Pig Islets Research Articles

Reluctance of French patients with type 1 diabetes to undergo pig pancreatic islet xenotransplantation

Type 1 diabetes could possibly be treated by transplantation of pig pancreatic islets. In addition to medical difficulties and ethical problems, social hurdles may need to be overcome. We have evaluated the attitude of patients with type 1 diabetes to the xenotransplantation of pig pancreatic islets and to the potential risks associated with such treatment. A survey of 214 patients with type 1 diabetes was carried out in France based on a multiple-choice questionnaire. At first, 52.0% of these patients indicated that they would agree to receive pig islet xenografts. The main sources of reluctance were the ''risk of disease transmission'' (55.5%) and ''risks not yet identified'' (48.7%). After they were told of the risk of cancer or infection associated with immunosuppression, 74.9% of the respondents chose to refuse the transplant, compared with 48.0% before they heard of such risks. A 68.1% would refuse the xenotransplant if it would not exempt them completely from being treated by insulin injections. Discontinuing insulin injections was the most important priority for diabetic patients (73.5%), rather than limitation of diabetes-related complications (52.5%) or increase in life expectancy (44.0%). After they were informed of all of the risks associated with the procedure, 70.5% of the respondents decided they would rather not take any risks, and said they would refuse pig islet transplantation. When diabetic patients learned about potential infectious risks and other risks associated with immunosuppression, reluctance to undergo xenotransplantation gained in significance or even led to refusal of the procedure.

Impact of Porcine Islet Size on Cellular Structure and Engraftment After Transplantation

To study the impact of porcine islet size on structural properties and cellular engraftment. The endocrine structure and collagen/vascular localization in pig islets were studied before and after enzymatic isolation on the pancreas from 6 young and 6 adult Landrace pigs. Isolated islets from both pig types were transplanted under the kidney capsula of diabetic nude rats to assess cellular engraftment. In comparison with adult pig pancreata, a significantly greater number of small beta cells (<100 microm) were observed before and after isolation (82% vs. 32%, respectively, P < 0.005) from young pig pancreata. Small islets (<100 microm) showed a peripheral vascular structure, whereas large islets showed a more centralized vascular organization, thereby providing protection during the enzymatic digestion procedure. The islet endocrine structure was not affected by the islet size, but a loss of glucagon cells (-7.9%, P < 0.005) was observed in large isolated islets. The purity of islet preparation was better with pancreata from adult than young donors (86% vs. 64%, respectively, P < 0.05). A lack of engraftment was observed for small islets from young pig donors as compared with large islets from adult donors. Large and well-structured islets, mainly found in adult pig pancreata, probably possess a better potential for cellular engraftment due to centralized vascularization and collagen distribution.

Survival of adult islet grafts from transgenic pigs with N‐acetylglucosaminyltransferase‐III (GnT‐III) in cynomolgus monkeys

Because of a severe shortage of human donor pancreases, pig islets are considered to be an attractive donor source. Our previous in vitro study revealed that adult pig islets have strong non-Galalpha1-3Galbeta1-4GlcNAc-R (alpha-Gal) antigenicity, including the Hanganutziu-Deicher (H-D) antigen, especially in N-linked sugars. In this study, the issue of whether islets from N-acetylglucosaminyltransferase-III (GnT-III) transgenic pigs can prolong their survival in cynomolgus monkeys was examined. Adult pig islets were isolated from transgenic pigs with GnT-III and wild-type genes. GnT-III enzyme activity in pig islets was measured by high performance liquid chromatography (HPLC). The antigenicity of the islets to human natural antibodies was examined by flow cytometry. Pig islets were transplanted under the kidney capsule of streptozotocin-induced diabetic monkeys. After transplantation, blood samples were obtained and plasma insulin levels were monitored on a daily basis. While GnT-III was barely expressed in wild-type islets, it was expressed at high levels in islets from transgenic pigs, and xenoantigenicity was significantly reduced. There was a trend for islets isolated from GnT-III-transgenic pigs to survive longer than those from wild-type pigs in cynomolgus monkeys (wild type: 1, 1, and 3 days; GnT-III: 1, >3, 4 and 5 days). Humoral and histological studies indicated up-regulated anti-pig islet antibodies and a relatively high deposition in islet grafts from wild-type pigs, respectively. A reduction in xenoantigenicity by GnT-III may have prolonged the survival of porcine islets, suggesting the importance of non-alpha-Gal and non-H-D antigens, as they relate to N-linked sugars in the early rejection of porcine islets in the monkey. This approach may be useful in the clinical xenotransplantation of islets in the future.

