Transplantation sites for porcine islets.

AIMS/HYPOTHESIS: Xenotransplantation has great potential to provide beta cell replacement and thereby provide a cure for large numbers of people with type 1 diabetes. Crucial to the success of xenotransplantation is establishment of the most viable sites for transplantation. METHODS: We compared porcine islet tissue transplanted into kidney, liver and spleen in pig recipients as assessed by blood glucose levels and IVGTT. RESULTS: Kidney was the superior site for porcine islet tissue transplantation, followed by liver then spleen. This was demonstrated by IVGTTs showing significant difference between the peak glucose levels: 22.8 ± 2.9 mmol/l for kidney compared with 26.8 ± 1.3 mmol/l for spleen and 24.7 ± 1.7 mmol/l for liver. CONCLUSIONS/INTERPRETATION: Kidney grafts are not as feasible in humans and liver results were relatively poorer than spleen. For islet transplantation to be viable and successful in the longer term, there remains a need for future investigation of alternative sites.

Pre-clinical model of composite foetal pig pancreas fragment/renal xenotransplantation to treat renal failure and diabetes.

UNLABELLED: BACKGROUND: Development of a limitless source of ß cells for xenotransplantation into patients suffering type 1 diabetes and renal failure that can control their diabetes and provide normal renal function in one procedure would be a major achievement. For the islet tissue to survive transplantation, as an islet-kidney composite graft this would have significant advantages. It would simplify the surgical procedure; remove the complications caused by the exocrine pancreas whilst reversing diabetes and uraemia. It was our hypothesis that a composite foetal porcine pancreas fragment (FPPF)/renal graft could achieve these objectives in a large pre-clinical animal model as a means to establish whether this would be feasible before moving to the clinic. METHODS: Inbred 'Westran' pig FPPF were transplanted under the kidney capsule of syngeneic Westran pig recipients without immunosuppression. Following maturation of the FPPF under the renal subcapsular space of this recipient, this kidney bearing the composite FPPF piggyback graft was removed and transplanted into another nephrectomized and pancreatectomized recipient to demonstrate function. RESULTS: Under the kidney capsule of the first transplant group (n = 6), the FPPF-transplanted tissue developed and matured to form islet cell nests. These composite FPPF/renal grafts were then successfully removed and transplanted into the second functional assessment recipient group. This second group of six composite FPPF/renal-grafted pigs had normal renal function for more than 44 days and normal glucose homoeostasis without exogenous insulin as assessed by normal glucose tolerance tests, K values and normal glucagon secretion. Histological analysis showed despite the ischaemic insult during the composite kidney transplant procedure, there was appropriate development of islet-like structures up to and beyond 224 days after the original transplantation under the kidney capsule. CONCLUSIONS: This study shows that the use of composite FPPF/renal grafts can cure both diabetes and renal failure with a single-transplant procedure. Using such composite grafts for xenotransplantation would simplify the surgical procedure and protect the islet graft from the immediate innate immune response.

Subcapsular fetal pig pancreas fragment transplantation provides normal blood glucose control in a preclinical model of diabetes.

OBJECTIVE: Identifying a limitless source of ß-cells that survive transplantation into a neovascularised site and provide normal blood glucose control remains an important goal in the development of pancreatic islet xenotransplantation. It was our hypothesis that fetal porcine pancreas fragments could achieve these objectives, and this was tested in a large preclinical animal model. RESEARCH DESIGN AND METHODS: Inbred "Westran Pig" fetal porcine pancreas fragments were transplanted beneath the splenic capsule into syngeneic Westran Pig recipients without immunosuppression, and 3 months later, a total native pancreatectomy was performed to demonstrate function. RESULTS: Histologic analysis showed appropriate development of islet-like structures up to and beyond 120 days after transplantation. After native pancreatectomy, recipients survived more than 100 days without exogenous insulin and with normal glucose homeostasis as assessed by normal glucose tolerance tests, K values, and normal glucagon secretion. CONCLUSIONS: This study confirms that fetal pig islet tissue has the potential to mature and function normally in a neovascularised site, hence, avoiding the innate immune destruction that occurs when islet tissue is exposed directly to the circulation.

In vitro suppression of xenoimmune-mediated macrophage activation by human CD4+CD25+ regulatory T cells.

BACKGROUND: Macrophages are important effector cells in T cell-mediated xenograft rejection. The aim of this study was to determine whether CD4+CD25+ regulatory T cells (Tregs) were capable of suppressing macrophage activation in vitro. METHODS: Porcine cell or xenoantigen-primed human peripheral blood mononuclear cells, CD4+ T cell-depleted peripheral blood mononuclear cells, or CD14+ macrophages plus autologous CD4+CD25- T cells were cultured with or without expanded autologous Tregs. Transwell cultures were used to separate the various components to determine the need for cell-cell contact. RESULTS: Pig cell primed CD14+ macrophages required the presence of CD4+CD25- T cells for activation and increased expression of CD40, interleukin-12, and tumor necrosis factor-alpha. This up-regulated expression of macrophage activation markers was reduced substantially in the presence of autologous Tregs. Coculture with Tregs did not alter macrophage viability but reduced the capacity of macrophages to stimulate proliferation of responder T cells. Tregs required direct contact with CD4+CD25- T cells to inhibit macrophage activation but activated macrophage phenotype was not altered by separating the stimulated human peripheral blood mononuclear cells or CD14+ macrophages from Tregs in a transwell system. Macrophages did not require direct cell contact with porcine stimulator cells for full activation by CD4+CD25- T cells. CONCLUSIONS: Human Tregs were able to suppress xenoantigen-primed and CD4+ T-cell-mediated macrophage activation and antigen-presenting cell function. However, Tregs had no direct effect on macrophages in vitro.

