Pancreatic islet transplantation

Abstract Background No formulation of exogenous insulin available to date has yet been able to mimic the physiological nictemeral rhythms of this hormone, and despite all engineering advancements, the theoretical proposal of developing a mechanical replacement for pancreatic β cell still has not been reached. Thus, the replacement of β cells through pancreas and pancreatic islet transplantation are the only concrete alternatives for re-establishing the endogenous insulin secretion in type 1 diabetic patients. Since only 1 to 1.5% of the pancreatic mass corresponds to endocrine tissue, pancreatic islets transplantation arises as a natural alternative. Data from the International Islet Transplant Registry (ITR) from 1983 to December 2000 document a total of 493 transplants performed around the world, with progressively worse rates of post-transplant insulin independence. In 2000, the "Edmonton Protocol" introduced several modifications to the transplantation procedure, such as the use of a steroid-free immunosuppression regimen and transplantation of a mean islet mass of 11,000 islet equivalents per kilogram, which significantly improved 1-year outcomes. Although the results of a 5-year follow-up in 65 patients demonstrated improvement in glycemic instability in a significant portion of them, only 7.5% of the patients have reached insulin independence, indicating the need of further advances in the preservation of the function of transplanted islet. In addition to the scarcity of organs available for transplantation, islets transplantation still faces major challenges, specially those related to cell loss during the process of islet isolation and the losses related to the graft site, apoptosis, allorejection, autoimmunity, and immunosuppression. The main strategies to optimize islet transplantation aim at improving all these aspects. Conclusion Human islet transplantation should be regarded as an intervention that can decrease the frequency of severe hypoglycemic episodes and improve glycemic control in selected patient for whom benefits of 4-5 years duration would be very valuable. Its limitations, however, indicate that the procedure in its current format is not suitable for all patients with type 1 diabetes.

HOXB7 mRNA is overexpressed in pancreatic ductal adenocarcinomas and its knockdown induces cell cycle arrest and apoptosis

Background Human homeobox genes encode nuclear proteins that act as transcription factors involved in the control of differentiation and proliferation. Currently, the role of these genes in development and tumor progression has been extensively studied. Recently, increased expression of HOXB7 homeobox gene (HOXB7) in pancreatic ductal adenocarcinomas (PDAC) was shown to correlate with an invasive phenotype, lymph node metastasis and worse survival outcomes, but no influence on cell proliferation or viability was detected. In the present study, the effects arising from the knockdown of HOXB7 in PDAC cell lines was investigated. Methods Real time quantitative PCR (qRT-PCR) (Taqman) was employed to assess HOXB7 mRNA expression in 29 PDAC, 6 metastatic tissues, 24 peritumoral tissues and two PDAC cell lines. siRNA was used to knockdown HOXB7 mRNA in the cell lines and its consequences on apoptosis rate and cell proliferation were measured by flow cytometry and MTT assay respectively. Results Overexpression of HOXB7 mRNA was observed in the tumoral tissues and in the cell lines MIA PaCa-2 and Capan-1. HOXB7 knockdown elicited (1) an increase in the expression of the pro-apoptotic proteins BAX and BAD in both cell lines; (2) a decrease in the expression of the anti-apoptotic protein BCL-2 and in cyclin D1 and an increase in the number of apoptotic cells in the MIA PaCa-2 cell line; (3) accumulation of cell in sub-G1 phase in both cell lines; (4) the modulation of several biological processes, especially in MIA PaCa-2, such as proteasomal ubiquitin-dependent catabolic process and cell cycle. Conclusion The present study confirms the overexpression of HOXB7 mRNA expression in PDAC and demonstrates that decreasing its protein level by siRNA could significantly increase apoptosis and modulate several biological processes. HOXB7 might be a promising target for future therapies.

