycemia when transplanted into a STZ-diabetic isograft mouse model. Although several technical problems remain with the mouse model, namely, the difficulty to collect enough islets from a single mouse because of animal size, the mouse isograft model is suitable for personalized islet development.

  3. Pig-islet xenotransplantation: recent progress and current perspectives.

    PubMed

    Zhu, Hai-Tao; Wang, Wan-Li; Yu, Liang; Wang, Bo

    2014-01-01

    Islet xenotransplantation is one prospective treatment to bridge the gap between available human cells and needs of patients with diabetes. Pig represents an ideal candidate for obtaining such available cells. However, potential clinical application of pig islet still faces obstacles including inadequate yield of high-quality functional islets and xenorejection of the transplants. Adequate amounts of available islets can be obtained by selection of a suitable pathogen-free source herd and the development of isolation and purification method. Several studies demonstrated the feasibility of successful preclinical 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 safety and efficacy data to translate these findings into clinic. PMID:25593932

  4. Protein isolation from the developing embryonic mouse heart valve region.

    PubMed

    Dyer, Laura A; Wu, Yaxu; Patterson, Cam

    2014-01-01

    Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 μg of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development. PMID:25285454

  5. Protein isolation from the developing embryonic mouse heart valve region.

    PubMed

    Dyer, Laura A; Wu, Yaxu; Patterson, Cam

    2014-09-23

    Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 μg of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

  6. The microsomal glucose-6-phosphatase enzyme of pancreatic islets.

    PubMed Central

    Waddell, I D; Burchell, A

    1988-01-01

    Microsomal fractions isolated from pancreatic islet cells were shown to contain high specific glucose-6-phosphatase activity. The islet-cell glucose-6-phosphatase enzyme has the same Mr (36,500), similar immunological properties and kinetic characteristics to the hepatic microsomal glucose-6-phosphatase enzyme. Images Fig. 1. Fig. 2. PMID:2849415

  7. Automated classification of mouse pup isolation syllables: from cluster analysis to an Excel-based "mouse pup syllable classification calculator".

    PubMed

    Grimsley, Jasmine M S; Gadziola, Marie A; Wenstrup, Jeffrey J

    2012-01-01

    Mouse pups vocalize at high rates when they are cold or isolated from the nest. The proportions of each syllable type produced carry information about disease state and are being used as behavioral markers for the internal state of animals. Manual classifications of these vocalizations identified 10 syllable types based on their spectro-temporal features. However, manual classification of mouse syllables is time consuming and vulnerable to experimenter bias. This study uses an automated cluster analysis to identify acoustically distinct syllable types produced by CBA/CaJ mouse pups, and then compares the results to prior manual classification methods. The cluster analysis identified two syllable types, based on their frequency bands, that have continuous frequency-time structure, and two syllable types featuring abrupt frequency transitions. Although cluster analysis computed fewer syllable types than manual classification, the clusters represented well the probability distributions of the acoustic features within syllables. These probability distributions indicate that some of the manually classified syllable types are not statistically distinct. The characteristics of the four classified clusters were used to generate a Microsoft Excel-based mouse syllable classifier that rapidly categorizes syllables, with over a 90% match, into the syllable types determined by cluster analysis.

  8. Establishment of mouse embryonic stem cells from isolated blastomeres and whole embryos using three derivation methods

    PubMed Central

    González, Sheyla; Ibáñez, Elena

    2010-01-01

    Purpose The aim of the present study is to compare three previously described mouse embryonic stem cell derivation methods to evaluate the influence of culture conditions, number of isolated blastomeres and embryonic stage in the derivation process. Methods Three embryonic stem cell derivation methods: standard, pre-adhesion and defined culture medium method, were compared in the derivation from isolated blastomeres and whole embryos at 4- and 8-cell stages. Results A total of 200 embryonic stem cell lines were obtained with an efficiency ranging from 1.9% to 72%. Conclusions Using either isolated blastomeres or whole embryos, the highest rates of mouse embryonic stem cell establishment were achieved with the defined culture medium method and efficiencies increased as development progressed. Using isolated blastomeres, efficiencies increased in parallel to the proportion of the embryo volume used to start the derivation process. PMID:20862536

  9. Biological and biomaterial approaches for improved islet transplantation.

    PubMed

    Narang, Ajit S; Mahato, Ram I

    2006-06-01

    Islet transplantation may be used to treat type I diabetes. Despite tremendous progress in islet isolation, culture, and preservation, the clinical use of this modality of treatment is limited due to post-transplantation challenges to the islets such as the failure to revascularize and immune destruction of the islet graft. In addition, the need for lifelong strong immunosuppressing agents restricts the use of this option to a limited subset of patients, which is further restricted by the unmet need for large numbers of islets. Inadequate islet supply issues are being addressed by regeneration therapy and xenotransplantation. Various strategies are being tried to prevent beta-cell death, including immunoisolation using semipermeable biocompatible polymeric capsules and induction of immune tolerance. Genetic modification of islets promises to complement all these strategies toward the success of islet transplantation. Furthermore, synergistic application of more than one strategy is required for improving the success of islet transplantation. This review will critically address various insights developed in each individual strategy and for multipronged approaches, which will be helpful in achieving better outcomes. PMID:16714486

  10. Isolation, culture and characterization of primary mouse RPE cells.

    PubMed

    Fernandez-Godino, Rosario; Garland, Donita L; Pierce, Eric A

    2016-07-01

    Mouse models are powerful tools for the study of ocular diseases. Alterations in the morphology and function of the retinal pigment epithelium (RPE) are common features shared by many ocular disorders. We report a detailed protocol to collect, seed, culture and characterize RPE cells from mice. We describe a reproducible method that we previously developed to collect and culture murine RPE cells on Transwells as functional polarized monolayers. The collection of RPE cells takes ∼3 h, and the cultures mimic in vivo RPE cell features within 1 week. This protocol also describes methods to characterize the cells on Transwells within 1-2 weeks by transmission and scanning electron microscopy (TEM and SEM, respectively), immunostaining of vibratome sections and flat mounts, and measurement of transepithelial electrical resistance. The RPE cell cultures are suitable to study the biology of the RPE from wild-type and genetically modified strains of mice between the ages of 10 d and 12 months. The RPE cells can also be manipulated to investigate molecular mechanisms underlying the RPE pathology in the numerous mouse models of ocular disorders. Furthermore, modeling the RPE pathology in vitro represents a new approach to testing drugs that will help accelerate the development of therapies for vision-threatening disorders such as macular degeneration (MD). PMID:27281648

  11. Transcriptional Regulation of the Pancreatic Islet: Implications for Islet Function

    PubMed Central

    Stitzel, Michael L.; Kycia, Ina; Kursawe, Romy; Ucar, Duygu

    2015-01-01

    Islets of Langerhans contain multiple hormone-producing endocrine cells controlling glucose homeostasis. Transcription establishes and maintains islet cellular fates and identities. Genetic and environmental disruption of islet transcription triggers cellular dysfunction and disease. Early transcriptional regulation studies of specific islet genes, including insulin (INS) and the transcription factor PDX1, identified the first cis-regulatory DNA sequences and trans-acting factors governing islet function. Here, we review how human islet “omics” studies are reshaping our understanding of transcriptional regulation in islet (dys)function and diabetes. First, we highlight the expansion of islet transcript number, form, and function and of DNA transcriptional regulatory elements controlling their production. Next, we cover islet transcriptional effects of genetic and environmental perturbation. Finally, we discuss how these studies’ emerging insights should empower our diabetes research community to build mechanistic understanding of diabetes pathophysiology and to equip clinicians with tailored, precision medicine options to prevent and treat islet dysfunction and diabetes. PMID:26272056

  12. A mouse chromosome 4 balancer ENU-mutagenesis screen isolates eleven lethal lines

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ENU-mutagenesis is a powerful technique to identify genes regulating mammalian development. To functionally annotate the distal region of mouse chromosome 4, we performed an ENU-mutagenesis screen using a balancer chromosome targeted to this region of the genome. We isolated 11 lethal lines that map...

  13. A Mouse Model for Characterization of Gastrointestinal Colonization Rates Among Environmental Aeromonas Isolates

    EPA Science Inventory

    The colonization rates of ten different environmental isolates of Aeromonas were determined using a novel mouse-streptomycin pre-treatment method. A novel streptomycin pre-treatment prepared animals with a transient alteration in colon flora that allowed colonization by Aeromon...

  14. Mechanisms of islet damage mediated by pancreas cold ischemia/rewarming.

    PubMed

    Omori, Keiko; Kobayashi, Eiji; Rawson, Jeffrey; Takahashi, Masafumi; Mullen, Yoko

    2016-10-01

    Prolonged pancreas cold ischemia is known to negatively correlate with islet isolation outcomes, hindering successful islet transplantation to treat Type-1 Diabetes. Due to poor islet isolation outcome, pancreata with over 16 h cold ischemia are currently not considered for islet transplantation. Mechanisms involved in pancreas cold ischemia/rewarming mediated islet damage during islet isolation and culture are not well understood. Using an en bloc cold preserved rat pancreas preparation, we attempted to clarify possible mechanisms of islet death associated with prolonged pancreas cold ischemia and subsequent rewarming. Cold ischemia lasting 16 h decreased post-isolation islet yield and increased islet death during the initial 6 h of culture. Electron micrographs revealed swelling and severe disruption of cellular and mitochondrial membranes, as well as an enlarged endoplasmic reticulum (ER) in β-cells isolated from cold preserved pancreata. Prolonged cold ischemia of the pancreas transiently activated mitogen-activated protein kinases (MAPKs) in isolated islets and increased lipid peroxidation products 4-hydroxynonenal (HNE) and heat shock protein (Hsp) 70 after culture, indicating the activation of oxidative stress signaling pathways. The islet isolation process, irrespective of pancreas cold ischemia, activated unfolded protein response (UPR), while the ER protective chaperon BiP was further upregulated by pancreas cold ischemia/rewarming. During the first 6 h of culture following islet isolation, p53 upregulated modulator of apoptosis (Puma) and caspase-3 activation were also upregulated. Our study indicates the involvement of both apoptosis and necrosis in islet death, and suggests oxidative stress and disruption of membranes are critical mechanisms mediated by pancreas cold ischemia/rewarming. PMID:27587006

  15. 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.

    PubMed

    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

  16. Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

    PubMed

    He, Chuan; Myers, Mark A; Forbes, Briony E; Grützner, Frank

    2015-04-01

    Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution.

  17. Immunohistochemical analysis of pancreatic islets of platypus (Ornithorhynchus anatinus) and echidna (Tachyglossus aculeatus ssp.).

    PubMed

    He, Chuan; Myers, Mark A; Forbes, Briony E; Grützner, Frank

    2015-04-01

    Monotremes have undergone remarkable changes to their digestive and metabolic control system; however, the monotreme pancreas remains poorly characterized. Previous work in echidna demonstrated the presence of pancreatic islets, but no information is available for platypus and the fine structure has not been described for either monotreme. Based on our recent finding that monotremes lack the ghrelin gene, which is expressed in mouse and human pancreatic islets, we investigated the structure of monotreme islets in more detail. Generally, as in birds, the islets of monotremes were smaller but greater in number compared with mouse. β-cells were the most abundant endocrine cell population in platypus islets and were located peripherally, while α-cells were observed both in the interior and periphery of the islets. δ-cells and pancreatic polypeptide (PP)-cells were mainly found in the islet periphery. Distinct PP-rich (PP-lobe) and PP-poor areas (non-PP-lobe) are present in therian mammals, and we identified these areas in echidna but not platypus pancreas. Interestingly, in some of the echidna islets, α- and β-cells tended to form two poles within the islets, which to our knowledge is the first time this has been observed in any species. Overall, monotreme pancreata share the feature of consisting of distinct PP-poor and PP-rich islets with other mammals. A higher number of islets and α- or β-cell only islets are shared between monotremes and birds. The islets of monotremes were larger than those of birds but smaller compared with therian mammals. This may indicate a trend of having fewer larger islets comprising several endocrine cell types during mammalian evolution. PMID:25682842

  18. A Simple Protocol for High Efficiency Protein Isolation After RNA Isolation from Mouse Thyroid and Other Very Small Tissue Samples.

    PubMed

    Ziros, Panos G; Chartoumpekis, Dionysios V; Sykiotis, Gerasimos P

    2016-01-01

    As a dedicated hormone-secreting organ, the thyroid gland possesses a complement of proteostatic systems, including antioxidant, unfolded protein, and autophagic responses. The vast majority of animal investigations of thyroid physiology and, more recently, proteostasis, have utilized as model the rat, rather than the mouse. This is due to the very small size of the thyroid gland in the latter, with a total weight of ~2 mg (~1 mg per thyroid lobe). However, this strategy has limited the utilization of genetic approaches, such as taking advantage of the various transgenic and knockout mouse models. Here, we describe a simple and highly efficient protocol for the simultaneous isolation of mRNA, micro-RNA and 150-200 μg of protein from as little as 1 mg of mouse thyroid tissue, the average weight of one of the two thyroid lobes, thus preserving the other lobe for immunohistochemical or other analyses. While our workflow is similar to other protocols published in the literature and/or proposed by commercial reagent providers, we have introduced a key modification that addresses efficiently the most challenging step of the protein isolation process: the solubilization of the protein pellet after RNA extraction and protein precipitation. We demonstrate the feasibility of our approach and its utility for downstream analyses (including Western blotting) that facilitate the comparative study of proteostatic pathways in the mouse thyroid. We have also successfully applied this protocol on samples from mouse liver, brown and white adipose tissue, as well as from rodent cell lines. PMID:27613051

  19. Mineral metabolism in isolated mouse long bones: Opposite effects of microgravity on mineralization and resorption

    NASA Technical Reports Server (NTRS)

    Veldhuijzen, Jean Paul; Vanloon, Jack J. W. A.

    1994-01-01

    An experiment using isolated skeletal tissues under microgravity, is reported. Fetal mouse long bones (metatarsals) were cultured for 4 days in the Biorack facility of Spacelab during the IML-1 (International Microgravity Laboratory) mission of the Space Shuttle. Overall growth was not affected, however glucose consumption was significantly reduced under microgravity. Mineralization of the diaphysis was also strongly reduced under microgravity as compared to the on-board 1 g group. In contrast, mineral resorption by osteoclasts was signficantly increased. These results indicate that these fetal mouse long bones are a sensitive and useful model to further study the cellular mechanisms involved in the changed mineral metabolism of skeletal tissues under microgravity.

  20. Endothelial cells modulate renin secretion from isolated mouse juxtaglomerular cells.

    PubMed Central

    Kurtz, A; Kaissling, B; Busse, R; Baier, W

    1991-01-01

    Utilizing cocultures of mouse renal juxtaglomerular cells with bovine microvascular endothelial cells, we have examined whether endothelial cells exert direct influence on renin secretion from renal juxtaglomerular cells. In the presence of endothelial cells both spontaneous and forskolin (10 microM) or isoproterenol (10 microM) stimulated renin release were markedly attenuated. The stimulatory effect of the calmodulin antagonist calmidazolium (10 microM) on renin secretion was not altered by endothelial cells, whereas the stimulatory effect of ethylisopropylamiloride (50 microM) an inhibitor of sodium-proton exchange was enhanced in the presence of endothelial cells. Indomethacin (10 microM) and NG-monomethyl-l-arginine (NMMA) (1 mM) used to inhibit cyclooxygenase activity and production of endothelium-derived relaxing factor (EDRF) decreased spontaneous renin release in the presence of endothelial cells only, but had no effect on forskolin stimulated renin secretion. Endothelin (1 microM) inhibited cAMP stimulated renin release both in the absence and in the presence of endothelial cells. ATP (10 microM) which acts on both endothelial and juxtaglomerular cells via purinergic P2 receptors inhibited cAMP stimulated renin release only in the absence but not in the presence of endothelial cells. This modulatory effect of endothelial cells was no altered by indomethacin nor by NMMA. Taken together, our findings provide first evidence for a local control function of the endothelium on cAMP stimulated renin secretion from renal juxtaglomerular cells, which could in part be mediated by endothelin. Images PMID:1717509

  1. Artificial islets from hybrid spheroids of three pancreatic cell lines.

    PubMed

    Jo, Y H; Jang, I J; Nemeno, J G; Lee, S; Kim, B Y; Nam, B M; Yang, W; Lee, K M; Kim, H; Takebe, T; Kim, Y S; Lee, J I

    2014-05-01

    Pancreatic islets have been the focus of recent studies exploring the pathologic mechanisms of diabetes mellitus as well as more effective and radical treatments for this disease. Islet transplantation is a promising therapeutic strategy; however, isolation of pancreatic islets for this purpose has been challenging, because the technique is time consuming and technically difficult, and tissue handling can be variable. Pseudo-islets can be used as an alternative to naïve islets, but require cellular sources or artificial materials. In this study, pancreas-derived cells were used to generate pseudo-islets. Because the pancreas is composed of a variety of cell types, namely α cells, β cells, δ cells, and other pancreatic cells that perform different functions, we used 3 different cell lines-NIT-1 (a β-cell line), α TC1 clone 6 (an α-cell line), and TGP52 (a pancreatic epithelial-like cell line)-which we cocultured in nonadhesive culture plates to produce hybrid cellular spheroids. These pseudo-islets had an oval shape and were morphologically similar to naïve islets; additionally, they expressed and secreted the pancreatic hormones insulin, glucagon, and somatostatin, as confirmed by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. The results demonstrate that pseudo-islets that mimic naïve islets can be successfully generated by a coculture method. These artificial islets can potentially be used for in vitro tests related to diabetes mellitus, specifically, in drug discovery or for investigating pathology. Moreover, they can be useful for examining basic questions pertaining to cell-cell interactions and tissue development. PMID:24815150

  2. Islet and Stem Cell Encapsulation for Clinical Transplantation

    PubMed Central

    Krishnan, Rahul; Alexander, Michael; Robles, Lourdes; Foster 3rd, Clarence E.; Lakey, Jonathan R.T.

    2014-01-01

    Over the last decade, improvements in islet isolation techniques have made islet transplantation an option for a certain subset of patients with long-standing diabetes. Although islet transplants have shown improved graft function, adequate function beyond the second year has not yet been demonstrated, and patients still require immunosuppression to prevent rejection. Since allogeneic islet transplants have experienced some success, the next step is to improve graft function while eliminating the need for systemic immunosuppressive therapy. Biomaterial encapsulation offers a strategy to avoid the need for toxic immunosuppression while increasing the chances of graft function and survival. Encapsulation entails coating cells or tissue in a semipermeable biocompatible material that allows for the passage of nutrients, oxygen, and hormones while blocking immune cells and regulatory substances from recognizing and destroying the cell, thus avoiding the need for systemic immunosuppressive therapy. Despite advances in encapsulation technology, these developments have not yet been meaningfully translated into clinical islet transplantation, for which several factors are to blame, including graft hypoxia, host inflammatory response, fibrosis, improper choice of biomaterial type, lack of standard guidelines, and post-transplantation device failure. Several new approaches, such as the use of porcine islets, stem cells, development of prevascularized implants, islet nanocoating, and multilayer encapsulation, continue to generate intense scientific interest in this rapidly expanding field. This review provides a comprehensive update on islet and stem cell encapsulation as a treatment modality in type 1 diabetes, including a historical outlook as well as current and future research avenues. PMID:25148368

  3. Update on islet cell transplantation for type 1 diabetes.

    PubMed

    Agarwal, Avinash; Brayman, Kenneth L

    2012-06-01

    Despite modern medical breakthroughs, diabetes mellitus is a worldwide leading cause of morbidity and mortality. Definitive surgical treatment of diabetes mellitus was established with the advent and refinement of clinical pancreas transplantation in the 1960s. During the following decades, critical discoveries involving islet isolation and engraftment took place. Clinical islet cell transplantation represents the potential for reduced insulin requirements and debilitating hypoglycemic episodes without the morbidity of surgery. Unfortunately, islet cell transplantation was unable to achieve comparable results with solid organ transplantation. This was until the Edmonton protocol (steroid-free immunosuppression) was described, which demonstrated that islet cell transplantation could be a viable alternative to pancreas transplantation. Significant advances in islet purification techniques and novel immunomodulatory agents have since renewed interest in islet cell transplantation. Yet the field is still challenged by a limited supply of islet cells, inadequate engraftment, and the deleterious effects of chronic immunosuppression. This article discusses the history and the current status of clinical islet cell transplantation. PMID:23729978

  4. Keep on rolling: optimizing human islet transport conditions using a perfused rotary system.

    PubMed

    Hermann, Martin; Wurm, Martin; Lubei, Verena; Pirkebner, Daniela; Draxl, Anna; Margreiter, Raimund; Hengster, Paul

    2012-01-01

    The setup of an islet isolation facility designed along the rules of good manufacturing practice (GMP) is a technically challenging, cost and time intensive process. ( 1) Consequently, several institutions have decided to perform transplantation of islets isolated at another center with an already standing expertise. Such a solution includes the necessity to transport the isolated islets from the isolation to the transplantation facility. In spite of its importance, an ideal solution for the transport of the isolated human islets has still not been established.   Here, we present an islet transport device suited to transport human islet cells under reproducible conditions and minimized cell stress. The transport simulation of the human islets was performed in a transfused "rotary transport system for islets" termed "ROTi." Besides measuring standard metabolic (LDH, lactate, glucose) and physical parameters (pH, dissolved oxygen and temperature), we used five different live stains in combination with real time live confocal microscopy to document islet quality parameters. As live stains we added tetramethylrhodamine methyl ester, cell permeant acetoxymethylester, propidium iodide, annexin-fitc and fluorescent wheat germ agglutinin, and assessed mitochondrial membrane potentials, calcium levels, cell death, apoptosis or cell morphology, respectively. We compared the viability of human islets after 24 h incubation in the ROTi device to an incubation simulating "standard" shipment of islets in 50 ml tubes. All cell viability parameters studied (mitochondrial membrane potentials, calcium content, apoptosis, cell death as well as cell morphology) documented a significantly better cell viability in the ROTi fraction compared with the simulated "standard" shipment fraction. Besides maintaining islet cell viability, the ROTi bears the advantage of a better reproducibility of islet transport conditions.

  5. Effect of nicotinamide on early graft failure following intraportal islet transplantation

    PubMed Central

    Jung, Da-Yeon; Park, Jae Berm; Joo, Sung-Yeon; Joh, Jae-Won; Kwon, Choon-Hyuck; Kwon, Ghee-Young

    2009-01-01

    Intraportal islet transplantation (IPIT) may potentially cure Type 1 diabetes mellitus; however, graft failure in the early post-transplantation period presents a major obstacle. In this study, we tested the ability of nicotinamide to prevent early islet destruction in a syngeneic mouse model. Mice (C57BL/6) with chemically-induced diabetes received intraportal transplants of syngeneic islet tissue in various doses. Islets were cultured for 24 h in medium with or without 10 mM nicotinamide supplementation. Following IPIT, islet function was confirmed by an intraperitoneal glucose tolerance test (IPGTT) and hepatectomy. The effects of nicotinamide were evaluated by blood glucose concentration, serum monocyte chemoattractant protein-1 (MCP-1) concentration, and immunohistology at 3 h and 24 h after IPIT. Among the various islet doses, an infusion of 300 syngeneic islets treated with nicotinamide exhibited the greatest differences in glucose tolerance between recipients of treated and untreated (i.e., control) islets. One day after 300 islet equivalent (IEQ) transplantation, islets treated with nicotinamide were better granulated than the untreated islets (P = 0.01), and the recipients displayed a slight decrease in serum MCP-1 concentration, as compared to controls. After 15 days, recipients of nicotinamide-pretreated islets showed higher levels of graft function (as measured by IPGTT) than controls. The pretreatment also prolonged graft survival (> 100 days) and function; these were confirmed by partial hepatectomy, which led to the recurrence of diabetes. Pretreatment of islet grafts with nicotinamide may prevent their deterioration on the early period following IPIT in a syngeneic mouse model. PMID:19641379

  6. Acrolein relaxes mouse isolated tracheal smooth muscle via a TRPA1-dependent mechanism.

    PubMed

    Cheah, Esther Y; Burcham, Philip C; Mann, Tracy S; Henry, Peter J

    2014-05-01

    Airway sensory C-fibres express TRPA1 channels which have recently been identified as a key chemosensory receptor for acrolein, a toxic and highly prevalent component of smoke. TRPA1 likely plays an intermediary role in eliciting a range of effects induced by acrolein including cough and neurogenic inflammation. Currently, it is not known whether acrolein-induced activation of TRPA1 produces other airway effects including relaxation of mouse airway smooth muscle. The aims of this study were to examine the effects of acrolein on airway smooth muscle tone in mouse isolated trachea, and to characterise the cellular and molecular mechanisms underpinning the effects of acrolein. Isometric tension recording studies were conducted on mouse isolated tracheal segments to characterise acrolein-induced relaxation responses. Release of the relaxant PGE₂ was measured by EIA to examine its role in the response. Use of selective antagonists/inhibitors permitted pharmacological characterisation of the molecular and cellular mechanisms underlying this relaxation response. Acrolein induced dose-dependent relaxation responses in mouse isolated tracheal segments. Importantly, these relaxation responses were significantly inhibited by the TRPA1 antagonists AP-18 and HC-030031, an NK₁ receptor antagonist RP-67580, and the EP₂ receptor antagonist PF-04418948, whilst completely abolished by the non-selective COX inhibitor indomethacin. Acrolein also caused rapid PGE₂ release which was suppressed by HC-030031. In summary, acrolein induced a novel bronchodilator response in mouse airways. Pharmacologic studies indicate that acrolein-induced relaxation likely involves interplay between TRPA1-expressing airway sensory C-fibres, NK₁ receptor-expressing epithelial cells, and EP₂-receptor expressing airway smooth muscle cells.

  7. Isolation of inhibitors of TPA-induced mouse ear edema from Hoelen, Poria cocos.

    PubMed

    Nukaya, H; Yamashiro, H; Fukazawa, H; Ishida, H; Tsuji, K

    1996-04-01

    Triterpene carboxylic acids were isolated from the methanol extract of Hoelen, Poria cocos, and found to inhibit 12-O-tetradecanoylphorbol 13-acetate (TAP)-induced mouse ear edema. Their chemical structures were identified as 3 beta,-16 alpha-dihydroxylanosta-7,9(11),24-trien-21-oic acid, 16 alpha-hydroxydehydropachymic acid, 16 alpha-hydroxytrametenolic acid and dehydrotumulosic acid. PMID:8681415

  8. Isolation of Mouse Hair Follicle Bulge Stem Cells and Their Functional Analysis in a Reconstitution Assay.

    PubMed

    Zheng, Ying; Hsieh, Jen-Chih; Escandon, Julia; Cotsarelis, George

    2016-01-01

    The hair follicle (HF) is a dynamic structure readily accessible within the skin, and contains various pools of stem cells that have a broad regenerative potential during normal homeostasis and in response to injury. Recent discoveries demonstrating the multipotent capabilities of hair follicle stem cells and the easy access to skin tissue make the HF an attractive source for isolating stem cells and their subsequent application in tissue engineering and regenerative medicine. Here, we describe the isolation and purification of hair follicle bulge stem cells from mouse skin, and hair reconstitution assays that allows the functional analysis of multipotent stem cells. PMID:27431247

  9. A highly selective fluorescent probe for direct detection and isolation of mouse embryonic stem cells.

    PubMed

    Chandran, Yogeswari; Kang, Nam-Young; Park, Sung-Jin; Alamudi, Samira Husen; Kim, Jun-Young; Sahu, Srikanta; Su, Dongdong; Lee, Jungyeol; Vendrell, Marc; Chang, Young-Tae

    2015-11-01

    Stem cell research has gathered immense attention in the past decade due to the remarkable ability of stem cells for self-renewal and tissue-specific differentiation. Despite having numerous advancements in stem cell isolation and manipulation techniques, there is a need for highly reliable probes for the specific detection of live stem cells. Herein we developed a new fluorescence probe (CDy9) with high selectivity for mouse embryonic stem cells. CDy9 allows the detection and isolation of intact stem cells with marginal impact on their function and capabilities. PMID:26115574

  10. Preservation of mitochondrial functional integrity in mitochondria isolated from small cryopreserved mouse brain areas.

    PubMed

    Valenti, Daniela; de Bari, Lidia; De Filippis, Bianca; Ricceri, Laura; Vacca, Rosa Anna

    2014-01-01

    Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and were compared with respect to their ability to generate membrane potential and produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency was successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics will significantly expand the study of mitochondrial defects in neurological pathologies, allowing large comparative studies and favoring interlaboratory and interdisciplinary analyses.

  11. The use of hydrogen gas clearance for blood flow measurements in single endogenous and transplanted pancreatic islets.

    PubMed

    Barbu, Andreea; Jansson, Leif; Sandberg, Monica; Quach, My; Palm, Fredrik

    2015-01-01

    The blood perfusion of pancreatic islets is regulated independently from that of the exocrine pancreas, and is of importance for multiple aspects of normal islet function, and probably also during impaired glucose tolerance. Single islet blood flow has been difficult to evaluate due to technical limitations. We therefore adapted a hydrogen gas washout technique using microelectrodes to allow such measurements. Platinum micro-electrodes monitored hydrogen gas clearance from individual endogenous and transplanted islets in the pancreas of male Lewis rats and in human and mouse islets implanted under the renal capsule of male athymic mice. Both in the rat endogenous pancreatic islets as well as in the intra-pancreatically transplanted islets, the vascular conductance and blood flow values displayed a highly heterogeneous distribution, varying by factors 6-10 within the same pancreas. The blood flow of human and mouse islet grafts transplanted in athymic mice was approximately 30% lower than that in the surrounding renal parenchyma. The present technique provides unique opportunities to study the islet vascular dysfunction seen after transplantation, but also allows for investigating the effects of genetic and environmental perturbations on islet blood flow at the single islet level in vivo.

  12. Oxygen supply by photosynthesis to an implantable islet cell device.

    PubMed

    Evron, Y; Zimermann, B; Ludwig, B; Barkai, U; Colton, C K; Weir, G C; Arieli, B; Maimon, S; Shalev, N; Yavriyants, K; Goldman, T; Gendler, Z; Eizen, L; Vardi, P; Bloch, K; Barthel, A; Bornstein, S R; Rotem, A

    2015-01-01

    Transplantation of islet cells is an effective treatment for type 1 diabetes with critically labile metabolic control. However, during islet isolation, blood supply is disrupted, and the transport of nutrients/metabolites to and from the islet cells occurs entirely by diffusion. Adequate oxygen supply is essential for function/survival of islet cells and is the limiting factor for graft integrity. Recently, we developed an immunoisolated chamber system for transplantation of human islets without immunosuppression. This system depended on daily oxygen supply. To provide independence from this external source, we incorporated a novel approach based on photosynthetically-generated oxygen. The chamber system was packed sandwich-like with a slab of immobilized photosynthetically active microorganisms (Synechococcus lividus) on top of a flat light source (LEDs, red light at 660 nm, intensity of 8 μE/m(2)/s). Islet cells immobilized in an alginate slab (500-1,000 islet equivalents/cm(2)) were mounted on the photosynthetic slab separated by a gas permeable silicone rubber-Teflon membrane, and the complete module was sealed with a microporous polytetrafluorethylene (Teflon) membrane (pore size: 0.4 μm) to protect the contents from the host immune cells. Upon illumination, oxygen produced by photosynthesis diffused via the silicone Teflon membrane into the islet compartment. Oxygen production from implanted encapsulated microorganisms was stable for 1 month. After implantation of the device into diabetic rats, normoglycemia was achieved for 1 week. Upon retrieval of the device, blood glucose levels returned to the diabetic state. Our results demonstrate that an implanted photosynthetic bioreactor can supply oxygen to transplanted islets and thus maintain islet viability/functionality. PMID:25365509

  13. Oxygen supply by photosynthesis to an implantable islet cell device.

    PubMed

    Evron, Y; Zimermann, B; Ludwig, B; Barkai, U; Colton, C K; Weir, G C; Arieli, B; Maimon, S; Shalev, N; Yavriyants, K; Goldman, T; Gendler, Z; Eizen, L; Vardi, P; Bloch, K; Barthel, A; Bornstein, S R; Rotem, A

    2015-01-01

    Transplantation of islet cells is an effective treatment for type 1 diabetes with critically labile metabolic control. However, during islet isolation, blood supply is disrupted, and the transport of nutrients/metabolites to and from the islet cells occurs entirely by diffusion. Adequate oxygen supply is essential for function/survival of islet cells and is the limiting factor for graft integrity. Recently, we developed an immunoisolated chamber system for transplantation of human islets without immunosuppression. This system depended on daily oxygen supply. To provide independence from this external source, we incorporated a novel approach based on photosynthetically-generated oxygen. The chamber system was packed sandwich-like with a slab of immobilized photosynthetically active microorganisms (Synechococcus lividus) on top of a flat light source (LEDs, red light at 660 nm, intensity of 8 μE/m(2)/s). Islet cells immobilized in an alginate slab (500-1,000 islet equivalents/cm(2)) were mounted on the photosynthetic slab separated by a gas permeable silicone rubber-Teflon membrane, and the complete module was sealed with a microporous polytetrafluorethylene (Teflon) membrane (pore size: 0.4 μm) to protect the contents from the host immune cells. Upon illumination, oxygen produced by photosynthesis diffused via the silicone Teflon membrane into the islet compartment. Oxygen production from implanted encapsulated microorganisms was stable for 1 month. After implantation of the device into diabetic rats, normoglycemia was achieved for 1 week. Upon retrieval of the device, blood glucose levels returned to the diabetic state. Our results demonstrate that an implanted photosynthetic bioreactor can supply oxygen to transplanted islets and thus maintain islet viability/functionality.

  14. Diffusion into human islets is limited to molecules below 10 kDa.

    PubMed

    Williams, S J; Schwasinger-Schmidt, T; Zamierowski, D; Stehno-Bittel, L

    2012-10-01

    Isolated islets are important tools in diabetes research and are used for islet transplantation as a treatment for type 1 diabetes. Yet these cell clusters have a dramatic diffusion barrier that leads to core cell death. Computer modeling has provided theoretical size limitations, but little has been done to measure the actual rate of diffusion in islets. The purpose of this study was to directly measure the diffusion barrier in intact human islets and determine its role in restricting insulin secretion. Impeded diffusion into islets was monitored with fluorescent dextran beads. Dextran beads of 10-70 kDa failed to diffuse into the core of the intact islets, while 0.9 kDa probe was observed within the core of smaller islets. Diffusion of the fluorescent form of glucose, 2-NBDG, had similar diffusion limitations as the beads, with an average intra-islet diffusion rate of 1.5 ± 0.2 μm/min. The poor diffusion properties were associated with core cell death from necrosis, not apoptosis. Short-term exposure to a mild papain/0 Ca(2+) cocktail, dramatically reduced the diffusion barrier so that all cells within islets were exposed to media components. Lowering the diffusion barrier increased the immediate and long-term viability of islet cells, and tended to increase the amount of insulin released, especially in low glucose conditions. However, it failed to improve the large islet's glucose-stimulated insulin secretion. Thus, the islet diffusion barrier leads to low viability and poor survival of large islets, but is not solely responsible for the reduced insulin secretion of large isolated islets.

  15. Automated separation of merged Langerhans islets

    NASA Astrophysics Data System (ADS)

    Švihlík, Jan; Kybic, Jan; Habart, David

    2016-03-01

    This paper deals with separation of merged Langerhans islets in segmentations in order to evaluate correct histogram of islet diameters. A distribution of islet diameters is useful for determining the feasibility of islet transplantation in diabetes. First, the merged islets at training segmentations are manually separated by medical experts. Based on the single islets, the merged islets are identified and the SVM classifier is trained on both classes (merged/single islets). The testing segmentations were over-segmented using watershed transform and the most probable back merging of islets were found using trained SVM classifier. Finally, the optimized segmentation is compared with ground truth segmentation (correctly separated islets).

  16. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    SciTech Connect

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-05-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 {sup o}C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  17. Endoscopic biopsy of islet transplants in the gastric submucosal space provides evidence of islet graft rejection in diabetic pigs.

    PubMed

    Tanaka, Takayuki; Fujita, Minoru; Bottino, Rita; Piganelli, Jon D; McGrath, Kevin; Li, Jiang; Lee, Whayoung; Iwase, Hayato; Wijkstrom, Martin; Bertera, Suzanne; Long, Cassandra; Landsittel, Douglas; Haruma, Ken; Cooper, David K C; Hara, Hidetaka

    2016-01-01

    Transplantation of islets into the gastric submucosal space (GSMS) has several advantages (e.g., avoidance of the instant blood-mediated inflammatory response [IBMIR], ability to biopsy). The aim of this study was to determine whether endoscopic biopsy of islet allografts transplanted into the GSMS in diabetic pigs can provide histopathological and immunohistochemical information that correlates with the clinical course (e.g.,, blood glucose level, insulin requirement). Islet allografts (Group1: 10,000 kIEq /kg [n = 4]; Group2: 15,000 kIEq /kg [n = 2]) were transplanted into the GSMS of diabetic pigs under immunosuppression. In Group2, the anti-oxidant, BMX-001 was applied during preservation, isolation, and culture of the islets, and at the time of transplantation. Endoscopic biopsies of the islet grafts were obtained one or 2 weeks after transplantation, and histopathological features were compared with the clinical course (e.g., blood glucose, insulin requirement). In Group1, in the absence of anti-oxidant therapy, most of the islets became fragmented, and there was no reduction in exogenous insulin requirement. In Group2, with an increased number of transplanted islets in the presence of BMX-001, more healthy insulin-positive islet masses were obtained at biopsy and necropsy (4 weeks), and these correlated with reductions in both blood glucose level and insulin requirement. In all cases, inflammatory cell infiltrates were present. After islet transplantation into the GSMS, endoscopic biopsy can provide information on graft rejection, which would be an immense advantage in clinical islet transplantation. PMID:26857703

  18. Anti-apoptotic Effects of Bone Marrow on Human Islets: A Preliminary Report

    PubMed Central

    Luo, Lu-Guang; Luo, John ZQ

    2015-01-01

    Apoptosis is one of the major factors contributing to the failure of human islet transplantation. Contributors to islet apoptosis exist in both the pre-transplantation and post transplantation stages. Factors include the islet isolation process, deterioration in vitro prior to transplantation, and immune rejection post transplantation. Previous studies have demonstrated that co-cultured bone marrow cells with human islets not only significantly enhanced the longevity of human islets but also maintained function. We hypothesized that the protective effects of bone marrow cells on human islets are through mechanisms related to preventing apoptosis. This study observed the levels of inflammatory factors such as interleukin-1β (IL-1β), the release of extracellular ATP in vitro, and expression levels of P2X7 ATP receptor (P2X7R), all of which lead to the occurrence of apoptosis in human islets. When human islets were co-cultured with human bone marrow, there was a reduction in the rate of apoptosis correlated with the reduction in inflammatory factors, extra cellular ATP accumulation, and ATP receptor P2X7R expression versus human islets cultured alone. These results suggest that co-culturing bone marrow cells with human islets inhibits inflammation and reduces apoptosis, thus protecting islets from self-deterioration. PMID:26229735

  19. Measurement of apoptosis of intact human islets by confocal optical sectioning and stereologic analysis of YO-PRO-1-stained islets.

