TNF-related apoptosis-inducing ligand (TRAIL) protects against diabetes and atherosclerosis in Apoe ⁻/⁻ mice.

AIMS/HYPOTHESIS: TNF-related apoptosis-inducing ligand (TRAIL) is implicated in the regulation of diabetes and is reduced in patients with cardiovascular disease. Although TRAIL receptors are widespread, and TRAIL can promote cell proliferation and apoptosis, it is not known how TRAIL might protect against diabetes and atherosclerosis. METHODS: We examined the development of atherosclerosis and diabetes in Apoe (-/-), Trail (also known as Tnfsf10)( -/- ) Apoe ( -/- ) and Trail ( -/- ) mice that were fed a high-fat diet. Plasma cholesterol, triacylglycerol, glucose and insulin, as well as changes in various metabolic enzymes and regulators were assessed. Glucose and insulin tolerance tests were performed. Pancreatic islets were examined for insulin and beta cell dysfunction (apoptosis and macrophage infiltration). RESULTS: Compared with Apoe ( -/- ) mice, Trail ( -/- ) Apoe ( -/- ) and Trail ( -/- ) mice exhibited several features of diabetes, including increased weight, hyperglycaemia, reduced plasma insulin, impaired glucose tolerance, beta cell dysfunction, reduced islet insulin, macrophage infiltration and increased apoptosis. Trail ( -/- ) Apoe ( -/- ) mice had increased plasma cholesterol, triacylglycerol, and VLDL- and LDL-cholesterol, and increased expression of genes involved in cholesterol synthesis and lipogenesis. Trail ( -/- ) Apoe ( -/- ) mice also had increased atherosclerosis, with several features of plaque instability. CONCLUSIONS/INTERPRETATION: We show for the first time that TRAIL deficiency promotes numerous features of diabetes that are typical of human disease, and are associated with reduced insulin and pancreatic inflammation/apoptosis. TRAIL also regulates cholesterol and triacylglycerol homeostasis in Apoe ( -/- ) mice by increasing the expression of genes involved in (1) cholesterol synthesis and absorption, and (2) triacylglycerol production.

Beneficial effects of desferrioxamine on encapsulated human islets--in vitro and in vivo study.

As many as 2000 IEQs (islet equivalent) of encapsulated human islets are required to normalize glucose levels in diabetic mice. To reduce this number, encapsulated islets were exposed to 100 µM desferrioxamine (DFO) prior to transplantation. Cell viability, glucose-induced insulin secretion, VEGF (Vascular endothelial growth factor), HIF-1α (Hypoxia inducible factor-1 alpha), caspase-3 and caspase-8 levels were assessed after exposure to DFO for 12, 24 or 72 h. Subsequently, 1000, 750 or 500 encapsulated IEQs were infused into peritoneal cavity of diabetic mice after 24 h exposure to DFO. Neither viability nor function in vitro was affected by DFO, and levels of caspase-3 and caspase-8 were unchanged. DFO significantly enhanced VEGF secretion by 1.6- and 2.5-fold at 24 and 72 h, respectively, with a concomitant increase in HIF-1α levels. Euglycemia was achieved in 100% mice receiving 1000 preconditioned IEQs, as compared to only 36% receiving unconditioned IEQs (p < 0.001). Similarly, with 750 IEQ, euglycemia was achieved in 50% mice receiving preconditioned islets as compared to 10% receiving unconditioned islets (p = 0.049). Mice receiving preconditioned islets had lower glucose levels than those receiving unconditioned islets. In summary, DFO treatment enhances HIF-1α and VEGF expression in encapsulated human islets and improves their ability to function when transplanted.

Glucagon-like peptide-1 enhances production of insulin in insulin-producing cells derived from mouse embryonic stem cells.

