Porcine Islet-like Cell Clusters Research Articles

Dual-targeted nano-encapsulation of neonatal porcine islet-like cell clusters with triiodothyronine-loaded bifunctional polymersomes

There is growing evidence that neonatal porcine islet-like cell clusters (NPCCs) isolated from piglets can be used to treat type 1 diabetes in humans. However, graft rejection is a common complication in humans owing to the prevalence of xenoantigens in porcine. Therefore, researchers have investigated various islet encapsulation techniques that could protect against these antigens. To this end, this study presents a robust nano-encapsulation method based on bifunctional polymersomes (PSomes), in which N-hydroxysuccinimide (NHS) and maleimide (Mal) groups conjugated to the PSomes terminal interact with the amine and thiol groups on the surface of NPCCs to induce dual targeting via two covalent bonds. The findings indicate that the ratio of NHS to Mal on PSomes is optimal for dual targeting. Moreover, triiodothyronine (T3) is known to promotes pancreatic islet maturation and differentiation of endocrine cells into beta cells. T3 encapsulated in PSomes is shown to increase the glucose sensitivity of NPCCs and enhance insulin secretion from NPCCs. Furthermore, improvements in the nano-encapsulation efficiency and insulin-secreting capability of NPCCs through dual targeting via dual-Psomes are demonstrated. In conclusion, the proposed nano-encapsulation technique could pave the way for significant advances in islet nano-encapsulation and the imprevement of NPCC immaturity via T3 release.

Formation of Re-Aggregated Neonatal Porcine Islet Clusters Improves In Vitro Function and Transplantation Outcome.

Neonatal porcine islet-like cell clusters (NPICCs) are a promising source for islet cell transplantation. Excellent islet quality is important to achieve a cure for type 1 diabetes. We investigated formation of cell clusters from dispersed NPICCs on microwell cell culture plates, evaluated the composition of re-aggregated porcine islets (REPIs) and compared in vivo function by transplantation into diabetic NOD-SCID IL2rγ-/- (NSG) mice with native NPICCs. Dissociation of NPICCs into single cells and re-aggregation resulted in the formation of uniform REPI clusters. A higher prevalence of normoglycemia was observed in diabetic NSG mice after transplantation with a limited number (n = 1500) of REPIs (85.7%) versus NPICCs (n = 1500) (33.3%) (p < 0.05). Transplanted REPIs and NPICCs displayed a similar architecture of endocrine and endothelial cells. Intraperitoneal glucose tolerance tests revealed an improved beta cell function after transplantation of 1500 REPIs (AUC glucose 0-120min 6260 ± 305.3) as compared to transplantation of 3000 native NPICCs (AUC glucose 0-120min 8073 ± 536.2) (p < 0.01). Re-aggregation of single cells from dissociated NPICCs generates cell clusters with excellent functionality and improved in vivo function as compared to native NPICCs.

Viability and Functionality of Neonatal Porcine Islet-like Cell Clusters Bioprinted in Alginate-Based Bioinks

The transplantation of pancreatic islets can prevent severe long-term complications in diabetes mellitus type 1 patients. With respect to a shortage of donor organs, the transplantation of xenogeneic islets is highly attractive. To avoid rejection, islets can be encapsulated in immuno-protective hydrogel-macrocapsules, whereby 3D bioprinted structures with macropores allow for a high surface-to-volume ratio and reduced diffusion distances. In the present study, we applied 3D bioprinting to encapsulate the potentially clinically applicable neonatal porcine islet-like cell clusters (NICC) in alginate-methylcellulose. The material was additionally supplemented with bovine serum albumin or the human blood plasma derivatives platelet lysate and fresh frozen plasma. NICC were analysed for viability, proliferation, the presence of hormones, and the release of insulin in reaction to glucose stimulation. Bioprinted NICC are homogeneously distributed, remain morphologically intact, and show a comparable viability and proliferation to control NICC. The number of insulin-positive cells is comparable between the groups and over time. The amount of insulin release increases over time and is released in response to glucose stimulation over 4 weeks. In summary, we show the successful bioprinting of NICC and could demonstrate functionality over the long-term in vitro. Supplementation resulted in a trend for higher viability, but no additional benefit on functionality was observed.

