Preliminary Single-Center Canadian Experience of Human Normothermic Ex Vivo Liver Perfusion: Results of a Clinical Trial.

After extensive experimentation, outcomes of a first clinical normothermic machine perfusion (NMP) liver trial in the United Kingdom demonstrated feasibility and clear safety, with improved liver function compared with standard static cold storage (SCS). We present a preliminary single-center North American experience using identical NMP technology. Ten donor liver grafts were procured, four (40%) from donation after circulatory death (DCD), of which nine were transplanted. One liver did not proceed because of a technical failure with portal cannulation and was discarded. Transplanted NMP grafts were matched 1:3 with transplanted SCS livers. Median NMP was 11.5 h (range 3.3-22.5 h) with one DCD liver perfused for 22.5 h. All transplanted livers functioned, and serum transaminases, bilirubin, international normalized ratio, and lactate levels corrected in NMP recipients similarly to controls. Graft survival at 30 days (primary outcome) was not statistically different between groups on an intent-to-treat basis (p = 0.25). Intensive care and hospital stays were significantly more prolonged in the NMP group. This preliminary experience demonstrates feasibility as well as potential technical risks of NMP in a North American setting and highlights a need for larger, randomized studies.

Liver transplantation for "very early" intrahepatic cholangiocarcinoma: International retrospective study supporting a prospective assessment.

UNLABELLED: The presence of an intrahepatic cholangiocarcinoma (iCCA) in a cirrhotic liver is a contraindication for liver transplantation in most centers worldwide. Recent investigations have shown that "very early" iCCA (single tumors ≤2 cm) may have acceptable results after liver transplantation. This study further evaluates this finding in a larger international multicenter cohort. The study group was composed of those patients who were transplanted for hepatocellular carcinoma or decompensated cirrhosis and found to have an iCCA at explant pathology. Patients were divided into those with "very early" iCCA and those with "advanced" disease (single tumor >2 cm or multifocal disease). Between January 2000 and December 2013, 81 patients were found to have an iCCA at explant; 33 had separate nodules of iCCA and hepatocellular carcinoma, and 48 had only iCCA (study group). Within the study group, 15/48 (31%) constituted the "very early" iCCA group and 33/48 (69%) the "advanced" group. There were no significant differences between groups in preoperative characteristics. At explant, the median size of the largest tumor was larger in the "advanced" group (3.1 [2.5-4.4] versus 1.6 [1.5-1.8]). After a median follow-up of 35 (13.5-76.4) months, the 1-year, 3-year, and 5-year cumulative risks of recurrence were, respectively, 7%, 18%, and 18% in the very early iCCA group versus 30%, 47%, and 61% in the advanced iCCA group, P = 0.01. The 1-year, 3-year, and 5-year actuarial survival rates were, respectively, 93%, 84%, and 65% in the very early iCCA group versus 79%, 50%, and 45% in the advanced iCCA group, P = 0.02. CONCLUSION: Patients with cirrhosis and very early iCCA may become candidates for liver transplantation; a prospective multicenter clinical trial is needed to further confirm these results. (Hepatology 2016;64:1178-1188).

De novo sirolimus and reduced-dose tacrolimus versus standard-dose tacrolimus after liver transplantation: the 2000-2003 phase II prospective randomized trial.

We studied whether the use of sirolimus with reduced-dose tacrolimus, as compared to standard-dose tacrolimus, after liver transplantation is safe, tolerated and efficacious. In an international multicenter, open-label, active-controlled randomized trial (2000-2003), adult primary liver transplant recipients (n=222) were randomly assigned immediately after transplantation to conventional-dose tacrolimus (trough: 7-15 ng/mL) or sirolimus (loading dose: 15 mg, initial dose: 5 mg titrated to a trough of 4-11 ng/mL) and reduced-dose tacrolimus (trough: 3-7 ng/mL). The study was terminated after 21 months due to imbalance in adverse events. The 24-month cumulative incidence of graft loss (26.4% vs. 12.5%, p=0.009) and patient death (20% vs. 8%, p=0.010) was higher in subjects receiving sirolimus. A numerically higher rate of hepatic artery thrombosis/portal vein thrombosis was observed in the sirolimus arm (8% vs. 3%, p=0.065). The incidence of sepsis was higher in the sirolimus arm (20.4% vs. 7.2%, p=0.006). Rates of acute cellular rejection were similar between the two groups. Early use of sirolimus using a loading dose followed by maintenance doses and reduced-dose tacrolimus in de novo liver transplant recipients is associated with higher rates of graft loss, death and sepsis when compared to the use of conventional-dose tacrolimus alone.

