Hyperinsulinemia and hyperglucagonemia following pancreatic islet transplantation in diabetic rats.

Fasting blood glucose (FBG), serum immunoreactive insulin (IRI), plasma immunoreactive glucagon (IRG), body weight, and caloric intake were measured in long-term islet-isografted rats eight to 10 months following intraperitoneal islet transplantation in in age-matched, sham-operated, concurrently followed normal and diabetic controls. Islet recipients had normal body weights, but they were significantly polyphagic, hyperinsulinemic, and hyperglucagonemic when compared with normals. Fasting blood glucose levels were reduced by 10 per cent. Several factors may be related to the occurrence of these abnormalities in long-term islet-isografted rats, including (1) the mass of islets transplanted, (2) the age of donor tissue, (3) the heterotopic location of islet grafts, and (4) the lack of normal innervation of transplanted islet cells.     

Revascularization and function of pancreatic islet isografts in diabetic rats following transplantation

In pancreatic islet transplantation, revascularization is crucial for the graft's survival and function. In this study, the endothelium of isolated islets and revascularization and function of islet isografts in diabetic rat were investigated. Islets were isolated from Lewis rats by collagenase digestion method and were examined using immunohistochemistry (CD31 stain) on days 0, 1, 3, and 7 after isolation. The number of CD31-positive cells in these isolated islets was counted (mean +/- SD %). Isografts (freshly isolated islets: group A, and islets cultured for 7 days: group B) transplanted in the renal subcapsule of streptozotocin-induced diabetic Lewis rats were examined using immunohistochemistry (CD31 stain) on days 3, 5, and 7 after transplantation. Intravenous glucose tolerance tests (IVGTT) were performed on days 3 and 7 after transplantation. The number of CD31-positive cells in the isolated islets on days 0, 1, 3, and 7 after isolation were: 17.3 +/- 4.1%, 8.2 +/- 0.7%, 2.1 +/- 0.8%, and 0.8 +/- 0.5%, respectively (p < 0.05). On day 5 after transplantation, CD31-positive cells were not detected in group A and B grafts, but were detected in both groups in periphery of the islets. On day 7, CD31-positive microvessels were present throughout the entire graft. IVGTT values in groups A and B on days 3 and 7 after transplantation did not show significant differences. In renal subcapsular isografts in diabetic rats, revascularization into islet grafts occurs from the surrounding host tissue 5 days after transplantation, but has no influence on the response to glucose during this period.     

Successful simultaneous transplantation of kidney and fetal pancreatic islet masses

This paper reports our experience with the successful simultaneous transplantation of kidney and fetal pancreatic islets in a 46-year-old diabetic man. No detectable C-peptide level was noted and the end-stage nephropathy required hemodialysis. The cadaver kidney and two masses of 8-week-cultured fetal islets were grafted simultaneously. After revascularization of the kidney, the islet masses were placed under the kidney capsule. Following transplantation, islet function was demonstrated by a higher C-peptide level, which subsequently persisted. Twenty-four months after grafting, islet function was provoked by glucagon and glucose, which led to elevations in the C-peptide and insulin levels. The insulin requirement fell from 58 to 24 U/day during the post-transplant period of 24 months. The mean value of HbA_1c (5.6%±0.3%) indicated a constantly normal carbohydrate metabolism. Improvements in retinopathy were also noted. Three periods of kidney rejection were diagnosed, but these proved reversible with high-dose steroid treatment. The serum and urine beta-2-microglobulin levels correlated well with rejection and recovery. More than 2 years after grafting, kidney function is in the normal range. On sonography, the transplanted islet masses were repeatedly clearly visible, and 24 months following transplantation the volume was twice the original one. The results indicate that simultaneous kidney and fetal pancreatic islet grafting is advantageous in end-stage nephropathy secondary to type I diabetes mellitus.     