Cell-mimic polymersome-shielded islets for long-term immune protection of neonatal porcine islet-like cell clusters.

Although islet cell transplantation has emerged as a promising treatment for type 1 diabetes, it remains an unmet clinical application due to the need for immunosuppression to prevent islet elimination and autoimmunity. To solve these problems, we developed novel nanoencapsulation of neonatal porcine islet-like cell clusters (NPCCs) with cell-mimic polymersomes (PSomes) based on PEG-b-PLA (poly(ethylene glycol)-b-poly(dl-lactic acid)). To accomplish this, we first formulated NHS-, NH2-, COOH-, and m(methoxy)-PSomes. This coating utilizes interactions involving NPCC surfaces and PSomes that have covalent bonds, electrostatic interactions, and hydrogen bonds. We extended the range of applicability by comparing the binding affinity of electrostatic attraction and hydrogen bonding, as well as covalent bonds. Our protocol can be used as an efficient hydrogen bonding method because it reduces cell membrane damage as well as the use of covalent bonding methods. We verified the selective permeability of NHS-, NH2-, COOH-, and m-PSome-shielded NPCCs. Furthermore, we showed that a novel nanoencapsulation did not affect insulin secretion from NPCCs. This study offers engineering advances in islet encapsulation technologies to be used for cell-based transplantation therapies.

LEA29Y expression in transgenic neonatal porcine islet-like cluster promotes long-lasting xenograft survival in humanized mice without immunosuppressive therapy

Genetically engineered pigs are a promising source for islet cell transplantation in type 1 diabetes, but the strong human anti-pig immune response prevents its successful clinical application. Here we studied the efficacy of neonatal porcine islet-like cell clusters (NPICCs) overexpressing LEA29Y, a high-affinity variant of the T cell co-stimulation inhibitor CTLA-4Ig, to engraft and restore normoglycemia after transplantation into streptozotocin-diabetic NOD-SCID IL2rγ−/− (NSG) mice stably reconstituted with a human immune system. Transplantation of INSLEA29Y expressing NPICCs resulted in development of normal glucose tolerance (70.4%) and long-term maintenance of normoglycemia without administration of immunosuppressive drugs. All animals transplanted with wild-type NPICCs remained diabetic. Immunohistological examinations revealed a strong peri- and intragraft infiltration of wild-type NPICCs with human CD45+ immune cells consisting of predominantly CD4+ and CD8+ lymphocytes and some CD68+ macrophages and FoxP3+ regulatory T cells. Significantly less infiltrating lymphocytes and only few macrophages were observed in animals transplanted with INSLEA29Y transgenic NPICCs. This is the first study providing evidence that beta cell-specific LEA29Y expression is effective for NPICC engraftment in the presence of a humanized immune system and it has a long-lasting protective effect on inhibition of human anti-pig xenoimmunity. Our findings may have important implications for the development of a low-toxic protocol for porcine islet transplantation in patients with type 1 diabetes.

Supplemental Analysis for N-linked Sugars in Adult Pig Islets

The pig pancreas is considered to be one of the most suitable sources of islets for clinical xenotransplantation. However, after producing α1-3galactosyltransferase knockout pigs, most of the organs of these pigs showed less antigenicity to the human body. Wild-type adult pig islets (APIs) that originally produced negligible levels of α-Gal, different from neonatal porcine islet-like cell clusters, showed a clear antigenicity to human serum. Concerning the so-called non-Gal epitopes, many studies related to glycoproteins and glycolipids are ongoing in efforts to identify them. However, our knowledge of non-Gal glycoantigens remains incomplete. In our previous study, N-glycans were isolated from APIs, and the structures of 28 of the N-glycans were detected. In this study, to identify additional structures, further analyses were performed by liquid chromatography−mass spectrometry (LC-MS). N-glycans were isolated from APIs by the method described by O'Neil et al with minor modifications and LC-MS−based structural analyses were then performed. The detected N-glycan peaks in the LC-MS spectra were selected using the FLexAnalysis software program and the structures of the glycans were predicted using the GlyocoMod Tool. The API preparation contained 11 peaks and 16 structures were then nominated as containing N-linked sugars. Among them, 5 sulfated glycans were estimated, confirming the existence of sulfate structures in N-glycans in API. In addition, these data may supplement several N-glycan structures that contain two deoxyhexose units, such as fucose, to our previous report. The data herein will be helpful for future studies of antigenicity associated with API.

