Caspase inhibitor therapy enhances marginal mass islet graft survival and preserves long-term function in islet transplantation

Islet transplantation can provide insulin independence in patients with type 1 diabetes, but islets derived from two or more donors are often required. A significant fraction of the functional islet mass is lost to apoptosis in the immediate posttransplant period. The caspase inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethyl ketone (zVAD-FMK) has been used therapeutically to prevent apoptosis in experimental animal models of ischemic injury, autoimmunity, and degenerative disease. In the current study, zVAD-FMK therapy was examined in a syngeneic islet transplant model to determine whether caspase inhibition could improve survival of transplanted islets. zVAD-FMK therapy significantly improved marginal islet mass function in renal subcapsular transplantation, where 90% of zVAD-FMK-treated mice became euglycemic with 250 islets, versus 27% of the control animals (P < 0.001). The benefit of zVAD-FMK therapy was further demonstrated after intraportal transplantation, where 75% of zVAD-FMK-treated animals established euglycemia with only 500 islets, and all of the controls remained severely diabetic (P < 0.001). zVAD-FMK pretreatment of isolated islets in the absence of systemic therapy resulted in no significant benefit compared with controls. Long-term follow-up of transplanted animals beyond 1 year posttransplant using glucose tolerance tests confirmed that a short course of zVAD-FMK therapy could prevent metabolic dysfunction of islet grafts over time. In addition, short-term zVAD-FMK treatment significantly reduced posttransplant apoptosis in islet grafts and resulted in preservation of graft insulin reserve over time. Our data suggest that caspase inhibitor therapy will reduce the islet mass required in clinical islet transplantation, perhaps to a level that would routinely allow for insulin independence after single-donor infusion.     

Gene transfection and expression of transforming growth factor-beta1 in nonobese diabetic mouse islets protects beta-cells in syngeneic islet grafts from autoimmune destruction

Nonobese diabetic (NOD) mice develop diabetes and destroy syngeneic islet grafts through an autoimmune response. Because transforming growth factor (TGF)-beta1 downregulates immune responses, we tested whether overexpression of TGF-beta1 by gene transfection of NOD mouse islets could protect beta-cells in islet grafts from autoimmune destruction. NOD mouse islet cells were transfected with an adenoviral DNA expression vector encoding porcine latent TGF-beta1 (Ad TGF-beta1) or the adenoviral vector alone (control Ad vector). The frequency of total islet cells expressing TGF-beta1 protein was increased from 12 +/- 1% in control Ad vector-transfected cells to 89 +/- 4% in Ad TGF-beta1-transfected islet cells, and the frequency of beta-cells that expressed TGF-beta1 was increased from 12 +/- 1% to 60 +/- 7%. Also, secretion of TGF-beta1 was significantly increased in islets that overexpressed TGF-beta1. Ad TGF-beta1-transfected NOD mouse islets that overexpressed TGF-beta1 prevented diabetes recurrence after transplantation into diabetic NOD mice for a median of 22 days compared with only 7 days for control Ad vector-transfected islets (p = 0.001). Immunohistochemical examination of the islet grafts revealed significantly more TGF-beta1+ cells and insulin+ cells and significantly fewer CD45+ leukocytes in Ad TGF-beta1-transfected islet grafts. Also, islet beta-cell apoptosis was significantly decreased whereas apoptosis of graft-infiltrating leukocytes was significantly increased in Ad TGF-beta1-transfected islet grafts. These observations demonstrate that overexpression of TGF-beta1, by gene transfection of NOD mouse islets, protects islet beta-cells from apoptosis and autoimmune destruction and delays diabetes recurrence after islet transplantation.     

Influence of postdiabetic onset time and immunosuppressive treatment on islet grafts in the spontaneous diabetic BB/W rat

1000 islets from one donor, isolated with a modified neutral red method, were intraportally transplanted from BB/Wistar or Lewis (RT1) donors to short- and long-term diabetic BB/Wistar recipients. Recipients received immunosuppression with cyclosporine i.m.--either postoperative 4 X 25 mg/kg body weight or pre- and postoperative 3 X 10 mg and 4 X 25 mg/kg body weight. Graft survival in short-term diabetic recipients was significantly shorter than in long-term diabetics, whether they were immunosuppressed or not. Especially in recent onset recipients, the additional preoperative immunosuppression with cyclosporine provided longer graft survival than postoperative cyclosporine alone. Histological examinations of islet grafts showed eosinophilic cells in all transplanted islets, in both functioning and clinically rejected grafts of allogenic and "pseudoisogenic" transplanted recipients. a coincidence of eosinophilic cells with the reenactment of the original autoimmune disease in islet grafts seems possible.     