Embryonic stem cells (ESCs) can be differentiated into insulin-producing cells by a five-stage procedure involving altering culture conditions and addition of nicotinamide. The amounts of insulin in these cells are lower than those found in pancreatic beta cells. Glucagon-like peptide-1 (GLP-1) induces the differentiation of beta cells from ductal progenitor cells. We examined the possibility of GLP-1, and its long-acting agonist exendin-4, enhancing the differentiation of insulin-producing cells from mouse ESCs (mESCs). A five-stage culturing strategy starting with embryoid bodies (EBs) was used in this study. mRNA for pancreatic duodenal homeobox gene 1 (PDX-1) and neurogenic differentiation (NeuroD) was detected from stage 1, hepatocyte nuclear factor 3 beta (HNF3beta) and insulin 2 from stage 2, Ngn3 and glucose transporter 2 (GLUT2) from stage 3, and insulin 1 and other beta-cell markers, at stages 4-5. Cells at stage 5 secreted C-peptide, being 0.68 +/- 0.01 pmol/10(6) cells per 2 days, and had an immunoreactive insulin content of 13.5 +/- 0.7 pmol/10(6) cells. Addition of GLP-1 (100 nM) and nicotinamide (10 mM) at stage 5 resulted in a 50% and 48% increase in insulin content and C-peptide secretion respectively compared with nicotinamide alone. Glucose-induced insulin secretion was enhanced 4-fold by addition of both growth factors. The GLP-1 receptor was present at all five stages of the culture. Addition of exendin-4 to cells at stage 2 resulted in a 4.9-fold increase in expression of the gene for insulin 1 and a 2-fold increase in insulin content compared with the effect of nicotinamide alone at stage 5. It is concluded that both GLP-1 and exendin-4 enhance the level of expression of insulin in glucose-responsive insulin-producing cells derived from the R1 mESC line.

Synthesis and release of human (pro)insulin in human BM progenitor cells.

BACKGROUND: Pancreatic insulin-producing g-cells are permanently destroyed in Type I diabetic patients, leading to hypoglycemica. Various somatic cells have been studied for their ability to deliver insulin as an alternative source of pancreatic g-cells. We investigated the potential of human BM progenitor cells for this purpose. METHODS: Two BM-derived hematopoietic cell lines, Tf-1 (CD34+) and K562 (CD34m) cell and primary human BM stromal cells were transduced with the human preproinsulin cDNA, and the ability of these cells to synthesize, store and release insulin was analyzed. RESULTS: All cells produce and released (pro)insulin at 116-295 wU/10(6) cells/day respectively. No storage of insulin was detected in either cell line or in stromal cells. DISCUSSION: We conclude that human BM-derived progenitor cells can be induced to produce and release basal levels of (pro)insulin.

Function of a genetically modified human liver cell line that stores, processes and secretes insulin.

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic beta cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose-response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for beta cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic beta cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic beta cells and become a therapy for the treatment of type I diabetes.

Susceptibility of insulin-secreting hepatocytes to the toxicity of pro-inflammatory cytokines.

The liver has been suggested as a suitable target organ for reversing type I diabetes by gene therapy. Whilst gene delivery systems to the hepatocyte have yet to be optimized in vivo, whether insulin-secreting hepatocytes are resistant to the autoimmune process that kills pancreatic beta-cells has never been addressed. One of the mechanisms by which beta-cells are killed in type I diabetes is by the release of the cytokines interleukin-1beta (IL-1beta), tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) by immune cells. To test the effect of the cytokines on insulin-secreting hepatocytes in vitro we exposed the betacyte, also called the HEP G2ins/g cell which possesses cytokine receptors and can synthesize, store and secrete insulin in a regulated fashion to a glucose stimulus, to the above mentioned cytokines for 14 days. Viability of the HEP G2ins/g cells was similar to that of other liver cell lines/primary cells which were more resistant to the cytokines than the beta-cell line NIT-1. The cytokines had no adverse effect for the first six days on insulin secretion, content and mRNA levels of the HEP G2ins/g cells and insulin secretion in response to 1-h exposure to 20 mM glucose was enhanced 14-fold. Our results indicate that genetically engineered hepatocytes and primary liver cells are more resistant than pancreatic beta-cells to the adverse effects of cytokines offering hope that insulin secreting hepatocytes in vivo made by gene therapy are less likely to be destroyed by cytokines released during autoimmune destruction.

Secretion of pancreatic icosapeptide from porcine pancreas.