    PubMed

    Boffa, Daniel J; Waka, John; Thomas, Dolca; Suh, Sungwook; Curran, Kevin; Sharma, Vijay K; Besada, Melissa; Muthukumar, Thangamani; Yang, Hua; Suthanthiran, Manikkam; Manova, Katia

    2005-04-15

    Apoptosis is an established pathway for islet cell demise. Current protocols for assessment of islet cell apoptosis are time-consuming (as with terminal deoxynucleotide transferase-mediated dUTP nick-end labeling reaction) and involve disruption of the islet architecture (as with flow cytometry) or destruction of cell integrity (as with enzyme-linked immunosorbent assay). The membranes of apoptotic cells, but not those of live cells, are permeant to the DNA-intercalant dye YO-PRO-1. We report a novel methodology for the rapid quantification of apoptosis of human islets: confocal laser optical sectioning and stereologic analysis of intact human islets stained with YO-PRO-1 and Hoechst 33342. The advantages include (1) rapid quantification of apoptosis without disrupting islet architecture and (2) identification of significant heterogeneity in the extent of apoptosis among islets from the same isolate. Confocal laser scanning microscopy microscopic imaging of YO-PRO-1-stained islets may advance investigation of islet cell apoptosis and help develop islet parameters predictive of posttransplant function. PMID:15818328

  20. Isolation of Dendritic Cell Progenitor and Bone Marrow Progenitor Cells from Mouse.

    PubMed

    Onai, Nobuyuki; Ohteki, Toshiaki

    2016-01-01

    Dendritic cells (DCs) comprise two major subsets, conventional DC (cDC) and plasmacytoid DC (pDC) in the steady-state lymphoid organ. These cells have a short half-life and therefore, require continuous generation from hematopoietic stem cells and progenitor cells. Recently, we identified DC-restricted progenitors called common DC progenitors (CDPs) in the bone marrow of mouse. The CDPs can be isolated from mouse bone marrow based on the hematopoietic cytokine receptors, such as Flt3 (Fms-related tyrosine kinase 3) (CD135), c-kit (CD117), M-CSF (macrophage colony-stimulating factor) receptor (CD115), and IL-7 (interleukin-7) receptor-α (CD127). The CDPs comprise of two progenitors, CD115(+) CDPs and CD115(-) CDPs, and give rise to only DC subsets in both in vitro and in vivo. The former CDPs are the main source of cDC, while the later CDPs are the main source of pDC in vivo. Here, we provide a protocol for the isolation of dendritic cell progenitor and bone marrow progenitor cells from mouse. PMID:27142008

  1. Human islet preparations distributed for research exhibit a variety of insulin-secretory profiles

    PubMed Central

    Kayton, Nora S.; Poffenberger, Gregory; Henske, Joseph; Dai, Chunhua; Thompson, Courtney; Aramandla, Radhika; Shostak, Alena; Nicholson, Wendell; Brissova, Marcela; Bush, William S.

    2015-01-01

    Human islet research is providing new insights into human islet biology and diabetes, using islets isolated at multiple US centers from donors with varying characteristics. This creates challenges for understanding, interpreting, and integrating research findings from the many laboratories that use these islets. In what is, to our knowledge, the first standardized assessment of human islet preparations from multiple isolation centers, we measured insulin secretion from 202 preparations isolated at 15 centers over 11 years and noted five distinct patterns of insulin secretion. Approximately three quarters were appropriately responsive to stimuli, but one quarter were dysfunctional, with unstable basal insulin secretion and/or an impairment in stimulated insulin secretion. Importantly, the patterns of insulin secretion by responsive human islet preparations (stable Baseline and Fold stimulation of insulin secretion) isolated at different centers were similar and improved slightly over the years studied. When all preparations studied were considered, basal and stimulated insulin secretion did not correlate with isolation center, biological differences of the islet donor, or differences in isolation, such as Cold Ischemia Time. Dysfunctional islet preparations could not be predicted from the information provided by the isolation center and had altered expression of genes encoding components of the glucose-sensing pathway, but not of insulin production or cell death. These results indicate that insulin secretion by most preparations from multiple centers is similar but that in vitro responsiveness of human islets cannot be predicted, necessitating preexperimental human islet assessment. These results should be considered when one is designing, interpreting, and integrating experiments using human islets. PMID:25648831

  2. Islet Transplantation in Pediatric Patients: Current Indications and Future Perspectives.

    PubMed

    Bertuzzi, Federico; Antonioli, Barbara; Tosca, Marta C; Galuzzi, Marta; Bonomo, Matteo; Marazzi, Mario; Colussi, Giacomo

    2016-01-01

    The first islet transplantation in diabetes mellitus was performed more than 20 years ago. Since then, clinical results have progressively improved. Nowadays, islet transplantation can be considered a real therapeutic option after pancreatectomy for painful chronic pancreatitis (autotransplantation) and in selected adult patients affected by type 1 diabetes mellitus (allotransplantation). Better results are mainly due to the advances in the standardization of islet isolation and purification procedures as well as in the pharmacological treatment of recipients. Anti-inflammatory treatments facilitate islet engraftment and prevent metabolic exhaustion and functional β-cell apoptosis; new strategies better control islet graft rejection. As a consequence, islet transplantation activities are no longer confined to few centers only, rather thousands of transplants are now performed all over the world. Many attempts are actually undertaken to find solutions to current problems of islets transplantation, from toxicity of immunosuppressive therapy to the limited engraftment, function and duration. There is general hope that these procedures will offer a safe and feasible therapeutic option for an increasing number of patients suffering from diabetes mellitus, including pediatric patients. PMID:26682915

  3. Labeling and Tracking of Human Pancreatic Islets Using Carbon Nanotubes.

    PubMed

    Syed, Farooq; Riggio, Cristina; Masini, Matilde; Bugliani, Marco; Battaglia, Valentina; Novelli, Michela; Suleiman, Mara; Vittorio, Orazio; Boggi, Ugo; Filipponi, Franco; Marselli, Lorella; Bartolozzi, Carlo; Masiello, Pellegrino; Raffa, Vittoria; Marchetti, Piero

    2015-04-01

    Limited tools are available for the non-invasive monitoring of transplanted islets. In this study, we have compared the widely used superparamagnetic iron oxide nanoparticle ferumoxide (Endorem) and multiwalled carbon nanotubes (MWCNTs) for islet cell labeling and tracking. INS-1 E cells and human pancreatic islets isolated from 12 non-diabetic cadaveric organ donors (age: 62 ±16 yr, BMI: 24.6 ± 3.3 kg/m2) were incubated with 50 μg/ml Endorem or 15 μg/ml MWCNTs and studied after 7 or 14 days to assess beta cell morphology, ultrastructure, function, cell survival and in-vitro and in-vivo magnetic resonance imaging (MRI). Light and electron (EM) microscopy showed the well-maintained morphology and ultrastructure of both INS-1 E and human islets during the incubation. EM also revealed the presence of Endorem and MWCNTs within the beta but not the alpha cells. The compounds did not affect beta cell function and viability, and in-vitro MRI showed that labeled INS-1 E cells and human islets could be imaged. Finally, MWCNT labeled human islets were successfully transplanted into the subcutis of rats localized in the desired site via magnetic field and tracked by MRI. These data suggest that MWCNTs can be an alternative labeling compound to be used with human islets for experimental and transplantation studies.

  4. Labeling and Tracking of Human Pancreatic Islets Using Carbon Nanotubes.

    PubMed

    Syed, Farooq; Riggio, Cristina; Masini, Matilde; Bugliani, Marco; Battaglia, Valentina; Novelli, Michela; Suleiman, Mara; Vittorio, Orazio; Boggi, Ugo; Filipponi, Franco; Marselli, Lorella; Bartolozzi, Carlo; Masiello, Pellegrino; Raffa, Vittoria; Marchetti, Piero

    2015-04-01

    Limited tools are available for the non-invasive monitoring of transplanted islets. In this study, we have compared the widely used superparamagnetic iron oxide nanoparticle ferumoxide (Endorem) and multiwalled carbon nanotubes (MWCNTs) for islet cell labeling and tracking. INS-1 E cells and human pancreatic islets isolated from 12 non-diabetic cadaveric organ donors (age: 62 ±16 yr, BMI: 24.6 ± 3.3 kg/m2) were incubated with 50 μg/ml Endorem or 15 μg/ml MWCNTs and studied after 7 or 14 days to assess beta cell morphology, ultrastructure, function, cell survival and in-vitro and in-vivo magnetic resonance imaging (MRI). Light and electron (EM) microscopy showed the well-maintained morphology and ultrastructure of both INS-1 E and human islets during the incubation. EM also revealed the presence of Endorem and MWCNTs within the beta but not the alpha cells. The compounds did not affect beta cell function and viability, and in-vitro MRI showed that labeled INS-1 E cells and human islets could be imaged. Finally, MWCNT labeled human islets were successfully transplanted into the subcutis of rats localized in the desired site via magnetic field and tracked by MRI. These data suggest that MWCNTs can be an alternative labeling compound to be used with human islets for experimental and transplantation studies. PMID:26310079

  5. Isolation and characterization of bovine and mouse terminal deoxynucleotidyltransferase cDNAs expressible in mammalian cells.

    PubMed Central

    Koiwai, O; Yokota, T; Kageyama, T; Hirose, T; Yoshida, S; Arai, K

    1986-01-01

    We have isolated nearly full-length cDNA clones of terminal deoxynucleotidyltransferase (TdT) from calf thymus and mouse lymphoma cDNA libraries. The libraries were constructed using the pcD vector system which permits the expression of cDNA inserts in mammalian cells. The bovine TdT cDNA clone contains an open reading frame coding for 520 amino acids, Mr 59,678. The mouse TdT cDNA clone contains an open reading frame of 1,587 bp, whose translated cDNA encodes a 60,004 dalton protein. The mouse TdT cDNA clone contains 60 bp in the 3' end region of the coding sequence not found in the bovine TdT cDNA sequence, otherwise, the clones share about 80% homology. A possible nuclear-localization-sequence (Pro-Arg-Lys-Lys-Arg-Pro-Arg) was conserved in the N-terminal region in the mouse and bovine cDNA clones. Bovine and mouse cDNAs transfected into COS7 monkey fibroblasts directed the synthesis of enzymatically active protein of Mr 60,000 which was detected immunologically using polyclonal rabbit antibody against bovine TdT. Bovine TdT expressed in COS7 cells by nearly full-length cDNA clone was localized in the nucleus and the translational product of pOK103 lacking the nuclear-localization-sequence was localized in the cytoplasm. Images PMID:3755527

  6. PDX-1 is a therapeutic target for pancreatic cancer, insulinoma and islet neoplasia using a novel RNA interference platform.

    PubMed

    Liu, Shi-He; Rao, Donald D; Nemunaitis, John; Senzer, Neil; Zhou, Guisheng; Dawson, David; Gingras, Marie-Claude; Wang, Zhaohui; Gibbs, Richard; Norman, Michael; Templeton, Nancy S; Demayo, Francesco J; O'Malley, Bert; Sanchez, Robbi; Fisher, William E; Brunicardi, F Charles

    2012-01-01

    Pancreatic and duodenal homeobox-1 (PDX-1) is a transcription factor that regulates insulin expression and islet maintenance in the adult pancreas. Our recent studies demonstrate that PDX-1 is an oncogene for pancreatic cancer and is overexpressed in pancreatic cancer. The purpose of this study was to demonstrate that PDX-1 is a therapeutic target for both hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Immunohistochemistry of human pancreatic and islet neoplasia specimens revealed marked PDX-1 overexpression, suggesting PDX-1 as a "drugable" target within these diseases. To do so, a novel RNA interference effector platform, bifunctional shRNA(PDX-1), was developed and studied in mouse and human cell lines as well as in mouse models of pancreatic cancer, insulinoma and islet neoplasia. Systemic delivery of bi-shRNA(humanPDX-1) lipoplexes resulted in marked reduction of tumor volume and improved survival in a human pancreatic cancer xenograft mouse model. bi-shRNA(mousePDX-1) lipoplexes prevented death from hyperinsulinemia and hypoglycemia in an insulinoma mouse model. shRNA(mousePDX-1) lipoplexes reversed hyperinsulinemia and hypoglycemia in an immune-competent mouse model of islet neoplasia. PDX-1 was overexpressed in pancreatic neuroendocrine tumors and nesidioblastosis. These data demonstrate that PDX-1 RNAi therapy controls hormonal symptoms and tumor volume in mouse models of pancreatic cancer, insulinoma and islet neoplasia, therefore, PDX-1 is a potential therapeutic target for these pancreatic diseases.

  7. Mouse models and techniques for the isolation of the diabetic endothelium.

    PubMed

    Darrow, April L; Maresh, J Gregory; Shohet, Ralph V

    2013-01-01

    Understanding the molecular mechanisms underlying diabetic endothelial dysfunction is necessary in order to improve the cardiovascular health of diabetic patients. Previously, we described an in vivo, murine model of insulin resistance induced by feeding a high-fat diet (HFD) whereby the endothelium may be isolated by fluorescence-activated cell sorting (FACS) based on Tie2-GFP expression and cell-surface staining. Here, we apply this model to two new strains of mice, ScN/Tie2-GFP and ApoE(-/-)/Tie2-GFP, and describe their metabolic responses and endothelial isolation. ScN/Tie2-GFP mice, which lack a functional toll-like receptor 4 (TLR4), display lower fasting glucose and insulin levels and improved glucose tolerance compared to Tie2-GFP mice, suggesting that TLR4 deficiency decreases susceptibility to the development of insulin resistance. ApoE(-/-)/Tie2-GFP mice display elevated glucose and cholesterol levels versus Tie2-GFP mice. Endothelial isolation by FACS achieves a pure population of endothelial cells that retain GFP fluorescence and endothelial functions. Transcriptional analysis of the aortic and muscle endothelium isolated from ApoE(-/-)/Tie2-GFP mice reveals a reduced endothelial response to HFD compared to Tie2-GFP mice, perhaps resulting from preexisting endothelial dysfunction in the hypercholesterolemic state. These mouse models and endothelial isolation techniques are valuable for assessing diabetic endothelial dysfunction and vascular responses in vivo.

  8. Sample preparation method for isolation of single-cell types from mouse liver for proteomic studies.

    PubMed

    Liu, Wei; Hou, Yufang; Chen, Huahai; Wei, Handong; Lin, Weiran; Li, Jichang; Zhang, Ming; He, Fuchu; Jiang, Ying

    2011-09-01

    It becomes increasingly clear that separation of pure cell populations provides a uniquely sensitive and accurate approach to protein profiling in biological systems and opens up a new area for proteomic analysis. The method we described could simultaneously isolate population of hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs) by a combination of collagenase-based density gradient centrifugation and magnetic activated cell sorting with high purity and yield for the first time. More than 98% of the isolated HCs were positive for cytokeratin 18, with a viability of 91%. Approximately 97% of the isolated HSCs expressed glial fibrillary acidic protein with a viability of 95%. Nearly 98% of isolated KCs expressed F4/80 with a viability of 94%. And the purity of LSECs reached up to 91% with a viability of 94%. And yield for HCs, HSCs, LSECs and KCs were 6.3, 1.3, 2.6 and 5.0 million per mouse. This systematic isolation method enables us to study the proteome profiling of different types of liver cells with high purity and yield, which is especially useful for sample preparation of Human Liver Proteome Project.

  9. Stress-impaired transcription factor expression and insulin secretion in transplanted human islets

    PubMed Central

    Dai, Chunhua; Kayton, Nora S.; Shostak, Alena; Poffenberger, Greg; Cyphert, Holly A.; Aramandla, Radhika; Thompson, Courtney; Papagiannis, Ioannis G.; Shiota, Masakazu; Stafford, John M.; Greiner, Dale L.; Herrera, Pedro L.; Shultz, Leonard D.; Stein, Roland; Powers, Alvin C.

    2016-01-01

    Type 2 diabetes is characterized by insulin resistance, hyperglycemia, and progressive β cell dysfunction. Excess glucose and lipid impair β cell function in islet cell lines, cultured rodent and human islets, and in vivo rodent models. Here, we examined the mechanistic consequences of glucotoxic and lipotoxic conditions on human islets in vivo and developed and/or used 3 complementary models that allowed comparison of the effects of hyperglycemic and/or insulin-resistant metabolic stress conditions on human and mouse islets, which responded quite differently to these challenges. Hyperglycemia and/or insulin resistance impaired insulin secretion only from human islets in vivo. In human grafts, chronic insulin resistance decreased antioxidant enzyme expression and increased superoxide and amyloid formation. In human islet grafts, expression of transcription factors NKX6.1 and MAFB was decreased by chronic insulin resistance, but only MAFB decreased under chronic hyperglycemia. Knockdown of NKX6.1 or MAFB expression in a human β cell line recapitulated the insulin secretion defect seen in vivo. Contrary to rodent islet studies, neither insulin resistance nor hyperglycemia led to human β cell proliferation or apoptosis. These results demonstrate profound differences in how excess glucose or lipid influence mouse and human insulin secretion and β cell activity and show that reduced expression of key islet-enriched transcription factors is an important mediator of glucotoxicity and lipotoxicity. PMID:27064285

  10. [Antimutagenic effect of chemosignals from isolated female house mouse on male germ cells (Mus musculus L)].

    PubMed

    Daev, E V; Bezruchko, Yu A; Dukelskaya, A V

    2014-06-01

    The influence of chemosignals from isolated mature females of the CBA line on level of spontaneous and radiation-induced meiotic disturbances in spermatocytes I of males of the same line was studied. Using an ana-telophase method, 24-hour exposure of males to soiled bedding containing isolated females chemosignals was shown to lead to a significantly lower frequency of chromosomal aberrations and other meiotic disturbances in spermatocytes I as compared to males kept on clean bedding. The same effect of female chemosignals was found in the reproductive cells of irradiated males (4 Gr). The mechanisms and importance of the revealed antimutagenic effect of mouse female chemosignals on the male reproductive cells in the reproduction process are discussed.

  11. Isolation and Fluorescence-Activated Cell Sorting of Mouse Keratinocytes Expressing β-Galactosidase.

    PubMed

    Kasper, Maria; Toftgård, Rune; Jaks, Viljar

    2016-01-01

    During the past decade, the rapid development of new transgenic and knock-in mouse models has propelled epidermal stem-cell research into "fast-forward mode". It has become possible to identify and visualize defined cell populations during normal tissue maintenance, and to follow their progeny during the processes of homeostasis, wound repair, and tumorigenesis. Moreover, these cells can be isolated using specific labels, and characterized in detail using an array of molecular and cell biology approaches. The bacterial enzyme, β-galactosidase (β-gal), the product of the LacZ gene, is one of the most commonly used in vivo cell labels in genetically-engineered mice. The protocol described in this chapter provides a guideline for the isolation of viable murine epidermal cells expressing β-gal, which can then be subjected to further characterization in vivo or in vitro. PMID:27431252

  12. Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus.

    PubMed

    Kryvalap, Yury; Lo, Chi-Wen; Manuylova, Ekaterina; Baldzizhar, Raman; Jospe, Nicholas; Czyzyk, Jan

    2016-01-01

    Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans.

  13. Antibody Response to Serpin B13 Induces Adaptive Changes in Mouse Pancreatic Islets and Slows Down the Decline in the Residual Beta Cell Function in Children with Recent Onset of Type 1 Diabetes Mellitus.

    PubMed

    Kryvalap, Yury; Lo, Chi-Wen; Manuylova, Ekaterina; Baldzizhar, Raman; Jospe, Nicholas; Czyzyk, Jan

    2016-01-01

    Type 1 diabetes mellitus (T1D) is characterized by a heightened antibody (Ab) response to pancreatic islet self-antigens, which is a biomarker of progressive islet pathology. We recently identified a novel antibody to clade B serpin that reduces islet-associated T cell accumulation and is linked to the delayed onset of T1D. As natural immunity to clade B arises early in life, we hypothesized that it may influence islet development during that time. To test this possibility healthy young Balb/c male mice were injected with serpin B13 mAb or IgG control and examined for the number and cellularity of pancreatic islets by immunofluorescence and FACS. Beta cell proliferation was assessed by measuring nucleotide analog 5-ethynyl-2'-deoxyuridine (5-EdU) incorporation into the DNA and islet Reg gene expression was measured by real time PCR. Human studies involved measuring anti-serpin B13 autoantibodies by Luminex. We found that injecting anti-serpin B13 monoclonal Ab enhanced beta cell proliferation and Reg gene expression, induced the generation of ∼80 pancreatic islets per animal, and ultimately led to increase in the beta cell mass. These findings are relevant to human T1D because our analysis of subjects just diagnosed with T1D revealed an association between baseline anti-serpin activity and slower residual beta cell function decline in the first year after the onset of diabetes. Our findings reveal a new role for the anti-serpin immunological response in promoting adaptive changes in the endocrine pancreas and suggests that enhancement of this response could potentially help impede the progression of T1D in humans. PMID:26578518

  14. Pancreatic islet plasticity: Interspecies comparison of islet architecture and composition

    PubMed Central

    Steiner, Donald J.; Kim, Abraham; Miller, Kevin; Hara, Manami

    2010-01-01

    The pancreatic islet displays diverse patterns of endocrine cell arrangement. The prototypic islet, with insulin-secreting β-cells forming the core surrounded by other endocrine cells in the periphery, is largely based on studies of normal rodent islets. Recent reports on large animals, including humans, show a difference in islet architecture, in which the endocrine cells are randomly distributed throughout the islet. This particular species difference has raised concerns regarding the interpretation of data based on rodent studies to humans. On the other hand, further variations have been reported in marsupials and some nonhuman primates, which possess an inverted ratio of β-cells to other endocrine cells. This review discusses the striking plasticity of islet architecture and cellular composition among various species including changes in response to metabolic states within a single species. We propose that this plasticity reflects evolutionary acquired adaptation induced by altered physiological conditions, rather than inherent disparities between species. PMID:20657742

  15. Matrix metalloproteinase-9 is essential for physiological Beta cell function and islet vascularization in adult mice.

    PubMed

    Christoffersson, Gustaf; Waldén, Tomas; Sandberg, Monica; Opdenakker, Ghislain; Carlsson, Per-Ola; Phillipson, Mia

    2015-04-01

    The availability of paracrine factors in the islets of Langerhans, and the constitution of the beta cell basement membrane can both be affected by proteolytic enzymes. This study aimed to investigate the effects of the extracellular matrix-degrading enzyme gelatinase B/matrix metalloproteinase-9 (Mmp-9) on islet function in mice. Islet function of Mmp9-deficient (Mmp9(-/-)) mice and their wild-type littermates was evaluated both in vivo and in vitro. The pancreata of Mmp9(-/-) mice did not differ from wild type in islet mass or distribution. However, Mmp9(-/-) mice had an impaired response to a glucose load in vivo, with lower serum insulin levels. The glucose-stimulated insulin secretion was reduced also in vitro in isolated Mmp9(-/-) islets. The vascular density of Mmp9(-/-) islets was lower, and the capillaries had fewer fenestrations, whereas the islet blood flow was threefold higher. These alterations could partly be explained by compensatory changes in the expression of matrix-related proteins. This in-depth investigation of the effects of the loss of MMP-9 function on pancreatic islets uncovers a deteriorated beta cell function that is primarily due to a shift in the beta cell phenotype, but also due to islet vascular aberrations. This likely reflects the importance of a normal islet matrix turnover exerted by MMP-9, and concomitant release of paracrine factors sequestered on the matrix.

  16. Comparison of atypical Brachyspira spp. clinical isolates and classic strains in a mouse model of swine dysentery.

    PubMed

    Burrough, Eric; Strait, Erin; Kinyon, Joann; Bower, Leslie; Madson, Darin; Schwartz, Kent; Frana, Timothy; Songer, J Glenn

    2012-12-01

    Multiple Brachyspira spp. can colonize the porcine colon, and the presence of the strongly beta-hemolytic Brachyspira hyodysenteriae is typically associated with clinical swine dysentery. Recently, several Brachyspira spp. have been isolated from the feces of pigs with clinical disease suggestive of swine dysentery, yet these isolates were not identified as B. hyodysenteriae by genotypic or phenotypic methods. This study used a mouse model of swine dysentery to compare the pathogenic potential of seventeen different Brachyspira isolates including eight atypical clinical isolates, six typical clinical isolates, the standard strain of B. hyodysenteriae (B204), and reference strains of Brachyspira intermedia and Brachyspira innocens. Results revealed that strongly beta-hemolytic isolates induced significantly greater cecal inflammation than weakly beta-hemolytic isolates regardless of the genetic identification of the isolate, and that strongly beta-hemolytic isolates identified as 'Brachyspira sp. SASK30446' and B. intermedia by PCR produced lesions indistinguishable from those caused by B. hyodysenteriae in this model.

  17. Cloning and primary structure of a human islet isoform of glutamic acid decarboxylase from chromosome 10

    SciTech Connect

    Karlsen, A.E.; Hagopian, W.A.; Grubin, C.E.; Dube, S.; Disteche, C.M.; Adler, D.A.; Baermeier, H.; Lernmark, A. ); Mathewes, S.; Grant, F.J.; Foster, D. )

    1991-10-01

    Glutamic acid decarboxylase which catalyzes formation of {gamma}-aminobutyric acid from L-glutamic acid, is detectable in different isoforms with distinct electrophoretic and kinetic characteristics. GAD has also been implicated as an autoantigen in the vastly differing autoimmune disease stiff-man syndrome and insulin-dependent diabetes mellitus. Despite the differing GAD isoforms, only one type of GAD cDNA (GAD-1), localized to a syntenic region of chromosome 2, has been isolated from rat, mouse, and cat. Using sequence information from GAD-1 to screen a human pancreatic islet cDNA library, the authors describe the isolation of an additional GAD cDNA (GAD-2), which was mapped to the short arm of human chromosome 10. Genomic Southern blotting with GAD-2 demonstrated a hybridization pattern different form that detected by GAD-1. GAD-2 recognizes a 5.6-kilobase transcript in both islets and brain, in contrast to GAD-1, which detects a 3.7-kilobase transcript in brain only. The deduced 585-amino acid sequence coded for by GAD-2 shows < 65% identify to previously published, highly conserved GAD-1 brain sequences, which show > 96% deduced amino acid sequence homology among the three species.

  18. Contractile effect of TRPA1 receptor agonists in the isolated mouse intestine.

    PubMed

    Penuelas, Angelica; Tashima, Kimihito; Tsuchiya, Shizuko; Matsumoto, Kenjiro; Nakamura, Tomonori; Horie, Syunji; Yano, Shingo

    2007-12-01

    TRPA1 is a member of the transient receptor potential (TRP) channel family expressed in sensory neurons. The present study focused on the effects of TRPA1 activation on contractile responses in isolated mouse intestine preparations. The jejunum, ileum, and proximal and distal colon were surgically isolated from male ddY mice. Intestinal motility was recorded as changes in isotonic tension. TRPA1, TRPM8, and TRPV1 expressions were examined by reverse transcription-polymerase chain reaction (RT-PCR). A TRPA1 agonist allyl isothiocyanate (AITC) dose-dependently induced contractions in the proximal and distal colon, whereas in the jejunum and ileum, even 100 muM AITC caused very little contraction. Likewise, a TRPA1 and TRPM8 agonist icilin, a TRPA1 agonist allicin, and a TRPV1 agonist capsaicin induced contractions in the colon. However, a TRPM8 agonist menthol induced long-lasting relaxation in the colon. Repeated exposure to AITC produced desensitization of its own contraction in the colon. Moreover, contractions induced by AITC generate cross-desensitization with icilin and capsaicin. Tetrodotoxin completely abolished AITC-induced contractions in the colon, whereas atropine significantly attenuated AITC-induced contractions in the distal colon, but not in the proximal colon. Menthol-induced relaxation in the colon was not inhibited by tetrodotoxin and atropine. RT-PCR analysis revealed the expression of TRPA1 and TRPV1, but not TRPM8, throughout the mouse intestine. These results suggest that TRPA1, but not TRPM8, are functionally expressed in the enteric nervous system throughout the mouse intestine on neurons that may also co-express TRPV1, yet the contractile responses to TRPA1 activation differ depending on their location along the intestine.

  19. Isolation of /sup 125/I-concanavalin A-labeled plasma membrane from unfertilized mouse eggs

    SciTech Connect

    Boldt, J.; Wolf, D.P.

    1987-04-01

    A procedure was developed for isolation of plasma membrane (PM) preparations from unfertilized mouse eggs. Zona-free mouse eggs prepared by the method of Boldt and Wolf (Gamete Res 13:213-222, 1986) were labeled with 125I-concanavalin A (ConA) prior to sonication and fractionation on iso-osmotic self-generated Percoll density gradients. Experiments using the ConA-specific sugar alpha-methylmannoside (alpha MM) indicated that 125I-ConA bound specifically to the egg PM. Greater than 95% of 125I-ConA binding to zona-free eggs was blocked in the presence of 0.1 M alpha MM, and incubation of eggs in alpha MM after 125I-ConA labeling caused release of 85-90% of bound label. Fractionation of 125I-ConA-labeled eggs by Percoll density gradient centrifugation yielded a single radioactive peak at density = 1.025, corresponding to egg PM material. Prolonged incubation of 125I-ConA-labeled eggs or egg sonicates prior to fractionation did not alter the location of the radioactive peak, indicating that 125I-ConA did not label other organelles. As a control, human erythrocytes were labeled with 125I-ConA and fractionated under identical experimental conditions and yielded a single radioactive peak at density (1.020) comparable to that observed for 125I-ConA-labeled eggs. These results indicate that 125I-ConA can be used as a specific marker to support PM isolation from small numbers of zona-free mouse eggs.

  20. Collagen IV-Modified Scaffolds Improve Islet Survival and Function and Reduce Time to Euglycemia

    PubMed Central

    Yap, Woon Teck; Salvay, David M.; Silliman, Michael A.; Zhang, Xiaomin; Bannon, Zachary G.; Kaufman, Dixon B.; Lowe, William L.

    2013-01-01

    Islet transplantation on extracellular matrix (ECM) protein-modified biodegradable microporous poly(lactide-co-glycolide) scaffolds is a potential curative treatment for type 1 diabetes mellitus (T1DM). Collagen IV-modified scaffolds, relative to control scaffolds, significantly decreased the time required to restore euglycemia from 17 to 3 days. We investigated the processes by which collagen IV-modified scaffolds enhanced islet function and mediated early restoration of euglycemia post-transplantation. We characterized the effect of collagen IV-modified scaffolds on islet survival, metabolism, and insulin secretion in vitro and early- and intermediate-term islet mass and vascular density post-transplantation and correlated these with early restoration of euglycemia in a syngeneic mouse model. Control scaffolds maintained native islet morphologies and architectures as well as collagen IV-modified scaffolds in vivo. The islet size and vascular density increased, while β-cell proliferation decreased from day 16 to 113 post-transplantation. Collagen IV-modified scaffolds promoted islet cell viability and decreased early-stage apoptosis in islet cells in vitro—phenomena that coincided with enhanced islet metabolic function and glucose-stimulated insulin secretion. These findings suggest that collagen IV-modified scaffolds promote the early restoration of euglycemia post-transplantation by enhancing islet metabolism and glucose-stimulated insulin secretion. These studies of ECM proteins, in particular collagen IV, and islet function provide key insights for the engineering of a microenvironment that would serve as a platform for enhancing islet transplantation as a viable clinical therapy for T1DM. PMID:23713524

  1. Streptococcus danieliae sp. nov., a novel bacterium isolated from the caecum of a mouse.

    PubMed

    Clavel, Thomas; Charrier, Cédric; Haller, Dirk

    2013-01-01

    We report the characterization of one novel bacterium, strain ERD01G(T), isolated from the cecum of a TNF(deltaARE) mouse. The strain was found to belong to the genus Streptococcus based on phylogenetic analysis of partial 16S rRNA gene sequences. The bacterial species with standing name in nomenclature that was most closely related to our isolate was Streptococcus alactolyticus (97 %). The two bacteria were characterized by a DNA-DNA hybridization similarity value of 35 %, demonstrating that they belong to different species. The new isolate was negative for acetoin production, esculin hydrolysis, urease, α-galactosidase and β-glucosidase, was able to produce acid from starch and trehalose, grew as beta-hemolytic coccobacilli on blood agar, did not grow at >40 °C, did not survive heat treatment at 60 °C for 20 min and showed negative agglutination in Lancefield tests. On the basis of these characteristics, strain ERD01G(T) differed from the most closely related species S. alactolyticus, Streptococcus gordonii, Streptococcus intermedius and Streptococcus sanguinis. Thus, based on genotypic and phenotypic evidence, we propose that the isolate belongs to a novel bacterial taxon within the genus Streptococcus, for which the name Streptococcus danieliae is proposed. The type strain is ERD01G(T) (= DSM 22233(T) = CCUG 57647(T)).

  2. Leptin rapidly suppresses insulin release from insulinoma cells, rat and human islets and, in vivo, in mice.

    PubMed Central

    Kulkarni, R N; Wang, Z L; Wang, R M; Hurley, J D; Smith, D M; Ghatei, M A; Withers, D J; Gardiner, J V; Bailey, C J; Bloom, S R

    1997-01-01

    Obesity is associated with diabetes, and leptin is known to be elevated in obesity. To investigate whether leptin has a direct effect on insulin secretion, isolated rat and human islets and cultured insulinoma cells were studied. In all cases, mouse leptin inhibited insulin secretion at concentrations within the plasma range reported in humans. Insulin mRNA expression was also suppressed in the cultured cells and rat islets. The long form of the leptin receptor (OB-Rb) mRNA was present in the islets and insulinoma cell lines. To determine the significance of these findings in vivo, normal fed mice were injected with two doses of leptin. A significant decrease in plasma insulin and associated rise in glucose concentration were observed. Fasted normal and leptin receptor-deficient db/db mice showed no response to leptin. A dose of leptin, which mimicked that found in normal mice, was administered to leptin-deficient, hyperinsulinemic ob/ob mice. This caused a marked lowering of plasma insulin concentration and a doubling of plasma glucose. Thus, leptin has a powerful acute inhibitory effect on insulin secretion. These results suggest that the action of leptin may be one mechanism by which excess adipose tissue could acutely impair carbohydrate metabolism. PMID:9389736

  3. Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China

    PubMed Central

    Liu, Daohua; Huo, Xingxing; Gao, Jiangmei; Song, Xiaorong; Xu, Xiucai; Huang, Kaiquan; Liu, Wenqi; Wang, Yong; Lu, Fangli; Lun, Zhao-Rong; Luo, Qingli; Wang, Xuelong; Shen, Jilong

    2013-01-01

    Background Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission. Methodology/Principal Findings Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7). Conclusion/Significance A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China. PMID:23308233

  4. Use of the BacT/alert system for rapid detection of microbial contamination in a pilot study using pancreatic islet cell products.

    PubMed

    Murray, Laura; McGowan, Neil; Fleming, John; Bailey, Laura

    2014-10-01

    At the Islet Isolation Laboratory of the Scottish National Blood Transfusion Service, manual sterility testing data show that contamination rates are 57.7% for pancreas transport fluid, 4.3% for postpurification islet samples, and 0% for pretransplant islet samples. This pilot study presents the BacT/Alert System as an alternative to manual testing to provide more rapid and sensitive sterility results for islet cell products.

  5. Data on morphometric analysis of the pancreatic islets from C57BL/6 and BALB/c mice.

    PubMed

    da Silva, Thiago Aparecido; Lemes, Robertha Mariana; Oliveira, Carlo Jose Freire; Almeida, Aline da Silva; Chica, Javier Emílio Lazo

    2016-09-01

    The endocrine portion of the pancreas, which is characterized by pancreatic islets, has been widely investigated among different species. The BALB/c and C57BL/6 mice are extensively used in experimental research, and the morphometric differences in the pancreatic islets of these animals have not been evaluated so far. Thus, our data have a comparative perspective related to the morphometric analysis of area, diameters, circularity, and density of pancreatic islets from BALB/c and C57BL/6 mice. The data presented here are focused to evaluate the differences in morphology of pancreatic islets of two common laboratory mouse strains.

  6. A pumpless microfluidic device driven by surface tension for pancreatic islet analysis.

    PubMed

    Xing, Yuan; Nourmohammadzadeh, Mohammad; Elias, Joshua E Mendoza; Chan, Manwai; Chen, Zequn; McGarrigle, James J; Oberholzer, José; Wang, Yong

    2016-10-01

    We present a novel pumpless microfluidic array driven by surface tension for studying the physiology of pancreatic islets of Langerhans. Efficient fluid flow in the array is achieved by surface tension-generated pressure as a result of inlet and outlet size differences. Flow properties are characterized in numerical simulation and further confirmed by experimental measurements. Using this device, we perform a set of biological assays, which include real-time fluorescent imaging and insulin secretion kinetics for both mouse and human islets. Our results demonstrate that this system not only drastically simplifies previously published experimental protocols for islet study by eliminating the need for external pumps/tubing and reducing the volume of solution consumption, but it also achieves a higher analytical spatiotemporal resolution due to efficient flow exchanges and the extremely small volume of solutions required. Overall, the microfluidic platform presented can be used as a potential powerful tool for understanding islet physiology, antidiabetic drug development, and islet transplantation.

