Successful long-term survival of pancreatic islet allografts in spontaneous or pancreatectomy-induced diabetes in dogs. Cyclosporine-induced immune unresponsiveness

Nineteen pancreatectomized beagles and three spontaneously diabetic dogs were recipients of canine islet allografts from one or more unrelated donors. The islets, enriched 30-45-fold for endocrine cells and contained in a packed cell volume of less than 1.5 ml, were engrafted in the livers of recipient animals. Treatment of diabetic recipients with cyclosporine (CsA) was begun 3-5 days before islet transplantation and the initial dosage was adjusted to attain and maintain CsA serum trough levels between 400 and 600 ng/ml. Five dogs with CsA levels less than this (155 +/- 35 SEM ng/ml) at the time of transplantation promptly rejected their grafts, whereas rejection was encountered in only 1 of 17 diabetic animals in which the initial level exceeded 400 ng/ml. CsA was discontinued 30, 60, or 90 days after continuous therapy in 10 animals. Graft failure was observed 2 mo after stopping CsA in 1 animal and 5 mo in the other. Eight other islet allograft recipients have sustained fasting euglycemia for 7 and 8 mo in 2 and for at least 2 mo in the remainder. These results demonstrate that short-term CsA therapy prolongs survival of islet allografts and induces a state of immune unresponsiveness to islet alloantigens in dogs with experimental and spontaneous diabetes. The findings are unique for a nonrodent mammal and thus hold promise that similar results may be achieved for islet allografts of other mammalian species, including humans.     

The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts

The effect of cyclosporin-A, low-temperature culture, and anti-Ia antibodies on prevention of rejection of rat islet allografts was determined. Wistar-Furth islets were isolated by the collagenase technique and transplanted via the portal vein into diabetic Lewis recipients. Cyclosporin-A (30 mg/kg) injected at 0, 1, and 2 days after transplantation produced a significant prolongation of survival of the islet allografts (MST greater than 35.7 +/- 7.0 days) when hand-picked donor islets were used, whereas only a modest prolongation of survival (14.0 +/- 1.6 days) was obtained using donor islets removed directly from Ficoll gradients. This difference in survival was apparently due to the large number of lymphoid, antigen-presenting cells that were present in the islet fraction removed directly from the Ficoll gradients. Treatment of donor, hand-picked islets with a mixture of cross-reactive anti-Ia antibodies and complement without cyclosporin-A therapy did not prolong the survival of islet allografts (MST, 6.5 +/- 0.4 days versus 7.0 +/- 0.5 days in controls). In contrast, treatment of the donor islets with the mixture of anti-Ia antibodies and complement in conjunction with the 3-day course of cyclosporin-A therapy produced an 83% survival of the islet allografts at 60 days after transplantation. In vitro culture of hand-picked donor islets at 24 degrees C for 7 days and the 3-day course of cyclosporin-A therapy produced a 100% survival of the allografts at 60 days after transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Long-term normoglycemia in pancreatectomized dogs following pancreatic islet allotransplantation and cyclosporine immunosuppression

Pancreatectomized dogs received intrasplenic autotransplants or allotransplants of unpurified islets prepared from the pancreata of unrelated outbred dogs, by a standard collagenase ductal perfusion method. Allograft immunosuppression consisted of tapering azathioprine-prednisone (AP), low level cyclosporine (CsA, through whole blood high-pressure liquid chromatography (HPLC) level 300-600 micrograms/L), or high CsA (600-1000 micrograms/L). While AP and low CsA failed to delay rejection, high CsA achieved prolonged (greater than 100 days) graft function in 5 of 12 dogs, with a median duration of 85.5 days. (P less than .01 vs. AP and low CsA). While no interference with islet engraftment was seen in CsA-treated dogs, late graft failure (greater than 30 days) was seen in 3 of 6 CsA autografts and 5 of 12 high CsA allografts. CsA at whole-blood HPLC levels of 600-1000 micrograms/L can achieve prolonged normoglycemia in pancreatectomized canine recipients of islet allografts. The effects of such doses of CsA on islet function may be substantial.     

