Survival Of Pancreatic Islets Research Articles

The collagen matrix regulates the survival and function of pancreatic islets.

The extracellular matrix (ECM) provides an appropriate microenvironment for many kinds of cells, including pancreatic cells. Collagens are the most abundant components of the ECM. Type I, IV, V and VI collagen has been detected in pancreatic islets, and each type plays important role in the proliferation, survival, function and differentiation of pancreatic cells. In some cases, collagens show behaviours similar to those of growth factors and regulate the biological behaviour of β cells by binding with certain growth factors, including IGFs, EGFs and FGFs. The transcriptional coactivator YAP/TAZ has been widely recognised as a mechanosensor that senses changes in the physical characteristics of the ECM and inhibition of YAP/TAZ enhances insulin production and secretion. Type 1 diabetes mellitus (T1DM) is an autoimmune disease characterised by the destruction of insulin-producing β cells. The crosstalk between collagens and immune cells plays a key role in the development and differentiation of immune cells. Further, Supplementation with collagens during islet transplantation is a promising strategy for improving the quality of the islets. But, excessive collagen deposition results in pancreatic fibrosis and pancreatic carcinoma. Targeting inhibit Piezo, autophagy or IL-6 may reduce excessive collagen deposition-induced pancreatic fibrosis and pancreatic carcinoma. This review provides insights into the treatment of T1DM to prolong life expectancy and provides the potential targets for treating collagen deposition-induced pancreatic fibrosis and pancreatic carcinoma.

Exploring lncRNAs associated with human pancreatic islet cell death induced by transfer of adoptive lymphocytes in a humanized mouse model.

Long noncoding RNA (lncRNA)-mediated posttranscriptional and epigenetic landscapes of gene regulation are associated with numerous human diseases. However, the regulatory mechanisms governing human β-cell function and survival remain unknown. Owing to technical and ethical constraints, studying the direct role of lncRNAs in β-cell function and survival in humans in vivo is difficult. Therefore, we utilized humanized mice with human islets to investigate lncRNA expression using whole transcriptome shotgun sequencing. Our study aimed to characterize lncRNAs that may be crucial for human islet cell function and survival. Human β-cell death was induced in humanized mice engrafted with functional human islets. Using these humanized mice harboring human islets with induced β-cell death, we investigated lncRNA expression through whole transcriptome shotgun sequencing. Additionally, we systematically identified, characterized, and explored the regulatory functions of lncRNAs that are potentially important for human pancreatic islet cell function and survival. Human islet cell death was induced in humanized mice engrafted with functional human islets. RNA sequencing analysis of isolated human islets, islet grafts from humanized mice with and without induced cell death, revealed aberrant expression of a distinct set of lncRNAs that are associated with the deregulated mRNAs important for cellular processes and molecular pathways related to β-cell function and survival. A total of 10 lncRNA isoforms (SCYL1-1:22, POLG2-1:1, CTRB1-1:1, SRPK1-1:1, GTF3C5-1:1, PPY-1:1, CTRB1-1:5, CPA5-1:1, BCAR1-2:1, and CTRB1-1:4) were identified as highly enriched and specific to human islets. These lncRNAs were deregulated in human islets from donors with different BMIs and with type 2 diabetes (T2D), as well as in cultured human islets with glucose stimulation and induced cell death induced by cytokines. Aberrant expression of these lncRNAs was detected in the exosomes from the medium used to culture islets with cytokines. Islet-enriched and specific human lncRNAs are deregulated in human islet grafts and cultured human islets with induced cell death. These lncRNAs may be crucial for human β-cell function and survival and could have an impact on identifying biomarkers for β-cell loss and discovering novel therapeutic targets to enhance β-cell function and survival.

Trimetazidine Preconditioning Potentiates the Effect of Mesenchymal Stem Cells Secretome on the Preservation of Rat Pancreatic Islet Survival and Function In Vitro.