Markedly decreased oxygen tension in transplanted rat pancreatic islets irrespective of the implantation site

In this study, we syngeneically transplanted islets to three different implantation sites of diabetic and nondiabetic rats, then 9-12 weeks later we measured the blood perfusion and compared the tissue partial pressure of oxygen (PO2) levels of these transplanted islets to endogenous islets. Modified Clark microelectrodes (outer tip diameter 2-6 microm) were used for the oxygen tension measurements, and islet transplant blood perfusion was recorded by laser-Doppler flowmetry (probe diameter 0.45 mm). The islet graft blood perfusion was similar in all islet grafts, irrespective of the implantation site. In comparison, the three implantation organs (the kidney cortex, liver, and spleen) differed markedly in their blood perfusion. There were no differences in islet graft blood perfusion between diabetic and nondiabetic recipients. Within native pancreatic islets, the mean PO2 was approximately 40 mmHg; however, all transplanted islets had a mean PO2 of approximately 5 mmHg. The oxygen tension of the grafts did not differ among the implantation sites. In diabetic recipients, an even lower PO2 level was recorded in the islet transplants. We conclude that the choice of implantation site seems less important than intrinsic properties of the transplanted islets with regard to the degree of revascularization and concomitant blood perfusion. Furthermore, the mean PO2 level in islets implanted to the kidney, liver, and spleen was markedly decreased at all three implantation sites when compared with native islets, especially in diabetic recipients. These results are suggestive of an insufficient oxygenization of revascularized transplanted islets, irrespective of the implantation site.     

Differences in suppressor of cytokine signaling-1 (SOCS-1) expressing islet allograft destruction in normal BALB/c and spontaneously-diabetic NOD recipient mice

BACKGROUND: The ability to block interferon signaling represents an important strategy in designing therapies to prevent beta-cell destruction during islet allograft rejection. METHODS: The SOCS proteins regulate cytokine signaling by blocking activation of JAK/STAT proteins. Using islets isolated from SOCS-1 transgenic mice (SOCS-1-Tg; these mice express SOCS-1 under the control of the human insulin promoter and are on the C57BL6/J background), we investigated whether SOCS proteins can prevent the destruction pancreatic islet cells transplanted beneath the kidney capsule of major histocompatibility complex mismatched normal BALB/c and spontaneously-diabetic NOD mouse recipients. RESULTS: Immunohistochemical staining for insulin confirmed the presence of donor SOCS-1-Tg islets in islet allografts harvested at 22 days posttransplant, whereas grafts of control non-Tg islets were destroyed by 14 days. In contrast, SOCS-1-Tg allogeneic islets were not protected from beta-cell destruction in clinically diabetic NOD mice. The islet allografts functioned for 1 week posttransplant; however, hyperglycemia returned after 2 weeks and the grafts were destroyed. Rejection of SOCS-1-Tg and non-Tg islets in autoimmune diabetic NOD mice was associated with an infiltrate of both CD4+ and CD8+ T cells and a T2-type cytokine response (IL-4) rather than the conventional T1-type cytokine response observed during islet allograft rejection. Self-antigen upregulation in response to IFN-gamma stimulation did not appear to be a factor in rejection of the islet allografts. CONCLUSIONS: These results demonstrate that expression of SOCS-1 in islets delays islet allograft rejection but cannot circumvent destruction of the islets by the recurrence of the tissue-specific autoimmune process of spontaneous diabetes.     

Effect of liver transplantation on islet allografts: up-regulation of Fas ligand and apoptosis of T lymphocytes are associated with islet graft tolerance