Successful Pancreas Preservation by a Perfluorocarbon-Based One-Layer Method For Subsequent Pig Islet Isolation

The oxygenation of human pancreas by the two-layer method (TLM) during cold storage was recently established for clinical islet transplantation. Simplification of TLM would facilitate the application of perfluorocarbon (PFC) as a regularly used preservation solution for subsequent islet transplantation. The present study examined whether PFC can be used in a one-layer method (OLM) for long-term pancreas preservation before isolation of adult pig islets. Resected pancreases were intraductally flushed with cold University of Wisconsin solution and immediately processed (n=6) or subjected to 7-hour storage by OLM (n=8) or TLM (n=10). Subsequently, pancreases were intraductally distended with collagenase NB-8 supplemented with neutral protease. Isolation and purification were performed as previously described. Compared with unstored pancreases (3,670+/-740 islet equivalents [IEQ]) purified islet yield in TLM-stored organs (2,080+/-290 IEQ, P<0.05) was significantly decreased in contrast with OLM-preserved pancreases (3,110+/-520 IEQ, NS). No differences were observed between groups regarding purity (>90%), trypan-blue exclusion (>95%), adenosine triphosphate content, and mitochondrial viability of islets. Stimulation index during static glucose incubation (20 vs. 2.8 mm) was decreased after storage by TLM (1.81+/-0.20, P<0.05) but not by OLM (2.27+/-0.57) if compared with unstored pancreases (2.47+/-0.36). However, transplantation into diabetic nude mice resulted in sustained normoglycemia of recipients of either group until nephrectomy of graft-bearing kidneys was performed. This study demonstrates that PFC alone can be used in a one-layer procedure for successful pig-pancreas preservation. This simplification can facilitate the broad application of PFC as pancreas preservation solution without reducing its benefits demonstrated by TLM.

Influence of Collagenase Loading on Long-Term Preservation of Pig Pancreas by the Two-Layer Method for Subsequent Islet Isolation

The introduction of the two-layer method (TLM) for long-term human pancreas preservation revealed the enormous potential of TLM to improve graft function of isolated islets. It is still unclear whether pig islets can be successfully isolated from pancreases after prolonged cold ischemia. To clarify this question, pig pancreases were subjected to 7-hour preservation by University of Wisconsin solution (UWS) storage or TLM. Another aim was to verify whether TLM can be synergistically combined with intraductal collagenase injection before cold storage. After intraductal flush with UWS, organs were distended with 4.4 PZ-U/g of UWS-dissolved collagenase NB-8 and neutral protease adjusted to respectively 1.1, 0.2, 0.5, or 0.8 DMC-U/g for pancreases freshly procured (n=6) or distended with enzymes before (TLM preloaded, n=7) or after cold storage (UWS storage, n=4; TLM postloaded, n=10). Purified islet yield decreased from 429,200+/-86,700 islet equivalents (IEQ) in unstored pancreases to respectively 37,670+/-19620, 210,400+/-22900 and 238,000+/-26600 IEQ in UWS-stored (P<0.01), TLM-preloaded, or postloaded organs (P<0.05). Purity (>90%), viability (>95%), and insulin content were not different between groups. Islets from UWS-stored pancreases fragmented extensively, preventing further assessment of in vivo function. Compared with other experimental groups, islets from TLM-preloaded organs were characterized by enhanced basal and stimulated insulin release. Sustained normoglycemia was observed in diabetic nude mice transplanted with islets from TLM-postloaded or unstored pancreases in contrast with transient function in TLM-preloaded islets. This study demonstrates that significant amounts of intact pig islets can be isolated after prolonged pancreas preservation by TLM. Enzyme administration before TLM preservation decreases islet graft function.

Is the expression of Gal-α1,3Gal on porcine pancreatic islets modified by isolation procedure?

Aim The aim of this study was to study the Galactosyl α(1,3) Gal expression and the vascular tissue distribution prior to and after isolation of porcine pancreatic islets. Methods Biopsies of native pancreas were carried out in young (12–15 weeks; n = 4) and adult Landrace pigs (2 years old; n = 7). These pancreases were then digested (Liberase Porcine Islets [PI]) to obtain isolated pancreatic islets from each pancreas. Alpha Gal-specific biotinylated BS-1 isolectin B4 and von Willebrand's Factor (vWF) staining were performed for Galactosyl and vascular structure analysis, respectively. Quantitative Galactosyl expression as well as location of the vascular structure were determined using image analysis in pig islets of different sizes. Results Vascular structures and Galactosyl expression varied following the islet sizes but not the pig age. In large islets (>100 μm), capillaries were mainly located within the islets, whereas in small islets (<100 μm), 4-fold more vessels were situated at the periphery of the islets. Galactosyl expression followed a comparable distribution than vascular tissue in small and large islets. After isolation, a significant decrease of Gal staining (−49%) was observed, but Galactosyl expression remained positive within both small and large islets. Conclusions Galactosyl expression is maintained within pancreatic islets after isolation procedure. Gal knock-out pigs could represent the solution to this hurdle.

