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Background: We report long-term outcomes and side effects after transplantation for hepatocellular carcinoma (HCC) using de novo, sirolimus-based immunosuppression (IS)., Methods: A total of 70 patients with HCC (mean age: 54.4+/-7 years, female/male: 12/58) were transplanted and included in the study. Immunosuppression included de novo sirolimus, low-dose calcineurin inhibitor for 6 to 12 months, with short-course (3 months) or no steroids., Results: After 49 months-median follow-up, eight patients have experienced an HCC recurrence, 2 of 34 when Milan criteria were respected (6%) and 6 of 36 when beyond Milan criteria (17%). One- and 4-year tumor-free survivals were 85 and 73%, when Milan criteria were respected and 82% and 75% when they were not, respectively. (P=0.9). After recurrence, mean survival was 23+/-28 months. Half (35 of 70) of the patients experienced a rejection. Incisional hernia (24 of 70, 34%), wound infection (12 of 70, 17%), anemia (39 of 70, 56%), leucopenia (39 of 70, 56%), high triglyceride (43 of 70, 61%), and cholesterol (28 of 70, 40%) levels and mouth ulcers (20 of 70, 29%) were among the most frequent complications. No hepatic artery thrombosis was observed., Conclusions: These data suggest that de novo sirolimus-based immunosuppression is associated with satisfactory outcomes after transplantation, even in selected patients beyond Milan criteria. The protocol has proven safe, with an acceptable side-effect profile. This study supports the conduct of larger randomized trials investigating sirolimus after transplantation for HCC.

The utility of formulas estimating glomerular filtration rate (GFR) in liver transplant patients has not been well described. The purpose is to determine the correlation between the radionuclide GFR (rGFR) with formulas commonly used to estimate GFR. This study represented a secondary outcome measure of a multicenter randomized trial comparing the effectiveness of two immunosuppressive regimens in adult liver transplant patients (n=148). A total of 68 rGFR were measured, 33 at baseline and at 35 at three months after transplantation. GFR was estimated using 1/Scr and Cockcroft-Gault, MDRD, and Nankivell equations. At both time points assessed, all correlations with rGFR were poor: 1/Scr (r: 0.17 and 0.25), Cockcroft-Gault (r: 0.31 and 0.35), MDRD (r: 0.27 and 0.35), and Nankivell (r: 0.11 and 0.20). Accepted formulas to estimate GFR correlate poorly with rGFR during the first three months after liver transplantation. Recalibration of these formulas is required to improve the estimation of GFR in liver transplant patients.

Background: The recent results of clinical islet transplantation have improved substantially with the introduction of a more potent but less diabetogenic immunosuppressant protocol. The successful development of this protocol was based in part on the outcomes of studies reported herein, addressing the diabetogenic potential of a series of immunosuppressant agents used alone or in combination in a canine islet autograft model. Although it is recognized that failure to achieve long-term insulin independence in human islet allotransplantation has been multifactorial, with low engraftment mass, acute or chronic rejection, autoimmune recurrence, loss of islet-acinar integrity, heterotopic site, denervation, and insulin resistance all being implicated to varying degrees, avoidance of diabetogenic immunosuppression has been pivotal to the enhanced outcomes of clinical islet transplantation. We here explore the effects of clinically relevant doses of cyclosporine or tacrolimus when given alone or in combination with glucocorticoids on long-term canine islet autograft function., Method: Dogs (n=8) underwent total pancreatectomy, islet isolation, and intrasplenic autotransplantation and were normoglycemic with stable long-term graft function 3 months to 8 years posttransplant. The frequently sampled intravenous glucose tolerance test (FSIGT) was performed predrug (baseline), at 1 month of therapy (on drug), and again 1 month after withdrawal of therapy (postdrug)., Results: Monotherapy treatments with low- or high-dose prednisone, Neoral, or tacrolimus had minimal impact on islet autograft function. The combination of Neoral and prednisone led to a marked impairment in glucose decay (25% decline from 1.77+/-0.2 to 1.24+/-0.2, P<0.05), without significant change in insulin responsiveness or glucose effectiveness. However, insulin sensitivity was markedly impaired while on therapy (7.10+/-1.2 to 3.10+/-0.5, P<0.01). Importantly, glucose decay and insulin sensitivity failed to return to baseline after withdrawal of therapy. The combination of tacrolimus and glucocorticoids led to permanent and irreversible diabetes in all recipients (n=6, P<0.001). Similar treatment of healthy control dogs led to a 44% decrease in glucose decay (P<0.01)., Conclusions: Immunosuppression must be specifically tailored for islet transplantation and be glucocorticoid free if insulin independence is to be sustained clinically.

