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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.

Acute rejection usually occurs within 3 months posttransplantation. Most centers reduce immunosuppression over 6 to 12 months to minimize opportunistic infection, malignancy, and drug toxicity. Pretransplant disease and low immunosuppression have been reported in association with late acute rejection (LAR). The objective of this study was to determine the incidence, predictive factors, and outcomes of LAR via retrospective review of adult liver transplant recipients in Western Canada from 1989 to 2000. LAR was defined as biopsy-proven acute rejection occurring more than 180 days posttransplantation. Patient characteristics, immunosuppression, and outcome were determined. Both a univariate and multiple logistic regression analysis were performed. LAR occurred in 97 (23%) of 415 patients more than 180 days posttransplantation. Median follow-up was 402 days (range, 180 to 3137 days); 79% of LAR episodes were graded mild. At the time of LAR, 33% were on a steroid taper. A total of 73% of LAR episodes were treated with pulse intravenous steroids, and 5% were steroid-resistant. In the univariate analysis, patients undergoing transplantation for viral etiologies and older age were associated with less LAR. Immunosuppression was significant in a multiple logistic regression model, but not with a proportional hazards model. On multivariate analysis, only patients undergoing transplantation for viral etiologies remained resistant to LAR (hazard ratio, 0.52; range, 0.34 to 0.93, P = .02). There was a trend toward increased chronic rejection in patients who developed LAR (P = .04). LAR is common and occurs after more than 1 year posttransplantation. Patients undergoing transplantation for viral etiologies seem to have a lower risk of LAR. There may be an increased risk of chronic rejection in those developing LAR.

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.

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.

The mucosal layer is the initial site of small bowel (SB) graft injury sustained during cold storage. Vascular administration of preservation solutions alone is unable to prevent ischemic injury of this layer during clinically relevant storage periods. The SB is unique in that it possesses both a vascular and a luminal route by which preservation solutions can be administered. We hypothesized that addition of a luminal-delivered solution, formulated on amino acid requirements for energy- and non-energy-related reactions, would provide site-specific preservation of mucosal energetics, barrier function and morphology throughout an extended period of cold storage. Of the three luminal solutions containing amino acids which were tested (UWG, AA1, AA2), only the two groups (AA1, AA2), containing glutamine plus 18 other amino acids, +/- osmotic agent (lactobionate) and buffer (BES), exhibited significant improvements in energetics, barrier function, and histology compared to the clinical standard of isolated vascular University of Wisconsin (UW) solution. Although the AA1 and AA2 groups preserved barrier function and morphology up to 24h better than all other solutions tested, AA2 proved to be the only luminal solution with values of permeability, conductance, and short-circuit current not significantly different from freshly isolated tissues. Furthermore, the greatest reduction in histologic injury was effected by AA2 treatment (median grade 2 compared to control, UW(v), grade 8). This study documents that a luminal-delivered solution, formulated on physiologic SB requirements, provides targeted preservation of the SB mucosa.

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.

Lack of a small animal model of the human hepatitis C virus (HCV) has impeded development of antiviral therapies against this epidemic infection. By transplanting normal human hepatocytes into SCID mice carrying a plasminogen activator transgene (Alb-uPA), we generated mice with chimeric human livers. Homozygosity of Alb-uPA was associated with significantly higher levels of human hepatocyte engraftment, and these mice developed prolonged HCV infections with high viral titers after inoculation with infected human serum. Initial increases in total viral load were up to 1950-fold, with replication confirmed by detection of negative-strand viral RNA in transplanted livers. HCV viral proteins were localized to human hepatocyte nodules, and infection was serially passaged through three generations of mice confirming both synthesis and release of infectious viral particles. These chimeric mice represent the first murine model suitable for studying the human hepatitis C virus in vivo.

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.

