Renal Subcapsular Space Research Articles

Induction of donor-specific tolerance to rat islet allografts by intrathymic inoculation of solubilized spleen cell membrane antigens.

The development of strategies that will allow permanent survival of islet allografts without continuous host immunosuppression continues to be the most important goal in the field of pancreatic islet cell transplantation. In our study, we demonstrated that intrathymic inoculation of allogeneic spleen cell membrane antigens with a single dose of anti-lymphocyte serum induces an unresponsive state that permits survival of a subsequent pancreatic islet allograft to an extrathymic site (renal subcapsular space). This effect is donor specific and cannot be reproduced by the intravenous injection of spleen cell membrane antigens. Our results offer a potential approach for establishing donor-specific allograft acceptance in adult recipients.

Cryopreservation of Rat Islets of Langerhans: A Comparison of Two Techniques

Although a number of different protocols have been described for the cryopreservation of pancreatic islets of Langerhans, most involve equilibration with 2 M Me 2SO followed by slow cooling and fast warming. This paper compares two methods with identical equilibration protocols: one with cooling at 0.25°C/min to - 40°C prior to transferring to liquid nitrogen and subsequent warming at 200°C/min and sucrose dilution of Me 2SO (Method A) and the other with cooling at 0.3°C/min to -70°C prior to transferring to liquid nitrogen and warming at 50°C/min followed by stepwise dilution of Me 2SO (Method B). Islet viability was assessed by syngeneic transplantation to the renal subcapsular space of streptozotocin-induced diabetic rats. Seven hundred fifty fresh islets in the size range 100-200 μm consistently reversed diabetes (blood glucose < 10 mmol/liter) whereas 1000 Method A cryopreserved islets and 2000 Method B cryopreserved islets were required. Cryopreservation by either method resulted in impaired islet function compared to that of fresh islets, although this functional loss could be partially compensated by the transplantation of greater numbers of cryopreserved islets.

Establishment and characterization of a new human renal cell carcinoma cell line (KG-2) and metastatic model in nude mice

A new cultured cell line (KG-2) derived from human renal cell carcinoma and a metastatic model in nude mice were studied. KG-2 was cultured from renal cell carcinoma (clear cell carcinoma) of the left kidney. In vitro doubling time of KG-2 was approximately 50 hours. KG-2 cells produced tumors in both the subcutaneous and renal sub-capsular space in nude mice, with tumorigenicity of 75%, showing no difference between the two sites. Histologically, tumors formed in the subcutaneous sites were hypovascular granular cell carcinoma. Moreover, each tumor was encapsulated by a thick fibrous capsule and never produced distant metastasis or invasion into the surrounding tissue. However, tumors formed in the subrenal capsular space were clear cell carcinoma. These tumors were hypervascular, and produced distant metastases. The most common metastatic site was the lung. Immunohistochemical analysis using anti-human collagenase type IV antibody on tumors formed in subcutaneous and subrenal capsular sites demonstrated that the expression of this enzyme in tumors formed in the subrenal capsular space was much higher than that in tumors formed in the subcutaneous site. Additionally, immunohistochemical study using anti-mouse collagen type IV antibody, a major components of the vascular wall, demonstrated many small densely growing vessels in tumors formed in the subrenal capsular space. In contrast, few vessels were produced in tumors formed in subcutaneous sites. These findings suggest that factors relating to the different injection sites may regulate the production of collagenase type IV secreted by KG-2 cells and neovascularity in nude mice. This metastatic model may be useful in the study of the mechanism of cancer metastases.

Reinnervation of syngeneic mouse pancreatic islets transplanted into renal subcapsular space

Reinnervation of syngeneic mouse pancreatic islets transplanted into renal subcapsular space

IN VIVO EFFECT OF FK506 ON HUMAN PANCREATIC ISLETS

The purpose of this study was to evaluate the in vivo effect of FK506 on human pancreatic islets. Twenty-five nude mice were made diabetic by one intravenous injection of streptozotocin. Approximately 600 islets were administered in the renal subcapsular space 3-5 days following streptozotocin administration. One week after transplantation, the mice were divided in four groups. In group 1, the animals received 1 injection of 0.5 ml of diluent i.p. daily for one week. In groups 2, 3, and 4 the treatments were daily i.p. injection of 0.3, 1, and 3 mg/kg FK506, respectively. After treatment, the functional integrity of the transplanted human islets was tested by measuring the plasma glucose and human C-peptide response to intraperitoneal glucose injection in groups 1 and 4. IPGTT alone was assessed in groups 2 and 3. The results indicate that i.p. administration of FK506 for one week at a dose 0.3 mg/kg/day did not result in any significant alteration of glucose disappearance and C-peptide response to IPGTT. Higher doses of FK506 produced a significant delay in glucose disappearance in groups 3 and 4, and a significant inhibition of glucose-mediated C-peptide response in group 4.