The pancreatic polypeptide cell, the only mature endocrine cell in the fetal pig pancreas, produces equimolar amounts of two peptides, pancreatic polypeptide and pancreatic icosapeptide, from the same precursor. The amino acid sequence of pancreatic polypeptide is more homogeneous among species, whereas pancreatic icosapeptide is heterogeneous. We determined the 19-amino acid sequence of porcine pancreatic icosapeptide, which is markedly different from that of known sequences (e.g. 47% homology with human). We developed an ELISA that can measure porcine pancreatic icosapeptide levels in the range of 7.2-480 pmol/liter. Actual levels of pancreatic icosapeptide in pig sera were 9.6-25 pmol/liter. The assay requires relatively small amounts of nonextracted samples, and human and mouse sera do not cross-react. Levels of pancreatic icosapeptide rose in response to hypoglycemia in pigs and to carbachol in fetal porcine pancreatic cells in vitro. When fetal porcine pancreatic tissue was transplanted into nonobese diabetic-severe combined immune deficiency mice, porcine pancreatic icosapeptide (but not C peptide) was detectable in mouse sera for up to 3 wk after transplantation, with levels highest on d 4. Porcine pancreatic icosapeptide and insulin were detectable in grafts removed from the mice. Therefore, porcine pancreatic icosapeptide may be used as a marker of the viability of xenotransplanted fetal pig pancreatic tissue in the immediate posttransplant period.

Role of pancreatic polypeptide as a market of transplanted insulin-producing fetal pig cells.

Transplantation of insulin-producing fetal pancreatic tissue into diabetic recipients has been shown to normalize blood glucose levels after several months. This time period is required for the growth and maturation of the fetal tissue so insulin levels cannot be used as a marker of graft function while the beta-cell is immature. Therefore, we have examined the use of another pancreatic endocrine hormone, pancreatic polypeptide (PP), to monitor graft function. The cell that produces this hormone has been shown to be the first mature endocrine cell in the fetal pancreas. Fetal pig pancreatic tissue, both in the form of 1 mm3 explants and islet-like cell clusters (ICCs), was transplanted into immunodeficient SCID mice and the levels of PP and insulin were measured in plasma and in the graft for up to 12 weeks. PP was detected in the untransplanted explants (0.58 pmol/mg) and ICCs (0.06 pmol/ICC) and the PP to insulin ratio was 2.7% and 5.8%, respectively. PP (but not porcine C-peptide, a marker of insulin secretion) was detectable in the plasma of SCID mice from 4 days to 3 weeks after transplantation, but not thereafter. The highest values were obtained at 4 days to 1 week. In the grafted tissue PP and insulin were present at all time points and the ratio of PP to insulin was 59%, 87%, 75%, 56%, 7%, 8%, and 7% at 4 days, 1, 2, 3, 6, 9, and 12 weeks, respectively. The decline in PP levels 3 weeks after transplantation was associated with beta-cell development in the graft. PP was also secreted by fetal pig pancreatic explants transplanted into diabetic NOD/SCID mice, with plasma levels measurable in the first week after the tissue was grafted. In immunocompetent BALB/c mice transplanted with the tissue, PP was detectable in plasma for 2 days after transplantation but not at 4 days, when cellular rejection commenced, or thereafter. We conclude that plasma PP levels can be used as a marker of the viability of fetal porcine pancreatic tissue in the first 3 weeks after it is transplanted into mice. These findings may have relevance to fetal pancreatic tissue transplanted into humans if suitable techniques can be developed to separate pig from human PP.

Lowering of blood glucose to nondiabetic levels in a hyperglycemic pig by allografting of fetal pig isletlike cell clusters.