We studied the effect of host IFN-gamma on the pathology of acute rejection of vascularized mouse heart and kidney allografts. Organs from CBA donors (H-2k) were transplanted into BALB/c (H-2d) hosts with wild-type (WT) or disrupted (GKO, BALB/c mice with disrupted IFN-gamma genes) IFN-gamma genes. In WT hosts, rejecting hearts and kidneys showed mononuclear cell infiltration, intense induction of donor MHC products, but little parenchymal necrosis at day 7. Rejecting allografts in GKO recipients showed infiltrate but little or no induction of donor MHC and developed extensive necrosis despite patent large vessels. The necrosis was immunologically mediated, since it developed during rejection, was absent in isografts, and was prevented by immunosuppressing the recipient with cyclosporine or mycophenolate mofetil. Rejecting kidneys in GKO hosts showed increased mRNA for heme oxygenase 1, and decreased mRNA for NO synthase 2 and monokine inducible by IFN-gamma (MIG). The mRNA levels for CTL genes (perforin, granzyme B, and Fas ligand) were similar in rejecting kidneys in WT and GKO hosts, and the host Ab responses were similar. The administration of recombinant IFN-gamma to GKO hosts reduced but did not fully prevent the effects of IFN-gamma deficiency: MHC was induced, but the prevention of necrosis and induction of MIG were incomplete compared with WT hosts. Thus, IFN-gamma has unique effects in vascularized allografts, including induction of MHC and MIG, and protection against parenchymal necrosis, probably at the level of the microcirculation. This is probably a local action of IFN-gamma produced in large quantities in the allograft.

Islet transplantation offers the prospect of good glycemic control without major surgical risks. After our initial report of successful islet transplantation, we now provide further data on 12 type 1 diabetic patients with brittle diabetes or problems with hypoglycemia previous to 1 November 2000. Details of metabolic control, acute complications associated with islet transplantation, and long-term complications related to immunosuppression therapy and diabetes were noted. Insulin secretion, both acute and over 30 min, was determined after intravenous glucose tolerance tests (IVGTTs). The median follow-up was 10.2 months (CI 6.5-17.4), and the longest was 20 months. Glucose control was stable, with pretransplant fasting and meal tolerance-stimulated glucose levels of 12.5+/-1.9 and 20.0+/-2.7 mmol/l, respectively, but decreased significantly, with posttransplant levels of 6.3+/-0.3 and 7.5+/-0.6 mmol/l, respectively (P < 0.006). All patients have sustained insulin production, as evidenced by the most current baseline C-peptide levels 0.66+/-0.06 nmol/l, increasing to 1.29+/-0.25 nmol/l 90 min after the meal-tolerance test. The mean HbA1c level decreased from 8.3+/-0.5% to the current level of 5.8+/-0.1% (P < 0.001). Presently, four patients have normal glucose tolerance, five have impaired glucose tolerance, and three have post-islet transplant diabetes (two of whom need oral hypoglycemic agents and low-dose insulin (<10 U/day). Three patients had a temporary increase in their liver-function tests. One patient had a thrombosis of a peripheral branch of the right portal vein, and two of the early patients had bleeding from the hepatic needle puncture site; but these technical problems were resolved. Two patients had transient vitreous hemorrhages. The two patients with elevated creatinine levels pretransplant had a significant increase in serum creatinine in the long term, although the mean serum creatinine of the group was unchanged. The cholesterol increased in five patients, and lipid-lowering therapy was required for three patients. No patient has developed cytomegalovirus infection or disease, posttransplant lymphoproliferative disorder, malignancies, or serious infection to date. None of the patients have been sensitized to donor antigen. In 11 of the 12 patients, insulin independence was achieved after 9,000 islet equivalents (IEs) per kilogram were transplanted. The acute insulin response and the insulin area under the curve (AUC) after IVGTT were consistently maintained over time. The insulin AUC from the IVGTT correlated to the number of islets transplanted, but more closely correlated when the cold ischemia time was taken into consideration (r = 0.83, P < 0.001). Islet transplantation has successfully corrected labile type 1 diabetes and problems with hypoglycemia, and our results show persistent insulin secretion. After a minimum of 9,000 IEs per kilogram are provided, insulin independence is usually attained. An elevation of creatinine appears to be a contraindication to this immunosuppressive regimen. For the subjects who had labile type 1 diabetes that was difficult to control, the risk-to-benefit ratio is in favor of islet transplantation.

