Sertoli cell-induced defects on functional and structural characteristics of isolated neonatal porcine islets.

A lack of a sufficient number of human donor pancreases has stimulated interest in isolation and cryopreservation techniques for islets from the porcine pancreas. But because of a poorly developed outer membrane porcine islets are particularly susceptible to damage during cryopreservation. The aims of this study were two-fold: 1) to develop a method for isolation and storage of islets from neonatal porcine pancreas and, 2) to examine effects of Sertoli cells on islet yield and function in Sertoli cell-islet cell cocultures. A total of 170 neonatal porcine pancreases were processed by means of a short period of digestion with collagenase and culture of the tissues at 32 degrees C for periods up to 7 days following isolation. Results were: The mean +/- SEM, number of viable islets, and percentage loss of cells following 7 days of culture were 29,442 +/- 1,119 and 22.2 +/- 1.2, respectively, Cryopreservation had a marked impact on recovery of viable islets: In absence of Sertoli cells an average of only 64% of islets remained viable; by contrast, when cryopreserved islets were cocultured with Sertoli cells, a mean of 82% was recovered. Glucose at 16.7 mmol/L had the capacity to elicit insulin release from 3-day-old cultured islets. The concentration in absence of Sertoli cells was 57.3 +/- 3.8 uU/mL/10 islets; in the presence of Sertoli cells the level increased to a mean +/- SEM of 112.8 +/- 17.7, uU/mL/10 islets. Similar results were obtained following cryopreservation: glucose at 16.7 mmol/L stimulated a mean +/- SEM of 27.9 +/- 6.6, uU/mL/10 islets, of insulin in absence of, and 44.9 +/- 9.9, uU/mL/10 islets, in presence of, Sertoli cells. Our results show that isolation and cryopreservation of neonatal porcine islets can be successfully accomplished. In addition, coculture with Sertoli cells significantly improves both the yield and functional capacity of islets following cryopreservation.

A role for CD95 ligand in preventing graft rejection.

Testis is a remarkable immune-privileged site, long known for its ability to support allogeneic and xenogeneic tissue transplants. Here we have investigated the molecular basis for testis immune privilege. Testis grafts derived from mice that can express functional CD95 (Fas or Apo-1) ligand survived indefinitely when transplanted under the kidney capsule of allogeneic animals, whereas testis grafts derived from mutant gld mice, which express non-functional ligand, were rejected. Further analysis of testis showed that CD95 ligand messenger RNA is constitutively expressed by testicular Sertoli cells, and that Sertoli cells from normal mice, but not gld mice, were accepted when transplanted into allogeneic recipients. CD95 ligand expression in the testis probably acts by inducing apoptotic cell death of CD95-expressing, recipient T cells activated in response to graft antigens. These findings indicate that CD95 ligand could be used to create immune-privileged tissue for a variety of transplant uses.

Assessment of proteinuria and neuropathy in the nonimmunosuppressed BB diabetic rat after abdominal intratesticular islet transplantation.

Only limited studies are available that assess diabetic complications following islet cell transplantation. Our objectives were to quantitate urine total protein, sural nerve morphometry, and sexual function in the diabetic BB/WOR male rat following islet cell transplantation into the abdominal testis. Success of islet cell transplantation was determined by nonfasting, morning, twice-weekly serum glucose and 12-hr fasting glucose, total glycosylated hemoglobin, and HbA1c after six months of diabetes and prior to death. Results showed that 9 of 16 rats were transplanted successfully for a period of at least six months. Pretransplant glucose was 21.9 +/- 4.67 (SD) mM/L and posttransplant glucose was 6.44 +/- 72 mM/L. The 12-hr fasting glucose ranged from 4.61 to 9.28 mM/L in animals prior to death, and glycosylated hemoglobins were not different from controls. Total urinary protein was significantly (P < 0.01) less than untreated diabetic rats (5.66 +/- 1.96 vs. 16.6 +/- 3.7 mg/24 hr) and not different from controls. Penile reflexes and serum testosterone remained normal in islet cell-transplanted animals. Sural nerve morphometry was normal, with 29.2% fewer abnormalities (paranodal swelling, paranodal demyelination, myelin wrinkling, Wallerian degeneration, and segmental demyelination) than untreated diabetic BB/WOR rats. We conclude that abdominal, intratesticular islet transplantation normalizes fasting blood glucose and glycosylated hemoglobin. In addition, the improvement in metabolic control at six months of diabetes was associated with normal total urinary protein, sural nerve morphometry, and sexual function.

