Renal Subcapsular Site Research Articles

An in vivo model for human fetal adipose tissue development

Adipogenesis in the developing fetus is a key determinant of postnatal adiposity and, by extension, the risk of developing obesity‐related disease later in life. A variety of environmental factors, including toxicants and maternal diet, can influence fetal adipogenesis. To study the effects of environmental factors on fetal adipogenesis, we have developed a xenotransplantation model. Mid‐gestation human fetal white adipose tissue (WAT), obtained from cases of spontaneous fetal loss, was implanted into immunocompromised rodents using the renal subcapsular site. In multiple experiments (7 fetal samples ranging in gestation from 20 to 22 weeks), we observed an initial loss of adipocytes within the first week. This was followed by development of obvious fat pads. Expansion of these fat pads continued for up to 6 months. Immunohistochemical analyses revealed the WAT foci to contain viable adipocytes that were heterogeneous in size, an indication of active adipogenesis. Staining for the endothelial cell marker CD31 demonstrated rich vascularity of the implants and invasion of vessels from the implant into the host kidney parenchyma. These results demonstrate that transplantation of human fetal WAT into immunocompromised mice is a model for marked healthy WAT expansion and remodeling. This model can be utilized to study the direct effect of nutrient manipulation and obesogens on fetal white adipose tissue.

The human fetal lung Xenograft: Validation as model of microvascular remodeling in the postglandular lung

Coordinated remodeling of epithelium and vasculature is essential for normal postglandular lung development. The value of the human-to-rodent lung xenograft as model of fetal microvascular development remains poorly defined. The aim of this study was to determine the fate of the endogenous (human-derived) microvasculature in fetal lung xenografts. Lung tissues were obtained from spontaneous pregnancy losses (14-22 weeks' gestation) and implanted in the renal subcapsular or dorsal subcutaneous space of SCID-beige mice (T, B, and NK-cell-deficient) and/or nude rats (T-cell-deficient). Informed parental consent was obtained. Lung morphogenesis, microvascular angiogenesis, and epithelial differentiation were assessed at 2 and 4 weeks post-transplantation by light microscopy, immunohistochemical, and gene expression studies. Archival age-matched postmortem lungs served as control. The vascular morphology, density, and proliferation of renal subcapsular grafts in SCID-beige mice were similar to age-matched control lungs, with preservation of the physiologic association between epithelium and vasculature. The microvasculature of subcutaneous grafts in SCID-beige mice was underdeveloped and dysmorphic, associated with significantly lower VEGF, endoglin, and angiopoietin-2 mRNA expression than renal grafts. Grafts at both sites displayed mild airspace dysplasia. Renal subcapsular grafts in nude rats showed frequent infiltration by host lymphocytes and obliterating bronchiolitis-like changes, associated with markedly decreased endogenous angiogenesis. This study demonstrates the critical importance of host and site selection to ensure optimal xenograft development. When transplanted to severely immune suppressed, NK-cell-deficient hosts and engrafted in the renal subcapsular site, the human-to-rodent fetal lung xenograft provides a valid model of postglandular microvascular lung remodeling.

High Vascular Density and Oxygenation of Pancreatic Islets Transplanted in Clusters into Striated Muscle

Pancreatic islet transplantation is presently almost exclusively performed using the intraportal route for transplantation into the liver. However, islets at this site are poorly revascularized and, when also considering the poor long-term results of clinical islet transplantation, there has in recent years emerged an increased interest to evaluate alternative sites for islet transplantation. Striated muscle is easily accessible and has for decades been used for autotransplantation of parathyroid glands. Moreover, it is almost the only tissue in the adult where physiological angiogenesis occurs. The present study tested the hypothesis that striated muscle would provide good conditions for revascularization and oxygenation of transplanted islets. Because we previously have observed similar revascularization of islets implanted to the renal subcapsular site and intraportally into the liver, islets grafted to the kidney were for simplicity besides native islets used for comparison. Islets grafted into muscle were found to have three times more blood vessels than corresponding islets at the renal subcapsular site at 2 month follow-up, but still less vascular numbers than native islets. The oxygen tension in 2-month-old intramuscular islet grafts was sixfold higher than in corresponding renal subcapsular grafts, and 70% of that in native islets. However, the oxygenation of surrounding muscle was only 50% of that in renal cortex, and connective tissue constituted a larger proportion of the intramuscular than the renal subcapsular grafts, suggesting exaggerated early islet cell death at the former site. We conclude that the intramuscular site provides excellent conditions for vascular engraftment, but that interventions to improve early islet survival likely are needed before clinical application. Such could include bioengineered matrices that not only spatially disperse the islet, but also could provide local supply of oxygen carriers, growth and survival factors, strategies that are much more easily applied at the intramuscular than the intrahepatic site.

