Pig Islets Research Articles

Cutting Edge: Pig Islet Xenografts Are Susceptible to “Anti-Pig” But Not Galα(1,3)Gal Antibody Plus Complement in Gal o/o Mice

Abstract Hyperacute rejection due to Galα(1,3)Gal (Gal) Ab plus complement (C′) is a major problem in xenografting vascularized organs from pigs to primates, but the fate of neovascularized xeno islets is unclear. Nonendocrine islet cells are Gal+, and there is a large rise in Gal Abs after transplantation, but graft remnants persist for some days in monkeys and humans. To define the role of αGal Ab plus C′ in porcine islet graft rejection, cultured porcine fetal islets were grafted to mice lacking the α(1,3)galactosyltransferase gene. Anti-Gal Ab plus C′ did not cause islet damage or rejection in mice lacking the α(1,3)galactosyltransferase gene, even when additional Ab plus C′ was given; in addition, hyperimmune mice (titer >1/20,000) did not reject pig islets, showing that islets are resistant to Gal Ab plus C′. However, islets can be destroyed by polyclonal mouse anti-pig Abs. Thus, the focus of islet xenografting should not be on Gal Ab plus C′.

LARGE SCALE ISOPYCNIC ISLET PURIFICATION UTILIZING NON-TOXIC, ENDOTOXIN-FREE MEDIA FACILITATES IMMEDIATE SINGLE-DONOR PIG ISLET ALLOGRAFT FUNCTION

120 Early graft function has been difficult to accomplish in clinical islet allotransplantation. It is becoming apparent that reagents used for islet preparation may stimulate the inflammatory response to islet grafts and may thereby interfere with early islet function and engraftment. The purpose of this study therefore was to develop islet purification density gradient media restricted to non-toxic, endotoxin-tested components and to subsequently test whether islets purified on the refined gradients immediately reverse diabetes in the relevant preclinical single-donor pig allograft model. Density gradients were constructed based on iodixanol x-ray contrast media and UW solution which are both approved for clinical use. In the first set of experiments, the osmolality most suitable for islet separation was identified. The effect of four different osmolalities (320; 350; 375; and 400 mosm/kg) on the density profile of islet and acinar tissue was studied by means of continuous test gradients. In the second part of the study, iodixanol/UW gradients were applied for large scale continuous islet density gradient purification on a Cobe 2991 and the percentage recovery of islet equivalents (IE) in fractions with purities ≥ 90% from iodixanol/UW was compared to Ficoll Na-diatrizoate gradients (previous standard). To test whether iodixanol/UW gradients interfere with immediate reversal of hyperglycemia posttransplant, 7134±1830 purified IE/kg body weight were intraportally transplanted into five streptozotocin-diabetic pigs. The percentage of exocrine tissue contaminating 95% yield of islets were 0.45±0.96%, 0.0±0.0%, 0.45±0.69%, and 0.80±1.00% for 320, 350, 375, and 400 mosm/kg iodixanol/UW gradients, respectively. The percentage recovery of pure islet equivalents from 350 mosm/kg iodixanol/UW (n=6) gradients was significantly higher compared to the standard Ficoll Na-diatrizoate (n=4) gradient (86.2±11.8% vs 67.0±9.3%; p=0.013). Four out of five consecutive diabetic single-donor pig islet allograft recipients became normoglycemic and insulin-independent within 24 hrs following transplantation. The data indicate that single donor pig islet allografts purified on non-toxic, endotoxin-free gradients establish insulin independence immediately. Osmolality adjustments should make iodixanol/UW gradients also suitable for human islet purification. Failure to achieve immediate insulin independence in the clinical setting would then point to non-technical obstacles.

In vitro xenorecognition of adult pig pancreatic islet cells by splenocytes from nonobese diabetic or non-diabetes-prone mice.