A structural analysis of N-glycans of neonatal porcine islet-like cell clusters (NPCC)

BackgroundN-glycans isolated from neonatal porcine islet-like cell clusters (NPCCs) were analyzed by a mapping technique, to examine the differences in glycosylation and antigenicity between adult pig islets (APIs) and NPCCs. MethodsNPCCs were isolated from 1-to-3day-old neonatal wild-type pigs and cultured for 9days, using the technique described by Korbutt et al. The extract was proteolyzed by treatment with a chymotrypsin and trypsin mixture and further digested with glycoamidase A to release the N-glycans. After the removal of the peptide materials, the reducing ends of the N-glycans were derivatized with 2-aminopyridine. This mixture was applied to DEAE, amide and ODS columns. PA-oligosaccharides were also subjected to MALDI TOF-MS analysis. ResultsThe NPCC glycans were comprised of 14 neutral, 5 mono-sialyl and 5 di-sialyl glycans. As a feature of the N-glycans of NPCC, NPCC contained large amounts of high mannose structures. On the other hand, all of the hybrid and complex types contained a Fucα1-6GlcNAc structure, but were not modified with sulfate residues. Among them, the NPCC preparation contained five neutral and two mono-sialyl glycans and two di-sialyl glycans that were not typically found in adult islets, and seven of these nine were not detected in human islets. Moreover, most of the structures could be clearly identified in this study. ConclusionsThe data herein will be helpful for future studies of the glycoantigen associated with NPCC.

Separation of empty microcapsules after microencapsulation of porcine neonatal islets

Pancreatic islet transplantation is used to treat diabetes mellitus that has minimal complications and avoids hypoglycemic shock. Conformal microencapsulation of pancreatic islets improves their function by blocking immunogenic molecules while protecting fragile islets. However, production of empty alginate capsules during microencapsulation causes enlargement of the transplantation volume of the encapsulated islets and interferes with efficient transfer of nutrients and insulin. In this study, empty alginate capsules were separated after microencapsulation of neonatal porcine islet-like cell clusters (NPCC) using density-gradient centrifugation. Densities of NPCC and alginate capsules were determined using Percoll. Encapsulation products following alginate removal were 97 % of products, with less than 10 % of the capsules remaining empty. The viability of this process compared with manually-selected encapsulated islets indicates the separation process does not harm islets.

A lectin microarray study of glycoantigens in neonatal porcine islet-like cell clusters

BackgroundBesides α-Gal expression, the differences of glycosylation and antigenicity between adult pig islets (APIs) and neonatal porcine islet-like cell clusters (NPCCs) are altogether unclear. In this study, lectin microarray analyses of NPCCs were performed and the results compared with the corresponding values for wild-type APIs and NPCCs from α-Gal transferase knockout (GalT-KO) pig. MethodsNPCCs were isolated from 1–3-d-old neonatal wild-type pigs and cultured for 1 d, 5 d, and 9 d, using a previously described technique. Alternatively, the isoration of APIs were isolated based on the method for human islets. ResultsIn a comparison between NPCCs and APIs, all of the NPCCs showed higher signals for Sambucus nigra, Sambucus sieboldiana, and Trichosanthes japonica I and the binding of α2,6 sialc acid, whereas the APIs showed stronger signals for Lotus tetragonolobus, Aleuria aurantia, Narcissus pseudonarcissus, and Galanthus nivalis, suggesting that APIs contain high levels of high-mannose forms. Among the NPCCs, NPCC (day1) appeared to be richer than the others in Lotus tetragonolobus, Narcissus pseudonarcissus, Galanthus nivalis, and Urtica dioica, implying the presence of high-mannose forms. However, as a whole, the signals for many lectins for NPCCs were very similar. The NPCCs from a GalT-KO pig indicated not only the downregulation of α-Gal expression but α-GalNAc as well, and α2-6 sialic acid was upregulated. ConclusionsThe results reported herein contain useful information for the future production of immunomodified pigs with less antigenicity than GalT-KO pigs toward clinical applications of NPCCs.