Background: Success of human pancreatic islet isolation depends largely on the techniques used during pancreas procurement and the quality of the gland. Warm ischemia of the pancreas of any duration during organ procurement may be detrimental to subsequent islet yield and functional viability of the islets. The aim of this study was to correlate pancreas procurement technique with the core temperature within the pancreas during in situ procurement to the recovery and in vitro function of isolated human islets., Methods: Alternative pancreata were recovered from human cadaveric organ donors with needle-point myocardial temperature probes placed within the body of the pancreas. After vascular flushing with University of Wisconsin organ preservation solution, the first group of human pancreata were removed after standard liver and kidney procurement followed by in situ dissection of the pancreas. The second group of pancreata were removed using a similar protocol, except that the lesser sac was opened (and the spleen was mobilized to the midline) rapidly at the time of aortic cross-clamp. An additional 3-4 L of ice slush solution was placed behind and in front of the pancreas and replenished throughout the procurement process for the liver and kidneys. Immediately after recovery, in both groups, the pancreas was placed in cold University of Wisconsin solution and transported to the islet isolation laboratory for processing. Islets were isolated using a standard protocol of Liberase perfusion via the duct, gentle mechanical dissociation, and Ficoll purification., Results: The absence of in situ ice-chilling of the entire pancreas during liver and kidney procurement caused the core pancreas temperature to rise to a peak of 18.2 degrees C; but when the pancreas was surrounded and replenished with iced saline slush, the core pancreas temperature was maintained at approximately 4 degrees C. The lower pancreas temperature correlated with a significantly higher recovery of islets in the group of pancreata surrounded with iced saline (223+/-35x10(3) IE vs. 103+/-26x10(3) IE; mean +/- SEM). Functional ability of the islets was also significantly improved in glucose static incubation, with a stimulation index of 4.4+/-0.3 in the iced-saline pancreas group versus 3.0+/-0.4 in the standard procurement group (P<0.05, paired t test)., Conclusions: Procurement technique and the maintenance of a low temperature of the pancreas are critical to subsequent islet recovery and function. Maintaining a low pancreas temperature during procurement through the addition and replenishment of iced saline slush surrounding the anterior and posterior aspects of the pancreas greatly improves islet yield and functional viability of the isolated islets and is essential for success in clinical islet transplantation.

Background: Glutamine (gln)-supplemented University of Wisconsin (UW) solution improves overall small bowel (SB) preservation. Sustained gln metabolism in a system devoid of hepatic detoxification will necessarily result in the accumulation of pH active end products leading to nonphysiologic pH shifts. We hypothesized that simultaneous addition of N,N-bis[2-hydroxyethyl]-2-aminoethane sulfonic acid (BES), a known buffering agent, would potentiate the beneficial effect of gln supplementation by addressing the fundamental metabolic principle of pH homeostasis., Methods: Sprague-Dawley SB rats were administered a vascular flush with one of four solutions: UW; UW+90 mM BES (UWB); UW+2% gln (UWG); or UW+2% gln+90 mM BES (UWBG). Indices of energetics, barrier function, gln catabolism, and histology (light and electron microscopy) were assessed over a 10-hr cold storage time course., Results: Superior gln utilization in the UWBG group was indicated by elevated levels of key catabolites (glutamate, aspartate, glycine, ammonia). The addition of BES and gln resulted in significantly higher levels of all energetic parameters (ATP, total adenylates) at 10 hr compared with UW, UWB, and/or UWG. Barrier function was markedly improved after 10 hr storage in the UWBG group; mannitol permeability was 169 nmol/cm2/hr versus 572 and 445 nmol/cm(2)/hr (for UW and UWG, respectively). Histologic injury at 10 hr was 5.5, 7.5, and 8 (Park's grade) for UWBG, UWG, and UW. Ultrastructural damage was markedly reduced with UWBG, as assessed by grade of mitochondria damage., Conclusion: This study strongly supports that the beneficial effects of gln-enriched UW solution can be amplified when combined with an effective buffering agent such as BES.

Background: We report a case of bile leak from an accessory duct of Luschka during cholecystectomy during liver transplantation., Methods: Radiological findings suggested that the collection was septated. An intra-operative cholangiogram was obtained by cannulation of the accessory hepatic duct., Results: An infected biloma with Clostridium perfringens was drained surgically. The bile leak that emanated from the gall bladder fossa was found to communicate with an accessory right hepatic duct draining a segmental duct in the right liver lobe. The bile leak resolved completely after direct suture of the accessory duct., Conclusions: Excessive use of electrocautery to the liver bed during donor cholecystectomy may injure subcapsular ducts in the gallbladder fossa. In liver transplantation, dissection should be kept close to the serosal lining of the gall bladder, preserving the areolar tissue in the gall bladder bed, to avoid injury to the duct of Luschka.