Repeated intraportal injections of subtherapeutic islet cell isografts restore normoglycemia in streptozotocin-diabetic rats

Poor engraftment and consequent loss of β-cell mass could be one of the factors that are responsible for function loss after intraportal islet transplantation (Tx). Streptozotocin-diabetic rats were transplanted with syngeneic islets, which were injected into the portal vein via an indwelling catheter connected to a subcutaneous port. In Group I (n = 6), 1,000 islets were injected in a single dose into the liver. In Group II (n = 6), five doses of 200 islets were repeatedly injected over a period of 14 days, for a total of 1,000 islets. In Group III (n = 4), five decreasing doses of islets were injected over a period of 14 days, for a total of 750 islets. Nonfasting blood glucose (n-FBG) and body weight (b.wt.) were determined twice a week and an intravenous glucose tolerance test (IVGTT) was performed at 30 and 90 days. In Group I, n-FBG decreased in 2 wk from the time of first islet injection, averaging 110 ± 21.9 mg/dL at 1 mo (p < 0.05 vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group II, n-FBG was normalized in 2 wk averaging 90.2 ± 25 mg/dL on day 12 (p = NS vs. normal controls) and 75.8 ± 14.6 mg/dL at 1 month (p = NS vs. normal controls); this value was maintained throughout the 3-mo duration of the study. In Group III, n-FBG decreased to normal values in 2 wk, averaging 77 ± 15.7 mg/dL at 1 mo (p = NS vs. normal controls), but normoglycemia was maintained for 40 days and then followed by a progressive increase. Only in Group II, K_G (percent/min decline in glucose level) was not significantly different from that of normal controls (1.702 ± 0.531 at 1 mo and 1.676 ± 0.891 at 3 mo), while it was significantly lower than normal controls in both Group I and III animals. Body weight increase after Tx correlated with the number of transplanted islets and at 90 days, Group III rats showed less increase than Groups I and II (p < 0.05), while no significant differences in b.wt. were recorded between Group I and II. The findings indicate that intraportal islet Tx, injected repeatedly and in small doses, produced better metabolic effects than injection of the same total number of islets in a single dose.     

Glucose tolerance normalization following transplantation of pig pancreatic primordia into non-immunosuppressed diabetic ZDF rats

Pancreas or pancreatic islet transplantation in humans is limited by organ availability, and success of the latter is negatively impacted upon by tissue loss post-transplantation and limited potential for expansion of beta cells. A way to overcome the supply and expansion problems is to xenotransplant embryonic tissue. Previously, we have shown that beta cells originating from embryonic day (E) 28 (E28) pig pancreatic primordia transplanted into the mesentery of streptozotocin-diabetic (type 1) Lewis rats engraft without the need for host immune-suppression and normalize glucose tolerance. Here we show long-term engraftment of pig beta cells within liver, pancreas and mesenteric lymph nodes post-transplantation of E28 pig pancreatic primordia into diabetic ZDF rats, a model for type 2 diabetes. Porcine insulin is present in circulation after an oral glucose load. Glucose tolerance is normalized in transplanted ZDF hosts and insulin sensitivity restored in formerly diabetic ZDF males. Release of porcine insulin in vitro from tissue originating in transplanted rats occurs within 1 min of glucose stimulation characteristic of first-phase secretion from beta cells. Of potential importance for application of this transplantation technology to treatment of type 2 diabetes in humans and confirmatory of our previous findings in Lewis rats, no host immunosuppression is required for engraftment of E28 pig pancreatic primordia.     

Insulin independence following isolated islet transplantation and single islet infusions

OBJECTIVE: To restore islet function in patients whose labile diabetes subjected them to frequent dangerous episodes of hypoglycemic unawareness, and to determine whether multiple transplants are always required to achieve insulin independence. SUMMARY BACKGROUND DATA: The recent report by the Edmonton group documenting restoration of insulin independence by islet transplantation in seven consecutive patients with type 1 diabetes differed from previous worldwide experience of only sporadic success. In the Edmonton patients, the transplanted islet mass critical for success was approximately more than 9,000 IEq/kg of recipient body weight and required two or three separate transplants of islets isolated from two to four cadaveric donors. Whether the success of the Edmonton group can be recapitulated by others, and whether repeated transplants using multiple donors will be a universal requirement for success have not been reported. METHODS: The authors report their treatment with islet transplantation of nine patients whose labile type 1 diabetes was characterized by frequent episodes of dangerous hypoglycemia. RESULTS: In each of the seven patients who have completed the treatment protocol (i.e., one or if necessary a second islet transplant), insulin independence has been achieved. In five of the seven patients only a single infusion of islets was required. To date, only one recipient has subsequently lost graft function, after an initially successful transplant. This patient suffered recurrent hyperglycemia 9 months after the transplant. CONCLUSIONS: This report confirms the efficacy of the Edmonton immunosuppressive regimen and indicates that insulin independence can often be achieved by a single transplant of sufficient islet mass.     