Sertoli cell-enriched fractions in successful islet cell transplantation.

Prolonged survival of Islet- allo- and xenografts can be induced following implantation of the islets into the abdominal testis of diabetic rats. We previously showed that a factor released by Sertoli cells appears to be responsible for the protection of the intratesticular islet allo- and xenografts against rejection. The aim of this study was to examine whether an immunologically privileged site can be established in an organ site in vivo, other than the testis, such as the renal, subcapsular space, to make feasible the grafting of female recipients as well. A total of 36 male and 21 female, diabetic, PVG rats were divided into six different treatment groups: 1) Six male rats were grafted with islets from Sprague-Dawley (S-D) donor rats only. 2) Ten male rats were grafted with islets from (S-D) donors and were then given a short course of cyclosporine (CsA) posttransplantation. 3) Ten male rats were grafted with islets from (S-D) donors and with Sertoli cell-enriched fractions (SEF) from PVG donors but without CsA. 4) Ten male rats were grafted with a combination of islets from (S-D) and SEF from (PVG), donors, respectively, and CsA. 5) Ten female rats were given an identical combination of cells and CsA as depicted for group 5. 6) Ten female rats were grafted with a combination of islets and SEF, both cell types from S-D donors, and CsA. The results showed that 70% to 100% of the grafted rats in groups 1, 2, and 3 remained hyperglycemic.(ABSTRACT TRUNCATED AT 250 WORDS)

Production of a factor, or factors, suppressing IL-2 production and T cell proliferation by Sertoli cell-enriched preparations. A potential role for islet transplantation in an immunologically privileged site.

Isolated islet allografts survive indefinitely in the abdominal testis of nonimmunosuppressed diabetic rats. The predominant feature of these testes is that the presence of Sertoli cells, but not Leydig cells, is required for extended survival of the islet allografts. Sertoli cells cultures were therefore established in vitro and we examined the effects of the conditioned media on Con A--stimulated spleen lymphocyte proliferation. These studies revealed that a product(s) secreted by Sertoli cells inhibits Con A-stimulated lymphocyte proliferation in a dose-dependent manner. The synthesis of this product is both temperature-dependent, occurring predominantly at 37 degrees C, and hormone-dependent, requiring the presence of follicle stimulating hormone, in the culture medium. We further examined the mechanism of inhibition of lymphocyte proliferation and showed that Sertoli cell-enriched media inhibit the production of IL-2 in a dose-dependent manner. Furthermore, the finding that the addition of exogenous IL-2 is not able to reverse this inhibition indicates that the Sertoli cell-enriched media inhibit both IL-2 production and IL-2 responsiveness of T lymphocytes.

Islet allografts in the cryptorchid testes of spontaneously diabetic BB/Wor dp rats: response to glucose, glipizide, and arginine.

The aim of this study was to examine insulin and glucagon secretory patterns in successfully transplanted spontaneously diabetic BB/Wor dp rats. Diabetic, BB/Wor dp rats received abdominal, intratesticular islet grafts of MHC-compatible BB/Wor dr donor rats without immunosuppression. After a period of 74 +/- 15 days of normoglycemia, they were given the following challenges: 1) glucose, by mouth, 2) a single oral dose of glipizide, with glucose, and 3) arginine, by iv infusion. The pertinent results included the mean fasting plasma glucose levels of control, Sprague-Dawley (C), of transplanted BB/Wor dp (T), and nontransplanted, insulin treated, diabetic BB/Wor dp (D), and they were, respectively, 97 +/- 4 mg/dl, 110 +/- 3 mg/dl, and 350 +/- 40 mg/dl. Fasting plasma insulin levels in C and T rats were 21.9 +/- 3 microU/ml, and 20.4 +/- 2 microU/ml, respectively. Fasting plasma glucagon levels in C, T, and D, were 37.8 +/- 5.7 pg/ml, 43.4 +/- 4.6 pg/ml, and 47.4 +/- 4.9 pg/ml, respectively. During oral glucose tolerance test, the pattern of insulin secretion in the C and T rats was identical with a peak attained at 15 min. Glucose caused a 70% suppression of plasma glucagon levels in C rats (P less than 0.01); T rats suppressed 14%, but this was not statistically significant; D rats failed to suppress. Glipizide plus glucose caused an improved glucose tolerance in T rats without significantly affecting insulin levels. In the same rats, glipizide resulted in a significant suppression of glucagon compared with levels in the presence of glucose alone. Arginine caused a minimal release of insulin in T rats and a major glucagon secretory response in D rats. Pancreatic glucagon content was significantly (P less than 0.03) lower in C and T, compared with D rats. Furthermore, the transplanted testes of T contained substantial amounts of glucagon. In summary, these data suggest that grafted testes in spontaneously diabetic BB/Wor dp rats contain both beta and alpha-cells and that these cells have the capacity to respond to specific secretagogues independently.