Adaption of neutral protease activity for islet isolation from the long-term two-layer method–stored pig pancreas

Background Islet release from the pancreas is mediated by both collagenase and neutral protease (NP), a critical effector of islet integrity. To prove the hypothesis that adjustment of NP reduces islet damage after prolonged ischemia, adult pig pancreata were digested after 7-hour preservation by the two-layer method (TLM) using a 2-component enzyme blend consisting of collagenase NB-8 and NP. Methods After intraductal University of Wisconsin (UW) flush resected pancreata were distended with 4.4 PZ-U/g of UW-dissolved Serva collagenase either before (TLM-preloaded, n = 7) or after (TLM-postloaded, n = 10) cold storage, or for immediate processing (n = 6). NP was adjusted after preliminary experiments to respectively 1.1, 0.2, or 0.8 DMC-U/g for unstored, TLM-preloaded, or postloaded organs. Results Purified islet yield decreased from 3670 ± 730 islet equivalents (IEQ)/g in unstored pancreata to 1800 ± 180 and 2080 ± 290 IEQ/g in TLM-preloaded or postloaded organs, respectively ( P < .05). Although purity was always >90%, IEQ recovery was significantly decreased in TLM-preloaded pancreata. Quality control revealed consistently high viability as determined using trypan-blue exclusion (>95%) or formazan production. Compared with unstored organs (2.47 ± 0.36; P < .05), glucose stimulation index was reduced in TLM-preloaded (1.48 ± 0.15) and TLM-postloaded pancreata (1.81 ± 0.20). Normoglycemia in diabetic nude mice transplanted with islets from TLM-preloaded pancreata was transient in contrast to sustained function in the other groups. Conclusions Significant amounts of viable pig islets can be isolated after prolonged TLM preservation by reducing NP activity. Nevertheless, early enzyme administration prior to long-term storage deteriorates islet graft function.

Long-term preservation of the pig pancreas by a one-layer method for successful islet isolation

Background Pancreas preservation by two-layer method (TLM) was recently established for clinical islet transplantation. The extensive use of TLM would require enormous efforts to solve logistical and technical problems. Omitting University of Wisconsin solution (UW) as second layer would facilitate the regular application of oxygenated perfluorocarbon; (PFC). To clarify whether long-term pancreas preservation is feasible by this simplified procedure, pancreases from retired breeder pigs were subjected to 7-hour preservation utilizing PFC alone in a one-layer method (OLM, n = 8) or in combination with UW (TLM, n = 10). Methods Resected pancreata were intraductally flushed with cold UW. Subsequently, pancreata were promptly processed ( n = 6) as previously described or stored by TLM or OLM. Results Compared to unstored (429200 ± 86700 IEQ) and OLM-stored pancreases (338600 ± 42100 IEQ), ( P = ns vs unstored) postpurification islet yield decreased after TLM storage (238000 ± 26600 IEQ, P < .05). No significant differences were found regarding purity (>90%), adenosine triphosphate (ATP) content, and viability as determined by formazan production and trypan-blue exclusion (>95%). Glucose stimulation index of freshly isolated islets (2.5 ± 0.4) was significantly decreased after TLM storage (1.8 ± 0.2, P < .05) but not after OLM storage (2.3 ± 0.6). Islet transplantation in diabetic nude mice demonstrated sustained graft function in all experimental groups. Conclusions This study demonstrates that viable pig islets can be successfully isolated after prolonged ischemia utilizing PFC alone for oxygenation of cold-stored pig pancreases. The easy handling of OLM could facilitate the regular application of PFC as pancreas preservation solution.

Survival of Microencapsulated Adult Pig Islets in Mice In Spite of an Antibody Response

The aim of this study was to assess the capacity of simple alginate capsules to protect adult pig islets in a model of xenotransplantation. Adult pig islets were microencapsulated in alginate, with either single alginate coats (SAC) or double alginate coats (DAC), and transplanted into the streptozotocin-induced diabetic B6AF1 mice. Normalization of glucose levels was associated with an improvement of the glucose clearance during intravenous glucose tolerance tests. After explantation, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated pig islets. Explanted capsules were mainly free of fibrotic reaction and encapsulated islets were still functional, responding to glucose stimulation with a 10-fold increase in insulin secretion. However, a significant decrease in the insulin content and insulin responses to glucose was observed for encapsulated islets explanted from hyperglycemic mice. An immune response of both IgG and IgM subtypes was detectable after transplantation. Interestingly, there were more newly formed antibodies in the serum of mice transplanted with SAC capsules than in the serum of mice transplanted with DAC capsules. In conclusion, alginate capsules can prolong the survival of adult pig islets transplanted into diabetic mice for up to 190 days, even in the presence of an antibody response.

Monitoring for Presence of Potentially Xenotic Viruses in Recipients of Pig Islet Xenotransplantation

This study represents a long-term follow-up of human patients receiving pig islet xenotransplantation. Eighteen patients had been monitored for up to 9 years for potentially xenotic pig viruses: pig endogenous retrovirus, pig cytomegalovirus, pig lymphotropic herpesvirus, and pig circovirus type 2. No evidence of viral infection was found.

Prolonged survival of fetal pig islet xenografts in mice lacking the capacity for an indirect response.