Natural history of multiple intrahepatic canine islet allografts during and following administration of cyclosporine

Cyclosporine (CsA) prevented acute rejection of intrahepatic canine islet allografts (IHIA) in 5/5 pancreatectomized dogs. Normal fasting blood glucose levels were sustained in these dogs for 210 +/- 78 days (mean +/- SEM) following withdrawal of CsA. We tested whether combining islets from more than one pancreas would improve function and prolong islet allograft survival during and following administration of CsA. Areas under the glucose disappearance (GDA) and C-peptide response (CPA) curves following i.v. glucose (0.5 g/kg), 1 month posttransplant were not significantly different using islets from 1 or 2 pancreases, whereas GDA and CPA approached normal if islet yields from 3 or more pancreases were combined. Mean islet allograft survival following interruption of CsA decreased with an increase in the number of donor pancreases (one: 210 +/- 78 days, vs. two: 113 +/- 23 days, vs. greater than 2: 57 +/- 5 days). These studies demonstrate that: (1) IHIA uniformly resulted in fasting euglycemia in 36 of 38 diabetic dogs treated with CsA; (2) normal i.v. glucose metabolism required the combined islet yield of 3 or more donor pancreases, suggesting that a substantial number of intrahepatic islet cells are functionally lost despite effective CsA-induced immunosuppression; (3) use of multiple donors to accumulate an increased mass of islets may immunologically compromise allograft survival following discontinuation of CsA (these experiments, however, do not exclude a direct relationship between the duration of CsA therapy and the duration of immune unresponsiveness following interruption of CsA in multidonor islet allografts as an independent variable); and (4) unmodified islets obtained from multiple donors seem to require continuous immunosuppression to prevent rejection.     

The use of direct ultraviolet irradiation and cyclosporine in facilitating indefinite pancreatic islet allograft acceptance

We have previously shown that direct ultraviolet (UV) irradiation of islets can reduce their immunogenicity without alteration of their endocrine function and effect prolonged survival of islet allografts in the Lewis-to-ACI strain combination without the use of immunosuppression. This study extends that work to investigate the survival of UV-irradiated Wistar-Furth islets in streptozotocin-diabetic Lewis rats, in which case the recipient is a relatively "high responder" to W/F alloantigens. Lewis recipients of 24-hr cultured W/F islets uniformly rejected islet allografts within one week of transplantation while the additional immunosuppression with cyclosporine (CsA) at 15 or 30 mg/kg on days 0, +1, and +2 was ineffective in prolonging islet allograft survival. Wistar-Furth islets, which were UV-irradiated at 850-900 J/m2 and cultured for 24 hr prior to transplantation, did not survive any longer than those in control animals receiving untreated islets (MST 5.5 +/- 1 day). When UV irradiation of islets was combined with recipient peritransplant treatment with CsA at 15 mg/kg on days 0, +1, and +2 islet allograft survival was markedly prolonged (MST of 18 +/- 4.1 days). Treatment of diabetic recipients of UV irradiated islets with an increased dose of CsA (30 mg/kg) during the same peri-transplant period (0, +1, +2 days) resulted in 100% islet allograft survival beyond 120 days. This data demonstrate the effectiveness and synergism between the use of the pretransplant UV irradiation of islet allograft and the peritransplant immunosuppression of the recipient with CsA in inducing prolonged islet allograft survival in "high responder" recipients, in which the singular use of either modality may be ineffective.     