Donor-derived bacteremia in liver transplant recipients despite antibiotic prophylaxis.

As the disparity between the number of candidates listed for transplant and the number of donors continues to grow, marginal organ donors are increasingly utilized. This includes bacteremic donors which may carry an increased risk of transmission of infection. It is recommended that recipients of organs from bacteremic donors receive antibiotic prophylaxis based on the susceptibilities of the donor isolate to prevent transmission. Here, we present four cases of donor-derived bacteremia, despite appropriate antimicrobial prophylaxis, in four liver transplant recipients. Transmitted pathogens included Staphylococcus aureus in two cases, and Escherichia coli and Group B Streptococcus each in one case. Interestingly, none of the nonhepatic organs (n=10) utilized from these bacteremic donors resulted in transmissions. These cases highlight the fact that risk of transmission from bacteremic donors is not eliminated with antimicrobial therapy in the donor and recipient. As no transmissions occurred in recipients of nonhepatic organs from these donors, these cases also suggest that liver recipients may be at higher risk of donor transmitted bacteremia.

Portal vein thrombosis is a potentially preventable complication in clinical islet transplantation.

Percutaneous transhepatic portal access avoids surgery but is rarely associated with bleeding or portal venous thrombosis (PVT). We herein report our large, single-center experience of percutaneous islet implantation and evaluate risk factors of PVT and graft function. Prospective data were collected on 268 intraportal islet transplants (122 subjects). A portal venous Doppler ultrasound was obtained on Days 1 and 7 posttransplant. Therapeutic heparinization, complete ablation of the portal catheter tract with Avitene paste and limiting packed cell volume (PCV) to <5 mL completely prevented any portal thrombosis in the most recent 101 islet transplant procedures over the past 5 years. In the previous cumulative experience, partial thrombosis did not affect islet function. Standard liver volume correlated negatively (r =-0.257, p < 0.001) and PCV correlated positively with portal pressure rise (r = 0.463, p < 0.001). Overall, partial portal thrombosis occurred after 10 procedures (overall incidence 3.7%, most recent 101 patient incidence 0%). There were no cases of complete thrombosis and no patient developed sequelae of portal hypertension. In conclusion, portal thrombosis is a preventable complication in clinical islet transplantation, provided therapeutic anticoagulation is maintained and PCV is limited to <5 mL.

Hepatitis C virus replication in mice with chimeric human livers.

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.

Cytopathology and ultrastructure identification of primary hepatic acinar cell carcinoma: Case report.

INTRODUCTION: A primary acinar cell carcinoma (ACC) of the liver was incidentally diagnosed in a clinically asymptomatic 80-year-old man. This study aimed to delineate critical diagnostic characteristics of an ACC originating uniquely from the liver to improve its future identification. PRESENTATION OF CASE: Enhanced MRI revealed a heterogenous, cystic 7.7 × 11.1 × 10.4 cm tumour occupying hepatic segments II and III. The mass demonstrated mild diffuse enhancement in hepatic arterial phase with minimal portal venous washout in a liver without cirrhotic features. A central stellate T2-hyperintense necrotic scar and outer capsule were apparent. No primary lesion or metastasis outside the liver was discernable. Post-left hepatic lobectomy, the tumour immunophenotype was atypical for presumptive diagnoses of hepatocellular carcinoma (HCC) or cholangiocarcinoma. Extensive morphologic workup on electron microscopy definitively diagnosed primary hepatic ACC by establishing presence of secretory zymogen-like granules, intracytoplasmic microvilli and acinar cell differentiation. Cytopathology revealed cellular lumen expressing PAS-positive diastase-resistant granular cytoplasmic contents. DISCUSSION: This case showcased the novel utility of electron microscopy that was crucial in yielding the definitive diagnosis. The previous literature on hepatic ACC was compiled here in context of the present case. The mechanism of hepatic acinar cell localization was also discussed. CONCLUSION: Primary hepatic ACC may easily be confused for other lesions due to nonspecific imaging patterns. Specifically, the presence of a central scar without risk factors for HCC can favour a diagnosis of benign entities such as focal nodular hyperplasia (FNH). Electron microscopy presents an important tool to identify primary hepatic ACC and may improve future patient outcomes.