Xenografts of islets from organ-cultured fetal pig pancreases transplanted into non-immunosuppressed mice are rejected within 10 days. Immunosuppression with anti-T cell (anti-CD4) monoclonal antibody alone delays rejection of these xenografts for about 28 days, but rejection eventually occurs despite marked depletion of T cells. To determine if the critical CD4+ T cells responsible for xenograft islet rejection function through the direct or indirect pathway, selective class II-deficient mice that express class II antigens only on their thymic epithelium (not on peripheral cells) with normal numbers of CD4+ T cells, (class II-, CD4+), were used as recipients of xenograft islets to test if rejection occurs in the absence of an indirect response. Control (C57BL/6) or class II-, CD4+ mice were transplanted under the kidney capsule with cultured fetal pig islets. Class II-, CD4+ mice have normal numbers of B cells, CD4+, gamma delta T cells, and slightly increased numbers of CD8+ T cells. Additional mice were thymectomized before receiving anti-CD4 or anti-CD8 monoclonal antibodies. Islet graft survival was determined histologically as fetal pig islets were too immature to secrete insulin. Xenograft survival in control animals was 7 to 14 days. In contrast, graft survival in class II-, CD4+ mice was significantly prolonged to greater than 35 days. Depletion of CD8+ T cells in class II-, CD4+ mice prolonged graft survival to about 70 days. Depletion of CD4+ T cells from these mice further prolonged xenograft survival to about 100 days. These results suggest that the rejection of pig islets by mice initially depends on a CD4 dependent indirect response. The CD4 direct response also contributes to graft destruction. CD8+ T cells also participate in graft destruction, albeit weakly.

Prevalent human coxsackie B-5 virus infects porcine islet cells primarily using the coxsackie-adenovirus receptor.

We have previously demonstrated that transplanting porcine encephalomyocarditis virus (EMCV)-infected porcine islet cells (PICs) results in transmission of the virus to recipient mice, which is manifested by acute fatal infection within 5 to 8 days. Here, we determined PIC susceptibility to a related and highly prevalent human picornavirus, coxsackie B-5 virus (CVB-5). PICs were inoculated with CVB-5 in vitro for up to 96 hours and infectivity, level of virus replication, and cellular function determined. Subsequently, monoclonal and polyclonal antibody blocking experiments were used to investigate the receptor CVB-5 uses to enter PICs, and the ability of CVB-5-infected islets to reverse diabetes analyzed in mice. Adult pig islets inoculated with CVB-5 in vitro showed a typical picornaviral replication cycle with a 2-h lag phase followed by a 4-h exponential phase during which the virus titer increased by 4 logs. However, CVB-5 was less cytolytic to PICs than EMCV, resulting in a persistent productive infection lasting for up to 96 h, with minimal evidence of cell lysis. Double immunostaining confirmed the presence of CVB-5 antigens in insulin-producing islets. Infection of PICs in the presence of antibodies against human coxsackie-adenovirus receptor (CAR) resulted in near complete blockage in production of infectious virus particles whereas blocking with anti-porcine decay-accelerating factor (DAF, also called CD55) or anti-porcine membrane cofactor protein (MCP, also called CD46) only slightly decreased the number of infectious CVB-5 particles produced. Immunofluoresence staining showed CAR and MCP expression on the islet surface, but not DAF. Transplanting CVB-5-infected PICs into diabetic C57BL/6 mice resulted in reversal of diabetes. Although PICs are susceptible to human CVB-5, the infection does not appear to affect xenograft function in vitro or in vivo in the short term.

Biological activity of pig islet-cell reactive IgG antibodies in xenotransplanted diabetic patients.

The IgG antibody response in type I diabetic patients, transplanted with fetal pig islet-like cell-clusters, was investigated using purified immunoglobulin G (IgG) fractions from sera collected 7 to 9 yr after transplantation. From our earlier studies, we knew that the immunological specificities of xenoreactive IgG1 and IgG2 antibodies are different, and that IgG1 antibodies, in contrast to the IgG2 population, are mainly directed against non-Galalpha1,3Gal epitopes. In this study our goal was to establish whether xenoreactive IgG1 and IgG2 antibodies react with pig islet cells and, if so, to identify the target cell type, the biological function as well as the specificity of such antibodies for islet cell antigens. Sera from xenotransplanted patients were compared with those of patients with diabetes, selected for high titres of islet-cell specific autoantibodies. IgG antibody fractions from patient sera were purified on a protein G column. Surface expression of target antigens was studied using flow cytometry as well as immunofluorescence microscopy. The biological function of islet-cell reactive sera was tested using antibody dependent cellular cytotoxicity with both xenogeneic adult pig islet cells and allogeneic human islet cells as targets. Antibody specificity was assessed using 2D Western blots with both fetal and adult pig islet as well as human islet cell antigenic preparations. Some of the diabetic patients, who have been transplanted with xenogeneic fetal pig islet cells, continue to produce xenospecific IgG1 and IgG2 antibodies for 7 to 9 yr post-transplantation. A separate analysis of IgG1 and IgG2 antibodies showed that IgG1 antibodies react with pig islet beta cells, whereas IgG2 antibodies mainly react with non-endocrine pig cells. Such antibodies are xenospecific, as they were found to mediate antibody-dependent cellular cytotoxicity of adult pig, but not human islet target cells. The reverse was true for antibodies from non-transplanted diabetic patients with high titres of autoantibodies against beta cells. Fluorescence analysis as well as 2D gel Western blots revealed that the reactivity was variable between patient samples, indicating that target antigens for non-Galalpha1,3Gal-specific antibodies are heterogeneous. Thus, xenotransplantation of diabetic patients induces islet-beta cell reactive xenospecific IgG1 antibodies, which are biologically active and can mediate antibody-dependent cellular cytotoxicity of pig islet cells.