Islet transplantation offers improved glycemic control in individuals with type 1 diabetes mellitus. However, in vitro islet culture is associated with islet apoptosis and eventually will lose their functionality prior to transplantation. In this study, we examined the effects of mesenchymal stem cells (MSCs) secretome preconditioned with diazoxide (DZ) and trimetazidine (TMZ) on rat islet cells during pre-transplant culture. With and without preconditioned hAD-MSCs' concentrated conditioned media (CCM) were added to the culture medium containing rat islets every 12h for 24 and 48h, after testing for selected cytokine concentrations (interleukin (IL)-4, IL-6, IL-13). Insulin content, glucose-stimulated insulin secretion, islet cell apoptosis, and mRNA expression of pro-apoptotic (BAX, BAK-1, and PUMA) and anti-apoptotic factors (BCL-2,BCL-xL, and XIAP) in rat islets were assessed after 24 and 48h of culture. The protein level of IL-6 and IL-4 was significantly higher in TMZ-MSC-CM compared to MSC-non-CM. In rat isolated islets, normalized secreted insulin in the presence of 16.7mM glucose was significantly higher in treated islet groups compared to control islets at both 24 and 48h cultivation. Also, the percentage of apoptotic islet cells TMZ-MSC-CCM-treated islets was significantly lower compared to MSC-CM and MSC-CCM-treated islets in both 24 and 48h cultivation. Consistent with the number of apoptotic cells, after 24h culture, the expression of BCL-2 and BCL-xL genes in the control islets was lower than all treatment islet groups and in 48h was lower than only TMZ-MSC-CM-treated islets. Also, the expression of the XIAP gene in control islets was significantly lower compared to the TMZ-MSC-CCM-treated islets at both at 24 and 48h. In addition, mRNA level of the BAX gene in TMZ-MSC-CCM-treated islets was significantly lower compared to other groups at 48h. Our findings revealed that TMZ proved to be more effective than DZ and could enhance the potential of hAD-MSCs-CM to improve the function and viability of islets prior to transplantation.

Intracutaneous Transplantation of Islets Within a Biodegradable Temporizing Matrix as an Alternative Site for Islet Transplantation.

Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model.

Albumin-binding DARPins as scaffold improve the hypoglycemic and anti-obesity effects of exendin-4 in vivo

Type 2 diabetes mellitus (T2DM) and obesity have been considered epidemics and threats to public health worldwide. Exendin-4 (Ex), a GLP-1R agonist, has potential for treating T2DM and obesity. However, Ex has a half-life of only 2.4 h in humans and needs to be administered twice daily, which hampers its clinical application. In this study, we synthesized four new GLP-1R agonists by genetically fusing Ex to the N-terminus of HSA-binding ankyrin repeat proteins (DARPins) via linkers of different lengths, denoted as Ex-DARPin-GSx fusion proteins (x = 0, 1, 2, and 3). The Ex-DARPin fusion proteins were substantially stable, resulting in incomplete denaturation even at 80 °C. The in vitro bioactivity results demonstrated that Ex-DARPin fusion proteins could bind to HSA and activate GLP-1R. The Ex-DARPin fusion proteins had a comparable half-life (29–32 h), which is much longer than that of native Ex (0.5 h in rats). Subcutaneous injection of 25 nmol/kg Ex-DARPin fusion protein normalized blood glucose (BG) levels for at least 72 h in mice. The Ex-DARPin fusion proteins, injected at 25 nmol/kg every three days, significantly lowered BG, inhibited food consumption, and reduced body weight (BW) for 30 days in STZ-induced diabetic mice. Histological analysis of pancreatic tissues using H&E staining revealed that Ex-DARPin fusion proteins significantly improved the survival of pancreatic islets in diabetic mice. The differences in in vivo bioactivity of fusion proteins with different linker lengths were not significant. According to the findings in this study, long-acting Ex-DARPin fusion proteins designed by us hold promise for further development as antidiabetic and antiobesity therapeutic agents. Our findings also indicate that DARPins are a universal platform for generating long-acting therapeutic proteins via genetic fusion, thus broadening the application scope of DARPins.

Three-dimensional bioprinting of functional β-islet-like constructs.

256Diabetes is an autoimmune disease that ensues when the pancreas does not deliver adequate insulin or when the body cannot react to the existing insulin. Type 1 diabetes is an autoimmune disease defined by continuous high blood sugar levels and insulin deficiency due to β-cell destruction in the islets of Langerhans (pancreatic islets). Long-term complications, such as vascular degeneration, blindness, and renal failure, result from periodic glucose-level fluctuations following exogenous insulin therapy. Nevertheless, the shortage of organ donors and the lifelong dependency on immunosuppressive drugs limit the transplantation of the entire pancreas or pancreas islet, which is the therapy for this disease. Although encapsulating pancreatic islets using multiple hydrogels creates a semi-privileged environment to prevent immune rejection, hypoxia that occurs in the core of the capsules is the main hindrance that should be solved. Bioprinting technology is an innovative process in advanced tissue engineering that allows the arranging of a wide array of cell types, biomaterials, and bioactive factors as a bioink to simulate the native tissue environment for fabricating clinically applicable bioartificial pancreatic islet tissue. Multipotent stem cells have the potential to be a possible solution for donor scarcity and can be a reliable source for generating autograft and allograft functional β-cells or even pancreatic islet-like tissue. The use of supporting cells, such as endothelial cells, regulatory T cells, and mesenchymal stem cells, in the bioprinting of pancreatic islet-like construct could enhance vasculogenesis and regulate immune activity. Moreover, scaffolds bioprinted using biomaterials that can release oxygen postprinting or enhance angiogenesis could increase the function of β-cells and the survival of pancreatic islets, which could represent a promising avenue.

