Pig Islet Xenotransplantation Research Articles

Pig islet xenotransplantation: activation of porcine endogenous retrovirus in the immediate post‐transplantation period

Porcine endogenous retroviruses (PERV) are considered as the main infectious barrier in islet xenotransplantation. PERV has been shown to infect, but not to cause symptomatic disease in mice after islet transplantation. In vivo activation of PERV have so far not been examined. Expression of PERV was examined in adult and fetal porcine islets with or without the presence of known retroviral inducers or after transplantation to rats. Isolated adult and fetal porcine islets were cultured under normal conditions or in the presence of dexamethasone or 5-azacytidine and 5-iodo-2-deoxyuridine. PERV mRNA content was analyzed by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and culture supernatants were analyzed for the presence of retroviral RT. Also, fetal islets were transplanted under the kidney capsule of immunocompetent or nude athymic rats. Expression of PERV mRNA in the grafts was evaluated by real-time quantitative RT-PCR. Infiltration of immunocompetent cells were evaluated by immunohistochemistry. Both fetal and adult islets in culture produced small or even undetectable amounts of PERV mRNA and retroviral RT. PERV expression was not enhanced by retroviral inducers. In contrast, activation of PERV expression was observed the first day after transplantation of fetal islet-like cell clusters in both athymic and normal rats. PERV expression peaked after 1 to 3 days and was then rapidly returned to background levels. PERV expression neither correlated with the innate immune response seen in athymic rats nor with the specific process of rejection in normal rats. Both fetal and adult islets produce low amounts of PERV mRNA in culture. After transplantation PERV expression is induced, seemingly independent of both the unspecific inflammatory response and the specific T-cell-mediated rejection process. It is speculated that PERV expression is correlated with the level of hypoxia in the islet xenograft.

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.

Intraportal pig islet xenotransplantation into athymic mice as an in vivo model for the study of the instant blood‐mediated inflammatory reaction

One of the main obstacles to successful intraportal islet transplantation is the instant blood-mediated inflammatory reaction (IBMIR) elicited by the isolated islets when exposed to fresh human blood. In the present study, we investigated whether intraportal transplantation of pig islets into diabetic athymic mice could be used as a small animal model to study xenogeneic IBMIR in vivo. Adult porcine islets (APIs) or rat islets were implanted into the portal vein or under the renal subcapsular space of diabetic athymic mice. Graft survival and morphology were evaluated by measuring blood glucose levels and by performing immunohistochemical staining, respectively. Transplantation of rat islets, irrespective of implantation site, cured all diabetic athymic mice. APIs transplanted subcapsularly also cured all diabetic athymic mice, while none of the animals transplanted with an equivalent amount of APIs via the portal vein remained normoglycemic for more than 10 days after transplantation. Immunohistochemical staining on day 7 showed that most of intraportally transplanted APIs were entrapped in clots and infiltrated with CD11b+ leukocytes. Intraportal transplantation of APIs into athymic mice induced IBMIR, thus providing a small animal model for studying xenogeneic IBMIR.

The role of chemokines and their receptors in the rejection of pig islet tissue xenografts.

