Search

Instant blood-mediated inflammatory reaction (IBMIR) is a serious obstacle to both clinical islet allotransplantation and future islet xenotransplantation via the portal vein. We have previously observed uniform long-term tilapia (fish) islet xenograft survival when islets were transplanted intraportally into nondiabetic nude mice (nDNM), but not in diabetic nude mice (DNM). In this study, we examined whether human islets (HI) and adult porcine islets (API) can tolerate intraportal transplantation into nDNM like tilapia islets. HI and API were transplanted intraportally into nDNM. Recipients were humanely killed either 14 or 28 days after transplantation and livers were processed for histology. Human insulin and human C-peptide were measured in the terminal serum samples of HI recipients. In six of seven HI and seven of seven API recipients, liver histology showed insulin-positive islet xenografts. In recipients with HI, the numbers of islets/ductal structures seen histologically correlated well with serum sample results. These results show that HI and API can survive and function long term after intraportal transplantation into nDNM recipients. Our previous and present data indicated that DNM and nDNM could be useful models to study "glucose toxicity" and the role of IBMIR in the fate of intraportal islet grafts.

Recent advances in human allogeneic islet transplantation have established beta-cell replacement therapy as a potentially viable treatment option for individuals afflicted with Type 1 diabetes. Two recent successes, one involving neonatal porcine islet xenografts transplanted into diabetic rhesus macaques treated with a costimulation blockade-based regimen and the other involving diabetic cynomolgus monkeys transplanted with adult porcine islet xenografts treated with an alternative multidrug immunosuppressive regimen have demonstrated the feasibility of porcine islet xenotransplantation in nonhuman primate models. In the current study, we assessed whether transplantation of adult porcine islet xenografts into pancreatectomized macaques, under the cover of a costimulation blockade-based immunosuppressive regimen (CD28 and CD154 blockade), could correct hyperglycemia. Our findings suggest that the adult porcine islets transplanted into rhesus macaques receiving a costimulation blockade-based regimen are not uniformly subject to hyperacute rejection, can engraft (2/5 recipients), and have the potential to provide sustained normoglycemia. These results provide further evidence to suggest that porcine islet xenotransplantation may be an attainable strategy to alleviate the islet supply crisis that is one of the principal obstacles to large-scale application of islet replacement therapy in the treatment of Type 1 diabetes.

Background: Transplantation of human islets has been successful clinically. Since human islets are scarce, we are studying microencapsulated porcine islet xenografts in nonobese diabetic (NOD) mice. We have evaluated the cellular immune response in NOD mice with and without dual costimulatory blockade., Methods: Alginate-poly-L-lysine-encapsulated adult porcine islets were transplanted i.p. in untreated diabetic NODs and NODs treated with CTLA4-Ig to block CD28/B7 and with anti-CD154 mAb to inhibit CD40/CD40-ligand interactions. Groups of mice were sacrificed on subsequent days; microcapsules were evaluated by histology; peritoneal cells were analyzed by FACS; and peritoneal cytokines were quantified by ELISA. Controls included immunoincompetent NOD-Scids and diabetic NODs given sham surgery or empty microcapsules., Results: Within 20 days, encapsulated porcine islets induced accumulation of large numbers of macrophages, eosinophils, and significant numbers of CD4 and CD8 T cells at the graft site, and all grafts were rejected. During rejection, IFNgamma, IL-12 and IL-5 were significantly elevated over sham-operated controls, whereas IL-2, TNFalpha, IL-4, IL-6, IL-10, IL-1beta and TGFbeta were unchanged. Treatment with CTLA4-Ig and anti-CD154 prevented graft destruction in all animals during the 26 days of the experiment, dramatically inhibited recruitment of host inflammatory cells, and inhibited peritoneal IFNgamma and IL-5 concentrations while delaying IL-12 production., Conclusions: When two different pathways of T cell costimulation were blocked, T cell-dependent inflammatory responses were inhibited, and survival of encapsulated islet xenografts was significantly prolonged. These findings suggest synergy between encapsulation of donor islets and simultaneous blockade of two host costimulatory pathways in prolonging xenoislet transplant survival.

Background: 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)., Methods: 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., Results: 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., Conclusion: 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.