  7. Effects of boldine on mouse diaphragm and sarcoplasmic reticulum vesicles isolated from skeletal muscle.

    PubMed

    Kang, J J; Cheng, Y W

    1998-02-01

    The effects of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine], a major alkaloid in the leaves and bark of boldo (Peumus boldus Mol.), on skeletal muscle were studied using mouse diaphragm and isolated sarcoplasmic reticulum membrane vesicles. Boldine, at 10-200 microM, has little effect on the muscle-evoked twitches; however, the ryanodine-induced contracture was potentiated dose-dependently. At higher concentrations of 300 microM, boldine by itself induced muscle contracture of two phases, which were caused by the influx of extracellular Ca2+ and induction of Ca2+ release from the internal Ca2+ storage site, the sarcoplasmic reticulum, respectively. When tested with isolated sarcoplasmic reticulum membrane vesicles, boldine dose-dependently induced Ca2+ release from actively loaded sarcoplasmic reticulum vesicles isolated from skeletal muscle of rabbit or rat which was inhibited by ruthenium red, suggesting that the release was through the Ca2+ release channel, also known as the ryanodine receptor. Boldine also dose-dependently increased apparent [3H]-ryanodine binding with the EC50 value of 50 microM. In conclusion, we have shown that boldine could sensitize the ryanodine receptor and induce Ca2+ release from the internal Ca2+ storage site of skeletal muscle. PMID:9491763

  8. Functional expression of a mouse H-2Kb gene isolated from non-expressing teratocarcinoma cells.

    PubMed Central

    Daniel-Vedele, F; Morello, D; Benicourt, C; Transy, C; Le Bail, O; Plata, F; Kourilsky, P

    1984-01-01

    Embryonal carcinoma cells do not express H-2 antigens or beta 2-microglobulin. Recent studies have suggested that the expression of these antigens is likely to be controlled at the level of transcription. To study the precise organization of the corresponding genes and their possible expression in adult mouse cells, we have isolated H-2-related genes from a genomic cosmid library constructed with PCC4-aza-RI from DNA of EC cells. Clones isolated from the library after stringent hybridization with an H-2 cDNA probe were tested for their ability to direct H-2 antigen synthesis after DNA-mediated gene transfer in a fibroblastic L cell. Four clones have been found to code for the major transplantation antigen H-2Kb. Structural analysis showed that these clones contained the same entire H-2Kb gene, identical to the corresponding gene isolated from differentiated C57Bl/10 cells. Furthermore, the present studies showed that this embryonal carcinoma gene was expressed and was functional when transfected into a differentiated cell. PMID:6714227

  9. Neuropathogenicity of Two Saffold Virus Type 3 Isolates in Mouse Models

    PubMed Central

    Kotani, Osamu; Naeem, Asif; Suzuki, Tadaki; Iwata-Yoshikawa, Naoko; Sato, Yuko; Nakajima, Noriko; Hosomi, Takushi; Tsukagoshi, Hiroyuki; Kozawa, Kunihisa; Hasegawa, Hideki; Taguchi, Fumihiro; Shimizu, Hiroyuki; Nagata, Noriyo

    2016-01-01

    Objective Saffold virus (SAFV), a picornavirus, is occasionally detected in children with acute flaccid paralysis, meningitis, and cerebellitis; however, the neuropathogenicity of SAFV remains undetermined. Methods The virulence of two clinical isolates of SAFV type 3 (SAFV-3) obtained from a patient with aseptic meningitis (AM strain) and acute upper respiratory inflammation (UR strain) was analyzed in neonatal and young mice utilizing virological, pathological, and immunological methods. Results The polyproteins of the strains differed in eight amino acids. Both clinical isolates were infective, exhibited neurotropism, and were mildly neurovirulent in neonatal ddY mice. Both strains pathologically infected neural progenitor cells and glial cells, but not large neurons, with the UR strain also infecting epithelial cells. UR infection resulted in longer inflammation in the brain and spinal cord because of demyelination, while the AM strain showed more infectivity in the cerebellum in neonatal ddY mice. Additionally, young BALB/c mice seroconverted following mucosal inoculation with the UR, but not the AM, strain. Conclusions Both SAFV-3 isolates had neurotropism and mild neurovirulence but showed different cell tropisms in both neonatal and young mouse models. This animal model has the potential to recapitulate the potential neuropathogenicity of SAFV-3. PMID:26828718

  10. A method for isolating high quality RNA from mouse cortical and cancellous bone.

    PubMed

    Kelly, Natalie H; Schimenti, John C; Patrick Ross, F; van der Meulen, Marjolein C H

    2014-11-01

    The high incidence of fragility fractures in cortico-cancellous bone locations, plus the fact that individual skeletal sites exhibit different responsiveness to load and disease, emphasizes the need to document separately gene expression in cortical and cancellous bone. A further confounding factor is marrow contamination since its high cellularity may effect gene expression measurements. We isolated RNA from cortical and cancellous bone of intact mouse tibiae, and also after marrow removal by flushing or centrifugation. RNA isolated from cancellous bone by each method was sufficient for gene expression analysis. Centrifugation removed contaminating cells more efficiently than flushing, as indexed by histology and decreased expression of Icam4, a highly expressed erythroid gene. In contrast, centrifuged cortical bone had 12- and 13- fold higher expression of the bone-related genes Col1a1 and Bglap, while levels in marrow-free cancellous bone were 30- and 31-fold higher when compared to bone where marrow was left intact. Furthermore, cortical bone had higher expression of Col1a1 and Bglap than cancellous bone. Thus, RNA isolated by this novel approach can reveal site-specific changes in gene expression in cortical and cancellous bone sites. PMID:25073031

  11. Isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface.

    PubMed

    Lam, Hilaire C; Choi, Augustine M K; Ryter, Stefan W

    2011-01-01

    Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems. PMID:21372793

  12. Evidence that ibogaine releases dopamine from the cytoplasmic pool in isolated mouse striatum.

    PubMed

    Harsing, L G; Sershen, H; Lajtha, A

    1994-01-01

    We measured the effect of ibogaine on the tritium efflux from isolated mouse striatum preloaded with [3H]dopamine ([3H]DA). Ibogaine increased the basal tritium outflow in a concentration-dependent manner, but it was without effect on electrical stimulation-induced tritium overflow. Separation of the released radioactivity after ibogaine administration showed that this drug increased the release of [3H]DA and [3H]-dihydroxyphenylacetic acid ([3H]DOPAC), but the efflux of O-methylated-deaminated metabolites was not changed. The dopamine (DA)-releasing effect of ibogaine was reduced by the DA uptake inhibitors cocaine and nomifensine. The tritium efflux evoked by ibogaine was not altered by omission of Ca2+ from the perfusion buffer or by inhibition of the voltage-sensitive Na+ channels with tetrodotoxin. Ibogaine maintained its effect on release from superfused striatum prepared from reserpine-pretreated mice. The ibogaine-induced tritium release measured from mouse striatum that was preloaded with [3H]DA was not affected by the D-2 DA receptor ligands (-)-quinpirole and (+/-)-sulpiride, indicating that the ibogaine-induced release is not subject to presynaptic autoreceptor regulation. Ibogaine failed to affect [3H]DA uptake and retention in mouse striatum. These data indicate that at the nerve terminal level ibogaine releases DA, and the primary source for the release is probably the cytoplasmic pool. The DA-releasing effect of ibogaine may have importance in mediation of its hallucinogenic action, as seen in a frequent practice in African cults. PMID:7826572

  13. Fetal endocannabinoids orchestrate the organization of pancreatic islet microarchitecture

    PubMed Central

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

    2015-01-01

    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. PMID:26494286

  14. Isolation and characterization of two mouse Pi-class glutathione S-transferase genes.

    PubMed Central

    Bammler, T K; Smith, C A; Wolf, C R

    1994-01-01

    Pi-class glutathione S-transferases (GSTs) play an important role in the detoxification of chemical toxins and mutagens and are implicated in neoplastic development and drug resistance. In all species characterized to date, only one functional Pi-class GST gene has been described. In this report we have identified two actively transcribed murine Pi-class GST genes, Gst p-1 and Gst p-2. The coding regions of Gst p-1 and the mouse Pi-class GST cDNA (GST-II) reported by Hatayama, Satoh and Satoh (1990) (Nucleic Acids Res. 18, 4606) are identical, whereas Gst p-2 encodes a protein that has not been described previously. The two genes are approximately 3 kb long and contain seven exons interrupted by six introns. In addition to a TATA box and a sequence motif matching the phorbol-ester-responsive element, the promoters of Gst p-1 and Gst p-2 exhibit one and two G+C boxes (GGGCGG) respectively. The cDNAs of the two genes were isolated from total liver RNA using reverse PCR. The peptide sequence deduced from the cDNAs share 97% identity and differ in six amino acids. Both genes are transcribed at significantly higher levels in male mouse liver than in female, and Gst p-1 mRNA is more abundant in both sexes than Gst p-2. Images Figure 4 Figure 5 PMID:8135745

  15. Expression of Innate Immunity Genes and Damage of Primary Human Pancreatic Islets by Epidemic Strains of Echovirus: Implication for Post-Virus Islet Autoimmunity

    PubMed Central

    Sarmiento, Luis; Frisk, Gun; Anagandula, Mahesh; Cabrera-Rode, Eduardo; Roivainen, Merja; Cilio, Corrado M.

    2013-01-01

    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. PMID:24223733

  16. PIG-TO-MONKEY ISLET XENOTRANSPLANTATION USING MULTI-TRANSGENIC PIGS

    PubMed Central

    Bottino, R.; Wijkstrom, M.; van der Windt, D.J.; Hara, H.; Ezzelarab, M.; Murase, N.; Bertera, S.; He, J.; Phelps, C.; Ayares, D.; Cooper, D.K.C.; Trucco, M.

    2014-01-01

    The generation of pigs with genetic modifications has significantly advanced the field of xenotransplantation. New genetically-engineered pigs were produced on an α1,3-galactosyltransferase gene-knockout background with ubiquitous expression of human CD46 (GTKO/CD46 pigs), with islet beta cell-specific expression of human tissue factor pathway inhibitor (hTFPI) and/or human CD39 and/or porcine CTLA4-lg. Isolated islets from pigs with 3, 4, or 5 genetic modifications were transplanted intraportally into streptozotocin-diabetic, immunosuppressed cynomolgus monkeys (n=5). Immunosuppression was based on anti-CD154mAb costimulation blockade. Monitoring included features of early islet destruction, glycemia, exogenous insulin requirement, and histopathology of the islets at necropsy. Using these modified pig islets, there was evidence of reduced islet destruction in the first hours after transplantation, compared with two series of historical controls that received identical therapy but were transplanted with islets from pigs with either no or only one genetic modification. Despite encouraging effects on early islet loss, these multi-transgenic islet grafts did not demonstrate consistency in regard to long-term success, with only 2 of 5 demonstrating function beyond 5 months. PMID:25220221

  17. Antiaging Glycopeptide Protects Human Islets Against Tacrolimus-Related Injury and Facilitates Engraftment in Mice.

    PubMed

    Gala-Lopez, Boris L; Pepper, Andrew R; Pawlick, Rena L; O'Gorman, Doug; Kin, Tatsuya; Bruni, Antonio; Abualhassan, Nasser; Bral, Mariusz; Bautista, Austin; Manning Fox, Jocelyn E; Young, Lachlan G; MacDonald, Patrick E; Shapiro, A M James

    2016-02-01

    Clinical islet transplantation has become an established treatment modality for selected patients with type 1 diabetes. However, a large proportion of transplanted islets is lost through multiple factors, including immunosuppressant-related toxicity, often requiring more than one donor to achieve insulin independence. On the basis of the cytoprotective capabilities of antifreeze proteins (AFPs), we hypothesized that supplementation of islets with synthetic AFP analog antiaging glycopeptide (AAGP) would enhance posttransplant engraftment and function and protect against tacrolimus (Tac) toxicity. In vitro and in vivo islet Tac exposure elicited significant but reversible reduction in insulin secretion in both mouse and human islets. Supplementation with AAGP resulted in improvement of islet survival (Tac(+) vs. Tac+AAGP, 31.5% vs. 67.6%, P < 0.01) coupled with better insulin secretion (area under the curve: Tac(+) vs. Tac+AAGP, 7.3 vs. 129.2 mmol/L/60 min, P < 0.001). The addition of AAGP reduced oxidative stress, enhanced insulin exocytosis, improved apoptosis, and improved engraftment in mice by decreasing expression of interleukin (IL)-1β, IL-6, keratinocyte chemokine, and tumor necrosis factor-α. Finally, transplant efficacy was superior in the Tac+AAGP group and was similar to islets not exposed to Tac, despite receiving continuous treatment for a limited time. Thus, supplementation with AAGP during culture improves islet potency and attenuates long-term Tac-induced graft dysfunction. PMID:26581595

  18. Mesenchymal stem cell and derived exosome as small RNA carrier and Immunomodulator to improve islet transplantation.

    PubMed

    Wen, Di; Peng, Yang; Liu, Di; Weizmann, Yossi; Mahato, Ram I

    2016-09-28

    Human bone marrow mesenchymal stem cells (hBMSCs) and their exosomes can suppress immune reaction and deliver small RNAs. Thus, they may improve islet transplantation by delivering small RNAs for promoting islet function and inhibiting immune rejection. Here, we proposed an hBMSC and its exosome-based therapy to overcome immune rejection and poor islet function, both of which hinder the success of islet transplantation. We found overexpressed siFas and anti-miR-375 in plasmid encoding shFas and anti-miR-375 transfected hBMSC-derived exosomes, which silenced Fas and miR-375 of human islets and improved their viability and function against inflammatory cytokines. This plasmid transfected hBMSCs downregulated Fas and miR-375 of human islets in a humanized NOD scid gamma (NSG) mouse model, whose immune reaction was inhibited by injecting hBMSC and peripheral blood mononuclear cell (PBMC) co-cultured exosomes. These exosomes suppressed immune reaction by inhibiting PBMC proliferation and enhancing regulatory T cell (Treg) function. Collectively, our studies elucidated the mechanisms of RNA delivery from hBMSCs to human islets and the immunosuppressive effect of hBMSC and peripheral blood mononuclear cell co-cultured exosomes for improving islet transplantation. PMID:27475298

  19. Differences in insulin biosynthesis pathway between small and large islets do not correspond to insulin secretion

    PubMed Central

    Huang, Han-Hung; Stehno-Bittel, Lisa

    2015-01-01

    In a variety of mammalian species, small islets secrete more insulin per volume than large islets. This difference may be due to diffusional limitations of large islets, or inherent differences in the insulin production pathways. The purpose of this study was to identify possible differences in the early phase of glucose-stimulated insulin biosynthesis between large and small islets. Isolated small and large rat islets were challenged with 30 minutes of high glucose. The expression of insulin gene transcription factors (MafA, NeuroD/ Beta2, and PDX-1), preproinsulin mRNA, proinsulin and insulin were compared between large and small islets. Under basal (low glucose) conditions, MafA and NeuroD had higher mRNA levels and greater protein amounts in large islets compared to small when normalized to GAPDH levels. 30 minutes of high glucose stimulation failed to alter the mRNA or subsequent protein levels of either gene. However, 30 minutes of high glucose suppressed activated PDX-1 protein levels in both small and large islets. High glucose stimulation did not statistically alter the preproinsulin mRNA (insulin 1 and insulin 2) levels. At the translational level, high glucose increased the proinsulin levels, and large islets showed a higher proinsulin content per cell than small islets. Insulin content per cell was not significantly different between small and large islets under basal or high glucose levels. The results fail to explain the higher level of insulin secretion noted in small versus large islets and may suggest that possible differences lie downstream in the secretory pathway rather than insulin biosynthesis. PMID:26752360

  20. Gene expression changes in human islets exposed to type 1 diabetic serum

    PubMed Central

    Jackson, Andrew M.; Kanak, Mazhar A.; Grishman, Ellen K.; Chaussabel, Damien; Levy, Marlon F.; Naziruddin, Bashoo

    2012-01-01

    A major obstacle to the success of islet cell transplantation as a standard treatment for labile type 1 diabetes mellitus is the immediate loss of up to 70% of the transplanted islet mass. Activation of the complement cascade and coagulation factors has been implicated in initiating the destruction of the islet graft. In this study, we analyzed the gene expression changes in islet cells following exposure to type 1 diabetes mellitus serum (T1DM). Isolated human pancreatic islet cells were cultured for 2 d to stabilize islet cell gene expression. Cultured islets were divided into three groups for treatment as follows: group 1 was treated with autologous donor serum, while groups two and three were treated with sera from ABO-matched allogeneic donors or autoantibody positive type 1 diabetic patient, respectively. Complement was detected using anti-C3 FITC and CH50 assay. Islet gene expression was analyzed using Illumina micro-array technology. Results were confirmed using real-time PCR. Immunofluorescent imaging demonstrated complement deposition only in the T1DM condition. Gene array and class prediction analysis generated a list of 50 genes that were able to predict the effect of T1DM serum on islets. Quantitative PCR corroborated microarray results. Both techniques demonstrated upregulation of MMP9 (243%), IL-1β (255%), IL-11 (220%), IL-12A (132%), RAD (343%) and a concomitant downregulation of IL-1RN (64%) in islets treated with T1DM serum. Islets treated with T1DM serum overexpressed genes associated with angiogenesis while decreasing transcription of genes that protect islets from inflammatory cytokines and reactive oxygen species. PMID:22885994

  1. Functional studies of rat, porcine, and human pancreatic islets cultured in ten commercially available media.

    PubMed

    Holmes, M A; Clayton, H A; Chadwick, D R; Bell, P R; London, N J; James, R F

    1995-10-27

    There have been no extensive studies investigating the effect of tissue culture media on the in vitro functional characteristics of rat, porcine and human Islets of Langerhans. We therefore aimed to compare ten commercially available tissue culture media on the basis of their ability to maintain islet viability. Following isolation, islets were cultured free-floating in the ten media (RPMI 1640-11mM glucose (control), RPMI 1640-2.2mM glucose, Dulbecco's MEM, TCM 199, CMRL 1066, Iscove's MEM, Waymouth's MEM, Serum-Free medium, Ex-cell 300, Ham's F-12) and viability was assessed after 24 hr, 3 days, and 7 days on the basis of macroscopic appearance, cell membrane integrity, and insulin secretion in response to glucose stimulation both by dynamic incubation and by perifusion. Each islet species demonstrated physiological insulin release characteristics in all media--however, it was possible to distinguish between the media by comparing the stimulation indices calculated from the insulin release studies. Significantly higher stimulation indices were produced in Iscove's MEM for rat islets, in Ham's F-12 for porcine islets and in CMRL 1066 for human islets. Over the entire culture period a significant deterioration in function was observed in all species cultured in the control media, although this was reversed when islets were cultured in the optimal media. Furthermore, in the case of porcine and human islets a significant improvement in function over the seven-day period was noted in the optimal media. In conclusion, of the commercially available media, the optimal tissue culture medium for rat islets is Iscove's MEM, for porcine islets is Ham's F-12, and for human islets is CMRL 1066. PMID:7482747

  2. Pancreas Transplantation: Solid Organ and Islet

    PubMed Central

    Mittal, Shruti; Johnson, Paul; Friend, Peter

    2014-01-01

    Transplantation of the pancreas, either as a solid organ or as isolated islets of Langerhans, is indicated in a small proportion of patients with insulin-dependent diabetes in whom severe complications develop, particularly severe glycemic instability and progressive secondary complications (usually renal failure). The potential to reverse diabetes has to be balanced against the morbidity of long-term immunosuppression. For a patient with renal failure, the treatment of choice is often a simultaneous transplant of the pancreas and kidney (SPK), whereas for a patient with glycemic instability, specifically hypoglycemic unawareness, the choice between a solid organ and an islet transplant has to be individual to the patient. Results of SPK transplantation are comparable to other solid-organ transplants (kidney, liver, heart) and there is evidence of improved quality of life and life expectancy, but the results of solitary pancreas transplantation and islets are inferior with respect to graft survival. There is some evidence of benefit with respect to the progression of secondary diabetic complications in patients with functioning transplants for several years. PMID:24616200

  3. 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.

    PubMed

    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

    2013-04-29

    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 / intra observer variability, which may induce false positive / negative islet counts. Here we describe a simple, reliable, automated digitalimage 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 automaticallyquantified using Nikon's inverted Eclipse Ti microscope, with built in NIS-ElementsAdvanced Research (AR) software.The AIDA method significantly enhanced the number of islet preparations eligible forengraftment compared to the standard manual method (P<0.001). Comparisons of individual methods showed good correlations between mean values of IEQ number (r(sup)2(/sup)≤0.91), and total islet number (r(sup)2(/sup)=0.88), and thus, increased to (r(sup)2(/sup)=0.93) when islet surface area was estimated comparatively with IEQ number. The ADIA method showed very high intra-observer reproducibility compared to the standard manual method (P<0.001). However, islet purity was routinely estimated as significantly higher with the manual method vs. the ADIA method(p<0.001). The ADIA method also detected small islets between 10-50 μm in size.Automated digital image analysis utilizing the Nikon Instruments (Nikon) software is anunbiased, simple, and reliable teaching tool to comprehensively assess the individual size ofeach islet cell preparation prior to transplantation. Implementation of

  4. Complete Genome Sequence of Turicibacter sp. Strain H121, Isolated from the Feces of a Contaminated Germ-Free Mouse

    PubMed Central

    Auchtung, T. A.; Holder, M. E.; Gesell, J. R.; Ajami, N. J.; Duarte, R. T. D.; Itoh, K.; Caspi, R. R.; Petrosino, J. F.; Horai, R.

    2016-01-01

    Turicibacter bacteria are commonly detected in the gastrointestinal tracts and feces of humans and animals, but their phylogeny, ecological role, and pathogenic potential remain unclear. We present here the first complete genome sequence of Turicibacter sp. strain H121, which was isolated from the feces of a mouse line contaminated following germ-free derivation. PMID:27013036

  5. Isolation of Sphere-Forming Stem Cells from the Mouse Inner Ear

    PubMed Central

    Oshima, Kazuo; Senn, Pascal; Heller, Stefan

    2010-01-01

    The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies. PMID:18839346

  6. Metabolic assessment prior to total pancreatectomy and islet autotransplant: utility, limitations and potential.

    PubMed

    Lundberg, R; Beilman, G J; Dunn, T B; Pruett, T L; Chinnakotla, S C; Radosevich, D M; Robertson, R P; Ptacek, P; Balamurugan, A N; Wilhelm, J J; Hering, B J; Sutherland, D E R; Moran, A; Bellin, M D

    2013-10-01

    Islet autotransplant (IAT) may ameliorate postsurgical diabetes following total pancreatectomy (TP), but outcomes are dependent upon islet mass, which is unknown prior to pancreatectomy. We evaluated whether preoperative metabolic testing could predict islet isolation outcomes and thus improve assessment of TPIAT candidates. We examined the relationship between measures from frequent sample IV glucose tolerance tests (FSIVGTT) and mixed meal tolerance tests (MMTT) and islet mass in 60 adult patients, with multivariate logistic regression modeling to identify predictors of islet mass ≥2500 IEQ/kg. The acute C-peptide response to glucose (ACRglu) and disposition index from FSIVGTT correlated modestly with the islet equivalents per kilogram body weight (IEQ/kg). Fasting and MMTT glucose levels and HbA1c correlated inversely with IEQ/kg (r values -0.33 to -0.40, p ≤ 0.05). In multivariate logistic regression modeling, normal fasting glucose (<100 mg/dL) and stimulated C-peptide on MMTT ≥4 ng/mL were associated with greater odds of receiving an islet mass ≥2500 IEQ/kg (OR 0.93 for fasting glucose, CI 0.87-1.0; OR 7.9 for C-peptide, CI 1.75-35.6). In conclusion, parameters obtained from FSIVGTT correlate modestly with islet isolation outcomes. Stimulated C-peptide ≥4 ng/mL on MMTT conveyed eight times the odds of receiving ≥2500 IEQ/kg, a threshold associated with reasonable metabolic control postoperatively.

  7. Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin.

    PubMed

    Sager, Martin; Benten, W Peter M; Engelhardt, Eva; Gougoula, Christina; Benga, Laurentiu

    2015-01-01

    [Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms

  8. Characterization of Biofilm Formation in [Pasteurella] pneumotropica and [Actinobacillus] muris Isolates of Mouse Origin

    PubMed Central

    Sager, Martin; Benten, W. Peter M.; Engelhardt, Eva; Gougoula, Christina; Benga, Laurentiu

    2015-01-01

    [Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of β-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of β-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms

  9. Effector-memory T cells develop in islets and report islet pathology in type 1 diabetes.

    PubMed

    Chee, Jonathan; Ko, Hyun-Ja; Skowera, Ania; Jhala, Gaurang; Catterall, Tara; Graham, Kate L; Sutherland, Robyn M; Thomas, Helen E; Lew, Andrew M; Peakman, Mark; Kay, Thomas W H; Krishnamurthy, Balasubramanian

    2014-01-15

    CD8(+) T cells are critical in human type 1 diabetes and in the NOD mouse. In this study, we elucidated the natural history of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)-specific CD8(+) T cells in NOD diabetes using MHC-tetramer technology. IGRP206-214-specific T cells in the peripheral lymphoid tissue increased with age, and their numbers correlated with insulitis progression. IGRP206-214-specific T cells in the peripheral lymphoid tissue expressed markers of chronic Ag stimulation, and their numbers were stable after diagnosis of diabetes, consistent with their memory phenotype. IGRP206-214-specific T cells in NOD mice expand, acquire the phenotype of effector-memory T cells in the islets, and emigrate to the peripheral lymphoid tissue. Our observations suggest that enumeration of effector-memory T cells of multiple autoantigen specificities in the periphery of type 1 diabetic subjects could be a reliable reporter for progression of islet pathology.

  10. Isolation of the etiologic agent of human granulocytic ehrlichiosis from the white-footed mouse (Peromyscus leucopus).

    PubMed

    Ravyn, M D; Kodner, C B; Carter, S E; Jarnefeld, J L; Johnson, R C

    2001-01-01

    We examined white-footed mice (Peromyscus leucopus) from Minnesota for infection with the etiologic agent of human granulocytic ehrlichiosis (HGE). From April to September 1997, we collected P. leucopus from Washington County, Minnesota, an area enzootic for HGE. Blood was cultivated in HL60 cells for isolation of the HGE agent. Of 59 mice examined, only a single mouse was culture positive for the HGE agent. The 16S ribosomal DNA sequence of the isolate was determined to be identical to that of the HGE agent. The isolate was reactive with monoclonal antibodies to the 44-kDa antigen of the HGE agent and was infectious for laboratory mice.

  11. Characterization of Pancreatic Ductal Cell in Human Islet Preparations

    PubMed Central

    Ichii, Hirohito; Miki, Atsushi; Yamamoto, Toshiyuki; Molano, R. Damaris; Barker, Scott; Mita, Atsuyoshi; Rodriguez-Diaz, Rayner; Klein, Dagmar; Pastori, Ricardo; Alejandro, Rodolfo; Inverardi, Luca; Pileggi, Antonello; Ricordi 6, Camillo

    2013-01-01

    Substantial amounts of non-endocrine cells are implanted as part of human islet grafts, and possible influence of non-endocrine cells on clinical islet transplantation outcome has been postulated. There are currently no product release criteria specific for non-endocrine cells due to lack of available methods. Aims of this study were to develop a method for the evaluation of pancreatic ductal cells (PDC) for clinical islet transplantation, and to characterize them regarding phenotype, viability and function. We assessed 161 human islet preparations using laser scanning cytometer (LSC/iCys) for phenotypic analysis of non-endocrine cells and flow cytometer (FACS) for PDC viability. PDC and β-cells obtained from different density fractions during the islet cell purification were compared in terms of viability. Furthermore, we examined PDC ability to produce pro-inflammatory cytokines/chemokines, vascular endothelial growth factor (VEGF) and tissue factor (TF), relevant to islet graft outcome. Phenotypic analysis by LSC/iCys indicated that single staining for CK19 or CA19-9 was not enough for identifying PDC, and that double staining for amylase and CK19 or CA19-9 allowed for quantitative evaluation of acinar cells and PDC content in human islet preparation. PDC showed a significantly higher viability than β-cells (PDC vs. β-cell: 75.5±13.9 and 62.7±18.7 %; p<0.0001). Although β-cells viability was independent from the density, that of PDC was higher as the density from which they were recovered increased. There was no correlation between PDC and β-cells viability (R2=0.0078). PDC sorted from high-density fractions produced significantly higher amount of pro-inflammatory mediators and VEGF, but not TF. PDC isolated from different fractions had different viability and function. The precise characterization and assessment of these cells in addition to β-cells in human islet cell products may be of assistance in understanding their contribution to islet

  12. The start of an islet transplantation program in Japan.

    PubMed

    Saito, T; Ise, K; Sato, Y; Gotoh, M; Matsumoto, S; Kenmochi, T; Kuroda, Y; Yasunami, Y; Inoue, K; Teraoka, S

    2005-10-01

    In Japan, pancreas donation had become possible from cadaveric donor sources, both heart-beating or non-heart-beating (NHB). Pancreas allografts have been distributed in the organ allocation system of the Japan Organ Transplant Network. Meanwhile, islet transplantation has been categorized as a tissue transplantation; it is free from legal restraints. Thus, pancreata for islet isolation must be obtained from NHB donors. Herein we report the starting program and preliminary results of islet transplantation in Japan. Selection and listing criteria for transplantation include regional priority, ABO blood type, previous islet transplant status with insulin independence, and a longer waiting time. Five institutes in Japan (Fukushima, Chiba, Kyoto, Kobe, and Fukuoka) are prepared to start programs. A two-layer cold storage method using perfluorocarbons and UW solution is recommended for pancreas preservation. Islet isolation and purification procedures are performed according to institute-specific protocol. Immunosuppression is based on sirolimus/tacrolimus combined with basiliximab induction. Two or three consecutive infusions of >5000 IE/kg are planned for each recipient until achieving insulin independence. Twenty-seven isolations and 14 transplants were performed in eight non-insulin-dependent diabetes mellitus (IDDM) recipients. Almost all (26 of 27) were NHB donors. All recipients are free from hypoglycemic episode after transplantation. One of these recipients is insulin independent; the others are currently on minimal doses of exogenous insulin. The feasibility of islet transplantation using NHB donors was confirmed using a two-layer cold storage method and a steroid-free immunosuppressive protocol, with a high rate of graft function.

  13. Magnetic resonance imaging and biological properties of pancreatic islets labeled with iron oxide nanoparticles.

    PubMed

    Kim, Hoe Suk; Choi, YoonSeok; Song, In Chan; Moon, Woo Kyung

    2009-10-01

    This study was undertaken to investigate the in vitro effect of islet labeling with iron oxide nanoparticles for MRI on islet viability, insulin secretion, and gene expression. Isolated rat islets were labeled with Resovist (25-200 microg Fe/mL, a clinically approved MRI contrast agent) in the presence or absence of poly-l-Lysine (PLL, 1.5 microg/mL) for 48 h. The iron content of labeled islets was found to increase in a dose-dependent manner. More than 90% of the islets were labeled with 100 microg Fe/mL. We confirmed the localizations of iron oxide nanoparticles within islet beta-cells by insulin immunostaining. As the concentration of Resovist increased, T(2) values as determined by T(2)-weighted MRI on a 1.5 Tesla MR scanner decreased. Labeling of 100 islets in a medium containing 100 microg Fe/mL of Resovist in the absence of PLL provided sufficient contrast for islet visualization on T(2)-weighted MRI. MTT assays showed that the viability of labeled islets was not different from that of unlabeled islets. No statistical difference was observed between labeled (2.91 +/- 0.36) and unlabeled islets (2.83 +/- 0.61) in terms of the ability to secrete insulin, as determined by the glucose stimulation index. We also evaluated the effect of iron oxide incorporation on the gene expressions in islet cells using RT-PCR (reverse transcriptase PCR). Insulin expression in labeled islets was significantly elevated (1.83 +/- 0.25 fold vs. unlabeled; p = 0.005), but not the expression of somatostatin (1.39 +/- 0.18 fold vs. unlabeled; p = 0.085) or glucagons (1.28 +/- 0.13 fold vs. unlabeled; p = 0.09). Expression of an important transcription factor for insulin gene transcription, BETA2 (beta-cell E-box trans-activator), was increased in labeled islets (1.67 +/- 0.15 fold vs. unlabeled; p = 0.029). The findings of this study indicate that Resovist provides a satisfactory means to image islets and has no deleterious effect on islet function or gene expression.

  14. Human amniotic epithelial cells induce localized cell-mediated immune privilege in vitro: implications for pancreatic islet transplantation.

    PubMed

    Qureshi, Khalid M; Oliver, Robert J; Paget, Michelle B; Murray, Hilary E; Bailey, Clifford J; Downing, Richard

    2011-01-01

    Chronic systemic immunosuppression in cell replacement therapy restricts its clinical application. This study sought to explore the potential of cell-based immune modulation as an alternative to immunosuppressive drug therapy in the context of pancreatic islet transplantation. Human amniotic epithelial cells (AEC) possess innate anti-inflammatory and immunosuppressive properties that were utilized to create localized immune privilege in an in vitro islet cell culture system. Cellular constructs composed of human islets and AEC (islet/AEC) were bioengineered under defined rotational cell culture conditions. Insulin secretory capacity was validated by glucose challenge and immunomodulatory potential characterized using a peripheral blood lymphocyte (PBL) proliferation assay. Results were compared to control constructs composed of islets or AEC cultured alone. Studies employing AEC-conditioned medium examined the role of soluble factors, and fluorescence immunocytochemistry was used to identify putative mediators of the immunosuppressive response in isolated AEC monocultures. Sustained, physiologically appropriate insulin secretion was observed in both islets and islet/AEC constructs. Activation of resting PBL proliferation occurred on exposure to human islets alone but this response was significantly (p < 0.05) attenuated by the presence of AEC and AEC-conditioned medium. Mitogen (phytohaemagglutinin, 5 μg/ml)-induced PBL proliferation was sustained on contact with isolated islets but abrogated by AEC, conditioned medium, and the islet/AEC constructs. Immunocytochemical analysis of AEC monocultures identified a subpopulation of cells that expressed the proapoptosis protein Fas ligand. This study demonstrates that human islet/AEC constructs exhibit localized immunosuppressive properties with no impairment of β-cell function. The data suggest that transplanted islets may benefit from the immune privilege status conferred on them as a consequence of their close

  15. Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins.

    PubMed

    Mathias, Rommel A; Chen, Yuan-Shou; Kapp, Eugene A; Greening, David W; Mathivanan, Suresh; Simpson, Richard J

    2011-08-01

    Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC-MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC-MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and

  16. Isles within islets: The lattice origin of small-world networks in pancreatic tissues

    NASA Astrophysics Data System (ADS)

    Barua, Amlan K.; Goel, Pranay

    2016-02-01

    The traditional computational model of the pancreatic islets of Langerhans is a lattice of β-cells connected with gap junctions. Numerous studies have investigated the behavior of networks of coupled β-cells and have shown that gap junctions synchronize bursting strongly. This simplistic architecture of islets, however, seems increasingly untenable at the face of recent experimental advances. In a microfluidics experiment on isolated islets, Rocheleau et al. (2004) showed a failure of penetration of excitation when one end received high glucose and other end was not excited sufficiently; this suggested that gap junctions may not be efficient at inducing synchrony throughout the islet. Recently, Stozer et al. (2013) have argued that the functional networks of β-cells in an islet are small world. Their results implicate the existence of a few long-range connections among cells in the network. The physiological reason underlying this claim is not well understood. These studies cast doubt on the original lattice model that largely predict an all-or-none synchrony among the cells. Here we have attempted to reconcile these observations in a unified framework. We assume that cells in the islet are coupled randomly to their nearest neighbors with some probability, p. We simulated detailed β-cell bursting in such islets. By varying p systematically we were led to network parameters similar to those obtained by Stozer et al. (2013). We find that the networks within islets break up into components giving rise to smaller isles within the super structure-isles-within-islets, as it were. This structure can also account for the partial excitation seen by Rocheleau et al. (2004). Our updated view of islet architecture thus explains the paradox how islets can have strongly synchronizing gap junctions, and be weakly coordinated at the same time.

  17. Pancreatic islet and stem cell transplantation: new strategies in cell therapy of diabetes mellitus.

    PubMed

    Bretzel, R G; Eckhard, M; Brendel, M D

    2004-03-01

    Long-term studies strongly suggest that tight control of blood glucose can prevent the development and retard the progression of chronic complications of type 1 diabetes mellitus. In contrast to conventional insulin treatment, replacement of a patient's islets of Langerhans either by pancreas organ transplantation of by isolated islet transplantation is the only treatment to achieve a constant normoglycemic state and avoiding hypoglycemic episodes, a typical adverse event of multiple daily insulin injections. However, the expense of this benefit is still the need for immunosuppressive treatment of the recipient with all its potential risks. Islet cell transplantation offers the advantage of being performed as a minimally invasive procedure, in which islets can be perfused percutaneously into the liver via the portal vein. As of June 2003, 705 pancreatic islet transplants worldwide have been reported to the International Islet Transplant Registry (ITR) at our Third Medical Department, University of Giessen/Germany. Data analysis shows at 1 year after adult islet transplantation a patient survival rate of 97%, a functioning islet graft in 54% of the cases, whereas insulin independence was meanwhile achieved in 20% of the cases. However, using a novel protocol established by the Edmonton Center/Canada, the insulin independence rates have improved significantly reaching meanwhile a 50-80% level. Finally, the concept of islet cell or stem cell transplantation is most attractive since it offers many perspectives: islet cell availability could become unlimited and islet or stem cells my be transplanted without life-long immunosuppressive treatment of the recipient, just to mention 2 of them. PMID:15238879

  18. Metabolic Oscillations in Pancreatic Islets Depend on the Intracellular Ca2+ Level but Not Ca2+ Oscillations

    PubMed Central

    Merrins, Matthew J.; Fendler, Bernard; Zhang, Min; Sherman, Arthur; Bertram, Richard; Satin, Leslie S.