BACKGROUND: Fetal pig isletlike cell clusters (ICCs) will differentiate when grafted into the thymus gland of outbred immunosuppressed nondiabetic pigs for up to 3 months. Whether these cells will survive for a similar period in a diabetic recipient and will mature with secretion of insulin to ameliorate the hyperglycemia is unknown. METHODS: Between 40,000 and 125,000 ICCs (7,000 to 11,400 ICCs/kg) were injected into the thymus gland of five juvenile pigs immunosuppressed with cyclosporine and deoxyspergualin, and the animals were subsequently made diabetic by the injection of streptozotocin. Insulin was administered subcutaneously, with one pig dying from hypoglycemia. The animal with the least number of ICCs transplanted was killed 81 days later, and the graft was analyzed histologically. Blood glucose levels and porcine C-peptide in the remaining animals were monitored for a median of 101 days. RESULTS: Histological analysis of the graft showed numerous epithelial cell clusters; the percentage of cells that contained insulin, glucagon, somatostatin, and pancreatic polypeptide were 61%, 64%, 25%, and 18%, respectively. Some cells contained more than one hormone. Porcine C-peptide was detected from 21 days after induction of diabetes but not before. In the pig receiving the most ICCs, blood glucose levels were lowered to nondiabetic levels 109 days after transplantation. Plasma C-peptide levels in response to glucagon in this pig steadily increased after grafting; peak levels were 0, 0.21, 0.45, and 0.52 ng/ml at 4, 21, 49, and 80 days after induction of diabetes compared to 0.09 ng/ml in control diabetic pigs. The secretion of C-peptide in response to oral and intravenous glucose and arginine also was greater than in untransplanted diabetic pigs, the pattern of secretion being consistent with developing fetal beta cells as the source of the C-peptide. Pancreatic insulin content was 0.1 mU/mg, 4% of that in nondiabetic pigs, and the number of beta cells per islet was 3 to 6 compared to 90 in nondiabetic controls. CONCLUSIONS: ICCs will differentiate and function for up to 111 days when transplanted into outbred immunosuppressed pigs rendered diabetic. Blood glucose levels can be lowered to nondiabetic levels when sufficient numbers of ICCs are grafted.

Cloning and characterization of porcine insulin gene.

The complete porcine preproinsulin cDNA and 1022 bp of its 5'-flanking region have been cloned by PCR-based technology and characterized. The porcine insulin gene has the same structure of three exons and two introns as that found in all insulin genes sequenced to date. Northern blot analysis of isolated adult porcine islets demonstrated an increase in steady-state insulin mRNA levels in response to high concentrations of glucose. Highly conserved cis-acting elements were found in the 5'-flanking region of the porcine insulin gene including multiple E and A elements as well as a cAMP responsive element (CRE). Tissue-specific activity of the proximal promoter was confirmed by transient transfection of the promoter/reporter gene constructs. This information now makes it possible for regulation and expression of the porcine insulin gene to be analyzed.

Development of human fetal pancreas after transplantation into SCID mice.

Only a small component of human fetal pancreas consists of beta cells, and yet this tissue is capable of normalizing the blood glucose levels of diabetic recipients when transplanted. The time taken to achieve this goal is several months, during which time the tissue proliferates and eventually differentiates into beta cells. The dynamics of beta cell development have not been described previously. We transplanted human fetal pancreas beneath the renal capsule of immunodeficient mice and analysed the grafts for a period of 12 weeks using antibodies against exocrine cells (lipase), endocrine cells and protodifferentiated duct cells. Exocrine cells constituted 48% of all epithelial cells in the untransplanted pancreas, with duct cells comprising 29% and endocrine cells 16% (beta cells 7%). The percentage of exocrine cells declined with time after transplantation, with only a small number undergoing apoptosis, and the duct cells increased, the values for these two cell types at 12 weeks being 20 and 57%, respectively. Both cell types appeared to proliferate equally for up to 8 weeks after transplantation, but only duct cells thereafter. Endocrine cells began to increase from 8 weeks after transplantation, representing 28% of epithelial cells (beta cells 11%) at this time. Intermediate cells, that is, cells expressing the characteristics of more than one type of mature pancreatic cell, were observed both in the ungrafted pancreas and after transplantation. The commonest intermediate cell type was duct/exocrine, with exocrine/endocrine and duct/endocrine cells also observed, suggesting active transdifferentiation from one cell type to another. We hypothesize that following the transplantation of human fetal pancreatic tissue, exocrine cells mostly transdifferentiate into duct cells and these eventually develop into endocrine cells, in particular beta cells.

In vitro dedifferentiation of fetal porcine pancreatic tissue prior to transplantation as islet-like cell clusters.