Background: The inability to diagnose early rejection of an islet allograft has previously proved to be a major impediment to progress in clinical islet transplantation. The need to detect early rejection will become even more relevant as new tolerance-inducing protocols are evaluated in the clinic. We explored three novel approaches toward development of early diagnostic markers of islet rejection after islet allotransplantation., Methods: (a) Canine islet allograft transplant recipients were immunosuppressed for 1 month, then therapy was withdrawn. Serum glutamic acid decarboxylase antigen (GAD65), an endogenous islet protein, was monitored daily with a CO2 release assay. (b) Rodent islets were genetically engineered to express a unique foreign protein (beta-galactosidase) by using adenoviral vectors, and after allograft transplantation, the viral-specific protein was measured in serum using optical luminescence. (c) Rodents receiving islet allografts were immunosuppressed temporarily, and daily glucose tolerance tests were followed until graft failure occurred., Results: (a) Although serum monitoring of GAD65 antigen demonstrated elevated levels preceding loss of graft function in preliminary studies, the effect was not reproducible in all animals. (b) Genetically engineered rodent islets demonstrated normal insulin kinetics in vitro (insulin stimulation index 2.57+/-0.2 vs. 2.95+/-0.3 for control islets, P=ns), and purified viral protein products had a stable half-life of 8 hr in vivo. After islet allotransplantation, there were two peak elevations in serum viral proteins, confirming that an intra-islet "sentinel signal" could be detected serologically during acute rejection. There was no lead-time ahead of hyperglycemia, however. (c) Daily sequential intravenous glucose tolerance (IVGT) tests demonstrated evidence of allograft dysfunction (decline in KG) with a 2-day lead time to hyperglycemia (2.58+/-0.3 vs. 1.63+/-0.2%/min, respectively, P<0.001), with an accuracy of 89%, sensitivity of 78%, and specificity of 95%., Conclusions: Of the three diagnostic tests, metabolic assessment with an abbreviated IVGT was the most effective method of demonstrating early islet dysfunction due to rejection.

Background: Although cyclosporine (CsA) has been a mainstay in liver transplantation immunosuppression the original formulation [Sandimmune (SIM)] has variable absorption, particularly in children. Neoral is a new formulation of CsA that may have improved biovailability that would be advantageous in children. This study was undertaken to assess the pharmacokinetics (PK) and effects on outcome of Neoral versus Sandimmune (SIM) in primary pediatric liver transplant recipients., Methods: Thirty-two patients were randomized to receive Neoral (17 patients) or SIM (15 patients) in the early posttransplant period (days 1-7) in a double-blind fashion. Intravenous CsA was instituted immediately posttransplant followed by Neoral or SIM as soon as the patient was tolerating oral fluids (days 1-7). PK were compared after the first dose (1-7 days), 3 weeks, and 6 and 12 months posttransplant. In addition, side effects, effect of age and food on absorption, and rejection episodes were assessed by intent to treat analysis. Notable characteristics of this study include the use of a central laboratory for all sample analyses and the assessment of renal function using radioisotopic evaluation of glomerular filtration rates., Results: At baseline the two groups were comparable. Neoral resulted in higher peak levels of CsA and total drug exposure with comparable time to peak drug levels at days 1-7 and week 3. This trend was maintained at 6 and 12 months. Time on i.v. CsA was reduced in the Neoral group (8.4 vs. 11.1 days) and the weight adjusted daily dose of SIM required to achieve target trough levels was about 2-fold more than Neoral from day 22 onward. In addition, biopsy proven and treated and steroid-resistant rejection episodes were fewer in the Neoral group (6 vs. 12; P=0.01 and 1 vs. 8: P=0.004, respectively). Side effects were comparable in both treatment groups., Conclusions: Neoral was well tolerated and had greater biovailability than SIM without any increase in the incidence of side effects. In addition fewer episodes of rejection were observed with Neoral versus SIM. We conclude that Neoral is the CsA formulation of choice for use in pediatric liver transplant recipients.

Background: This study was designed to investigate the effects of a modified University of Wisconsin (UW) solution supplemented with one of four buffering agents (histidine, bicine [N,N-bis(2-hydroxyethyl)glycine], tricine [N-tris(hydroxymethyl)methylglycine], and Tris) on liver metabolism during cold ischemic storage., Methods: Rat livers were flushed and stored for a maximum period of 24 hr at 4 degrees C, and tissue energetics, substrate, and anaerobic end-products were assessed; the group exhibiting the best results during storage was recovered in a 60-min period of warm reperfusion. Relative buffering capacities of the experimental solutions (measured over physiological pH range, in mM H+/L) were: UW, 4.1; histidine+UW, 9.8; Tris+UW, 19.0; bicine+UW, 22.5; tricine+UW, 26.8., Results: In the UW group, ATP levels dropped rapidly over the first 4 hr; 1.0 micromol/g (40% of initial) remained after 4 hr of storage. By 2 hr, ATP levels in bicine- and tricine-treated groups were 0.5 and 1.1 micromol/g greater than in the UW-stored livers and by 10 hr, ATP in bicine-treated livers was twofold that of the control (UW) group. Total adenylate levels also reflected a superior elevation of cellular energetics; even after 24 hr, quantities were 1.4 and 2.0 micromol/g higher than the UW group in bicine- and histidine-supplemented organs. The increase in energetics occurred as a result of increased flux through the major anaerobic energy-producing pathway, glycolysis. The glycolytic rate was significantly greater at storage times > 10 hr with solutions supplemented with bicine, histidine, and tricine. Final values for net lactate accumulation over the entire 24-hr storage period were: UW, 10.1 micromol/g; histidine, 14.3 micromol/g; bicine, 15.2 micromol/g; tricine, 13.8 micromol/g. Activities of glycogen phosphorylase revealed that the activity of this enzyme dropped by 50% within 2 hr of storage in UW. However, histidine and bicine supplementation resulted in a substantial elevation of phosphorylase "a" over 4 hr and 10 hr, respectively. The best buffer of the four examined in this study was bicine; energetics, glycolytic flux, and patterns of adenylate regeneration upon reperfusion were markedly superior to modified UW solution., Conclusion: The results of this study suggest that supplementing the "gold standard" UW solution with an additional buffering agent (in order of efficacy: bicine>tricine>histidine) may improve the metabolic status of livers during clinical organ retrieval/storage.