    2010-01-01

    Abstract Plasma insulin is pulsatile and reflects oscillatory insulin secretion from pancreatic islets. Although both islet Ca2+ and metabolism oscillate, there is disagreement over their interrelationship, and whether they can be dissociated. In some models of islet oscillations, Ca2+ must oscillate for metabolic oscillations to occur, whereas in others metabolic oscillations can occur without Ca2+ oscillations. We used NAD(P)H fluorescence to assay oscillatory metabolism in mouse islets stimulated by 11.1 mM glucose. After abolishing Ca2+ oscillations with 200 μM diazoxide, we observed that oscillations in NAD(P)H persisted in 34% of islets (n = 101). In the remainder of the islets (66%) both Ca2+ and NAD(P)H oscillations were eliminated by diazoxide. However, in most of these islets NAD(P)H oscillations could be restored and amplified by raising extracellular KCl, which elevated the intracellular Ca2+ level but did not restore Ca2+ oscillations. Comparatively, we examined islets from ATP-sensitive K+ (KATP) channel-deficient SUR1−/− mice. Again NAD(P)H oscillations were evident even though Ca2+ and membrane potential oscillations were abolished. These observations are predicted by the dual oscillator model, in which intrinsic metabolic oscillations and Ca2+ feedback both contribute to the oscillatory islet behavior, but argue against other models that depend on Ca2+ oscillations for metabolic oscillations to occur. PMID:20655835

  19. In vitro maturation of viable islets from partially digested young pig pancreas.

    PubMed

    Lamb, Morgan; Laugenour, Kelly; Liang, Ouwen; Alexander, Michael; Foster, Clarence E; Lakey, Jonathan R T

    2014-03-01

    Isolation of islets from market-sized pigs is costly, with considerable islet losses from fragmentation occurring during isolation and tissue culture. Fetal and neonatal pigs yield insulin unresponsive islet-like cell clusters that become glucose-responsive after extended periods of time. Both issues impact clinical applicability and commercial scale-up. We have focused our efforts on a cost-effective scalable method of isolating viable insulin-responsive islets. Young Yorkshire pigs (mean age 20 days, range 4-30 days) underwent rapid pancreatectomy (<5 min) and partial digestion using low-dose collagenase, followed by in vitro culture at 37°C and 5% CO2 for up to 14 days. Islet viability was assessed using FDA/PI or Newport Green, and function was assessed using a glucose-stimulated insulin release (GSIR) assay. Islet yield was performed using enumeration of dithizone-stained aliquots. The young porcine (YP) islet yield at dissociation was 12.6 ± 2.1 × 10(3) IEQ (mean ± SEM) per organ and increased to 33.3 ± 6.4 × 10(3) IEQ after 7 days of in vitro culture. Viability was 97.3 ± 7% at dissociation and remained over 90% viable after 11 days in tissue culture (n = ns). Glucose responsiveness increased throughout maturation in culture. The stimulation index (SI) of the islets increased from 1.7 ± 2 on culture day 3 to 2.58 ± 0.5 on culture day 7. These results suggest that this method is both efficient and scalable for isolating and maturing insulin-responsive porcine islets in culture. PMID:23394130

  20. Pancreatic islet blood flow and its measurement

    PubMed Central

    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

    2016-01-01

    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. PMID:27124642

  1. Human isolates of dengue type 1 virus induce apoptosis in mouse neuroblastoma cells.

    PubMed Central

    Desprès, P; Flamand, M; Ceccaldi, P E; Deubel, V

    1996-01-01

    Human isolates of dengue (DEN) type 1 viruses FGA/89 and BR/90 differ in their membrane fusion properties in mosquito cell lines (P. Desprès et al., Virology 196:209-216, 1993). FGA/89 and BR/90 were assayed for their neurovirulence in newborn mice, and neurons were the major target cells for both DEN-1 virus strains within the central nervous system. To study the susceptibility of neurons to DEN virus infection, DEN virus replication was analyzed in the murine neuroblastoma cell line Neuro 2a. Infection of Neuro 2a cells with FGA/89 or BR/90 induced apoptotic DNA degradation after 25 h of infection. Studies of DEN protein synthesis revealed that accumulation of viral proteins leads to apoptotic cell death. The apoptotic process progressed more rapidly following BR/90 infection than it did after FGA/89 infection. The higher cytotoxicity of BR/90 for Neuro 2a cells was linked to an incomplete maturation of the envelope proteins, resulting in abortive virus assembly. Accumulation of viral proteins in the endoplasmic reticulum may induce stress and thereby activate the apoptotic pathway in mouse neuroblastoma cells. PMID:8648748

  2. Conversion of Daidzein and Genistein by an Anaerobic Bacterium Newly Isolated from the Mouse Intestine▿

    PubMed Central

    Matthies, Anastasia; Clavel, Thomas; Gütschow, Michael; Engst, Wolfram; Haller, Dirk; Blaut, Michael; Braune, Annett

    2008-01-01

    The metabolism of isoflavones by gut bacteria plays a key role in the availability and bioactivation of these compounds in the intestine. Daidzein and genistein are the most common dietary soy isoflavones. While daidzein conversion yielding equol has been known for some time, the corresponding formation of 5-hydroxy-equol from genistein has not been reported previously. We isolated a strictly anaerobic bacterium (Mt1B8) from the mouse intestine which converted daidzein via dihydrodaidzein to equol as well as genistein via dihydrogenistein to 5-hydroxy-equol. Strain Mt1B8 was a gram-positive, rod-shaped bacterium identified as a member of the Coriobacteriaceae. Strain Mt1B8 also transformed dihydrodaidzein and dihydrogenistein to equol and 5-hydroxy-equol, respectively. The conversion of daidzein, genistein, dihydrodaidzein, and dihydrogenistein in the stationary growth phase depended on preincubation with the corresponding isoflavonoid, indicating enzyme induction. Moreover, dihydrogenistein was transformed even more rapidly in the stationary phase when strain Mt1B8 was grown on either genistein or daidzein. Growing the cells on daidzein also enabled conversion of genistein. This suggests that the same enzymes are involved in the conversion of the two isoflavones. PMID:18539813

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

    SciTech Connect

    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.

  4. In Vivo Imaging of Transplanted Islets Labeled with a Novel Cationic Nanoparticle

    PubMed Central

    Oishi, Koichi; Miyamoto, Yoshitaka; Saito, Hiroaki; Murase, Katsutoshi; Ono, Kenji; Sawada, Makoto; Watanabe, Masami; Noguchi, Yasufumi; Fujiwara, Toshiyoshi; Hayashi, Shuji; Noguchi, Hirofumi

    2013-01-01

    To monitor pancreatic islet transplantation efficiency, reliable noninvasive imaging methods, such as magnetic resonance imaging (MRI) are needed. Although an efficient uptake of MRI contrast agent is required for islet cell labeling, commercially-available magnetic nanoparticles are not efficiently transduced into cells. We herein report the in vivo detection of transplanted islets labeled with a novel cationic nanoparticle that allowed for noninvasive monitoring of islet grafts in diabetic mice in real time. The positively-charged nanoparticles were transduced into a β-cell line, MIN6 cells, and into isolated islets for 1 hr. MRI showed a marked decrease in the signal intensity on T1- and T2-weighted images at the implantation site of the labeled MIN 6 cells or islets in the left kidneys of mice. These data suggest that the novel positively-charged nanoparticle could be useful to detect and monitor islet engraftment, which would greatly aid in the clinical management of islet transplant patients. PMID:23451139

  5. 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.

    PubMed

    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

  6. FRET-based voltage probes for confocal imaging: membrane potential oscillations throughout pancreatic islets.

    PubMed

    Kuznetsov, Andrey; Bindokas, Vytautas P; Marks, Jeremy D; Philipson, Louis H

    2005-07-01

    Insulin secretion is dependent on coordinated pancreatic islet physiology. In the present study, we found a way to overcome the limitations of cellular electrophysiology to optically determine cell membrane potential (V(m)) throughout an islet by using a fast voltage optical dye pair. Using laser scanning confocal microscopy (LSCM), we observed fluorescence (Förster) resonance energy transfer (FRET) with the fluorescent donor N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidyl-ethanolamine and the acceptor bis-(1,3-diethylthiobarbiturate) trimethine oxonol in the plasma membrane of essentially every cell within an islet. The FRET signal was approximately linear from V(m) -70 to +50 mV with a 2.5-fold change in amplitude. We evaluated the responses of islet cells to glucose and tetraethylammonium. Essentially, every responding cell in a mouse islet displayed similar time-dependent changes in V(m). When V(m) was measured simultaneously with intracellular Ca2+, all active cells showed tight coupling of V(m) to islet cell Ca2+ changes. Our findings indicate that FRET-based, voltage-sensitive dyes used in conjunction with LSCM imaging could be extremely useful in studies of excitation-secretion coupling in intact islets of Langerhans. PMID:15758044

  7. Light scattering as an intrinsic indicator for pancreatic islet cell mass and secretion.

    PubMed

    Ilegems, E; van Krieken, P P; Edlund, P K; Dicker, A; Alanentalo, T; Eriksson, M; Mandic, S; Ahlgren, U; Berggren, P-O

    2015-01-01

    The pancreatic islet of Langerhans is composed of endocrine cells producing and releasing hormones from secretory granules in response to various stimuli for maintenance of blood glucose homeostasis. In order to adapt to a variation in functional demands, these islets are capable of modulating their hormone secretion by increasing the number of endocrine cells as well as the functional response of individual cells. A failure in adaptive mechanisms will lead to inadequate blood glucose regulation and thereby to the development of diabetes. It is therefore necessary to develop tools for the assessment of both pancreatic islet mass and function, with the aim of understanding cellular regulatory mechanisms and factors guiding islet plasticity. Although most of the existing techniques rely on the use of artificial indicators, we present an imaging methodology based on intrinsic optical properties originating from mature insulin secretory granules within endocrine cells that reveals both pancreatic islet mass and function. We demonstrate the advantage of using this imaging strategy by monitoring in vivo scattering signal from pancreatic islets engrafted into the anterior chamber of the mouse eye, and how this versatile and noninvasive methodology permits the characterization of islet morphology and plasticity as well as hormone secretory status.

  8. Structure and expression of the mouse beta 2-microglobulin gene isolated from somatic and non-expressing teratocarcinoma cells.

    PubMed Central

    Daniel, F; Morello, D; Le Bail, O; Chambon, P; Cayre, Y; Kourilsky, P

    1983-01-01

    Mouse teratocarcinoma cells express neither H-2 heavy chains nor beta 2-microglobulin (beta 2-m). We have constructed two genomic libraries, one from PCC4-aza-RI embryonal carcinoma cells and the other from their adult syngenic counterpart 129/Sv liver cells (H-2bc). The libraries were screened with a full length mouse beta 2-m cDNA probe which we isolated and sequenced. Two cosmid clones carrying the entire beta 2-m gene were isolated, one from each library. There was no detectable difference in structure between the two genes. Furthermore, both were shown to be active and to restore beta 2-m synthesis upon transfer into mutant cells deficient in beta 2-m. Irreversible DNA alterations in or around the beta 2-m gene are thus unlikely to account for the lack of beta 2-m gene expression in embryonal teratocarcinoma cells. Images Fig. 3. Fig. 4. Fig. 6. PMID:6354707

  9. Inhibitory effect of quercetin isolated from rose hip (Rosa canina L.) against melanogenesis by mouse melanoma cells.

    PubMed

    Fujii, Takashi; Saito, Morio

    2009-09-01

    We investigated the effects of compounds isolated from a methanolic extract of rose hips on melanin biosynthesis in B16 mouse melanoma cells and the possible mechanisms responsible for the inhibition of melanin biosynthesis. We found that, among the isolated compounds, quercetin was a particularly potent melanogenesis inhibitor. To reveal the mechanism for this inhibition, the effects on tyrosinase of B16 mouse melanoma were measured. Quercetin decreased the intracellular tyrosinase activity as well as the tyrosinase activity in a cell culture-free system. We also examined the cellular level of tyrosinase protein and found that quercetin dose-dependently inhibited tyrosinase protein expression. We consider from these results that the inhibition of melanogenesis by quercetin was due to the inhibition of both tyrosinase activity and of the protein expression.

  10. Unraveling pancreatic islet biology by quantitative proteomics

    SciTech Connect

    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.

  11. The use of biomaterials in islet transplantation.

    PubMed

    Borg, Danielle J; Bonifacio, Ezio

    2011-10-01

    Pancreatic islet transplantation is a therapeutic option to replace destroyed β cells in autoimmune diabetes. Islets are transplanted into the liver via the portal vein; however, inflammation, the required immunosuppression, and lack of vasculature decrease early islet viability and function. Therefore, the use of accessory therapy and biomaterials to protect islets and improve islet function has definite therapeutic potential. Here we review the application of niche accessory cells and factors, as well as the use of biomaterials as carriers or capsules, for pancreatic islet transplantation. PMID:21748257

  12. 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.

    PubMed

    Bruni, Antonio; Pepper, Andrew R; Gala-Lopez, Boris; Pawlick, Rena; Abualhassan, Nasser; Crapo, James D; Piganelli, Jon D; Shapiro, A M James

    2016-07-01

    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 < 0 .05). Marginal, syngeneic islets (200 islets) transplanted under the renal capsule exhibited similar in vivo outcomes regardless of WI or 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. PMID:27220256

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

    NASA Technical Reports Server (NTRS)

    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.

  14. Step-by-step protocol to perfuse and dissect the mouse parotid gland and isolation of high-quality RNA from murine and human parotid tissue.

    PubMed

    Watermann, Christoph; Valerius, Klaus Peter; Wagner, Steffen; Wittekindt, Claus; Klussmann, Jens Peter; Baumgart-Vogt, Eveline; Karnati, Srikanth

    2016-04-01

    Macroscopic identification and surgical removal of the mouse parotid gland is demanding because of its anatomic location and size. Moreover, the mouse parotid gland contains high concentrations of RNases, making it difficult to isolate high-quality RNA. So far, appropriate methods for optimal perfusion-fixation and dissection of mouse parotid glands, as well as the isolation of high quality RNA from this tissue, are not available. Here we present a simple, optimized, step-by-step surgical method to perfuse and isolate murine parotid glands. We also compared two common RNA extraction methods (RNeasy Mini Kit versus TRIzol) for their yields of high-quality, intact RNA from human and murine parotid gland tissues that were either snap-frozen or immersed in RNAlater stabilization solution. Mouse parotid tissue that was perfused and immersed in RNAlater and human samples immersed in RNAlater exhibited the best RNA quality, independent of the isolation method. PMID:27071609

  15. Human fetal pancreatic islet-like structures as source material to treat type 1 diabetes.

    PubMed

    Ikeda, Yasuhiro; Kudva, Yogish C

    2013-01-01

    The incidence of type 1 diabetes is increasing worldwide. Current therapy continues to be suboptimal. An exciting therapeutic advance in the short term is closed loop technology development and application. However, cell and tissue therapy continues to be an unmet need for the disorder. Human islets isolated from deceased donors will be clinically available to treat type 1 diabetes within the next 1 to 2 years. Other approaches such as xenotransplantation and islet products derived from human embryonic stem cells and induced pluripotent stem cells are currently being pursued. The current commentary provides context and discusses future endeavors for transplantation of islet-like structures derived from fetal pancreas. PMID:24377429

  16. Pancreatic islet-specific overexpression of Reg3β protein induced the expression of pro-islet genes and protected the mice against streptozotocin-induced diabetes mellitus.

    PubMed

    Xiong, Xiaoquan; Wang, Xiao; Li, Bing; Chowdhury, Subrata; Lu, Yarong; Srikant, Coimbatore B; Ning, Guang; Liu, Jun-Li

    2011-04-01

    Reg family proteins have been implicated in islet β-cell proliferation, survival, and regeneration. The expression of Reg3β (pancreatitis-associated protein) is highly induced in experimental diabetes and acute pancreatitis, but its precise role has not been established. Through knockout studies, this protein was shown to be mitogenic, antiapoptotic, and anti-inflammatory in the liver and pancreatic acinars. To test whether it can promote islet cell growth or survival against experimental damage, we developed β-cell-specific overexpression using rat insulin I promoter, evaluated the changes in normal islet function, gene expression profile, and the response to streptozotocin-induced diabetes. Significant and specific overexpression of Reg3β was achieved in the pancreatic islets of RIP-I/Reg3β mice, which exhibited normal islet histology, β-cell mass, and in vivo and in vitro insulin secretion in response to high glucose yet were slightly hyperglycemic and low in islet GLUT2 level. Upon streptozotocin treatment, in contrast to wild-type littermates that became hyperglycemic in 3 days and lost 15% of their weight, RIP-I/Reg3β mice were significantly protected from hyperglycemia and weight loss. To identify specific targets affected by Reg3β overexpression, a whole genome DNA microarray on islet RNA isolated from the transgenic mice revealed more than 45 genes significantly either up- or downregulated. Among them, islet-protective osteopontin/SPP1 and acute responsive nuclear protein p8/NUPR1 were significantly induced, a result further confirmed by real-time PCR, Western blots, and immunohistochemistry. Our results suggest that Reg3β is unlikely an islet growth factor but a putative protector that prevents streptozotocin-induced damage by inducing the expression of specific genes.

  17. Expression of nucleoside transporter in freshly isolated neurons and astrocytes from mouse brain.

    PubMed

    Li, B; Gu, L; Hertz, L; Peng, L

    2013-11-01

    Nucleoside transporters comprise equilibrative ENT1-4 and concentrative CNT1-3. CNTs transport against an intracellular/extracellular gradient and are essential for transmitter removal, independently of metabolic need. ENT1-4 mediate transport until intracellular/extracellular equilibrium of the transported compound, but are very efficient, when the accumulated nucleoside or nucleobase is rapidly eliminated by metabolism. Most nucleoside transporters are membrane-bound, but ENT3 is mainly intracellular. This study uses freshly isolated neurons and astrocytes from two adult mouse strains. In one transgenic strain the neuronal marker Thy1 was associated with a compound fluorescing at one wavelength, and in the other the astrocytic marker GFAP was associated with a compound fluorescent at a different wavelength. Highly purified astrocytic and neuronal populations (as determined by presence/absence of cell-specific genes) were obtained from these mice by fluorescence-activated cell sorting. In each population mRNA analysis was performed by reverse-transcription polymerase chain reaction. CNT1 was absent in both cell types; all other nucleoside transporters were expressed to at least a similar degree (in relation to applied amount of RNA and to a house-keeping gene) in astrocytes as in neurons. Astrocytic ENT3 enrichment was dramatic, but it was not up-regulated after fluoxetine-mediated increase in DNA synthesis. A comparison with results obtained in cultured astrocytes shows that the latter are generally compatible with the present findings and suggests that many observations obtained in intact tissue, mainly by in situ hybridization (which also determines mRNA expression) may underestimate astrocytic nucleoside transporter expression.

  18. Histamine-dependent prolongation by aldosterone of vasoconstriction in isolated small mesenteric arteries of the mouse.

    PubMed

    Schjerning, Jeppe; Uhrenholt, Torben R; Svenningsen, Per; Vanhoutte, Paul M; Skøtt, Ole; Jensen, Boye L; Hansen, Pernille B L

    2013-04-15

    In arterioles, aldosterone counteracts the rapid dilatation (recovery) following depolarization-induced contraction. The hypothesis was tested that this effect of aldosterone depends on cyclooxygenase (COX)-derived products and/or nitric oxide (NO) synthase (NOS) inhibition. Recovery of the response to high K(+) was observed in mesenteric arteries of wild-type and COX-2(-/-) mice but it was significantly diminished in preparations from endothelial NOS (eNOS)(-/-) mice. Aldosterone pretreatment inhibited recovery from wild-type and COX-2(-/-) mice. The NO donor sodium nitroprusside (SNP) restored recovery in arteries from eNOS(-/-) mice, and this was inhibited by aldosterone. Actinomycin-D abolished the effect of aldosterone, indicating a genomic effect. The effect was blocked by indomethacin and by the COX-1 inhibitor valeryl salicylate but not by NS-398 (10(-6) mol/l) or the TP-receptor antagonist S18886 (10(-7) mol/l). The effect of aldosterone on recovery in arteries from wild-type mice and the SNP-mediated dilatation in arteries from eNOS(-/-) mice was inhibited by the histamine H2 receptor antagonist cimetidine. RT-PCR showed expression of mast cell markers in mouse mesenteric arteries. The adventitia displayed granular cells positive for toluidine blue vital stain. Confocal microscopy of live mast cells showed loss of quinacrine fluorescence and swelling after aldosterone treatment, indicating degranulation. RT-PCR showed expression of mineralocorticoid receptors in mesenteric arteries and in isolated mast cells. These findings suggest that aldosterone inhibits recovery by stimulation of histamine release from mast cells along mesenteric arteries. The resulting activation of H2 receptors decreases the sensitivity to NO of vascular smooth muscle cells. Aldosterone may chronically affect vascular function through paracrine release of histamine.

  19. Transgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates.

    PubMed

    Vidal, Enric; Fernández-Borges, Natalia; Pintado, Belén; Eraña, Hasier; Ordóñez, Montserrat; Márquez, Mercedes; Chianini, Francesca; Fondevila, Dolors; Sánchez-Martín, Manuel A; Andreoletti, Olivier; Dagleish, Mark P; Pumarola, Martí; Castilla, Joaquín

    2015-08-01

    Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrPC was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrPd) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrPd originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits.

  20. Transgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates

    PubMed Central

    Pintado, Belén; Eraña, Hasier; Ordóñez, Montserrat; Márquez, Mercedes; Chianini, Francesca; Fondevila, Dolors; Sánchez-Martín, Manuel A.; Andreoletti, Olivier; Dagleish, Mark P.; Pumarola, Martí; Castilla, Joaquín

    2015-01-01

    Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrPC was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrPd) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrPd originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits. PMID:26247589

  1. Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

    PubMed

    Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

    2016-10-01

    The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets.

  2. Fluorescent protein vectors for pancreatic islet cell identification in live-cell imaging.

    PubMed

    Shuai, Hongyan; Xu, Yunjian; Yu, Qian; Gylfe, Erik; Tengholm, Anders

    2016-10-01

    The islets of Langerhans contain different types of endocrine cells, which are crucial for glucose homeostasis. β- and α-cells that release insulin and glucagon, respectively, are most abundant, whereas somatostatin-producing δ-cells and particularly pancreatic polypeptide-releasing PP-cells are more scarce. Studies of islet cell function are hampered by difficulties to identify the different cell types, especially in live-cell imaging experiments when immunostaining is unsuitable. The aim of the present study was to create a set of vectors for fluorescent protein expression with cell-type-specific promoters and evaluate their applicability in functional islet imaging. We constructed six adenoviral vectors for expression of red and green fluorescent proteins controlled by the insulin, preproglucagon, somatostatin, or pancreatic polypeptide promoters. After transduction of mouse and human islets or dispersed islet cells, a majority of the fluorescent cells also immunostained for the appropriate hormone. Recordings of the sub-plasma membrane Ca(2+) and cAMP concentrations with a fluorescent indicator and a protein biosensor, respectively, showed that labeled cells respond to glucose and other modulators of secretion and revealed a striking variability in Ca(2+) signaling among α-cells. The measurements allowed comparison of the phase relationship of Ca(2+) oscillations between different types of cells within intact islets. We conclude that the fluorescent protein vectors allow easy identification of specific islet cell types and can be used in live-cell imaging together with organic dyes and genetically encoded biosensors. This approach will facilitate studies of normal islet physiology and help to clarify molecular defects and disturbed cell interactions in diabetic islets. PMID:27539300

  3. Effect of social odor context on the emission of isolation-induced ultrasonic vocalizations in the BTBR T+tf/J mouse model for autism

    PubMed Central

    Wöhr, Markus

    2015-01-01

    An important diagnostic criterion for social communication deficits in autism spectrum disorders (ASD) are difficulties in adjusting behavior to suit different social contexts. While the BTBR T+tf/J (BTBR) inbred strain of mice is one of the most commonly used mouse models for ASD, little is known about whether BTBR mice display deficits in detecting changes in social context and their ability to adjust to them. Here, it was tested therefore whether the emission of isolation-induced ultrasonic vocalizations (USV) in BTBR mouse pups is affected by the social odor context, in comparison to the standard control strain with high sociability, C57BL/6J (B6). It is known that the presence of odors from mothers and littermates leads to a calming of the isolated mouse pup, and hence to a reduction in isolation-induced USV emission. In accordance with their behavioral phenotypes with relevance to all diagnostic core symptoms of ASD, it was predicted that BTBR mouse pups would not display a calming response when tested under soiled bedding conditions with home cage bedding material containing maternal odors, and that similar isolation-induced USV emission rates would be seen in BTBR mice tested under clean and soiled bedding conditions. Unexpectedly, however, the present findings show that BTBR mouse pups display such a calming response and emit fewer isolation-induced USV when tested under soiled as compared to clean bedding conditions, similar to B6 mouse pups. Yet, in contrast to B6 mouse pups, which emitted isolation-induced USV with shorter call durations and lower levels of frequency modulation under soiled bedding conditions, social odor context had no effect on acoustic call features in BTBR mouse pups. This indicates that the BTBR mouse model for ASD does not display deficits in detecting changes in social context, but has a limited ability and/or reduced motivation to adjust to them. PMID:25852455

  4. Novel role for matricellular proteins in the regulation of islet β cell survival: the effect of SPARC on survival, proliferation, and signaling.

    PubMed

    Ryall, Claire L; Viloria, Katrina; Lhaf, Fadel; Walker, Anthony J; King, Aileen; Jones, Peter; Mackintosh, David; McNeice, Rosemary; Kocher, Hemant; Flodstrom-Tullberg, Malin; Edling, Charlotte; Hill, Natasha J

    2014-10-31

    Understanding the mechanisms regulating islet growth and survival is critical for developing novel approaches to increasing or sustaining β cell mass in both type 1 and type 2 diabetes patients. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular protein that is important for the regulation of cell growth and adhesion. Increased SPARC can be detected in the serum of type 2 diabetes patients. The aim of this study was to investigate the role of SPARC in the regulation of β cell growth and survival. We show using immunohistochemistry that SPARC is expressed by stromal cells within islets and can be detected in primary mouse islets by Western blot. SPARC is secreted at high levels by pancreatic stellate cells and is regulated by metabolic parameters in these cells, but SPARC expression was not detectable in β cells. In islets, SPARC expression is highest in young mice, and is also elevated in the islets of non-obese diabetic (NOD) mice compared with controls. Purified SPARC inhibits growth factor-induced signaling in both INS-1 β cells and primary mouse islets, and inhibits IGF-1-induced proliferation of INS-1 β cells. Similarly, exogenous SPARC prevents IGF-1-induced survival of primary mouse islet cells. This study identifies the stromal-derived matricellular protein SPARC as a novel regulator of islet survival and β cell growth.

  5. Prophylactically Decontaminating Human Islet Product for Safe Clinical Application: Effective and Potent Method

    PubMed Central

    Qi, Meirigeng; Omori, Keiko; Mullen, Yoko; McFadden, Brian; Valiente, Luis; Juan, Jemily; Bilbao, Shiela; Tegtmeier, Bernard R.; Dafoe, Donald; Kandeel, Fouad; Al-Abdullah, Ismail H.

    2016-01-01

    Background Transplanting pancreatic islets into recipients must be safe and effective to treat type 1 diabetes. Islet quality and quantity are important; however, the final product must also be free from microbial contamination and low endotoxin levels. Methods This study explored a method to eliminate contamination in manufacturing islets for transplantation. A simple (single antibiotic n = 164) and refined (triple antimicrobial agents, n = 279) pancreas decontaminating methods were used to test their effects on reducing the contamination rates in the islet final product. A total of 443 pancreata were processed for islet isolations. Three samples for microbial tests (Gram stain, aerobic, and anaerobic culture) were taken at preprocess (pancreas preservation), postisolation, and postculture. Endotoxin levels were measured only for islets considered for transplantation. Results Of 443 pancreata used for islet isolation, 79 (17.8%) showed signs of contamination in preprocess samples; 10 (2.3%) were contaminated in both preprocess and in the final product (postisolation and postculture) samples. Contamination rates in which preprocess and final product samples were positive for contamination was significantly lower using the refined method (refined vs simple method: 5% vs 20.5%, P = 0.045). Identical microbial species were present in both preprocess and in the final product. Conclusions This study demonstrated that the refined method reduces the rate of contamination of the islet final product and is safe for clinical application. Moreover, it may be used as a standard method during human islet manufacturing facilitating the application of a biological license agreement from United States Food and Drug Administration. PMID:26894230

  6. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.

    PubMed

    Martin, G R

    1981-12-01

    This report describes the establishment directly from normal preimplantation mouse embryos of a cell line that forms teratocarcinomas when injected into mice. The pluripotency of these embryonic stem cells was demonstrated conclusively by the observation that subclonal cultures, derived from isolated single cells, can differentiate into a wide variety of cell types. Such embryonic stem cells were isolated from inner cell masses of late blastocysts cultured in medium conditioned by an established teratocarcinoma stem cell line. This suggests that such conditioned medium might contain a growth factor that stimulates the proliferation or inhibits the differentiation of normal pluripotent embryonic cells, or both. This method of obtaining embryonic stem cells makes feasible the isolation of pluripotent cells lines from various types of noninbred embryo, including those carrying mutant genes. The availability of such cell lines should made possible new approaches to the study of early mammalian development.

  7. Human islet purification: a prospective comparison of Euro-Ficoll and bovine serum albumin density gradients.

    PubMed

    Chadwick, D R; Robertson, G S; Contractor, H; Swift, S; Rose, S; Thirdborough, S T; Chamberlain, R; James, R F; Bell, P R; London, N J

    1993-01-01

    Euro-Ficoll (EF) and bovine serum albumin (BSA) are the two most commonly used media for the density gradient purification of human pancreatic islets. The aim of this study was to compare these two media with respect to the efficiency of human islet isolation. Ten human pancreata were collagenase-digested, and samples of digest were separated on either a continuous linear density gradient of BSA or a discontinuous gradient of EF (1.108/1.096/1.037/Euro-Collins). Efficiency of islet purification was assessed by insulin and amylase assay of aliquots aspirated from the BSA gradients, and from the interfaces of the EF gradients. Islets were obtained from two interfaces in the EF gradients. Islet yield from the upper interface was generally poor (median 28% of total insulin; range 2-71%), but purity was better than for an equivalent yield using BSA [1% (0-3%) amylase contamination for EF versus 6% (0-37%) for BSA; P = 0.013]. Pooling both EF interfaces increased yield to 66% (17-81%) but markedly reduced purity [46% (0-50%) amylase for EF versus 31% (0-52%) for BSA]. In conclusion, the efficiency of human islet purification is similar, though disappointingly low, with BSA and with EF. Considerable scope exists, therefore, for improvement in the density gradient purification of human islets. PMID:8329732

  8. Isolation and Culture of Cells from the Nephrogenic Zone of the Embryonic Mouse Kidney

    PubMed Central

    Brown, Aaron C.; Blank, Ulrika; Adams, Derek C.; Karolak, Michele J.; Fetting, Jennifer L.; Hill, Beth L.; Oxburgh, Leif

    2011-01-01

    Embryonic development of the kidney has been extensively studied both as a model for epithelial-mesenchymal interaction in organogenesis and to gain understanding of the origins of congenital kidney disease. More recently, the possibility of steering naïve embryonic stem cells toward nephrogenic fates has been explored in the emerging field of regenerative medicine. Genetic studies in the mouse have identified several pathways required for kidney development, and a global catalog of gene transcription in the organ has recently been generated http://www.gudmap.org/, providing numerous candidate regulators of essential developmental functions. Organogenesis of the rodent kidney can be studied in organ culture, and many reports have used this approach to analyze outcomes of either applying candidate proteins or knocking down the expression of candidate genes using siRNA or morpholinos. However, the applicability of organ culture to the study of signaling that regulates stem/progenitor cell differentiation versus renewal in the developing kidney is limited as cultured organs contain a compact extracellular matrix limiting diffusion of macromolecules and virus particles. To study the cell signaling events that influence the stem/progenitor cell niche in the kidney we have developed a primary cell system that establishes the nephrogenic zone or progenitor cell niche of the developing kidney ex vivo in isolation from the epithelial inducer of differentiation. Using limited enzymatic digestion, nephrogenic zone cells can be selectively liberated from developing kidneys at E17.5. Following filtration, these cells can be cultured as an irregular monolayer using optimized conditions. Marker gene analysis demonstrates that these cultures contain a distribution of cell types characteristic of the nephrogenic zone in vivo, and that they maintain appropriate marker gene expression during the culture period. These cells are highly accessible to small molecule and recombinant

  9. Effects of N-acetylcysteine on isolated mouse skeletal muscle: contractile properties, temperature dependence, and metabolism.

    PubMed

    Katz, Abram; Hernández, Andrés; Caballero, Diana Marcela Ramos; Briceno, Javier Fernando Bonilla; Amezquita, Laura Victoria Rivera; Kosterina, Natalia; Bruton, Joseph D; Westerblad, Håkan

    2014-03-01

    The effects of the general antioxidant N-acetylcysteine (NAC) on muscle function and metabolism were examined. Isolated paired mouse extensor digitorum longus muscles were studied in the absence or presence of 20 mM NAC. Muscles were electrically stimulated to perform 100 isometric tetanic contractions (300 ms duration) at frequencies resulting in ∼85% of maximal force (70-150 Hz at 25-40 °C). NAC did not significantly affect peak force in the unfatigued state at any temperature but significantly slowed tetanic force development in a temperature-dependent fashion (e.g., time to 50% of peak tension averaged 35 ± 2 ms [control] and 37 ± 1 ms [NAC] at 25 °C vs. 21 ± 1 ms [control] and 52 ± 6 ms [NAC, P < 0.01] at 40 °C). During repeated contractions, NAC maximally enhanced peak force by the fifth tetanus at all temperatures (by ∼30%). Thereafter, the effect of NAC disappeared rapidly at high temperatures (35-40 °C) and more slowly at the lower temperatures (25-30 °C). At all temperatures, the enhancing effect of NAC on peak force was associated with a slowing of relaxation. NAC did not significantly affect myosin light chain phosphorylation at rest or after five contractions (∼50% increase vs. rest). After five tetani, lactate and inorganic phosphate increased about 20-fold and 2-fold, respectively, both in control and NAC-treated muscles. Interestingly, after five tetani, the increase in glucose 6-P was ∼2-fold greater, whereas the increase in malate was inhibited by ∼75% with NAC vs. control, illustrating the metabolic effects of NAC. NAC slightly decreased the maximum shortening velocity in early fatigue (five to seven repeated tetani). These data demonstrate that the antioxidant NAC transiently enhances muscle force generation by a mechanism that is independent of changes in myosin light chain phosphorylation and inorganic phosphate. The slowing of relaxation suggests that NAC enhances isometric force by facilitating fusion

  10. Transplantation of islet cells across major histocompatibility barriers after total lymphoid irradiation and infusion of allogeneic bone marrow cells

    SciTech Connect

    Britt, L.D.; Scharp, D.W.; Lacy, P.E.; Slavin, S.

    1982-08-01

    Diabetic Lewis rats (AgB1/L) were evaluated as recipients of allogeneic Wistar-Furth (AgB2/2) isolated adult islets without the use of standard recipient immunosuppression. One group was treated with fractionated total lymphoid irradiation (TLI) and Wistar-Furth bone marrow cell reconstitution to proven chimerism prior to islet transplantation. This group returned to a prediabetic state following Wistar-Furth islet transplantation without any evidence of rejection for 100 days posttransplant. A second group of Lewis rats received only TLI without bone marrow treatment. They gave a varying result following islet transplantation with one recipient showing evidence of prolonged islet survival. A third chimeric control group did not receive isolated islets and did not alter their diabetic state. A fourth group was not given TLI nor donor bone marrow cells and uniformly rejected their allogeneic islets by 7 days. Thus, allogeneic adult islets will survive across major rat histocompatibility barriers using TLI and donor bone marrow chimerism as the only form of immunosuppression.

  11. Isolation of cDNA clones specifying the fourth component of mouse complement and its isotype, sex-limited protein.

    PubMed Central

    Nonaka, M; Takahashi, M; Natsuume-Sakai, S; Nonaka, M; Tanaka, S; Shimizu, A; Honjo, T

    1984-01-01

    cDNA clones specific for the fourth component of mouse complement (C4) and its hormonally regulated isotype, sex-linked protein (Slp), were isolated using as a probe a 20-mer synthetic oligonucleotide corresponding to a known sequence of human C4 cDNA. Two types of clones, one specific for C4 (pFC4/10, with a 3.7 kilobase insert) and one specific for Slp (pFSlp/1, with a 4.7 kilobase insert), were isolated from liver cDNA libraries constructed from the Slp-producing FM mouse strain. The cDNA inserts of these clones shared 70% of the restriction sites determined. Only one type of clone was isolated from the Slp-negative DBA/1 strain; this type showed restriction maps indistinguishable from that of pFC4/10. pFC4/10 and pFSlp/1 displayed extensive homology: 94% nucleotide homology and 89% derived amino acid homology in the C4a region and 92% nucleotide homology and 89% derived amino acid homology in the thiol-ester region. An Arg-Gln-Lys-Arg sequence in the beta-alpha junction and a Cys-Ala-Glu-Gln sequence in the thiol-ester site were identified for both proteins. A remarkable divergency between C4 and Slp sequences was recognized in the region immediately following the C4a sequence. PMID:6208559

  12. Animal Models of Diabetes Mellitus for Islet Transplantation

    PubMed Central

    Sakata, Naoaki; Yoshimatsu, Gumpei; Tsuchiya, Haruyuki; Egawa, Shinichi; Unno, Michiaki

    2012-01-01

    Due to current improvements in techniques for islet isolation and transplantation and protocols for immunosuppressants, islet transplantation has become an effective treatment for severe diabetes patients. Many diabetic animal models have contributed to such improvements. In this paper, we focus on 3 types of models with different mechanisms for inducing diabetes mellitus (DM): models induced by drugs including streptozotocin (STZ), pancreatomized models, and spontaneous models due to autoimmunity. STZ-induced diabetes is one of the most commonly used experimental diabetic models and is employed using many specimens including rodents, pigs or monkeys. The management of STZ models is well established for islet studies. Pancreatomized models reveal different aspects compared to STZ-induced models in terms of loss of function in the increase and decrease of blood glucose and therefore are useful for evaluating the condition in total pancreatomized patients. Spontaneous models are useful for preclinical studies including the assessment of immunosuppressants because such models involve the same mechanisms as type 1 DM in the clinical setting. In conclusion, islet researchers should select suitable diabetic animal models according to the aim of the study. PMID:23346100

  13. Animal models of diabetes mellitus for islet transplantation.

    PubMed

    Sakata, Naoaki; Yoshimatsu, Gumpei; Tsuchiya, Haruyuki; Egawa, Shinichi; Unno, Michiaki

    2012-01-01

    Due to current improvements in techniques for islet isolation and transplantation and protocols for immunosuppressants, islet transplantation has become an effective treatment for severe diabetes patients. Many diabetic animal models have contributed to such improvements. In this paper, we focus on 3 types of models with different mechanisms for inducing diabetes mellitus (DM): models induced by drugs including streptozotocin (STZ), pancreatomized models, and spontaneous models due to autoimmunity. STZ-induced diabetes is one of the most commonly used experimental diabetic models and is employed using many specimens including rodents, pigs or monkeys. The management of STZ models is well established for islet studies. Pancreatomized models reveal different aspects compared to STZ-induced models in terms of loss of function in the increase and decrease of blood glucose and therefore are useful for evaluating the condition in total pancreatomized patients. Spontaneous models are useful for preclinical studies including the assessment of immunosuppressants because such models involve the same mechanisms as type 1 DM in the clinical setting. In conclusion, islet researchers should select suitable diabetic animal models according to the aim of the study. PMID:23346100

  14. Young capillary vessels rejuvenate aged pancreatic islets.

    PubMed

    Almaça, Joana; Molina, Judith; Arrojo E Drigo, Rafael; Abdulreda, Midhat H; Jeon, Won Bae; Berggren, Per-Olof; Caicedo, Alejandro; Nam, Hong Gil

    2014-12-01

    Pancreatic islets secrete hormones that play a key role in regulating blood glucose levels (glycemia). Age-dependent impairment of islet function and concomitant dysregulation of glycemia are major health threats in aged populations. However, the major causes of the age-dependent decline of islet function are still disputed. Here we demonstrate that aging of pancreatic islets in mice and humans is notably associated with inflammation and fibrosis of islet blood vessels but does not affect glucose sensing and the insulin secretory capacity of islet beta cells. Accordingly, when transplanted into the anterior chamber of the eye of young mice with diabetes, islets from old mice are revascularized with healthy blood vessels, show strong islet cell proliferation, and fully restore control of glycemia. Our results indicate that beta cell function does not decline with age and suggest that islet function is threatened by an age-dependent impairment of islet vascular function. Strategies to mitigate age-dependent dysregulation in glycemia should therefore target systemic and/or local inflammation and fibrosis of the aged islet vasculature. PMID:25404292

  15. Hair Follicle Dermal Sheath Derived Cells Improve Islet Allograft Survival without Systemic Immunosuppression

    PubMed Central

    Wang, Xiaojie; Hao, Jianqiang; Leung, Gigi; Breitkopf, Trisia; Wang, Eddy; Kwong, Nicole; Akhoundsadegh, Noushin; Warnock, Garth L.; Shapiro, Jerry; McElwee, Kevin J.