The mechanism by which inflammatory cells are recruited to pig islet tissue (proislet) xenografts was investigated by examining the intragraft mRNA expression of murine alpha- and beta-chemokines in CBA/H mice from days 3 to 10 post-transplant. Xenograft rejection was associated with early intragraft transcript expression for monocyte chemotactic protein-1 (MCP-1) (3 to 5 days), IP-10 (3 to 4 days) and macrophage inflammatory protein-1alpha (MIP-1alpha) (3 to 5 days) and subsequent expression of eotaxin (days 4 to 10), MIP-1beta (days 4 and 5) and regulated on activation, normal T cell expressed and secreted (RANTES) (days 4 to 6) mRNA. This pattern was consistent with the early recruitment of macrophages (MCP-1, MIP-1alpha), the influx of CD4 T cells (MCP-1, MIP-1alpha, MIP-1beta, IP-10 and RANTES) and the characteristic infiltrate of eosinophils (eotaxin and RANTES) associated with islet xenograft rejection. Inhibition of beta-chemokine signaling in CCR2-/- mice (which lack the major co-receptor for MCP-1) resulted in retarded macrophage and CD4 T cell recruitment, enhanced eosinophil influx and a minor delay in rejection, compared with wildtype mice; there was little effect on leukocyte infiltration in xenografts harvested from CCR5-/- mice (lacking the co-receptor for MIP-1alpha, MIP-1beta and RANTES). The impeded migration of leukocytes into xenografts in CCR2-/- hosts was associated with delayed intragraft expression of MCP-1 and RANTES mRNA; absence of MCP-1/CCR2-mediated signaling led to enhanced intragraft expression of MCP-1, MIP-1alpha and MIP-1beta mRNA. These findings suggest that MCP-1 plays an important role in regulating macrophage and CD4 T cell infiltration to xenograft sites via the CCR2 signaling pathway. Additional treatment of xenografted CCR2-/- transplant recipients with anti-interleukin-(IL)-4 and anti-IL-5 mAbs further delayed xenograft rejection demonstrating the potential for combined antirejection strategies in facilitating pig islet xenotransplantation.

Xenotransplantation of pig islets in diabetic dogs with use of a microcapsule composed of agarose and polystyrene sulfonic acid mixed gel.

The authors have designed a microcapsule composed of agarose and polystyrene sulfonic acid (PSSa) mixed gel that provides a protective barrier against complement attack. Xenografts of islets, encapsulated in an agarose-PSSa microcapsule, have been shown to normalize blood glucose in rodents with chemically induced diabetes for extended periods of time without immunosuppression. To investigate the efficacy of agarose-PSSa microencapsulated pig islets in reversing diabetes in a large animal model. Diabetes was induced in beagle recipients by total pancreatectomy. Each recipient received three to five intraperitoneal injections of either encapsulated (n = 5) or nonencapsulated pig islets (n = 2). In all dogs receiving microencapsulated islets, the graft function was achieved for 7.4 +/- 3.1 weeks (mean +/- standard error), as determined by elimination or reduction of exogenous insulin requirement. In three recipients, the fasting blood glucose levels were maintained at < or = 200 mg/dL without any exogenous insulin for a period of 6, 50, and 119 days. Circulating porcine C-peptide was detected in the sera of all dogs after transplantation of encapsulated islets. Immunohistologic examination revealed the presence of insulin-positive cells in the microcapsules. In contrast, in two dogs receiving nonencapsulated islets there was no graft function. This preliminary study demonstrates that agarose-PSSa microencapsulated pig islets can survive and function for weeks or months in totally pancreatectomized diabetic dogs without immunosuppression.

Rapid failure of pig islet transplantation in non human primates.

We have previously demonstrated that adult pig islets of Langerhans are not destroyed in vitro by primate sera. Whether these islets can function when placed into the liver of non-human primates is not known. We now report on the outcome of pig islet xenotransplantation into five non diabetic primates (four baboons and one macacus fascicularis) receiving intraportally purified adult pig islets. The average number of islet-equivalent per graft was 110,000 (60-180,000). All animals received associations of ATG, cyclosporine or LF 195 (a deoxyspergualin analog), mycophenolate mofetil and corticosteroids. A specific porcine C-peptide (C-pep) RIA test was used to monitor insulin secretion. Two hours after grafting, porcine C-peptide was positive (from 0.37 to 4.25 ng/ml) in all monkeys except one. Primate C-pep was normal in all cases. Only two monkeys had detectable levels of porcine C-pep on day 1 or 2 with undetectable levels thereafter, even after glucagon challenge between days 6 and 10. Several normal islets with moderate inflammatory infiltration were observed in one animal liver on day 2 (the time of necropsy) as well as islets with IgM and complement deposition. Among animals sacrificed on days 14, 16 and 38, some residual islet cells could be identified only in livers collected on day 14. Partial glycaemic control was achieved in some rats receiving islets from the same preparations. In conclusion, adult pig islets are not able to maintain insulin secretion for more than 24 h when injected intraportally into non diabetic immunosuppressed monkeys. suggesting immediate islet xenograft destruction.