In transplant rejection interferon (IFN)-gamma regulates the recipient immune response but also acts directly on IFN-gamma receptors in the graft. We investigated these direct actions by comparing rejecting kidneys from donors lacking IFN-gamma receptors (GRKO mice) or control donors (129Sv/J) in CBA recipients. Beginning day 5, 129Sv/J kidneys displayed high major histocompatibility complex (MHC) expression, progressive infiltration by inflammatory cells, but no thrombosis and little necrosis, even at day 21. GRKO kidneys showed increasing fibrin thrombi in small veins, peritubular capillary congestion, hyaline casts, and patchy parenchymal necrosis, progressing to near total necrosis at day 10. Terminal dUTP nick-end labeling assays were positive only in the interstitial infiltrate, confirming that massive cell death in GRKO transplants was not apoptotic. Paradoxically, GRKO kidneys showed little donor MHC induction and less inflammatory infiltration. Both GRKO and 129Sv/J allografts evoked vigorous host immune responses including alloantibody and mRNA for cytotoxic T cell genes (perforin, granzyme B, Fas ligand), and displayed similar expression of complement inhibitors (CD46, CD55, CD59). GRKO kidneys displayed less mRNA for inducible nitric oxide synthase and monokine inducible by IFN-gamma but increased heme oxygenase-1 mRNA. Thus IFN-gamma acting on IFN-gamma receptors in allografts promotes infiltration and MHC induction but prevents early thrombosis, congestion, and necrosis.

We investigated the metabolic effects of buffering agents alpha-amino-4-imidazole-propionic acid (Histidine), N, N-bis(2-hydroxyethyl)glycine (bicine), N, N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) on anaerobic energy production (via glycolysis) and conservation of key regulatory enzyme activity, and phosphofructokinase (PFK) throughout prolonged hypothermic hypoxia in porcine hearts. Hearts from 35 to 40 kg pigs were flushed with one of the following five solutions: St. Thomas' Hospital solution (STHS); modified University of Wisconsin (UW) solution; and three solutions containing modified UW plus 90 mM of histidine, bicine, or BES. The hearts were then stored at 4 degrees C for 10 h. After 10 h of hypothermic hypoxia, lactate values were 6.7-12.9 micromol/g higher than control; this reflected an increase in anaerobic end product of 35-67%. The consequences of enhanced anaerobic metabolism were higher ATP, total adenylate, Energy Charge, and ATP/ADP ratios in most of the buffered groups after 4-10 h cold storage; effectiveness of the buffers employed correlated with buffering capacity (BES proved to be the most effective). PFK remained activated throughout most of the 10-h period in hearts stored with buffers and did not undergo the rapid inactivation experienced by hearts stored in STHS. Conservation of PFK integrity with buffering agents was not related to a pH-mediated event; changes in kinetic parameters suggested that this protection was due to an irreversible posttranslational modification, specifically a dephosphorylation event.

Background: Registry data on patients with type 1 diabetes mellitus who undergo pancreatic islet transplantation indicate that only 8 percent are free of the need for insulin therapy at one year., Methods: Seven consecutive patients with type 1 diabetes and a history of severe hypoglycemia and metabolic instability underwent islet transplantation in conjunction with a glucocorticoid-free immunosuppressive regimen consisting of sirolimus, tacrolimus, and daclizumab. Islets were isolated by ductal perfusion with cold, purified collagenase, digested and purified in xenoprotein-free medium, and transplanted immediately by means of a percutaneous transhepatic portal embolization., Results: All seven patients quickly attained sustained insulin independence after transplantation of a mean (+/-SD) islet mass of 11,547+/-1604 islet equivalents per kilogram of body weight (median follow-up, 11.9 months; range, 4.4 to 14.9). All recipients required islets from two donor pancreases, and one required a third transplant from two donors to achieve sustained insulin independence. The mean glycosylated hemoglobin values were normal after transplantation in all recipients. The mean amplitude of glycemic excursions (a measure of fluctuations in blood glucose concentrations) was significantly decreased after the attainment of insulin independence (from 198+/-32 mg per deciliter [11.1+/-1.8 mmol per liter] before transplantation to 119+/-37 mg per deciliter [6.7+/-2.1 mmol per liter] after the first transplantation and 51+/-30 mg per deciliter [2.8+/-1.7 mmol per liter] after the attainment of insulin independence; P<0.001). There were no further episodes of hypoglycemic coma. Complications were minor, and there were no significant increases in lipid concentrations during follow-up., Conclusions: Our observations in patients with type 1 diabetes indicate that islet transplantation can result in insulin independence with excellent metabolic control when glucocorticoid-free immunosuppression is combined with the infusion of an adequate islet mass.