Previous studies demonstrated that porcine encephalomyocarditis virus (EMCV) caused acute and persistent infection in the myocardium, central nervous system, and spleen of non-human primates (cynomolgus macaques); and it productively infected primary human cardiomyocytes, suggesting that the virus may pose a risk in pig-to-human transplantation. Recently, transplantation of myocardial and pancreatic tissues from acutely infected pigs transmitted the virus to recipient mice, resulting in acute fatal EMCV disease. Here, we examined whether porcine islet cells (PICs), which are under clinical trial for treatment of type I diabetes in humans, are susceptible to porcine EMCV, and whether EMCV-infected PICs could function in vivo to reverse diabetes. PICs were infected with EMCV in vitro for 5 h, and resulting insulin production compared with that produced by uninfected PICs. Subsequently, infected PICs were transplanted intra-abdominally or under the kidney capsule of C57BL/6 mice, and both virus transmission and PIC function analyzed. PICs were highly susceptible to porcine EMCV, resulting in a 1500-fold increase in production of infectious virus within 5 h of inoculation and cytolysis that destroyed up to 50% of cells within 96 h. However, as long as they were viable, infected PICs produced insulin at levels comparable with uninfected PICs. Intra-abdominal transplantation of 2000 PICs, infected with one plaque forming unit (pfu) per cell of porcine EMCV, into C57BL/6 mice transmitted the virus resulting in acute fatal EMCV disease characterized by hind limb paresis and paralysis and acute respiratory distress in 40% of recipient mice. More importantly, transplantation of 2500 EMCV-infected PICs under the kidney capsule of diabetic C57BL/6 mice (glucose level > or =350 mg/dl) reversed diabetes in 83% of recipient mice (glucose level < or =170 mg/dl); however these mice succumbed to acute EMCV disease transmitted by the xenograft 5 days after transplantation. EMCV infection does not appear to affect insulin production by PICs, but infected xenografts can transmit the virus to recipient animals, resulting in severe disease.

Background: T-cell activation and the subsequent induction of effector functions require not only the recognition of antigen peptides bound to MHC molecules by T-cell receptor (TCR) for antigen but also a costimulatory signal provided by antigen presenting cells. CD4 T-cell activation and function require the CD4 molecule as a coreceptor of TCR. The CD28/B7 pathway is a major costimulatory signal for T-cell activation and differentiation., Methods: The effect of targeting CD4 by nondepleting anti-CD4 monoclonal antibodies (mAbs) versus blocking CD28/B7 by CTLA4Ig, anti-CD80 mAbs, and anti-CD86 mAbs on the prevention of recurrence of autoimmune diabetes after MHC-matched nonobese diabetes-resistant (NOR) islet transplantation in nonobese diabetic (NOD) mice were compared. Whether nondepleting anti-CD4 mAbs prolong allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice were studied. Furthermore, the effect of nondepleting anti-CD4 mAbs combined with CTLA4Ig on allogeneic islet graft survival in NOD mice was investigated., Results: Recurrence of autoimmune diabetes can be prevented by nondepleting anti-CD4 mAbs. Blocking the CD28/B7 costimulatory pathway by CTLA4Ig or by anti-CD80 mAbs and anti-CD86 mAbs cannot prevent recurrence of autoimmune diabetes after islet transplantation. Short-term treatment with nondepleting anti-CD4 mAbs significantly prolongs allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice. But nondepleting anti-CD4 mAbs combined with CTLA4Ig decreased allogeneic islet graft survival., Conclusions: Nondepleting anti-CD4 mAbs but not CD28 antagonists protect islet grafts in diabetic NOD mice from autoimmune destruction and allogeneic and xenogeneic graft rejection. The efficacy of nondepleting anti-CD4 mAbs is compromised when it combines with CTLA4Ig.

Animal donors such as pigs could provide an alternative source of organs for transplantation. However, the promise of xenotransplantation is offset by the possible public health risk of a cross-species infection. All pigs contain several copies of porcine endogenous retroviruses (PERV), and at least three variants of PERV can infect human cell lines in vitro in co-culture, infectivity and pseudotyping experiments. Thus, if xenotransplantation of pig tissues results in PERV viral replication, there is a risk of spreading and adaptation of this retrovirus to the human host. C-type retroviruses related to PERV are associated with malignancies of haematopoietic lineage cells in their natural hosts. Here we show that pig pancreatic islets produce PERV and can infect human cells in culture. After transplantation into NOD/SCID (non-obese diabetic, severe combined immunodeficiency) mice, we detect ongoing viral expression and several tissue compartments become infected. This is the first evidence that PERV is transcriptionally active and infectious cross-species in vivo after transplantation of pig tissues. These results show that a concern for PERV infection risk associated with pig islet xenotransplantation in immunosuppressed human patients may be justified.