Low Revascularization of Human Islets When Experimentally Transplanted Into the Liver

Previously, we have found that human islets experimentally transplanted beneath the kidney capsule have lower vascular density than native islets. This study aimed to investigate whether human islets experimentally transplanted into the liver are also poorly revascularized in the same manner as islets at the renal subcapsular site. Human islets were transplanted to nude mice. One month posttransplantation, the islet graft-bearing livers or kidneys were removed, formalin-fixed, and stained with the lectin Bandeiraea (Griffonia) simplicifolia (BS-1) to visualize endothelium. The vascular density in the intraportally transplanted human islets was found to be similarly low as in human islets transplanted beneath the kidney capsule. The intrahepatic human islets were coated with numerous vessels, but few vessels could be seen within the islets. Human islets transplanted intraportally into the liver become poorly revascularized. This could contribute to the loss of function in human islets transplanted into the liver over time.

Novel In Vivo Murine Model to Study Islet Potency: Engraftment and Function

Standard islet potency testing uses transplantation of islets under the kidney capsule in diabetic severe combined immunodeficient (d-SCID) mice. Even though it is possible to achieve normoglycemia in the majority of recipients by this method, the surgical procedure, by itself, is technically difficult and associated with an appreciable mortality of animals. In addition, the spatially limited renal subcapsular site restricts the mass of islet tissue that can be transplanted. Matrigel basement membrane matrix (MATRIGEL), extracted from a mouse sarcoma, is rich in angiogenic growth factors and has been shown to support the growth of mammalian cells using murine models. In this report we demonstrate that subcutaneous islet transplantation with MATRIGEL can effectively achieve normoglycemia and that this is a simple and reproducible model for in vivo islet potency testing in d-SCID mice that overcomes many drawbacks of the conventional method of kidney subcapsular islet transplantation.

PH is decreased in transplanted rat pancreatic islets.

Recent studies of transplanted pancreatic islets have indicated incomplete revascularization. We investigated the pH, in relation to oxygen tension (Po(2)), in endogenous islets and islets syngeneically transplanted to the renal subcapsular site of nondiabetic and streptozotocin-diabetic recipients. Tissue pH and Po(2) were measured using microelectrodes. In the endogenous islets, tissue pH was similar to that in arterial blood. In the transplanted islets, tissue pH was 0.11-0.15 pH units lower. No differences in islet graft pH were seen between nondiabetic and diabetic animals, and none if the islet grafts were investigated 1 day or 1 mo posttransplantation. The Po(2) in the endogenous islets was approximately 35 mmHg. Transplanted islets had a markedly lower tissue Po(2) both 1 day and 1 mo after transplantation. A negative correlation between the tissue Po(2) and the hydrogen ion concentration was seen in the 1-mo-old islet transplants in diabetic animals. In conclusion, decreased Po(2) in transplanted islets is associated with a decreased tissue pH, suggesting a shift toward more anaerobic glucose metabolism after transplantation.

Decreased survival of islet allografts in rats with advanced chronic complications of diabetes.

Successful islet transplantation has been possible in experimental animals in contrast to humans. One difference between animal models of diabetes and human islet transplantation is the presence of advanced chronic complications in humans. Even longer-term follow-up of islet transplantation in humans according to the Edmonton protocol suggests that advanced chronic complications may adversely affect allograft survival with the glucocorticoid-free immunosuppressive regimen as well. We developed a rat model of chronic complications of diabetes and compared islet allograft survival in rats with advanced chronic complications to age-matched control rats with acute onset diabetes. Islets were transplanted at either the renal supcapsular, intrahepatic, or intramuscular location. The survival of islet allografts in rats with chronic complications was decreased at all sites compared with the age-matched controls. The best survival in the rats with advanced chronic complications occurred at the renal subcapsular site. Blood sugar measurements indicated impaired glucose tolerance in most of the rats with chronic complications and surviving renal subcapsular islet allograft. Histological and gross examination of the surviving renal subcapsular islet allografts indicated disordered angiogenesis in the rats with chronic complications. Rats with successful intrahepatic islet allografts and the respective age-matched controls had comparable blood sugars. Survival of islet allografts at the intramuscular site was poor in rats with chronic complications or acute onset diabetes. We conclude that the structural or metabolic abnormalities associated with chronic poor control of diabetes impair islet allograft survival and function. This should be considered as a possible explanation for failure of islet allograft survival in human islet transplantation.