Successful islet transplantation: continued insulin reserve provides long-term glycemic control

Clinical islet transplantation is gaining acceptance as a potential therapy, particularly for subjects who have labile diabetes or problems with hypoglycemic awareness. The risks of the procedure and long-term outcomes are still not fully known. We have performed 54 islet transplantation procedures on 30 subjects and have detailed follow-up in 17 consecutive Edmonton protocol-treated subjects who attained insulin independence after transplantation of adequate numbers of islets. Subjects were assessed pretransplant and followed prospectively posttransplant for immediate and long-term complications related to the procedure or immunosuppressive therapy. The 17 patients all became insulin independent after a minimum of 9,000 islets/kg were transplanted. Of 15 consecutive patients with at least 1 year of follow-up after the initial transplant, 12 (80%) were insulin independent at 1 year. In 14 subjects who have maintained demonstrable C-peptide secretion, glucose control has been stable and glycemic lability and problems with hypoglycemic reactions have been corrected. After 2 of the 54 procedures, some thrombosis was detected in the portal vein circulation. Five subjects had bleeding related to the percutaneous portal vein access procedures: three required transfusion alone, and in one subject, who had a partial thrombosis of the portal vein, an expanding intrahepatic and subscapular hemorrhage occurred while on anticoagulation, requiring transfusion and surgery. Elevated liver function test results were found in 46% of subjects but resolved in all. Complications related to the therapy have been hypercholesterolemia requiring statin therapy in 65%; a rise in creatinine in two patients, both of whom had preexisting renal disease; a rise in protein in four, all of whom had preexisting proteinuria; and antihypertensive therapy increased or started in 53%. Three of the 17 patients have required retinal laser photocoagulation. There have been no cases of posttransplant lymphoproliferative disorder or cytomegalovirus infection, and no deaths. The acute insulin response to arginine correlated better with transplanted islet mass than acute insulin response to glucose (AIR(g)) and area under the curve for insulin (AUC(i)), but the AIR(g) and AUC(i) were more closely related to glycemic control. The AUC(i) directly posttransplant was lower in those who eventually became C-peptide deficient. Our results, with a maximum follow-up of 34 months, indicate that prolonged insulin independence can be achieved after islet transplantation. There are some risks associated acutely with the procedure, and hypercholesterolemia and hypertension are treatable concerns on longer-term follow-up. All patients with persisting C-peptide secretion have had a resolution of both glycemic lability and problems with hypoglycemic reactions. Apart from the rise in serum creatinine in two subjects, no serious consequences of immunosuppressive therapy have been encountered. Islet transplantation is a reasonable option in those with severe problems with glycemic lability or hypoglycemia.     

Syngeneic islet transplantation into seminal vesicles of diabetic rats.

Pancreatic islet transplantation has been proposed as an attractive option for the treatment of type I diabetes. Transplantation into different sites has been investigated, among them those that are immuno-logically privileged (e.g., thymus, uterus, brain, anterior eye chamber, and testicle). Because of their characteristics, seminal vesicles could be considered as immunologically privileged organs, but there is no worldwide experience that can confirm it. The purpose of the present study is to assess the viability and functionality of islet transplantation into seminal vesicles of diabetic rats. One hundred ninety inbred adult male syngeneic Lewis rats were used as donors (n = 72), receptors (n = 36), and controls(n = 11). Diabetes was chemically induced through a single intraperitoneal injection of streptozotocin. Groups of 1200 purified islets were introduced in the right seminal vesicle of diabetic rats. Diabetic control rats were sham transplanted. Body weight and glycemia were monitored every 2 d. Of transplanted rats, 16.7% achieved a good function due to islet engraftment, while 30.6% achieved a partially good response, and 52.7% were considered as nonresponding. This is the first report about islet transplantation into seminal vesicles of diabetic animals. Our results indicate that islet transplantation into rat seminal vesicles is technically possible, and that islets can function normally after engraftment into the wall of the seminal vesicle.     