BACKGROUND: Liver allografts in spontaneously tolerant strain combinations can protect other organs of the same donor origin from rejection and reverse ongoing rejection in previously placed grafts. The aims of this study were to examine whether liver allografts have the same protective effect on islet allografts and to investigate the underlying mechanisms. METHODS: PVG islets were transplanted beneath the kidney capsule of streptozotocin-induced diabetic DA rats with or without liver allografting. The cellular infiltrate, and the extent of apoptosis and of Fas ligand (FasL) expression in the islet grafts were evaluated on days 2, 4, and 7 after transplantation by means of immunostaining and the in situ terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay. Donor and recipient mixed lymphocyte reactions (MLR) were determined at 7 days or 100 days after islet transplantation. RESULTS: Islet allografts transplanted alone were rapidly rejected within 5-8 days. Rejection was delayed, but not prevented, when islets were transplanted simultaneously with the liver. Liver transplantation 1 month before islet transplantation resulted in long-term survival (>100 days) of islet grafts in three of seven animals, whereas the other four died of liver rejection with functional islet grafts. Liver transplantation on day 4 after islet grafting reversed ongoing islet rejection and led to indefinite islet graft survival in three of seven cases. There was a progressive increase of cellular infiltration in all of the islet allografts, but the intensity of the infiltrate did not correlate with the outcome of the islet allografts. Islet rejection was characterized by an early dominance of monocytes/macrophages and CD25+ T cells in the infiltrates, a high incidence of apoptotic beta cells in grafts, and a sensitized status in the MLR. Tolerance of islet allografts was associated with increased numbers of dendritic cells in the graft infiltrates, up-regulation of FasL, and prominent apoptosis of alloreactive leukocytes in the islet grafts, as well as donor-specific MLR suppression in long-term survivors. CONCLUSIONS: These results demonstrate that the extent of the protective effect of liver transplantation on islet allografts varies with the time of liver grafting, ranging from delay in islet rejection to complete islet acceptance. Islet graft tolerance induced by liver transplantation is the result of an immune process that involves up-regulation of Fas ligand expression on, and apoptosis of, islet graft infiltrating lymphocytes.     

Multiple donor allotransplantation. A new approach to pancreatic islet transplantation

Currently it is not feasible to isolate sufficient numbers of islets from a single pancreas for clinical transplantation. We examined whether small numbers of islets obtained from multiple donors could be used for transplantation. Islets were isolated from four inbred strains of mice (DBA/2, DBA/1, C3H, and A.SW) by a stationary collagenase digestion and Ficoll separation and transplanted into the renal subcapsular space of streptozotocin-induced diabetic B6AF1 mice. At least 200 handpicked islets were required to produce normoglycemia in syngeneic and allogeneic diabetic recipient mice. None of the mice given 50 islets became normoglycemic within 2 weeks postgrafting. When various numbers of purified islets from a single donor were transplanted, the survival was significantly better for 200-islet allografts than for 800-islet and 400-islet allografts. When a 200-islet composite graft was prepared from four donors (50 fresh handpicked islets from each donor), the survival of the composite graft as measured by sustained normoglycemia in nonimmunosuppressed recipients was dramatic, with 17 of 18 recipients maintaining normoglycemia indefinitely (greater than 200 days). Similarly, when islets isolated from four donors and cultured for various periods were mixed and transplanted (200 islets/recipient) all recipient mice (n = 8) enjoyed indefinite graft survival. Use of higher numbers of purified islets or crude islets in a composite multiple-donor islet allograft was less effective in achieving indefinite graft survival. Thus, transplantation of a composite graft made up with subtherapeutic numbers of islets from multiple histoincompatible donors to provide adequate therapeutic numbers is a practical solution to the lack of islet availability. In addition, composite islet grafts appear to possess immunological advantages, with significantly prolonged survival over that produced by single-donor islet grafts.     

Gene expression of VEGF and its receptors Flk-1/KDR and Flt-1 in cultured and transplanted rat islets

BACKGROUND: Vascular endothelial growth factor (VEGF) and its two receptor tyrosine kinases, Flk-1/KDR and Flt-1, may play an important role in mediating the revascularization of transplanted pancreatic islets. METHODS: Using semiquantitative multiplex reverse-transcribed polymerase chain reaction we determined the gene expression of VEGF and its receptors in cultured and transplanted rat islets. RESULTS: After exposure of islet cells to hypoxia in vitro, increases were found in the gene expression of the VEGF120 and VEGF164 isoforms, with simultaneous increases in VE-cadherin, Flk-1/KDR, and Flt-1. In vivo studies consisted of analysis of islet grafts transplanted into both normal and diabetic recipients. Expression of both VEGF120 and VEGF164 in grafts was up-regulated for the first 2-3 days after transplantation, with the response being more prolonged in the diabetic rats. These increases were followed by reduced expression of VEGF on days 5, 7, and 9. Increases in the expression of VE-cadherin in islet grafts in normal and diabetic recipients tended to parallel VEGF expression, with the increases in both probably being caused by hypoxia. The early increases of VEGF expression were followed by a rise in the expression of VEGF receptors, which probably represents the early stages of angiogenesis. Graft expression of Flk-1/KDR and Flt-1 was enhanced at 3 and 5 days in the normoglycemic recipients, while in the diabetic recipients increases were found later on days 5, 7, and 14. CONCLUSIONS: The delayed expression of VEGF receptors in the diabetic recipients could reflect impaired angiogenesis caused by the diabetic milieu; this delay could contribute to the less outcomes of grafts transplanted into a hyperglycemic environment.     