Ultraviolet light immunomodulation of canine islets for prolongation of allograft survival

Ultraviolet (UV) light treatment of donor islets has been shown to be effective for the prolongation of islet allograft survival in rodent models. This study evaluated UV as an immunomodulator of canine islets. The effects of UV irradiation on islet secretory function in vitro revealed a trend of increasing basal insulin release with increasing doses of UV and a corresponding significant decrease in glucose-mediated insulin release (expressed as percentage of basal fractional insulin release) beginning at UV light exposures of 200-300 J/m2 (n = 3, P less than 0.05). Proliferative responses to UV-irradiated allogeneic peripheral blood leukocytes and islets were significantly decreased by 53-112% (P less than 0.05) in 27 of 29 mixed-lymphocyte cultures and by 35-74% (P less than 0.05) in 4 of 5 mixed-lymphocyte islet culture experiments, respectively, beginning at 200-600 J/m2. Autotransplantation of nonirradiated (n = 8) and irradiated islets (600 J/m2, n = 6) resulted in a 1-mo graft survival rate of 75% for the control group and 50% for the irradiated group. Allotransplantation of irradiated islets (600 J/m2) into either nonimmunosuppressed recipients (1 donor to 1 recipient, n = 8) or recipients of subimmunosuppressive doses of cyclosporin (2 donors to 1 recipient, n = 4) resulted in 100% rejection by day 10. In contrast, when islets were cultured for 24 h postirradiation and transplanted into cyclosporin-treated pancreatectomized recipients (2 donors to 1 recipient), 3 of 7 grafts were prolonged beyond day 10 to days 16, 26, and greater than 100.(ABSTRACT TRUNCATED AT 250 WORDS)     

Extension of survival of rat parathyroid allografts by depletion of Ia donor cells plus preoperative cyclosporine

Methods that avoid chronic immunosuppression of transplant recipients must be developed to eliminate the various risk factors associated with such treatment (e.g., increased infections and malignancies). Pretransplant treatment of the graft with anti-Ia serum plus complement to eliminate "passenger cells" is one such method. An alternative approach is short-term treatment of the recipients with cyclosporine (CsA). In this study, parathyroid glands from Lewis X Brown Norway rats were cultured for one week at 37 degrees C and treated with anti-Ia and complement. Treated glands were transplanted into parathyroidectomized, hypocalcemic Wistar-Furth recipients that had received 30 mg/kg of CsA once a day for the three days prior to transplant. At 1 year posttransplant, 67% of the recipients had functional parathyroid allografts. Control rats (no CsA; fresh, untreated glands) rejected their grafts within 28 days. Controls given three days of CsA and transplanted with fresh, untreated glands all had functional grafts for greater than 56 days (median survival: 80.5 days). Prolongation of allograft survival with short-term, preoperative CsA demonstrates the efficacy of immunosuppression given only at the time of antigen presentation. This course of CsA allowed for indefinite graft survival when the recipient received a graft previously cultured and treated with Ia antiserum. These results are encouraging and should be evaluated further to determine whether similar approaches will be useful in human transplants.     

Prolongation of allograft survival in diabetic rats treated with cyclosporine by deoxyguanosine pretreatment of pancreatic islets of Langerhans

In vitro pretreatment of islets of Langerhans with deoxyguanosine (dGuo) has been shown to be effective for the prolongation of islet allograft survival in rats. [This study evaluates the effect of pretreatment of islets with dGuo transplanted into CsA-treated recipients.] Transplantation of dGuo-treated islets from Wistar rats into diabetic hooded (PVG) rats resulted in 36% graft survival without immunosuppression (dGuo-group) and 89% islet survival after a short course of cyclosporine was used in recipients (dGuo + CsA group). In contrast, transplantation of untreated islets into rats without immunosuppression (controls) and with CsA (CsA group) immunosuppression resulted in 0 and 56% survival, respectively. The differences in graft survival between dGuo versus control group (P less than 0.001), (dGuo + CsA) versus control group (P less than 0.0001), and CsA versus control group (P less than 0.002) are statistically significant. Donor-strain skin-graft challenge failed to induce rejection of transplanted normoglycemic rats in (dGuo) and (dGuo + CsA) groups. The results indicate that a state of immunologic unresponsiveness may have been induced in the recipients of dGuo-treated islets, and further treatment with CsA synergistically prolongs islet survival in fully mismatched rats.     