Deciphering the gene regulatory network associated with anti-apoptosis in the pancreatic islets of type 2 diabetes mice using computational approaches

<abstract> <p>Type 2 diabetes (T2D) is a major global health problem often caused by the inability of pancreatic islets to compensate for the high insulin demand due to apoptosis. However, the complex mechanisms underlying the activation of apoptosis and its counter process, anti-apoptosis, during T2D remain unclear. In this study, we employed bioinformatics and systems biology approaches to understand the anti-apoptosis-associated gene expression and the biological network in the pancreatic islets of T2D mice. First, gene expression data from four peripheral tissues (islets, liver, muscle and adipose) were used to identify differentially expressed genes (DEGs) in T2D compared to non-T2D mouse strains. Our comparative analysis revealed that <italic>Gm2036</italic> is upregulated across all four tissues in T2D and is functionally associated with increased cytosolic Ca<sup>2+</sup> levels, which may alter the signal transduction pathways controlling metabolic processes. Next, our study focused on islets and performed functional enrichment analysis, which revealed that upregulated genes are significantly associated with sucrose and fructose metabolic processes, as well as negative regulation of neuron apoptosis. Using the Ingenuity Pathway Analysis (IPA) tool of QIAGEN, gene regulatory networks and their biological effects were analyzed, which revealed that glucose is associated with the underlying change in gene expression in the islets of T2D; and an activated gene regulatory network—containing upregulated <italic>CCK, ATF3, JUNB, NR4A1, GAST</italic> and downregulated <italic>DPP4</italic>—is possibly inhibiting apoptosis of islets and β-cells in T2D. Our computational-based study has identified a putative regulatory network that may facilitate the survival of pancreatic islets in T2D; however, further validation in a larger sample size is needed. Our results provide valuable insights into the underlying mechanisms of T2D and may offer potential targets for developing more efficacious treatments.</p> </abstract>

334.3: Presentation of FasL Protein by PEG Microgels Within Graft Site Leads to Survival of Pancreatic Islets in Nonhuman Primates Without the Need for Chronic Immunosuppression

334.3: Presentation of FasL Protein by PEG Microgels Within Graft Site Leads to Survival of Pancreatic Islets in Nonhuman Primates Without the Need for Chronic Immunosuppression

Indirect and Direct Effects of SARS-CoV-2 on Human Pancreatic Islets.

Recent studies have shown that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may induce metabolic distress, leading to hyperglycemia in patients affected by coronavirus disease 19 (COVID-19). We investigated the potential indirect and direct effects of SARS-CoV-2 on human pancreatic islets in 10 patients who became hyperglycemic after COVID-19. Although there was no evidence of peripheral anti-islet autoimmunity, the serum of these patients displayed toxicity on human pancreatic islets, which could be abrogated by the use of anti-interleukin-1β (IL-1β), anti-IL-6, and anti-tumor necrosis factor α, cytokines known to be highly upregulated during COVID-19. Interestingly, the receptors of those aforementioned cytokines were highly expressed on human pancreatic islets. An increase in peripheral unmethylated INS DNA, a marker of cell death, was evident in several patients with COVID-19. Pathology of the pancreas from deceased hyperglycemic patients who had COVID-19 revealed mild lymphocytic infiltration of pancreatic islets and pancreatic lymph nodes. Moreover, SARS-CoV-2-specific viral RNA, along with the presence of several immature insulin granules or proinsulin, was detected in postmortem pancreatic tissues, suggestive of β-cell-altered proinsulin processing, as well as β-cell degeneration and hyperstimulation. These data demonstrate that SARS-CoV-2 may negatively affect human pancreatic islet function and survival by creating inflammatory conditions, possibly with a direct tropism, which may in turn lead to metabolic abnormalities observed in patients with COVID-19.