In vitro studies of the nonobese diabetic (NOD) mouse prone to type 1 autoimmune diabetes were conducted in order to investigate the mechanisms possibly involved in cell-mediated rejection of adult pig islet xenografts. Mouse cellular proliferation in discordant situations was previously investigated only with stimulator lymphocytes and found to be low in intensity and due to an indirect recognition mechanism involving murine antigen-presenting cells (APC). It was also important to characterize murine anti-pig islet response. In the present study, mouse splenocytes responded to pig islet cells since primary proliferations were detected in non-diabetes-prone Balb/c (P<0.04) or NOD (P<0.001) mice. Moreover, NOD mice displayed a higher (P<0.003) splenocyte response to pig islet cells (stimulation index: 5.8+/-0.7) than did Balb/c mice (stimulation index: 2.3+/-0.3), whereas responses to pig stimulator splenocytes were similar in both strains. The proliferation of NOD splenocytes to pig islet cells was lower (P<0.0001) than the allogeneic response to Balb/c islet cells but similar to syngeneic proliferation to NOD islet cells. In both NOD and Balb/c mice, splenocyte proliferation to pig islet cells was abolished (P<0.01) when CD4+ cells were blocked with antibodies, whereas the blocking of CD8+ cells had a nonsignificant effect. The main T-splenocyte subsets involved were restricted to mouse MHC class II molecules as they did not proliferate in the presence of monoclonal antibodies directed at I-A molecules. NOD and Balb/c splenocyte proliferation to pig islet cells was abolished after removal of plastic-adherent APC, which indicates that the major activation pathway was indirect. Purified CD4+ or CD8- cells alone did not proliferate in response to pig islet cells but recovered a proliferative ability when mixed with APC. CD4- cells, alone or in the presence of APC, were not capable of responding to pig islet cells. Both Th1 and Th2 splenocytes were involved in response to pig islet cells since interferon-gamma (IFN-gamma) and interleukin (IL-)-4 production increased significantly (300-fold and 11-fold, respectively, P<0.02 for both), whereas the increase in IL-10 production was much lower (only 1.5-fold). The IFN-gamma/IL-4 and IFN-gamma/IL-10 ratios stimulated by pig islet cells were not different with NOD and Balb/c splenocytes. In conclusion, mouse cell-mediated reaction against xenogeneic adult pig islet cells mainly involves class II-restricted CD4+ T lymphocytes of Th1 and Th2 subtypes, with an indirect pathway for the recognition. Although of low intensity, this cell-mediated reaction constitutes an obstacle to pig islet engraftment in the mouse, although one not necessarily more insurmountable than alloreactivity. The peculiarity of NOD mouse splenocytes, in terms of proliferation against pig islets, suggests that the study of islet xenograft rejection should take the immunogenetic context of diabetes into account, in which case the use of non-diabetes-prone mice has its limitations.

Adenovirus-mediated catalase gene transfer reduces oxidant stress in human, porcine and rat pancreatic islets.

Susceptibility of pancreatic islets to oxidant stress may affect islet viability and contribute to primary non function of allo- or xenogenic grafts. We investigated the influence of overexpression of catalase (CAT) on the viability of human, porcine and rat islets, as well as INS-1 beta-cell line. Islets were transfected with a replication-deficient adenovirus vector containing human CAT cDNA under the control of the adenovirus major late promoter (AdCAT) or a vector containing no foreign gene (AdNull) and used as a control. Oxidant stress was induced 48 h later by xanthine oxidase-hypoxanthine (XO 25 mU/ml, HX 0.5 mmol/l) or hydrogen peroxide (100 or 250 micromol/l). Islet cell viability was assessed 72 h after CAT transfer by 4-[3-(4-Idophenyl)-2-(4 nitrophenyl)-2H-5-tetrazolio]-1,2,benzene disulphonate (WST-1) test. Baseline catalase activity was three to fourfold lower in porcine than in human islets. CAT activity was reproducibly increased 2.5- to 7-fold in AdCAT infected islets, at least for 13 days. Overall, AdCAT conferred on human and pig islets a protection of 26.1 +/- 6.1 and 21.2 +/- 9.8% on XOHX injury and 35.4 +/- 4.2 and 57.9 +/- 10.5% on H2O2 stress. Similarly, rat islet cells and INS-1 cells were protected on XOHX stress by 17.8 +/- 2.3 and 30.8 +/- 8.7%, respectively. AdNull had no effect. Basal and stimulated insulin secretion was preserved in AdCAT-transfected human islets despite a XOHX challenge. This study validates adenovirus-mediated catalase gene transfer as a realistic approach to reduce non specific inflammation effects on human or porcine islet grafts. Moreover the relevance of defense mechanisms, previously suggested in human islets, is here illustrated in porcine islets.

Xenotransplantation of adult porcine islets in diabetic mice. A study of UVB irradiation, cryopreservation and immunosuppression on graft survival time.