The fetal porcine pancreas under experimental conditions can be transplanted in the form of explants or islet-like cell clusters (ICCs) to normalize blood glucose levels in diabetic recipients. ICCs are released from the collagenase-digested pancreas and require a 4- to 5-day culture period for their complete formation. In order to maximize insulin producing beta cell differentiation following transplantation, an understanding of ICC development is essential to utilize this alternative treatment for type 1 diabetes. In this study a role is proposed for exocrine cells in the generation of the multipotent pancreatic precursor cells during the culture period. Acinar cells undergo dedifferentiation during the initial stages of the culture period into multipotent pancreatic precursor cells, previously called protodifferentiated cells. The progressive loss of exocrine differentiation appears to involve rapid degranulation of zymogen granules by exocytosis and loss of the prominent secretory apparatus. These processes occur in parallel with a significant reduction in the expression of lipase in the period from day 0 to day 5 and simultaneously there is an increase in the epithelioid/ductal cell marker, cytokeratin 20. Using proliferating cell nuclear antigen, cell proliferation during the culture period does not appear to account for the increase in epithelioid/ductal cells. Further the rates of apoptosis and necrosis which were identified using the TUNEL technique and propidium iodide, respectively, do not appear to account for the reduction in exocrine cell numbers. Exocrine cell dedifferentiation appears to increase the pool of protodifferentiated cells which have the potential to develop into the insulin-producing beta-cell population following transplantation into the diabetic recipient

Mapping dispersed repetitive loci using semi-specific PCR cloning and somatic cell hybrid mapping.

A simple and effective method based upon semi-specific PCR followed by cloning has been developed. Chromosomal mapping of the generated fragment on a somatic cell hybrid panel identifies the chromosomal position, and yields a unique sequence tag for the site. Using this method, the chromosomal location of one porcine endogenous retrovirus (PERV) was determined. The porcine genomic sequences were first amplified by PCR using a PERV-specific primer and a porcine short interspersed nuclear element (SINE)-specific primer. PCR products were cloned, and those sequences that contained PERV plus flanking regions were selected using a second round of PCR and cloning. Sequences flanking the PERV were determined and a PERV-B was physically mapped on porcine chromosome 17 using a somatic hybrid panel. The general utility of the method was subsequently demonstrated by locating PERVs in the genome of PERV infected human 293 cells. This method obviates the need for individual library construction or linker/adaptor ligation, and can be used to quickly locate individual sites of moderately repeated, dispersed DNA sequences in any genome.

Transmission of porcine endogenous retroviruses in severe combined immunodeficient mice xenotransplanted with fetal porcine pancreatic cells.

BACKGROUND: Xenotransplantation using pig organs or tissues may alleviate the human donor organ shortage. However, one concern is the potential transmission of pig pathogens to humans, especially pig endogenous retroviruses (PERV), which infect human cell lines in vitro. In this report, the cross-species in vivo transmission of PERV by xenotransplantation was studied using a severe combined immunodeficient (SCID) mouse model. METHODS: Twenty-one SCID mice were transplanted with fetal pig pancreatic cells and left for periods from three to 41 weeks before being killed. DNA and RNA were extracted from liver, spleen, and brain of these mice, and examined for PERV using nested polymerase chain reaction (PCR) and reverse transcriptase-PCR. The pig mitochondrial cytochrome oxidase II subunit gene (COII) was also amplified to monitor the presence of pig cell microchimerism in xenotransplanted tissues, and a housekeeping gene was included to monitor the DNA quality and quantity. RESULTS: Examination of 39 DNA samples from tissues of the 21 xenografted mice identified two mouse tissues (M4-liver and M19-spleen) that were positive for PERV but negative for COII. A total of 23 (59%) of the mouse tissues were positive for both PERV and COII, 6 (16%) were negative for both, and 8 (20%) were positive for COII only. PCR and direct sequencing of the PCR products identified three PERV variants, which were different from the PERV sequence detected by PCR direct sequencing from the pig donor cells. CONCLUSIONS: The PERV+/COII- results from M4-liver and M19-spleen indicated the presence of PERV transmission from pig to mouse tissue. The PERV variants detected in the mouse tissues indicated that different PERVs were transmissible from the pig to mouse tissue during xenotransplantation. The negative reverse transcriptase-PCR results for PERV from three mouse samples including M4-liver and M19-spleen suggest there was no active PERV transcription in the mouse tissues, although this would need to be studied further.

Effect of interleukin-10 on human anti-porcine xenogeneic cellular response in vitro.