A Study of the Glycoantigens of Neonatal Porcine Islet-Like Cell Clusters Using a Lectin Microarray

BackgroundThe pig pancreas is considered to be the most suitable source of islets for clinical xenotransplantation. Two types of islet transplantation are: adult pig islets and neonatal porcine islet-like cell clusters (NPCC). However, besides a-Gal expression, differences in glycosylation and xenoantigenicity between both types were not clear so fat to date. In this study, we performed lectin microarray analyses of NPCCs cultured for 1, 5, or 9 days. MethodsWe studied differences in gycoantigens among several kinds of wild-type NPCCs isolated from 1- to 3-day-old neonatal wild-type pigs (Large White/Landrace × Duroc) and cultured for 1, 5 and 9 days in Ham's 10 in the presence of nicotinamide, using a previously published technique. After sonication and centrifugation, supernatant proteins from each islet were labeled with Cy3, applied to a lectin array and scanned with an SC-Profiler for evaluation using an Array Pro Analyzer. ResultsThe overall signals of NPCC at days 5 and 9, showed almost the same values to most lectins, whereas those on day 1 showed differences, suggesting that the NPCC on day 1 contain immature cells that gradually turn to mature NPCCs in culture.

Adenovirus-mediated heme oxygenase-1 gene transfer to neonatal porcine islet-like cluster cells: the effects on gene expression and protection from cell stress

Porcine islet xenotransplantation is a promising strategy for the treatment of diabetes that overcomes donor shortages. However, islet xenografts are susceptible to oxidative stress and apoptosis. Heme oxygenase-1 (HO-1) has been shown to protect cells from oxidative stress, apoptosis and inflammation. Here, we investigated whether introduction of human HO-1 (hHO-1) into neonatal porcine islet-like cell clusters (NPCCs) can induce beneficial transcriptional changes in NPCCs against cellular stress. NPCCs were transduced with either adenovirus-HO-1 (Ad-HO-1) or control adenovirus-GFP (Ad-GFP). After treatment with hydrogen peroxide (H2O2) for 24 hours, nitrite oxide (NO) production assays were performed to detect oxidative stress. Microarray analysis was performed using a pig oligonucleotide 44 K gene chip. We profiled transcriptional changes to apoptosis, oxidant and inflammatory genes, and real-time PCR analysis was also performed to confirm the microarray results. Survival of NPCCs after treatment with H2O2 was significantly higher in the Ad-HO-1 group (p<0.001), and NO production also decreased in the Ad-HO-1 group (p<0.01). The microarray results showed that the expression of pro-apoptosis genes such as CASP3, CASP7, CASP10, CIDE-B and CIDE-C was significantly decreased in the Ad-HO-1 virus group (CASP10; p<0.05, CASP3, CIDE-C; p<0.01, CASP7, CIDE-B; p<0.001). We also found that the expression of oxidative stresses genes including COX1, COX2, CYB5A, SDHD and NOS2 was decreased, and that the anti-oxidant genes Gpx1 and SOD2 were increased in the Ad-HO-1 group (NOS2; p<0.05, COXI, COX2, CYB5A, SDHD, SOD2, GPX1; p<0.001). However, inflammatory gene expression was not significantly changed. Realtime PCR analysis confirmed the results of the microarray analysis. These results shed light on the underlying mechanisms of the protective effects of hHO-1 on porcine islets from cellular stresses and suggest that hHO-1 could be a promising target gene for the production of transgenic pigs that confer improved islet xenograft survival.