Xenotransplantation of encapsulated islets of Langerhans is a possibility to overcome problems of human organ donor shortage in islet transplantation. Preexisting natural xenoantibodies are known to play a major role in the rejection of vascularized xenografts. Only little is known about the mechanism of rejection of non-vascularized cellular xenotransplants. In this study we introduce a method for the characterization of xenograft rejection of encapsulated islets by FACS analysis of peritoneal cells. Pig islets were transplanted intraperitoneally into non-diabetic Lewis rats either encapsulated or non-encapsulated. Animals receiving empty capsules and sham-operated animals served as controls. After 7 days a peritoneal lavage was performed. The total cell number and the viability of the cells were determined. Cells were analysed after staining with a panel of antibodies for the detection of T-lymphocytes, B-lymphocytes, macrophages, MHC class II molecules. Total cell number was highest after microencapsulated transplantation (149.4+/-30.1x10(6)) compared with empty capsules (41.4+/-19.7x10(6)) and non-encapsulated porcine islets (18.1+/-3.3x10(6)). The percentage of CD 3 positive T-lymphocytes rose to 44.5+/-11.5% in case of microencapsulated xenografts compared with 19.2+/-8.2% for non-encapsulated xenografts and 4.9+/-2.4% for empty controls. B-lymphocytes were detected in only small amounts. MHC class II expression on macrophages as activation marker was significantly increased after encapsulated transplantation (60.2+/-8.9% vs 15.2+/-7.0% for free islets and 4.9+/-1.2% for empty controls). The discrepancy between the macrophage activation due to encapsulated xenogeneic islets in comparison to empty capsules made from the same material clearly indicates that the reaction is not only material related but that a recognition of the encapsulated islet takes place despite the effective inhibition of a direct cell-to-cell contact. This recognition occurs on a T-cell level as well as on the macrophage level. 7 days after transplantation the reaction towards encapsulated xenografts is even more intense than to non-encapsulated xenografts. This might be due either to the time course of the rejection process or to a prolongation of the activation because antigen elimination is hindered by the capsule.

Cryopreservation of islets of Langerhans offers advantages for the transplantation into diabetic patients. In this study two different methods of cryopreservation were compared with respect to islet viability and recovery after cryostorage. It was also investigated whether human islet survival in mice was affected by cryopreservation. Aliquots of human islets were cryopreserved conventionally or vitrified, respectively. After rapid thawing, islet viability and islet equivalent (IEQ) recovery rate were determined. Aliquots of freshly isolated or conventionally cryopreserved islets were transplanted beneath the kidney capsule of non-diabetic C57BL/6 mice. After three days renal insulin content was determined. Islet cell viability was 17.3+/-8.0% for vitrified and 51.8+/-3.0% for conventionally cryopreserved islets; the recovery rate was 84.8+/-12.2% and 92.8+/-12.4%, respectively. Insulin recovery after transplantation was 25.6+/-7.3% for fresh and 24.1+/-7.4% for cryopreserved islets. This study suggests that the conventional method of cryopreservation is superior to vitrification with respect to islet viability after thawing. We found no significant difference between fresh and cryopreserved islets with respect to insulin recovery after transplantation into mice.

Highly purified porcine islets were prepared by isokinetic gradients performed subsequently to isopycnic gradients. This additional purification step separates ductal, vascular, and lymphoid tissue effectively from endocrine tissue. Although ductal, vascular, and lymphoid tissue comprises only a minor contamination of the islet suspensions, a significant prolongation of the survival of porcine islets xenografted into streptozotocin diabetic C57BL/6 mice can be achieved by the elimination of the non-endocrine tissue. Rejection after islet transplantation is delayed from 2.2+/-0.4 days (n=27) to 13.1+/-2.1 days (n=36), respectively, when conventionally purified and highly purified islets are compared. Irrespective of the purification state, pretreatment of islets by low temperature culture had no effect on xenograft survival.