Experimental pancreatic allotransplantation in large animals. The role of donor kidney and cyclosporine in modifying rejection

Experiments were carried out in outbred dogs and pigs to evaluate the relative immunogenicity of pancreatic islets and segmental pancreas grafts, and whether these could be ameliorated by transplanting a kidney simultaneously from the same donor animal. Various immunosuppressive regimens were also studied. Pancreatic islet allografts never normalized blood glucose in totally pancreatectomized recipients despite the use of cyclosporine (CsA) in high doses (40 mg/kg/day) and the simultaneous transplantation of a kidney from the same donor. These grafts which never "took" contrast sharply with the experience of pancreatic islet autografts prepared in the same way and inoculated into the spleen, which in all nine instances normalized blood glucose in pancreatectomized recipients. Segmental transplants were performed in swine with duct drainage into the jejunum. Totally pancreatectomized pigs died at 7.8 +/- 1.0 days. In recipients suppressed with low-dose azathioprine (Az) and prednisone (Pred) pancreas grafts alone were rejected in 12.9 +/- 10 days. Synchronous pancreas and kidney transplants treated similarly extended the mean survival of pancreatic grafts to 20 +/- 10 days--which, however, was not significant (P less than 0.1 greater than 0.05). Mean survival time of pancreatic grafts in recipients receiving CsA at 20 mg/kg/day and prednisone 1 mg/kg/day was 14 +/- 6.3 days. The combination of CsA 20 mg/kg/day, Az 2 mg/kg/day, and Pred 1 mg/kg/day prolonged the mean survival time to 39.8 +/- 22 days. These results allow us to conclude that: crude preparations of islet tissue invariably capable of normalizing blood sugar at day 4 when used as autografts failed to "take" despite the existence of alternative sources of antigen present in a well vascularized kidney from the same donor, and despite very high dosages of CsA; triple immunosuppressive therapy had synergistic effects on pancreatic allograft survival; and simultaneous transplantation of kidney and pancreas had little effect on survival times of the pancreas or the kidney.     

Renal subcapsular islet cell transplantation

Islet cell transplantation has been associated with ultimate graft rejection. This preliminary study investigates the use of the renal subcapsular region as a site for placement of canine islet cell allografts. A new noncollagenase mechanical technique was used for preparation of the allografts. Animals in group I (N = 6) died of hyperglycemia in 4.0 +/- 1.89 days (X +/- SD) after pancreatectomy without subsequent islet cell transplant. Normoglycemia and excellent survival (greater than 60 days) was obtained in pancreatectomized animals in group II (N = 6) and in group III (N = 6), who received an islet cell allograft to the renal subcapsular site. Group II recipients were given no immunosuppression, and animals in group III received minimal immunosuppression with azathioprine. Dependence on the islet cell allograft for maintenance of normoglycemia was confirmed in the majority of the recipients by nephrectomy, to remove the graft, with resulting hyperglycemia and death. One normoglycemic animal in group II died on day 6 from peritonitis. One recipient in group II was normoglycemic at greater than 1 mo after removal of the first graft by nephrectomy, followed by retransplantation of islet cells from a third-party donor. Two other recipients are being studied on a long-term basis, and have been normoglycemic for greater than 6 mo and greater than 4 mo after transplantation. These studies encourage further investigation in this area for application of islet cell transplantation in man, and elucidation of the possible mechanisms for prolongation of islet cell allograft survival at the renal subcapsular site.     

Prolongation of segmental and pancreaticoduodenal allografts in the primate with total-lymphoid irradiation and cyclosporine