THREE MONTH IMMUNE PROTECTION OF MICROENCAPSULATED PORCINE ISLETS TRANSPLANTED IN PRIMATES WITHOUT IMMUNOSUPPRESSION: PRELIMINARY RESULTS

P1072 Aims: Survival of xenogeneic pig pancreatic islets into primates has been casuistically reported. Although the major hurdle for pig islets graft remains the control of primary non-function, acute vascular and cellular rejection, there is no uniform regimen which is able to overcome all immunological problems. The aim of this study was to assess the ability of alginate micro-encapsulation to protect islets in the gold standard discordant pig to primate model. Methods: Pancreases were obtained from adult (>2 years) Landrace pigs. All pig islets isolations were performed with Liberase PI following a static digestion method. Pig islets encapsulation with alginate solution (1% w/v), was carried out by vibrating nozzle and cross-linked with calcium ions (100mM). The mean size of capsules was 600-800μm and contained an average of three pig islets. After microencapsulation, pig islets (15,000 islets equivalent/kg) were transplanted under the capsule of both kidneys of non-diabetic Cynomolgus (n=2) and streptozotocin-induced diabetic primates (n=2). Porcine C-peptide and anti-pig IgM and IgG antibodies were assessed in serum prior and up to 90 days after transplantation by radioimmunoassay and flow cytometry, respectively. As controls, two animals received either non-encapsulated pig islets or empty capsules. Primate kidneys were removed after 30, 60 and 90 days post-transplantation, fixed in Bouin solution and immunostained for insulin. Viability of islets was assessed by Trypan blue and dithyzone staining. Results: The first non-diabetic animal underwent a total pancreatectomy 30 days after graft and serum porcine C-peptide was detected at 0.121ng/ml thereby demonstrating that islets could still produce C-peptide even without immunosuppression. This animal unfortunately died three days after surgery for unknown reasons. The two kidneys of the second primate were removed 60 and 90 days after graft. The alginate capsules were intact and a majority of islets were positive for dithyzone coloration and insulin immunostaining as well as negative by trypan blue. To induce diabetes in the third animal, high dose of STZ (130mg/kg) was used. After encapsulated pig islet graft, the non-fasting blood glucose decreased from 600 to 214 mg/dl day 2 after transplantation and the porcine C-peptide level went from 0 to 3.6 ng/ml. Unfortunately, this animal died on post-graft day 3 due toxic STZ side-effects (liver steatosis and acute tubular necrosis (creatinine 9mg/dl)). In the fourth primate, diabetes was induced with a lower STZ dose (55mg/kg). The kidneys were removed 30 and 60 days after graft. The alginate capsules were intact and insulin and dithyzone positive cells could be evidenced. One additional animal received empty capsules and the kidney removed 146 days after transplantation demonstrated the integrity of the capsules. The animal which received non-encapsulated pig islets demonstrated 10 days after the graft a complete destruction of the cells, a cellular infiltration and major fibrosis. In all these animals, but one which received empty capsules, there was a strong anti-porcine IgM and IgG antibody response thereby demonstrating the xenogeneic sensitization. Conclusions: A calcium-alginate microencapsulation is able to protect, for at least 90 days, pig islets again a strong discordant humoral immune response without any immunosuppression. Functional beta-cells were present after one month in two animals since swine C-peptide could be detected. This method must be confirmed in additional diabetic primates.

DOES ISOLATION PROCEDURE MODIFY THE EXPRESSION OF GAL ALPHA 1,3GAL ON PORCINE PANCREATIC ISLETS?

P1081 Aims: Gal alpha 1,3Gal (Gal) is the first target for antibody-mediated rejection in pig-to-non-human primate xenograft. The Galactosyl expression on porcine islets remains controversial but is usually reported on vascular endothelial cells in pig pancreas. Although the Gal expression in adult pigs has been reported as low or absent, it seems important to assess whether the Gal expression changed on pig islets from adult pigs prior to and after isolation procedure. In fact, the isolation procedure might damage the vascular structure in pig islets thereby eliminating the expression of Gal. Methods: This work was performed using adult pig (Landrace, 2 years old, n=9) pancreases. Biopsies, prior and after islets isolation were realized. The immunoperoxydase technique using the alpha Gal-specific biotinylated BS-1 isolectin B4 (1:25 dilution; Rayat, 1998) and von Willebrand’s Factor (vWF) were performed respectively for Gal and vasculature structure analysis. Transmission electron microscopy (TEM) was performed to analyse the endothelial ultrastructure prior to and after isolation. Image analysis was undertaken with Scion Image, Beta 4.02, acquisition and analysis software (SCION CORPORATION, Maryland, USA) to determine the percentage of Gal positive proportion and locate the vascular structure in pig islets. Islets were isolated by Liberase PI digestion following a static digestion method. Results: Vascular structure analysis of pig islets demonstrated that capillaries of large islets (>100 μm), essentially found in adult pig donors (mean diameter:169±10μm), were mainly located within the islets between the pancreatic beta cells. After isolation, immunohistochemistry in fact showed that vWF positive staining cells were located in the middle of freshly isolated large islets. Gal staining confirmed that islets endothelial cells essentially expressed Gal epitope and represented a proportion of 16±2% of total islets area in native pig pancreases. After isolation, however, a significant decrease of Gal staining (- 49%) was observed in pig islets but the expression was still positive even in adult pig islets. The decrease of Gal expression could be the consequence of enzymatic pancreatic digestion process at the level of peripheral capillaries (in common with exocrine tissue). Although vWF staining cells were found within islets after isolation, TEM has demonstrated that continuous capillaries structure was lost during isolation process and endothelial cells appeared as single spindle shapes cells. Conclusions: Adult pig islets still expressed the Gal epitope after isolation procedure. The availability of pig deficient in Gal expression as a source of islets may be beneficial for extending islets graft survival in non-human primate models and in humans.