Studies on Autoimmunity for Initiation of β-Cell Destruction: VII. Evidence for Antigenic Changes on β-Cells Leading to Autoimmune Destruction of β-Cells in BB Rats

The diabetic syndrome in BioBreeding (BB) rats is believed to result from the destruction of beta-cells by autoimmune responses. However, the initial events that cause the autoimmune destruction of beta-cells remain largely unknown. This investigation was initiated to see whether there are any antigenic changes on the beta-cells from neonatal to adult BB rats that may lead to the autoimmune destruction of beta-cells. Pancreatic grafts from neonatal BB rats remained largely intact without insulitis when transplanted into the renal subcapsular space of acutely diabetic BB rats. Similarly transplanted islet grafts from neonatal BB rats were also not subject to autoimmune destruction. In contrast, islet grafts obtained from adult BB rats, which had been treated with silica to prevent insulitis, were rapidly destroyed in diabetic recipients. These results indicate that beta-cells from neonatal BB rats are different from beta-cells from adult BB rats, at least regarding their recognition by immunologic effectors. Considering our observations and previous information on the initial role of macrophages/dendritic cells in the development of insulitis in BB rats, we suggest that beta-cell-specific antigenic changes that precede insulitis may result in the autoimmune destruction of beta-cells in BB rats.

The effect of capsule composition on the biocompatibility of alginate-poly-1-lysine capsules

The encapsulation of islets of Langerhans in alginate-poly-l-lysine has been proposed as a method for the immunoprotection of transplanted islets. Although several capsule compositions have been reported, there has been no published study concerning the effect of capsule composition on the severity of the foreign body reaction. Empty capsules were prepared from high mannuronic acid alginate and were coated with: (1) poly-l-lysine alone, (2) poly-l-lysine plus high guluronic acid alginate, or (3) poly-l-lysine plus high mannuronic acid alginate. The capsules were placed in the renal subcapsular space or the peritoneal cavity, and retrieved after three weeks of histological examination. The recipients were WAG/01a, nude (athymic), diabetic BB, and non-diabetes prone BB rats. The severity of reaction to the capsules was determined by measuring the thickness of the pericapsular cell infiltrate or by a scoring system. The severity of the reaction to the capsules was strain-dependent in both the renal and peritoneal sites, with the BB and nude rats displaying the most severe responses. The degree of response was not affected by capsule composition in the renal subcapsular space, but in the peritoneum, the high mannuronic acid alginate capsules provoked the weakest response, and this type of capsule will be used for future transplantation work. The infiltrating cells were characterised by immunohistochemistry and electron microscopy and found to be mostly fibroblasts and macrophages.

Studies on Autoimmunity for Initiation of β-Cell Destruction: VI. Macrophages Essential for Development of β-Cell–Specific Cytotoxic Effectors and Insulitis in NOD Mice

NOD mice were treated with silica (which is selectively toxic to macrophages) from 4 or 20.5 wk of age. Syngeneic neonatal pancreases were transplanted into the renal subcapsular space of the NOD mice at 21 wk of age. Silica treatment was continued until 24 wk of age, and then the mice were killed for examination of islet morphology. Neither the islets in transplanted pancreases nor the host pancreatic islets from the early long-term silica-treated animals revealed insulitis. In contrast, most of the islets in transplanted pancreases from the late short-term silica-treated animals showed severe insulitis and beta-cell necrosis, as did the host islets. A further experiment was performed to compare the effect of late short-term silica treatment with that of anti-L3T4-antibody treatment of the same time and duration. In contrast to the late short-term silica-treated animals, the transplanted pancreases in the anti-L3T4-antibody-treated animals revealed intact islets, although most of the host islets showed insulitis. The control group, which received no treatment but did receive neonatal pancreases, revealed severe insulitis and beta-cell necrosis of both transplanted and host islets. These results suggest that early macrophage depletion can abolish the development of beta-cell-specific immunologic effectors but that late macrophage depletion, after the development of insulitis, does not affect the destruction of beta-cells by preexisting effectors other than macrophages. We conclude that macrophages are essential for the development of beta-cell-specific cytotoxic effectors in the initial phase of insulitis in NOD mice.