In vitro expanded human CD4+CD25+ regulatory T cells are potent suppressors of T-cell-mediated xenogeneic responses.

BACKGROUND: Cellular rejection of xenografts is predominantly mediated by CD4 T cells. Little is known of the effectiveness of CD4CD25 T regulatory (Treg) cells at suppressing this strong T-cell mediated immune response. In this study, we evaluated the activity of fresh Treg cells and expanded Treg cells to suppress the xeno immune response in vitro. METHODS: Human Treg cells were preferentially expanded by CD3/CD28 expand beads, interleukin (IL)-2, and rapamycin. Human CD4CD25 T cells were stimulated with irradiated porcine peripheral blood mononuclear cells in the presence or absence of fresh or expanded human Treg cells for 5 days before proliferation assay. In a separate experiment, the porcine xenoantigen-stimulated CD4CD25 T cells were separated from Treg cells by transwells and assessed for cytotoxicity of porcine peripheral blood mononuclear cells target cells. Cytokine-producing cells and cytokine release in the cocultures were examined by enzyme-linked immunosorbent spot and enzyme-linked immonosorbent assay, respectively. RESULTS: Human Treg were expanded up to 3,500-fold after 14 days in culture. The addition of fresh Treg suppressed the T-cell mediated xenoimmune response. Compared with fresh Treg cells, expanded Treg cells were more potent at suppressing CD4CD25 T-cell-mediated antiporcine xenogeneic responses. This suppression required cell contact. However, the enhanced suppression by expanded Treg cells was associated with increased secretion of IL-4 and IL-10 when compared with their nonexpanded Treg counterparts. CONCLUSION: This study shows that expanded human Treg cells were capable of suppressing antiporcine xenogeneic responses in vitro and involve both contact dependent and cytokine mediated mechanisms.

Characterization of the swine major histocompatibility complex alleles at eight loci in Westran pigs.

BACKGROUND: Pigs are an important large animal model for transplantation and a potential source of xenografts. Swine leukocyte antigen (SLA) molecules are strong mediators of alloreactive and xenoreactive immune responses. We have characterized the SLA alleles of a new pig line bred for transplantation research, the Westran (Westmead Hospital transplantation) pig, described in a companion paper. METHODS: Three sixth generation inbred Westran pigs and a Large White pig control were used to assess SLA alleles. We examined the SLA-1, SLA-3, SLA-6, SLA-2, DQA1, DQB1, DRA1 and DRB1 loci using reverse transcription-polymerase chain reaction and sequencing-based method. RESULTS: All of the Westran pigs had a single allele at each locus, except for the SLA-1 locus. Typing of the SLA-1 locus in additional animals indicated that this is most likely the result of a duplication of the SLA-1 locus rather than heterozygosity. The lack of SLA heterozygosity is consistent with the previous finding of low microsatellite marker heterozygosity and is the result of both the recent deliberate inbreeding of these pigs and their derivation from a feral stock from Kangaroo Island, South Australia, established by the release of a single pair in 1803. CONCLUSIONS: After comparing DNA and protein sequences of the Westran SLA alleles with published GenBank SLA sequences, the SLA class I alleles found in the Westran pigs were all novel, while the SLA-DR and DQB1 alleles have been previously described in other pig breeds. Characterization of the SLA alleles in the Westran pigs has identified novel alleles and will be useful for designing protocols for modulation of immune responses to allografts and xenografts.

Genetic and functional evaluation of the level of inbreeding of the Westran pig: a herd with potential for use in xenotransplantation.

BACKGROUND: The Westran pig has been purposely inbred for use in xenotransplantation. The herd originated in the wild from a limited gene pool and has been inbred by repeated full-sib matings for nine generations. METHODS: The aim of this study was to evaluate the level of inbreeding by functional assays, such as bi-directional MLR and reciprocal skin grafts between herd members, and by genetic analysis using highly polymorphic genetic markers to calculate the level of inbreeding. RESULTS: The MLR between herd members were non-reactive whereas there was a prompt response to third party pig lymphocytes, indicative of a normal immune responsiveness in Westran pigs but isogenicity of the major histocompatibility complex. Skin grafts between male siblings or female sibling skin grafts on male recipients showed prolonged survival but with few exceptions did not survive beyond 100 days suggesting that by the fifth generation the Westran herd was still mismatched at minor histocompatibility antigens. This level of functional inbreeding was confirmed by microsatellite analysis of highly polymorphic markers, which showed that 52 of 53 chromosomally dispersed markers were fixed by the ninth generation. This level of fixation was consistent with 19 to 20 generations of full-sibling inbreeding. The calculated inbreeding coefficient at generation 10 was 0.98159. CONCLUSIONS: This analysis confirms that the Westran pig is highly inbred and we propose that analysis of chromosomally dispersed highly polymorphic markers is an accurate and reproducible method for assessing the level of inbreeding of a pig herd.