Liver transplantation for endstage hepatitis B virus (HBV) infection has been associated with survival inferior to that of liver transplantation in other chronic liver diseases due to HBV reinfection of the graft. Lamivudine is a new nucleoside analog with potent antiviral effects against hepatitis B. Our aim was to test its efficacy when used pre- and posttransplantation in HBV-DNA positive patients with endstage liver disease. Patients received oral lamivudine 100 mg daily both pretransplant and posttransplant. Viral serology, serum and tissue HBV-DNA and liver histology were assessed sequentially. Five consecutive patients with endstage hepatitis B were entered into the trial. Serum HBV-DNA was cleared pretransplant in all patients. Three of four transplanted patients cleared HBeAg and HBsAg postoperatively, whereas all four became negative for serum HBV-DNA (dot-blot and PCR). Liver biopsies were negative for HBV-DNA by PCR in 3 of 4 cases. Lymphocytes were negative for HBV-DNA by PCR in all cases. With follow-up of 3, 14, 16, and 26 months, two patients have normal liver enzymes and normal liver histology and two have developed recurrent hepatitis B. No significant side effects were seen. This pilot study shows that lamivudine can effectively inhibit hepatitis B virus in cirrhotic patients pretransplant and posttransplant. A lamivudine resistant mutant developed in two patients. Transplant recipients with actively replicating HBV related cirrhosis may achieve a good outcome after liver transplantation using lamivudine, but viral resistance is likely to be a significant problem.

Chylous ascites is the accumulation of chylomicronrich lymphatic fluid within the peritoneal cavity. It is a rare complication of retroperitoneal surgery, and may occur spontaneously in 0.5% of patients with cirrhosis. Its management is controversial, and despite a variety of treatment options with limited efficacy, the course is usually indolent. In this article, we report a case of rapid resolution of chylous ascites after liver transplantation following 10 days of treatment using somatostatin analog (Octreotide, 100 micrograms sc. t.i.d.) and total parenteral nutrition (TPN). A 55-year-old man underwent liver transplantation for hepatitis C cirrhosis, and developed an infected chylous fistula on the 10th day. Treatment by fasting, TPN, and somatostatin analog resulted in a rapid falloff in fistula output, with complete resolution of ascites within 2 days. This is the first report, to our knowledge, of somatostatin analog and TPN used in combination for rapid and successful closure of a chylous fistula.

The objective of this study was to analyze the impact of rapamycin and/or cyclosporine on the metabolic efficiency of intrasplenic islet autografts in dogs. An insulin modified frequently sampled intravenous glucose tolerance test was carried out before, on the last of 30 days treatment with drug and 30 days after cessation of drug therapy in dogs with stable function 1 to 7 years after total pancreatectomy and intrasplenic islet autografting. Analyses were performed for glucose clearance, insulin release, insulin sensitivity, and other variables. Rapamycin treatment was associated with increased glucose clearance, increased total and stimulated insulin release in response to glucose, and increased fasting plasma insulin level, as well as reduced insulin clearance. Cyclosporine at 300 micrograms/L had little impact on the measured variables. Treatment with rapamycin and cyclosporine showed a similar (although less-marked) pattern of changes to rapamycin alone. Rapamycin, with or without concomitant cyclosporine, was not associated with adverse impact on islet function or glucose metabolism in this canine model of pancreatic islet transplantation.

In an attempt to reduce the variability in the yields of human islets isolations and to identify donor factors that were potentially deleterious, we retrospectively reviewed 153 human islets isolations in our center over a 3-year period. Isolations were performed using controlled collagenase perfusion via the duct, automated dissociation, and Ficoll purification. Factors leading to successful isolations (recovery of >100,000 islet equivalents at a purity >50%) were analyzed retrospectively using univariate and multivariate analysis. Critical factors in the multiorgan cadaveric donors that were identified using univariate analysis included donor age (P<0.01), body mass index (BMI)(P<0.01), cause of death (P<0.01), and prolonged hypotensive episodes (systolic blood pressure <90 mmHg or mean arterial pressure <60 mmHg for > 15 min) requiring high vasopressors (>15 microgram/kg/min dopamine or >5 microgram/kg/min Levophed) (P>0.01). Independent analysis of 19 donor variables using multivariate logistic stepwise regression showed six factors were statistically significant. Odds ratio (OR) showed that donor age (OR 1.1, P<0.01), local procurement team (OR 10.9, P<0.01), and high BMI (OR 1.4, P<0.01) had a positive correlation with islet recovery. In contrast, hyperglycemia (all blood glucose >10 mmol/L) (OR 0.63, P<0.01), frequency and duration of cardiac arrest (OR 0.7, P<0.01), and increased duration of cold storage before islet isolation (OR 0.83, P<0.01) had negative correlation. Using these combinations of factors, the prediction of success was 85% accurate. By donor age, success was 13% for 2.5- to 18-year-old donors (n=23), 37% for 19- to 28-year-old donors (n=30), 65% for 29- to 50-year-old donors (n=70), and 83% for 51- to 65-year-old (n=29) donors. However, when vitro function was assessed by perifusion, the insulin secretory capabilities of islets isolated from the >50-year-old donor group was significantly reduced as compared with the 2.5- to 18-year-old group (P<0.02). Multiple regression analysis using postdigestion and postpurification islet recovery as outcome variables identified BMI, procurement team, pancreas weight, and collagenase digestion time factors tht can affect the recovery of human islets. Locally procured pancreases and donors with elevated minimum blood glucose levels were identified as factors that affect the insulin secretory capabilities of the isolated islets. This review of parameters suggests an improved approach to the prediction of successful islet isolation from human pancreases. Selection of suitable pancreases for processing may improve consistency in human islet isolation and thereby decrease costs.