PURIFIED CANINE ISLET AUTOGRAFTS

Using a modification of the basic principles of pancreatic intraductal collagenase digestion and density gradient purification to isolate canine islets, in conjunction with simultaneous fluorogenic and dithizone islet staining, we quantified the yield, purity, and viability of the isolated islets. We then determined the combined influences of total and weight-corrected islet counts and implantation site on immediate and long-term functional outcome of purified canine islet autografts. Weight-corrected islet counts were 100% sensitive and specific in differentiating successful and unsuccessful islet autografts implanted to the liver (n = 10) and spleen (n = 10) of pancreatectomized dogs. The threshold number of islets required to achieve normoglycemia in the liver (4400 islets/kg) and spleen (4650 islets/kg) were nearly identical. Islet autografts failed to ameliorate hyperglycemia when implanted to the renal subcapsular space (n = 5) at counts of 4400 to 5500 islets/kg. The mean one- and three-month intravenous glucose tolerance test K-values of dogs with purified islet autografts to the liver (-1.43 +/- 0.27 and -1.69 +/- 0.27, respectively) and spleen (-1.78 +/- 0.36 and -1.64 +/- 0.3, respectively) were also similar. Time needed to achieve normoglycemia , however, was significantly (P less than 0.02) shorter for intrahepatic islets (1.0 +/- 0.0 days posttransplant) than intrasplenic islets (6.8 +/- 2.3 days posttransplant). The long-term durability of islet autograft function was not unlimited. Overall, thirteen canine islet autograft recipients have been followed for greater than or equal to 12 months posttransplant (range 12-18 months), seven canine islet autograft recipients (five intrahepatic and two intrasplenic) have had spontaneous recurrence of hyperglycemia at 2, 6, 11, 13, 14, 8, and 16 months, respectively. The phenomenon depended only on the number of islets implanted. The data underscore the significance of quantitatively defined islet preparations and the importance of islet number and implantation site on immediate and long-term functional outcome of canine islet autografts.

Formation and malformation of the enteric nervous system in mice: An organ culture study

Despite some progress in the treatment of congenital malformations of the enteric nervous system, there is no knowledge about the pathogenesis. The study of the normal formation of the enteric nervous system is hampered by the difficulty of manipulating and culturing mammalian embryos and their organs. Three methods to culture bowel explants of murine embryos, (choriollantoic membrane grafting, organotypic tissue culture, and renal subcapsular space grafting) were compared. The three-dimensional cytoarchitecture of the bowel developed almost normally in the renal subcapsular space cultures. Using this culture system, it was found that neural crest cells colonize the murine bowel in distinct phases. The distal bowel was colonized at the 13th day of development. In a spontaneous mouse mutant model for intestinal aganglionosis, the lethal spotted mouse, the colonization of the distal 2 mm of the bowel did not occur at E13.

Histological Studies on the Therapeutic Effect of Sustained-Release Microspheres of a Potent LHRH Agonist (Leuprorelin Acetate) in an Experimental Endometriosis Model in Rats.

The therapeutic effect of sustained-release microspheres of a potent LHRH agonist (leuprorelin acetate) on experimental endometriosis in female rats was examined histologically. Endometriosis was produced in rats by autotransplantation of endometrial tissue obtained from the left uterine horn into the renal subcapsular space. In the nontreated rats, the transplants were well established and had formed large cysts containing fluid. The walls of the cysts were composed of epithelium and stroma resembling that of normal endometrium. In the rats which received the microspheres of leuprorelin acetate, growth of the transplant was markedly suppressed as evidenced by the reduced size of the cystic cavity and the flattened and pyknotic epithelium. Also, the uterine and ovarian weight decreased significantly. In the ovariectomized rats, growth of the transplant was also markedly suppressed, and the uterine weight decreased. The present results clearly indicate that a single injection of the sustained-release microspheres of leuprorelin acetate markedly suppresses growth of the transplant and produces uterine and ovarian atrophy in the rats.