    2015-01-01

    Immunosuppressive drugs successfully prevent rejection of islet allografts in the treatment of type I diabetes. However, the drugs also suppress systemic immunity increasing the risk of opportunistic infection and cancer development in allograft recipients. In this study, we investigated a new treatment for autoimmune diabetes using naturally immune privileged, hair follicle derived, autologous cells to provide localized immune protection of islet allotransplants. Islets from Balb/c mouse donors were cotransplanted with syngeneic hair follicle dermal sheath cup cells (DSCC, group 1) or fibroblasts (FB, group 2) under the kidney capsule of immune-competent, streptozotocin induced, diabetic C57BL/6 recipients. Group 1 allografts survived significantly longer than group 2 (32.2 ± 12.2 versus 14.1 ± 3.3 days, P < 0.001) without administration of any systemic immunosuppressive agents. DSCC reduced T cell activation in the renal lymph node, prevented graft infiltrates, modulated inflammatory chemokine and cytokine profiles, and preserved better beta cell function in the islet allografts, but no systemic immunosuppression was observed. In summary, DSCC prolong islet allograft survival without systemic immunosuppression by local modulation of alloimmune responses, enhancing of beta cell survival, and promoting of graft revascularization. This novel finding demonstrates the capacity of easily accessible hair follicle cells to be used as local immunosuppression agents in islet transplantation. PMID:26000314

  16. Hair follicle dermal sheath derived cells improve islet allograft survival without systemic immunosuppression.

    PubMed

    Wang, Xiaojie; Hao, Jianqiang; Leung, Gigi; Breitkopf, Trisia; Wang, Eddy; Kwong, Nicole; Akhoundsadegh, Noushin; Warnock, Garth L; Shapiro, Jerry; McElwee, Kevin J

    2015-01-01

    Immunosuppressive drugs successfully prevent rejection of islet allografts in the treatment of type I diabetes. However, the drugs also suppress systemic immunity increasing the risk of opportunistic infection and cancer development in allograft recipients. In this study, we investigated a new treatment for autoimmune diabetes using naturally immune privileged, hair follicle derived, autologous cells to provide localized immune protection of islet allotransplants. Islets from Balb/c mouse donors were cotransplanted with syngeneic hair follicle dermal sheath cup cells (DSCC, group 1) or fibroblasts (FB, group 2) under the kidney capsule of immune-competent, streptozotocin induced, diabetic C57BL/6 recipients. Group 1 allografts survived significantly longer than group 2 (32.2 ± 12.2 versus 14.1 ± 3.3 days, P < 0.001) without administration of any systemic immunosuppressive agents. DSCC reduced T cell activation in the renal lymph node, prevented graft infiltrates, modulated inflammatory chemokine and cytokine profiles, and preserved better beta cell function in the islet allografts, but no systemic immunosuppression was observed. In summary, DSCC prolong islet allograft survival without systemic immunosuppression by local modulation of alloimmune responses, enhancing of beta cell survival, and promoting of graft revascularization. This novel finding demonstrates the capacity of easily accessible hair follicle cells to be used as local immunosuppression agents in islet transplantation.

  17. Design Principles of Pancreatic Islets: Glucose-Dependent Coordination of Hormone Pulses.

    PubMed

    Hoang, Danh-Tai; Hara, Manami; Jo, Junghyo

    2016-01-01

    Pancreatic islets are functional units involved in glucose homeostasis. The multicellular system comprises three main cell types; β and α cells reciprocally decrease and increase blood glucose by producing insulin and glucagon pulses, while the role of δ cells is less clear. Although their spatial organization and the paracrine/autocrine interactions between them have been extensively studied, the functional implications of the design principles are still lacking. In this study, we formulated a mathematical model that integrates the pulsatility of hormone secretion and the interactions and organization of islet cells and examined the effects of different cellular compositions and organizations in mouse and human islets. A common feature of both species was that islet cells produced synchronous hormone pulses under low- and high-glucose conditions, while they produced asynchronous hormone pulses under normal glucose conditions. However, the synchronous coordination of insulin and glucagon pulses at low glucose was more pronounced in human islets that had more α cells. When β cells were selectively removed to mimic diabetic conditions, the anti-synchronicity of insulin and glucagon pulses was deteriorated at high glucose, but it could be partially recovered when the re-aggregation of remaining cells was considered. Finally, the third cell type, δ cells, which introduced additional complexity in the multicellular system, prevented the excessive synchronization of hormone pulses. Our computational study suggests that controllable synchronization is a design principle of pancreatic islets. PMID:27035570

  18. Design Principles of Pancreatic Islets: Glucose-Dependent Coordination of Hormone Pulses

    PubMed Central

    Hoang, Danh-Tai; Hara, Manami; Jo, Junghyo

    2016-01-01

    Pancreatic islets are functional units involved in glucose homeostasis. The multicellular system comprises three main cell types; β and α cells reciprocally decrease and increase blood glucose by producing insulin and glucagon pulses, while the role of δ cells is less clear. Although their spatial organization and the paracrine/autocrine interactions between them have been extensively studied, the functional implications of the design principles are still lacking. In this study, we formulated a mathematical model that integrates the pulsatility of hormone secretion and the interactions and organization of islet cells and examined the effects of different cellular compositions and organizations in mouse and human islets. A common feature of both species was that islet cells produced synchronous hormone pulses under low- and high-glucose conditions, while they produced asynchronous hormone pulses under normal glucose conditions. However, the synchronous coordination of insulin and glucagon pulses at low glucose was more pronounced in human islets that had more α cells. When β cells were selectively removed to mimic diabetic conditions, the anti-synchronicity of insulin and glucagon pulses was deteriorated at high glucose, but it could be partially recovered when the re-aggregation of remaining cells was considered. Finally, the third cell type, δ cells, which introduced additional complexity in the multicellular system, prevented the excessive synchronization of hormone pulses. Our computational study suggests that controllable synchronization is a design principle of pancreatic islets. PMID:27035570

  19. Fractal spatial distribution of pancreatic islets in three dimensions: a self-avoiding growth model

    PubMed Central

    Jo, Junghyo; Hörnblad, Andreas; Kilimnik, German; Hara, Manami; Ahlgren, Ulf; Periwal, Vipul

    2013-01-01

    The islets of Langerhans, responsible for controlling blood glucose levels, are dispersed within the pancreas. A universal power law governing the fractal spatial distribution of islets in two-dimensional pancreatic sections has been reported. However, the fractal geometry in the actual three-dimensional pancreas volume, and the developmental process that gives rise to such a self-similar structure, have not been investigated. Here, we examined the three-dimensional spatial distribution of islets in intact mouse pancreata using optical projection tomography and found a power law with a fractal dimension, 2.1. Furthermore, based on two-dimensional pancreatic sections of human autopsies, we found that the distribution of human islets also follows a universal power law with fractal dimension 1.5 in adult pancreata, which agrees with the value previously reported in smaller mammalian pancreas sections. Finally, we developed a self-avoiding growth model for the development of the islet distribution and found that the fractal nature of the spatial islet distribution may be associated with the self-avoidance in the branching process of vascularization in the pancreas. PMID:23629025

  20. PAX4 Defines an Expandable β-Cell Subpopulation in the Adult Pancreatic Islet

    PubMed Central

    Lorenzo, Petra I.; Fuente-Martín, Esther; Brun, Thierry; Cobo-Vuilleumier, Nadia; Jimenez-Moreno, Carmen María; G. Herrera Gomez, Irene; López Noriega, Livia; Mellado-Gil, José Manuel; Martin-Montalvo, Alejandro; Soria, Bernat; Gauthier, Benoit R.

    2015-01-01

    PAX4 is a key regulator of pancreatic islet development whilst in adult acute overexpression protects β-cells against stress-induced apoptosis and stimulates proliferation. Nonetheless, sustained PAX4 expression promotes β-cell dedifferentiation and hyperglycemia, mimicking β-cell failure in diabetic patients. Herein, we study mechanisms that allow stringent PAX4 regulation endowing favorable β-cell adaptation in response to changing environment without loss of identity. To this end, PAX4 expression was monitored using a mouse bearing the enhanced green fluorescent protein (GFP) and cre recombinase construct under the control of the islet specific pax4 promoter. GFP was detected in 30% of islet cells predominantly composed of PAX4-enriched β-cells that responded to glucose-induced insulin secretion. Lineage tracing demonstrated that all islet cells were derived from PAX4+ progenitor cells but that GFP expression was confined to a subpopulation at birth which declined with age correlating with reduced replication. However, this GFP+ subpopulation expanded during pregnancy, a state of active β-cell replication. Accordingly, enhanced proliferation was exclusively detected in GFP+ cells consistent with cell cycle genes being stimulated in PAX4-overexpressing islets. Under stress conditions, GFP+ cells were more resistant to apoptosis than their GFP- counterparts. Our data suggest PAX4 defines an expandable β-cell sub population within adult islets. PMID:26503027

  1. Proangiogenic Hydrogels Within Macroporous Scaffolds Enhance Islet Engraftment in an Extrahepatic Site

    PubMed Central

    Brady, Ann-Christina; Martino, Mikaël M.; Pedraza, Eileen; Sukert, Steve; Pileggi, Antonello; Ricordi, Camillo; Hubbell, Jeffrey A.

    2013-01-01

    The transplantation of allogeneic islets in recent clinical trials has shown substantial promise as a therapy for type 1 diabetes; however, long-term insulin independence remains inadequate. This has been largely attributed to the current intravascular, hepatic transplant site, which exposes islets to mechanical and inflammatory stresses. A highly macroporous scaffold, housed within an alternative transplant site, can support an ideal environment for islet transplantation by providing three-dimensional distribution of islets, while permitting the infiltration of host vasculature. In the present study, we sought to evaluate the synergistic effect of a proangiogenic hydrogel loaded within the void space of a macroporous poly(dimethylsiloxane) (PDMS) scaffold on islet engraftment. The fibrin-based proangiogenic hydrogel tested presents platelet derived growth factor (PDGF-BB), via a fibronectin (FN) fragment containing growth factor and major integrin binding sites in close proximity. The combination of the proangiogenic hydrogel with PDMS scaffolds resulted in a significant decrease in the time to normoglycemia for syngeneic mouse islet transplants. This benefit was associated with an observed increase in competent vessel branching, as well as mature intraislet vessels. Overall, the addition of the proangiogenic factor PDGF-BB, delivered via the FN fragment-functionalized hydrogel, positively influenced the efficiency of engraftment. These characteristics, along with its ease of retrieval, make this combination of a biostable macroporous scaffold and a degradable proangiogenic hydrogel a supportive structure for insulin-producing cells implanted in extrahepatic sites. PMID:23790218

  2. Regulation of PDK mRNA by high fatty acid and glucose in pancreatic islets.

    PubMed

    Xu, Jianxiang; Han, Junying; Epstein, Paul N; Liu, Ye Q

    2006-06-01

    Pyruvate dehydrogenase (PDH) converts pyruvate to acetyl-CoA, links glycolysis to the Krebs cycle, and plays an important role in glucose metabolism and insulin secretion in pancreatic beta cells. In beta cells from obese and Type 2 diabetic animals, PDH activity is significantly reduced. PDH is negatively regulated by multiple pyruvate dehydrogenase kinase (PDK) isotypes (PDK subtypes 1-4). However, we do not know whether fatty acids or high glucose modulate PDKs in islets. To test this we determined PDH and PDK activities and PDK gene and protein expression in C57BL/6 mouse islets. Both high palmitate and high glucose reduced active PDH activity and increased PDK activity. The gene and protein for PDK3 were not expressed in islets. Palmitate up-regulated mRNA expression of PDK1 (2.9-fold), PDK2 (1.9-fold), and PDK4 (3.1-fold). High glucose increased PDK1 (1.8-fold) and PDK2 (2.7-fold) mRNA expression but reduced PDK4 mRNA expression by 40 percent in cultured islets. Changed PDK expression was confirmed by Western blotting. These results demonstrate that in islet cells both fat and glucose regulate PDK gene and protein expression and indicate that hyperglycemia and hyperlipidemia contribute to the decline in diabetic islet PDH activity by increasing mRNA and protein expression of PDK. PMID:16631612

  3. The fractal spatial distribution of pancreatic islets in three dimensions: a self-avoiding growth model

    NASA Astrophysics Data System (ADS)

    Jo, Junghyo; Hörnblad, Andreas; Kilimnik, German; Hara, Manami; Ahlgren, Ulf; Periwal, Vipul

    2013-06-01

    The islets of Langerhans, responsible for controlling blood glucose levels, are dispersed within the pancreas. A universal power law governing the fractal spatial distribution of islets in two-dimensional pancreatic sections has been reported. However, the fractal geometry in the actual three-dimensional pancreas volume, and the developmental process that gives rise to such a self-similar structure, has not been investigated. Here, we examined the three-dimensional spatial distribution of islets in intact mouse pancreata using optical projection tomography and found a power law with a fractal dimension of 2.1. Furthermore, based on two-dimensional pancreatic sections of human autopsies, we found that the distribution of human islets also follows a universal power law with a fractal dimension of 1.5 in adult pancreata, which agrees with the value previously reported in smaller mammalian pancreas sections. Finally, we developed a self-avoiding growth model for the development of the islet distribution and found that the fractal nature of the spatial islet distribution may be associated with the self-avoidance in the branching process of vascularization in the pancreas.

  4. Proangiogenic hydrogels within macroporous scaffolds enhance islet engraftment in an extrahepatic site.

    PubMed

    Brady, Ann-Christina; Martino, Mikaël M; Pedraza, Eileen; Sukert, Steve; Pileggi, Antonello; Ricordi, Camillo; Hubbell, Jeffrey A; Stabler, Cherie L

    2013-12-01

    The transplantation of allogeneic islets in recent clinical trials has shown substantial promise as a therapy for type 1 diabetes; however, long-term insulin independence remains inadequate. This has been largely attributed to the current intravascular, hepatic transplant site, which exposes islets to mechanical and inflammatory stresses. A highly macroporous scaffold, housed within an alternative transplant site, can support an ideal environment for islet transplantation by providing three-dimensional distribution of islets, while permitting the infiltration of host vasculature. In the present study, we sought to evaluate the synergistic effect of a proangiogenic hydrogel loaded within the void space of a macroporous poly(dimethylsiloxane) (PDMS) scaffold on islet engraftment. The fibrin-based proangiogenic hydrogel tested presents platelet derived growth factor (PDGF-BB), via a fibronectin (FN) fragment containing growth factor and major integrin binding sites in close proximity. The combination of the proangiogenic hydrogel with PDMS scaffolds resulted in a significant decrease in the time to normoglycemia for syngeneic mouse islet transplants. This benefit was associated with an observed increase in competent vessel branching, as well as mature intraislet vessels. Overall, the addition of the proangiogenic factor PDGF-BB, delivered via the FN fragment-functionalized hydrogel, positively influenced the efficiency of engraftment. These characteristics, along with its ease of retrieval, make this combination of a biostable macroporous scaffold and a degradable proangiogenic hydrogel a supportive structure for insulin-producing cells implanted in extrahepatic sites. PMID:23790218

  5. Intramacrophage survival of uropathogenic Escherichia coli: differences between diverse clinical isolates and between mouse and human macrophages.

    PubMed

    Bokil, Nilesh J; Totsika, Makrina; Carey, Alison J; Stacey, Katryn J; Hancock, Viktoria; Saunders, Bernadette M; Ravasi, Timothy; Ulett, Glen C; Schembri, Mark A; Sweet, Matthew J

    2011-11-01

    Uropathogenic E. coli (UPEC) are the primary cause of urinary tract infections. Recent studies have demonstrated that UPEC can invade and replicate within epithelial cells, suggesting that this bacterial pathogen may occupy an intracellular niche within the host. Given that many intracellular pathogens target macrophages, we assessed the interactions between UPEC and macrophages. Colonization of the mouse bladder by UPEC strain CFT073 resulted in increased expression of myeloid-restricted genes, consistent with the recruitment of inflammatory macrophages to the site of infection. In in vitro assays, CFT073 was able to survive within primary mouse bone marrow-derived macrophages (BMM) up to 24h post-infection. Three additional well-characterized clinical UPEC isolates associated with distinct UTI symptomatologies displayed variable long-term survival within BMM. UPEC strains UTI89 and VR50, originally isolated from patients with cystitis and asymptomatic bacteriuria respectively, showed elevated bacterial loads in BMM at 24h post-infection as compared to CFT073 and the asymptomatic bacteriuria strain 83972. These differences did not correlate with differential effects on macrophage survival or initial uptake of bacteria. E. coli UTI89 localized to a Lamp1(+) vesicular compartment within BMM. In contrast to survival within mouse BMM, intracellular bacterial loads of VR50 were low in both human monocyte-derived macrophages (HMDM) and in human T24 bladder epithelial cells. Collectively, these data suggest that some UPEC isolates may subvert macrophage anti-microbial pathways, and that host species differences may impact on intracellular UPEC survival.

  6. Classification of microscopy images of Langerhans islets

    NASA Astrophysics Data System (ADS)

    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.

  7. Gap junctions and other mechanisms of cell-cell communication regulate basal insulin secretion in the pancreatic islet.

    PubMed

    Benninger, R K P; Head, W Steven; Zhang, Min; Satin, Leslie S; Piston, David W

    2011-11-15

    Cell-cell communication in the islet of Langerhans is important for the regulation of insulin secretion. Gap-junctions coordinate oscillations in intracellular free-calcium ([Ca(2+)](i)) and insulin secretion in the islet following elevated glucose. Gap-junctions can also ensure that oscillatory [Ca(2+)](i) ceases when glucose is at a basal levels. We determine the roles of gap-junctions and other cell-cell communication pathways in the suppression of insulin secretion under basal conditions. Metabolic, electrical and insulin secretion levels were measured from islets lacking gap-junction coupling following deletion of connexion36 (Cx36(-/-)), and these results were compared to those obtained using fully isolated β-cells. K(ATP) loss-of-function islets provide a further experimental model to specifically study gap-junction mediated suppression of electrical activity. In isolated β-cells or Cx36(-/-) islets, elevations in [Ca(2+)](i) persisted in a subset of cells even at basal glucose. Isolated β-cells showed elevated insulin secretion at basal glucose; however, insulin secretion from Cx36(-/-) islets was minimally altered. [Ca(2+)](i) was further elevated under basal conditions, but insulin release still suppressed in K(ATP) loss-of-function islets. Forced elevation of cAMP led to PKA-mediated increases in insulin secretion from islets lacking gap-junctions, but not from islets expressing Cx36 gap junctions. We conclude there is a redundancy in how cell-cell communication in the islet suppresses insulin release. Gap junctions suppress cellular heterogeneity and spontaneous [Ca(2+)](i) signals, while other juxtacrine mechanisms, regulated by PKA and glucose, suppress more distal steps in exocytosis. Each mechanism is sufficiently robust to compensate for a loss of the other and still suppress basal insulin secretion.

  8. Activation of antioxidant response element in mouse primary cortical cultures with sesquiterpene lactones isolated from Tanacetum parthenium

    PubMed Central

    Fischedick, Justin T; Standiford, Miranda; Johnson, Delinda A.; De Vos, Ric C.H.; Todorović, Slađana; Banjanac, Tijana; Verpoorte, Rob; Johnson, Jeffrey A.

    2012-01-01

    Tanacetum parthenium (Asteraceae) produces biologically active sesquiterpene lactones (SL). Nuclear factor E2-related factor 2 (Nrf2) is a transcription factor known to activate a series of genes termed the antioxidant response element (ARE). Activation of the Nrf2/ARE may be useful for the treatment of neurodegenerative disease. In this study we isolated 11 sesquiterpene lactones from T. parthenium with centrifugal partition chromatography and semi-preparative HPLC. Compounds were screened in-vitro for their ability to activate the ARE on primary mouse cortical cultures as well as for their toxicity towards the cultures. All sesquiterpene lactones containing the α-methylene-γ-lactone moiety were able to activate the ARE although a number of compounds displayed significant cellular toxicity towards the cultures. The structure activity relationship of the sesquiterpene lactones indicate that the guaianolides isolated were more active and less toxic then the germacranolides. PMID:22923197

  9. Extensive Hair Shaft Growth after Mouse Whisker Follicle Isolation, Cryopreservation and Transplantation in Nude Mice.

    PubMed

    Cao, Wenluo; Li, Lingna; Tran, Benjamin; Kajiura, Satoshi; Amoh, Yasuyuki; Liu, Fang; Hoffman, Robert M

    2015-01-01

    We previously demonstrated that whole hair follicles could be cryopreserved to maintain their stem-cells differentation potential. In the present study, we demonstrated that cryopreserved mouse whisker hair follicles maintain their hair growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. Cryopreserved hair follicles also maintained the hair follicle-associated-pluripotent (HAP) stem cells, evidenced by P75NTR expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair shaft growth of cryopreserved hair follicles.

  10. Extensive Hair Shaft Growth after Mouse Whisker Follicle Isolation, Cryopreservation and Transplantation in Nude Mice.

    PubMed

    Cao, Wenluo; Li, Lingna; Tran, Benjamin; Kajiura, Satoshi; Amoh, Yasuyuki; Liu, Fang; Hoffman, Robert M

    2015-01-01

    We previously demonstrated that whole hair follicles could be cryopreserved to maintain their stem-cells differentation potential. In the present study, we demonstrated that cryopreserved mouse whisker hair follicles maintain their hair growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. Cryopreserved hair follicles also maintained the hair follicle-associated-pluripotent (HAP) stem cells, evidenced by P75NTR expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair shaft growth of cryopreserved hair follicles. PMID:26716690

  11. Uranyl nitrate inhibits lactate gluconeogenesis in isolated human and mouse renal proximal tubules: A {sup 13}C-NMR study

    SciTech Connect

    Renault, Sophie; Faiz, Hassan; Gadet, Rudy; Ferrier, Bernard; Martin, Guy; Baverel, Gabriel; Conjard-Duplany, Agnes

    2010-01-01

    As part of a study on uranium nephrotoxicity, we investigated the effect of uranyl nitrate in isolated human and mouse kidney cortex tubules metabolizing the physiological substrate lactate. In the millimolar range, uranyl nitrate reduced lactate removal and gluconeogenesis and the cellular ATP level in a dose-dependent fashion. After incubation in phosphate-free Krebs-Henseleit medium with 5 mM L-[1-{sup 13}C]-, or L-[2-{sup 13}C]-, or L-[3-{sup 13}C]lactate, substrate utilization and product formation were measured by enzymatic and NMR spectroscopic methods. In the presence of 3 mM uranyl nitrate, glucose production and the intracellular ATP content were significantly reduced in both human and mouse tubules. Combination of enzymatic and NMR measurements with a mathematical model of lactate metabolism revealed an inhibition of fluxes through lactate dehydrogenase and the gluconeogenic enzymes in the presence of 3 mM uranyl nitrate; in human and mouse tubules, fluxes were lowered by 20% and 14% (lactate dehydrogenase), 27% and 32% (pyruvate carboxylase), 35% and 36% (phosphoenolpyruvate carboxykinase), and 39% and 45% (glucose-6-phosphatase), respectively. These results indicate that natural uranium is an inhibitor of renal lactate gluconeogenesis in both humans and mice.

  12. Development of in vitro 3D TissueFlex® islet model for diabetic drug efficacy testing.

    PubMed

    Li, Zhaohui; Sun, He; Zhang, Jianbin; Zhang, Haijiao; Meng, Fanyu; Cui, Zhanfeng

    2013-01-01

    Increasing individuals diagnosed with type II diabetes pose a strong demand for the development of more effective anti-diabetic drugs. However, expensive, ethically controversial animal-based screening for anti-diabetic compounds is not always predictive of the human response. The use of in vitro cell-based models in research presents obviously ethical and cost advantages over in vivo models. This study was to develop an in vitro three-dimensional (3D) perfused culture model of islets (Islet TF) for maintaining viability and functionality longer for diabetic drug efficacy tests. Briefly fresh isolated rat islets were encapsulated in ultrapure alginate and the encapsulated islets were cultured in TissueFlex(®), a multiple, parallel perfused microbioreactor system for 7 days. The encapsulated islets cultured statically in cell culture plates (3D static) and islets cultured in suspension (2D) were used as the comparisons. In this study we demonstrate for the first time that Islet TF model can maintain the in vitro islet viability, and more importantly, the elevated functionality in terms of insulin release and dynamic responses over a 7-day culture period. The Islet TF displays a high sensitivity in responding to drugs and drug dosages over conventional 2D and 3D static models. Actual drug administration in clinics could be simulated using the developed Islet TF model, and the patterns of insulin release response to the tested drugs were in agreement with the data obtained in vivo. Islet TF could be a more predictive in vitro model for routine short- and long-term anti-diabetic drug efficacy testing.

  13. IFN-{gamma} gene expression in pancreatic islet-infiltrating mononuclear cells correlates with autoimmune diabetes in nonobese diabetic mice

    SciTech Connect

    Rabinovitch, A.; Suarez-Pinzon, W.L.; Sorensen, O.

    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.

  14. Feasibility of islet magnetic resonance imaging using ferumoxytol in intraportal islet transplantation.

    PubMed

    Jin, Sang-Man; Oh, Seung-Hoon; Oh, Bae Jun; Shim, Wooyoung; Choi, Jin Myung; Yoo, Dongkyeom; Hwang, Yong Hwa; Lee, Jung Hee; Lee, Dong Yun; Kim, Jae Hyeon

    2015-06-01

    There is a clinical need for an alternative labeling agent for magnetic resonance imaging (MRI) in islet transplantation. We aimed to evaluate the feasibility of islet MRI using ferumoxytol, which is the only clinically-available ultrasmall superparamagnetic iron oxide. We compared islet function and viability of control islets and islets labeled with ferumoxytol and/or a heparin-protamine complex (HPF). Efficacy of ferumoxytol labeling was assessed in both ex vivo and in vivo models. Labeling for 48 h with HPF, but not up to 800 μg/mL ferumoxytol, deranged ex vivo islet viability and function. The T2∗ relaxation time was optimal when islets were labeled with 800 μg/mL of ferumoxytol for 48 h. Prussian blue stain, iron content assay, transmission electron microscopy (TEM) supported internalization of ferumoxytol particles. However, the labeling intensity in the ex vivo MRI of islets labeled with ferumoxytol was much weaker than that of islets labeled with ferucarbotran. In syngeneic intraportal islet transplantation, there was a correlation between the total area of visualized islets and the transplanted islet mass. In conclusion, islet MRI using ferumoxytol was feasible in terms of in vitro and in vivo efficacy and safety. However, the weak labeling efficacy is still a hurdle for the clinical application.

  15. Isolation of Anaerobic Bacteria from Human Gingiva and Mouse Cecum by Means of a Simplified Glove Box Procedure1

    PubMed Central

    Aranki, Alexander; Syed, Salam A.; Kenney, Ernest B.; Freter, Rolf

    1969-01-01

    An anaerobic glove box constructed of clear flexible vinyl plastic is described. It is sufficiently inexpensive and simple in operation to be used not only in research but also in a clinical laboratory by technicians without special training. Conventional bacteriological techniques may be used inside the glove box for culturing and transferring anaerobic bacteria. The box may be heated to 37 C and thus serve as an anaerobic incubator as well, permitting inspection of cultures at any time. Media may be prepared and agar plates may be poured on the laboratory bench in the conventional manner. An overlay of trace amounts of palladium black catalyst over plated agar media reduces the medium to an oxidation-reduction (O-R) potential of - 300 mv within 2 days after introduction into the glove box. In spite of its greater simplicity, the system matched or excelled the roll tube method with respect to all parameters tested, including O-R potential obtainable in the media, O2 concentration in the gas phase, and efficiency in isolating anaerobic bacteria from the mouse cecum. Comparative studies indicate that the conventional anaerobic jar method was inadequate for the isolation of strict anaerobes from human gingival specimens and from the mouse cecum. This was due to the exposure of specimens and media to air during plating on the open laboratory bench. Anaerobic jars were adequate for maintaining the proper conditions for growth of anaerobic bacteria once these had been established in the glove box. Images PMID:4890748

  16. Alterations of pancreatic islet structure, metabolism and gene expression in diet-induced obese C57BL/6J mice.

    PubMed

    Roat, Regan; Rao, Vandana; Doliba, Nicolai M; Matschinsky, Franz M; Tobias, John W; Garcia, Eden; Ahima, Rexford S; Imai, Yumi

    2014-01-01

    The reduction of functional β cell mass is a key feature of type 2 diabetes. Here, we studied metabolic functions and islet gene expression profiles of C57BL/6J mice with naturally occurring nicotinamide nucleotide transhydrogenase (NNT) deletion mutation, a widely used model of diet-induced obesity and diabetes. On high fat diet (HF), the mice developed obesity and hyperinsulinemia, while blood glucose levels were only mildly elevated indicating a substantial capacity to compensate for insulin resistance. The basal serum insulin levels were elevated in HF mice, but insulin secretion in response to glucose load was significantly blunted. Hyperinsulinemia in HF fed mice was associated with an increase in islet mass and size along with higher BrdU incorporation to β cells. The temporal profiles of glucose-stimulated insulin secretion (GSIS) of isolated islets were comparable in HF and normal chow fed mice. Islets isolated from HF fed mice had elevated basal oxygen consumption per islet but failed to increase oxygen consumption further in response to glucose or carbonyl cyanide-4-trifluoromethoxyphenylhydrazone (FCCP). To obtain an unbiased assessment of metabolic pathways in islets, we performed microarray analysis comparing gene expression in islets from HF to normal chow-fed mice. A few genes, for example, those genes involved in the protection against oxidative stress (hypoxia upregulated protein 1) and Pgc1α were up-regulated in HF islets. In contrast, several genes in extracellular matrix and other pathways were suppressed in HF islets. These results indicate that islets from C57BL/6J mice with NNT deletion mutation develop structural, metabolic and gene expression features consistent with compensation and decompensation in response to HF diet. PMID:24505268

  17. Evaluation of two cell surface modification methods for proteomic analysis of plasma membrane from isolated mouse hepatocytes.

    PubMed

    Li, Xuanwen; Jin, Qihui; Cao, Jia; Xie, Chunliang; Cao, Rui; Liu, Zhen; Xiong, Jixian; Li, Jianglin; Yang, Xiaoxu; Chen, Ping; Liang, Songping

    2009-01-01

    The hepatocyte is a highly polarized cell with a heterogeneous distribution of plasma-membrane (PM) proteins. To reduce the complexity of the proteome of liver tissue and give a comprehensive profile of hepatocyte PM, two PM purification methods based on cell surface modification, named the biotin-avidin (BA) and cationic silica-polyanion (CSP) strategies were evaluated and compared with the traditional cell fractionation method to prepare highly enriched PM from freshly isolated C57 mouse hepatocytes. Employing different principles for PM modification, both methods were effective in the isolation of general and purified PM fraction. The CSP strategy showed better yield for the PM purification from freshly isolated hepatocytes. 189 non-redundant proteins were identified, including 49 from the BA method and 185 from CSP strategy. Many known and novel PM-associated proteins were also found. Our evaluation here should give implications for PM preparation from other freshly isolated tissue-derived cells. The hepatocyte PM proteins identified here should be taken as a references for the PM-related functional and diseases research.

  18. Reduced insulin secretion in response to nutrients in islets from malnourished young rats is associated with a diminished calcium uptake.

    PubMed

    Latorraca, M Q; Carneiro, E M; Mello, M A; Boschero, A C

    1999-01-01

    Changes in (45)Ca uptake and insulin secretion in response to glucose, leucine, and arginine were measured in isolated islets derived from 4-week-old rats born of mothers maintained with normal protein (NP, 17%) or low protein (LP, 6%) diet during pregnancy and lactation. Glucose provoked a dose-dependent stimulation of insulin secretion in both groups of islets, with basal (2.8 mmol/L glucose) and maximal release (27.7 mmol/L glucose) significantly reduced in LP compared with NP islets. In the LP group the concentration-response curve to glucose was shifted to the right compared with the NP group, with the half-maximal response occurring at 16.9 and 13.3 mmol/L glucose, respectively. In LP islets, glucose-induced first and second phases of insulin secretions were drastically reduced. In addition, insulin response to individual amino acids, or in association with glucose, was also significantly reduced in the LP group compared with NP islets. Finally, in LP islets the (45)Ca uptake after 5 minutes or 90 minutes of incubation (which reflect mainly the entry and retention, respectively, of Ca(2+)), was lower than in NP islets. These data indicate that in malnourished rats both initial and sustained phases of insulin secretion in response to glucose were reduced. This poor secretory response to nutrients seems to be the consequence of an altered Ca(2+) handling by malnourished islet cells. PMID:15539248

  19. The role of endothelial cells on islet function and revascularization after islet transplantation

    PubMed Central

    Del Toro-Arreola, Alicia; Robles-Murillo, Ana Karina; Daneri-Navarro, Adrian; Rivas-Carrillo, Jorge David

    2016-01-01

    ABSTRACT Islet transplantation has become a widely accepted therapeutic option for selected patients with type 1 diabetes mellitus. However, in order to achieve insulin independence a great number of islets are often pooled from 2 to 4 pancreata donors. Mostly, it is due to the massive loss of islets immediately after transplant. The endothelium plays a key role in the function of native islets and during the revascularization process after islet transplantation. However, if a delayed revascularization occurs, even the remaining islets will also undergo to cell death and late graft dysfunction. Therefore, it is essential to understand how the signals are released from endothelial cells, which might regulate both differentiation of pancreatic progenitors and thereby maintenance of the graft function. New strategies to facilitate islet engraftment and a prompt revascularization could be designed to intervene and might lead to improve future results of islet transplantation. PMID:27002241

  20. Co-culture with mature islet cells augments the differentiation of insulin-producing cells from pluripotent stem cells.

    PubMed

    Oh, Bea Jun; Oh, Seung-Hoon; Choi, Jin Myung; Jin, Sang-Man; Shim, Woo-Young; Lee, Myung-Shik; Lee, Moon-Kyu; Kim, Kwang-Won; Kim, Jae Hyeon

    2015-02-01

    Islet transplantation has been hampered by the shortage of islet donors available for diabetes therapy. However, pluripotent stem cells (PSCs) can be an alternative source of insulin-producing cells (IPCs) because of their capacity for self-renewal and differentiation. We described a method to efficiently differentiate PSCs into IPCs by co-culturing mature islets with directed-differentiated pancreatic endoderm (PE) cells from mouse and human PSCs. PE cells co-cultured with islet cells or islet cell-derived conditioned medium (CM) showed increased expression levels of β-cell markers; significantly higher levels of proinsulin- and Newport Green (NG)-positive cells, which revealed the characteristics of insulin producing cells; and increased insulin secretion upon glucose stimulation. Co-culturing human PE cells with islet cells was also effective to differentiate PE cells into IPCs. Diabetic nude mice transplanted with co-cultured cells exhibited restored euglycemia, human C-peptide release, and improved glucose tolerance. Immunohistochemistry revealed that insulin+/C-peptide + cells existed in the grafted tissues. These results suggest that mature islet cells can increase the differentiation efficiency of PE cells into mature IPCs via paracrine effects.

  1. Improving efficacy of clinical islet transplantation with iodixanol-based islet purification, thymoglobulin induction, and blockage of IL-1β and TNF-α.

    PubMed

    Matsumoto, Shinichi; Takita, Morihito; Chaussabel, Damien; Noguchi, Hirofumi; Shimoda, Masayuki; Sugimoto, Koji; Itoh, Takeshi; Chujo, Daisuke; SoRelle, Jeff; Onaca, Nicholas; Naziruddin, Bashoo; Levy, Marlon F

    2011-01-01

    Poor efficacy is one of the issues for clinical islet transplantation. Recently, we demonstrated that pancreatic ductal preservation significantly improved the success rate of islet isolation; however, two transplants were necessary to achieve insulin independence. In this study, we introduced iodixanol-based purification, thymoglobulin induction, and double blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression to improve the efficacy of clinical islet transplantation. Nine clinical-grade human pancreata were procured. Pancreatic ductal preservation was performed using ET-Kyoto solution in all cases. When the isolated islets met the clinical criteria, they were transplanted. We utilized two methods of immunosuppression and anti-inflammation. The first protocol prescribed daclizumab for induction, then sirolimus and tacrolimus to maintain immunosuppression. The second protocol used thymoglobulin for induction and tacrolimus and mycophenolate mofetil to maintain immunosuppression. Eternacept and anakinra were administered as anti-inflammatory drugs. The total amount of insulin required, HbA1c, and the SUITO index were determined to analyze and compare the results of transplantation. All isolated islet preparations (9/9) met the criteria for clinical transplantation, and they were transplanted into six type 1 diabetic patients. All patients achieved insulin independence with normal HbA1c levels; however, the first protocol required two islet infusions (N = 3) and the second protocol only required a single infusion (N = 3). The average SUITO index, at 1 month after a single-donor islet transplantation, was significantly higher in the second protocol (49.6 ± 8.3 vs. 19.3 ± 6.3, p < 0.05). Pancreatic ductal preservation, iodixanol-based purification combined with thymoglobulin induction, and blockage of IL-1β and TNF-α as well as sirolimus-free immunosuppression dramatically improved the efficacy of clinical islet transplantations. This protocol

  2. Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells.