Cyclosporine-induced tolerance to intratesticular islet xenografts.

Survival of highly immunogenic hamster islet xenografts can be achieved in rats if the graft is transplanted into the abdominal testis. Permanent survival requires the administration of cyclosporine during the first thirty days after grafting. The majority of grafts will survive indefinitely beyond this point if the grafted animals receive a once-weekly maintenance dose of CsA until day 100, when CsA is no longer necessary. Hamster islet xenografts transplanted under the kidney capsule or into the portal vein are rejected, regardless of CsA treatment. Animals maintaining long-term primary intratesticular xenografts accept secondary contralateral testicular xenografts. CsA is not required. Primary grafts are also resistant to the adoptive transfer of lymphocytes from rat donors primed to hamster xenoantigens. Secondary hepatic and renal islet xenograft survival is also extended--some hepatic grafts long-term. Therefore, the combination of CsA and the privileged status of the abdominal testis leads not only to the acceptance of primary intratesticular islet xenografts but also to partial immunological unresponsiveness of subsequent grafts in other sites.

Successful islet/abdominal testis transplantation does not require Leydig cells.

Pancreatic islet allo- and xenografts are not rejected and exhibit long-term beta-cell function if transplanted into the abdominal testis of the diabetic host. Successful transplantation appears dependent on local factors unique to the abdominal testis. Because Leydig cells remain viable in abdominal testes, which also retain high levels of testosterone, the following question was addressed: do Leydig cells and/or their secretory products influence islet transplantability in the successful islet/abdominal testis transplantation model? Streptozotocin-induced diabetic rats (Sprague-Dawley) were injected with 75 mg/kg ethane dimethanesulfonate (EDS) to selectively eliminate Leydig cells prior to or following transplantation with islets isolated from the BBWORdr rat. Subcutaneous silastic tubes packed with estradiol prevented Leydig cell repopulation in the EDS-treated recipient. Grafted diabetic animals, including the EDS-treated rats with serum testosterone at castration levels, became nornoglycemic following islet transplantation and remained so far for up to ten months. Leydig cells were not observed in testes of the EDS- or EDS/estradiol-treated rats, whereas the transplanted islets within these testes appeared structurally normal and highly vascularized. Islets resided within the testicular interstitial compartment and contained alpha-, beta and delta-cells, as identified by electron microscopy. Beta cells were most prominent, contained secretory granules and exhibited a close structural and functional relationship with adjacent intraislet capillaries. We conclude that Leydig cells and Leydig cell secretory products, including testosterone, are not necessary for protecting islets against rejection and they do not play an obligatory role in the success of the islet/abdominal testis transplantation protocol. Leydig cells and Leydig cell secretory products do not promote long-term beta-cell function and are not required for the return to and maintenance of normoglycemia in the grafted diabetic rat.

Abdominal intratesticular islet-xenograft survival in rats.

We investigated the survival of islet xenografts in the abdominal testes of normal, diabetes-resistant ACl and Wistar-Lewis (W-L) and in diabetes-prone BB/Wor rats. Islets were isolated from hamster donors, and after a period in tissue culture, they were injected into the abdominal testes of diabetic rats. Postoperatively, the rats were divided into two treatment groups. Four ACl rats received antilymphocyte serum (ALS) injections over a period of 30 days, and 13 ACl, 15 W-L, and 13 BB/Wor rats were given cyclosporin (CsA) according to the following regimen: 25 mg/kg i.p. for 7 days, then 7 mg/kg i.p. for 23 days. On day 30, immunosuppression was stopped. The results showed that none of the ALS-treated ACl rats remained normoglycemic for greater than 9 days. However, CsA therapy resulted in an extended mean duration of normoglycemia in the ACl and W-L rats of 160.0 +/- 36.0 and 131.0 +/- 31 days, respectively. By contrast, the mean duration of normoglycemia in the BB/Wor rats was 33.0 +/- 4.0 days. Furthermore, all of the BB/Wor rats reverted to diabetes after CsA was stopped. Therefore, the concomitant administration of an antigenic islet xenograft with CsA led to a state of unresponsiveness only in diabetes-resistant but not in diabetes-prone rats. Because the BB/Wor rats have demonstrable T-lymphocyte dysfunction, they may be unable to generate the CsA-induced suppressor T-lymphocytes required for the long-term acceptance of the graft by the host.