Islet Transplantation to the Renal Subcapsular Space Improves Late Complications in Streptozotocin-Diabetic Rats

Transplantation of isolated islets is a promising approach in the treatment of diabetes. We have examined the long-term effects on the late complications of islet transplantation in an experimental diabetes model in the rat. Diabetes was induced by streptozotocin (70 mg/kg i.v.) and the rats were treated with either insulin (daily injection of 40 U) or transplantation of 1,000 freshly isolated, hand-picked, islets into the left renal subcapsular space. Both islet transplantation and insulin treatment completely normalized the increased levels of blood glucose, urine volume and water intake that were observed in the diabetic rats. The decreased growth rate of the diabetic rats was almost normalized by both treatment protocols. As for late complications, after 3 months, all untreated diabetic rats had cataract. They also had swelling and vacuolation of renal tubular cells, and, consistent with this, very high levels of urinary beta 2-microglobulin excretion. Both islet transplantation and insulin treatment completely prevented these late complications. Thus, islet transplantation to the renal subcapsular space is in this experimental model as good as insulin treatment in treating the clinical signs of diabetes and in preventing diabetic complications in the eye and kidney.

Effect of Low-Temperature Culture and L3T4 Antibody on the Survival of Pancreatic Islets Xenograft (Fish to Mouse)

Catfish (Ictalurus punctatus) islets were transplanted into the renal subcapsular space of diabetic C57BL/6J mice. The xenograft mean survival time (MST) of islets cultured for 6 days at 24 degrees C was 4.9 +/- 0.5 days, while xenotransplantation of 1-hour-cultured islets did not restore normoglycemia in the diabetic mice. A modest but significant prolongation of the MST was obtained by administration of anti-L3T4 monoclonal antibody to the recipients (13.4 +/- 2.1 days). Anti-L3T4 in conjunction with low-temperature culture did not produce a further increase of the MST (9.1 +/- 2.7 days). These findings indicate that catfish islets may be a source of islet tissue for transplantation in diabetic recipients. However, procedures that demonstrated to be effective in prolonging the MST of xenografts transplanted across wide species barrier, such as low-temperature culture and anti-L3T4 treatment, failed to prolong markedly the xenograft survival. Further experimental work is needed to study the possibility of improving islets survival after xenotransplantation across distant species barrier (fish to mouse).

Demonstration of donor specific unresponsiveness in rat islet allografts: importance of transplant site for induction by cyclosporin A and maintenance

The purpose of the present study was to evaluate the effects of transplant site (the liver via the portal vein vs the renal subcapsular space) on islet allograft survival using cyclosporin A and also to determine whether or not the unresponsiveness obtained is donor specific and how the site selected is related to the maintenance of unresponsiveness. Prolongation of islet allograft survival (WKA/Qdj:RT1u----Lewis:RT1l) was obtained by using hand-picked clean fresh islets as donors and by s.c. administration of cyclosporin A (30 mg/kg, day 0, 1, 2) when the islets were transplanted into the liver. Eleven out of 15 recipients were normoglycaemic for more than 90 days after transplantation (control mean survival time: 4.4 +/- 1.1 days, n = 5). However, all the renal subcapsular grafts were rejected within 18 days after transplantation. Two normoglycaemic recipients bearing intrahepatic grafts were challenged with full-thickness donor-strain (WKA/Qdj) skin grafts at 120 days after the islet transplantation. Both recipients became diabetic and the skin grafts rejected within 16 days. The nine normoglycaemic recipients were made diabetic again with streptozotocin (40 mg/kg) between 90 and 120 days after the initial islet transplantation. Re-transplants of fresh donor-strain (WKA/Qdj) islets into the liver (n = 3) in the absence of cyclosporin A maintained normoglycaemia in the recipients for more than 60 days, whereas re-transplants of donor-strain islets beneath the kidney capsule were rejected within 20 days. Intrahepatic re-transplants of third party grafts (PVG/c: RTlc) (n = 3) were rejected after seven days.(ABSTRACT TRUNCATED AT 250 WORDS)

Islet transplantation to the renal subcapsular space in streptozotocin-diabetic rats: short-term effects on glucose-stimulated insulin secretion

The immediate and short-term effects of transplantation of freshly isolated islets on glucose-stimulated insulin secretion were studied in streptozotocin-diabetic rats. At 2 days after the intravenous injection of streptozotocin (70 mg/kg), either 400 or 1000 freshly isolated, hand-picked islets were transplanted to the left renal subcapsular space. Intravenous glucose infusion (10 mg/min) performed 1, 3, 6, and 10 days after transplantation revealed that already at day 3, rats transplanted with 1000 islets had a sinnificant plasma insulin response to glucose, though the response was lower than in healthy controls. At 6 and 10 days after transplantation, the 1000 islet-transplanted group of rats had a near-normal plasma insulin response to glucose. Basal plasma glucose levels were, however, still slightly elevated. Rats transplanted with 400 islets had a significant plasma insulin response at 10 days after transplantation, though the response was lower than in those transplanted with 1000 islets. It is concluded that a near-normal in vivo insulin secretory response to glycose is already obtained 6 days after transplantation with 1000 freshly isolated islets to the left renal subcapsular space in streptozotocin-diabetic rats. However, a slight hyperglycemia persists.