    PubMed

    Stuelsatz, Pascal; Yablonka-Reuveni, Zipora

    2016-01-01

    The extraocular muscles (EOMs) comprise a group of highly specialized skeletal muscles controlling eye movements. Although a number of unique features of EOMs including their sparing in Duchenne muscular dystrophy have drawn a continuous interest, knowledge about these hard to reach muscles is still limited. The goal of this chapter is to provide detailed methods for the isolation and histological analysis of mouse EOMs. We first introduce in brief the basic anatomy and established nomenclature of the extraocular primary and accessory muscles. We then provide a detailed description with step-by-step images of our procedure for isolating (and subsequently cryosectioning) EOMs while preserving the integrity of their original structural organization. Next, we present several useful histological protocols frequently used by us, including: (1) a method for highlighting the general organization of periocular tissue, using the MyoD(Cre) × R26(mTmG) reporter mouse that elegantly distinguishes muscle (MyoD(Cre)-driven GFP(+)) from the non-myogenic constituents (Tomato(+)); (2) analysis by H&E staining, allowing for example, detection of the pathological features of the dystrophin-null phenotype in affected limb and diaphragm muscles that are absent in EOMs; (3) detection of the myogenic progenitors (i.e., satellite cells) in their native position underneath the myofiber basal lamina using Pax7/laminin double immunostaining. The EOM tissue harvesting procedure described here can also be adapted for isolating and studying satellite cells and other cell types. Overall, the methods described in this chapter should provide investigators the necessary tools for entering the EOM research field and contribute to a better understanding of this highly specialized muscle group and its complex micro-anatomy.

  3. Isolation of Mouse Periocular Tissue for Histological and Immunostaining Analyses of the Extraocular Muscles and Their Satellite Cells.

    PubMed

    Stuelsatz, Pascal; Yablonka-Reuveni, Zipora

    2016-01-01

    The extraocular muscles (EOMs) comprise a group of highly specialized skeletal muscles controlling eye movements. Although a number of unique features of EOMs including their sparing in Duchenne muscular dystrophy have drawn a continuous interest, knowledge about these hard to reach muscles is still limited. The goal of this chapter is to provide detailed methods for the isolation and histological analysis of mouse EOMs. We first introduce in brief the basic anatomy and established nomenclature of the extraocular primary and accessory muscles. We then provide a detailed description with step-by-step images of our procedure for isolating (and subsequently cryosectioning) EOMs while preserving the integrity of their original structural organization. Next, we present several useful histological protocols frequently used by us, including: (1) a method for highlighting the general organization of periocular tissue, using the MyoD(Cre) × R26(mTmG) reporter mouse that elegantly distinguishes muscle (MyoD(Cre)-driven GFP(+)) from the non-myogenic constituents (Tomato(+)); (2) analysis by H&E staining, allowing for example, detection of the pathological features of the dystrophin-null phenotype in affected limb and diaphragm muscles that are absent in EOMs; (3) detection of the myogenic progenitors (i.e., satellite cells) in their native position underneath the myofiber basal lamina using Pax7/laminin double immunostaining. The EOM tissue harvesting procedure described here can also be adapted for isolating and studying satellite cells and other cell types. Overall, the methods described in this chapter should provide investigators the necessary tools for entering the EOM research field and contribute to a better understanding of this highly specialized muscle group and its complex micro-anatomy. PMID:27492169

  4. Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates.

    PubMed

    Obara, Chizuka; Tomiyama, Ken-Ichi; Takizawa, Kazuya; Islam, Rafiqul; Yasuda, Takeshi; Gotoh, Takaya; Tajima, Katsushi

    2016-10-01

    Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair.

  5. Characteristics of three-dimensional prospectively isolated mouse bone marrow mesenchymal stem/stromal cell aggregates on nanoculture plates.

    PubMed

    Obara, Chizuka; Tomiyama, Ken-Ichi; Takizawa, Kazuya; Islam, Rafiqul; Yasuda, Takeshi; Gotoh, Takaya; Tajima, Katsushi

    2016-10-01

    Three-dimensional (3-D) aggregate culturing is useful for investigating the functional properties of mesenchymal stem/stromal cells (MSCs). For 3-D MSC analysis, however, pre-expansion of MSCs with two-dimensional (2-D) monolayer culturing must first be performed, which might abolish their endogenous properties. To avoid the need for 2-D expansion, we used prospectively isolated mouse bone marrow (BM)-MSCs and examined the differences in the biological properties of 2-D and 3-D MSC cultures. The BM-MSCs self-assembled into aggregates on nanoculture plates (NCP) that have nanoimprinted patterns with a low-cellular binding texture. The 3-D MSCs proliferated at the same rate as 2-D-cultured cells by only diffusion culture and secreted higher levels of pro-angiogenic factors such as vascular endothelial growth factor and hepatocyte growth factor (HGF). Conditioned medium from 3-D MSC cultures promoted more capillary formation than that of 2-D MSCs in an in vitro tube formation assay. Matrigel-implanted 3-D MSC aggregates tended to induce angiogenesis in host mice. The 3-D culturing on NCP induced alpha-fetoprotein (AFP) expression in MSCs without the application of AFP- or endodermal-inducible factors, possibly via an HGF-autocrine mechanism, and maintained their differentiation ability for adipocytes, osteocytes, and chondrocytes. Prospectively isolated mouse BM-MSCs expressed low/negative stemness-related genes including Oct3/4, Nanog, and Sox2, which were not enhanced by NCP-based 3-D culturing, suggesting that some of these cells differentiate into meso-endodermal layer cells. Culturing of prospectively isolated MSCs on NCP in 3-D allows the analysis of the biological properties of more closely endogenous BM-MSCs and might contribute to tissue engineering and repair. PMID:27100525

  6. Social isolation induces autophagy in the mouse mammary gland: link to increased mammary cancer risk.

    PubMed

    Sumis, Allison; Cook, Katherine L; Andrade, Fabia O; Hu, Rong; Kidney, Emma; Zhang, Xiyuan; Kim, Dominic; Carney, Elissa; Nguyen, Nguyen; Yu, Wei; Bouker, Kerrie B; Cruz, Idalia; Clarke, Robert; Hilakivi-Clarke, Leena

    2016-10-01

    Social isolation is a strong predictor of early all-cause mortality and consistently increases breast cancer risk in both women and animal models. Because social isolation increases body weight, we compared its effects to those caused by a consumption of obesity-inducing diet (OID) in C57BL/6 mice. Social isolation and OID impaired insulin and glucose sensitivity. In socially isolated, OID-fed mice (I-OID), insulin resistance was linked to reduced Pparg expression and increased neuropeptide Y levels, but in group-housed OID fed mice (G-OID), it was linked to increased leptin and reduced adiponectin levels, indicating that the pathways leading to insulin resistance are different. Carcinogen-induced mammary tumorigenesis was significantly higher in I-OID mice than in the other groups, but cancer risk was also increased in socially isolated, control diet-fed mice (I-C) and G-OID mice compared with that in controls. Unfolded protein response (UPR) signaling (GRP78; IRE1) was upregulated in the mammary glands of OID-fed mice, but not in control diet-fed, socially isolated I-C mice. In contrast, expression of BECLIN1, ATG7 and LC3II were increased, and p62 was downregulated by social isolation, indicating increased autophagy. In the mammary glands of socially isolated mice, but not in G-OID mice, mRNA expressions of p53 and the p53-regulated autophagy inducer Dram1 were upregulated, and nuclear p53 staining was strong. Our findings further indicated that autophagy and tumorigenesis were not increased in Atg7(+/-) mice kept in social isolation and fed OID. Thus, social isolation may increase breast cancer risk by inducing autophagy, independent of changes in body weight. PMID:27550962

  7. Social isolation induces autophagy in the mouse mammary gland: link to increased mammary cancer risk.

    PubMed

    Sumis, Allison; Cook, Katherine L; Andrade, Fabia O; Hu, Rong; Kidney, Emma; Zhang, Xiyuan; Kim, Dominic; Carney, Elissa; Nguyen, Nguyen; Yu, Wei; Bouker, Kerrie B; Cruz, Idalia; Clarke, Robert; Hilakivi-Clarke, Leena

    2016-10-01

    Social isolation is a strong predictor of early all-cause mortality and consistently increases breast cancer risk in both women and animal models. Because social isolation increases body weight, we compared its effects to those caused by a consumption of obesity-inducing diet (OID) in C57BL/6 mice. Social isolation and OID impaired insulin and glucose sensitivity. In socially isolated, OID-fed mice (I-OID), insulin resistance was linked to reduced Pparg expression and increased neuropeptide Y levels, but in group-housed OID fed mice (G-OID), it was linked to increased leptin and reduced adiponectin levels, indicating that the pathways leading to insulin resistance are different. Carcinogen-induced mammary tumorigenesis was significantly higher in I-OID mice than in the other groups, but cancer risk was also increased in socially isolated, control diet-fed mice (I-C) and G-OID mice compared with that in controls. Unfolded protein response (UPR) signaling (GRP78; IRE1) was upregulated in the mammary glands of OID-fed mice, but not in control diet-fed, socially isolated I-C mice. In contrast, expression of BECLIN1, ATG7 and LC3II were increased, and p62 was downregulated by social isolation, indicating increased autophagy. In the mammary glands of socially isolated mice, but not in G-OID mice, mRNA expressions of p53 and the p53-regulated autophagy inducer Dram1 were upregulated, and nuclear p53 staining was strong. Our findings further indicated that autophagy and tumorigenesis were not increased in Atg7(+/-) mice kept in social isolation and fed OID. Thus, social isolation may increase breast cancer risk by inducing autophagy, independent of changes in body weight.

  8. An Islet-Targeted Genome-Wide Association Scan Identifies Novel Genes Implicated in Cytokine-Mediated Islet Stress in Type 2 Diabetes.

    PubMed

    Sharma, Poonam R; Mackey, Aaron J; Dejene, Eden A; Ramadan, James W; Langefeld, Carl D; Palmer, Nicholette D; Taylor, Kent D; Wagenknecht, Lynne E; Watanabe, Richard M; Rich, Stephen S; Nunemaker, Craig S

    2015-09-01

    Genome-wide association studies in human type 2 diabetes (T2D) have renewed interest in the pancreatic islet as a contributor to T2D risk. Chronic low-grade inflammation resulting from obesity is a risk factor for T2D and a possible trigger of β-cell failure. In this study, microarray data were collected from mouse islets after overnight treatment with cytokines at concentrations consistent with the chronic low-grade inflammation in T2D. Genes with a cytokine-induced change of >2-fold were then examined for associations between single nucleotide polymorphisms and the acute insulin response to glucose (AIRg) using data from the Genetics Underlying Diabetes in Hispanics (GUARDIAN) Consortium. Significant evidence of association was found between AIRg and single nucleotide polymorphisms in Arap3 (5q31.3), F13a1 (6p25.3), Klhl6 (3q27.1), Nid1 (1q42.3), Pamr1 (11p13), Ripk2 (8q21.3), and Steap4 (7q21.12). To assess the potential relevance to islet function, mouse islets were exposed to conditions modeling low-grade inflammation, mitochondrial stress, endoplasmic reticulum (ER) stress, glucotoxicity, and lipotoxicity. RT-PCR revealed that one or more forms of stress significantly altered expression levels of all genes except Arap3. Thapsigargin-induced ER stress up-regulated both Pamr1 and Klhl6. Three genes confirmed microarray predictions of significant cytokine sensitivity: F13a1 was down-regulated 3.3-fold by cytokines, Ripk2 was up-regulated 1.5- to 3-fold by all stressors, and Steap4 was profoundly cytokine sensitive (167-fold up-regulation). Three genes were thus closely associated with low-grade inflammation in murine islets and also with a marker for islet function (AIRg) in a diabetes-prone human population. This islet-targeted genome-wide association scan identified several previously unrecognized candidate genes related to islet dysfunction during the development of T2D. PMID:26018251

  9. Metabolomics applied to the pancreatic islet

    PubMed Central

    Gooding, Jessica R.; Jensen, Mette V.; Newgard, Christopher B.

    2016-01-01

    Metabolomics, the characterization of the set of small molecules in a biological system, is advancing research in multiple areas of islet biology. Measuring a breadth of metabolites simultaneously provides a broad perspective on metabolic changes as the islets respond dynamically to metabolic fuels, hormones, or environmental stressors. As a result, metabolomics has the potential to provide new mechanistic insights into islet physiology and pathophysiology. Here we summarize advances in our understanding of islet physiology and the etiologies of type-1 and type-2 diabetes gained from metabolomics studies. PMID:26116790

  10. D-Alanine in the islets of Langerhans of rat pancreas.

    PubMed

    Ota, Nobutoshi; Rubakhin, Stanislav S; Sweedler, Jonathan V

    2014-05-01

    Relatively high levels of D-alanine (D-Ala), an endogenous D-amino acid, have been found in the endocrine systems of several animals, especially in the anterior pituitary; however, its functional importance remains largely unknown. We observed D-Ala in islets of Langerhans isolated from rat pancreas in significantly higher levels than in the anterior/intermediate pituitary; specifically, 180±60 fmol D-Ala per islet (300±100 nmol/gislet), and 10±2.5 nmol/g of wet tissue in pituitary. Additionally, 12±5% of the free Ala in the islets was in the d form, almost an order of magnitude higher than the percentage of D-Ala found in the pituitary. Surprisingly, glucose stimulation of the islets resulted in D-Ala release of 0.6±0.5 fmol per islet. As D-Ala is stored in islets and released in response to changes in extracellular glucose, D-Ala may have a hormonal role.

  11. Silk matrices promote formation of insulin-secreting islet-like clusters.

    PubMed

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

    2016-06-01

    Ex vivo expansion of endocrine cells constitutes an interesting alternative to be able to match the unmet need of transplantable pancreatic islets. However, endocrine cells become fragile once removed from their extracellular matrix (ECM) and typically become senescent and loose insulin expression during conventional 2D culture. Herein we develop a protocol where 3D silk matrices functionalized with ECM-derived motifs are used for generation of insulin-secreting islet-like clusters from mouse and human primary cells. The obtained clusters were shown to attain an islet-like spheroid shape and to maintain functional insulin release upon glucose stimulation in vitro. Furthermore, in vivo imaging of transplanted murine clusters showed engraftment with increasing vessel formation during time. There was no sign of cell death and the clusters maintained or increased in size throughout the period, thus suggesting a suitable cluster size for transplantation. PMID:26986856

  12. Isolation rearing reduces mechanical allodynia in a mouse model of chronic inflammatory pain.

    PubMed

    Horiguchi, Naotaka; Ago, Yukio; Hasebe, Shigeru; Higashino, Kosuke; Asada, Kazuki; Kita, Yuki; Takuma, Kazuhiro; Matsuda, Toshio

    2013-11-15

    Social isolation rearing in mice after weaning reduces pain sensitivity to acute pain, and this hypoalgesia is mediated by the descending serotonergic pain inhibitory system in which the spinal serotonin (5-HT)1A receptor is involved. However, it is not known whether isolation rearing affects pain sensitivity to neuropathic or inflammatory chronic pain. In this study, we examined the effects of isolation rearing on chronic pain induced by Freund's complete adjuvant (FCA) and partial sciatic nerve ligation using the von Frey test (to assess mechanical allodynia) and the plantar test (to assess thermal hyperalgesia). In the FCA model, isolation rearing reduced mechanical allodynia, but not thermal hyperalgesia. In contrast, isolation rearing had no effect on allodynia or hyperalgesia in the sciatic nerve ligation model. The isolation rearing-induced inhibition of allodynia was alleviated by intrathecal injection of WAY100635, a selective 5-HT1A receptor antagonist. FCA increased 5-HT turnover and decreased 5-HT1A receptor expression in the spinal cord of group-reared mice, while it did not have these effects in isolation-reared mice. These results suggest that FCA suppresses the serotonergic pain inhibitory system selectively in group-reared mice. Moreover, systemic administration of osemozotan, a selective 5-HT1A receptor agonist, inhibited FCA-induced mechanical allodynia in group-reared mice, and this effect of the drug was suppressed by intrathecal injection of WAY100635. Collectively, these findings suggest that isolation rearing selectively reduces FCA-induced mechanical allodynia in mice and that this effect is mediated by the activation of spinal 5-HT1A receptors.

  13. Enterovirus infection of human islets of Langerhans affects β-cell function resulting in disintegrated islets, decreased glucose stimulated insulin secretion and loss of Golgi structure

    PubMed Central

    Hodik, M; Skog, O; Lukinius, A; Isaza-Correa, J M; Kuipers, J; Giepmans, B N G; Frisk, G

    2016-01-01

    Aims/hypothesis In type 1 diabetes (T1D), most insulin-producing β cells are destroyed, but the trigger is unknown. One of the possible triggers is a virus infection and the aim of this study was to test if enterovirus infection affects glucose stimulated insulin secretion and the effect of virus replication on cellular macromolecules and organelles involved in insulin secretion. Methods Isolated human islets were infected with different strains of coxsackievirus B (CVB) virus and the glucose-stimulated insulin release (GSIS) was measured in a dynamic perifusion system. Classical morphological electron microscopy, large-scale electron microscopy, so-called nanotomy, and immunohistochemistry were used to study to what extent virus-infected β cells contained insulin, and real-time PCR was used to analyze virus induced changes of islet specific genes. Results In islets infected with CVB, GSIS was reduced in correlation with the degree of virus-induced islet disintegration. The expression of the gene encoding insulin was decreased in infected islets, whereas the expression of glucagon was not affected. Also, in islets that were somewhat disintegrated, there were uninfected β cells. Ultrastructural analysis revealed that virus particles and virus replication complexes were only present in β cells. There was a significant number of insulin granules remaining in the virus-infected β cells, despite decreased expression of insulin mRNA. In addition, no typical Golgi apparatus was detected in these cells. Exposure of islets to synthetic dsRNA potentiated glucose-stimulated insulin secretion. Conclusions/interpretation Glucose-stimulated insulin secretion; organelles involved in insulin secretion and gene expression were all affected by CVB replication in β cells. PMID:27547409

  14. Mold-casted non-degradable, islet macro-encapsulating hydrogel devices for restoration of normoglycemia in diabetic mice.

    PubMed

    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

  15. Isolation of the stromal-vascular fraction of mouse bone marrow markedly enhances the yield of clonogenic stromal progenitors

    PubMed Central

    Suire, Colby; Brouard, Nathalie; Hirschi, Karen

    2012-01-01

    The low incidence of CFU-F significantly complicates the isolation of homogeneous populations of mouse bone marrow stromal cells (BMSCs), a common problem being contamination with hematopoietic cells. Taking advantage of burgeoning evidence demonstrating the perivascular location of stromal cell stem/progenitors, we hypothesized that a potential reason for the low yield of mouse BMSCs is the flushing of the marrow used to remove single-cell suspensions and the consequent destruction of the marrow vasculature, which may adversely affect recovery of BMSCs physically associated with the abluminal surface of blood vessels. Herein, we describe a simple methodology based on preparation and enzymatic disaggregation of intact marrow plugs, which yields distinct populations of both stromal and endothelial cells. The recovery of CFU-F obtained by pooling the product of each digestion (1631.8 + 199) reproducibly exceeds that obtained using the standard BM flushing technique (14.32 + 1.9) by at least 2 orders of magnitude (P < .001; N = 8) with an accompanying 113.95-fold enrichment of CFU-F frequency when plated at low oxygen (5%). Purified BMSC populations devoid of hematopoietic contamination are readily obtained by FACS at P0 and from freshly prepared single-cell suspensions. Furthermore, this population demonstrates robust multilineage differentiation using standard in vivo and in vitro bioassays. PMID:22262767

  16. External Application of Apo-9'-fucoxanthinone, Isolated from Sargassum muticum, Suppresses Inflammatory Responses in a Mouse Model of Atopic Dermatitis.

    PubMed

    Han, Sang-Chul; Kang, Na-Jin; Yoon, Weon-Jong; Kim, Sejin; Na, Min-Chull; Koh, Young-Sang; Hyun, Jin-Won; Lee, Nam-Ho; Ko, Mi-Hee; Kang, Hee-Kyoung; Yoo, Eun-Sook

    2016-04-01

    Allergic skin inflammation such as atopic dermatitis is characterized by skin barrier dysfunction, edema, and infiltration with various inflammatory cells. The anti-inflammatory effects of Apo-9'-fucoxanthinone, isolated from Sargassum muticum, have been described in many diseases, but the mechanism by which it modulates the immune system is poorly understood. In this study, the ability of Apo-9'-fucoxanthinone to suppress allergic reactions was investigated using a mouse model of atopic dermatitis. The Apo-9'-fucoxanthinone-treated group showed significantly decreased immunoglobulin E in serum. Also, Apo-9'-fucoxanthinone treatment resulted in a smaller lymph node size with reduced the thickness and length compared to the induction group. In addition, Apo-9'-fucoxanthinone inhibited the expression of interleukin-4, interferon-gamma and tumor necrosis factor-alpha by phorbol 12-myristate 13-acetate and ionomycin-stimulated lymphocytes. These results suggest that Apo-9'-fucoxanthinone may be a useful therapeutic strategy for treating chronic inflammatory diseases. PMID:27123161

  17. Antidepressant-like effects of psoralen isolated from the seeds of Psoralea corylifolia in the mouse forced swimming test.

    PubMed

    Xu, Qun; Pan, Ying; Yi, Li-Tao; Li, Yu-Cheng; Mo, Shi-Fu; Jiang, Fu-Xin; Qiao, Chun-Feng; Xu, Hong-Xi; Lu, Xiao-Bo; Kong, Ling-Dong; Kung, Hsiang-Fu

    2008-06-01

    The forced swimming test (FST) is suggested to produce abnormalities in the serotonergic and hypothalamic-pituitary-adrenal (HPA) axis systems. Therefore, compounds that attenuate these neurobiological alterations may have potential as antidepressants. The behavioral and biochemical effects of psoralen, a major furocoumarin isolated from Psoralea corylifolia, were investigated in the FST model of depression in male mice. Psoralen significantly reduced immobility and increased swimming without altering climbing in the mouse FST. Psoralen remarkably reversed FST-induced alterations in serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels in frontal cortex and hippocampus in mice. Furthermore, psoralen attenuated FST-induced elevations in serum corticotropin-releasing factor (CRF) and corticosterone concentrations to normalize the HPA axis activity. These results suggested that psoralen possessed potent antidepressant-like properties which were at least in part mediated by improving the abnormalities in the serotonergic and the HPA axis systems.

  18. Isolation of the mouse (MFH-1) and human (FKHL14) mesenchyme fork head-1 genes reveals conservation of their gene and protein structures

    SciTech Connect

    Miura, Naoyuki; Iida, Kiyoshi; Yang, Xiao-Li

    1997-05-01

    The very recently found evolutionarily conserved DNA-binding domain of 100 amino acids, termed the fork head domain, emerged from a sequence comparison of the rat hepatocyte transcription factor HNF-3{alpha} and the homeotic gene fork head of Drosophila. We previously isolated a new member of this family, the mesenchyme fork head-1 (MFH-1) gene, which is expressed in developing mesenchyme. Here we describe the isolation of the mouse (MFH-1) and human (FKHL14) chromosomal MFH-1 genes and the determination of the gene and protein structures of MFH-1. We found that the MFH-1 gene has no introns and that the identity of the amino acid sequences of mouse and human MFH-1 proteins is 94%. We also investigated the transcriptional activity of the mouse and human MFH-1 proteins and found that both proteins act as positive transactivators. 31 refs., 3 figs.

  19. A new atypical genotype mouse virulent strain of Toxoplasma gondii isolated from the heart of a wild caught puma (Felis concolor) from Durango, Mexico

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Nothing is known of the genetic diversity of Toxoplasma gondii circulating in wildlife in Mexico. In the present study, a mouse virulent T. gondii strain was isolated from the heart of a wild puma (Felis concolor). The puma was found roaming in outskirt of Durango City, Mexico and tranquailized for ...

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

    PubMed

    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.

  1. Culture of bovine embryos in intermediate host oviducts with emphasis on the isolated mouse oviduct.

    PubMed

    Rizos, D; Ramirez, M A; Pintado, B; Lonergan, P; Gutierrez-Adan, A

    2010-04-01

    The oviduct provides the optimal environment for the transport of sperm and oocyte at the earliest stages of mammalian embryo development. During the early postfertilization period, several major developmental events occur in the embryo including (i) the first cleavage division, (ii) activation of the embryonic genome, (iii) compaction of the morula, and (iv) formation of the blastocyst. Most of these events are initiated in the oviduct. The absence of assistance from the oviduct may compromise the developmental ability of the cattle embryo under in vitro culture conditions. The oviducts of several mammalian species, including rabbits, cow, sheep (in situ), and mice (organ culture), can sustain early bovine embryos and yield blastocysts of better quality compared with those of culture conditions in vitro, leading to normal pregnancy rates in recipient animals. This review focuses on the use of oviducts in vitro or in vivo as intermediate hosts for postfertilization culture environment of bovine in vitro-produced zygotes with emphasis on the mouse model.

  2. Control of insulin secretion by cytochrome C and calcium signaling in islets with impaired metabolism.

    PubMed

    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.

  3. Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA

    SciTech Connect

    Permutt, M.A.; Koranyi, L.; Keller, K.; Lacy, P.E.; Scharp, D.W.; Mueckler, M. )

    1989-11-01

    Previous studies have suggested that pancreatic islet glucose transport is mediated by a high-K{sub m}, low-affinity facilitated transporter similar to that expressed in liver. To determine the relationship between islet and liver glucose transporters, liver-type glucose-transporter cDNA clones were isolated from a human liver cDNA library. The liver-type glucose-transporter cDNA clone hybridized to mRNA transcripts of the same size in human liver and pancreatic islet RNA. A cDNA library was prepared from purified human pancreatic islet tissue and screened with human liver-type glucose-transporter cDNA. The authors isolated two overlapping cDNA clones encompassing 2600 base pairs, which encode a pancreatic islet protein identical in sequence to that of the putative liver-type glucose-transporter protein. Xenopus oocytes injected with synthetic mRNA transcribed from a full-length cDNA construct exhibited increased uptake of 2-deoxyglucose, confirming the functional identity of the clone. These cDNA clones can now be used to study regulation of expression of the gene and to assess the role of inherited defects in this gene as a candidate for inherited susceptibility to non-insulin-dependent diabetes mellitus.

  4. Altered hepatic clearance and killing of Candida albicans in the isolated perfused mouse liver model.

    PubMed

    Sawyer, R T; Horst, M N; Garner, R E; Hudson, J; Jenkins, P R; Richardson, A L

    1990-09-01

    The adherence of Candida albicans was studied in situ by using the perfused mouse liver model. After exhaustive washing, 10(6) C. albicans were infused into mouse livers. At the time of recovery, 62 +/- 5% (mean +/- standard error of the mean) of the infused C. albicans were recovered from the liver and 14 +/- 3% were recovered from the effluent for a total recovery of 76 +/- 4%. This indicates that 86 +/- 3% of the original inoculum was trapped by the liver and that 24 +/- 4% was killed within the liver. Chemical pretreatment of C. albicans with 8 M urea, 12 mM dithiothreitol, 2% beta-mercaptoethanol, 1% sodium dodecyl sulfate, 10% Triton X-100, or 3 M potassium chloride or enzyme pretreatment with alpha-mannosidase, alpha-chymotrypsin, subtilisin, beta-N-acetyl-glucosaminidase, pronase, trypsin, papain, or lipase did not alter adherence of C. albicans to hepatic tissue. By contrast, pepsin pretreatment significantly decreased hepatic trapping. Simultaneous perfusion with either 100 mg of C. albicans glycoprotein per liter or 100 mg of C. albicans mannan per liter also decreased trapping. Furthermore, both substances eluted previously trapped C. albicans from hepatic tissue. Chemical pretreatment with 8 M urea, 12 mM dithiothreitol, or 3 M KCI or enzymatic pretreatment with alpha-mannosidase, subtilisin, alpha-chymotrypsin, or papain increased killing of C. albicans three- to fivefold within hepatic tissue. The data suggest that mannose-containing structures on the surface of C. albicans, for example. mannans or glucomannoproteins, mediate adherence of C. albicans within the liver. Indirectly, chemical and enzymatic pretreatment renders C. albicans more susceptible to hepatic killing.

  5. Comparative cytotoxicity of alkyl gallates on mouse tumor cell lines and isolated rat hepatocytes.

    PubMed

    Frey, Christian; Pavani, Mario; Cordano, Gianni; Muñoz, Sergio; Rivera, Enrique; Medina, Jorge; Morello, Antonio; Diego Maya, Juan; Ferreira, Jorge

    2007-04-01

    Alkyl esters of gallic acid inhibited the respiration rate of mouse sarcoma 786A and mouse mammary adenocarcinoma TA3 cell lines and its multiresistant variant TA3-MTX-R more effectively than gallic acid, both in the absence and in the presence of the uncoupler CCCP. The order of inhibition of the respiration rate by gallates in intact cells was n-octyl- approximately iso-amyl- approximately n-amyl- approximately iso-butyl->n-butyl->iso-propyl->n-propyl-gallate>gallic acid. Sarcoma 786A was significantly more susceptible to all seven esters than the TA3 cell line. Respiration rates of the TA3-MTX-R cell line showed almost the same sensitivity to these esters as the TA3 cell line. However, hepatocytes were significantly less sensitive than all tumor cells tested. These alkyl gallates blocked mitochondrial electron flow, mainly at the NADH-CoQ segment, preventing ATP synthesis, which would lead to cellular death. These esters also inhibited, in the same order of potencies as respiration, the growth of 786A, TA3 and TA3-MTX-R cells in culture. In mice carrying TA3 or TA3-MTX-R tumor cells, an important decrease of the tumor growth rate and an increase of survival were observed when mice were treated with iso-butyl gallate alone or in combination with doxorubicin. These results indicate that alkyl gallates are selectively cytotoxic to tumor cells, which may be due to the mitochondrial dysfunctions of these cells.

  6. Using pancreas tissue slices for in situ studies of islet of Langerhans and acinar cell biology.

    PubMed

    Marciniak, Anja; Cohrs, Christian M; Tsata, Vasiliki; Chouinard, Julie A; Selck, Claudia; Stertmann, Julia; Reichelt, Saskia; Rose, Tobias; Ehehalt, Florian; Weitz, Jürgen; Solimena, Michele; Slak Rupnik, Marjan; Speier, Stephan

    2014-12-01

    Studies on the cellular function of the pancreas are typically performed in vitro on its isolated functional units, the endocrine islets of Langerhans and the exocrine acini. However, these approaches are hampered by preparation-induced changes of cell physiology and the lack of an intact surrounding. We present here a detailed protocol for the preparation of pancreas tissue slices. This procedure is less damaging to the tissue and faster than alternative approaches, and it enables the in situ study of pancreatic endocrine and exocrine cell physiology in a conserved environment. Pancreas tissue slices facilitate the investigation of cellular mechanisms underlying the function, pathology and interaction of the endocrine and exocrine components of the pancreas. We provide examples for several experimental applications of pancreas tissue slices to study various aspects of pancreas cell biology. Furthermore, we describe the preparation of human and porcine pancreas tissue slices for the validation and translation of research findings obtained in the mouse model. Preparation of pancreas tissue slices according to the protocol described here takes less than 45 min from tissue preparation to receipt of the first slices.

  7. Lipoteichoic Acid Isolated from Weissella cibaria Increases Cytokine Production in Human Monocyte-Like THP-1 Cells and Mouse Splenocytes.

    PubMed

    Hong, Yi-Fan; Lee, Yoon-Doo; Park, Jae-Yeon; Kim, Seongjae; Lee, Youn-Woo; Jeon, Boram; Jagdish, Deepa; Kim, Hangeun; Chung, Dae Kyun

    2016-07-28

    Lactic acid bacteria (LAB) have beneficial effects on intestinal health and skin diseases. Lipoteichoic acid (LTA), a cell wall component of gram-positive bacteria, is known to induce the production of several cytokines such as TNF-α, IL-1β, and IL-8 and affect the intestinal microflora, anti-aging, sepsis, and cholesterol level. In this study, Weissella cibaria was isolated from Indian dairy products, and we examined its immune-enhancing effects. Live and heatkilled W. cibaria did not induce the secretion of immune-related cytokines, whereas LTA isolated from W. cibaria (cLTA) significantly increased the secretion of TNF-α, IL-1β, and IL-6 in a dose-dependent manner. cLTA increased the phosphorylation of nuclear factor kappalight-chain-enhancer of activated B cells, p38 mitogen-activated protein kinases, and c-Jun N-terminal kinases in THP-1 cells. The secretion of TNF-α and IL-6 was also increased in the cLTA-treated mouse splenocytes. These results suggest that cLTA, but not W. cibaria whole cells, has immune-boosting potential and can be used to treat immunosuppression diseases. PMID:27012236

  8. Isolation and Molecular Profiling of Primary Mouse Retinal Ganglion Cells: Comparison of Phenotypes from Healthy and Glaucomatous Retinas

    PubMed Central

    Chintalapudi, Sumana R.; Djenderedjian, Levon; Stiemke, Andrew B.; Steinle, Jena J.; Jablonski, Monica M.; Morales-Tirado, Vanessa M.

    2016-01-01

    Loss of functional retinal ganglion cells (RGC) is an element of retinal degeneration that is poorly understood. This is in part due to the lack of a reliable and validated protocol for the isolation of primary RGCs. Here we optimize a feasible, reproducible, standardized flow cytometry-based protocol for the isolation and enrichment of homogeneous RGC with the Thy1.2hiCD48negCD15negCD57neg surface phenotype. A three-step validation process was performed by: (1) genomic profiling of 25-genes associated with retinal cells; (2) intracellular labeling of homogeneous sorted cells for the intracellular RGC-markers SNCG, brain-specific homeobox/POU domain protein 3A (BRN3A), TUJ1, and RNA-binding protein with multiple splicing (RBPMS); and (3) by applying the methodology on RGC from a mouse model with elevated intraocular pressure (IOP) and optic nerve damage. Use of primary RGC cultures will allow for future careful assessment of important cell specific pathways in RGC to provide mechanistic insights into the declining of visual acuity in aged populations and those suffering from retinal neurodegenerative diseases. PMID:27242509

  9. Taurine supplementation modulates glucose homeostasis and islet function.

    PubMed

    Carneiro, Everardo M; Latorraca, Marcia Q; Araujo, Eliana; Beltrá, Marta; Oliveras, Maria J; Navarro, Mónica; Berná, Genoveva; Bedoya, Francisco J; Velloso, Licio A; Soria, Bernat; Martín, Franz

    2009-07-01

    Taurine is a conditionally essential amino acid for human that is involved in the control of glucose homeostasis; however, the mechanisms by which the amino acid affects blood glucose levels are unknown. Using an animal model, we have studied these mechanisms. Mice were supplemented with taurine for 30 d. Blood glucose homeostasis was assessed by intraperitoneal glucose tolerance tests (IPGTT). Islet cell function was determined by insulin secretion, cytosolic Ca2+ measurements and glucose metabolism from isolated islets. Islet cell gene expression and translocation was examined via immunohistochemistry and quantitative real-time polymerase chain reaction. Insulin signaling was studied by Western blot. Islets from taurine-supplemented mice had: (i) significantly higher insulin content, (ii) increased insulin secretion at stimulatory glucose concentrations, (iii) significantly displaced the dose-response curve for glucose-induced insulin release to the left, (iv) increased glucose metabolism at 5.6 and 11.1-mmol/L concentrations; (v) slowed cytosolic Ca2+ concentration ([Ca2+]i) oscillations in response to stimulatory glucose concentrations; (vi) increased insulin, sulfonylurea receptor-1, glucokinase, Glut-2, proconvertase and pancreas duodenum homeobox-1 (PDX-1) gene expression and (vii) increased PDX-1 expression in the nucleus. Moreover, taurine supplementation significantly increased both basal and insulin stimulated tyrosine phosphorylation of the insulin receptor in skeletal muscle and liver tissues. Finally, taurine supplemented mice showed an improved IPGTT. These results indicate that taurine controls glucose homeostasis by regulating the expression of genes required for glucose-stimulated insulin secretion. In addition, taurine enhances peripheral insulin sensitivity. PMID:18708284

  10. Mouse Y-specific repeats isolated by whole chromosome representational difference analysis

    SciTech Connect

    Navin, A.; Prekeris, R.; Sonti, M.M.

    1996-09-01

    Representational difference analysis (RDA) was used to generate Y-specific probes by enriching for and cloning the differences between the male (XY) and the female (XX) C57BL/6J mouse genomes. Characterization of 35 clones revealed 12 families related by sequence similarity. One clone from each family was chosen for detailed analysis by Southern blot hybridization, polymerase chain reaction (PCR) on normal and aberrant genomes (Sxr), and fluorescence in situ hybridization. From one difference product we have characterized 12 Y-specific probes for hybridization, created seven male-specific PCR assays, mapped all repeat families, and identified one repeat with a distinct XY homology. We report the first cloning of a Y-specific long interspersed repeat element (LINE) fragment. In total, RDA has identified six novel Y Chromosome repeat families and allowed us to extend the characterization of six known Y repeats. We conclude that this novel use of RDA for whole chromosome subtraction successfully enriches chromosome subtraction successfully enriches chromosome-specific sequences and it suitable for the rapid generation of new Y Chromosome-specific probes. 16 refs., 2 figs., 1 tab.

  11. Intracellular ATP measured with luciferin/luciferase in isolated single mouse skeletal muscle fibres.

    PubMed

    Allen, David G; Lännergren, Jan; Westerblad, Håkan

    2002-03-01

    The firefly luciferin/luciferase reaction was utilized to monitor intracellular ATP concentration ([ATP](i)). Single fibres of mouse skeletal muscle were dissected and injected with luciferase. Luciferin was added to the perfusate and light emission from the fibres was monitored as an indication of [ATP](i). Inhibition of oxidative phosphorylation with cyanide and anaerobic glycolysis with iodoacetate caused light emission to fall to zero within 10 min and the fibres developed a rigor contraction. Inhibition of creatine kinase with 2,4-dinitro-1-fluorobenzene produced a small transient fall in light emission in association with each tetanus. Muscle fibres were fatigued by repeated tetani and 5/12 fibres showed a fall in light emission in the late phase of fatigue. If fibres were allowed to recover from fatigue in the absence of glucose and then restimulated in the absence of glucose they fatigued much more rapidly. However, such fibres showed no obvious change in light emission. We conclude that the luciferin/luciferase system can be used to monitor [ATP](i) in functioning single skeletal muscle cells. A depletion of global [ATP](i) is not observed in all fatiguing fibres and cannot be the sole cause of the final phase of fatigue.