The prolongation of segmental and pancreaticoduodenal allografts (PDA) by total lymphoid irradiation (TLI) and in combination with cyclosporine (CsA) was assessed in a well established total pancreatectomy, diabetic, primate transplantation model. Pancreatic transplantation was performed in 119 pancreatectomized baboons (Papio ursinus). Of a total of 109 allografts performed, 71 were segmental allografts (open duct drainage) and 38 PDA. Of 119 graft recipients, 10 received segmental pancreatic autografts. TLI and CsA administered separately to segmental allograft recipients resulted in modest allograft survival and indefinite graft survival was not observed. 8 of 17 (47%) segmental allograft recipients that received TLI and CsA had graft survival beyond 100 days, indicating highly significant pancreatic allograft survival. All long-term segmental allograft recipients were rendered normoglycemic (plasma glucose less than 8 mmol/L) by this immunosuppressive regimen. In contrast, poor results were observed in PDA recipients treated with TLI and CsA. Mean survival in 18 treated PDA recipients was 23.8 days, 8 survived longer than 20 days (44.4%), and 1 greater than 100 days (5.5%). Despite treatment, early rejection of the duodenum in PDA recipients frequently resulted in necrosis and perforation and contributed to a high morbidity and mortality. This study indicates that, in contrast to the significant prolongation of segmental allografts by TLI and CsA, poor immunosuppression was achieved by this regimen in PDA recipients and was associated with a high morbidity and mortality caused by early rejection of the duodenum.     

Reversal of diabetes in outbred mice by islet allotransplantation

The combination of donor pretreatment with cyclophosphamide, organ culture in 95% O2:5% CO2 for 7-10 days, and short-term immunosuppression of recipients with cyclosporin A (CsA) were necessary to obtain 100% survival of single-cluster BALB/c islet allografts in outbred mice. In vivo and in vitro pretreatment of the donor tissue alone resulted in the acceptance of 45% of the islet allografts in nonimmunosuppressed outbred mice. CsA treatment of recipients alone yielded 40% survival of the untreated allografts. CsA treatment played an important role in maintaining the capacity of islet allografts to function in outbred mice. During CsA treatment, 88% of streptozocin-treated mice showed graft-dependent reversal of diabetes; the remainder showed no evidence of graft function, and CsA treatment failed to prevent acute graft rejection. After withdrawal of CsA immunosuppression, 38% of this total group remained normoglycemic. These findings suggest that modulation of both donor-tissue immunogenicity and recipient responsiveness will be required for successful pancreatic islet transplantation in diabetic humans.     

Effect of a New Immunosuppressive Agent,FTY720, on Survival of Islet Allografts

A newly developed immunosuppressant, FTY720, has a unique mechanism that is quite different from those of conventional immunosuppressants, and is presumed to be mediated through decreases in the number of peripheral lymphocytes, especially helper T cells. This study was performed to ascertain whether this innovative drug could prolong islet allograft survival. The donors were inbred Lewis rats and the recipients were ACI rats rendered hyperglycemic with intravenous streptozotocin. In the study group, FTY720 dissolved in distilled water was orally administered at a dose of 5 mg/kg to the recipient ACI rats 1 day before and on the day of grafting. In the control group, only distilled water was orally administered to the recipient ACI rats on the day before and the day of grafting. Two thousand islets were transplanted into the portal vein of the recipient rats in the study and control groups immediately after isolation. The graft survival time in the study group was significantly longer than that in the control group, indicating that FTY720 retains a potent effect on the prolongation of islet allograft survival. FTY720 could become a useful immunosuppressant for future clinical islet allotransplantation.     

Prolongation of intraperitoneal segmental pancreatic allografts in primates receiving cyclosporin A

In this study the efficacy of the new immunosuppressive agent, cyclosporin A (CYA), was examined in a model of segmental, intraperitoneal pancreatic allotransplantation with free duct drainage in totally pancreatectomized, outbred Chacma baboons. CYA, in doses of 25 to 50 mg/kg/day administered to recipients of heterotopic segmental (tail) allografts, produced a slight but significant prolongation of graft survival. CYA (25 to 85 mg/kg/day), administered orally after pancreatic transplantation gave daily serum trough levels of CYA that ranged from 300 to 600 ng/ml. Mean serum trough levels on the first postoperative day in recipients of 50 mg/kg/day were 121.1 +/- 61.6 ng/ml. There was a wide variation in daily serum trough levels exhibited between primates on the same daily oral dose, and there was no correlation between absolute serum trough levels of CYA and rejection. It is postulated that adequate serum CYA levels were not achieved by the oral administration of the drug to ensure allograft survival beyond 60 days in pancreatectomized recipients. Adverse effects occurred frequently and included anorexia, diarrhea, and tremors and were in direct proportion to the quantity of CYA required to prolong graft survival. Free duct drainage into the abdominal cavity frequently resulted in pancreatic ascites, which necessitated paracentesis, indicating that this method of duct drainage has limited clinical application. Although heterotopic autotransplantation or allotransplantation of the tail of the pancreas in the baboon was capable of maintaining normoglycemia in pancreatectomized baboons, glucose intolerance, reduced K values, and hypoinsulinemia were consistent findings during glucose tolerance tests, suggesting that an insufficient islet cell mass had been transplanted.     