Infection by porcine endogenous retrovirus after islet xenotransplantation in SCID mice.

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.

Isolation of adult porcine islets of Langerhans.

While human islet allotransplantation is limited due to the shortage of donors, pig islet xenotransplantation appears to be a possible alternative. Enzymatic digestion is the method most commonly used for adult pig islet isolation, but since the description of the semiautomated isolation technique in the late 1980s, yields of pig islet isolations have not increased significantly. This review article is intended to collate published information on adult pig islet isolation to provide the reader with a clear overview of the relevant issues in this field, and to detail the current state of the art.

Intact pancreatic islet function despite humoral xenorecognition in the pig-to-monkey combination.

The aim of this study was to analyze humoral xenoreactivity of various Old World primate species sera against pig islets and the effects of these sera on pig islet viability and function after culture. Freshly isolated or cultured adult pig islets were analyzed by immunohistology or by cytofluorimetry for Old World primate xenoreactive natural antibody (XNA) binding and complement deposition. Complement-mediated cytotoxicity was evaluated by 51Cr release assays. After 4 days of culture in 50% sera from Old World primates, the morphology and in vitro metabolic function of pig islets were also analyzed. Chimpanzee, Macaca mulatta (rhesus), or baboon XNA binding was detectable only on intra-islet endothelial cells (ECs). Incubation of pig islets with sera from all Old World primate species tested showed C3 and C4 deposition on ECs and on some surrounding endocrine cells. However, membrane attack complex (MAC) showed a pattern of positivity similar to XNA binding, i.e., restricted to ECs only. No deposition of factor B was detected. Although complement cascade was activated, no cytotoxicity was observed after incubation of islets with chimpanzee serum, whereas between 10% and 35% 51Cr specific release was obtained with rhesus, baboon, or Macaca fascicularis sera. Despite this cytotoxic effect, purified pig islets showed a normal morphology and a well-preserved insulin release in response to an acute glucose stimulus, after prolonged culture with 50% serum obtained from all primate species considered. Despite the fact that pig beta-cell function was not affected by the serum of any of the primate species tested, some of them yielded significant lysis of islet cells, presumably as a result of a cytotoxic effect on intra-islet ECs. These data show that Old World primate sera from different species do not have equivalent effect on pig islets; these differences should be taken into account in preclinical trials of pig islet xenotransplantation.

Prolongation of pig islet xenograft survival in rats immunosuppressed with FK506

FK506 a new and potent immunosuppressive agent has been shown to be effective in prolonging pancreatic islet allograft survival. The present study was to determine its efficacy in prolonging pig islet xenotransplantation in two different strains of rat recipients. A total of two dosages of FK506 at 1 or 2 mg/kg per day for 2 weeks and then at weekly intervals were tested as monotherapy for their effect on the survival of renal subcapsular xenografts of purified or impure adult pig islets in inbred ACI and outbred Wistar rats. Histological assessment indicated that FK506 at 2 mg/kg per day significantly prolonged purified pig islet xenograft survival and to 7.5 months in two of three ACI recipients. Monotherapy with a lower dosage of FK506 or transplantation with impure pig islets resulted in increased graft survival time over controls, but less than that with the 2 mg/kg per day FK506. The viable pig islet xenografts showed a normal appearance and were readily identified by immunohistochemical staining for insulin and glucagon and further confirmed by immunohistochemical staining with anti-pig islet specific monoclonal antibody clone P44, developed in our laboratory. Mononuclear cell infiltration, mainly of the CD8-positive T-cell subset, increased with the duration of the graft in the recipient. By 7.5 months the majority of the xenografted islet cells were enclosed by the cellular infiltrate. The in vitro perfusion study of pig islets that had survived for 1 or 2 months in vivo showed that they were responsive to glucose stimulation with increase in insulin secretion into the perfusate. The results demonstrated that FK506 significantly prolonged pig islet survival in two rat strains and suggested that it is an effective immunosuppressant for the xenotransplantation model.