  12. FEM-based oxygen consumption and cell viability models for avascular pancreatic islets

    PubMed Central

    Buchwald, Peter

    2009-01-01

    Background The function and viability of cultured, transplanted, or encapsulated pancreatic islets is often limited by hypoxia because these islets have lost their vasculature during the isolation process and have to rely on gradient-driven passive diffusion, which cannot provide adequate oxygen transport. Pancreatic islets (islets of Langerhans) are particularly susceptible due to their relatively large size, large metabolic demand, and increased sensitivity to hypoxia. Here, finite element method (FEM) based multiphysics models are explored to describe oxygen transport and cell viability in avascular islets both in static and in moving culture media. Methods Two- and three-dimensional models were built in COMSOL Multiphysics using the convection and diffusion as well as the incompressible Navier-Stokes fluid dynamics application modes. Oxygen consumption was assumed to follow Michaelis-Menten-type kinetics and to cease when local concentrations fell below a critical threshold; in a dynamic model, it was also allowed to increase with increasing glucose concentration. Results Partial differential equation (PDE) based exploratory cellular-level oxygen consumption and cell viability models incorporating physiologically realistic assumptions have been implemented for fully scaled cell culture geometries with 100, 150, and 200 μm diameter islets as representative. Calculated oxygen concentrations and intra-islet regions likely to suffer from hypoxia-related necrosis obtained for traditional flask-type cultures, oxygen-permeable silicone-rubber membrane bottom cultures, and perifusion chambers with flowing media and varying incoming glucose levels are presented in detail illustrated with corresponding colour-coded figures and animations. Conclusion Results of the computational models are, as a first estimate, in good quantitative agreement with existing experimental evidence, and they confirm that during culture, hypoxia is often a problem for non-vascularised islet

  13. Isolation of plasma membrane vesicles from mouse placenta at term and measurement of system A and system beta amino acid transporter activity.

    PubMed

    Kusinski, L C; Jones, C J P; Baker, P N; Sibley, C P; Glazier, J D

    2010-01-01

    Placental amino acid transport is essential for optimal fetal growth and development, with a reduced fetal provision of amino acids being implicated as a potential cause of fetal growth restriction (FGR). Understanding placental insufficiency related FGR has been aided by the development of mouse models that have features of the human disease. However, to take maximal advantage of these, methods are required to study placental function in the mouse. Here, we report a method to isolate plasma membrane vesicles from mouse placenta near-term and have used these to investigate two amino acid transporters, systems A and beta, the activities of which are reduced in human placental microvillous plasma membrane (MVM) vesicles from FGR pregnancies. Plasma membrane vesicles were isolated at embryonic day 18 by a protocol involving homogenisation, MgCl(2) precipitation and centrifugation. Vesicles were enriched 11.3+/-0.5-fold in alkaline phosphatase activity as compared to initial homogenate, with minimal intracellular organelle contamination as judged by marker analyses. Cytochemistry revealed alkaline phosphatase was localised between trophoblast layers I and II, with intense reaction product deposited on the maternal-facing plasma membrane of layer II, suggesting that vesicles were derived from this trophoblast membrane. System A and system beta activity in mouse placental vesicles, measured as Na(+)-dependent uptake of (14)C-methylaminoisobutyric acid (MeAIB) and (3)H-taurine respectively confirmed localisation of these transporters to the maternal-facing plasma membrane of layer II. Comparison to human placental MVM showed that system A activity was comparable at initial rate between species whilst system beta activity was significantly lower in mouse. This mirrored the lower expression of TAUT observed in mouse placental vesicles. We conclude that syncytiotrophoblast layer II-derived plasma membrane vesicles can be isolated and used to examine transporter function.

  14. Total Pancreatectomy With Islet Autotransplantation

    PubMed Central

    Bellin, Melena D.; Gelrud, Andres; Arreaza-Rubin, Guillermo; Dunn, Ty B.; Humar, Abhinav; Morgan, Katherine A.; Naziruddin, Bashoo; Rastellini, Cristiana; Rickels, Michael R.; Schwarzenberg, Sarah J.; Andersen, Dana K.

    2015-01-01

    A workshop sponsored by the National Institute of Diabetes and Digestive and Kidney Diseases focused on research gaps and opportunities in total pancreatectomy with islet autotransplantation (TPIAT) for the management of chronic pancreatitis. The session was held on July 23, 2014 and structured into 5 sessions: (1) patient selection, indications, and timing; (2) technical aspects of TPIAT; (3) improving success of islet autotransplantation; (4) improving outcomes after total pancreatectomy; and (5) registry considerations for TPIAT. The current state of knowledge was reviewed; knowledge gaps and research needs were specifically highlighted. Common themes included the need to identify which patients best benefit from and when to intervene with TPIAT, current limitations of the surgical procedure, diabetes remission and the potential for improvement, opportunities to better address pain remission, GI complications in this population, and unique features of children with chronic pancreatitis considered for TPIAT. The need for a multicenter patient registry that specifically addresses the complexities of chronic pancreatitis and total pancreatectomy outcomes and postsurgical diabetes outcomes was repeatedly emphasized. PMID:25599324

  15. Arg-Gly-Asp (RGD) peptides alter hepatic killing of Candida albicans in the isolated perfused mouse liver model.

    PubMed

    Sawyer, R T; Garner, R E; Hudson, J A

    1992-01-01

    The isolated perfused mouse liver model was used to study the effect of Arg-Gly-Asp (RGD)-containing peptides on hepatic trapping and killing of Candida albicans. After extensive washing, 10(6) C. albicans CFU were infused into mouse livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicates that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/- 2% was killed within the liver. Prior to their infusion into livers, 10(7) CFU of C. albicans were incubated at 37 degrees C for 30 min in the presence of various RGD peptides (0.1 mg/ml). Repeatedly, more than 90% of the infused RGD-treated C. albicans was trapped by the perfused liver. In comparison with the 23% killing rate observed in control livers, perfused livers killed approximately 40 to 50% of the infused C. albicans treated either with fibronectin, PepTite 2000, RGD, or RGDS. Hepatic killing of C. albicans treated with PepTite 2000 or fibronectin was dose dependent. Treatment of C. albicans with GRGDTP, GRGDSP, GRADSP, or GRGESP did not alter the ability of the perfused liver to kill C. albicans, suggesting that a degree of specificity for RGD peptides is associated with an increased ability of liver to kill RGD-treated C. albicans. Together, the data suggest that RGD peptides bind to a receptor on the surface of C. albicans, thereby increasing hepatic, and presumably Kupffer cell, killing of C. albicans. Natural or synthetic RGD peptides may serve as opsonins promoting C. albicans killing by Kupffer cells.

  16. Lipid composition of membrane rafts, isolated with and without detergent, from the spleen of a mouse model of Gaucher disease.

    PubMed

    Hattersley, Kathryn J; Hein, Leanne K; Fuller, Maria

    2013-12-01

    Biological membranes are composed of functionally relevant liquid-ordered and liquid-disordered domains that coexist. Within the liquid-ordered domains are low-density microdomains known as rafts with a unique lipid composition that is crucial for their structure and function. Lipid raft composition is altered in sphingolipid storage disorders, and here we determined the lipid composition using a detergent and detergent-free method in spleen tissue, the primary site of pathology, in a mouse model of the sphingolipid storage disorder, Gaucher disease. The accumulating lipid, glucosylceramide, was 30- and 50-fold elevated in the rafts with the detergent and detergent-free method, respectively. Secondary accumulation of di- and trihexosylceramide resided primarily in the rafts with both methods. The phospholipids distributed differently with more than half residing in the rafts with the detergent-free method and less than 10% with the detergent method, with the exception of the fully saturated species that were primarily in the rafts. Individual isoforms of sphingomyelin correlated with detergent-free extraction and more than half resided in the raft fractions. However, this correlation was not seen with the detergent extraction method as sphingomyelin species were spread across both the raft and non-raft domains. Therefore caution must be exercised when interpreting phospholipid distribution in raft domains as it differs considerably depending on the method of isolation. Importantly, both methods revealed the same lipid alterations in the raft domains in the spleen of the Gaucher disease mouse model highlighting that either method is appropriate to determine membrane lipid changes in the diseased state.

  17. Effect of lectins on hepatic clearance and killing of Candida albicans by the isolated perfused mouse liver.

    PubMed Central

    Sawyer, R T; Garner, R E; Hudson, J A

    1992-01-01

    The isolated perfused mouse liver model was used to study the effects of various lectins on hepatic trapping and killing of Candida albicans. After mouse livers were washed with 20 to 30 ml of perfusion buffer, 10(6) C. albicans CFU were infused into the livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicated that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/- 2% was killed within the liver. When included in both preperfusion and postperfusion buffers (0.2 mg of lectin per ml), Ulex europeaus lectin (binding specificity for fucose) decreased hepatic trapping of C. albicans by 37% and eluted trapped C. albicans from the liver only when included in postperfusion buffer. By comparison, treatment of C. albicans with U. europeaus lectin before infusion had no effect on the trapping or killing of yeast cells. When Lens culinaris lectin (binding specificity for mannose) was included in the perfusion buffers, hepatic killing of C. albicans increased by 16% with no significant effect on hepatic killing when yeast cells were treated with L. culinaris lectin before infusion. Forty to 55% of the infused C. albicans were killed when concanavalin A (binding specificities for mannose and glucose), Glycine max (binding specificity for N-acetylgalactosamine), or Arachis hypogea (binding specificity for galactose) lectin was included in the perfusion buffer or when yeast cells were treated with these lectins before their infusion. When C. albicans was treated with concanavalin A at a concentration of less than 0.02 mg/ml, hepatic killing of yeast cells was not significantly increased. The data suggest that a fucose-containing receptor on the surface of either sinusoidal endothelial cells or Kupffer cells is involved in the trapping of C. albicans by the perfused mouse

  18. Effect of lectins on hepatic clearance and killing of Candida albicans by the isolated perfused mouse liver.

    PubMed

    Sawyer, R T; Garner, R E; Hudson, J A

    1992-03-01

    The isolated perfused mouse liver model was used to study the effects of various lectins on hepatic trapping and killing of Candida albicans. After mouse livers were washed with 20 to 30 ml of perfusion buffer, 10(6) C. albicans CFU were infused into the livers. At the time of recovery, 63% +/- 2% (mean +/- standard error of the mean) of the infused C. albicans CFU were recovered from the liver and 14% +/- 1% were recovered from the effluent for a total recovery of 77% +/- 2%. This indicated that 86% +/- 9% of the original inoculum was trapped by the liver and that 23% +/- 2% was killed within the liver. When included in both preperfusion and postperfusion buffers (0.2 mg of lectin per ml), Ulex europeaus lectin (binding specificity for fucose) decreased hepatic trapping of C. albicans by 37% and eluted trapped C. albicans from the liver only when included in postperfusion buffer. By comparison, treatment of C. albicans with U. europeaus lectin before infusion had no effect on the trapping or killing of yeast cells. When Lens culinaris lectin (binding specificity for mannose) was included in the perfusion buffers, hepatic killing of C. albicans increased by 16% with no significant effect on hepatic killing when yeast cells were treated with L. culinaris lectin before infusion. Forty to 55% of the infused C. albicans were killed when concanavalin A (binding specificities for mannose and glucose), Glycine max (binding specificity for N-acetylgalactosamine), or Arachis hypogea (binding specificity for galactose) lectin was included in the perfusion buffer or when yeast cells were treated with these lectins before their infusion. When C. albicans was treated with concanavalin A at a concentration of less than 0.02 mg/ml, hepatic killing of yeast cells was not significantly increased. The data suggest that a fucose-containing receptor on the surface of either sinusoidal endothelial cells or Kupffer cells is involved in the trapping of C. albicans by the perfused mouse

  19. Isolation and characterization of the mouse heme oxygenase-1 gene. Distal 5' sequences are required for induction by heme or heavy metals.

    PubMed

    Alam, J; Cai, J; Smith, A

    1994-01-14

    Mouse genomic fragments encoding heme oxygenase-1 (HO-1) were isolated from a recombinant lambda library by in situ plaque hybridization. The mouse HO-1 gene, approximately 7 kilobase pairs (kbp) in length, is organized into 5 exons and 4 introns. The primary structure of the exons and 1287 base pairs (bp) of the 5'-flanking region was determined. The deduced amino acid sequence of the mouse HO-1 gene is identical to that of p32, initially identified as a stress-induced protein in mouse BALBc/3T3 cells. A single, major transcription initiation site is utilized for constitutive and heme- or metal-induced expression of the HO-1 gene in mouse hepatoma (Hepa) cells. The transcriptional activity of the 5'-flanking region was examined by transient expression assays using the chloramphenicol acetyltransferase gene as the reporter gene. Basal promoter activity in several cell lines was localized to within 149 bp of the upstream sequence by deletion analysis. This proximal promoter region of the mouse HO-1 gene contains several sequence elements that are not only conserved in both the rat and human HO-1 genes but also resemble consensus binding sites of various transcription factors including AP-1, AP-4, C/EBP and c-Myc:Max/USF. Heavy metals activate HO-1 gene transcription and the rat gene contains a putative metal regulatory element (Müller, R. M., Taguchi, H., and Shibahara, S. (1987) J. Biol. Chem. 262, 6795-6802) that is completely conserved in the mouse gene. Transient expression analyses, however, indicate that this sequence, which contains a core heptanucleotide, TGCACTC, identical to that of the strongest metal regulatory element of the mouse metallothionein-1 gene, is not responsive to Cd2+ or Zn2+. Stable transfection of constructs containing the entire mouse HO-1 gene and various portions of the 5'-flanking region into rat C6 glioma cells and simultaneous, quantitative analysis of the mouse and rat HO-1 mRNAs indicate that distal 5' sequences, between positions

  20. Compensatory hyperinsulinemia in high-fat diet-induced obese mice is associated with enhanced insulin translation in islets

    SciTech Connect

    Kanno, Ayumi; Asahara, Shun-ichiro; Masuda, Katsuhisa; Matsuda, Tomokazu; Kimura-Koyanagi, Maki; Seino, Susumu; Ogawa, Wataru; Kido, Yoshiaki

    2015-03-13

    A high-fat diet (HF) is associated with obesity, insulin resistance, and hyperglycemia. Animal studies have shown compensatory mechanisms in pancreatic β-cells after high fat load, such as increased pancreatic β-cell mass, enhanced insulin secretion, and exocytosis. However, the effects of high fat intake on insulin synthesis are obscure. Here, we investigated whether insulin synthesis was altered in correlation with an HF diet, for the purpose of obtaining further understanding of the compensatory mechanisms in pancreatic β-cells. Mice fed an HF diet are obese, insulin resistant, hyperinsulinemic, and glucose intolerant. In islets of mice fed an HF diet, more storage of insulin was identified. We analyzed insulin translation in mouse islets, as well as in INS-1 cells, using non-radioisotope chemicals. We found that insulin translational levels were significantly increased in islets of mice fed an HF diet to meet systemic demand, without altering its transcriptional levels. Our data showed that not only increased pancreatic β-cell mass and insulin secretion but also elevated insulin translation is the major compensatory mechanism of pancreatic β-cells. - Highlights: • More stored insulin was recognized in islets of mice fed a high-fat diet. • Insulin translation was not enhanced by fatty acids, but by insulin demand. • Insulin transcription was not altered in islets of mice fed a high-fat diet. • Insulin translation was markedly enhanced in islets of mice fed a high-fat diet. • Non-radioisotope chemicals were used to measure insulin translation in mouse islets.

  1. Protein synthesis inhibitors prevent both spontaneous and hormone-dependent maturation of isolated mouse oocytes

    SciTech Connect

    Downs, S.M. )

    1990-11-01

    The present study was carried out to examine the role of protein synthesis in mouse oocyte maturation in vitro. In the first part of this study, the effects of cycloheximide (CX) were tested on spontaneous meiotic maturation when oocytes were cultured in inhibitor-free medium. CX reversibly suppressed maturation of oocytes as long as maturation was either initially prevented by the phosphodiesterase inhibitor, 3-isobutyl-1-methyl-xanthine (IBMX), or delayed by follicle-stimulating hormone (FSH). In the second part of this study, the actions of protein synthesis inhibitors were tested on hormone-induced maturation. CEO were maintained in meiotic arrest for 21-22 h with hypoxanthine, and germinal vesicle breakdown (GVB) was induced with follicle-stimulating hormone (FSH). Three different protein synthesis inhibitors (CX, emetine (EM), and puromycin (PUR)) each prevented the stimulatory action of FSH on GVB in a dose-dependent fashion. This was accompanied by a dose-dependent suppression of 3H-leucine incorporation by oocyte-cumulus cell complexes. The action of these inhibitors on FSH- and epidermal growth factor (EGF)-induced GVB was next compared. All three drugs lowered the frequency of GVB in the FSH-treated groups, below even that of the controls (drug + hypoxanthine); the drugs maintained meiotic arrest at the control frequencies in the EGF-treated groups. Puromycin aminonucleoside, an analog of PUR with no inhibitory action on protein synthesis, had no effect. The three inhibitors also suppressed the stimulatory action of FSH on oocyte maturation when meiotic arrest was maintained with the cAMP analog, dbcAMP.

  2. Delineation of a frequency-organized region isolated from the mouse primary auditory cortex

    PubMed Central

    Horie, Masao; Bo, Takeshi; Uchimura, Arikuni; Hishida, Ryuichi; Kudoh, Masaharu; Takahashi, Kuniyuki; Takebayashi, Hirohide; Shibuki, Katsuei

    2015-01-01

    The primary auditory cortex (AI) is the representative recipient of information from the ears in the mammalian cortex. However, the delineation of the AI is still controversial in a mouse. Recently, it was reported, using optical imaging, that two distinct areas of the AI, located ventrally and dorsally, are activated by high-frequency tones, whereas only one area is activated by low-frequency tones. Here, we show that the dorsal high-frequency area is an independent region that is separated from the rest of the AI. We could visualize the two distinct high-frequency areas using flavoprotein fluorescence imaging, as reported previously. SMI-32 immunolabeling revealed that the dorsal region had a different cytoarchitectural pattern from the rest of the AI. Specifically, the ratio of SMI-32-positive pyramidal neurons to nonpyramidal neurons was larger in the dorsal high-frequency area than the rest of the AI. We named this new region the dorsomedial field (DM). Retrograde tracing showed that neurons projecting to the DM were localized in the rostral part of the ventral division of the medial geniculate body with a distinct frequency organization, where few neurons projected to the AI. Furthermore, the responses of the DM to ultrasonic courtship songs presented by males were significantly greater in females than in males; in contrast, there was no sex difference in response to artificial pure tones. Our findings offer a basic outline on the processing of ultrasonic vocal information on the basis of the precisely subdivided, multiple frequency-organized auditory cortex map in mice. PMID:25695649

  3. Accumulation of M1-like macrophages in type 2 diabetic islets is followed by a systemic shift in macrophage polarization.

    PubMed

    Cucak, Helena; Grunnet, Lars Groth; Rosendahl, Alexander

    2014-01-01

    Human T2D is characterized by a low-grade systemic inflammation, loss of β-cells, and diminished insulin production. Local islet immunity is still poorly understood, and hence, we evaluated macrophage subpopulations in pancreatic islets in the well-established murine model of T2D, the db/db mouse. Already at 8 weeks of disease, on average, 12 macrophages were observed in the diabetic islets, whereas only two were recorded in the nondiabetic littermates. On a detailed level, the islet resident macrophages increased fourfold compared with nondiabetic littermates, whereas a pronounced recruitment (eightfold) of a novel subset of macrophages (CD68+F4/80-) was observed. The majority of the CD68+F4/80+ but only 40% of the CD68+F4/80- islet macrophages expressed CD11b. Both islet-derived macrophage subsets expressed moderate MHC-II, high galectin-3, and low CD80/CD86 levels, suggesting the cells to be macrophages rather than DCs. On a functional level, the vast majority of the macrophages in the diabetic islets was of the proinflammatory, M1-like phenotype. The systemic immunity in diabetic animals was characterized by a low-grade inflammation with elevated cytokine levels and increase of splenic cytokine, producing CD68+F4/80- macrophages. In late-stage diabetes, the cytokine signature changed toward a TGF-β-dominated profile, coinciding with a significant increase of galectin-3-positive macrophages in the spleen. In summary, our results show that proinflammatory M1-like galectin-3+ CD80/CD86(low) macrophages invade diabetic islets. Moreover, the innate immunity matures in a diabetes-dependent manner from an initial proinflammatory toward a profibrotic phenotype, supporting the concept that T2D is an inflammatory disease. PMID:24009176

  4. Accumulation of M1-like macrophages in type 2 diabetic islets is followed by a systemic shift in macrophage polarization.

    PubMed

    Cucak, Helena; Grunnet, Lars Groth; Rosendahl, Alexander

    2014-01-01

    Human T2D is characterized by a low-grade systemic inflammation, loss of β-cells, and diminished insulin production. Local islet immunity is still poorly understood, and hence, we evaluated macrophage subpopulations in pancreatic islets in the well-established murine model of T2D, the db/db mouse. Already at 8 weeks of disease, on average, 12 macrophages were observed in the diabetic islets, whereas only two were recorded in the nondiabetic littermates. On a detailed level, the islet resident macrophages increased fourfold compared with nondiabetic littermates, whereas a pronounced recruitment (eightfold) of a novel subset of macrophages (CD68+F4/80-) was observed. The majority of the CD68+F4/80+ but only 40% of the CD68+F4/80- islet macrophages expressed CD11b. Both islet-derived macrophage subsets expressed moderate MHC-II, high galectin-3, and low CD80/CD86 levels, suggesting the cells to be macrophages rather than DCs. On a functional level, the vast majority of the macrophages in the diabetic islets was of the proinflammatory, M1-like phenotype. The systemic immunity in diabetic animals was characterized by a low-grade inflammation with elevated cytokine levels and increase of splenic cytokine, producing CD68+F4/80- macrophages. In late-stage diabetes, the cytokine signature changed toward a TGF-β-dominated profile, coinciding with a significant increase of galectin-3-positive macrophages in the spleen. In summary, our results show that proinflammatory M1-like galectin-3+ CD80/CD86(low) macrophages invade diabetic islets. Moreover, the innate immunity matures in a diabetes-dependent manner from an initial proinflammatory toward a profibrotic phenotype, supporting the concept that T2D is an inflammatory disease.

  5. Microfluidics-generated pancreatic islet microfibers for enhanced immunoprotection.

    PubMed

    Jun, Yesl; Kim, Min Jun; Hwang, Yong Hwa; Jeon, Eun Ae; Kang, Ah Ran; Lee, Sang-Hoon; Lee, Dong Yun

    2013-11-01

    Pancreatic islet transplantation is a promising method for treatment of type 1 diabetes mellitus. However, transplanted islets can be destroyed due to host immune reactions. To immunologically protect transplanted islets, here an immunoprotective microfiber including islets by using a polydimethylsiloxane (PDMS)-based microfluidic device is newly designed. A cylindrical-flow channel in the microfluidic platform is used for producing collagen-alginate composite (CAC) fibers. This enables mass production and uniform diameter distribution (<250 μm) without protruding islets. Collagen, which is the main extracellular matrix component, is added to alginate to mimic the native islet microenvironment. Compared to free islets (control) and alginate-fiber-encapsulated islets, CAC-fiber-encapsulated islets show higher viability and normal insulin secretion. When CAC-fiber-encapsulated islets (1200 islet equivalent) are implanted into the intraperitoneal cavity of streptozotocin-induced diabetic BALB/C mice, the blood glucose levels of all mice return to normoglycemia. Moreover, intraperitoneal glucose tolerance tests demonstrate that islets in the CAC-fiber have similar glucose responsiveness to those of non-diabetic normal mice. These results are attributed to the immunoprotection of the transplanted islets from host immune reactions. On the other hand, all free islets are completely rejected within a week due to severe immune responses. Collectively, fabrication of CAC fibers using microfluidic devices can be used for successful islet transplantation. PMID:23927952

  6. Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting

    PubMed Central

    Biesemann, Christoph; Grønborg, Mads; Luquet, Elisa; Wichert, Sven P; Bernard, Véronique; Bungers, Simon R; Cooper, Ben; Varoqueaux, Frédérique; Li, Liyi; Byrne, Jennifer A; Urlaub, Henning; Jahn, Olaf; Brose, Nils; Herzog, Etienne

    2014-01-01

    For decades, neuroscientists have used enriched preparations of synaptic particles called synaptosomes to study synapse function. However, the interpretation of corresponding data is problematic as synaptosome preparations contain multiple types of synapses and non-synaptic neuronal and glial contaminants. We established a novel Fluorescence Activated Synaptosome Sorting (FASS) method that substantially improves conventional synaptosome enrichment protocols and enables high-resolution biochemical analyses of specific synapse subpopulations. Employing knock-in mice with fluorescent glutamatergic synapses, we show that FASS isolates intact ultrapure synaptosomes composed of a resealed presynaptic terminal and a postsynaptic density as assessed by light and electron microscopy. FASS synaptosomes contain bona fide glutamatergic synapse proteins but are almost devoid of other synapse types and extrasynaptic or glial contaminants. We identified 163 enriched proteins in FASS samples, of which FXYD6 and Tpd52 were validated as new synaptic proteins. FASS purification thus enables high-resolution biochemical analyses of specific synapse subpopulations in health and disease. PMID:24413018

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

    PubMed

    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. PMID:27225179

  8. Islet-intrinsic effects of CFTR mutation.

    PubMed

    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

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

    PubMed

    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.

  10. Total Pancreatectomy with Islet Autologous Transplantation: The Cure for Chronic Pancreatitis?

    PubMed Central

    Kesseli, Samuel J; Smith, Kerrington A; Gardner, Timothy B

    2015-01-01

    Chronic pancreatitis (CP) is a debilitating disease that leads to varying degrees of pancreatic endocrine and exocrine dysfunction. One of the most difficult symptoms of CP is severe abdominal pain, which is often challenging to control with available analgesics and therapies. In the last decade, total pancreatectomy with autologous islet cell transplantation has emerged as a promising treatment for the refractory pain of CP and is currently performed at approximately a dozen centers in the United States. While total pancreatectomy is not a new procedure, the endocrine function-preserving autologous islet cell isolation and re-implantation have made the prospect of total pancreatectomy more acceptable to patients and clinicians. This review will focus on the current status of total pancreatectomy with autologous islet cell transplant including patient selection, technical considerations, and outcomes. As the procedure is performed at an increasing number of centers, this review will highlight opportunities for quality improvement and outcome optimization. PMID:25630865

  11. Pancreatic stem cells: building blocks for a better surrogate islet to treat type 1 diabetes.

    PubMed

    Peck, A B; Chaudhari, M; Cornelius, J G; Ramiya, V K

    2001-04-01

    Type 1, insulin-dependent, diabetes is one of the more costly chronic diseases of children, adolescents and adults in Europe and North America. While routine insulin injections currently provide diabetic patients with their daily insulin requirements, blood glucose excursions are common, leading eventually to microvascular and macrovascular complications and early death. A 'cure' for Type 1 diabetes relies on replacement of the beta-cell mass which, today, is accomplished by pancreas transplants or islets of Langerhans implants. Recent advances in the isolation of stem cells that possess the capacity to differentiate to functional endocrine pancreas provide new opportunities to produce large numbers of islets, even autologous islets, that can be used as implants. We discuss briefly this new technology and its meaning for diabetes. PMID:11370772

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

    PubMed

    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.

  13. Silicon nanopore membrane (SNM) for islet encapsulation and immunoisolation under convective transport.

    PubMed

    Song, Shang; Faleo, Gaetano; Yeung, Raymond; Kant, Rishi; Posselt, Andrew M; Desai, Tejal A; Tang, Qizhi; Roy, Shuvo

    2016-01-01

    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/m(2)/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. PMID:27009429

  14. Silicon nanopore membrane (SNM) for islet encapsulation and immunoisolation under convective transport

    PubMed Central

    Song, Shang; Faleo, Gaetano; Yeung, Raymond; Kant, Rishi; Posselt, Andrew M; Desai, Tejal A; Tang, Qizhi; Roy, Shuvo

    2016-01-01

    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. PMID:27009429

  15. Silicon nanopore membrane (SNM) for islet encapsulation and immunoisolation under convective transport

    NASA Astrophysics Data System (ADS)

    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.

  16. Decreased cholinergic stimulation of insulin secretion by islets from rats fed a low protein diet is associated with reduced protein kinase calpha expression.

    PubMed

    Ferreira, Fabiano; Filiputti, Eliane; Arantes, Vanessa C; Stoppiglia, Luis F; Araújo, Eliana P; Delghingaro-Augusto, Viviane; Latorraca, Márcia Q; Toyama, Marcos H; Boschero, Antonio C; Carneiro, Everardo M

    2003-03-01

    Undernutrition has been shown to affect the autonomic nervous system, leading to permanent alterations in insulin secretion. To understand these interactions better, we investigated the effects of carbamylcholine (CCh) and phorbol 12-myristate 13-acetate (PMA) on insulin secretion in pancreatic islets from rats fed a normal (17%; NP) or low (6%; LP) protein diet for 8 wk. Isolated islets were incubated for 1 h in Krebs-bicarbonate solution containing 8.3 mmol glucose/L, with or without PMA (400 nmol/L) and CCh. Increasing concentrations of CCh (0.1-1000 micro mol/L) dose dependently increased insulin secretion by islets from both groups of rats. However, insulin secretion by islets from rats fed the NP diet was significantly higher than that of rats fed the LP diet, and the dose-response curve to CCh was shifted to the right in islets from rats fed LP with a 50% effective concentration (EC(50)) of 2.15 +/- 0.7 and 4.64 +/- 0.1 micro mol CCh/L in islets of rats fed NP and LP diets, respectively (P < 0.05). PMA-induced insulin secretion was higher in islets of rats fed NP compared with those fed LP. Western blotting revealed that the protein kinase (PK)Calpha and phospholipase (PL)Cbeta(1) contents of islets of rats fed LP were 30% lower than those of islets of rats fed NP (P < 0.05). In addition, PKCalpha mRNA expression was reduced by 50% in islets from rats fed LP. In conclusion, a reduced expression of PKCalpha and PLCbeta(1) may be involved in the decreased insulin secretion by islets from LP rats after stimulation with CCh and PMA. PMID:12612139

  17. Taurine supplementation increases K(ATP) channel protein content, improving Ca2+ handling and insulin secretion in islets from malnourished mice fed on a high-fat diet.

    PubMed

    Vettorazzi, Jean F; Ribeiro, Rosane A; Santos-Silva, Junia C; Borck, Patricia C; Batista, Thiago M; Nardelli, Tarlliza R; Boschero, Antonio C; Carneiro, Everardo M

    2014-09-01

    Pancreatic β-cells are highly sensitive to suboptimal or excess nutrients, as occurs in protein-malnutrition and obesity. Taurine (Tau) improves insulin secretion in response to nutrients and depolarizing agents. Here, we assessed the expression and function of Cav and KATP channels in islets from malnourished mice fed on a high-fat diet (HFD) and supplemented with Tau. Weaned mice received a normal (C) or a low-protein diet (R) for 6 weeks. Half of each group were fed a HFD for 8 weeks without (CH, RH) or with 5% Tau since weaning (CHT, RHT). Isolated islets from R mice showed lower insulin release with glucose and depolarizing stimuli. In CH islets, insulin secretion was increased and this was associated with enhanced KATP inhibition and Cav activity. RH islets secreted less insulin at high K(+) concentration and showed enhanced KATP activity. Tau supplementation normalized K(+)-induced secretion and enhanced glucose-induced Ca(2+) influx in RHT islets. R islets presented lower Ca(2+) influx in response to tolbutamide, and higher protein content and activity of the Kir6.2 subunit of the KATP. Tau increased the protein content of the α1.2 subunit of the Cav channels and the SNARE proteins SNAP-25 and Synt-1 in CHT islets, whereas in RHT, Kir6.2 and Synt-1 proteins were increased. In conclusion, impaired islet function in R islets is related to higher content and activity of the KATP channels. Tau treatment enhanced RHT islet secretory capacity by improving the protein expression and inhibition of the KATP channels and enhancing Synt-1 islet content.

  18. Asiatic acid uncouples respiration in isolated mouse liver mitochondria and induces HepG2 cells death.

    PubMed

    Lu, Yapeng; Liu, Siyuan; Wang, Ying; Wang, Dang; Gao, Jing; Zhu, Li

    2016-09-01

    Asiatic acid, one of the triterpenoid components isolated from Centella asiatica, has received increasing attention due to a wide variety of biological activities. To date, little is known about its mechanisms of action. Here we examined the cytotoxic effect of asiatic acid on HepG2 cells and elucidated some of the underlying mechanisms. Asiatic acid induced rapid cell death, as well as mitochondrial membrane potential (MMP) dissipation, ATP depletion and cytochrome c release from mitochondria to the cytosol in HepG2 cells. In mitochondria isolated from mouse liver, asiatic acid treatment significantly stimulated the succinate-supported state 4 respiration rate, dissipated the MMP, increased Ca(2+) release from Ca(2+)-loaded mitochondria, decreased ATP content and promoted cytochrome c release, indicating the uncoupling effect of asiatic acid. Hydrogen peroxide (H2O2) produced by succinate-supported mitochondrial respiration was also significantly inhibited by asiatic acid. In addition, asiatic acid inhibited Ca(2+)-induced mitochondrial swelling but did not induce mitochondrial swelling in hyposmotic potassium acetate medium which suggested that asiatic acid may not act as a protonophoric uncoupler. Inhibition of uncoupling proteins (UCPs) or blockade of adenine nucleotide transporter (ANT) attenuated the effect of asiatic acid on MMP dissipation, Ca(2+) release, mitochondrial respiration and HepG2 cell death. When combined inhibition of UCPs and ANT, asiatic acid-mediated uncoupling effect was noticeably alleviated. These results suggested that both UCPs and ANT partially contribute to the uncoupling properties of asiatic acid. In conclusion, asiatic acid is a novel mitochondrial uncoupler and this property is potentially involved in its toxicity on HepG2 cells.

  19. Antiinflammatory and Antiphotodamaging Effects of Ergostatrien-3β-ol, Isolated from Antrodia camphorata, on Hairless Mouse Skin.

    PubMed

    Kuo, Yueh-Hsiung; Lin, Tzu-Yu; You, Ya-Jhen; Wen, Kuo-Ching; Sung, Ping-Jyun; Chiang, Hsiu-Mei

    2016-01-01

    Ergostatrien-3β-ol (EK100), isolated from the submerged whole broth of Antrodia camphorata, has antidiabetic, hyperlipidemic, and hepatoprotective activities. However, the antiphotodamage activity of EK100 has still not been revealed. Inflammation and collagen degradation contribute to skin photodamage and premature aging. In the present study, in vivo experiments were designed to investigate the antiinflammatory and antiphotodamaging activities of EK100 in hairless mice by physiological and histological analysis of the skin. Results indicated that topical application of EK100 (25 and 100 μM) for 10 weeks efficiently inhibited ultraviolet B (UVB)-induced wrinkle formation, erythema, and epidermal thickness in the mice skin. EK100 also restored UVB-induced collagen content reduction in hairless mice skin. In addition, the immunohistochemistry results indicated that EK100 significantly inhibited the UVB-induced expression of matrix metalloproteinase-1 (MMP-1), interleukin-6 (IL-6), inducible nitric oxide synthase (iNOS), and nuclear factor kappaB (NF-κB) in the mouse skin. The expression of these proteins was similar to the Normal group after 100 μM EK100 treatment. EK100 inhibited collagen degradation in the skin through MMP-1 inhibition and antiinflammation. EK100 significantly reduced the transepidermal water loss (TEWL), indicating that EK100 protected skin from UVB-induced damage. Our findings strongly suggest that EK100 has significant beneficial antiinflammatory and antiphotoaging activities and that EK100 can be developed as an antiphotodamaging agent. PMID:27626393

  20. An Optimized Method for Isolating and Expanding Invariant Natural Killer T Cells from Mouse Spleen.

    PubMed

    Govindarajan, Srinath; Elewaut, Dirk; Drennan, Michael

    2015-01-01

    The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells are therefore characterized as an innate T cell population capable of activating and steering adaptive immune responses. The development of improved techniques for the culture and expansion of murine iNKT cells facilitates the study of iNKT cell biology in in vitro and in vivo model systems. Here we describe an optimized procedure for the isolation and expansion of murine splenic iNKT cells. Spleens from C57Bl/6 mice are removed, dissected and strained and the resulting cellular suspension is layered over density gradient media. Following centrifugation, splenic mononuclear cells (MNCs) are collected and CD5-positive (CD5(+)) lymphocytes are enriched for using magnetic beads. iNKT cells within the CD5(+) fraction are subsequently stained with αGalCer-loaded CD1d tetramer and purified by fluorescence activated cell sorting (FACS). FACS sorted iNKT cells are then initially cultured in vitro using a combination of recombinant murine cytokines and plate-bound T cell receptor (TCR) stimuli before being expanded in the presence of murine recombinant IL-7. Using this technique, approximately 10(8) iNKT cells can be generated within 18-20 days of culture, after which they can be used for functional assays in vitro, or for in vivo transfer experiments in mice. PMID:26555769

  1. Isolation of homozygous mutant mouse embryonic stem cells using a dual selection system

    PubMed Central

    Huang, Yue; Pettitt, Stephen J.; Guo, Ge; Liu, Guang; Li, Meng Amy; Yang, Fengtang; Bradley, Allan

    2012-01-01

    Obtaining random homozygous mutants in mammalian cells for forward genetic studies has always been problematic due to the diploid genome. With one mutation per cell, only one allele of an autosomal gene can be disrupted, and the resulting heterozygous mutant is unlikely to display a phenotype. In cells with a genetic background deficient for the Bloom's syndrome helicase, such heterozygous mutants segregate homozygous daughter cells at a low frequency due to an elevated rate of crossover following mitotic recombination between homologous chromosomes. We constructed DNA vectors that are selectable based on their copy number and used these to isolate these rare homozygous mutant cells independent of their phenotype. We use the piggyBac transposon to limit the initial mutagenesis to one copy per cell, and select for cells that have increased the transposon copy number to two or more. This yields homozygous mutants with two allelic mutations, but also cells that have duplicated the mutant chromosome and become aneuploid during culture. On average, 26% of the copy number gain events occur by the mitotic recombination pathway. We obtained homozygous cells from 40% of the heterozygous mutants tested. This method can provide homozygous mammalian loss-of-function mutants for forward genetic applications. PMID:22127858

  2. In vivo imaging of pancreatic endocrine islets

    NASA Astrophysics Data System (ADS)

    Villiger, Martin; Goulley, Joan; Pache, Christophe; Friedrich, Michael; Grapin-Botton, Anne; Meda, Paolo; Leitgeb, Rainer; Lasser, Theo

    2009-07-01

    Extended focus optical coherence microscope (xfOCM) circumvents the compromise between lateral resolution and depth of field by us of a Bessel-like illumination beam. The high sensitivity and parallel depth profiling of Fourier domain optical coherence tomography are preserved, and combined with high isotropic resolution of 1.5 - 2 μm. To comply with the requirements for in vivo measurements, beam scanning had to be implemented. We then performed measurements, first of excised pancreas, validated by standard immunohistochemistry, to investigate the structures that can be observed. For a quantitative analysis, a semi-automatic islet detection algorithm evaluated the islet size, position, contrast and homogeneity. The influence of streptozotocin on the signature of the islets was investigated in a next step. Finally, xfOCM was applied to make measurements of the murine pancreas in situ and in vivo, visualizing pancreatic lobules, ducts, blood vessels and individual islets of Langerhans.