Graft-versus-host disease after liver transplantation complicated by systemic aspergillosis with pancarditis. Can J Gastroenterol 2000;14(7):637-640. Acute graft-versus-host disease (GVHD) is a common complication after bone marrow transplantation, with characteristic rash and diarrhea being the most common features. After liver transplantation, however, this phenomenon is very rare. Most transplant patients are on a variety of medications, including immunosuppressants; therefore, the differential diagnosis of skin rash or diarrhea is broad. A 37-year-old man who underwent liver transplantation for primary biliary cirrhosis, and developed a rash and watery diarrhea, is presented. Skin and colonic biopsies confirmed acute GVHD. A pulse of intravenous steroids was given. The skin rash improved, but he developed pancytopenia. His course was complicated by central line infection, jugular and subclavian vein thrombosis, pseudomembranous colitis, recurrent bacteremia, cholestasis on total parenteral nutrition and cytomegalovirus infection. After the onset of pleuritic chest pain and clinical sepsis, spiral computed tomography scan of his chest and abdomen revealed septic infarcts in multiple organs. Despite empirical treatment with amphotericin B, he died of multiorgan dysfunction syndrome within 72 h. Autopsy revealed systemic aspergillosis with pancarditis, endocardial vegetations, and septic pulmonary, splenic, hepatic and renal infarcts. The pathogenesis and experience with this rare, but often fatal, complication of liver transplantation are reviewed. In contrast to GVHD after bone marrow transplantation, pancytopenia is common and liver dysfunction is rare. One should have a high level of suspicion in the liver transplant recipient presenting with rash and/or diarrhea.

Background/aim: During cold liver storage in University of Wisconsin solution, glycolysis is inhibited by declining intracellular pH and a reduction in glycogen phosphorylase activity. The current study investigated the effects of a histidine-buffered, modified University of Wisconsin solution with cyclic-AMP analogue plus phosphodiesterase inhibitors to optimize both pH and PK A-mediated limits on glycolytic energy production., Methods: In an isolated rodent-liver system, dioctanoyl-cAMP was supplemented with each phosphodiesterase inhibitor (isobutylmethylxanthine, papaverine, Ro 20-1724, dipyridamole). Once the most efficacious combination was determined, a separate group of livers was cold-stored for 24 h and then reperfused at 37 degrees C to examine regeneration of high energy adenylates., Results: Lactate accumulation in the histidine-lactobionate-raffinose group was 8.7 micromol/g; net increases were greater with all four phosphodiesterase inhibitors with dioctanoyl-cAMP; dipyridamole resulted in a maximum increase of 16.7 micromol/g. ATP was consistently higher in all treatment groups with phosphodiesterase inhibitors throughout 24 h; even after 10-24 h, levels with dipyridamole-treatment were 250-280% higher than with University of Wisconsin (p<0.05). Assessment of glycogen phosphorylase activity in the dipyridamole-treatment group indicated that increased glycolytic activity over the first 4 h was a direct consequence of elevated enzyme levels. However, between 4-10 h, phosphofructokinase underwent a phosphorylation, leading to an inhibition at this point in glycolysis. Upon reperfusion, the higher ATP/ADP and ADP/ AMP ratios found with phosphodiesterase inhibitor treatment suggested that adenylate regeneration was superior with dipyridamole+dioctanoyl-cAMP., Conclusion: Dipyridamole plus dioctanoyl-cAMP treatment achieved increased glycogenolysis throughout 24 h storage by maintaining glycogen phosphorylase in a phosphorylated (active) state; however, a PK A-mediated phosphorylation (inhibition) of phosphofructokinase resulted in decreased glycolytic ATP production between 4-10 h.