NR5A2/LRH-1 regulates the PTGS2-PGE2-PTGER1 pathway contributing to pancreatic islet survival and function

SummaryLRH-1/NR5A2 is implicated in islet morphogenesis postnatally, and its activation using the agonist BL001 protects islets against apoptosis, reverting hyperglycemia in mouse models of Type 1 Diabetes Mellitus. Islet transcriptome profiling revealed that the expression of PTGS2/COX2 is increased by BL001. Herein, we sought to define the role of LRH-1 in postnatal islet morphogenesis and chart the BL001 mode of action conferring beta cell protection. LRH-1 ablation within developing beta cells impeded beta cell proliferation, correlating with mouse growth retardation, weight loss, and hypoglycemia leading to lethality. LRH-1 deletion in adult beta cells abolished the BL001 antidiabetic action, correlating with beta cell destruction and blunted Ptgs2 induction. Islet PTGS2 inactivation led to reduced PGE2 levels and loss of BL001 protection against cytokines as evidenced by increased cytochrome c release and cleaved-PARP. The PTGER1 antagonist—ONO-8130—negated BL001-mediated islet survival. Our results define the LRH-1/PTGS2/PGE2/PTGER1 signaling axis as a key pathway mediating BL001 survival properties.

Generating pancreatic islets organoids: Langerhanoids

The extension of islet transplantation to a wider number of Type 1 diabetic patients is compromised by the scarcity of donors, the reduced ex vivo survival of pancreatic islets and the use of immunosuppressive treatments. Islets of Langerhans isolated from brain-dead donors are currently the only cell source for transplantation. Thus, it is crucial to find an alternative and an abundant source of functional insulin secreting cells not only for clinical use but also for the development of research dedicated to the screening of drugs and to the development of new therapeutic targets. Several groups around the world, including ours, develop 3D culture models as Langerhanoids that closely mimick human pancreatic islets physiology. In this review, we describe recent advances to mimic the pancreatic niche (extracellular matrix, vascularization, microfluidics) allowing better functionality of Langerhanoids.

Immune‐Modulating Mucin Hydrogel Microdroplets for the Encapsulation of Cell and Microtissue

AbstractImmune‐modulating biomaterials used to encapsulate cells and microtissue transplants can be engineered to dampen the immune reaction and increase treatment efficacy. Mucin‐derived materials have gained attention for their ability to modulate macrophage and dendritic cell activity, and to trigger mild foreign body response when implanted in vivo. In this study, the potential of mucin hydrogels (Muc‐gels) as cell‐encapsulating materials is investigated. When placed in contact with blood, Muc‐gels trigger significantly lower complement activation, compared to clinical grade alginate hydrogels. Muc‐gel is a size‐selective barrier strongly hindering the diffusion of molecules with a hydrodynamic radius larger than 6 nm such as immunoglobulins. Muc‐gels support the growth of MIN6m9 insulin‐secreting cells into islet‐like organoids and the survival of primary human pancreatic islets, which maintained glucose responsiveness. Muc‐gels can be shaped into microdroplets in which MIN6m9 cells or cell aggregates can be encapsulated without loss of viability. Microdroplet encapsulation will allow transplants to be easily injected and improve their survival by favoring mass transport through the capsule. The combination of strong immune modulatory properties, appropriate selective barrier profile, biocompatibility for embedded cells Muc‐gels of particular value for microencapsulating cells or microtissues for transplantation.

Global Deletion of the Prolactin Receptor Aggravates Streptozotocin-Induced Diabetes in Mice.

Prolactin (PRL) levels are reduced in the circulation of rats with diabetes or obesity, and lower circulating levels of PRL correlate with increased prevalence of diabetes and a higher risk of metabolic alterations in the clinic. Furthermore, PRL stimulates β-cell proliferation, survival, and insulin production and pregnant mice lacking PRL receptors in β-cells develop gestational diabetes. To investigate the protective effect of endogenous PRL against diabetes outside pregnancy, we compared the number of cases and severity of streptozotocin (STZ)-induced hyperglycemia between C57BL/6 mice null for the PRL receptor gene (Prlr-/-) and wild-type mice (Prlr+/+). STZ-treated diabetic Prlr-/- mice showed a higher number of cases and later recovery from hyperglycemia, exacerbated glucose levels, and glucose intolerance compared to the Prlr+/+ mice counterparts. Consistent with the worsening of hyperglycemia, pancreatic islet density, β-cell number, proliferation, and survival, as well as circulating insulin levels were reduced, whereas α-cell number and pancreatic inflammation were increased in the absence of PRL signaling. Deletion of the PRL receptor did not alter the metabolic parameters in vehicle-treated animals. We conclude that PRL protects whole body glucose homeostasis by reducing β-cell loss and pancreatic inflammation in STZ-induced diabetes. Medications elevating PRL circulating levels may prove to be beneficial in diabetes.