Effects of islet grafts of MHC-compatible donors on glucose metabolism in the spontaneously diabetic BB/Wor rat.

Complete recovery of the diabetic process occurred in spontaneously diabetic BB/Wor rats after the transplantation of islets of MHC-compatible donor rats. Islets were isolated from diabetes-resistant BB/Wor rats and were cultured for 4 days at 37 degrees C. The islets were then hand-picked and each BB/Wor rat with spontaneous diabetes received a total of 10 islets per g of body weight injected either into the immunologically privileged abdominal testis or into the non-immunologically favored renal subcapsular space. No immunosuppression was given to the recipients. After a period of at least 80 days of normoglycemia, the effects of an intravenous glucose injection on serum glucose and insulin levels were assessed. The grafts and the pancreases were then surgically removed and the insulin content of each organ site determined. The results showed that normoglycemia and a rapid weight gain were induced whether the rats were given an intratesticular or a renal, subcapsular islet graft. The amounts of extracted insulin recovered from the grafts after 83-259 days of normoglycemia were not significantly different from the pancreatic insulin content of age-matched control rats. But, despite the survival of a large mass of insulin-producing cells, glucose tolerance was impaired with a blunted insulin response and a delayed return of serum glucose to basal levels. Histologic examination of the islet grafts between 214 and 269 days after transplantation showed remarkable preservation of the islets with no evidence of an inflammatory reaction. Successful transplantation did not, however, lead to a recovery of the native pancreas of the BB/Wor recipient.(ABSTRACT TRUNCATED AT 250 WORDS)

Prolonged intratesticular islet allograft survival is not dependent on local steroidogenesis.

The mechanisms that control the privileged survival of intratesticular organ allografts are not known. It had been postulated that the elevated levels of testicular steroid hormones, testosterone and/or progesterone, could be responsible for the inhibition of the local immune response. The goals of this study were to examine intratesticular islet allograft survival in rats in which both germ cell and Leydig cell function had been selectively destroyed. A chemical castration was induced in diabetic Sprague-Dawley rats with the chronic administration of a GnRH analog, leuprolide. In addition, both germ cell function and steroidogenesis were severely impaired by means of the surgical removal of the pituitary in diabetic rats. Pancreatic islets were isolated from Wistar-Lewis rats and were then implanted into the abdominal testes of leuprolide-treated and of hypophysectomized rats. No immunosuppression was given to the grafted rats. The results showed that long-term allograft survival occurred in the abdominal testes deprived of germ cells, of testosterone and of progesterone.

Intratesticular islet xenograft survival in relation to tissue cyclosporine levels.

A study of the comparative survival of islet xenografts using a combination of treatment modalities was carried out in the spontaneously diabetic BB/Wor rat. Islets were isolated from hamster donors, and after 4 to 6 days of incubation the cells were injected either into the immunologically privileged abdominal testis or into the nonimmunologically favored renal subcapsular space. Postoperatively the rats were given cyclosporine at two different dose schedules. The results showed that islets injected into the abdominal testis of nonimmunosuppressed rats caused the induction of normoglycemia with a mean duration of 8.3 +/- 1.3 days. A significant prolongation of normoglycemia to a mean of 36.1 +/- 4.5 days occurred in rats that were given abdominal, intratesticular islet xenografts and cyclosporine for 30 days. The longest average survival in excess of a mean of 90.1 +/- 6.5 days was achieved in rats that were given abdominal, intratesticular islet xenografts and cyclosporine continuously, every other day. All of the grafted rats reverted to diabetes upon the cessation of cyclosporine. A similar cyclosporine regimen failed to prolong islet xenograft survival for longer than a mean of 9.0 +/- 2.2 days in rats that were given islet xenografts injected into the renal subcapsular space. Extended survival of abdominal, intratesticular islet xenografts corresponded with trough plasma and testis cyclosporine levels of 457 +/- 46 ng/mL and 643 +/- 45 ng/g, of wet weight, respectively. It is concluded that islet xenografts are protected against immune destruction in the BB/Wor rat with type 1 diabetes only as long as the cells are injected into an immunologically privileged site and the host is continuously immunosuppressed with cyclosporine.