BACKGROUND: Pigs are being used as an alternative source of tissues for humans and we are interested in the xenotransplantation of fetal pig islet-like cell clusters (ICC) into type 1 diabetic patients. Interleukin-(IL) 10 is a Th2 cytokine with immunosuppressive properties that down-regulate the cell-mediated response. In this study, we evaluated the effects of recombinant human IL-10 on human anti-pig xenogeneic cellular response in mixed lymphocyte culture (MLC) and in mixed islet lymphocyte culture (MILC). METHODS: Human peripheral blood mononuclear cells as responder cells were cultured in one-way MLC with pig and human peripheral blood mononuclear cells as stimulant cells in xeno and allo-MLC, respectively, and also with fetal pig ICCs in MILC. IL-10 was added at the time of culture. RESULTS: The addition of IL-10 significantly inhibited the xeno-MLC (human anti-pig) in a dose-dependent manner, the percentage inhibition being 36, 60, and 73% at 1, 10, and 50 ng/ml, respectively. Inhibition in xeno-MLC was significantly lower than that of the allo-MLC (human anti-human) at all concentrations used, the percentage inhibition of the latter being 58, 84, and 92% at 1, 10, and 50 ng/ml, respectively. Further, the addition of IL-10 also significantly inhibited the proliferation of human peripheral blood mononuclear cells when they were cocultured with fetal pig ICCs, the inhibition being 59, 72, and 80% at 1, 10, and 50 ng/ml, respectively. IL-10 was not toxic to ICCs as determined by 3H-thymidine incorporation over 5 days culture. Preincubation of IL-10 with the pig stimulant cells or the human responder cells did not confer additional benefit in the inhibition of xeno-MLC. IL-10 needs to be present at the start or at an early stage (within 4 hr) in the xeno-MLC because if the addition of IL-10 was delayed by 4 hr, the effect was lost. Next, the production of cytokines was examined in MLC and MILC. In xeno-MLC, levels (pg/ml) of tumor necrosis factor-alpha (TNF-alpha) (163+/-17), interferon-gamma (IFN-gamma) (278+/-60), IL-5 (24+/-10), IL-6 (2959+/-923), and IL-10 (17+/-2) were produced in greater amounts than autologous controls (P<0.05). The levels of TNF-alpha, IFN-gamma, IL-6, and IL-10 but not IL-5 were significantly (P<0.05) lower in xeno-MLC than those produced in allo-MLC. All of these cytokines were also produced in MILC when human peripheral blood mononuclear cells (PBMC) were cocultured with ICCs, levels (pg/ml) being TNF-alpha (308+/-47), IFN-gamma (93+/-17), IL-5 (6.2+/-3), IL-6 (5649+/-421), and IL-10 (122+/-18). No detectable levels of IL-2 and IL-4 were produced in the MLC and in MILC. Addition of IL-10 significantly inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 76, 96, 100, and 93%, respectively, in xeno-MLC. Addition of IL-10 also significantly (P<0.05) inhibited the production of TNF-alpha, IFN-gamma, IL-5, and IL-6 by 88, 91, 100, and 96%, respectively, in MILC. Exogenous addition of IL-2 was partially able to reverse the effect of IL-10 although addition of TNF-alpha had no effect on xeno and allo-MLC. Synergism was seen between IL-10 and cyclosporine in the inhibition of xeno and allo-MLC. CONCLUSION: Taken together, the results demonstrated that IL-10 has an immunomodulatory role to play in the inhibition of cellular immune responses associated with the xenotransplantation of fetal pig ICCs.

A comparison of the sensitivity of pig and human peripheral blood mononuclear cells to the antiproliferative effects of traditional and newer immunosuppressive agents.

Difficulty in preventing rejection of fetal pig islet-like cell clusters (ICCs) transplanted into pigs using traditional forms of immunotherapy has been reported. An in vitro study of the efficacy of seven different immunosuppressive agents to inhibit proliferation of pig peripheral blood mononuclear cells (PBMC) was performed, and a comparison was made between the human and pig to determine if the efficacy of these agents differed between species. The efficacy of cyclosporine (CsA), azathioprine (Aza), methylprednisolone (MP), FK506, rapamycin (RAP), mycophenolate mofetil (MMF) and deoxymethylspergualin (MeDSG) to inhibit pig and human PBMC proliferation in mitogenic experiments using phytohaemagglutinin (PHA) as a stimulus was performed. Further, allogeneic pig mixed lymphocyte reactions (MLR) were used to determine the activity of these agents in a model more comparable to the allograft rejection process. It was found that pig PBMC stimulated with PHA or in a MLR were inhibited by the agents tested, with the exception of MeDSG that was ineffective in mitogenic experiments. The inhibitory effects of these agents differed between PHA and MLR, the respective (50% inhibitory concentration) IC50 values for pig PBMC being 1.7 and 0.08 microg/ml for CsA, 1.4 and 4.4 microg/ml for Aza, 0.11 and 0.002 microg/ml for MP, 3.0 and 2.8 ng/ml for FK506, 2.1 and 0.3 ng/ml for RAP and 10.8 and 454 ng/ml for MME Pig PBMC were less sensitive than human PBMC to the antiproliferative effects of CsA, Aza, FK506, RAP and MMF, but not MP on PHA stimulation, the ratio of the pig to human IC50 values being 19, 11, 13, 2.3, 1.4, and 0.4, respectively. These data suggest that the doses of most immunosuppressive agents administered to prevent rejection in pigs need to be higher than those used to achieve therapeutic benefit in humans.