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.

Despite recent advances in hepatic surgery, resection of the cirrhotic liver continues to be fraught with high morbidity and mortality rates. As a result, for many patients requiring resection of HCC the postoperative course is complicated and the probability of cure is diminished by coexisting cirrhosis. In this review, we discuss the characteristics of the cirrhotic liver which make it poorly tolerant of resection and the most common complications that follow such surgery. The main purpose of this paper is to review recent attempts to identify interventions that might be beneficial to cirrhotic patients undergoing resection. These interventions include assessment of liver reserve, advances in surgical technique, and improvement in liver function and regeneration.

Ogilvie's syndrome, or acute colonic pseudo-obstruction, is a rare complication following liver transplantation. We describe two cases in which the onset of Ogilvie's syndrome is strongly temporally associated with acute cytomegaloviral (CMV) infection in immunosuppressed liver transplant recipients. The pseudo-obstruction resolved rapidly in both cases following treatment with intravenous ganciclovir. Acute CMV infection therefore appeared to be causally linked to pathogenesis of Ogilvie's syndrome in these two cases. This association has not been described previously to our knowledge, and should be considered in any transplant patient presenting with Ogilvie's syndrome.

Effective intraductal delivery of the enzyme collagenase into the pancreas is crucial to the subsequent ability to isolate viable islets. Most clinical islet transplant centers load the enzyme into the pancreas by retrograde injection using a syringe following cannulation of the pancreatic duct. An alternative approach is to perfuse the pancreas via the pancreatic duct with collagenase solution using a recirculating perfusion device system. This provides control over perfusion pressures and collagenase temperature. This study reports on our evaluation of the delivery of Liberase-HI into the pancreas of 14 consecutive adult multiorgan cadaveric donors. Alternate glands were procured and processed using an identical protocol with the exception of collagenase delivery. The first group of pancreases was loaded using the perfusion technique where cold (4 degrees C) Liberase-HI was perfused at 80 mmHg for 5 min after which the pressure was increased to 180 mmHg. The collagenase solution was then slowly warmed to 35 degrees C, transferred to the dissociation chamber and mechanically dissociated, and then purified using discontinuous gradients of Ficoll. Pancreases in the second group were loaded with collagenase (28-32 degrees C) using the syringe technique before mechanical dissociation and purification. There were no significant differences in pancreas cold ischemia, donor age, body mass index, maximum blood glucose, or serum amylase of the donors between the two groups. Mean collagenase digestion time in the digestion chamber was not different between the two groups; however, the amount of undigested tissue remaining after dissociation was significantly higher in the syringe-loaded group (15.3 +/- 2.6 g vs. 4.6 +/- 2.1 g, mean +/- SEM, p < 0.05). Postdigestion recovery of islets was 471 +/- 83 x 10(3) IE in the perfusion group compared with 391 +/- 57 x 10(3) IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 +/- 45 vs. 251 +/- 28 x 10(3) IE, p < 0.05, two-tailed paired t-test). No difference in in vitro islet viability was observed between the two groups following glucose perifusion with the calculated stimulation index of 4.6 +/- 0.6 for the perfusion group and 4.2 +/- 0.7 for the syringe-loaded group. Controlled perfusion via the pancreatic duct allows the effective delivery of the enzyme achieving maximal distension to all regions of the pancreas leading to an increased recovery of the islets with no detrimental effect on subsequent in vitro islet function.