The major obstacle for successful xenotransplantation of islets to large animals and human diabetics is the host rejection. To address the rejection problem, we studied the efficacy of UV-B irradiation, cryopreservation and immunosuppression on the in vivo functional time and immunogenicity of adult porcine islets (PI) in outbred CD1 mice. Exposure of PI to UV-B irradiation between 300-1800J/M2 did not affect the cellular viability as assessed by fluorescein diacetate or their daily insulin secretion in vitro. Fresh PI normalized the blood glucose (BG) of diabetic CD1 mice for 3.1+/-0.6 (n = 8, mean+/-SEM) days. Islets treated with 600J/M2 UV-B irradiation or cryopreservation had similar graft functional times to fresh islets upon transplantation in diabetic CD1 mice. Immunosuppression with cyclosporin A (CsA), antilymphocyte serum (ALS) and FK506 prolonged the functional time of fresh pig islets to 7.9+/-0.9 (n = 9), 6.2+/-1.3 (n = 5) and 24.2+/-10.4 (n = 12) days, respectively. However, additional pretransplant treatment with either UV-B irradiation or cryopreservation did not further increase the functional time of pig islets in mice immunosuppressed with CsA. Furthermore, there was no apparent difference in the frequency of appearance of cytotoxic antibodies and antibody titers in the recipients of UV-B irradiated or cryopreserved pig islet compared with non-treated islets. The UV-B irradiation and cryopreservation of PI before transplantation with the present protocols did not appear to have significant effect on the islet immunogenicity when assessed by in vivo survival duration and anti-donor antibody titer production.

Cyclophosphamide, but not CTLA4Ig, prolongs survival of fetal pig islet grafts in anti-T cell monoclonal antibody-treated NOD mice.

Fetal pig islets, xenografted after organ culture into non-immunosuppressed prediabetic NOD mice, are rejected within 10 days. Immunosuppression with anti-T cell (anti-CD4 and anti-CD3) monoclonal antibodies alone is highly effective in delaying graft rejection in this discordant model, but rejection eventually occurs, usually within 80 days, despite marked depletion of T cells. In an attempt to prevent rejection, we used cyclophosphamide (CP), a powerful anti-B cell agent, or CTLA4Ig, an inhibitor of T-cell co-stimulation [via B7-1 (CD80) and B7-2 (CD86)], either given in combination with anti-CD4 (GK1.5) or anti-CD3 (KT3) MAb to the recipient mice. The addition of cyclophosphamide in a dose that significantly depleted B cells in peripheral blood was highly effective in preventing rejection, with xenografts surviving for at least 112 days, when the experiment was terminated. CTLA4Ig, administered alone, did not prevent delayed rejection (rejection occurred in <60 days) and, in contrast to CP, did not prevent delayed rejection when used in combination with GK1.5 and KT3 treatment. Thus, immunosuppressive agents found to be highly effective in other strains, e.g., CTLA4Ig and anti-T cell MAbs, had a lesser effect in NOD mice but the addition of an anti-B cell drug, CP, was useful. This finding may be applicable to patients with IDDM.

Spleen cells of non-obese diabetic mice fed with pig splenocytes display modified proliferation and reduced aggressiveness in vitro against pig islet cells.

A new means of modifying xenogeneic reaction to pig islet cells, which involves pre-feeding with pig spleen cells, was investigated for the first time in the non-obese diabetic (NOD) mouse. Compared with controls, mice fed with pig spleen cells displayed much higher splenocyte proliferation in response to pig spleen and islet cells (p < 0.0001). This enhanced proliferation was specific for the species providing the fed cells. Positive relationships (p < 0.01) were found between increased splenocyte proliferation in response to pig spleen or islet cells and the number of cells per feeding or the number of daily feedings. Concomitantly, while co-incubation with splenocytes from control mice led to inhibition of both basal and stimulated insulin releases from pig islet cells (p < 0.001), this aggressiveness was abolished (p < 0.001) after co-culture with splenocytes from mice fed with pig spleen cells. The proliferative responses of splenocytes from fed or control mice to pig islet or spleen cells were abolished after removal of plastic-adherent cells, indicating that the major indirect pathway of T-cell activation was unchanged by pig spleen cell feeding. The main T-splenocyte subsets involved were restricted to MHC class II as they did not proliferate in the presence of monoclonal antibodies (mAbs) directed at I-A molecules. In mice fed with pig spleen cells, as well as in control mice, the blocking of CD4 + T cells with mAbs led to abolition of proliferation (p < 0.002), while the blocking of CD8 + led to a less marked effect. However, an increase in the blocking effect of anti-CD8 mAbs was noted in mice fed with pig spleen cells (p < 0.02). In control mice, the main splenocyte subset involved during proliferation in response to pig islet cells was Thl, since interferon gamma (IFNgamma) production increased significantly (p < 0.01) while that of interleukin-10 (IL-10) increased only slightly. The main change observed in mice fed with pig spleen cells was a marked increase in basal IL-10 production (p < 0.01) and the basal IL-10/IFNgamma ratio (p < 0.001). It seems likely that feeding with pig spleen cells shifted the Th1/Th2 balance towards a dominance of Th2-type class II-restricted CD4 + T cells, which may have been conducive to activating CD8 + suppressor T cells. In any event, oral administration of pig cells modified xenogeneic cellular response, which may have implications for xenografts of pig islets. In a more general sense, physiological feeding of cells from xenogeneic species would appear to have certain effects on the immune system.