Encapsulated pancreatic progenitors derived from human embryonic stem cells as a therapy for insulin‐dependent diabetes

Cellular-based therapies for insulin-dependent diabetes are potential means of achieving and maintaining normal blood glucose levels (BGL) without the need for insulin administration. Islets isolated from donor pancreases have been the most common tissue used to date, but supply is a limiting factor. The use of human embryonic stem cells (hESC) as a therapy became a possibility with the report that these cells could be differentiated to pancreatic progenitors (PP) over 12 days in vitro. Conversion of PP to glucose-responsive insulin-secreting cells can be achieved by transplanting the progenitors in vivo where cell maturation occurs. To date this step has not been shown under in vitro conditions. Prior to transplanting, cells are encapsulated in alginate to prevent the immune cells of recipient attacking the graft. The alginate capsules have pores with a molecular weight cut-off of 250 kDa. These are too small to allow entry of immune cells, but large enough for passage of nutrients and insulin. Encapsulated insulin-producing cells survive and function when transplanted, and have been shown to normalize BGL when allografted into diabetic mice. As few as 750 encapsulated human islets are sufficient to normalize BGL of diabetic non-obese diabetic severe combined immunodeficient (NOD/SCID) recipient mice for at least 2 months. The safety of transplanting encapsulated human islets as demonstrated by the lack of major adverse events and infection was recently shown in a first-in-human clinical trial. Finally, fetal porcine islet-like cell clusters, which are akin to PP derived from ESC, mature and normalize BGL of diabetic recipient mice with the same efficiency as non-encapsulated clusters placed under the kidney capsule. Transplanting encapsulated PP, derived from hESCs, into diabetic recipients is the strategy that is now being explored in the Australia Diabetes Therapy Project.

Anticarbohydrate Antibody Repertoires in Patients Transplanted with Fetal Pig Islets Revealed by Glycan Arrays

Ten patients with type I diabetes were transplanted with porcine fetal islet-like cell clusters (ICC) between 1990 and 1993. A significant rise in the anti-α-Gal antibody titers was seen posttransplant, but also non-α-Gal-specific antibodies were detected in some patients. We have reanalyzed the carbohydrate specificity of antibodies in the sera from seven of these patients taken before transplantation, 1, 6 and 12 months posttransplantation using a glycan array with 200 structurally defined glycans. The main findings were: (i) prepig ICC transplantation patients had antibodies reactive with terminal α-GalNAc (e.g. the Forssman antigen, but not the blood group A determinant in blood group A patients), α-Gal (except blood group B determinants in B individuals), β3-linked Gal especially Galβ1,3GlcNAc even if terminally sulfated or sialylated, β-GlcNAc except if β1,3-linked and oligomannosyl compounds; (ii) the titers of all carbohydrate-specific antibodies detected before transplantation rose after transplantation; (iii) the kinetics of the antibody responses differed between patients; (iv) in some patients antibodies reacting with Galα1,3Lex and several structures terminated with Neu5Gc appeared after transplantation. In conclusion, anti-α-Gal antibodies are the predominant anticarbohydrate antibodies detected after porcine ICC transplantation, with some patients also developing Neu5Gc-specific antibodies. Their clinical significance needs to be established.

Cobalt Protoporpyhrin Reduces Caspase-3,-7 Enzyme Activity in Neonatal Porcine Islets, But Does Not Inhibit Cell Death Induced by TNF-α