  3. A new atypical genotype mouse virulent strain of Toxoplasma gondii isolated from the heart of a wild caught puma (Felis concolor) from Durango, Mexico.

    PubMed

    Dubey, J P; Alvarado-Esquivel, C; Herrera-Valenzuela, V H; Ortiz-Diaz, J J; Oliveira, S; Verma, S K; Choudhary, S; Kwok, O C H; Su, C

    2013-11-01

    Nothing is known of the genetic diversity of Toxoplasma gondii circulating in wildlife in Mexico. In the present study, a mouse virulent T. gondii strain was isolated from the heart of a wild puma (Felis concolor). The puma was found roaming in outskirt of Durango City, Mexico and tranquilized for moving to a zoo. The puma died during translocation and a necropsy examination was performed. The puma had an antibody titer for T. gondii of 200 by the modified agglutination test. Its heart and brain tissue were bioassayed into 2 outbred Swiss Webster (SW) and 1 gamma interferon gene knockout (KO) mouse. The KO mouse and the 2 SW mice that became infected after inoculation with homogenate of puma heart died of acute toxoplasmosis 12, 19 and 20 days p.i. respectively and tachyzoites were found in lungs of all 3 mice. None of the 4 SW and 1 KO mouse inoculated with digest of the puma brain became infected with T. gondii. Tachyzoites from the lungs of mice were propagated in cell cultures. Tachyzoites from cell culture were inoculated into 5 SW; the mice died or had to be killed 14 days p.i. and a cat fed tissues of these mice shed T. gondii oocysts. Results of mortality and infectivity of tachyzoites and oocysts in SW mice indicated that the puma T. gondii strain (designated TgPumaMe1) was virulent for outbred mice. DNA isolated from culture-derived tachyzoites was characterized using 11 PCR-RFLP markers (SAG1, 5'- and 3'-SAG2, alt.SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed a new genotype (ToxoDB PCR-RFLP #222). Isolation of atypical genotype T. gondii from wild puma indicates that mouse virulent strains are circulating in wildlife in Mexico.

  4. The pancreatic islet as a signaling hub.

    PubMed

    Barker, Christopher J; Leibiger, Ingo B; Berggren, Per-Olof

    2013-01-01

    Over the last two decades we have focused on beta cell signal transduction, bringing many new insights, especially in the context of insulin signal transduction, the role of inositol polyphosphates and the regulation of cytoplasmic free Ca(2+) concentration. However, there has been a growing awareness that the beta cell, which is mandatory for insulin secretion, has a unique context within the micro-organ of the pancreatic Islet of Langerhans. In this environment the beta cell both mediates and receives paracrine regulation, critical for the control of blood glucose homeostasis. Failure of an appropriate beta cell function leads to the development of diabetes mellitus. In our quest to understand the molecular events maintaining beta cell function we have faced two key challenges. Firstly, whilst there are many similarities between signal transduction in pancreatic islets between the much used rodent models and humans there are some notable differences. Critical distinctions between rodent and primate can be made in the structure of the islet, including the arrangement of the islet cells, the innervation pattern and the microcirculation. This means that important signaling interactions between islets cells, mediated through for example insulin, glucagon, GABA, glutamate and ATP, will have a unique human framework. The second challenge was to be able to take the discoveries we have made using in vitro systems and examine them in an in vivo context. Advances in in vivo imaging achieved by utilizing the anterior chamber of the eye as a transplantation site for pancreatic islets make it possible for non-invasive, longitudinal studies at single cell resolution in real time of islet cell physiology and pathology. Thus it is becoming possible to study the insulin secreting pancreatic beta cell within the framework of the unique micro-organ, the Islet of Langerhans, for the first time in a physiological context, i.e. when being innervated and connected to the blood supply.

  5. Spontaneous pancreatic islet amyloidosis in 40 baboons.

    PubMed

    Hubbard, G B; Steele, K E; Davis, K J; Leland, M M

    2002-04-01

    Spontaneous amyloidosis occurs in many nonhuman primate species but remains difficult to diagnose and treat. Nonhuman primates continue to offer promise as animal models in which to study amyloidosis in humans. Amyloidosis was not diagnosed clinically but was found histologically in four male and 36 female baboons. The baboons averaged 18 years of age at death (range, 7-28 years). Clinical signs, if present, were hyperglycemia and cachexia. Blood glucose values were elevated in 12 of 30 baboons with available clinical pathology data. Four baboons had been clinically diagnosed as diabetic and three were treated with insulin. Amyloid was found in the islets of Langerhans of the pancreas in 40 baboons; 35 baboons had amyloid only in the islets of Langerhans. Amyloid was found in nonislet tissue of baboons as follows: five, nonislet pancreas; four, intestine and adrenal; three, kidney; two, prostate and spleen; and one each, lymph node, liver, gall bladder, stomach, tongue, urinary bladder, and salivary gland. Sections of paraffin-embedded tissues were evaluated for amyloid with hematoxylin and eosin (HE) and congo red (CR) staining, and using immunohistochemistry for human islet amyloid polypeptide (IAPP), calcitonin gene-related peptide (CGRP), glucagon, pancreatic polypeptide (PP), somatostatin (SS), and porcine insulin. Islet amyloid was positive with HE in 40 baboons, with CR in 39 baboons, and with IAPP and CGRP in 35 baboons. IAPP and CGRP only stained islet amyloid. PP, SS, glucagon, and porcine insulin did not stain amyloid. Islet amyloidosis in the baboon appears to be difficult to diagnose clinically, age-related, and similar to islet amyloidosis in other species. The baboon may be a good model for the study of islet amyloidosis in humans.

  6. A Method to Identify and Isolate Pluripotent Human Stem Cells and Mouse Epiblast Stem Cells Using Lipid Body-Associated Retinyl Ester Fluorescence

    PubMed Central

    Muthusamy, Thangaselvam; Mukherjee, Odity; Menon, Radhika; Megha, P.B.; Panicker, Mitradas M.

    2014-01-01

    Summary We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450–500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies. PMID:25068130

  7. A method to identify and isolate pluripotent human stem cells and mouse epiblast stem cells using lipid body-associated retinyl ester fluorescence.

    PubMed

    Muthusamy, Thangaselvam; Mukherjee, Odity; Menon, Radhika; Megha, P B; Panicker, Mitradas M

    2014-07-01

    We describe the use of a characteristic blue fluorescence to identify and isolate pluripotent human embryonic stem cells and human-induced pluripotent stem cells. The blue fluorescence emission (450-500 nm) is readily observed by fluorescence microscopy and correlates with the expression of pluripotency markers (OCT4, SOX2, and NANOG). It allows easy identification and isolation of undifferentiated human pluripotent stem cells, high-throughput fluorescence sorting and subsequent propagation. The fluorescence appears early during somatic reprogramming. We show that the blue fluorescence arises from the sequestration of retinyl esters in cytoplasmic lipid bodies. The retinoid-sequestering lipid bodies are specific to human and mouse pluripotent stem cells of the primed or epiblast-like state and absent in naive mouse embryonic stem cells. Retinol, present in widely used stem cell culture media, is sequestered as retinyl ester specifically by primed pluripotent cells and also can induce the formation of these lipid bodies.

  8. HLA Class I Sensitization in Islet Transplant Recipients – Report from the Collaborative Islet Transplant Registry

    PubMed Central

    Naziruddin, Bashoo; Wease, Steve; Stablein, Donald; Barton, Franca B.; Berney, Thierry; Rickels, Michael R.; Alejandro, Rodolfo

    2015-01-01

    Pancreatic islet transplantation is a promising treatment option for patients severely affected with type 1 diabetes. This report from CITR presents pre- and post-transplant human leukocyte antigen (HLA) class I sensitization rates in islet alone transplantation. Data came from 303 recipients transplanted with islet alone between January 1999 and December 2008. HLA class I sensitization was determined by the presence of anti-HLA class I antibodies. Panel-reactive antibodies (PRA) from prior to islet infusion and at 6 months, and yearly post-transplant was correlated to measures of islet graft failure. The cumulative number of mismatched HLA alleles increased with each additional islet infusion from a median of 3 for one infusion to 9 for three infusions. Pre-transplant PRA was not predictive of islet graft failure. However, development of PRA ≥20% post-transplant was associated with 3.6 fold (p=.001) increased hazard ratio for graft failure. Patients with complete graft loss who had discontinued immunosuppression had significantly higher rate of PRA ≥ 20% compared to those with functioning grafts who remained on immunosuppression. Exposure to repeat HLA class I mismatch at second or third islet infusions resulted in less frequent development of de novo HLA class I antibodies when compared to increased class I mismatch. The development of HLA class I antibodies while on immunosuppression is associated with subsequent islet graft failure. The risk of sensitization may be reduced by minimizing the number of islet donors used per recipient, and in the absence of donor-specific anti-HLA antibodies, repeating HLA class I mismatches with subsequent islet infusions. PMID:22080832

  9. Justifying clinical trials for porcine islet xenotransplantation.

    PubMed

    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.

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

    PubMed Central

    Nair, Gopika; Hebrok, Matthias

    2015-01-01

    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. PMID:25909383

  11. Immunodeficient Mouse Models with Different Disease Profiles by In Vivo Infection with the Same Clinical Isolate of Enterovirus 71

    PubMed Central

    Liao, Chun-Che; Liou, An-Ting; Chang, Ya-Shu; Wu, Szu-Yao; Chang, Chih-Shin; Lee, Chien-Kuo; Kung, John T.; Tu, Pang-Hsien; Yu, Ya-Yen; Lin, Chi-Yung; Lin, Jen-Shiou

    2014-01-01

    ABSTRACT Like poliovirus infection, severe infection with enterovirus 71 (EV71) can cause neuropathology. Unlike poliovirus, EV71 is often associated with hand-foot-and-mouth disease (HFMD). Here we established three mouse models for experimental infection with the same clinical isolate of EV71. The NOD/SCID mouse model is unique for the development of skin rash, an HFMD-like symptom. While the NOD/SCID mice developed limb paralysis and death at near-100% efficiency, the gamma interferon receptor knockout (ifngr KO) and stat-1 knockout mice exhibited paralysis and death rates near 78% and 30%, respectively. Productive infection with EV71 depends on the viral dose, host age, and inoculation route. Levels of infectious EV71, and levels of VP1-specific RNA and protein in muscle, brain, and spinal cord, were compared side by side between the NOD/SCID and stat-1 knockout models before, during, and after disease onset. Spleen fibrosis and muscle degeneration are common in the NOD/SCID and stat-1 knockout models. The main differences between these two models include their disease manifestations and cytokine/chemokine profiles. The pathology of the NOD/SCID model includes (i) inflammation and expression of viral VP1 antigen in muscle, (ii) increased neutrophil levels and decreased eosinophil and lymphocyte levels, and (iii) hair loss and skin rash. The characteristic pathology of the stat-1 knockout model includes (i) a strong tropism of EV71 for the central nervous system, (ii) detection of VP1 protein in the Purkinje layer of cerebellar cortex, pons, brain stem, and spinal cord, (iii) amplification of microglial cells, and (iv) dystrophy of intestinal villi. Our comparative studies on these new models with oral or intraperitoneal (i.p.) infection underscored the contribution of host immunity, including the gamma interferon receptor, to EV71 pathogenesis. IMPORTANCE In the past decade, enterovirus 71 (EV71) has emerged as a major threat to public health in the Asia

  12. Inhibition of Insulin-Degrading Enzyme Does Not Increase Islet Amyloid Deposition in Vitro.

    PubMed

    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. PMID:27404391

  13. Inhibition of Insulin-Degrading Enzyme Does Not Increase Islet Amyloid Deposition in Vitro.

    PubMed

    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.

  14. Spontaneous diabetes mellitus in transgenic mice expressing human islet amyloid polypeptide.

    PubMed Central

    Janson, J; Soeller, W C; Roche, P C; Nelson, R T; Torchia, A J; Kreutter, D K; Butler, P C

    1996-01-01

    The islet in non-insulin-dependent diabetes mellitus (NIDDM) is characterized by loss of beta cells and large local deposits of amyloid derived from the 37-amino acid protein, islet amyloid polypeptide (IAPP). We have hypothesized that IAPP amyloid forms intracellularly causing beta-cell destruction under conditions of high rates of expression. To test this we developed a homozygous transgenic mouse model with high rates of expression of human IAPP. Male transgenic mice spontaneously developed diabetes mellitus by 8 weeks of age, which was associated with selective beta-cell death and impaired insulin secretion. Small intra- and extracellular amorphous IAPP aggregates were present in islets of transgenic mice during the development of diabetes mellitus. However, IAPP derived amyloid deposits were found in only a minority of islets at approximately 20 weeks of age, notably after development of diabetes mellitus in male transgenic mice. Approximately 20% of female transgenic mice spontaneously developed diabetes mellitus at 30+ weeks of age, when beta-cell degeneration and both amorphous and amyloid deposits of IAPP were present. We conclude that overexpression of human IAPP causes beta-cell death, impaired insulin secretion, and diabetes mellitus. Large deposits of IAPP derived amyloid do not appear to be important in this cytotoxicity, but early, small amorphous intra- and extracellular aggregates of human IAPP were consistently present at the time of beta-cell death and therefore may be the most cytotoxic form of IAPP. Images Fig. 1 Fig. 3 Fig. 4 Fig. 5 PMID:8692984

  15. Virulence of patient and water isolates of Legionella pneumophila in guinea pigs and mouse L929 cells varies with bacterial genotype.

    PubMed

    Bezanson, G; Fernandez, R; Haldane, D; Burbridge, S; Marrie, T

    1994-06-01

    Thirteen isolates of Legionella pneumophila serogroup 1 (five from patients, eight from water) were screened for their virulence in guinea pigs after intraperitoneal injection and for their infectivity in L929 mouse cells. Since these isolates included three monoclonal antibody subtypes and four genotypes, the relative influence of these parameters on the pathogenicity of naturally occurring L. pneumophila could be assessed. There was no correlation between infectivity in the L929 assay and virulence for guinea pigs. The source of the isolate, patient or environmental, as well as the isolate's monoclonal antibody subtype did not correlate with virulence. At the p < 0.05 level, isolates with genotype IIb (20-MDa plasmid and EcoRI fragmentation pattern b) were significantly more virulent (mean log LD50 6.84) than genotype VIb (100-MDa plasmid, pattern b), IIId (72- and 96-MDa plasmids, pattern d) or Oc (no detectable plasmid, pattern c) isolates. Genotype IIId isolates were the least virulent (mean log LD50 9.49). Plasmid-containing isolates were more infective than plasmidless ones in L929 cells (p = 0.0001). We conclude that our strain types of L. pneumophila exhibit a gradation in virulence for guinea pigs and that infectivity in L929 cells does not correlate with virulence for guinea pigs. PMID:8050062

  16. Insulin release and insulin mRNA levels in rat islets of Langerhans cultured on extracellular matrix.

    PubMed

    Perfetti, R; Henderson, T E; Wang, Y; Montrose-Rafizadeh, C; Egan, J M

    1996-07-01

    Primary culture of rat islets of Langerhans lose glucose responsiveness and eventually die when cultured for a long period of time. In this study we evaluated the effect of matrigel, a basement membrane extract, on (i) islet cell survival, (ii) cell responsiveness following a glucose challenge, and (iii) mRNA levels for insulin, glucagon, and somatostatin. Pancreatic islets were isolated by collagenase digestion and plated in culture dishes either coated or not with a matrigel layer. Using the reverse hemolytic plaque assay, we determined the total number of insulin-secreting cells and the amount of insulin secreted by individual beta cells. After 1 h of exposure to 5 mM glucose, beta cells from 6-month-old rat islets cultured for 6 weeks on matrigel showed an equal number of insulin-secreting cells compared to freshly isolated islets cultured for only 3 days in the absence of matrigel (39.5 +/- 2.5 vs. 37.1 +/- 2.6%). Furthermore, the release of insulin by cells cultured on matrigel for 6 weeks increased in a glucose-dependent manner (p < 0.001) and showed an ED50 of 7 mM. However, the amount of insulin released per single beta cell was reduced by 40-60% (p < 0.02) compared to that released from isolated beta cells derived from a 3-day culture of islets. Finally, there was a 35-55% increase (p < 0.05) in the levels of insulin, glucagon, and somatostatin mRNAs in cells cultured for 6 weeks on matrigel. These data suggest a trophic effect of matrigel on the maintenance of normal beta-cell activity and function and may lead the way to the development of a new model for the study of pancreatic islets in long-term culture.

  17. A brain-specific gene cluster isolated from the region of the mouse obesity locus is expressed in the adult hypothalamus and during mouse development

    SciTech Connect

    Laig-Webster, M.; Lim, M.E.; Chehab, F.F.

    1994-09-01

    The molecular defect underlying an autosomal recessive form of genetic obesity in a classical mouse model C57 BL/6J-ob/ob has not yet been elucidated. Whereas metabolic and physiological disturbances such as diabetes and hypertension are associated with obesity, the site of expression and the nature of the primary lesion responsible for this cascade of events remains elusive. Our efforts aimed at the positional cloning of the ob gene by YAC contig mapping and gene identification have resulted in the cloning of a brain-specific gene cluster from the ob critical region. The expression of this gene cluster is remarkably complex owing to the multitude of brain-specific mRNA transcripts detected on Northern blots. cDNA cloning of these transcripts suggests that they are expressed from different genes as well as by alternate splicing mechanisms. Furthermore, the genomic organization of the cluster appears to consist of at least two identical promoters displaying CpG islands characteristic of housekeeping genes, yet clearly involving tissue-specific expression. Sense and anti-sense synthetic RNA probes were derived from a common DNA sequence on 3 cDNA clones and hybridized to 8-16 days mouse embryonic stages and mouse adult brain sections. Expression in development was noticeable as of the 11th day of gestation and confined to the central nervous system mainly in the telencephalon and spinal cord. Coronal and sagittal sections of the adult mouse brain showed expression only in 3 different regions of the brain stem. In situ hybridization to mouse hypothalamus sections revealed the presence of a localized and specialized group of cells expressing high levels of mRNA, suggesting that this gene cluster may also be involved in the regulation of hypothalamic activities. The hypothalamus has long been hypothesized as a primary candidate tissue for the expression of the obesity gene mainly because of its well-established role in the regulation of energy metabolism and food intake.

  18. /sup 3/H-cyclosporine internalization and secretion by human fetal pancreatic islets

    SciTech Connect

    Formby, B.; Walker, L.; Peterson, C.M.

    1988-10-01

    Human fetal pancreatic islets were isolated from 16- to 20-week-old fetuses by a collagenase technique and cultured 48 hr in RPMI 1640 containing 10% human adult serum and unlabeled 0 to 5 micrograms cyclosporine A (CsA)/ml. Insulin secretory capacity of human fetal islets was expressed as a fractional stimulatory ratio FSR = F2/F1 of the fractional secretion rates during two successive 1 hr static incubations first with 2 mM glucose (F1) to stabilize secretion followed by maximal stimulus, i.e., 25 mM glucose plus 10 mM L-leucine and 10 mM L-arginine (F2). Unlabeled CsA at the above concentrations had no significant effects on the insulin secretory capacity expressed by FSR-values. Studies of net uptake of 3H-CsA by islets cultured for varying periods up to 40 hr and expressed as picomole 3H-CsA per picomole islet insulin content demonstrated that uptake rate was slow and did not reach isotopic equilibrium over the 40 hr of culture. When isolated fetal islets were cultured for 48 hr in the presence of 3H-CsA and varying concentrations of unlabeled CsA it was found during two successive 1 hr static incubations that fetal islets secrete insulin concomitantly with 3H-CsA following maximal stimulus for secretion. An optimal secretory molar ratio of 3H-CsA to insulin of 4.0 +/- 1.3 (n = 7) was found after islets were cultured 48 hr in the presence of a saturating 2.128 micrograms 3H-CsA per milliliter culture medium. In three successive 30-min static incubations of 3H-CsA loaded islets, first with low glucose, followed by high glucose plus L-arginine and L-leucine, and finally with high glucose plus L-arginine and L-leucine and 10 mM theophylline, the proportional fractional secretion rates of insulin and 3H-CsA were of the same magnitude.

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

    PubMed

    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. PMID:24945458

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

    PubMed

    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.

  1. Low protein diet confers resistance to the inhibitory effects of interleukin 1beta on insulin secretion in pancreatic islets*

    PubMed

    Vieira, E C.; Carneiro, E M.; Latorraca, M Q.; Delguingaro-Augusto, V; Amaral, M E.C.; Bosqueiro, J R.; Boschero, A C.

    2001-05-01

    High protein content in the diet during childhood and adolescence has been associated to the onset insulin-dependent diabetes mellitus. We investigated the effect of interleukin-1beta (IL-1beta) on insulin secretion, glucose metabolism, and nitrite formation by islets isolated from rats fed with normal protein (NP, 17%) or low protein (LP, 6%) after weaning. Pretreatment of islets with IL-1beta for 1 h or 24 h inhibited the insulin secretion induced by glucose in both groups, but it was less marked in LP than in NP group. Islets from LP rats exhibited a decreased IL-1beta-induced nitric oxide (NO) production, lower inhibition of D-[U(14)C]-glucose oxidation to (14)CO(2) and less pronounced effect of IL-1beta on alpha-ketoisocaproic acid-induced insulin secretion than NP islets. However, when the islets were stimulated by high concentrations of K(+) the inhibitory effect of IL-1beta on insulin secretion was not different between groups. In conclusion, protein restriction protects beta-cells of the deleterious effect of IL-1beta, apparently, by decreasing NO production. The lower NO generation in islets from protein deprived rats may be due to increased free fatty acids oxidation and consequent alteration in Ca(2+) homeostasis. PMID:11382546

  2. Islet Oxygen Consumption Rate (OCR) Dose Predicts Insulin Independence in Clinical Islet Autotransplantation

    PubMed Central

    Papas, Klearchos K.; Bellin, Melena D.; Sutherland, David E. R.; Suszynski, Thomas M.; Kitzmann, Jennifer P.; Avgoustiniatos, Efstathios S.; Gruessner, Angelika C.; Mueller, Kathryn R.; Beilman, Gregory J.; Balamurugan, Appakalai N.; Loganathan, Gopalakrishnan; Colton, Clark K.; Koulmanda, Maria; Weir, Gordon C.; Wilhelm, Josh J.; Qian, Dajun; Niland, Joyce C.; Hering, Bernhard J.

    2015-01-01

    Background 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. Methods 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. Results 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). Conclusions 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. PMID:26258815

  3. Ca(2+) handling in isolated brain mitochondria and cultured neurons derived from the YAC128 mouse model of Huntington's disease.

    PubMed

    Pellman, Jessica J; Hamilton, James; Brustovetsky, Tatiana; Brustovetsky, Nickolay

    2015-08-01

    We investigated Ca(2+) handling in isolated brain synaptic and non-synaptic mitochondria and in cultured striatal neurons from the YAC128 mouse model of Huntington's disease. Both synaptic and non-synaptic mitochondria from 2- and 12-month-old YAC128 mice had larger Ca(2+) uptake capacity than mitochondria from YAC18 and wild-type FVB/NJ mice. Synaptic mitochondria from 12-month-old YAC128 mice had further augmented Ca(2+) capacity compared with mitochondria from 2-month-old YAC128 mice and age-matched YAC18 and FVB/NJ mice. This increase in Ca(2+) uptake capacity correlated with an increase in the amount of mutant huntingtin protein (mHtt) associated with mitochondria from 12-month-old YAC128 mice. We speculate that this may happen because of mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage. In experiments with striatal neurons from YAC128 and FVB/NJ mice, brief exposure to 25 or 100 μM glutamate produced transient elevations in cytosolic Ca(2+) followed by recovery to near resting levels. Following recovery of cytosolic Ca(2+), mitochondrial depolarization with FCCP produced comparable elevations in cytosolic Ca(2+), suggesting similar Ca(2+) release and, consequently, Ca(2+) loads in neuronal mitochondria from YAC128 and FVB/NJ mice. Together, our data argue against a detrimental effect of mHtt on Ca(2+) handling in brain mitochondria of YAC128 mice. We demonstrate that mutant huntingtin (mHtt) binds to brain synaptic and nonsynaptic mitochondria and the amount of mitochondria-bound mHtt correlates with increased mitochondrial Ca(2+) uptake capacity. We propose that this may happen due to mHtt-mediated sequestration of free fatty acids thereby increasing resistance of mitochondria to Ca(2+)-induced damage.

  4. Expression, purification, crystallization and preliminary crystallographic study of isolated modules of the mouse coactivator-associated arginine methyltransferase 1

    SciTech Connect

    Troffer-Charlier, Nathalie; Cura, Vincent; Hassenboehler, Pierre; Moras, Dino; Cavarelli, Jean

    2007-04-01

    Isolated modules of mouse coactivator-associated arginine methyltransferase 1 encompassing the protein arginine N-methyltransferase catalytic domain have been overexpressed, purified and crystallized. X-ray diffraction data have been collected and have enabled determination of the structures by multiple isomorphous replacement using anomalous scattering. Coactivator-associated arginine methyltransferase 1 (CARM1) plays a crucial role in gene expression as a coactivator of several nuclear hormone receptors and also of non-nuclear receptor systems. Its recruitment by the transcriptional machinery induces protein methylation, leading to chromatin remodelling and gene activation. CARM1{sub 28–507} and two structural states of CARM1{sub 140–480} were expressed, purified and crystallized. Crystals of CARM1{sub 28–507} belong to space group P6{sub 2}22, with unit-cell parameters a = b = 136.0, c = 125.3 Å; they diffract to beyond 2.5 Å resolution using synchrotron radiation and contain one monomer in the asymmetric unit. The structure of CARM1{sub 28–507} was solved by multiple isomorphous replacement and anomalous scattering methods. Crystals of apo CARM1{sub 140–480} belong to space group I222, with unit-cell parameters a = 74.6, b = 99.0, c = 207.4 Å; they diffract to beyond 2.7 Å resolution and contain two monomers in the asymmetric unit. Crystals of CARM1{sub 140–480} in complex with S-adenosyl-l-homocysteine belong to space P2{sub 1}2{sub 1}2, with unit-cell parameters a = 74.6, b = 98.65, c = 206.08 Å; they diffract to beyond 2.6 Å resolution and contain four monomers in the asymmetric unit. The structures of apo and holo CARM1{sub 140–480} were solved by molecular-replacement techniques from the structure of CARM1{sub 28–507}.

  5. Prediction of Liver Injury Induced by Chemicals in Human With a Multiparametric Assay on Isolated Mouse Liver Mitochondria

    PubMed Central

    Porceddu, Mathieu; Buron, Nelly; Borgne-Sanchez, Annie

    2012-01-01

    Drug-induced liver injury (DILI) in humans is difficult to predict using classical in vitro cytotoxicity screening and regulatory animal studies. This explains why numerous compounds are stopped during clinical trials or withdrawn from the market due to hepatotoxicity. Thus, it is important to improve early prediction of DILI in human. In this study, we hypothesized that this goal could be achieved by investigating drug-induced mitochondrial dysfunction as this toxic effect is a major mechanism of DILI. To this end, we developed a high-throughput screening platform using isolated mouse liver mitochondria. Our broad spectrum multiparametric assay was designed to detect the global mitochondrial membrane permeabilization (swelling), inner membrane permeabilization (transmembrane potential), outer membrane permeabilization (cytochrome c release), and alteration of mitochondrial respiration driven by succinate or malate/glutamate. A pool of 124 chemicals (mainly drugs) was selected, including 87 with documented DILI and 37 without reported clinical hepatotoxicity. Our screening assay revealed an excellent sensitivity for clinical outcome of DILI (94 or 92% depending on cutoff) and a high positive predictive value (89 or 82%). A highly significant relationship between drug-induced mitochondrial toxicity and DILI occurrence in patients was calculated (p < 0.001). Moreover, this multiparametric assay allowed identifying several compounds for which mitochondrial toxicity had never been described before and even helped to clarify mechanisms with some drugs already known to be mitochondriotoxic. Investigation of drug-induced loss of mitochondrial integrity and function with this multiparametric assay should be considered for integration into basic screening processes at early stage to select drug candidates with lower risk of DILI in human. This assay is also a valuable tool for assessing the mitochondrial toxicity profile and investigating the mechanism of action of new

  6. Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts.

    PubMed

    Farthing, Don; Gehr, Lynne; Karnes, H Thomas; Sica, Domenic; Gehr, Todd; Larus, Terri; Farthing, Christine; Xi, Lei

    2007-01-01

    Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5-2.5 mM), anti-inflammatory (0.5-5.0 mM) or antiplatelet (0.18-0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0-10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575+/-3319 vs. 1437+/-348 ng ml(-1) min(-1), mean+/-SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo

  7. Effects of salicylic acid on post-ischaemic ventricular function and purine efflux in isolated mouse hearts.

    PubMed

    Farthing, Don; Gehr, Lynne; Karnes, H Thomas; Sica, Domenic; Gehr, Todd; Larus, Terri; Farthing, Christine; Xi, Lei

    2007-01-01

    Acetyl salicylic acid (aspirin) is one of the most widely used drugs in the world. Various plasma concentrations of aspirin and its predominant metabolite, salicylic acid, are required for its antiarthritic (1.5-2.5 mM), anti-inflammatory (0.5-5.0 mM) or antiplatelet (0.18-0.36 mM) actions. A recent study demonstrated the inhibitory effects of both aspirin and salicylic acid on oxidative phosphorylation and ATP synthesis in isolated rat cardiac mitochondria in a dose-dependent manner (0-10 mM concentration range). In this context, the present study was conducted to determine the effects of salicylic acid on inosine efflux (a potential biomarker of acute cardiac ischaemia) as well as cardiac contractile function in the isolated mouse heart following 20 min of zero-flow global ischaemia. Inosine efflux was found at significantly higher concentrations in ischaemic hearts perfused with Krebs buffer fortified with 1.0 mM salicylic acid compared with those without salicylic acid (12575+/-3319 vs. 1437+/-348 ng ml(-1) min(-1), mean+/-SEM, n=6 per group, p<0.01). These results indicate that 1.0 mM salicylic acid potentiates 8.8-fold ATP nucleotide purine catabolism into its metabolites (e.g. inosine, hypoxanthine). Salicylic acid (0.1 or 1.0 mM) did not appreciably inhibit purine nucleoside phosphorylase (the enzyme converts inosine to hypoxanthine) suggesting the augmented inosine efflux was due to the salicylic acid effect on upstream elements of cellular respiration. Whereas post-ischaemic cardiac function was further depressed by 1.0 mM salicylic acid, perfusion with 0.1 mM salicylic acid led to a remarkable functional improvement despite moderately increased inosine efflux (2.7-fold). We conclude that inosine is a sensitive biomarker for detecting cardiac ischaemia and salicylic acid-induced effects on cellular respiration. However, the inosine efflux level appears to be a poor predictor of the individual post-ischaemic cardiac functional recovery in this ex vivo

  8. PDX-1 mRNA-induced reprogramming of mouse pancreas-derived mesenchymal stem cells into insulin-producing cells in vitro.

    PubMed

    Guo, Xing Rong; Wang, Xiao Li; Li, Man Chol; Yuan, Ya Hong; Chen, Yun; Zou, Dan Dan; Bian, Liu Jiao; Li, Dong Sheng

    2015-11-01

    Pancreatic islet transplantation has remained an effective therapy for type 1 diabetes since 2000. Its widespread use has been prohibited by the shortage of suitable donors. It is critical to explore an applicable alternative for β-cell replacement. This study was performed to generate insulin-producing cells (IPCs) from pancreas-derived mesenchymal stem cells (pMSCs). pMSCs were isolated from discarded pancreatic tissue in the filter liquor during islet isolation procedure in mice and ex vivo expanded in culture. IPCs were induced by transfection of pancreas and duodenal transcription factor 1 (PDX-1) mRNA in vitro. Some islet characteristics were identified on pMSC-derived IPCs in mRNA and protein levels. Our results demonstrated that mouse pMSCs can be transdifferentiated into effective glucose-responsive insulin-producing cells through transfecting synthetic modified PDX-1 mRNA in vitro. The study of PDX-1 mRNA-induced pMSC reprogramming may pave the way toward the development of a novel β-cell source for the treatment of diabetes.

  9. Brevinema andersonii gen. nov., sp. nov., an infectious spirochete isolated from the short-tailed shrew (Blarina brevicauda) and the white-footed mouse (Peromyscus leucopus).

    PubMed

    Defosse, D L; Johnson, R C; Paster, B J; Dewhirst, F E; Fraser, G J

    1995-01-01

    A spirochete which infects short-tailed shrews (Blarina brevicauda) and white-footed mice (Peromyscus leucopus) has been shown previously to be ultrastructurally and serologically distinct from other spirochetes. Two of the original isolates from Connecticut and Minnesota and 16 new isolates obtained from shrews captured in Minnesota were characterized phenotypically and genetically in this study. A comparative analysis of the 16S rRNA sequences of two shrew isolates and one mouse isolate and the 16S rRNA sequences of 16 other spirochetes and Escherichia coli revealed that these organisms exhibited low levels of similarity (range of similarity values, 73.9 to 77.8%; average level of similarity, 74.7%). The shrew and mouse isolates which we examined formed a deeply branching subgroup that was clearly distinct from the other genera of spirochetes examined. These and other results indicated that the new spirochetes represent a unique taxon in the order Spirochaetales. Accordingly, we propose that they should be classified as members of a new genus, Brevinema. The three strains of Brevinema which we examined had 16S rRNA sequences that were nearly identical. We also compared these isolates by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fatty acid and enzyme analyses, restriction endonuclease analysis, and Southern hybridization and found that the levels of genetic and phenotypic homogeneity among the strains were very high. We concluded that the isolates which we examined were members of a single species, for which we propose the name Brevinema andersonii. The type strain of Brevinema andersonii is CT11616 (= ATCC 43811).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7857811

  10. Social isolation stress-induced oxidative damage in mouse brain and its modulation by majonoside-R2, a Vietnamese ginseng saponin.

    PubMed

    Huong, Nguyen Thi Thu; Murakami, Yukihisa; Tohda, Michihisa; Watanabe, Hiroshi; Matsumoto, Kinzo

    2005-08-01

    Stressors with a physical factor such as immobilization, electric foot shock, cold swim, etc., have been shown to produce oxidative damage to membrane lipids in the brain. In this study, we investigated the effect of protracted social isolation stress on lipid peroxidation activity in the mouse brain and elucidated the protective effect of majonoside-R2, a major saponin component of Vietnamese ginseng, in mice exposed to social isolation stress. Thiobarbituric acid reactive substance levels, one of the end products of lipid peroxidation reaction, were increased in the brains of mice subjected to 6-8 weeks of social isolation stress. Measurements of nitric oxide (NO) metabolites (NO(x)(-)) also revealed a significant increase of NO production in the brains of socially isolated mice. Moreover, the depletion of brain glutathione content, an endogenous antioxidant, in socially isolated animals occurred in association with the rise in lipid peroxidation. The intraperitoneal administration of majonoside-R2 (10-50 mg/kg) had no effect on thiobarbituric acid reactive substances (TBARS), NO, or glutathione levels in the brains of group-housed control mice but it significantly suppressed the increase in TBARS and NO levels and the decrease in glutathione levels caused by social isolation stress. These results suggest that mice subjected to 6-8 weeks of social isolation stress produces oxidative damage in the brain partly via enhancement of NO production, and that majonoside-R2 exerts a protective effect by modulating NO and glutathione systems in the brain.

  11. Expression of PDX-1 is reduced in pancreatic islets from pups of rat dams fed a low protein diet during gestation and lactation.

    PubMed

    Arantes, Vanessa C; Teixeira, Vicente P A; Reis, Marise A B; Latorraca, Márcia Q; Leite, Adriana R; Carneiro, Everardo M; Yamada, Aureo T; Boschero, Antonio C

    2002-10-01

    Intrauterine and early postnatal malnutrition has profound consequences on fetal and postnatal development in both humans and animals. In addition, low birth weight has been reported to be associated with impaired insulin secretion, insulin resistance and diminished area of pancreatic islets. Because the transcription factor pancreatic and duodenal homeobox 1 (PDX-1) is important for the maintenance of B-cell physiology, PDX-1 expression and islet area were assessed in neonatal rats of dams fed low (6%) or normal (17%) protein diets during pregnancy. PDX-1 protein and mRNA levels, as well as insulin secretion and islet area, were measured after 28 d of life in normal, low protein and recovered rats whose dams consumed a normal protein diet after delivery. Insulin secretion by isolated islets in response to 2.8 and 16.7 mmol glucose/L was reduced in 28-d-old low protein rats compared with the control (P < 0.05). At birth and after 28 d of life, the islet area and PDX-1 protein expression were also reduced (P < 0.05). In contrast, PDX-1 mRNA levels in islets from 28-d-old low protein rats were not different from control rats. PDX-1 protein expression in pancreatic islets, the area of islets and insulin secretion were restored in recovered rats, whereas PDX-1 mRNA levels were higher than in normal rats (P < 0.05). These results suggest a link among diminished PDX-1 protein expression, a reduction in islet area and impaired insulin secretion in low protein rats. The reintroduction of a normal diet early in life restored islet area and cell physiology. PMID:12368391

  12. General Information about Pancreatic Neuroendocrine Tumors (Islet Cell Tumors)

    MedlinePlus

    ... Islet Cell Tumors) Treatment (PDQ®)–Patient Version General Information About Pancreatic Neuroendocrine Tumors (Islet Cell Tumors) Go ... the PDQ Adult Treatment Editorial Board . Clinical Trial Information A clinical trial is a study to answer ...

  13. Protein phosphatases in pancreatic islets

    PubMed Central

    Ortsäter, Henrik; Grankvist, Nina; Honkanen, Richard E.; Sjöholm1, Åke

    2014-01-01

    The prevalence of diabetes is increasing rapidly world-wide. A cardinal feature of most forms of diabetes is the lack of insulin-producing capability, due to the loss of insulin-producing β-cells, impaired glucose-sensitive insulin secretion from the β-cell, or a combination thereof, the reasons for which largely remain elusive. Reversible phosphorylation is an important and versatile mechanism for regulating the biological activity of many intracellular proteins, which, in turn, controls a variety of cellular functions. For instance, significant changes in protein kinase activities and in protein phosphorylation patterns occur subsequent to stimulation of insulin release by glucose. Therefore, the molecular mechanisms regulating phosphorylation of proteins involved in the insulin secretory process by the β-cell have been extensively investigated. How