AN IMPROVED TECHNIQUE FOR ISOLATING PIG ISLETS: THE IMPORTANCE OF TENSEGRITY IN ISLET LONG TERM CULTURE VIABILITY

176 Pig Islets (PI) are clearly the xenogeneic donor islet of choice for human islet transplant. Pig insulin is known to work well in the human without antibody problems. Furthermore, newer short-term immunosuppression can markedly reduce early graft loss with PI and tolerance induction can even be demonstrated in some discordant xenografts. Isolation of PI remains a major problem with islet fragmentation and islet loss during digestion. In addition, PI undergo a high rate of apoptosis following isolation in culture which can result in 50-95% loss of PI in 24-48 hours. We initially observed that PI embedded in extracellular adherent matrix exhibited an Anchorage-Dependent cell growth and survival with viability of >80%(Ethidium Bromide/Acridine Orange Assay - EB/AO) at 20 days (p<0.01 with controls). We tested the so-called "Tensegrity Concept" that cell viability and growth in culture depends on adhesion and extracellular integrin structuring and "scaffolding". Tensegrity is optimal when the cell cytoskeleton and the extracellular matrix integrate to achieve a physiological balance of intra and extracellular mechanical tension. By shortening collagenase digestion to yield PI attached to acinar matrix cell remnants, we sought to provide a potential extracellular structure to develop tensegrity. Control PI digestions showed 95% purity with pure islets free of attached acinar and extracellular matrix elements, but a loss of EB/AO viability to 40% and 10% in 24 and 48 hours. In contrast, the matrix-embedded PI had a purity of only 52% but an EB/AO viability of 93% and 86% at 24 and 48 hours and 65% at 30-35 days(p<0.05) At 1 month, the acinar tissue had largely died but fibrous strands remained attached to the cells. Islet function in static incubation was excellent with a insulin rise of 420% in response to a increased glucose incubation solution (normal for fresh islets=320%,p=>0.10). Thus, incomplete digestion of PI with retention of attached elements of the acinar and extracellular matrices apparently supplies a tensegrity (extracellular matrix scaffolding) which promotes long term growth and viability of PI in culture. We conclude that incomplete digestion of islets and/or imbedding islets in extracellular matrix markedly enhances the viability and long term yield of PI and maybe the technique of choice for PI digestion and preparation of PI for culture and/or transplantation.

LONG TERM (10 MONTH) PIG ISLET XENOGRAFT (PIX) TOLERANCE USING MEASURED-TOTAL LYMPHOID IRRADIATION, SPLENECTOMY SHORT TERM RABBIT ANTITHYMOCYTE GLOBULIN AND DEOXYSPERGUALIN WITHOUT CHRONIC IMMUNOSUPPRESSION