Up-regulation of fas and fasL pro-apoptotic genes expression in type 1 diabetes patients after autologous haematopoietic stem cell transplantation

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T cell-mediated destruction of pancreatic beta cells, resulting in insulin deficiency and hyperglycaemia. Recent studies have described that apoptosis impairment during central and peripheral tolerance is involved in T1D pathogenesis. In this study, the apoptosis-related gene expression in T1D patients was evaluated before and after treatment with high-dose immunosuppression followed by autologous haematopoietic stem cell transplantation (HDI-AHSCT). We also correlated gene expression results with clinical response to HDI-AHSCT. We observed a decreased expression of bad, bax and fasL pro-apoptotic genes and an increased expression of a1, bcl-xL and cIAP-2 anti-apoptotic genes in patients' peripheral blood mononuclear cells (PBMCs) compared to controls. After HDI-AHSCT, we found an up-regulation of fas and fasL and a down-regulation of anti-apoptotic bcl-xL genes expression in post-HDI-AHSCT periods compared to pre-transplantation. Additionally, the levels of bad, bax, bok, fasL, bcl-xL and cIAP-1 genes expression were found similar to controls 2 years after HDI-AHSCT. Furthermore, over-expression of pro-apoptotic noxa at 540 days post-HDI-AHSCT correlated positively with insulin-free patients and conversely with glutamic acid decarboxylase autoantibodies (GAD65) autoantibody levels. Taken together, the results suggest that apoptosis-related genes deregulation in patients' PBMCs might be involved in breakdown of immune tolerance and consequently contribute to T1D pathogenesis. Furthermore, HDI-AHSCT modulated the expression of some apoptotic genes towards the levels similar to controls. Possibly, the expression of these apoptotic molecules could be applied as biomarkers of clinical remission of T1D patients treated with HDI-AHSCT therapy.

Angiotensin II-induced JNK activation is mediated by NAD(P)H oxidase in isolated rat pancreatic islets

Angiotensin II (All), the active component of the renin angiotensin system (RAS), plays a vital role in the regulation of physiological processes of the cardiovascular system, but also has autocrine and paracrine actions in various tissues and organs. Many studies have shown the existence of RAS in the pancreas of humans and rodents. The aim of this study was to evaluate potential signaling pathways mediated by All in isolated pancreatic islets of rats. Phosphorylation of MAPKs (ERK1/2, JNK and p38MAPK), and the interaction between proteins JAK/STAT were evaluated. All increased JAK2/STAT1 (42%) and JAK2/STAT3 (100%) interaction without altering the total content of JAK2. Analyzing the activation of MAPKs (ERK1/2, JNK and p38MAPK) in isolated pancreatic islets from rats we observed that All rapidly (3 min) promoted a significant increase in the phosphorylation degree of these proteins after incubation with the hormone. Curiously JNK protein phosphorylation was inhibited by DPI, suggesting the involvement of NAD(P)H oxidase in the activation of protein. (C) 2012 Elsevier B.V. All rights reserved.

Expression of NADPH oxidase in human pancreatic islets

Aims: NADPH oxidase (NOX) is a known source of superoxide anions in phagocytic and non-phagocytic cells. In this study, the presence of this enzyme in human pancreatic islets and the importance of NADPH oxidase in human beta-cell function were investigated. Main methods and key findings: In isolated human pancreatic islets, the expression of NADPH oxidase components was evidenced by real-time PCR (p22(PHOX), p47(PHOX) and p67(PHOX)), Western blotting (p47(PHOX) and p67(PHOX)) and immunohistochemistry (p47(PHOX), p67(PHOX) and gp91(PHOX)). Immunohistochemistry experiments showed co-localization of p47(PHOX), p67(PHOX) and gp91(PHOX) (isoform 2 of NADPH oxidase-NOX2) with insulin secreting cells. Inhibition of NADPH oxidase activity impaired glucose metabolism and glucose-stimulated insulin secretion. Significance: These findings demonstrate the presence of the main intrinsic components of NADPH oxidase comprising the NOX2 isoform in human pancreatic islets, whose activity also contributes to human beta-cell function. (C) 2012 Elsevier Inc. All rights reserved.

Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

Chronic administration of glucocorticoids (GC) leads to characteristic features of type 2 diabetes in mammals. The main action of dexamethasone in target cells occurs through modulation of gene expression, although the exact mechanisms are still unknown. We therefore investigated the gene expression profile of pancreatic islets from rats treated with dexamethasone using a cDNA array screening analysis. The expression of selected genes and proteins involved in mitochondria] apoptosis was further analyzed by PCR and immunoblotting. Insulin, triglyceride and free fatty acid plasma levels, as well as glucose-induced insulin secretion, were significantly higher in dexamethasone-treated rats compared with controls. Out of 1176 genes, 60 were up-regulated and 28 were down-regulated by dexamethasone treatment. Some of the modulated genes are involved in apoptosis, stress response, and proliferation pathways. RT-PCR confirmed the cDNA array results for 6 selected genes. Bax alpha protein expression was increased, while Bcl-2 was decreased. In vivo dexamethasone treatment decreased the mitochondrial production of NAD(P)H, and increased ROS production. Concluding, our data indicate that dexamethasone modulates the expression of genes and proteins involved in several pathways of pancreatic-islet cells, and mitochondria dysfunction might be involved in the deleterious effects after long-term GC treatment.

Protective effects of bone marrow mononuclear cell therapy on lung and heart in an elastase-induced emphysema model

We hypothesized that bone marrow-derived mononuclear cell (BMDMC) therapy protects the lung and consequently the heart in experimental elastase-induced emphysema. Twenty-four female C57BL/6 mice were intratracheally instilled with saline (C group) or porcine pancreatic elastase (E group) once a week during 4 weeks. C and E groups were randomized into subgroups receiving saline (SAL) or male BMDMCs (2 x 10(6), CELL) intravenously 3 h after the first saline or elastase instillation. Compared to E-SAL group, E-CELL mice showed, at 5 weeks: lower mean linear intercept, neutrophil infiltration, elastolysis, collagen fiber deposition in alveolar septa and pulmonary vessel wall, lung cell apoptosis, right ventricle wall thickness and area, higher endothelial growth factor and insulin-like growth factor mRNA expressions in lung tissue, and reduced platelet-derived growth factor, transforming growth factor-beta, and caspase-3 expressions. In conclusion, BMDMC therapy was effective at modulating the inflammatory and remodeling processes in the present model of elastase-induced emphysema. (c) 2012 Elsevier B.V. All rights reserved.

Endurance training stimulates growth and survival pathways and the redox balance in rat pancreatic islets

Calegari VC, Abrantes JL, Silveira LR, Paula FM, Costa JM Jr, Rafacho A, Velloso LA, Carneiro EM, Bosqueiro JR, Boschero AC, Zoppi CC. Endurance training stimulates growth and survival pathways and the redox balance in rat pancreatic islets. J Appl Physiol 112: 711-718, 2012. First published December 15, 2011; doi:10.1152/japplphysiol.00318.2011.-Endurance training has been shown to increase pancreatic beta-cell function and mass. However, whether exercise modulates beta-cell growth and survival pathways signaling is not completely understood. This study investigated the effects of exercise on growth and apoptotic markers levels in rat pancreatic islets. Male Wistar rats were randomly assigned to 8-wk endurance training or to a sedentary control group. After that, pancreatic islets were isolated; gene expression and the total content and phosphorylation of several proteins related to growth and apoptotic pathways as well as the main antioxidant enzymes were determined by real-time polymerase chain reaction and Western blot analysis, respectively. Reactive oxygen species (ROS) production was measured by fluorescence. Endurance training increased the time to reach fatigue by 50%. Endurance training resulted in increased protein phosphorylation content of AKT (75%), AKT substrate (AS160; 100%), mTOR (60%), p70s6k (90%), and ERK1/2 (50%), compared with islets from control group. Catalase protein content was 50% higher, whereas ROS production was 49 and 77% lower in islets from trained rats under basal and stimulating glucose conditions, respectively. Bcl-2 mRNA and protein levels increased by 46 and 100%, respectively. Bax and cleaved caspase-3 protein contents were reduced by 25 and 50% in islets from trained rats, respectively. In conclusion, these results demonstrate that endurance training favors the beta-cell growth and survival by activating AKT and ERK1/2 pathways, enhancing antioxidant capacity, and reducing ROS production and apoptotic proteins content.