Non-invasive evaluation of the location, the functional integrity and the oxygen supply of implants: 19F nuclear magnetic resonance imaging of perfluorocarbon-loaded Ba2+-alginate beads.

19F nuclear magnetic resonance imaging (MRI) can be used as a non-invasive tool to simultaneously determine the location, the integrity and the oxygen supply of Ba2+-alginate implants. This requires that the beads (implants) are pre-loaded with the perfluorocarbon compound F-44E. Implantation of solid 19F-labelled beads into the peritoneum, below the kidney capsule or into the muscle of Wistar WU rats demonstrated that these beads could be detected by 19F-MRI for up to 18 months after implantation. This indicated that F-44E is not considerably released from the beads during implantation. The signal to noise ratio of liquid-core beads was higher by a factor of 4 than the signal to noise ratio of solid beads, but liquid-core beads were more fragile and also too large for implantation under the kidney capsule and into the intramuscular tissue. Quantitative 2-dimensional 19F-T1 maps (resolution 0.5 x 0.5 mm) could be deduced from 19F-MRI measurements. These T1-maps correlated to the local pO2-values. The partial oxygen pressure estimated in F-44E-loaded Ba2+-alginate beads showed that the oxygen supply inside the beads was very poor when they were implanted below the kidney capsule or into the peritoneal cavity. These low pO2-values obtained for the renal subcapsular site and the peritoneum may explain the failure of previous immunoisolated islet transplantation studies using these locations.

CD40/CD40L (CD154) BLOCKADE ALONE IS SUFFICIENT TO PREVENT ISLET ALLOGRAFT REJECTION IN THE RAT.

882 The CD40-CD154 pathway plays a critical role in T cell activation. Blockade of the T cell co-stimulatory molecule CD154 alone is reported to be effective in producing long-term allograft survival in non-human primates while additional treatments are required to produce the same effect in the mouse. The recent availability of anti-rat CD154 mAb [(C.D. Benjamin et al, manuscript in preparation)] has allowed for testing in the rat model for comparison. The goal of this project was to characterize the immunosuppressive effects of anti-CD154 on the rate of rejection of islet allografts in the high responder rat strain combination, DA (RT1aa) to DR-BB (RT1uu). DR-BB rats were made diabetic by an injection of streptozotocin (65 mg/kg). Reversal of diabetes was accomplished by transplanting 1200 (islet equivalency) DA islets to the renal subcapsular site. Each treatment was given IV peri-transplant (days −1, 0, +1), with weekly maintenance dosing post-transplant through week 5 and then stopped. Blood glucose levels were monitored and a return to hyperglycemia (3 consecutive days of blood glucose levels > 250 mg/dl) was an indication of rejection. All control animals promptly rejected their DA islets by day 8 post-transplant. Eight of 9 anti-CD154 treated recipients have indefinite allograft survival and currently are 95 days post-transplant. Recipients bearing long-term allografts showed a significant decrease in T cell proliferation compared to naive rats in response to donor antigen in a MLR. We conclude from these data that anti-CD154 mAb is effective as a single agent in promoting long-term islet allograft survival in the rat.

Clinical experience with autologous and allogeneic pancreatic islet transplantation.