ADULT PORCINE ISLET TRANSPLANTATION FROM N-ACETYLGLUCOSAMINYL-TRANSFERASE-III (GNT-III) TRANSGENIC PIGS IN CYNOMOLGUS MONKEYS

P1074 Introduction: Because of the severe shortage of human donor pancreases, pig islets has been considered as an attractive donor source. The findings of our previous in vitro study indicated that the level of expression of alpha-Gal by adult pig islets is very low, and that adult pig islets have non-alpha-Gal antigenicity, including H-D antigen, especially in terms of N-linked sugars. In addition, pig islets can be injured by both the classical and the alternative complement pathways in human serum. In the present study, we examined the issue of whether these humoral factors are involved in the early graft loss of islets in the monkey, using N-acetylglucosaminyltransferase-III (GnT-III) transgenic (Tg) pigs which revealed a significant down-regulation in xenoantigenicity and in cynomolgus monkeys.. Material and Methods: Preparation of pig islets. The pancreatic glands were removed from adult pigs. After removed, the gland was infused with a collagenase solution and distended. The gland was digested at 37 degrees C with static incubation. After digestion, purification was carried out by centrifugation on a Ficoll discontinuous gradient. The purified islets were cultured in Medium 199 supplemented with 10% porcine serum and antibiotics. GnT-III enzyme activity. GnT-III enzyme activity in pig islets was measured by high performance liquid chromatography (HPLC). Transplantation procedure. Recipient cynomolgus monkeys were injected with streptozotocin (STZ) at a dose of 150 mg/Kg/body weight (BW) 3 days before transplantation. Diabetes was then confirmed by checking the endogenous insulin production in the monkeys. After laparotomy, pelleted pig islets were transplanted under the left kidney capsule of the monkeys. Three monkeys were transplanted with islets from naive pigs (Group 1: naive), and five from GnT-III Tg pigs (Group 2: Tg). After transplantation, blood was sampled daily and plasma insulin was monitored to evaluate the graft function. Results: GnT-III was expressed at high levels in islets from Tg pigs (1628 684 p mol/h/mg protein) and xenoantigenicity, was reduced, while it was barely expressed in naive islets (10 p mol/h/mg protein). The survival time of the islets was estimated by the detection of insulin production in the recipient monkeys. Islets isolated from GnT-III Tg pigs survived longer than those from naive pigs in cynomolgus monkey, as follows: Group 1: < 1, < 1, and 3 day; Group 2: 1, 3, 4 and 5 day. Changes in anti-pig antibodies in the monkeys and histological analyses of the rejected islets are now under investigation. Conclusion: The data suggest that a reduction in xenoantigenicity by GnT-III can prolong the survival time of porcine islets, and that a humoral response is involved in the early rejection of porcine islets in the monkey.