The role of macrophages in xenograft rejection.

Safe and effective xenotransplantation would provide a valuable answer to many of the limitations of allogenic transplantation. Such limitations include scarcity of organ supply and morbidity to donors in cases of living-related donor transplantation. The main hurdle to the efficacious application of xenotransplantation in clinical medicine is the fierce host immune response to xenografts. This immune response is embodied in 3 different types of xenograft rejection. Both hyperacute rejection and delayed xenograft rejection are mediated by natural antibodies and are concerned primarily with whole organ rejection. Cellular xenograft rejection (CXR), on the other hand, is concerned with both whole organ and CXR and is mediated by innate immunity rather than natural antibodies. Macrophages, which are cells of the innate immune system, play a role in all 3 types of xenograft rejection (not just CXR). They impart their effects both directly and through T-cell activation.

Prolonged cryopreservation of purified human pancreatic islets.

Long-term islet storage would facilitate many aspects of islet research and clinical islet transplantation. Collagenase-isolated, Ficoll-purified islets from eight cadaveric pancreases were stored in liquid nitrogen for 44 +/- 9 days after dimethyl sulfoxide equilibration and slow cooling. Rapid rewarming and 48 h of culture preceded repeat evaluation of recovery by islet counts, insulin extraction, and glucose-stimulated perifusion. Islet recovery was 94 +/- 4% by count and 90 +/- 22% by insulin extraction immediately after thawing. After an additional 48 h in culture, recovery was 74 +/- 12% by insulin extraction and 79% by quantitative perifusion culture. Perifusion demonstrated normal baseline and first-phase insulin secretion with decreased second-phase insulin secretion after cryopreservation. Insulin-stained sections and electron microscopy revealed preserved islet morphology and ultrastructure. Granulated islets with preserved morphology were recovered 14 days after renal subcapsular xenografting into nude mice. This study demonstrates high recovery and good functional activity of human islets after prolonged cryopreservation.

Long-term cryogenic storage of purified adult human islets of Langerhans.

Reliable high-recovery human islet storage would facilitate tissue matching, organ sharing, and immune manipulation of donor islets and prospective diabetic recipients. Collagenase-isolated, Ficoll-purified pancreatic islets (median 21,000, 15% of total islet yield) from eight cadaver pancreases were cultured in vitro for 24 h, equilibrated in three steps with dimethyl sulfoxide (DMSO) to a 2-M concentration, supercooled, nucleated, and cooled at 0.25 degree C/min to -40 degrees C before storage at -196 degrees C for 44.25 +/- 8.75 days. Rewarming at 200 degrees C/min and removal of DMSO with 0.75 M sucrose preceded 48 h of culture and retesting. Recovery postthaw by microscope count on duplicate aliquots was 94.2 +/- 3.5% of prefreeze counts and by triplicate assay of extractable insulin was 90.0 +/- 22.3% on day 0 and 74.1 +/- 12.6% after a 48-h culture. Nonfrozen islets increased basal insulin secretion 7.7 +/- 2.8 times after stimulation with 300 mg/dl glucose in perifusion, whereas islets frozen-thawed and cultured 48 h increased 6.2 +/- 0.8 times (NS). Peak stimulated insulin release was 0.92 +/- 0.14 microU.islet-1.min-1 before storage and 0.73 +/- 0.14 microU.islet-1.min-1 (79% of control, NS) after freeze-thaw and a 48-h culture. Total insulin secretion (area under curve) was 66% of prefreeze values at 48 h. Immunocytochemical stains revealed preservation of islet morphology postthaw. Electron microscopy showed intact cellular and nuclear membranes and intracellular organelles. Frozen-thawed islets harvested 14 days after renal subcapsular xenografting in nude mice were revascularized and well granulated. Cryopreservation can achieve prolonged storage of large numbers of human islets with high recovery numerically and functionally, making this a feasible approach for future trials of human islet transplantation.