Background. We initiated a pancreatic islet transplantation program using fresh, single-donor tissue. Methods. Group 1 (n=3) received cadaver kidney and islets from the same donor; islets were placed beneath the capsule of the transplanted kidney. Group 2 (n=6) received islets via percutaneous portal vein puncture subsequent to successful kidney transplant. Group 1 and 2 recipients had type 1 diabetes mellitus. Group 3 (n=6) received autologous islet transplantation after total or near-total pancreatectomy (for chronic pancreatitis and intractable pain). Implantation and graft function was considered successful if C-peptide exceeded 0.10 ng/ml. Sustacal challenge test was done before and after transplant at day 1, week 1, and months 1, 3, 6, and 12. We also measured glycated hemoglobin and insulin requirements at regular time intervals. Results. Post-Ficoll yield was 402,000 (SD 174,000) equivalent islet number, average purity of 67% (range, 50-90%). Group 1 had initial primary nonfunction followed by occasional evidence of C-peptide at months 3 and 6. Group 2 recipients had significant C-peptide levels early after transplantation which gradually declined over several months. However, all recipients had graft function at 6 months and 50% recipients still maintained graft function at 1 year. Group 3 recipients are off exogenous insulin (mean follow-up of 5.5 months). Insulin requirement was unchanged in group 1, while a reduction of approximately 20% was noted in four of six patients in group 2. Glycated hemoglobin was reduced in four out of six recipients of group 2, while in group 3, glycated hemoglobin remained within normal limits. Pain relief was sustained when patients (group 3) received total pancreatectomy; near-total pancreatectomy resulted in recurrence of pain within 3 months. Conclusions. Autologous islet transplantation is uniformly successful; total excision of pancreas was superior to partial pancreatectomy for pain relief. For allogeneic islet transplantation, the intrahepatic site is superior to the renal subcapsular site. Insulin independence was not achieved in allogeneic islet transplants; however, continued graft function resulted in reduction of glycated hemoglobin in some recipients.

Angiogenesis in cultured and cryopreserved pancreatic islet grafts.

The ability of rat pancreatic islets to revascularize after transplantation was examined via in vitro and in vivo imaging of the microvasculature using laser scanning confocal microscopy (LSCM). Cultured or cryoprocessed islets were transplanted at the renal subcapsular site in rats. At various time intervals after transplantation, three-dimensional imaging of the graft was performed by LSCM. In vitro studies were conducted via microvascular corrosion casting of the grafted kidney in situations where it was difficult to obtain in vivo confocal data due to surgical complications. The vascular morphology of the islet grafts was evaluated quantitatively via digital image analysis algorithms to determine the morphology of the neovascular ingrowth and the rate of revascularization. In cultured islet grafts, the initiation of angiogenesis was observed within 1 week, characterized by the presence of capillary sprouts, tortuous vessels, and blood vessels with blind ends. The revascularization of the graft was typically completed within 2 weeks and could be distinguished as a network of completely perfused blood vessels consisting of intertwining capillaries, with surrounding arterioles and venules. The angiogenesis process in cryopreserved islet grafts required a longer time period to initiate (approximately 2 weeks), and the revascularization was completed in 1 week after the initiation. These results successfully demonstrate the potential of the described in vivo and in vitro LSCM techniques to measure the angiogenesis process in pancreatic islet grafts.

In-vivo analysis of angiogenesis and revascularization of transplanted pancreatic islets using confocal microscopy.

A technique to measure angiogenesis and revascularization in pancreatic islets transplanted at the renal subcapsular site in the rat has been developed. In-vivo imaging of the microcirculation of transplanted pancreatic islets was conducted using a confocal scanning laser microscope (CSLM) to achieve optical sectioning through the graft in order to perform a computer reconstruction of the three-dimensional neovascular morphology. Individual islets were harvested by enzymatic digestion of excised pancreas from Fischer 344 rats. Isolated islets were cultured for 24 h, and approximately 300-350 islets were transplanted at the renal subcapsular site of the left kidney in an anaesthetized rat. Six to 14 days post-transplantation, the animal was anaesthetized and prepared for in-vivo imaging of the microvasculature on a Zeiss LSM-10. Optical contrast of the microvasculature was enhanced by the administration of fluorescein-labelled dextran into the circulating blood. The transplant site was identified and serial sections were obtained through the vascular bed at varying z-intervals. Complementary fluorescence video images were also obtained via a silicon intensifier tube camera mounted on the CSLM. At completion of the imaging procedure, the kidney was returned into the body cavity, the area was sutured and the animal was allowed to recuperate for subsequent examinations. Image processing algorithms, such as grey-level thresholding, median filtering, skeletonization and template matching, were applied to compute the vessel density and diameters and extrapolated to measure 3-D vessel lengths and the tortousity index of the neovasculature.

Müllerian inhibiting substance production associated with loss of oocytes and testicular differentiation in the transplanted mouse XX gonadal primordium.