Magnetic resonance imaging study of mouse islet allotransplantation

Although only 10% of islet transplant recipients maintain insulin independence, 80% of them are C-peptide positive at 5 years. To better understand the fate of transplanted islets, a magnetic resonance imaging (MRI) technique has been used to detect superparamagnetic iron oxide (SPIO)–labeled transplanted islets. Recently, we successfully used a novel MRI contrast agent, chitosan-coated SPIO (CSPIO) nanoparticles, to monitor mouse islet isografts for 18 weeks after transplantation. In the present study, we tested whether CSPIO could be applied to monitor islet allografts, which are supposedly rejected without immune interventions. Male C57BL/6 and Balb/c mice were used as donors and recipients of islet transplantation, respectively. After overnight incubation with or without CSPIO (10 μg/mL), 300 C57BL/6 islets were transplanted under the left kidney capsule of each Balb/c mouse. Starting from day 10 after transplantation, 3.0-Tesla MRI of the recipients was performed weekly. Four mice were followed for ≥38 days. At 38 and 45 days, 1 islet graft was removed for insulin and Prussian blue staining, respectively. From days 10 to 45 after transplantation, CSPIO-labeled islet grafts were visualized on MRI scans as sustained distinct hypointense spots homogeneously located at the upper pole of left kidney, the site of transplantation. At days 38 and 45, the histology of CSPIO-labeled islet grafts revealed insulin and iron staining colocalized in the same areas. Our results in a mouse allotransplantation model indicated that CSPIO-labeled islets survived as long as 45 days with positive MRI.     

Magnetic Resonance Imaging of Transplanted Mouse Islets Labeled With Chitosan-Coated Superparamagnetic Iron Oxide Nanoparticles

Although only 10% of islet recipients maintain insulin independence, 80% of them are C-peptide positive at 5 years after transplantation. To better understand the fate of transplanted islets, a magnetic resonance imaging (MRI) technique has been used to detect Feridex-labeled islet grafts in rodents. In this study, we used a novel MRI contrast agent, chitosan-coated superparamagnetic iron oxide (CSPIO) nanoparticles, to monitor mouse islet grafts. Male inbred C57BL/6 mice were used as donors and recipients of islet transplantation. The islet cytotoxicity was evaluated by fluorescein diacetate and propidium iodide staining for RAW cells incubated with CSPIO. After being incubated overnight with and without CSPIO (10 mg/mL), 300 islets were transplanted under the left kidney capsule of each mouse. After transplantation, 3.0-Tesla MRI of the recipients was performed biweekly until 19 weeks. At the end of study, the islet graft was removed for insulin and Prussian blue staining. The cell death rates in RAW cells did not increase with increasing CSPIO concentrations or incubation time. The grafts of CSPIO-labeled islets were visualized on MRI scans as distinct hypointense spots homogeneously located at the upper pole of left kidney. Their MRI signal was 30%-50% that of control islets and was maintained throughout the follow-up period. At 18 weeks, the histology of CSPIO-labeled islet graft revealed the insulin- and iron-stained areas to be almost identical. Our results indicate that isolated mouse islets labeled with CSPIO nanoparticles can be effectively and safely imaged by using MRI as long as 18 weeks after transplantation.     

Rejection of islets versus immediately vascularized pancreatic allografts: a quantitative comparison

It has been suggested that free grafts of islets are rejected more vigorously than immediately vascularized intact organs grafts. However, the physiological manifestations of rejection depend, in part, upon the functional reserve of the transplanted tissue. If the number of islets transplanted is just adequate to maintain normoglycemia, the immune destruction of only a few islets will be manifested by hyperglycemia. Thus, differences in rejection time could be an artifact of the islet mass transplanted. We compared the onset of rejection of immediately vascularized segmental pancreatic grafts and of free grafts of islets under conditions in which the β cell mass transplanted, as determined by tissue insulin content, was equivalent. Lewis rats, made diabetic (plasma glucose > 400 mg/dl) by streptozotocin, received either free islet allografts by portal embolization or vascularized segmental pancreatic allografts derived from Fischer donors. Identical pancreatic segments that were not transplanted had a mean (± SE) total tissue insulin content of 33 ± 3 μg. The mean total insulin content of Fischer islets prepared by collagenase digestion in a quantity identical to that used for transplantation to single recipients was 35 ± 7 μg. Similar measurements were made in Fischer to Fischer and Lewis to Lewis isograft control groups. Recipients of both segmental pancreas and free islet grafts became normoglycemic after transplantation and this state was sustained indefinitely in recipients of syngeneic grafts. In rats receiving allografts, the day of rejection, defined as an elevation of plasma glucose to >200 mg/dl, occurred at a mean of 12.1 ± 0.3 days for recipients of pancreatic grafts (n = 17) and 5.2 ± 0.3 days in recipients of islet grafts (n = 17) (P < 0.001). The functional survival of free grafts of allogeneic islets is less than that of islets contained within immediately vascularized pancreatic grafts, even when the transplanted β cell mass is equivalent. However, this difference could still be due to nonimmunologic, quantitative factors that influenced the rate with which hyperglycemia occurred after initiation of the rejection process. The insulin content in the livers of islet isograft recipients showed that only 53 to 71% of the transplanted islets survived. Further experiments that compensate for this factor are needed to determine whether or not there are differences in susceptibility to rejection of the two types of grafts. Nevertheless, on the basis of the number of donors used per recipient, islet allotransplantation is less efficient than pancreas allotransplantation.     