In vitro activation of human lymphocytes by crude and purified alginate microcapsules

Xenotransplantation of pig islets under the kidney capsule (KC) of diabetic rats was performed. Natural preformed ACI rat anti-pig leukocytotoxicity, leukoagglutination and hemagglutination antibody titers ranged from Neat-1 : 16, 1 : 8-1 : 32 and 1 : 128-1 : 256, respectively (n = 14). Normal ACI sera were non-toxic to pig islets during short term incubation. Pig islet xenograft survival times in the non-immunosuppressed ACI rats, ACI rats immunosuppressed with antithymocyte serum (ATS) or cyclosporin A were 3.8 ± 0.4 (mean ± SE; n = 5), 10.4 ± 0.7 (n = 13) and 6.0 ± 1.0 (n = 5) days, respectively. Pig islets implanted in the abdominal testis of ACI recipients immunosuppressed with 5 doses ATS survived for a mean of 6.4 ± 1.0 days (n = 7). The mean K rate following an intravenous glucose tolerance test (IVGTT) in ACI rats 1 week after transplantation with pig islet under the KC was 2.2 ± 0.4 (n = 10) compared to that of 2.91 ± 0.30 found in normal control rats (n = 8). Peak insulin at 1 min was 60.1 ± 3.9 μU/ml (n = 4). Histological and immunohistochemical examination showed that the xenograft from recipients treated with 5 doses of ATS still contained well-preserved islet tissue with many insulin- and glucagon-containing cells on the day of graft removal when blood glucose had returned to hyperglycemic level. Both CD4 and CD8 positive cells were in the vicinity of the graft tissue. This study has shown that hyperacute rejection of islet xenograft did not occur in this discordant xenograft model despite the presence of preformed anti-pig antibodies in the rat recipients. Further islet xenotransplantation using this discordant model would be useful in the design of an effective immunosuppressive regimen.

Transplantation of discordant pig islet xenografts in diabetic rats

Xenotransplantation of pig islets under the kidney capsule (KC) of diabetic rats was performed. Natural preformed ACI rat anti-pig leukocytotoxicity, leukoagglutination and hemagglutination antibody titers ranged from Neat-1 : 16, 1 : 8-1 : 32 and 1 : 128-1 : 256, respectively ( n = 14). Normal ACI sera were non-toxic to pig islets during short term incubation. Pig islet xenograft survival times in the non-immunosuppressed ACI rats, ACI rats immunosuppressed with antithymocyte serum (ATS) or cyclosporin A were 3.8 ± 0.4 (mean ± SE; n = 5), 10.4 ± 0.7 ( n = 13) and 6.0 ± 1.0 ( n = 5) days, respectively. Pig islets implanted in the abdominal testis of ACI recipients immunosuppressed with 5 doses ATS survived for a mean of 6.4 ± 1.0 days ( n = 7). The mean K rate following an intravenous glucose tolerance test (IVGTT) in ACI rats 1 week after transplantation with pig islet under the KC was 2.2 ± 0.4 ( n = 10) compared to that of 2.91 ± 0.30 found in normal control rats ( n = 8). Peak insulin at 1 min was 60.1 ± 3.9 μU/ml ( n = 4). Histological and immunohistochemical examination showed that the xenograft from recipients treated with 5 doses of ATS still contained well-preserved islet tissue with many insulin- and glucagon-containing cells on the day of graft removal when blood glucose had returned to hyperglycemic level. Both CD4 and CD8 positive cells were in the vicinity of the graft tissue. This study has shown that hyperacute rejection of islet xenograft did not occur in this discordant xenograft model despite the presence of preformed anti-pig antibodies in the rat recipients. Further islet xenotransplantation using this discordant model would be useful in the design of an effective immunosuppressive regimen.