The mouse XX gonadal primordium develops seminiferous-like tubules after transplantation into the renal subcapsular site of the adult male or female mouse. We examined the ontogeny of Sertoli cell differentiation in XX gonadal grafts by immunocytochemical staining and organ culture bioassay for Müllerian Inhibiting Substance (MIS). During normal in situ development of the XY gonad, MIS staining was first detected in fetal Sertoli cells at 12 days of gestation (d.g.) and remained intense until 4 days postpartum (d.pp.), after which it gradually diminished with progressive testicular development. In the normal in situ XX gonad, MIS was detected in granulosa cells of growing follicles at 7 d.pp. and thereafter. When the XX gonad at 12 d.g. was grafted beneath the renal capsule, a few testicular cords composed of MIS-positive cells appeared on Day 7 post-transplantation (equivalent to 19 d.g.), much earlier than the normal appearance of MIS production in the intact XX ovary. The ovarian region containing germ cells at the meiotic prophase was unstained for MIS in the same sections. The incidence of XX gonadal grafts containing MIS-positive testicular cords and the number of such cords per gonadal graft steadily increased from Day 7 to Day 14 post-transplantation. Germ cells were absent or scarce inside the MIS-positive testicular cords. The MIS bioactivity in both control gonads and gonadal grafts coincided with the immunocytochemical staining for MIS. These results support the hypothesis that XX cells differentiate into Sertoli cells as a consequence of oocyte loss in the gonadal graft.

The Concept of Metabolic Clearance Rate in Endocrinology

Disappearance of growth hormone from blood (plasma) was studied in young broiler chickens in two experiments: a) following single injection of GH (50 micrograms or 250 micrograms/kg) to anesthetized chicks; b) injection of GH (50 micrograms/kg) (under anesthesia) to two lines of broilers selected for either rapid growth or feed efficiency. Clearance of GH never followed a single negative exponential curve. Characterization of the disappearance by a single metabolic clearance rate and half-life time were found to be inaccurate and inappropriate on methodological grounds. High doses of hormone might disturb receptor or catabolism equilibria and result in aberrant values of MCR which could probably be because of the very dynamic nature of the GH clearance system, which appeared to be influenced by dose, line (or genetics of the animal) and by some indices of clearance. Therefore, physiological significance of such data must be interpreted carefully.

EFFECTS OF COTRANSPLANTED FETAL LIVER ON FETAL PANCREAS ISOGRAFTS 1, 2.

Ricordi et al. described a hepatotrophic effect mediated by pancreatic islets on cotransplanted hepatocytes. We found a reciprocal salutary effect of fetal liver (FL) on fetal pancreas (FP) in the intramural small bowel (ISB) site. To further investigate this intriguing finding, composite FP/FL isografts were transplanted to the conventional renal subcapsular (RSC) site and the accessible but historically inhospitable intramuscular site in streptozotocin-diabetic Lewis rats. A comparison of recipients of FP/FL and FP alone found the proportion rendered normoglycemic was site dependent. All recipients of either composite FP/FL grafts or FP alone transplanted in the ISB site became normoglycemic. The proportion of normoglycemic recipients was lower in the RSC site (71% FP and 40% FP/FL) and the i.m. site (14% FP and 67% FP/FL). Importantly, regardless of site, normoglycemia was established with an accelerated time course in recipients of FP/FL versus FP alone (24 +/- 8 vs. 67 +/- 43 days; P = 0.001). Normal (or more rapid) glucose clearance after challenge was achieved in all normoglycemic recipients except those transplanted in the RSC site. On histological examination of excised FP/FL grafts, hepatocytes were present in association with islets. Cyclosporine-induced islet toxicity could not be overcome in 6 recipients of FP alone, but 6 of 8 recipients of FP/FL became normoglycemic (P less than 0.01). To assess the effect of FP on hepatocytes, allografts (Wistar donors) of FP or FP/FL were cotransplanted in the ISB of enzyme-deficient jaundiced Gunn rats. Immunosuppression consisted of rapamycin (0.8 mg/kg/day) infused intravenously for 4 weeks. In the FP/FL group (n = 4), the mean serum bilirubin level decreased from 8.6 to 4.9 mg/dl at 6 weeks after transplantation. This was a significant difference as compared with the increased mean serum bilirubin from 6.9 to 7.8 mg/dl (P less than 0.05; paired Student's t test) in recipients of FL alone (n = 4). In conclusion, we found a mutual paracrine effect on islets and hepatocytes transplanted as a composite FP/FL graft. FL hastened the establishment of normoglycemia following transplantation of FP in diabetic rats, and FP enhanced FL transplant function in Gunn rats.