LONG-TERM PRESERVATION OF THE PIG PANCREAS BY THE ONE-LAYER METHOD FOR SUCCESSFUL ISLET ISOLATION

P33 Aims: The oxygenation of the human pancreas during cold storage was recently established for clinical islet transplantation by introduction of the two-layer method (TLM). Nevertheless, simplification of TLM would facilitate its application as a broadly used pancreas preservation method for subsequent islet transplantation. Preliminary data about vascularized pancreas transplantation in rats and dogs indicate that successful preservation by a one-layer method (OLM) omitting UW solution (UWS) as the second layer is also possible. The purpose of the present study was therefore to examine the efficiency of OLM for pig islet isolation after 7 hours of cold ischemia. Methods: After resection adult pig pancreata were intraductally flushed with 2 mL/g of cold UWS. Subsequently, pancreata were either continuously oxygenated during cold ischemia by the TLM (n=10) or OLM (n=8), or processed promptly (n=6). Immediately before digestion-filtration pancreata were intraductally distended with cold UWS supplemented with Collagenase NB 8 and Neutral Protease NB (SERVA). Isolated islets were isolated and purified as previously described. Quality control included determination of islet yield expressed as islet equivalents (IEQ), isolation index (IEQ/islet number), trypan-blue exclusion, glucose stimulation index during static incubation (20 vs 2.8 mM glucose), insulin and ATP content, and mitochondrial activity measured as formazan production at 490 nm and expressed as optical density (OD). Islet in vivo function was evaluated after transplantation into diabetic nude mice. Results: No significant differences were found regarding islet purity (>90%) and viability (>95%). Compared to freshly procured pancreata (429200±86700 IEQ) postpurification islet yield was significantly decreased (P<0.05) after prolonged cold storage by TLM (238000±26600 IEQ). In contrast, islet yield was significantly higher (P<0.05) in cold-stored organs using OLM (338600±42100 IEQ, NS vs fresh). Metabolic islet integrity was not different between unstored pancreata and organs preserved by OLM or TLM as indicated by the intracellular insulin content (710±130 vs 830±120 vs 750±70 μU/IEQ), formazan production (0.7±0.1 vs 0.6±0.1 vs 0.7±0.1 OD/1000 IEQ) and ATP content (390±70 vs 370±80 vs 410±170 pg/IEQ). In contrast, glucose stimulation index was significantly decreased in islets isolated after TLM-storage (1.8±0.2, P<0.05) but not after OLM-storage (2.3±0.6, NS) compared to freshly isolated islets (2.5±0.4). Transplantation into diabetic nude mice demonstrated islet graft function in all experimental groups. Conclusions: The present study demonstrates clearly that viable pig islets can be successfully isolated from long-term stored pig pancreata utilizing perfluorochemicals as a single layer for oxygenation of pancreatic tissue during cold storage. This methodological simplification of the TLM might be of relevance for broad application in clinical islet transplantation.

ADAPTION OF NEUTRAL PROTEASE ACTIVITY FOR ISLET ISOLATION FROM THE LONG-TERM TLM-STORED PIG PANCREAS

P32 Aims: The enzymatic dissociation of pancreatic tissue by collagenase is a key step in the islet isolation procedure. Previous investigations clearly showed that purified collagenase needs additional protease activities to successfully release islets from the acinar tissue. We speculate that adjustment of neutral protease (NP) activity as a critical effector of islet integrity might be useful to reduce islet damage during digestion after long-term storage of the pancreas. To prove this hypothesis adult pig pancreata were digested by means of a new two-component collagenase blend consisting of Collagenase NB 8 and Neutral Protease NB (SERVA) after 7 hours of cold ischemia. Methods: After resection pancreata were intraductally flushed with 2 mL/g cold UW solution (UWS). Subsequently, pancreata were intraductally distended with Collagenase NB-8 (4.3 PZ-U/g) dissolved in cold UWS either before or after cold storage by the two-layer method (TLM). NP activity was adjusted according to collagenase administration to respectively 1.1, 0.2 or 0.8 DMC-U/g for pancreata freshly procured (n=6) or distended with collagenase before (TLM-preloaded, n=7) or after TLM storage (TLM-postloaded, n=10). Islets were isolated as previously described. Quality control included determination of islet yield expressed as islet equivalents (IEQ), isolation index (IEQ/islet number), trypan-blue exclusion, glucose stimulation index during static incubation (20 vs 2.8 mM glucose), insulin content, and mitochondrial activity measured as formazan production at 490 nm and expressed as optical density (OD). Islet in vivo function was evaluated after transplantation into diabetic nude mice. Results: Postpurification islet yield decreased significantly (P<0.05) from 429200±86700 IEQ in freshly procured pancreata to respectively 210400±22900 and 238000±26600 IEQ in TLM-preloaded or postloaded organs. As indicated by the isolation index (IEQ/islet number) islet integrity was preserved in TLM-preloaded (0.7±0.1) as well as in postloaded organs (0.7±0.1) compared to freshly procured pancreata (0.9±0.1). No significant differences were found regarding islet purity (>90%) and viability (>95%). Cold storage significantly reduced glucose stimulation index of islets isolated from TLM-preloaded (1.5±0.2) and postloaded organs (1.8±0.2) compared to freshly isolated islets (2.5±0.4, P<0.05). No differences were observed between unstored organs, TLM-preloaded or postloaded pancreata regarding insulin content (710±130 vs 680±30 vs 750±70 μU/IEQ) and mitochondrial activity (0.7±0.1 vs 0.8±0.1 vs 0.7±0.1 OD/1000 IEQ). Transplantation into diabetic nude mice revealed islet graft function in all experimental groups. Conclusions: To our knowledge pig pancreata have never been subjected to 7 hours of cold storage prior to islet isolation before. The present study demonstrates that pig islets can be successfully isolated after prolonged periods of cold storage by the TLM if proteolytic activity is adapted according to the timing of collagenase administration.