Apoptotic phenomena observed in vitro following isolation and following transplantation contribute significantly to islet graft loss. Strategies to reduce apoptosis of islet tissue prior to and posttransplantation may improve graft survival and function and reduce the amount of tissue necessary to achieve insulin independence. The expression of cytoprotective proteins is one such strategy that may prolong islet survival. In this light, heme-oxygenase 1 (HO-1) upregulation has been studied in both allo- and xenotransplantation models. In this study, the effect of HO-1 on apoptosis in neonatal porcine islet-like cell clusters (NPICC) was assessed. In in vitro assessments of NPICC apoptosis, NPICC showed a high sensitivity to apoptotic stimulation using a combination of TNF-alpha and cycloheximide. Stimulation with TNF-alpha alone was sufficient to induce reproducible apoptotic responses as demonstrated by caspase-3,-7 activation and subdiploid DNA analysis. Dose-dependent, high-level HO-1 protein expression was achieved following culture of NPICC in medium containing either cobalt protoporphyrin (CoPP) or cobalt mesoporphyrin (CoMP). CoPP treatment resulted in the reduction of caspase-3,-7 enzyme activity following TNF-alpha stimulation. However, such an effect was not associated with a reduction in the levels of cell death. Indeed, the inhibition of caspase enzyme activity resulted in decreased PARP-1 cleavage, which may lead to heightened levels of necrosis in treated NPICC cultures, possibly explaining the observed commitment of NPICC to the death pathway.

A study of the xenoantigenicity of neonatal porcine islet‐like cell clusters (NPCC) and the efficiency of adenovirus‐mediated DAF (CD55) expression

The pig pancreas is considered to be the most suitable source of islets for xenotransplantation in patients with type I diabetes. The objective of this study was to assess the antigenicity of neonatal porcine islet-like cell clusters (NPCC), including the Galalpha1-3Galbeta1-4GlcNAc-R (alpha-Gal) and Hanganutziu-Deicher (H-D) antigens, and the pathway involved in human complement activation. The efficiency of expression of human decay-accelerating factor (DAF: CD55) on NPCC by adenoviral transduction was also examined, and the functional capacity of DAF was also estimated. The deposition of human natural antibodies, immunoglobulin (Ig)G and IgM, and the expression of alpha-Gal and H-D antigens on NPCC were investigated by FACS analysis. The downregulation in the antigenicity to human natural antibodies, including the alpha-Gal and H-D antigens on NPCC by treatment with tunicamycin, PDMP and neuraminidase were also examined. In addition, complement-mediated islet lysis was examined using factor D-deficient and C1-deficient sera. An adenovirus encoding DAF under the control of the cytomegalovirus promoter, Ad.pCMV-DAF, was then constructed, and used for transducing NPCC. The amelioration of complement-dependent cytotoxicity of the NPCC by the transduced DAF was assessed as an in vitro hyperacute rejection model of a pig to human xenograft. The NPCC clearly expressed the alpha-Gal epitope, and the human natural antibodies, IgG and IgM, and the anti-H-D antibody also reacted with the NPCC. Treatment of NPCC with tunicamycin led to a drastic reduction in the extent of deposition of IgG, indicating the importance of N-linked sugars on the islets, presumably related to alpha-Gal expression on N-linked sugars. Neuraminidase treatment indicated the presence of, not only the H-D antigen, but also other sialic acid antigens which reacted with the human natural antibody, especially IgG. The complement deposition of factor B on NPCC was clear, and the alternative pathway-mediated NPCC killing accounted for approximately 30% of that by the total complement pathway. On the other hand, approximately 90% of the NPCC could be transduced to express DAF by the adenovector, Ad.pCMV-DAF. The expressed DAF showed an approximately 50-62% suppression in complement-dependent NPCC lysis. The origin of the antigenicity of NPCC is mainly N-linked sugars including alpha-Gal and sialic acid antigens, and NPCC expressed the transduced molecule in high efficiency by the adenovector.

The Efficacy of CD40 Ligand Blockade in Discordant Pig-to-Rat Islet Xenotransplantation Is Correlated with an Immunosuppressive Effect of Immunoglobulin