FETAL ISLET ALLOTRANSPLANTATION IN RABBITS

Haploidentical fetal rabbit islets were transplanted into the renal subcapsular space of outbred maternal hosts. Islets were prepared by a mild collagenase digestion and were cultured in air and a high oxygen environment for 10 days prior to implantation. Grafts were examined at 14 days and later. No grafts survived in the absence of host immunosuppression, even when treatment of the donor tissue included both culture and ultraviolet-B irradiation (groups 1 and 3). With a high dose of oral cyclosporine, 30 mg/kg/day on days 0-3, viable but disrupted islets were present in 1/7 recipients of islets treated by culture alone. Only with high-dose peritransplant cyclosporine or chronic low-dose immunosuppression, and islet treatment with UVB and culture, was regular graft survival achieved (groups 4A and 5). These results demonstrate that haploidentical outbred hosts can be made to accept fetal islet allografts pretreated by UVB irradiation and culture in a high oxygen environment only if the recipient is given a high peritransplant dose of oral cyclosporine (30 mg/kg/day) or chronic low-dose cyclosporine, (10 mg/kg/day).

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)

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.

In vivo assessment of isolated pancreatic islet viability using the streptozotocin-induced diabetic nude rat.

In order to establish a model for the in vivo assessment of islet function we have used the Rowett nude rat with transplantation of allogeneic and xenogeneic (mouse) islets into the renal subcapsular space following a minimal period of diabetic induction. Thirty-one nude rats were given streptozotocin and 30 became diabetic with blood glucose levels of greater than 20 mmol/l at 48 h. Rat and mouse islets were prepared by intraductal collagenase and bovine serum albumin density gradient isolation. Eight rats received transplants of freshly prepared allogeneic islets and 8 rats received transplants of 48 h cultured allogeneic islets. Seven rats received transplants of 48 h cultured mouse islets. Diabetes was reversed in all animals and all remained normoglycaemic for 21 days. Graft removal by nephrectomy resulted in hyperglycaemia in 22 out of 23 animals. Histological examination of the grafts showed a band of endocrine tissue beneath the renal capsule which stained strongly positive for insulin and there was no evidence of lymphocytic infiltration/rejection. One rat remained normoglycaemic after graft removal, which may represent recovery of the animal's own islets from the streptozotocin-induced diabetes. Control rats remained diabetic until death. In conclusion, the athymic nude rat can be used for the assessment of allogeneic and xenogeneic islet function when a short (48 h) period of streptozotocin-induced diabetes is used. This model offers a potential method for assessing in vivo function of isolated human islets.

MULTIPLE DONOR ALLOTRANSPLANTATION

Currently it is not feasible to isolate sufficient numbers of islets from a single pancreas for clinical transplantation. We examined whether small numbers of islets obtained from multiple donors could be used for transplantation. Islets were isolated from four inbred strains of mice (DBA/2, DBA/1, C3H, and A.SW) by a stationary collagenase digestion and Ficoll separation and transplanted into the renal subcapsular space of streptozotocin-induced diabetic B6AF1 mice. At least 200 handpicked islets were required to produce normoglycemia in syngeneic and allogeneic diabetic recipient mice. None of the mice given 50 islets became normoglycemic within 2 weeks postgrafting. When various numbers of purified islets from a single donor were transplanted, the survival was significantly better for 200-islet allografts than for 800-islet and 400-islet allografts. When a 200-islet composite graft was prepared from four donors (50 fresh handpicked islets from each donor), the survival of the composite graft as measured by sustained normoglycemia in nonimmunosuppressed recipients was dramatic, with 17 of 18 recipients maintaining normoglycemia indefinitely (greater than 200 days). Similarly, when islets isolated from four donors and cultured for various periods were mixed and transplanted (200 islets/recipient) all recipient mice (n = 8) enjoyed indefinite graft survival. Use of higher numbers of purified islets or crude islets in a composite multiple-donor islet allograft was less effective in achieving indefinite graft survival. Thus, transplantation of a composite graft made up with subtherapeutic numbers of islets from multiple histoincompatible donors to provide adequate therapeutic numbers is a practical solution to the lack of islet availability. In addition, composite islet grafts appear to possess immunological advantages, with significantly prolonged survival over that produced by single-donor islet grafts.