320 Tolerance of pig islet xenografts without chronic immunosuppression is an attractive protocol for islet transplantation since allograft islets are in short supply and chronic immunosuppression induced morbidity and mortality is not an acceptable trade off for diabetic morbidity, especially in the juvenile age group when islet replacement should be performed ideally. In this study, 30 Lewis Rats (STZ diabetes) were transplanted with 2000 pig islet xenografts (PIX). All 30 animals had measured-total lymphoid irradiation with implanted quantitative radiosensors, splenectomy, a 3-day course of rabbit-antithymocyte globulin and deoxyspergualin (21 days at 2.5 mg/kg). The incidence of early graft loss was 0% and all pig islet xenografts survived 200 days or more. The mean pig islet xenograft survival, as determined by euglycemia and differential radioimmunoassay (All animals had circulating Pig Insulin @ 300 days) was 302 +/-42 days with euglycemia to 14 months in 23 animals.In addition, nephrectomy of the host to remove the functioning islet tissue produced a return of the diabetic state in 9 animals. This long term function and tolerance of pig islet xenograft was associated with minimal changes in T cell reactivity assayed by MLC and CML testing. In contrast, studies of anti donor CDC and ADCC showed a complete antibody block in long term function and tolerance in 10 animals (1:8 and 1:128) A separate Control group who rejected their PIX early had high levels of CDC and ADCC to donor (1:64 and 1:1024, p<0.05). The long term function and tolerance of PIX was associated closely with a block of anti-donor CDC and ADCC antibodies. The superior long term function and tolerance appears due to the unique immunosuppression which permits variations in the measured-total lymphoid irradiation to deliver uniform measured doses of TLI as well as short term effective anti-B cell suppression with splenectomy and rabbit-antithymocyte globulin-deoxyspergualin short-term treatment. Animals remained in a robust state of health presumably because they were off all drug immunosuppression for over 9 months. This suppressive regimen seems superior to all others we have tested because of the length of islet survival and the excellent health of the animal and suggests that Chronic Immunosuppression with Drugs is the major debilitating effect of multiple agent suppressive protocols.

LONG TERM (10 MONTH) PIG ISLET XENOGRAFT (PIX) TOLERANCE USING MEASURED-TOTAL LYMPHOID IRRADIATION, SPLENECTOMY SHORT TERM RABBIT ANTITHYMOCYTE GLOBULIN AND DEOXYSPERGUALIN WITHOUT CHRONIC IMMUNOSUPPRESSION

320 Tolerance of pig islet xenografts without chronic immunosuppression is an attractive protocol for islet transplantation since allograft islets are in short supply and chronic immunosuppression induced morbidity and mortality is not an acceptable trade off for diabetic morbidity, especially in the juvenile age group when islet replacement should be performed ideally. In this study, 30 Lewis Rats (STZ diabetes) were transplanted with 2000 pig islet xenografts(PIX). All 30 animals had measured-total lymphoid irradiation with implanted quantitative radiosensors, splenectomy, a 3-day course of rabbit-antithymocyte globulin and deoxyspergualin (21 days at 2.5 mg/kg). The incidence of early graft loss was 0% and all pig islet xenografts survived 200 days or more. The mean pig islet xenograft survival, as determined by euglycemia and, differential radioimmunoassay (All animals had circulating Pig Insulin @ 300 days) was 302 +/- 42 days with euglycemia to 14 months in 23 animals. In addition, nephrectomy of the host to remove the functioning islet tissue produced a return of the diabetic state in 9 animals. This long term function and tolerance of pig islet xenograft was associated with minimal changes in T cell reactivity assayed by MLC and CML testing. In contrast, studies of anti donor CDC and ADCC showed a complete antibody block in long term function and tolerance in 10 animals (1:8 and 1:128) A separate Control group who rejected their PIX early had high levels of CDC and ADCC to donor (1:64 and 1:1024, p<0.05). The long term function and tolerance of PIX was associated closely with a block of anti-donor CDC and ADCC antibodies. The superior long term function and tolerance appears due to the unique immunosuppression which permits variations in the measured-total lymphoid irradiation to deliver uniform measured doses of TLI as well as short term effective anti-B cell suppression with splenectomy and rabbit-antithymocyte globulin-deoxyspergualin short-term treatment. Animals remained in a robust state of health presumably because they were off all drug immunosuppression for over 9 months. This suppressive regimen seems superior to all others we have tested because of the length of islet survival and the excellent health of the animal and suggests that Chronic Immunosuppression with Drugs is the major debilitating effect of multiple agent suppressive protocols.