Many lower vertebrates (reptilian and amphibian species) are capable of surviving natural episodes of hypoxia and hypothermia. It is by specific metabolic adaptations that anurans are able to tolerate prolonged exposure to harsh environmental stresses. In this study, it was hypothesized that livers from an aquatic frog would possess an inherent metabolic ability to sustain high levels of ATP in an isolated organ system, providing insight into a metabolic system that is well-adapted for low temperature in vitro organ storage. Frogs of the species, R. pipiens were acclimated at 20 degrees C and at 5 degrees C. Livers were preserved using a clinical preservation solution after flushing. Livers from 20 degrees C-acclimated frogs were stored at 20 degrees C and 5 degrees C and livers from 5 degrees C-acclimated frogs were stored at 5 degrees C. The results indicated that hepatic adenylate status was maintained for 96 h during 5 degrees C storage, but not longer than 4-10 h during 20 degrees C storage. In livers from 5 degrees C-acclimated animals subjected to 5 degrees C storage, ATP was maintained at 100% throughout the 96-h period. Warm acclimation (20 degrees C) and 20 degrees C storage resulted in poorer maintenance of ATP; energy charge values dropped to 0.50 within 2 h and by 24 h, only 24% of control ATP remained. Lactate levels remained less than 25 mumol/g dry weight in all 5 degrees C-stored livers; 20 degrees C-stored livers exhibited greater accumulation of this anaerobic endproduct (lactate reached 45-50 mumol/g by 10 h). The data imply that hepatic adenylate status is largely dependent on exposure to hypothermic hypoxia and although small amounts of ATP were accounted for by anaerobic glycolysis, there must have been either a substantial reduction in cellular energy-utilization or an efficient use of low oxygen tensions.

We compared the efficacy of fresh and frozen/thawed islets by determining the minimum number required to consistently reverse diabetes in mice. Defined numbers of islets, isolated from Balb/c (H-2d) and CBA/J (H-2k) mice, were transplanted into streptozotocin-induced diabetic Balb/c mice. Frozen/thawed grafts were cooled slowly to -40 degrees C, stored at -196 degrees C, and thawed rapidly. At 100 days after transplantation, isografts were recovered for measurement of insulin content. Mean (+/-SD) recovery of cryopreserved islets after thawing was 80 +/- 3% (range 67-89%). For both fresh and frozen/thawed isografts and allografts, 200 islets were required to establish normoglycemia. The degree of metabolic function provided by equivalent quantities of fresh and frozen/thawed grafts was similar; and all normoglycemic isograft recipients remained so until graft nephrectomy. The insulin content of fresh and frozen/thawed isografts containing 200 and 300 islets were 151 +/- 25 and 126 +/- 8 mU and 259 +/- 36 and 278 +/- 20 mU, respectively. Among allograft recipients, median survival ranged from 15 to 20 days, and was not influenced by cryopreservation or graft size. The results of this study demonstrate a high rate of recovery of viable islets following cryopreservation. The function of equivalent quantities of fresh and cryopreserved islet isografts and allografts in nonimmunosuppressed recipients is similar.

This study was designed to determine whether the metabolic adaptations developed by frogs to tolerate natural events of hypothermic hypoxia would precondition its liver for ex vivo organ storage. The metabolic responses of the frog, Rana castabiena, were compared to those of a mammalian system (rat) throughout a prolonged period of organ storage. Livers from rats and frogs were flushed and stored in UW solution at 5 degrees C for periods of 24-96 h. In frog livers, ATP was maintained high and constant over the first 24 h of storage; values ranged from 2.7 to 3.0 micro mol/g. Even after 96 h cold storage, ATP remained > 1.0 micro mol/g. In contrast, ATP levels in stored rat livers dropped rapidly, and by 4 h ATP was 1.2 micro mol/g. In terms of anaerobic endproduct accumulation, lactate levels rose 5.8 micro mol/g in frog liver (over 96 h) and by 8.6 micro mol/g in rat liver (over 24 h). This difference in flux through glycolysis was also reflected in relative rates of carbohydrate catabolism (i.e., glucose + lactate production). The rate of carbohydrate catabolism for frog liver was 0.74 micro mol/g/h compared to 2.26 micro mol/g/h for rat liver; a Q10 value of 6.2 was estimated for livers from R. castabiena. An assessment of glycolytic enzyme activities revealed that key differences in the responsiveness of pyruvate kinase to allosteric modifiers may have been responsible for the marked drop in the rate of anaerobic energy production in frog tissues. Although the concept of depressed metabolism in a lower vertebrate is not new, the data presented in this study demonstrate that a depressed metabolic state can be achieved in isolated livers from R. castabiena simply through cold exposure. With respect to clinical relevance, the results of this study indicate that energetics of stored livers can be maintained effectively through an efficient reduction in energy use in combination with a slow, yet continuous, rate of energy production facilitated by glycolysis., (Copyright 1998 Academic Press.)