Transplantation of purified single-donor canine islet allografts with cyclosporine

We evaluated the survival of highly purified freshly isolated pancreatic islets transplanted from single canine donors into 20 outbred mongrel dogs immunosuppressed with cyclosporine or untreated. The grafts (mean weight +/- SE, 0.5 +/- 0.1 g, containing 122 +/- 8 X 10(3) islets; purity 91% by electron microscopy) were transplanted into 3 groups of dogs: group 1, autograft without CsA (5444 +/- 688 islets/kg body weight, n = 6); group 2, allograft without CsA (6669 +/- 1744, n = 4); and group 3, allograft with CsA (8645 +/- 1149, n = 10). The CsA was injected i.m. daily for 4 days before and 30 days after transplantation. Fasting plasma glucose (PG, mg/dl) and serum CsA trough values were determined daily. Intravenous glucose tolerance tests were done before and after transplantation, for calculation of K values (decline in glucose, %/min; preoperatively, mean K = 3.9 +/- 0.2). In group 1 all 6 dogs were normoglycemic (PG = 98 +/- 2 and K = 1.8 +/- 0.2) at 1 month; in group 2 the graft failed in all 4 dogs, at 4 +/- 1.2 days; in group 3 all 10 dogs were normoglycemic initially. Of the group 3 dogs, 4 died (intussusception developed in 2, and the graft failed at 3 and 9 days in 2 the CsA values of which were less than 300 micrograms/L preoperatively), but the other 6 were still normoglycemic when the CsA was stopped at 30 days (mean PG = 132 +/- 16 and K = 0.9 +/- 0.2; P less than 0.05 vs. group 1). Their CsA values were 708 +/- 197 before and 359 +/- 41 micrograms/L during the third week after transplantation; their grafts failed 12.3 +/- 3.4 days after the cessation of CsA. This data is unique in demonstrating prolonged function of purified allogeneic islets transplanted from individual outbred canine donors, but glucose tolerance was impaired. CsA at serum levels greater than 300 micrograms/L induced prolonged survival of purified canine islets and rejection was prompt when it was stopped.     

Gamma irradiation of isolated rat islets pretransplantation produces indefinite allograft survival in cyclosporine-treated recipients

In this study we have examined the use of low-dose gamma-irradiation for the reduction of islet immunogenicity in the strong allogeneic combination of WAG rat islets transplanted into diabetic AUG recipients. First, we determined that gamma-irradiation reduced immunogenicity in vitro by use of a modified MLR with WAG islets as stimulators and AUG splenocytes as responders. We then determined the maximum dose of gamma-irradiation that could be used (250 rads) before islet function was affected. As 250 rads islet pretreatment alone was ineffective in prolonging allograft survival, we combined the pretreatment with a short course (days 0, 1, 2; 30 mg/kg) of cyclosporine. We found that CsA was only effective in significantly prolonging allograft survival when given subcutaneously in olive oil. The CsA treatment alone gave a significantly prolonged survival time for the islet allografts (median, 37 days vs. 6 days for controls), but when combined with the 250 rads islet pretreatment a synergistic effect was seen with 100% becoming long-term survivors (greater than 100 days). The long-term surviving AUG rats from both the CsA alone group and the CsA plus 250 rads pretreated islets group were challenged with WAG dendritic cells (DC). The islets from the 250 rads pretreated group were subsequently rejected (day 6) while the CsA alone group were not affected. The role of low dose gamma-irradiation when combined with CsA treatment of islet graft recipients in inducing specific unresponsiveness will be discussed.     