Insulin sensitivity and glucose effectiveness in long-term islet-autotransplanted dogs.

Parameters determining glucose tolerance were quantified with Bergman's minimal-model method applied to frequently sampled intravenous glucose tolerance tests in five normal and five islet-autotransplanted mongrel dogs 8-10 mo posttransplantation. Despite normal fasting glucose and insulin levels, glucose tolerance was reduced in the islet-transplanted dogs (1.5 +/- 0.4 vs. 4.2 +/- 0.4%/min in normal controls, P less than .002). The reduction in glucose tolerance was due to a reduced insulin response to glucose injection (4 +/- 1 vs. 32 +/- 5 microU/ml, P less than .001) and reduced glucose effectiveness (1.7 +/- 0.5 vs. 5.3 +/- 0.6 x 10(2) min-1, P less than .005) but not to a reduction in insulin sensitivity (8.4 +/- 0.6 vs. 7.8 +/- 0.7 x 10(4) min-1.microU-1.ml-1, P greater than .5). Our results suggest that reduced insulin secretory response as a result of islet transplantation may result in a defect in glucose's ability to promote its own disposal but not necessarily in a defect in insulin sensitivity.

Viable purified islets of Langerhans from collagenase-perfused human pancreas.

Human islets of Langerhans were isolated with the principles of collagenase perfusion via the pancreatic duct and gentle dissociation of tissue. The number of islets released was 161 x 10(3), distributed as 76 x 10(3) large (greater than 100-micron) and 85 x 10(3) small (less than 100-micron) islets. Recovery after Ficoll-gradient purification was 61% for the large islets and 42% for the small islets. The final islet volume was 240 microliter, with purity of 70-90% (large islets) and 20-40% (small islets). Perifusion with glucose elicited a biphasic release of insulin, with the response rising sixfold from basal secretion. Implantation of pure islets under the kidney capsule of normal or streptozocin-induced diabetic nude mice resulted in human C-peptide secretion and partial or complete reversal of hyperglycemia, confirmed by histological recovery. The data show that these methods provide large quantities of viable purified human islets.

Clinical outcomes and insulin secretion after islet transplantation with the Edmonton protocol.

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.

Treatment of unresectable intrahepatic cholangiocarcinoma with yttrium-90 radioembolization: a systematic review and pooled analysis.

Radioembolization with yttrium-90 microspheres offers an alternative treatment option for patients with unresectable intrahepatic cholangiocarcinoma (ICC). However, the rarity and heterogeneity of ICC makes it difficult to draw firm conclusions about treatment efficacy. Therefore, the goal of the current study is to systematically review the existing literature surrounding treatment of unresectable ICCs with yttrium-90 microspheres and provide a comprehensive review of the current experience and clinical outcome of this treatment modality. We performed a comprehensive search of electronic databases for ICC treatment and identified 12 studies with relevant data regarding radioembolization therapy with yttrium-90 microspheres. Based on pooled analysis, the overall weighted median survival was 15.5 months. Tumour response based on radiological studies demonstrated a partial response in 28% and stable disease in 54% of patients at three months. Seven patients were able to be downstaged to surgical resection. The complication profile of radioembolization is similar to that of other intra-arterial treatment modalities. Overall survival of patients with ICC after treatment with yttrium-90 microspheres is higher than historical survival rates and shows similar survival to those patients treated with systemic chemotherapy and/or trans-arterial chemoembolization therapy. Therefore, the use of yttrium-90 microspheres should be considered in the list of available treatment options for ICC. However, future randomized trials comparing systemic chemotherapy, TACE and local radiation will be required to identify the optimal treatment modality for unresectable ICC.

Investigation of a primary requirement of organ preservation solutions: supplemental buffering agents improve hepatic energy production during cold storage.

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.

Long-term normoglycemia in pancreatectomized dogs following pancreatic islet allotransplantation and cyclosporine immunosuppression.

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.

Reduction of nutrient absorption in normal rats by cyclosporine.

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.

Bile leak from duct of Luschka after liver transplantation.

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.

The metabolic impact of rapamycin (sirolimus) in chronic canine islet graft recipients.

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.

Reversal of diabetes in dogs by transplantation of pure cryopreserved islets.

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.