Lamivudine is a nucleoside analogue with efficacy in the suppression of hepatitis B viral (HBV) replication. In a previously reported study, lamivudine was administered to patients with chronic, actively replicating HBV infection who subsequently underwent liver transplantation. Patients became serum HBV DNA-negative in response to lamivudine before transplantation, which was continued in the post-transplant period. Two of four patients surviving the immediate postoperative period developed allograft reinfection 240 and 409 days post-transplant. The strain of the reinfecting virus was analyzed, and a mutation in the YMDD region of the viral polymerase conferring resistance to lamivudine was discovered. The long term follow-up of these two patients is reported. The first patient developed ascites 16.5 months after allograft reinfection. A transjugular liver biopsy performed 18 months after the emergence of the lamivudine-resistant strain revealed cirrhosis and lobular hepatitis without rejection. The gradient between hepatic vein wedged and free pressures was 13 mmHg, consistent with portal hypertension. The second patient, 16 months after allograft reinfection with the lamivudine-resistant strain, is without clinical evidence of portal hypertension, although liver enzymes remain elevated. Both patients were given a trial of famciclovir, which did not significantly suppress HBV viremia. In conclusion, lamivudine-resistant HBV strains with the YMDD mutation may have an aggressive clinical course with rapid progression to cirrhosis. Famciclovir did not appear to be an effective rescue agent in these two patients.

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.

Despite two decades of use, there are limited data on the best way to administer and monitor cyclosporine for orthotopic liver transplantation (OLT). The present study was undertaken: 1) to establish the safety of a new formulation of cyclosporine, Neoral, 2) to determine if treatment with Neoral will improve the results of liver transplantation and 3) to study the relationship between pharmacokinetic parameters and clinical outcomes after OLT. A double-blind, randomized, comparison of Sandimmune and Neoral was conducted at 5 Canadian centers in 188 consecutive adults undergoing OLT. Patients were induced with intravenous cyclosporine (CsA) then switched to Neoral or Sandimmune. Dose adjustments were made daily, or as needed, to reach a target trough CsA level (C0) of 350 ng/mL in both groups. Pharmacokinetic studies were performed on days 5, 10, 15 and 30 after transplantation. The Neoral group stopped intravenous CsA earlier (p < 0.0001), and these patients required a lower median daily oral dose (p < 0.01) to maintain comparable trough CsA levels. Five Sandimmune patients, but no Neoral patients discontinued the study because of the inability to reach target trough levels of CsA within the prescribed time (p < 0.05). At 4 months, there were no differences between the two groups with respect to patient survival, graft survival or rejection-free survival. The incidence of serious adverse events was also similar and did not correlate with CsA profiles. The Neoral group had a higher area under the drug concentration curve (AUC) and peak CsA levels (Cmax). There was a correlation between freedom from graft rejection and both AUC and Cmax at days 5 and 10 post-transplant. In contrast, there was a poor correlation between C0 and graft rejection. In summary, Neoral appears to be safe and well tolerated by patients. Cmax and/or AUC maybe better markers for monitoring cyclosporine based immunosuppression after liver transplantation.

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.