Reduced NO production improves early canine islet xenograft function: a role for nitric oxide in islet xenograft primary nonfunction

Isolated canine islets transplanted to hyperglycemic rats fail to restore euglycemia in almost all cases, although the grafted islet tissue appears to be morphologically intact for up to 48 h following transplantation. Cytokines typically produced in the xenograft environment (e.g., IL-1 and TNF) inhibit insulin biosynthesis and secretion from isolated pancreatic islets, and are associated with the production of nitric oxide (NO). To further define the relationship between NO production and islet xenotransplantation, the inhibition of NO in a splenocyte/islet coculture system, and the in vivo effect of this inhibition on canine islet xenotransplantation, was investigated. Splenocytes (SPLC) from Lewis rats were cocultured with canine islets (freshly isolated or cultured 7 days), supernatant removed, and NO concentration (NO2) determined by optical density (Griess reaction, 550 nm, expressed as nmol nitrite/10(6) cells/18 h). Lipopolysaccharide (LPS) was used as a positive control of SPLC production of NO. Stimulation by LPS resulted in maximal NO production (2.20 +/- 0.16 nmol/10(6) cells/18 h, p < 0.001 compared to baseline values of 0.73 +/- 0.04 nmol/10(6) cells/18 h). In the presence of NO inhibitors (NMA, polymyxin B, hydrocortisone, aminoguanidine, DMSO), nitrite levels did not significantly rise above unstimulated values. Freshly isolated canine islets did stimulate NO production (1.26 +/- 0.12 nmol/10(6) cells/18 h, p < 0.001). In contrast, cultured canine islets did not stimulate NO production (0.84 +/- 0.09 nmol/10(6) cells/18 h). Transplantation of freshly isolated canine islets to STZ-diabetic recipient Lewis rats resulted in amelioration of hyperglycemia in only 50% (n = 6) of recipients 12 h posttransplant, with a return to hyperglycemia at all subsequent time points. Transplantation of 7-day cultured canine islets resulted in amelioration of hyperglycemia in 88% of recipients 12 h posttransplant and 63% of recipients 24 h posttransplant [p = 0.028, mean survival time (MST) = 1.0 days, n = 8]. Transplantation of canine islet xenografts with aminoguanidine therapy (BID, n = 11) resulted in amelioration of hyperglycemia in 100% of recipients at 12 h posttransplant, decreasing to 82% by 24 h following transplantation (p = 0.002, MST = 0.9 days). These results demonstrate that freshly isolated canine islets are potent stimulators of NO production by rat SPLC in vitro, and that culture of canine islets, or addition of NO inhibitors, abrogates stimulated NO production. These results also demonstrate a statistically significant improvement (p < 0.001) in early function of canine islet xenografts following 7 days of islet culture prior to transplant, and following recipient treatment with aminoguanidine. These studies suggest that the production of NO in the microenvironment of the graft site may adversely affect engraftment and function of canine islets, and suggest that the abrogation of islet-stimulated NO production may improve engraftment following islet xenotransplantation.     

Transplantation of purified frozen/thawed canine pancreatic islet allografts with cyclosporine

We studied the survival of defined volumes of highly purified frozen/thawed islets transplanted into the spleen of 4 groups of apancreatic mongrel dogs (total, 21), with or without cyclosporine from day -4 to day 30: group 1 (controls), without CsA, autograft of mean (+/- SE) islet volume 5 +/- 1 microliters/kg body weight; group 2, single-donor allograft, and group 3, multiple-donor allograft (both, 6 +/- 1 microliters/kg), both with CsA; and group 4, large-volume multiple-donor allograft (24 +/- 1 microliters/kg) with CsA. Grafts were cooled slowly to -40 degrees C, stored at -196 degrees C, and thawed rapidly. The CsA dose was adjusted to maintain whole-blood trough values of 600-800 micrograms/L, and allograft recipients manifesting early hyperglycemia were given insulin to maintain plasma glucose concentration below 150 mg/dl. In group 1, two dogs died early (graft failure in 1, intussusception in 1), but the remaining five were normoglycemic at 30 days. In groups 2 and 3, hyperglycemia ensued from about day 2.5, but 4 of 5 grafts in group 2 and all grafts in group 3 secreted insulin into the splenic vein at 30 days. In group 4, normoglycemia ensued within 24 hr of transplantation and was maintained in all 6 dogs for 30 days; the grafts failed 15 +/- 2 days after CsA was stopped. The data demonstrate prolonged function of purified frozen/thawed islets after transplantation into these outbred dogs but indicate a need for a greater volume of allogeneic islets from multiple donors than of autologous islets to achieve normoglycemia.     