The authors' aim was to evaluate the efficacy of immunosuppression with monoclonal anti-CD40 ligand antibodies (aCD40L) or nonspecific polyclonal intravenous immunoglobulin (IVIG) in the pig-to-rat islet xenotransplantation model. Fetal porcine islet-like cell clusters were transplanted under the kidney capsule of nondiabetic rats. All antibodies were administered alone or in combination with cyclosporine A (CsA). In addition, some animals were administered antibodies plus tacrolimus (TAC) or sirolimus (SIR). Twelve days after transplantation, islet xenograft survival and rejection were evaluated using immunohistochemistry. aCD40L plus CsA had a pronounced inhibitory effect on islet xenograft rejection for up to 12 days after transplantation. Unexpectedly, treatment with a monoclonal control antibody (anti-keyhole limpet hemocyanin [aKLH]) plus CsA had a similar inhibitory effect. Furthermore, a similar inhibition of islet xenograft rejection was observed also in animals administered IVIG plus CsA. Monotherapy with aCD40L, aKLH, IVIG, or CsA had no effect on the rejection process. Also, when aCD40L or aKLH was administered together with TAC, islet xenograft rejection was inhibited. There was no marked difference compared with rats treated with aCD40L or aKLH and CsA. Immunosuppression with aCD40L or aKLH in combination with SIR also inhibited pig-to-rat islet xenograft rejection, but the protective effect was not as pronounced. Immunosuppression with high doses of antibodies, monoclonal or polyclonal, in combination with CsA or TAC inhibits pig-to-rat islet xenograft rejection. No specific effect of co-stimulatory blockade with aCD40L could be observed. Instead, the results indicate a nonspecific immunosuppressive effect of high doses of antibodies in this model.

MyD88-dependent toll-like receptor signalling is not a requirement for fetal islet xenograft rejection in mice.

Rejection of pancreatic islet xenografts in mice shares immunopathological features with a Th1-associated delayed-type hypersensitivity (DTH) reaction. The aim of the present study was to investigate the mechanism of acute cellular xenograft rejection in a strain of mice with a targeted gene disruption of the toll-like receptor (TLR) signal adaptor protein MyD88. These mice have been shown to have markedly impaired Th1 immunity. The MyD88-/- and normal mice were transplanted with 2 microl of fetal porcine islet-like cell clusters (ICC) under the left kidney capsule. On days 3, 6 or 12 after transplantation the mice were killed and the grafts either prepared for immunohistochemistry or real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The number of remaining ICC and infiltrating cells with different phenotypic characteristics was assessed semi-quantitatively. Grafts used for quantitative RT-PCR were analysed for content of murine mRNA of interferon (IFN)-gamma, interleukin (IL)-12p40, IL-4 and IL-10. On day 3, the rejection process was initiated in both MyD88-/- and normal mice as characterized by a moderate infiltration of F4/80+ and MAC-1+ macrophages and occasional CD3+ and CD4+ cells. Expression of IFN-gamma and IL-12p40 was lower but still detectable in the MyD88-/- mice, when compared with control animals. By day 6, rejection was almost completed in all animals with only few ICC remaining. 12 days after transplantation all grafts were completely destroyed and heavily infiltrated by macrophages. Moderate numbers of CD3+ and CD4+ and occasional CD8+ cells were also present. Islet xenograft rejection was found to persist in MyD88-/- mice. Despite a relatively lower expression of the Th1-associated cytokines IFN-gamma and IL12-p40 within the xenograft area, both the time course and morphological pattern of the rejection were essentially similar to that found in normal animals. Hence, MyD88-dependent TLR signalling does not appear to be a crucial component of acute cellular xenograft rejection.

Importance of the Gal alpha1-3 Gal antigen in discordant islet xenotransplantation: immunosuppression, which inhibits porcine islet xenograft rejection in ordinary mice, is equally effective in Gal-knockout mice.