A novel prevascularized tissue-engineered chamber as a site for allogeneic and xenogeneic islet transplantation to establish a bioartificial pancreas.

Although sites for clinical or experimental islet transplantation are well established, pancreatic islet survival and function in these locations remain unsatisfactory. A possible factor that might account for this outcome is local hypoxia caused by the limited blood supply. Here, we modified a prevascularized tissue-engineered chamber (TEC) that facilitated the viability and function of the seeded islets in vivo by providing a microvascular network prior to transplantation. TECs were created, filled with Growth Factor-Matrigel™ (Matrigel™) and then implanted into the groins of mice with streptozotocin-induced diabetes. The degree of microvascularization in each TECs was analyzed by histology, real-time PCR, and Western blotting. Three hundred syngeneic islets were seeded into each chamber on days 0, 14, and 28 post-chamber implantation, and 300, 200, or 100 syngeneic islets were seeded into additional chambers on day 28 post-implantation, respectively. Furthermore, allogeneic or xenogeneic islet transplantation is a potential solution for organ shortage. The feasibility of TECs as transplantation sites for islet allografts or xenografts and treatment with anti-CD45RB and/or anti-CD40L (MR-1) was therefore explored. A highly developed microvascularized network was established in each TEC on day 28 post-implantation. Normalization of blood glucose levels in diabetic mice was negatively correlated with the duration of prevascularization and the number of seeded syngeneic islets. Combined treatment with anti-CD45RB and MR-1 resulted in long-term survival of the grafts following allotransplantation (5/5, 100%) and xenotransplantation (16/20, 80%). Flow cytometry demonstrated that the frequency of CD4+Foxp3-Treg and CD4+IL-4+-Th2 cells increased significantly after tolerogenic xenograft transplantation, while the number of CD4+IFN-γ-Th1 cells decreased. These findings demonstrate that highly developed microvascularized constructs can facilitate the survival of transplanted islets in a TECs, implying its potential application as artificial pancreas in the future.

A tale of two pancreases: exocrine pathology and endocrine dysfunction.

The islets of Langerhans are well embedded within the exocrine pancreas (the latter comprised of ducts and acini), but the nature of interactions between these pancreatic compartments and their role in determining normal islet function and survival are poorly understood. However, these interactions appear to be critical, as when pancreatic exocrine disease occurs, islet function and insulin secretion frequently decline to the point that diabetes ensues, termed pancreatogenic diabetes. The most common forms of pancreatogenic diabetes involve sustained exocrine disease leading to ductal obstruction, acinar inflammation, and fibro-fatty replacement of the exocrine pancreas that predates the development of dysfunction of the endocrine pancreas, as seen in chronic pancreatitis-associated diabetes and cystic fibrosis-related diabetes and, more rarely, MODY type 8. Intriguingly, a form of tumour-induced diabetes has been described that is associated with pancreatic ductal adenocarcinoma. Here, we review the similarities and differences among these forms of pancreatogenic diabetes, with the goal of highlighting the importance of exocrine/ductal homeostasis for the maintenance of pancreatic islet function and survival and to highlight the need for a better understanding of the mechanisms underlying these diverse conditions. Graphical abstract.

A NOVEL PRE-VASCULARIZED TISSUE ENGINEERING CHAMBER SERVES AS A BIO-ARTIFICIAL PANCREAS FOR AN ATTRACTIVE SITE OF ALLOGENIC AND XENOGENEIC ISLET TRANSPLANTATION