Exercise induces hypoglycemia in rats with islet transplantation

Recently, islet transplantation in patients with type 1 diabetes has had greater success than in the past, but the important question of whether the kinetics of islet secretion are able to accommodate the metabolic demands of special conditions such as exercise remains unanswered. Syngeneic rat islets (4,000 islet equivalents/rat) were transplanted into the liver, kidney, and peritoneal cavity (encapsulated or nonencapsulated) of rats with streptozocin-induced diabetes. Normoglycemic transplanted rats and age-matched controls were subjected to 30 min of moderate exercise on a treadmill 5 weeks after transplantation. Although control rats maintained near normoglycemia during and after exercise, the rats with islet transplants had significantly lower blood glucose levels. For the rats with islets in the liver, increased C-peptide levels were found at 30 min (790 +/- 125 and 1,450 +/- 250 pmol/l at 0 and 30 min, respectively; P < 0.01), whereas a decrease was found in controls and in rats with islets transplanted into the peritoneal cavity or under the kidney capsule. Moreover, increased glucagon levels were found after exercise in the rats with islets transplanted into the liver (62 +/- 6, 165 +/- 29, 155 +/- 27, and 97 +/- 13 pg/ml at 0, 30, 60, and 90 min, respectively; P < 0.05), whereas no changes in glucagon levels were observed in controls. In conclusion, moderate exercise caused hypoglycemia in rats with islet transplants in different sites including liver, kidney, and peritoneal cavity. C-peptide and glucagon responses to exercise were very different in rats with transplanted islets compared with controls. This islet dysfunction led to exercise-induced hypoglycemia.     

mPEG包裹大鼠胰岛在同种胰岛移植中的作用分析

目的探讨单甲氧基聚乙二醇(mPEG)对同种大鼠胰岛移植物存活的影响及作用机制。方法分离纯化Wistar大鼠胰岛,处理组胰岛用30%mPEG包裹,对照组不作处理,植入糖尿病SD大鼠的右后肢肌肉,空白对照组在相同部位注入生理盐水,动态监测血糖变化,病理切片检测移植物存活,通过淋巴细胞混合培养检测免疫排斥反应。结果mPEG包裹的胰岛存活时间明显延长[(75.8±4.5)d vs(7.2±1.1)d,P<0.05],免疫组化染色呈强阳性,对照组仅见少量阳性颗粒;处理组淋巴细胞刺激指数明显低于对照组[(1.27±0.22)vs(3.78±0.32),P<0.05)]。结论mPEG包裹大鼠胰岛可有效延长胰岛存活时间,其机制与免疫修饰作用有关。     

Dynamics of erythropoiesis following renal transplantation

We examined the temporal dynamics of the correction of anemia following renal transplantation in 65 recipients using a sensitive radioimmunoassay for erythropoietin to determine the effects of modern immunosuppressive agents, delayed graft function, and early acute rejection. Pretransplant mean erythropoietin (25.6 +/- 3.3 mU/ml) was only 25% of the expected value at the mean hematocrit of 27.2 +/- 0.7, and erythropoietin correlated positively with hematocrit (r = 0.37, P less than 0.05). Following onset of graft function, erythropoietin increased to 109 +/- 13 mU/ml and then decreased in a negative feedback fashion over the next several months. Delayed graft function was associated with delay in the assumption of this orderly process irrespective of the immunosuppressive regimen used. Cyclosporine A produced a biphasic response despite delayed graft function in recipients with underlying adult polycystic kidney disease. Correction of anemia required resumption of graft function. Onset of acute graft rejection within the first month posttransplantation (14 episodes in 11 patients) abrogated the hematopoietic response until the rejection was successfully reversed. We conclude that a major cause for the anemia of renal failure is subnormal production of erythropoietin. Following transplantation, anemia corrects in an orderly manner with restoration of the normal biofeedback process between erythropoietin and red cell mass. This process is delayed by failure of graft to function initially and interrupted by acute early rejection, re-commencing following successful reversal.     

Three cases of cytomegalovirus infection following pancreatic islet transplantation

Transmission of cytomegalovirus (CMV) is uncommon in patients transplanted with CMV-mismatched pancreatic islets, and CMV-seropositive recipients rarely experience reactivation of the virus or reinfection from a CMV-positive graft. This study describes three cases of CMV infection following islet transplantation for type 1 diabetes despite prophylaxis with valganciclovir. Further studies are needed to evaluate risk factors for CMV transmission and reactivation in this patient population.