Immunomodulation of fetal pig islet-like cell clusters by gamma-irradiation.

Pretreatment of tissues to reduce their immunogenicity is an attractive option, and exposure of donor islets to gamma-irradiation has previously been shown to result in their prolonged survival when transplanted into rodents. Fetal pig islet-like cell clusters (ICCs) are currently under trial as a potential xenogeneic tissue for the treatment of type 1 diabetes. The purpose of this study was to examine in vivo and in vitro the immunomodulatory effects of gamma-irradiation on ICCs in a xenogeneic situation. The immunogenicity of gamma-irradiated ICCs was determined in a mixed islet lymphocyte culture (MILC), in which fetal pigs ICCs were able to stimulate human peripheral blood mononuclear cells (PBMCs). Exposure of the ICCs to gamma-irradiation significantly reduced their ability to stimulate PBMCs in a MILC when 10 Gy but not lower doses of irradiation were applied. However, this effect of gamma-irradiation was variable and was present only in those experiments in which the stimulation index was relatively low. Gamma-irradiation was toxic to ICCs in vitro, causing a reduction in the [3H]-thymidine incorporation of 82-94% at 5-20 Gy. This toxic effect of gamma-irradiation was also demonstrated in vivo: the insulin content of ICCs beneath the renal capsule in SCID mice treated with 5-20 Gy significantly was reduced (P < 0.05) 6 weeks after transplantation. Exposure of ICCs to gamma-irradiation (2.5 Gy) alone in vitro or in combination with injection of cyclosporine (12.5 mg/kg per day) did not prevent the rejection of ICCs transplanted beneath the renal capsule of BALB/c mice. We conclude that gamma-irradiation is toxic to fetal pig ICCs at a higher dose and at a lower dose, alone or in combination with cyclosporine, and was unable to prolong discordant islet xenograft survival in mice.

Outcome of xenografted fetal porcine pancreatic tissue is superior in inbred scid (C.B-17/Icr-scid/scid) compared to outbred nude (CD-1-nu/nu) mice.

Nude mice are used as recipients of foreign tissue because of their inability to reject these grafts. Our experience has been that there is variable rejection of fetal porcine insulin-producing tissue transplanted into CD-1 (athymic) outbred nude mice. To examine the suitability of this line of nude mouse as a recipient of the tissue, fetal porcine pancreas was grafted either into these outbred animals or into an inbred mutant strain of mice, the more immunocompromised severe combined immunodeficient (scid) mouse. Eight weeks after transplantation grafts were recovered from recipients and assayed for insulin content. Mean insulin levels were not significantly different between the two groups of mice, but a wider range of values was obtained from grafts recovered from nude (CD-1-nu/nu) mice. Reversal of diabetes in hyperglycemic recipients was achieved in 4 of 8 nude mice and 8 of 8 scid (C.B-17/lcr-scid/scid) mice. The time taken to achieve this was longer in the nudes than the scid mice, 121 +/- 12 vs. 44 +/- 2 days, the grafts increasing in size at a slower rate in the nude mice. Time taken for the weight of the grafts to double in size was 94 +/- 17 vs. 32 +/- 1 days, respectively. Histologically the grafts in the scid mice contained mostly epithelial cell clusters, a majority of which were insulin containing. In the nude mice that achieved normoglycemia, a similar pattern was observed and, as well, there was a localized lymphoid infiltrate. In those nude mice that remained diabetic fibrous tissue predominated together with a lymphoid infiltrate. In summary, fetal porcine pancreatic tissue grows and develops more efficiently when xenografted into scid rather than outbred nude mice.