Background: Although the sites for clinical or experimental islet transplantation have been well established, the pancreatic islet survival and function in homing locations still remain unsatisfied[1]. One of possible reasons that account for this is local hypoxia due to the shortage of blood supply[2]. Here we modified a pre-vascularized tissue engineering chamber (TEC) in vivo which facilitated the vitality and function of seeded islets by providing a microvascular network prior to transplantation. Method: A TEC filled with MatrigelTM and bFGF-2 was created and embedded in the groin of streptozotocin-induced diabetic mice. The micro-vascularization in the TEC was analyzed by histology, real-time PCR and western blotting. Three hundreds of syngeneic islets were seeded into the chamber on day 0, 14, and 28 post-chamber implantation to research the degree of pre-vascularized in the TEC. In addition, different numbers of syngenic islets (300, 200, and 100) were seeded into the chamber on day 28 post implantation to study the TEC function influenced by islets quantities. Furthermore, we explore the feasibility of TEC as a transplantation site for islet allograft and xenograft which was treated by anti-CD45RB and/or anti-CD40L (MR-1). Results and Discussion: A highly developed micro-vascularized network was established in the TEC on day 28 post implantation. The normalization of blood glucose levels of diabetic mice was negatively correlated with the time required for pre-vascularization and the number of seeded syngenic islets. The combined treatment of anti-CD45RB and MR-1 guaranteed the grafts long-term survival after allotransplantation (5/5, 100%) and xenotransplantation (16/20, 80%). Flow cytometry showed the frequency of CD4+Foxp3 Tregs and CD4+IL-4+Th2 cells significantly increased in tolegenic xenograft transplantation, compared with the decrease of CD4+IFN-γTh1 cells. Conclusion: These findings highlight that the highly developed micro-vascularized construct facilitates islet transplantation in the TEC, implicating the possibility of application as an artificial pancreas in the future.Fig. 1. (A) HE images (1st row; scale bars: 10 μm) and histology analysis (2nd and 3rd rows; scale bars: 40 μm) of pre-vascularization TECs on day 7, 14, 28, and 35. Blood vessels were marked by black arrows in HE (1st row; scale bars: 10 μm). The micro-vascularize were stained by anti-VEGF and anti-CD31, and marked by brown and black arrows seperately (2nd and 3rd rows; scale bars: 40 μm). (B)The level of VEGF mRNA was quantified. The extent of pre-vascularization confirmed a significant increase in the 28d group compared with VEGF levels in the 7d and 14d groups (p**<0.001, p***<0.001), and no significant difference between the 28d group and the 35day group (P > 0.05). Sham of inguinal adipose tissue set as control. Data was shown as the mean ± SD and representative of three separate experiments. (C) Western blot analysis showed the protein levels of VEGF at different time points in TEC.Fig. 2. The time required for the blood glucose returning to normal level (11.1mol/dl) in diabetic mice was negatively correlated with the pre-vascularization time (A) and the seeding quantity of syngeneic islets (B). (C) Non-fasting blood glucose maintained normoglycemia after syngeneic islets transplanted in the TEC of diabetic mice, and became hyperglycaemia when the TEC removed (black arrow). (D) Insulin stained donor islet in recipient TEC at 90d posttransplant (shown brown), the islet was pointed by the red arrow (Scale bars:40 μm).Txpl: Transplantation post-implantation.Fig. 3. (A) Islet allografts (Balb/c to C57BL/6) were rejected in the untreated group within 22±2.2 d. Islet allografts treated with anti-CD45RB or anti-CD40L alone were also rejected in recipients within 24±3.12 d, 26±1.74 d. Islet allografts with the dual antibody treatment of anti-CD45RB and anti-CD40L presented long-term graft survival in all recipients. The Kaplan–Meier curves were compared with the log-rank test. (B) Non-fasting blood glucose maintained normoglycemia (5/5, 80%) in the diabetic mice after allogeneic islets transplanted, and became hyperglycaemia when the TEC removed (black arrow) (C) Islet xenograft (SD rat to C57BL/6) rejected in the untreated group, treated-CD45RB group and treated-CD40L group within 13±3.1d, 16±3.12d, 18±1.74d, respectively. Islet xenografts with the dual antibody treatment of anti-CD45RB and anti-CD40L resulted in long-term graft survival in all recipients. The Kaplan–Meier curves were compared with the log-rank test. (D)Non-fasting blood glucose maintained normoglycemia (16/20, 80%) after xenogeneic islets transplanted in the diabetic mice, and showed hyperglycaemia when the TEC removed (black arrow).(E) Another 300 xenogeneic islets were re-transplanted into diabetic mice again, which were failures for the acceptance of the xenograft. Three of four diabetic mice showing that hyperglycemia was ameliorated until the TEC was removed (black arrow). (F)Insulin stained donor allograft and xenograft in recipients TEC at 90d of posttransplant (shown brown), the islets were pointed by the red arrow (Scale bars:40 μm). National Natural Science Foundation of China (nos. 81172832 and 81771723). Special Program for Sichuan Youth Science and Technology Innovation (no. 2014TD0010). Sichuan Youth Science and Technology Foundation (no. 2013JQ0020). The Health and Family Planning Commission of Sichuan Province (no. 110190).