Pancreatic islet transplantation. Induction of graft acceptance by ultraviolet irradiation of donor tissue.

The effect of ultraviolet (UV) irradiation on the immunogenicity of rat pancreatic islets was examined in allograft and xenograft models. Direct UV irradiation (900 J/m2) of Lewis islets, isolated and hand-picked, does not alter pancreatic islet endocrine function in isograft experiments and results in indefinite islet allograft survival in streptozocin diabetic ACI rats without chronic immunosuppression. Direct UV irradiation, at an appropriate dose, also leads to indefinite islet xenograft survival of Lewis islets in B10-BR diabetic mice and prolonged survival of rat islets in Balb/C mice. When direct UV irradiation of islet allografts did not result in indefinite islet allograft prolongation [Wistar/Furth (W/F) to diabetic Lewis], the addition of brief peritransplant immunosuppression with cyclosporine (days 0, +1, and +2) resulted in permanent acceptance of islet allografts, a result not achieved by cyclosporine alone. The effectiveness of UV irradiation in abrogating islet allograft rejection in several experimental models is supported by in vitro studies showing that UV irradiation of stimulator cells, peripheral blood lymphocytes, splenocytes, and isolated rat dendritic cells abolishes any significant stimulation by such cells of totally histoincompatible thoracic duct responder lymphocytes. In vitro nonreactivity of mixed lymphocyte culture (MLC) with UV-irradiated stimulator cells and in vivo permanent allograft acceptance are reversed by the addition of a small number of untreated donor-type dendritic cells to either the MLC or the recipient bearing the permanent graft. The authors suggest that the primary effect of UV irradiation on immune alteration of islet allografts and xenografts is due to induction of a major metabolic change in the dendritic cells in the graft. This then leads to defective antigen presentation and results in either permanent or prolonged allograft and xenograft acceptance, depending on the degree of MLC stimulation between the islet donor and the diabetic recipient.     

Elimination of acute GVHD and prolongation of rat pancreas allograft survival with DST cyclosporine, and spleen transplantation

Allogeneic spleen transplantation has been shown to have a tolerizing effect on pancreas allograft survival in rats. In this study we examined the effect of blood transfusions and cyclosporine administration on both rat pancreas allograft survival and acute graft-versus-host disease in BN recipients of Lewis pancreas-spleen allografts. We found that in this strain combination significant pancreas allograft prolongation occurred when the spleen was included en bloc with the pancreas graft. However, 50% of these recipients developed GVHD and died. A single donor-specific transfusion delivered to BN recipients of Lewis pancreas and pancreas-spleen allografts did not extend graft survival, but precluded the development of GVHD. A short, 6-day, and long, 26-day, course of CsA to recipients of pancreas and pancreas-spleen allografts extended graft and animal survival times, although not indefinitely. All pancreas-spleen recipients of both CsA protocols died following acute GVHD. Combined DST and CsA (short and long-course) administration in pancreas-only allograft recipients was deleterious to graft survival, compared with CsA administration alone. However, in pancreas-spleen recipients, combined DST and CsA had an additive effect. Moreover, in DST and long-course CsA-treated pancreas-spleen recipients, indefinite graft survival occurred with no signs of acute GVHD. The beneficial effect of the spleen allograft on pancreas graft survival was not dependent upon GVHD, since splenic-induced prolongation of pancreas graft survival was observed in the F1-donor to parent-recipient combination.