BALB/cマウス自然発生腎細胞癌に対するBRMs (Biological Response Modifiers) の抗腫瘍効果

The effect of the streptococcal preparation OK-432, which is one of the biological response modifiers, was examined in BALB/c mice using a transplantable murine renal cell carcinoma (Renca) of spontaneous origin, and an analysis of effector cells was performed. The tumor grew progressively and metastasized consistently to the abdominal lymph nodes and then to distant organs following the inoculation of Renca cells in the left renal subcapsular site in BALB/c mice, and the survival time of the mice was under 42 days. In this tumor model, i.p. administration of OK-432 after tumor inoculation significantly extended the survival time and significantly inhibited the formation of the inoculated tumor itself. Removal of the left kidney on the 7th day after tumor inoculation neither extended the survival time nor augmented the effect of OK-432. Splenic cells obtained on the 7th day after tumor inoculation from Renca-bearing mice treated with OK-432 were capable of lysing syngeneic Renca cells, NK-sensitive allogenic YAC-1 cells, and LAK-sensitive EL-4 cells in a 4-hour 51Cr-release assay in vitro. Those obtained from healthy mice treated with OK-432 also showed cytotoxic activity against Renca cells. The cytotoxicity of splenic cells from Renca-bearing mice treated with OK-432 was lost almost completely for both Renca and YAC-1 cells after in vitro treatment with anti-asialo GM1 antibody, and was partially lost after in vitro treatment with anti-Thy-1,2 antibody. Additionally, in vivo i.p. administration of anti-asialo GM1 antibody significantly counteracted the effect of OK-432 on survival. These findings demonstrated that Renca cells were NK-sensitive and that the i.p. administration of OK-432 was beneficial for the prevention of the spontaneous metastasis of Renca carcinoma. As the effectors, NK cells played a dominant role and activated T cells were also involved.

REJECTION OF CULTURED KERATINOCYTE ALLOGRAFTS IN THE RAT

We have made a detailed analysis of the fate of Langerhans cell-free cultured keratinocyte allografts in two rat strain combinations, DA-to-PVG and DA-to-LEW, and compared the results with the rejection of conventional skin allografts in these strain combinations. The cultured keratinocyte layers were grafted both to the body surface using a technique to prevent wound contraction, and to the renal subcapsular site. Histological examination of grafts was made on days 2, 7, 10, 14, and 28 after transplantation. Donor-specific anti-class I MHC monoclonal antibodies were used to verify the donor origin of the keratinocytes. We report that the keratinocyte allografts are acutely rejected but, in contrast to the conventional allografts, do not evoke alloantibody responses. Rejection of the keratinocytes at the renal subcapsular site was as rapid as that of conventional skin grafts. However, rejection of keratinocyte grafts on the body surface was delayed by a few days when compared with conventional skin grafts. Immunosuppression with cyclosporine prevented the rejection of DA keratinocyte layers placed at the renal subcapsular site of PVG rats, but rejection followed soon after cessation of cyclosporine therapy. These data suggest that rejection is a major constraint for the clinical application of cultured keratinocytes, and that autografts must be used if permanent cover is required. Moreover, the findings have interesting theoretical implications relating to the much greater vulnerability to rejection of skin grafts compared with organ grafts. Our current and previous data also suggest that class II positive dendritic cells are the major stimulus to alloantibody production after tissue transplantation.

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 islet xenograft survival (rat to mouse) by in vitro culture at 37 C.

Meticulously selected rat islets were maintained in vitro for 7 days in a minimal volume of tissue culture medium at 37 C in air and 5% CO2. The cultured islets were transplanted into diabetic mice, either into the liver via the portal vein, or beneath the renal capsule. The survival of the cultured islets, following intrahepatic or renal subcapsular transplantation, was significantly prolonged compared with that of fresh islets. The renal subcapsular site apparently provides some additional immunologic advantage, because the survival time of the cultured islets in this site was approximately twice as long as in the intrahepatic transplants.

A histological investigation of isografts of immature mouse molars to an intrabony and extrabony site

Partially-developed teeth with remnants of their periodontal ligaments isografted to the tibial shaft medulla and renal subcapsular site were capable of progressive regeneration characterized by renewed tubular dentine formation. Bone formation occurred in relation to grafts at the renal site as well as the tibial shaft; evidence of periodontal fibre reorganization was observed in grafts at both sites. Despite post-transplantation dentine and cementum formation, continued development of roots of normal morphology did not occur, perhaps due to disappearance of epithelial root sheath.