Extended survival of MHC-compatible islet grafts from diabetes-resistant donors in spontaneously diabetic BB/W rat.

Transplantation of a large inoculum of incubated islets of MHC-compatible donors led to an extended survival of the grafts to an average of greater than 86 days in 71% of male diabetic BB/W recipients. Identical results were obtained whether the immunologically privileged abdominal testis or the nonimmunologically favored renal subcapsular space was used as the organ site for the injection of the islets. Survival of the islet grafts was also independent of the duration of diabetes in the BB/W rats at the time of transplantation. These results showed that under our experimental conditions the grafted islets were able to become established and survive for extended periods in nonimmunosuppressed spontaneously diabetic BB/W hosts.

Effects of intratesticular islet transplantation on hepatic glycogen metabolism in the rat.

Pancreatic islet transplantation into cryptorchid testes resulted in near-complete normalization of hepatic enzymic parameters associated with glycogen metabolism. Measurements of plasma glucose levels and of immunoreactive insulin levels indicated that islet transplantation also resulted in improved control of glycemia in diabetic animals receiving these grafts. Electron microscopic examination of cryptorchid testes revealed the presence of islet cells in the interstitial spaces outside of the seminiferous tubules. These islet cells both had granules identified as B granules by morphologic criteria and appeared to be actively secreting the contents of these granules. This site of islet transplantation appeared to provide a protected site which facilitated long-term survival and continued functioning of islet grafts.

Intratesticular islet allografts in the spontaneously diabetic BB/W rat.

UNLABELLED: Thirty-three male BB/W rats with diabetes of 11-145 days duration were divided into 3 groups: six received abdominal, intratesticular islet allografts and no immunosuppression posttransplantation; 15 were similarly grafted and in addition were given four injections of ALS regularly at 10-day intervals for 30 days after transplantation; and 12 rats received scrotal, intratesticular islet allografts and four injections of ALS. THE RESULTS: in the absence of immunosuppression all six of the rats with abdominal, intratesticular islet allografts became normoglycemic within 2 days after transplantation but in none did graft survival exceed 17 days; a marked prolongation of graft survival of greater than 65-441 days occurred in 13 of the 15 animals with identical intratesticular allografts; sustained immunosuppression was not needed for prolonged islet allograft survival in rats with cryptorchid islet allografts; and (4) only one of the 12 rats with scrotal, intratesticular allografts became normoglycemic whereas 11 remained severely glycosuric. However, on surgical translocation of the grafted testes from the scrotum into the abdominal cavity, the rats promptly became normoglycemic in the absence of any additional therapy.

Regulation of hepatic glycogen metabolism: effects of diabetes, insulin infusion, and pancreatic islet transplantation.

Insulin-deficient diabetes mellitus results in diminished capacity of the liver to accumulate glycogen. One site of metabolic lesion in the diabetic liver is at the level of the synthase-activating enzyme, synthase phosphatase. This activity is progressively diminished with increasing severity of chemically induced diabetes in both soluble and smooth endoplasmic reticulum (SER) associated subfractions. Insulin administration via an implanted miniosmotic pump or via intrahepatic islet transplantation increased synthase phosphatase activity, particularly in SER. Hepatic glycogen synthesis and accumulation was enhanced as well. The data support a role for insulin in maintenance of the ability of the liver to synthesize and accumulate glycogen mediated either directly or indirectly through SER-synthase phosphatase activity.

Extended allograft survival of islets grafted into intra-abdominally placed testis.

Isolated islets of ACI donor rats were cultured for 4 days at 37 degrees C and then grafted into male diabetic Wistar-Lewis rats into three different organ sites without adjuvant immunosuppression. None of 6 recipients of the intraportal injection of 10 islets per gram of body weight became normoglycemic. Similarly, six rats that received islets injected under the renal capsule remained diabetic. None of six rats that received an intratesticular islet allograft became normoglycemic while these organs remained within the scrotum. By contrast, six rats that were transplanted with an identical number of islets into the testis, which were then surgically placed into the abdominal cavity, promptly became aglycosuric and have remained so for more than 50 days.

Improved allograft survival using highly enriched populations of rat islets.

A method is described for the purification of islets before the cells are placed in tissue culture, thus permitting the transplantation of islets cultured for three days against major histocompatibility barriers without adjuvant immunosuppression. Mixed lymphocyte culture reactions were carried out with three rat strain combinations and the in vitro responses were correlated with the in vivo survival of islet allografts. These results showed that islet allograft acceptance is independent of the degree of histoincompatibility between different rat strains.