Increased numbers of low-oxygenated pancreatic islets after intraportal islet transplantation

OBJECTIVE

No previous study has measured the oxygenation of intraportally transplanted islets, although recent data suggest that insufficient engraftment may result in hypoxia and loss of islet cells.

RESEARCH DESIGN AND METHODS

After intraportal infusion into syngeneic mice, islet oxygenation was investigated in 1-day-old, 1-month-old, or 3-month-old grafts and compared with renal subcapsular grafts and native islets. Animals received an intravenous injection of pimonidazole for immunohistochemical detection of low-oxygenated islet cells (pO₂ <10 mmHg), and caspase-3 immunostaining was performed to assess apoptosis rates in adjacent tissue sections.

RESULTS

In the native pancreas of nontransplanted animals, ∼30% of the islets stained positive for pimonidazole. In 1-day-old and 1-month-old grafts, the percentage of pimonidazole-positive islets in the liver was twice that of native islets, whereas this increase was abolished in 3-month-old grafts. Beneath the renal capsule, pimonidazole accumulation was, however, similar to native islets at all time points. Apoptosis rates were markedly increased in 1-day-old intrahepatic grafts compared with corresponding renal islet grafts, which were slightly increased compared with native islets. One month posttransplantation renal subcapsular grafts had similar frequencies of apoptosis as native islets, whereas apoptosis in intraportally implanted islets was still high. In the liver, islet graft vascular density increased between 1 and 3 months posttransplantation, and apoptosis rates simultaneously dropped to values similar to those observed in native islets.

CONCLUSIONS

The vascular engraftment of intraportally transplanted islets is markedly delayed compared with renal islet grafts. The prolonged ischemia of intraportally transplanted islets may favor an alternative implantation site.Long-term outcome after clinical islet transplantation still remains poor, with few patients being insulin-independent 5 years posttransplantation (1). Several factors contribute to the deterioration of islet graft function, including instant death of infused islets (2), recurrence of disease (3), acute or chronic rejection (4), and insufficient revascularization (5,6).The endogenous islets are richly vascularized, with no cell more than one cell away from arterial blood (7). The islet organ has a high blood flow, although the blood perfusion of different islets varies (8). Moreover, the islet β-cells are highly dependent on their vasculature for nutrient metabolism and insulin release (9), and pretransplantation islet isolation and culture result in rapid degeneration of islet microvasculature (10).At transplantation, islets completely depend on diffusion of nutrients and oxygen from the surrounding tissue. Thus, islet size, graft composition, and implantation organ oxygen levels may influence graft oxygenation. Posttransplantation new islet microvessels are formed, but the vascular density of native islets is not restored (5,6), which results in anaerobic metabolism in renal subcapsular grafts (11). In clinical islet transplantation, islets are, however, infused into the portal vein and dispersed into the liver. The oxygen diffusion properties are probably different between 200–250 islets implanted subcapsularly (12) and the intraportal route, where only one or a few islets engraft in the same area of liver tissue. Hitherto, vascular density has been reported to be decreased after intraportal islet transplantation (5,6), but the islet graft oxygen tension has only been recorded in liver subcapsular multi-islet grafts (12).The current study aimed to investigate the oxygenation of intraportally transplanted islets. For this purpose, we used the oxygen-dependent bioreductive metabolism of pimonidazole to localize endogenous and transplanted islets with low oxygenation.     