Tacrolimus (FK506) is at present the mainstay of immunosuppression for small intestinal transplantation. This study investigates the effects of chronic treatment with varying dosages of tacrolimus on animal well-being, weight gain, intestinal permeability, and the active transport of nutrients as measured by in vitro studies quantifying glucose flux. The effect of acute treatment with high-dose tacrolimus on glucose flux was also investigated. In the chronic studies, juvenile male Lewis rats were given tacrolimus in a dosage of 0.1 mg/kg, 0.5 mg/kg, and 2 mg/kg q. second day by subcutaneous injection for five weeks. In the acute studies, animals were treated with 2 mg/kg given q. 24 hr [mult] 48 hr, 24 hr and 12 hr prior to sacrifice. In the acute treatment groups, tacrolimus caused no change in glucose flux. In the chronically treated animals, FK506 levels were within the clinically relevant range. Chronic treatment with 0.5 and 2 mg/kg caused a significant reduction in weight gain. These same groups of animals had a significant increase in intestinal permeability as measured by absorption of 99Te-DTPA. Glucose flux was affected in all chronically treated groups, with net flux increasing in the jejunum and decreasing in the ileum. These findings show that chronic treatment with low-dose tacrolimus is well tolerated, but in higher doses there are significant effects in intestinal permeability and nutrient uptake, and animal weight gain. We suggest that these changes are due to alterations in intestinal permeability that do not appear to be mediated by an acute drug effect and more likely represent chronic changes, possibly from alterations in gene expression. These findings suggest that further studies regarding the effects of tacrolimus on nutrient transport, intestinal permeability, and the known immunologically related functions of tacrolimus should be done.

The combination of pharmacokinetic and pharmacodynamic (measurement of the biological effect) monitoring of immunosuppressive drugs provides a method for the optimization of drug dosing. We chose to investigate this using mycophenolic acid (MPA), an immunosuppressive drug that mediates its effect by the inhibition of inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de novo biosynthesis of purines. Using an assay developed for measurement of IMPDH activity in whole blood, the concentration required for 50% inhibition of IMPDH activity was approximately 200 mg/L (58 +/- 8.3% for whole blood [n = 6] and 55 +/- 10.0% for isolated lymphocytes). To ascertain the relationship between MPA concentration and IMPDH inhibition in vivo, dogs were administered a single dose of mycophenolate mofetil, the pro-drug of MPA, at 20 or 40 mg/kg orally. Pharmacokinetic analysis revealed that the Cmax of the 40-mg/kg group was statistically greater than that of the 20-mg/kg group (P < 0.05). There were no statistical differences in the other parameters investigated (area under the curve, beta half-life, mean residence time, volume of distribution at steady state, and clearance) between the two treatment groups. The half-life was calculated at approximately 8 hr for both dose groups. There was also substantial variability among the dogs in the absorption and clearance of MPA. An inverse relationship was found between the MPA concentration and IMPDH. Maximal inhibition of IMPDH activity of 30-40% occurs approximately 2-4 hr after dosing, followed by a gradual restoration in enzyme activity. After 24 hr, there is an increase in IMPDH activity that exceeds the pre-dosing levels in some cases by 3-fold. Evaluation of the pharmacokinetic and the pharmacodynamic responses to MPA in the canine model suggests that the drug should be administered ever 8 hr to optimize its immunosuppressive efficacy. This combined approach can be used for optimization of doses of this and other immunosuppressive drugs.

The small intestine and its mesentery contain a large amount of lymphoid tissue that can mediate graft-versus-host disease (GVHD) in small intestinal transplant recipients. To assess the impact of surgical technique and the retention of the recipient's small intestine on GVHD intensity, 12 adult Lewis rats received heterotopic small bowel transplants and 12 received orthotopic small bowel transplants from Brown Norway donors. Twelve Lewis to Lewis heterotopic small-bowel-transplanted animals served as the control group. All recipients were given cyclosporine (10 mg/kg/alternate days) subcutaneously. The parameters followed were: weight gain and feed intake; clinical signs of GVHD; relative spleen weight; popliteal lymph node enlargement assay; and histological evaluation of spleen, liver, skin, native intestine, and transplanted intestine. According to the clinical scoring system, heterotopically transplanted animals were found to have a more severe GVHD than the orthotopic group. There were statistically significant differences between the relative spleen weights of the heterotopic transplant group and the control group (P = 0.001, 0.004, and 0.007 on days 7, 14, and 21, respectively) and between the heterotopic and orthotopic groups at 7 days (P = 0.037). Lymph node enlargement assays were statistically different between heterotopic and orthotopic groups (P = 0.019, 0.020, and 0.007 on days 7, 14, and 21, respectively). Histological evaluation of skin biopsy specimens also demonstrated that GVHD was indeed more severe in the heterotopic transplanted group when compared with orthotopically transplanted animals. These findings confirm that retention of the native small intestine in the heterotopic intestinal transplant model significantly increases the severity of GVHD following transplantation.