Islet xenotransplantation will most likely be performed in diabetic patients treated with immunosuppressive drugs. The importance of the galactosyl alpha(1-3) galactose (Galalpha1-3Gal) antigen in immunosuppressed islet xenograft recipients has not been studied. Fetal porcine islet-like cell clusters (ICCs) were transplanted into the renal subcapsular space of both Gal-knockout mice and ordinary mice. Transplantations were performed in untreated mice and mice immunosuppressed with cyclosporine A (CsA) plus 15-deoxyspergualin (DSG). Studies were also performed in immunosuppressed Gal-knockout mice that had been actively immunized against Galalpha1-3Gal. Evaluation was performed 12 days after transplantation using morphologic techniques. The levels of serum immunoglobulin (Ig)G and IgM to the Galalpha1-3Gal antigen or to the ICCs were determined. No difference in the morphologic appearance could be seen between ordinary mice and Gal-knockout mice. No deposits of IgG, IgM, or C3 could be detected. Almost no difference could be seen between immunosuppressed Gal-knockout mice and immunosuppressed ordinary mice. In immunosuppressed, immunized Gal-knockout mice, the results were similar. In ordinary mice treated with CsA+DSG, the levels of anti-Gal IgM were lower than they were in untreated mice, whereas the levels of anti-Gal IgG were similar. In Gal-knockout mice (including immunized animals) treated with CsA+DSG, the levels of anti-Gal IgG and IgM were lower than they were in untreated Gal-knockout mice. After renal subcapsular transplantation, antibodies against Galalpha1-3Gal have no major influence on islet xenograft rejection in the pig-to-mouse model. Immunosuppression, which inhibits rejection in the pig-to-mouse model, is equally effective when transplantation is performed across the Galalpha1-3Gal barrier.

Tacrolimus inhibits discordant islet xenograft rejection: a study in the pig-to-rat model.

The aim of the present study was to evaluate the immunosuppressive effect of tacrolimus (TAC) in discordant islet xenotransplantation. Fetal porcine islet-like cell clusters (ICCs) were transplanted under the kidney capsule in normoglycemic rats treated with TAC monotherapy, TAC plus other immunosuppressive drugs or cyclosporin A (CsA) monotherapy. Twelve or 24 days after transplantation, the extent of a cellular infiltration in the xenografts was evaluated using immunohistochemistry. In some animals, the grafts were examined for antibody and complement deposition and the levels of xenoreactive antibodies in serum were determined. In untreated rats, the xenografts were completely rejected after 12 days and no intact ICCs remained. TAC monotherapy (at 0.5 and 1.0 mg/kg b.w.) almost completely inhibited rejection for up to 12 days. In animals treated with TAC monotherapy (at 0.5 mg/kg b.w.), rejection was markedly inhibited for up to 24 days. However, the effect after 24 days was not consistent and in some grafts there were signs of rejection. The protective effect of TAC observed in this study is in contrast to the findings in rats given CsA monotherapy in which no or only a marginal effect on islet xenograft rejection was observed. Only when CsA was given at 20 mg/kg b.w., an inhibitory effect could be observed. Immunosuppression with TAC at a suboptimal dose (0.3 mg/kg b.w.) plus 15-deoxyspergualin or brequinar also had an inhibitory effect on the rejection. In animals given TAC plus mycophenolate mofetil, a protective effect was observed as well; however, this effect was not consistent.

Insulin secretagogues, but not glucose, stimulate an increase in [Ca2+]i in the fetal human and porcine beta-cell.

Fetal pancreatic beta-cells release insulin poorly in response to glucose; however, the cellular mechanism for this is unknown. By using fura-2 to measure changes in the cytoplasmic free Ca(2+) concentration in beta-cells, we examined human/porcine fetal islet-like cell clusters (ICCs) and human adult islets for the presence of functional K(+)(ATP) and voltage-activated Ca(2+) ion channels. The effects of glucose, glyceraldehyde, leucine, KCl, and the channel effectors glipizide and BAY K8644 were studied. In fetal human/porcine ICCs and adult islets, KCl, glipizide, and BAY K8644 increased [Ca(2+)](i). Both glucose and glyceraldehyde increased [Ca(2+)](i) in islets but had no effect on ICCs. Leucine increased [Ca(2+)](i) in islets and porcine but not human ICCs. We hypothesize that the beneficial effect of leucine in fetal porcine, but not human ICCs, is attributable to time-dependent maturation of the beta-cells, because porcine ICCs examined were at 87% of the gestational period, and human ICCs were at 42%. Our data demonstrate that both K(+)(ATP) and voltage-activated Ca(2+) channels, required for glucose-stimulated increase in [Ca(2+)](i), are functional early in gestation. This suggests that the cause of the immaturity of fetal human/porcine beta-cells is at a more proximal step of glucose-induced metabolism than the channels on the cell surface.