TLR4 Mediates Early Graft Failure After Intraportal Islet Transplantation

We have previously shown that islet emboli in the portal vein block blood flow and induce local inflammatory reaction, resulting in functional loss of islet grafts following intraportal transplantation. This study was designed to test whether Toll‐like receptor (TLR) activation mediates early islet graft failure. Syngeneic islet grafts were transplanted into chemically induced diabetic mice, and TLR deficient mice were used as donors and/or recipients of islet grafts. Islet viability, proinflammatory cytokines, high‐mobility group box‐1 (HMGB1) and NF‐κB activation were analyzed by bioluminesce imaging (BLI), quantitative RT‐PCR (qRT‐PCR) and histology. Early islet graft failure was observed in mice with intraportal islet engrafts with increased proinflammatory cytokines, HMGB1 expression, NF‐κB activation, caspase‐3 and TUNEL positive cells. Deficiency of TLR4 in donor, but not in recipient, inhibited NF‐κB activation, reduced proinflammatory cytokines and improved viability of islet grafts. Blockade of HMGB1 with anti‐HMGB1 monoclonal antibody (mAb, 2g7) inhibited inflammatory reactions, as evidenced by reduced TNFα and IL‐1ß production, and improved islet viability. We conclude that TLR4 activation mediates early graft failure following intraportal islet transplantation. Inhibition of TLR4 activation represents a novel strategy to attenuate early graft failure following intraportal islet transplantation.     

Ex vivo and systemic transfer of adenovirus-mediated CTLA4Ig gene combined with a short course of FK506 therapy prolongs islet graft survival

Adenovirus-mediated CTLA4Ig gene transfer has been reported to enhance graft survival in several rodent transplantation models. In this study, we investigated the efficacy of ex vivo and systemic transfer of the CTLA4Ig gene by adenoviral vectors in pancreatic islet allo-transplantation. Islet grafts from BN rats were transplanted to chemically induced diabetic LEW rats. First, ex vivo CTLA4Ig gene transfer into isolated islets was performed prior to transplantation. Survival of transduced grafts under the kidney capsule was slightly prolonged (8.6+/-1.3 days) compared with survival of untransduced grafts (6.7+/-1.2 days); when combined with a short course of FK506, graft survival was further extended (32.6+/-10.7 days vs. 13.7+/-1.0 days with FK506 alone). Secondly, systemic gene transfer was accomplished by intravenous administration immediately after the transplantation procedure. In these animals, islet grafts under the kidney capsule survived longer (15.2+/-3.3 days) than in controls (6.7+/-1.2 days), and when FK506 was administered perioperatively, all the islet grafts survived for more than 100 days. In systemically transduced recipients, the survival of islet grafts transplanted into the liver was not significantly different from that of the grafts placed under the kidney capsule. In order to examine organ-specific immunogenicity, heterotopic BN cardiac grafts were transplanted to LEW rats intra-abdominally, with the virus transferred systemically as in the islet model. In contrast to the islet grafts, all the cardiac grafts were accepted for longer than 100 days, even without FK506 therapy. Finally, the LEW recipients with long-surviving islet or cardiac grafts were re-transplanted with islet grafts from the same donor strain (BN) on day 100. The second islet grafts survived longer than 100 days in half of the cardiac recipients, but consistently failed in the islet recipients. We conclude that in this transplant model, CTLA4Ig gene transfer and FK506 treatment synergistically improved islet graft survival, systemic transfer of the gene was more effective than ex vivo transfer to the islets, and donor-specific tolerance could not be achieved for islet transplantation but was achieved for cardiac transplantation.     

Effect of glucose toxicity on intraportal tilapia islet xenotransplantation in nude mice

BACKGROUND: Discordant xenogeneic islets transplanted intraportally into athymic nude rats experience primary non-function and are rapidly destroyed. Recently, it has been reported that adult porcine islets transplanted intraportally into nude mice are also rapidly destroyed and that this constitutes a new model for instant blood-mediated inflammatory reaction (IBMIR). METHODS: Tilapia (fish) islets were harvested, mechanically broken into mammalian islet-sized fragments, cultured for 48 h, and transplanted via the portal vein into athymic or euthymic mice. RESULTS: There were several groups of recipient mice. Streptozotocin-diabetic nude mice received 400 islets via the portal vein (n = 12). Recipients were killed when hyperglycemic (>200 mg/dl); livers and native pancreases were examined histologically. Mean graft survival time, based on function, was 5.4 +/- 1.2 days; at autopsy, histology showed occasional viable islets. We also performed a group of transplants in non-diabetic nude mice (n = 6) and then killed the recipients 2 or 4 weeks later; all had abundant viable, well-granulated islet grafts based on histology. Therefore, the intraportal environs in nude mice are not incompatible with discordant fish islets; rather, it appears as if hyperglycemia adversely affects the intraportal islet grafts (i.e. 'glucose toxicity'). To test this hypothesis, transplants were performed into non-diabetic nude mice and allowed to engraft for either 3 days (n = 6) or 10 days (n = 8) prior to injection of streptozotocin (200 to 220 mg/kg i.v.) to destroy the beta-cells in the recipients' native islets (n.b. tilapia islets are exceedingly resistant to streptozotocin); these recipients were followed for 28 days post-transplantation (or until hyperglycemic) and then killed for histology. Mean graft function exceeded 25 days for both groups and viable well-granulated, tilapia islets grafts were readily identified in all recipients; in all but one, the native pancreases were markedly beta-cell depleted -- confirming that normoglycemia was due to functional fish islet xenografts. CONCLUSIONS: Our results suggest that 'glucose toxicity' plays a role in the immediate demise of intraportal tilapia islet xenografts.     