Effects of platelet-rich plasma on the pancreatic islet survival and function, islet transplantation outcome and pancreatic pdx1 and insulin gene expression in streptozotocin-induced diabetic rats

Platelet-rich plasma (PRP) is a therapeutic option in different fields based on its growth factors. We investigated influence of PRP on islet survival, function, transplantation outcomes, and pancreatic genes expression in diabetic rats. In vitro: pancreatic isolated islets were incubated with/without PRP then viability, insulin secretion, and content were assessed. In vivo: Series 1 were designed to determine whether islet treatment with PRP improves transplantation outcome in diabetic rats by evaluating plasma glucose and insulin concentrations and oxidative parameters. Series 2, effects of PRP subcutaneous injection were evaluated on pancreatic genes expression and glucose tolerance test in diabetic rats. PRP enhanced viability and secretary function of islet. Reduced glucose and malondialdehyde levels as well as increased insulin levels, superoxide dismutase activity, and expressions of pdx1 and insulin were observed in diabetic rats. PRP treatment has positive effects on islet viability, function, transplantation outcome, and pancreatic genes expression in diabetic rats.

2094-P: Effects of Irisin on Human Pancreatic Islets from Type 2 Diabetic Subjects

Irisin is a hormone secreted by skeletal muscle following physical activity and excess of saturated fatty acids, able to improve metabolic homeostasis and promote energy expenditure. In a previous study, we have demonstrated that, in beta-cells, irisin promotes proliferation and glucose-stimulated insulin secretion (GSIS), increases insulin content levels, and reduces lipotoxicity-induced apoptosis. The aim of this study was to evaluate the effects of irisin on the function and survival of pancreatic islets isolated from patients with type 2 diabetes mellitus (T2DM), characterized by reduced ability to secrete insulin and high levels of apoptosis, and to investigate the molecular mechanisms underlying this action. Pancreatic islets isolated from T2DM patients (n=7) and nondiabetic subjects (ND) (n=3), as well as INS-1E cells, were exposed to 100 nM irisin for different times. GSIS, insulin content, apoptosis and cytoplasmic calcium levels were measured by ELISA assays; the activation of the main mediators of beta-cell intracellular signalling (AKT, CREB and PKA) was evaluated by immunoblotting. Irisin increased GSIS approximately 2-fold (p&amp;lt;0.05) in both ND and T2DM islets. Notably, in T2DM islets, characterized by reduced GSIS, this was restored to the levels of ND islets. In addition, irisin increased insulin content (1.6-fold in ND, 1.9-fold in T2DM, p&amp;lt;0.05) and tendentially decreased apoptosis levels only in T2DM islets. Moreover, irisin was able to activate AKT and CREB under conditions of high glucose in the ND islets, without changing cytoplasmic calcium levels. In contrast, in the T2DM islets, irisin did not induce AKT or CREB activation. In conclusion, the myokine irisin is able to increase the secretory function and survival of human pancreatic islets and to improve the functional defects typical of the T2DM islets. The mechanisms underlying the secretagogue effects of irisin in the T2DM islets do not apparently involve the known signaling machinery of this novel hormone. Disclosure N. Marrano: None. A. Natalicchio: Consultant; Self; AstraZeneca, Novo Nordisk Inc., Sanofi-Aventis. G. Biondi: None. A. Cignarelli: None. L. Vincenti: None. L.G. Lupo: None. S. Perrini: None. L. Laviola: Advisory Panel; Self; Abbott, AstraZeneca, Boehringer Ingelheim International GmbH, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi-Aventis. Speaker’s Bureau; Self; Abbott, Medtronic. F. Giorgino: Advisory Panel; Self; AstraZeneca, Eli Lilly and Company, Novo Nordisk Inc., Roche Diabetes Care, Sanofi. Consultant; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., LifeScan, Inc., MedImmune, Merck Sharp &amp; Dohme Corp., Roche Diabetes Care, Sanofi. Research Support; Self; Eli Lilly and Company, LifeScan, Inc., Takeda Development Centre Europe Ltd. Funding European Union (AIM1810057)