Assessment of the In Vivo Function of Pig Islets Encapsulated in Uncoated Alginate Microspheres

RECENT REPORTS showed that xenogeneic islets microencapsulated in uncoated alginate microspheres prevented rejection in rodents. Our previous report indicated that uncoated alginate microspheres protected the xenogeneic pig islets (PI) in vitro when incubated in mouse sera with low titers of preformed antibodies. However, PI were not protected when they were incubated in xenogeneic sera with preformed cytotoxic antibodies. In this study we compared the in vivo functional period in diabetic mice of PI encapsulated in uncoated alginate microspheres with nonencapsulated PI transplanted under the kidney capsule as free graft.

Similar Effect of Enzymes on Pig and Rat Islets

Similar Effect of Enzymes on Pig and Rat Islets

Improved Pig Islet Yield and Post-Culture Recovery Using Liberase PI Purified Enzyme Blend

Improved Pig Islet Yield and Post-Culture Recovery Using Liberase PI Purified Enzyme Blend

Cellular Immune Response in the Liver of Mice Rejecting Intrahepatic Rat and Guinea Pig Islet Xenografts: Expansion of Intermediate TCR Cells

Cellular Immune Response in the Liver of Mice Rejecting Intrahepatic Rat and Guinea Pig Islet Xenografts: Expansion of Intermediate TCR Cells

Use of polyethyleneglycol for porcine islet cryopreservation

The aim of this work was to determine whether polyethylene glycol 20000 Da (PEG) could be used as protective agent in porcine islet cryopreservation. Cryopreservation was performed on 1-wk cultured pig islets and consisted in an overnight storage in liquid nitrogen. In a first set of experiments, we compared the in vitro function of PEG-cryopreserved islets to that of porcine islets cryopreserved under the standard procedure using dimethylsulfoxide (DMSO), by incubating the islets over 45 min in Krebs buffer containing either 2.8 or 10 mmol/L glucose. Insulin secretion of both types of islets reached a maximum at day 10 postthawing and had stimulation indices above 2 up to 3 wk after thawing. PEG-cryopreserved islets secreted more insulin than DMSO-treated islets and showed glucosedependency insulin secretion in a 0–16.6 mmol/L glucose range. We also established that PEG-cryopreserved islets were as functional in vitro as nonfrozen tissue and that they could reverse experimental diabetes of the mouse for longer periods of time than noncryopreserved islets (p < 0.005 3 wk after transplantation) when implanted in the peritoneal cavity, being immunoprotected in a semipermeable hollow fiber. PEG can, therefore, be considered as a suitable cryoprotective compound for porcine islet storage.

The role of cytokines during rejection of foetal pig and foetal mouse pancreas grafts in nonobese diabetic mice

The rejection of discordant foetal pig islet xenografts in nomimmunosupressed nonobese diabetic (NOD) mice is dominated by polymorphonuclear cell infiltration whereas allografts are almost exclusively infiltrated by mononuclear cells. To determine if this variation is due to different proinflammatory factors generated at the graft site, we analysed graft-site mRNA expression of various cytokines, and the eosinophil attractant chemokine, eotaxin, in a renal subcapsular islet transplant model using organ cultured foetal pig (xenograft) and foetal BALB/c (allograft) pancreas in prediabetic NOD mice. Using semiquantitative RT-PCR on samples recovered at multiple time points during the first 15 post-transplantation days from mice transplanted with either allogeneic or xenogeneic tissue, we found increased expression of IL-2, IL-4, TNF-β and IL-10 mRNAs at the peak of the cellular infiltrate (on day 5) in both xenografts and allografts but, in contrast to the allografts, no enhanced transcription of IFN-γ mRNA in the rejecting xenografts. When an allograft and a xenograft were placed at the opposite pole of the same kidney the histological appearance of the rejecting allograft site resembled the xenograft site with significant numbers of eosinophils in both, and enhanced expression of eotaxin and iNOS. Additionally, the xenograft response, unlike the allograft response, was marked by an early increased expression of TNF-α and IL-S (day 3) and an almost complete absence of IFN-γ expression. The results suggest a distinct cell-mediated mechanism for rejection of local pancreas xenografts compared to the rejection of foetal pancreas allografts.

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 function of pig islets isolated from bleach-treated pancreata

In vitro function of pig islets isolated from bleach-treated pancreata

Insulin secretory studies on isolated pig islets

Insulin secretory studies on isolated pig islets

Effect of molecular weight exclusion of polysulfone fibers on macroencapsulated pig islet xenograft function in diabetic mice

Effect of molecular weight exclusion of polysulfone fibers on macroencapsulated pig islet xenograft function in diabetic mice