Adenovirus Transduction Induces Expression of Multiple Chemokines and Chemokine Receptors in Murine β Cells and Pancreatic Islets

Adenoviral vectors are highly efficient for transferring genes to islets. However, the inflammatory and immune responses stimulated by adenovirus may be detrimental to islet survival. Given the role of chemokines and their receptors in inflammation, we analyzed their expression in isolated murine islets, in a murine beta cell line and in syngeneic islet grafts after adenovirus transduction (AdRSVLacZ). AdRSVLacZ transduction enhanced and induced the expression of a variety of chemokines. Transduced syngeneic transplanted islets showed significantly enhanced expression of multiple chemokines and receptors, including monocyte chemoattractant protein-1 (MCP-1), CC chemokine receptor 2 (CCR2) and regulated upon activation, normal T cell expressed and secreted (RANTES), compared with untransduced islet grafts. AdRSVLacZ-transduced islet grafts had significant mononuclear infiltrates, and in situ hybridization demonstrated intragraft expression of MCP-1, CCR2 and RANTES. Although adenovirus transduction did not impair in vitro insulin secretion, diabetes was reversed in only one of six recipients of a marginal mass of AdRSVLacZ-transduced islets, compared with six of six control recipients. In conclusion, multiple chemokines and chemokine receptors are expressed by murine islets constitutively and in response to adenovirus transduction. Adenovirus transduction impairs engraftment of marginal mass of transplanted islets. This is not because of direct vector toxicity of islet secretory capacity, but may be related to host innate immunity in response to adenovirus vector.     

Prevention of recurrence of IDDM in islet-transplanted diabetic NOD mice by adjuvant immunotherapy.

Insulin-dependent diabetes mellitus (IDDM) involves the destruction of the insulin-producing cells in the islets of Langerhans. One possible cure is by transplanting the islet cells; however, transplanted islets, even between identical twins, are subject to autoimmune destruction by the disease process, resulting in diabetes recurrence. We recently reported that complete Freund's adjuvant (CFA), an immunomodulating agent, prevented development of autoimmune diabetes in the NOD mouse. In this study, we evaluated adjuvant therapy in prevention of autoimmune destruction and rejection of transplanted islets in diabetic NOD mice. After transplantation, untreated syngeneic islet recipients (n = 16) initially became normoglycemic and then hyperglycemic, with a median survival time (MST) of the graft of 17 days. When CFA was administered at the time of transplantation, 11 of 13 CFA-treated syngeneic islet recipients remained normoglycemic long term (greater than 100 days) with an MST greater than 107 days. Ten of 11 mice maintained indefinite normoglycemia until the conclusion of follow-up (101 to 172 days). When adjuvant therapy was used in conjunction with allogeneic islet transplantation, graft survival was not extended, with MST being similar to the untreated allogeneic islet recipients (12 [n = 5] and 13 [n = 5] days, respectively). The extended acceptance of second syngeneic islet grafts by CFA-treated mice indicates that the persistent autoimmunity against the transplanted islets can be reversed in the diabetic NOD mice after CFA treatment.     