Expression of Pancreatic Endocrine Markers by Mesenchymal Stem Cells From Human Adipose Tissue

Mesenchymal stem cells (MSCs) from human adipose tissue have a great potential for use in cell therapy due to their ease of isolation, expansion, and differentiation, besides the relative acceptance from the ethical point of view. Our intention was to isolate and promote in vitro expansion and differentiation of MSCs from human adipose tissue into cells with a pancreatic endocrine phenotype. Human adipose tissue obtained from patients undergoing abdominal dermolipectomy was digested with type I collagenase. MSCs isolated by plastic adherence and characterized by cytochemistry and FACS were expanded in vitro. MSC differentiation into an endocrine phenotype was induced over 2 to 4 months with high glucose (25 mmol/L) media containing nicotinamide, exendin-4, and 2-mercaptoethanol. Insulin and glucagon expressions were analyzed by immunofluorescence. Cells isolated from human adipose tissue and expanded in vitro expressed MSC markers as confirmed by FACS and cytochemistry. Insulin but not glucagon production by differentiated cells was demonstrated by irnmunofluorescence. MSCs isolated from human adipose tissue were induced to differentiate in vitro into an endocrine phenotype that expressed insulin

Risks, Benefits, and Therapeutic Potential of Hematopoietic Stem Cell Transplantation for Autoimmune Diabetes

Type 1 diabetes mellitus is a chronic disease that results from the autoimmune response against pancreatic insulin producing beta cells. Apart of several insulin regimens, since the decade of 80s various immunomodulatory regimens were tested aiming at blocking some steps of the autoimmune process against beta cell mass and at promoting beta cell preservation. In the last years, some independent research groups tried to cure type 1 diabetes with an "immunologic reset" provided by autologous hematopoietic stem cell transplantation in newly diagnosed patients, and the majority of patients became free form insulin with increasing levels of C-peptide along the time. In this review, we discuss the biology of hematopoietic stem cells and the possible advantages and disadvantages related to the high dose immunosuppression followed by autologous hematopoietic stem cell transplantation.

Effect of Cholecalciferol as Adjunctive Therapy With Insulin on Protective Immunologic Profile and Decline of Residual beta-Cell Function in New-Onset Type 1 Diabetes Mellitus

Objective: To evaluate the effect of vitamin D-3 on cytokine levels, regulatory T cells, and residual beta-cell function decline when cholecalciferol (vitamin D-3 administered therapeutically) is given as adjunctive therapy with insulin in new-onset type 1 diabetes mellitus (T1DM). Design and Setting: An 18-month (March 10, 2006, to October 28, 2010) randomized, double-blind, placebo-controlled trial was conducted at the Diabetes Center of Sao Paulo Federal University, Sao Paulo, Brazil. Participants: Thirty-eight patients with new-onset T1DM with fasting serum C-peptide levels greater than or equal to 0.6 ng/mL were randomly assigned to receive daily oral therapy of cholecalciferol, 2000 IU, or placebo. Main Outcome Measure: Levels of proinflammatory and anti-inflammatory cytokines, chemokines, regulatory T cells, hemoglobin A(1c), and C-peptide; body mass index; and insulin daily dose. Results: Mean (SD) chemokine ligand 2 (monocyte chemoattractant protein 1) levels were significantly higher (184.6 [101.1] vs 121.4 [55.8] pg/mL) at 12 months, as well as the increase in regulatory T-cell percentage (4.55%[1.5%] vs 3.34%[1.8%]) with cholecalciferol vs placebo. The cumulative incidence of progression to undetectable (<= 0.1 ng/mL) fasting C-peptide reached 18.7% in the cholecalciferol group and 62.5% in the placebo group; stimulated C-peptide reached 6.2% in the cholecalciferol group and 37.5% in the placebo group at 18 months. Body mass index, hemoglobin A(1c) level, and insulin requirements were similar between the 2 groups. Conclusions: Cholecalciferol used as adjunctive therapy with insulin is safe and associated with a protective immunologic effect and slow decline of residual beta-cell function in patients with new-onset T1DM. Cholecalciferol may be an interesting adjuvant in T1DM prevention trials.