We have retrospectively evaluated islet isolation records collected from 230 consecutive adult pancreases with 146 pancreases fulfilling all criteria for our evaluation. Fifty-six pancreases procured by our local organ procurement team (33 before in situ vascular flush and 23 after in situ vascular flush with University of Wisconsin [UW] solution) were compared with 90 pancreases received from distant centers that were shipped in UW solution after in situ vascular flushing. Cold storage of 3-26 hr preceded islet isolation using collagenase digestion and Ficoll purification. Recoveries of islets were assessed by duplicate counts of dithizone-stained aliquots before and after purification. Isolations were considered successful if > 100,000 viable islets (islet equivalents to 150 microns) were recovered after purification. Islet function was assessed by in vitro glucose-stimulated perifusion and islets were considered viable if the stimulation index (glucose stimulated over basal insulin secretion) was > 2. Eighty-three percent of the isolations from locally procured, UW-flushed pancreases were successful, as compared with 86% for pancreases that were stored for 3 to 8 hr, 73% for 8 to 16 hr of storage, and 38% for isolations following > 16 hr of cold storage. Increasing the duration of cold storage prior to islet isolation was associated with an increased proportion of failed isolations and decreased measures of islet viability. The actual numbers of islets per gram of pancreas before and after purification were significantly reduced if the hypothermic cold storage was > 16 hr. In vitro viability of isolated islets during glucose-stimulated perifusion showed a higher percentage of viable islets from pancreases with shorter durations of cold storage. For pancreases with > 16 hr of cold storage prior to islet isolation, islet viability was significantly reduced. We conclude that, with the current methods available to recover and store cadaver donor pancreases and the methods currently used to isolate human islets, yields of purified islets decline significantly from human pancreases that have been subjected to cold storage of > 16-18 hr.

Previous studies in rodent and canine animal models have suggested a detrimental impact on islet recovery and function when pancreas excision is preceded by in situ vascular flushing with cold preservation solutions. We studied the efficacy of islet isolation from 19 consecutive cadaver pancreases procured alternately by initial pancreatectomy before in situ flush (group 1, our standard procurement technique, n = 9) or pancreas removal following in situ vascular flushing with cold University of Wisconsin solution (group 2, n = 10). Once procured, pancreases were weighed, the main pancreatic duct was cannulated, and 150 ml of collagenase solution was injected. The pancreases were transported to the isolation laboratory and processed within 2 hr. Islets were counted and sized after dithizone staining, and the islet equivalents were calculated. Aliquots of isolated islets were cryopreserved using established techniques. Islet function of both freshly isolated and frozen-thawed islets was assessed using a glucose-stimulated perifusion system. Significantly more pancreas was harvested after University of Wisconsin flush (90.6 +/- 6.9 g for group 1 versus 66.7 +/- 4 for group 2, P < 0.05). The quantity of islets per gram of processed pancreas released during enzymatic digestion from each of the experimental groups did not differ significantly (4.5 +/- 0.6 x 10(3) islet equivalents per gram for primary pancreatectomy versus 4.0 +/- 0.4 x 10(3) University of Wisconsin flush). Similarly, following Ficoll purification, the overall yields of islets did not differ significantly. Total islet yield in the primary pancreatectomy group was 181 +/- 25 x 10(3) islet equivalents (2.7 +/- 0.3 x 10(3) IE/g) versus 217 +/- 41 x 10(3) for the University of Wisconsin flush group (2.9 +/- 0.8 x 10(3) islet equivalents/g; P not significant). No differences were observed in in vitro viability. Perifusion stimulation indexes (peak/basal insulin release) were 5.9 +/- 1.3 for group 1 and 7.1 +/- 1.5 for group 2. These results conflict with published results in animal models and indicate that large numbers of viable islets can be recovered from cadaver pancreas utilizing either procurement technique. The decreased operating time, simplicity, and safety favor the use of total pancreatectomy after limited in situ vascular flushing as the method of choice for pancreas procurement for subsequent islet isolation.