66-LB: Combined Immunotherapy with T Regulatory Cells and Anti-CD20 Antibody Prolongs Survival of Pancreatic Islets in Type 1 Diabetes

Background: Immunotherapy using T regulatory cells (Tregs, Tr) prolongs remission in type 1 diabetes (T1DM) but the effect fades with the time. Here, we report phase II trial in which Tregs therapy was combined with anti-CD20 antibody (TrCD20) to make the effect more sustained. Methods: Thirty-six children with newly diagnosed T1DM were randomized 1:1:1 into the following groups: Tr, TrCD20 and control. The treatment in Tr group consisted of autologous expanded CD3+CD4+CD25highCD127- Tregs in the two doses (30x106 of Tregs/kg b.w. each), three months apart. TrCD20 group received additionally 4 doses of rituximab (375 mg/m2, each) between the first and second dose of Tregs. Control group was left without the treatment and all patients were followed for 2 years. Results: The best results were found in TrCD20 group, who was characterized by stable serum levels of both fasting C-peptide and stimulated C-peptide in glucagon and MMT tests throughout the entire trial (for fasting C-peptide only 20% decrease at +24months when compared to baseline). Tr group was characterized by stable levels of fasting and stimulated C-peptide up to +12months and then it dropped gradually until the end of the trial. As compared to control group, there was a better glycemic control measured as HbA1c and mean fasting glucose in both interventional groups throughout the whole follow up. TrCD20 group was in partial remission defined as insulin dose below 0.5IU/kg up to +21 months. The longest insulin independent follow up lasted 18months. The end of the remission in Tr group was noted at +18month and in controls at +12months. At the +24months daily insulin requirement and HbA1c levels were significantly lower in TrCD20 as compared to control group. Conclusions: The combined immunotherapy with Tregs and anti-CD20 antibody prolonged the most significantly survival of pancreatic islets in T1DM children. Trial registration: EudraCT: 2014-004319-35. Disclosure M. Zielinski: None. M. Zalinska: None. D. Iwaszkiewicz-Grzes: None. M. Gliwinski: None. M. Hennig: None. A. Jazwinska-Curyllo: None. H. Kaminska: None. J. Sakowska: Employee; Self; Poltreg S.A. R. Owczuk: None. W. Mlynarski: None. P. Jarosz-Chobot: None. A. Bossowski: None. A. Szadkowska: None. J. Siebert: None. M. Mysliwiec: Stock/Shareholder; Self; POLTREG SA. N.M. Marek-Trzonkowska: Stock/Shareholder; Self; Poltreg SA. P. Trzonkowski: Board Member; Self; Poltreg S.A. Stock/Shareholder; Self; Poltreg S.A. Funding National Centre for Research and Development in Poland (STRATEGMED1/233368/1/NCBR/2014)

Pilot study: deficiency of mannose-binding lectin-dependent lectin pathway, a novel modulator in outcome from pancreatic islet auto-transplantation.

BackgroundNumerous factors influence pancreatic islet survival following auto-transplantation. Of these, the host immune response in the early peri-operative period is one of the most important. In this study we investigated the role of the mannose-binding lectin (MBL)-dependent pathway in a group of total pancreatectomy (TP) islet auto-transplantation (TPIAT) patients and classified them as competent or deficient in MBL activity. Complement pathway activities, MBL protein and inflammatory cytokine concentrations were evaluated from eleven pancreatic islet auto-transplant patients from two institutions.MethodsEleven patients from two institutions were prospectively recruited. Serum was screened at different time points for 29 different cytokines and compared according to their MBL deficient or competent status. Twelve patients from previous TPIAT patients also underwent screening of MBL pathway activity.ResultsA total nine of twenty three patients (39%) were MBL pathway deficient. MCP-1, IL-7 and IL-1a concentrations were significantly lower in the MBL deficient cohort compared to the normal MBL group (P=0.0237, 0.0001 and 0.0051 respectively). IL-6 and IL-8 concentrations were significantly raised in the normal MBL group. MBL functional activity was lower in insulin-independent group compared to the insulin-dependent group.ConclusionsComplement activation is an important, possibly damaging response during intra-portal islet infusion. MBL pathway deficiency appears common in this population and the cytokine response was attenuated in MBL pathway deficient patients. Therapeutic MBL pathway blockade during and following islet auto-transplantation (IAT) may improve islet survival and function and thereby clinical outcome.