Intraductal distention of the pancreas with collagenase followed by stationary warm incubation improves the recovery of islets of Langerhans in the rat, but controlled studies are needed for valid comparison with standard isolation methods. We have modified Gotoh's technique of stationary digestion for high-yield isolation in the rat (Stationary). The method is subjected herein to rigorous blinded comparison with the standard chopped tissue (Chopped) technique, based on Lacy et al., as performed in our laboratory for over 10 yr. Islet recovery was determined by a single observe 'blinded' to the method of isolation used, and only intact islets of diameter > or = 100 microns were included. Stationary gave 719 +/- 114 islets per pancreas (mean +/- SD, n = 21 isolations) vs. 487.5 +/- 69 for Chopped (n = 36 isolations), a 47.5% increment in yield (p < 0.0001). In vitro islet perifusion showed no statistical difference in stimulation index (SI) or stimulated area under the curve (SAUC) between the two methods, but Stationary showed a trend towards improved phase II insulin release. In vivo function was assessed by isogeneic transplantation of 2,000 islets beneath the renal capsule of streptozotocin diabetic recipients (65 mg/kg Sigma); Stationary recipients (n = 7) became normoglycemic (< or = 8 mmol/L) by 3.3 +/- 4.8 days vs. 1.6 +/- 1.5 days for Chopped recipients (p = 0.4 ns, mean +/- SEM). IVGTT performed at 1 mo posttransplant gave K-values for Stationary of 2.64 +/- 0.8 vs. 2.62 +/- 0.8 for Chopped (mean +/- SD, p = 0.9 ns, n = 6, unpaired t-test), which were not distinguishable from normal control rats (2.59 +/- 0.8) (p = 0.9 ns, n = 10). Graft function remained stable until graft bearing nephrectomy induced hyperglycemia uniformly within 1 day. Graft histology showed a healthy well-preserved structure on light microscopy, with well-granulated beta cells on EM. Economic costs of rat, collagenase, and Ficoll were 26% ($50.82) lower per recipient for Stationary. We conclude that modified stationary digestion significantly improves islet recovery with excellent in vitro and in vivo function, and is cost effective.

The (-) enantiomer of 3'-thiacytidine (lamivudine) has been found to be a potent inhibitor of hepatitis B virus (HBV) and human immunodeficiency virus (HIV) replication. Mutation of methionine to valine or isoleucine at the YMDD (tyrosine, methionine, aspartate, aspartate) motif of the HIV reverse transcriptase has been shown to be responsible for lamivudine resistance in HIV. The hepadnaviruses also have the YMDD motif in their DNA polymerase. Therefore, it is possible that hepadnaviruses could develop lamivudine resistance by a similar mutation at this motif. We analyzed the HBV from a liver transplantation patient who developed recurrent HBV viremia during lamivudine treatment. The polymerase gene was amplified by polymerase chain reaction (PCR), and the region coding for the YMDD motif was sequenced. The pretreatment HBV sequence coded for YMDD, while the lamivudine-resistant mutant HBV coded for YIDD (tyrosine, isoleucine, aspartate, aspartate). With the documented changes in the YMDD motif of lamivudine-resistant HIV, it is likely that the methionine-to-isoleucine mutation in the YMDD motif of the HBV polymerase contributes significantly to the lamivudine-resistance of HBV isolated from this patient.

Objectives: Transplantation of the small intestine would be an attractive therapeutic option for treatment of short bowel syndrome if effective, nontoxic immunosuppressive agents could be developed. This study examines the effect of three newly developed immuno-suppressive agents: rapamycin, deoxyspergualin, and mycophenolate mofetil, on the nutritional status and intestinal function of normal juvenile rats., Design & Methods: Rapamycin (2 mg/kg every second day), deoxyspergualin (2 mg/kg every second day) and mycophenolate mofetil (MM) (25 mg/kg every second day) were injected subcutaneously for six weeks., Results: Rapamycin and deoxyspergualin caused significant reductions in weight gain without impairing feed intake. Both drugs caused small decreases in fat absorption; treatment with DSG induced an increase in permeability to 99Tc-DTPA. However, the permeability to other markers, such as mannitol and lactulose, was decreased in the rapamycin and mycophenolate mofetil-treated animals. Intestinal function in vitro was quantified using glucose flux (absorption). In the rapamycin group, there was a significant decrease in ileal uptake of glucose, with the net flux (absorption) being zero; there was an associated loss of villous size histologically. In the deoxyspergualin-treated groups, there was a decrease in the jejunal glucose flux. In the mycophenolate mofetil-treated animals, there was a decrease in jejunal with a compensatory increase in ileal glucose absorption. There were minor variations in intestinal morphology, but these were not consistent., Conclusions: Rapamycin and deoxyspergualin in these doses cause a significant reduction in weight gain in healthy juvenile animals, and all the drugs caused changes in the active transport characteristics of the intestine. Accordingly, the use of these drugs for intestinal transplantation should be evaluated carefully for their nutritional impact.

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.