Survival of an islet allograft deficient in iNOS after implantation into diabetic NOD mice

Proinflammatory cytokines play a major role in rejection of pancreatic islet allografts and in type 1 diabetes (T1D). In rodent islets, exposure to IL-1beta alone or combined with IFN-gamma induces expression of inducible nitric oxide synthase (iNOS). Inhibition of iNOS or a deletion of the iNOS gene has been shown to be protective in animal models of T1D. In the present study we tested the hypothesis that transplantation of pancreatic islets deficient in iNOS (iNOS-/-) would permit increased graft survival. Pancreatic islets isolated from wild-type (wt) mice and iNOS-/- mice were allogeneically transplanted beneath the kidney capsule of spontaneously diabetic NOD mice. When blood glucose increased above 12.0 mM after preceding normalization of hyperglycemia, animals were sacrificed. Histological examinations of grafts were performed and graft gene expression was analyzed by real-time PCR. Transplantations of the two types of islets could reverse hyperglycemia and the grafts functioned for on average 1 week posttransplantation. Morphological examination of both types of islet grafts showed immune cell infiltration around and within the grafts. Remaining endocrine cells could be observed in wt and iNOS-/- islet grafts. In the removed grafts iNOS-/- islet tissue contained higher mRNA levels of insulin, proinsulin convertases (PC-1 and PC-2), and IL-1beta compared to transplanted wt islets. The assessments of insulin, PC-1 and PC-2 mRNAs of the grafts suggest that the iNOS-/- islets may be more resistant to destruction in the transplantation model used; however, this was not sufficient to prolong the period of normoglycemia posttransplantation.     

Local blood flow regulation in transplanted rat pancreatic islets: influence of adenosine, angiotensin II, and nitric oxide inhibition

BACKGROUND: Transplanted islets lack endothelial cells immediately after implantation and therefore depend on an adequate revascularization for their survival and function. However, the functional properties of the newly formed islet graft microvessels are largely unknown. This study aimed to investigate the blood flow regulation of transplanted pancreatic islets. METHODS: Pancreatic islets were syngeneically transplanted beneath the renal capsule of control and streptozotocin-diabetic rats. Blood flow measurements were performed 4 weeks later using laser-Doppler flowmetry. Adenosine (0.6 mg x kg(-1) x min(-1), angiotensin II (AT II; 0.17 microg x kg(-1) x min(-1)) and the nitric oxide synthase inhibitor NG-nitro-L-arginine (25 mg/ kg) were given to each animal. RESULTS: An increased basal blood flow and basal vascular conductance in the islet grafts, but not in the renal cortex, were seen in diabetic rats compared with control rats. Adenosine increased, and AT II decreased, the vascular conductance of the islet grafts in both nondiabetic and diabetic animals. A more pronounced circulatory response to AT II was observed in kidneys of diabetic animals, whereas there was no difference in the islet graft blood flow response between nondiabetic and diabetic animals. NG-Nitro-L-arginine decreased islet graft blood flow and vascular conductance in both nondiabetic and diabetic recipients, but the effect was more pronounced in the non-diabetic animals. CONCLUSIONS: Islet graft blood flow was influenced by adenosine, AT II, and nitric oxide inhibition in all animals. However, diabetic animals were less dependent on nitric oxide to maintain a basal blood flow in the islet graft.     

Evidence of endoplasmic reticulum stress mediating cell death in transplanted human islets

A key limitation to the success of islet transplantation is islet cell exhaustion and cell death during islet isolation and following transplantation. Endoplasmic reticulum (ER) stress has been identified as an important mechanism in the development of β-cell dysfunction, cell death, and diabetes. This study investigated the role of ER stress in islet loss during human islet isolation and posttransplantation in a diabetic athymic mouse model. Islets were isolated from human organ donor pancreata using intraductal enzymatic dissociation and continuous density gradient purification. ER stress mediators were assessed by Western blot and by RT-PCR. Caspase-3 activity was quantified by a bioluminescent peptide cleavage assay. Normal and streptozotocin-treated diabetic nude mice were transplanted with 2,000 IEQ of human islets under the kidney capsule and the grafts were harvested 3 or 28 days after transplantation. The grafts were analyzed for the presence for ER stress signals by immunohistochemistry. Isolated islets demonstrated higher levels of ER chaperone Bip, ER stress mediators eIF2α, ATF, spliced XBP-1, and CHOP, and also ER stress-associated apoptotic signals like JNK, caspase-3/7, and cleaved PARP. Donor pancreatic tissue did not show expression of any of these ER stress mediators. After transplantation, low expression of only protective ER stress mediators was evident in the grafts from the normal recipients. In contrast, both protective and apoptotic ER stress mediators were highly expressed in the grafts of hyperglycemic mice. ER stress mediators were induced during islet isolation and may contribute to islet apoptosis and cell death. Islet isolation activates ER stress and apoptotic pathways in isolated islets. Hyperglycemia may prolong this ER stress signal in engrafted islets, converting the protective aspects of the ER stress response to a proapoptotic response and thus contribute to deterioration of β-cell function and survival.