Comparison of Fetal Porcine Aggregates of Purified β-Cells versus Islet-Like Cell Clusters as a Treatment of Diabetes

Fetal pig islet-like cell clusters (ICCs) have the potential to reverse diabetes 1-5 months after transplantation. In a fetal ICC, however, beta-cells constitute only 6-8% of the cells, in contrast to 65% in an adult pig islet. Attempts to purify fetal beta-cells from cell clusters and compare their function to that of ICCs have not been shown previously. Beta-cells were purified from ICCs isolated from the fetal pig pancreas. These were then aggregated and maintained in culture for 3 days. ICCs were isolated from fetal pig pancreas and allowed to round up in culture for 3 days. Transplantation of aggregates and ICCs (10,000 and 12,600, respectively) into diabetic immunoincompetent mice resulted in normoglycemia at 18 +/- 2 and 8 +/- 1 weeks, respectively (p = 0.0006). Removal of grafts after normalization of blood glucose levels resulted in rapid return of hyperglycemia in both groups. In conclusion, a purified population of immature beta-cells can be produced from the fetal pig pancreas. The reason these cells take longer than ICCs to reverse diabetes when transplanted is postulated to be because of the relative lack of precursor cells from which beta-cells differentiate. This finding may have implications for stem cell therapy, as other cell types, other than purified beta-cells, may be necessary for appropriate function in vivo.

IN JUVENILE PIGS, DIABETES INDUCTION BY SREPTOZOTOCIN IS NOT AN APPROPRIATE MODEL FOR ISLETS TRANSPLANTATION BUT IS AN INTERESTING “IN VIVO” MODEL FOR PANCREATIC DUCTAL TRANSDIFFERENTIATION

P1191 Aims: In order to investigate the function of pancreatic islets transplantation in allogeneic and xenogeneic large animal models, induction of diabetes mellitus is needed. In this experimental study, we searched the most appropriate tool for induction of diabetes in both pig and primate models: Streptozotocin (STZ) or total pancreatectomy (TP). Methods: In juvenile pigs (20 weeks), multiple (2-3) low doses STZ (40mg/kg iv, n=4), one high dose STZ (150mg/kg iv, n=9) and total pancreatectomy (n=3) were tested to induce diabetes. Two different intravenous STZ doses were tested in Cynomolgus monkeys (50mg/kg (n=5) vs. 135mg/kg (n=2)). Hepatic enzymes (AST, ALT), renal function (creatinine), intra-venous glucose tolerance test (IVGTT: Area under the curve, AUC), non-fasting blood glucose and animal weight were assessed prior to, one and 4 weeks after diabetes induction. Beta cell destruction was evaluated by immunohistochemistry with insulin staining and non-beta cells by somatostatin, glucagon and pancreatic polypeptide staining. Results: Single or multiple low doses of STZ did not induce diabetes or side-effects in four in juvenile pigs (normal AST, ALT and creatinine). The three pigs which underwent TP became rapidly and definitively (4-5 days) hyperglycaemic and polyuric. Total pancreatectomy provoked an irreversible and pathological IVGTT (AUC: 45,513 ± 3,021 mg/dl/min) as compared to controls (n=9) (8,094 ± 1,902 mg/dl/min) (p<0.005). Similarly, one high dose STZ induced in pigs a significantly pathological IVGTT after one week (n=5) (32,134 ±7,037 mg/dl/min) (p<0.05). Four weeks after one high dose STZ injection, however, a significant decrease of AUC calculated after IVGTT was observed in six pigs tested (20,562±7,261 mg/dl/min) (p<0.005), thereby suggesting the survival or regeneration of beta cells. In fact, all the pancreas from STZ-treated pigs analysed after 4 weeks demonstrated the presence of small beta cells located at distance of vascular structure (co-staining insulin/von Willebrand factor). Moreover, a significant beta cellular hypertrophy could be evidenced between one and 4 weeks after STZ (98±7 μm2 vs. 120±12μm2) (p<0.05). Last but not least, a clear ductal-beta cell transdifferentiation (co-staining insulin/cytokeratin 19) was broadly observed in pancreas harvested 4 weeks after STZ. Cynomolgus treated with high dose STZ (135mg/kg (n=2) rapidly demonstrated an increase of hepatic enzymes (>200 IU/l)) and creatinine (>6mg/dl) due to liver steatosis and kidney tubular necrosis. These animals eventually died within two weeks after injection. Low dose of STZ (55mg/kg) induced an increase of non-fasting blood glucose (>200mg/dl), a pathological IVGTT (AUC: 5,534±747 mg/dl/min) as compared with untreated animals (13,005±3,084 mg/dl/min) (p<0.05). This STZ regimen did not induce any hepatic or renal deterioration and no residual serum C-peptide could be detected. In these animals, pathological IVGTT was confirmed 3 months after low dose STZ injection and the insulin-deficient state could be related to massive amyloid polypeptide deposits in islets. Conclusions: Low-dose STZ successfully induces irreversible diabetes in monkeys but not in young pigs. High dose STZ induces transient hyperglycaemic state in pigs but no irreversible diabetes due to a hypertrophy of beta cells and ductal transdifferentiation. This result suggests that high dose STZ could be used as a model of “in vivo” ductal differentiation. In juvenile pigs, TP seems to be the only choice for diabetes induction model for studying pig islets transplantation.