We studied the survival of 5 groups of apancreatic mongrel dogs that received 30 days of treatment with CsA adjusted to 300 micrograms/L, rapamycin (0.05 mg/kg/day), both, or no immunosuppression after intrasplenic allotransplantation with purified pancreatic islets. Autografts survived indefinitely. Neither CsA nor rapamycin alone at low doses showed significant increase in islet allograft survival: 6.2 +/- 1.7 and 5.0 +/- 1.1, respectively, versus 3.4 +/- 1.0 days in controls. Dogs treated with low doses of both CsA and rapamycin demonstrated prolongation of graft function to 23.6 +/- 13.2 days (P < 0.05). These findings support synergism between these 2 agents, especially as CsA was not shown to increase trough rapamycin blood concentration when given together. In the combined treatment group, a significant (r = 0.90, P < 0.001) relationship was found between rapamycin blood levels and graft survival. Animals having trough rapamycin concentrations > 10 micrograms/L had significantly (P < 0.05) prolonged graft survival, which suggests that dosing of rapamycin according to blood levels may optimize the effectiveness of the drug. Given at these low doses, combination CsA and rapamycin gave no evidence of adverse effects as measured by hepatic and renal function tests, histology, or electron microscopy.

We performed an in vivo and in vitro dose-response study of the novel immunosuppressive macrolide antibiotic rapamycin looking at murine islet allograft survival, impact on glucose homeostasis, and possible tissue toxicity. A total of 300 islets were isolated from CBA/J mice (H-2k) and transplanted beneath the renal capsule of streptozotocin-induced diabetic BALB/c (H-2d) recipients. Seven groups of allografted mice received intraperitoneally for 7 days post-transplant: no immunosuppression (n = 8); vehicle only (carboxymethyl-cellulose) (n = 6); or rapamycin at dosages of 0.05 (n = 8), 0.1 (n = 8), 0.3 (n = 8), 1.0 (n = 8), or 5.0 (n = 6) mg/kg/day. Blood glucose was monitored on alternate days, with graft failure defined by the first day of persistently high blood glucose (> 14 mmol/L). The 0.1 and 0.3 mg/kg/day groups showed statistically significant prolongation of diet allograft survival (P < 0.01) when compared to the controls and vehicle-treated mice. Three mice in both the 0.1 and 0.3 mg/kg/day groups and one mouse in the 0.05 mg group reached 100 days normoglycemia and, following nephrectomy of the islet-bearing kidney, returned to hyperglycemia. The 0.05, 1.0, and 5.0 mg/kg/day groups showed no statistically significant prolongation of graft survival. In addition, the higher dosage (1.0 and 5.0 mg/kg/day) groups had erratic blood glucose control. Histologically, there was no evidence of toxicity seen in any of the multiple organ samples. In the in vitro analysis, BALB/c (H-2k) islets cultured in either 0, 10, 30, or 100 ng/ml rapamycin had no significant differences in insulin secretion following a 24-hr culture period; however, there was a significant deterioration in glucose stimulated insulin release after 72 hr culture at high rapamycin concentration (100 ng/ml). Rapamycin significantly prolonged murine islet allograft survival. At doses 10 to 50 times the effective antirejection dosage, we demonstrated adverse impact on glucose homeostasis without histological evidence of end-organ toxicity. We also demonstrated an adverse impact on insulin release in vitro following prolonged culture (72 hr) in a high concentration of rapamycin.

Cryopreservation is an effective method of islet storage that can facilitate clinical trials of islet transplantation. In the present study we examined the effect of cryopreservation on the survival of islet allografts and the quantity of islet MHC antigen expression. Islets isolated from CBA/J (H-2k) mice were transplanted into streptozotocin-induced diabetic BALB/c (H-2d) mice treated with or without antilymphocyte serum (ALS). Frozen/thawed (F/T) grafts were cooled slowly to -40 degrees C, stored at -196 degrees C, and thawed rapidly. Fresh and F/T isograft controls reversed diabetes promptly and maintained normoglycemia > 100 days. Allografts of fresh and F/T islets induced normoglycemia initially, but graft failure ensued at 18.2 +/- 1.5 and 16.4 +/- 1.9 days, respectively. ALS treatment prolonged allograft survival significantly to 35.3 +/- 3.9 and 37.5 +/- 6.3 days for fresh and F/T islets, respectively. Following cryopreservation, the quantity of class I antigen expression was reduced by 40%, while the quantity of class II expression was variable. These data indicate that murine islet MHC class I expression is reduced after cryopreservation. This decrease was not associated with altered survival of allogeneic grafts.

The indications for using cyclosporine are expanding rapidly beyond immune suppression for transplantation. We have previously described reduced active glucose uptake by small bowel following CsA treatment in rats. This study examined the effect of varying the dose and route of administration of CsA on bowel function. Male Lewis rats were given CsA via subcutaneous injection at doses of 5 mg/kg or 30 mg/kg on alternate days, or orally via gavage at 0 (control solvent oil), 7.5 mg/kg, or 30 mg/kg daily. Weight gain and feed intake were followed for 1 month when a balance study was performed to quantify in vivo nutrient absorption from the feed. In vitro studies of glucose and fatty acid uptake studies were then performed. Weight gain was reduced by high-dose CsA whether given orally or by subcutaneous injection. Oral CsA reduced in vivo fat and energy absorption from the diet, and all doses and routes of administration of CsA caused a reduction in both active glucose uptake and passive fatty acid absorption by the bowel in vitro. Thus, CsA has significant effects on bowel function in the normal rat. We suggest that further studies are indicated to determine the effects of CsA in man, especially in conditions with already-impaired bowel function.