Physical exercise and pancreatic islets Acute and chronic actions on insulin secretion

Diabetes mellitus (DM) is a great public health problem, which attacks part of the world population, being characterized by an imbalance in body glucose homeostasis. Physical exercise is pointed as a protective agent and is also recommended to people with DM. As pancreatic islets present an important role in glucose homeostasis, we aim to study the role of physical exercise (chronic adaptations and acute responses) in pancreatic islets functionality in Wistar male rats. First, animals were divided into two groups: sedentary (S) and aerobic trained (T). At the end of 8 weeks, half of them (S and T) were submitted to an acute exercise session (exercise until exhaustion), being subdivided as acute sedentary (AS) and acute trained (AT). After the experimental period, periepididymal, retroperitoneal and subcutaneous fat pads, blood, soleus muscle and pancreatic islets were collected and prepared for further analysis. From the pancreatic islets, total insulin content, insulin secretion stimulated by glucose, leucine, arginine and carbachol were analyzed. Our results pointed that body adiposity and glucose homeostasis improved with chronic physical exercise. In addition, total insulin content was reduced in group AT, insulin secretion stimulated by glucose was reduced in trained groups (T and AT) and insulin secretion stimulated by carbachol was increased in group AT. There were no significant differences in insulin secretion stimulated by arginine and leucine. We identified a possible modulating action on insulin secretion, probably related to the association of chronic adaptation with an acute response on cholinergic activity in pancreatic islets.

Apoptosis rate and transcriptional response of pancreatic islets exposed to the PPAR gamma agonist Pioglitazone

To explore the molecular pathways underlying thiazolidinediones effects on pancreatic islets in conditions mimicking normo- and hyperglycemia, apoptosis rate and transcriptional response to Pioglitazone at both physiological and supraphysiological glucose concentrations were evaluated. Adult rat islets were cultured at physiological (5.6 mM) and supraphysiological (23 mM) glucose concentrations in presence of 10 μM Pioglitazone or vehicle. RNA expression profiling was evaluated with the PancChip 13k cDNA microarray after 24-h, and expression results for some selected genes were validated by qRT-PCR. The effects of Pioglitazone were investigated regarding apoptosis rate after 24-, 48- and 72-h. At 5.6 mM glucose, 101 genes were modulated by Pioglitazone, while 1,235 genes were affected at 23 mM glucose. Gene networks related to lipid metabolism were identified as altered by Pioglitazone at both glucose concentrations. At 23 mM glucose, cell cycle and cell death pathways were significantly regulated as well. At 5.6 mM glucose, Pioglitazone elicited a transient reduction in islets apoptosis rate while at 23 mM, Bcl2 expression was reduced and apoptosis rate was increased by Pioglitazone. Our data demonstrate that the effect of Pioglitazone on gene expression profile and apoptosis rate depends on the glucose concentration. The modulation of genes related to cell death and the increased apoptosis rate observed at supraphysiological glucose concentration raise concerns about Pioglitazone’s direct effects in conditions of hyperglycemia and reinforce the necessity of additional studies designed to evaluate TZDs effects on the preservation of β-cell function in situations where glucotoxicity might be more relevant than lipotoxicity.