This study tests the hypothesis that small bowel transplantation alters the function of the intestine. The function of the small intestine was investigated after syngeneic (BN----BN or Lew----Lew) and fully allogeneic (BN----Lew) orthotopic total small intestinal transplantation (SIT) using a two-stage model. All animals were treated with cyclosporine A throughout the 60-day study period. Syngeneic transplantation reduced weight gain in the (BN----BN) rats, but not in the (Lew----Lew) animals. Allogeneic transplantation caused a reduction in weight gain for the first 30 days posttransplantation, which may have been associated with graft-versus-host disease. Thereafter, the rate of growth of allogeneic SIT animals was normal. Dietary fat absorption was reduced in all groups of transplanted animals. Intestinal permeability to mannitol and polyethylene glycol 400 (PEG-400) was increased by syngeneic transplantation in all groups, with further permeability increases to mannitol, lactulose, PEG-400, and 51Cr-EDTA after allogeneic SIT. The glucose-stimulated intestinal short circuit current was reduced by both syngeneic and allogeneic SIT, but the maximal active transport rate for glucose uptake was increased, as was the passive uptake of fatty acids. These functional alterations were not associated with changes in intestinal morphology or evidence of rejection. These findings demonstrate that: (1) SIT results in significant changes in the transport characteristics of the bowel, but these have a minimal impact on the well-being of the animal overall; (2) SIT induces an increase in intestinal permeability to mannitol and PEG-400, with a further increase in permeability to all markers following allogeneic SIT; (3) following SIT, and the immune events associated with allogeneic SIT, significant adaptation of the transplanted intestine occurs. We suggest that denervation of the small intestine after SIT is the underlying cause of the changes observed.

Immunomodulation of rodent islets can significantly prolong allograft survival. We utilized a murine model of primary islet transplantation to study the relationship between allograft survival and the quantitative pretransplant expression of class I and II MHC antigens in freshly isolated CBA/J islets, and islets subjected to 37 degrees C tissue culture, brief culture at 7 degrees C, or exposure of the donor to lipopolysaccharide. Seven-day culture resulted in decreased class II expression, a tendency to decreased class I expression, and a significant prolongation of allograft survival. Brief culture at 7 degrees C resulted in increased class I expression, a trend to decreased class II expression, and no significant change in allograft survival. Donor pretreatment with LPS resulted in increased class I expression without significant change in class II expression and was correlated with prolongation of allograft survival. These studies demonstrate that an upregulation of MHC class I by in vitro or in vivo islet pretreatment is not associated with an acceleration of islet rejection. Reduction of class II was associated with delayed rejection. These results do not support a major role of the indirect pathway of antigen presentation in islet rejection in vivo. Certain protocols that alter the usual expression of class I and II on pancreatic islets are associated with alteration in the initiation and/or propagation of the normal cell-mediated rejection process, suggesting that the concept of pretransplant treatment should continue to be pursued.

We have autotransplanted highly purified cryopreserved canine islets into pancreatectomized dogs. Islets were frozen by slow cooling (0.25 degrees C/min) to -40 degrees C, stored at -196 degrees C and thawed rapidly (200 degrees C/min). A total of 7868 +/- 665 (means +/- SE) cryopreserved islets/kg body weight were implanted to the spleen (n = 7) by venous reflux, and seven other control dogs received 6811 +/- 653 fresh islets/kg (P versus cryo., NS). Fasting plasma glucose [( PG] mg/dl, +/- SEM) and intravenous glucose tolerance were determined before and at 1 and 3 months postimplantation. Six dogs that received cryopreserved islets and all seven control animals promptly became normoglycemic, with PGs of 133 +/- 23 and 110 +/- 4, respectively, at 1 week postimplantation (NS). During follow-up one normoglycemic animal from each group died due to small bowel volvulus and one recipient of fresh islets became diabetic at 14 days. The remaining dogs remained normoglycemic with PGs of 89 +/- 3 and 92 +/- 3 in dogs receiving cryopreserved and fresh islets, respectively, at 3 months postimplantation (NS). Mean K value (decline of glucose, %/min +/- SEM) at ivGTT for all dogs was 2.5 +/- 0.3 preoperatively. At 1 and 3 months postimplantation, the K values of dogs receiving cryopreserved islets were 1.3 +/- 0.1 and 1.4 +/- 0.2, respectively, compared with 1.3 +/- 0.2 and 2.0 +/- 0.2 for control dogs (NS). These data demonstrate prompt and sustained function of a defined quantity of frozen-thawed purified islets in a large mammal. Cryopreservation is an effective method for the long-term storage of purified islet tissue.

The long-term viability and function of pancreatic islets transplanted as nonvascularized dispersed grafts has not been well established. We report maintenance of nondiabetic carbohydrate metabolism for up to 36 months (17.8 +/- 1.4 months) in 77% of 40 consecutive canine recipients of nonpurified islet grafts transplanted to the spleen by venous reflux. Spontaneous loss of graft function occurred in 9 dogs during the follow-up period. Failure usually occurred within 1 year of implantation and was predicted by low K value and low insulin output on intravenous glucose tolerance tests 1 and 3 months postimplant. High K values and insulin output correlated strongly with maintenance of function beyond 24 months. A sufficient mass of implanted islets can provide satisfactory metabolic control for prolonged periods.

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

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