Norman M. Kneteman

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.

Islet isolation assessment in man and large animals

SummaryRecent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity andin vitro andin vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 µ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason,in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.

Variables in organ donors that affect the recovery of human islets of Langerhans.

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.

Sirolimus‐based immunosuppression is associated with increased survival after liver transplantation for hepatocellular carcinoma

Liver transplantation is an important treatment option for selected patients with nonresectable hepatocellular carcinoma (HCC). Several reports have suggested a lower risk of posttransplant tumor recurrence with the use of sirolimus and a higher one with calcineurin inhibitors, but the selection of an ideal immunosuppression protocol is still a matter of debate. The aim of this study was to define the immunosuppression associated with the best survival after liver transplantation for HCC. It was based on the Scientific Registry of Transplant Recipients and included 2,491 adult recipients of isolated liver transplantation for HCC and 12,167 for non‐HCC diagnoses between March 2002 and March 2009. All patients remained on stable maintenance immunosuppression protocols for at least 6 months posttransplant. In a multivariate analysis, only anti‐CD25 antibody induction and sirolimus‐based maintenance therapy were associated with improved survivals after transplantation for HCC (hazard ratio [HR] 0.64, 95% confidence interval [CI]: 0.45–0.9, P ≤ 0.01; HR 0.53, 95% CI: 0.31–0.92, P ≤ 0.05, respectively). The other studied drugs, including calcineurin inhibitors, did not demonstrate a significant impact. In an effort to understand whether the observed effects were due to a direct impact of the drug on tumor or more on liver transplant in general, we conducted a similar analysis on non‐HCC patients. Although anti‐CD25 induction was again associated with a trend toward improved survival, sirolimus showed a trend toward lower rates of survival in non‐HCC recipients, confirming the specificity of its beneficial impact to cancer patients. Conclusion: According to these data, sirolimus‐based immunosuppression has unique posttransplant effects on HCC patients that lead to improved survival. (HEPATOLOGY 2010.)

Reassessing selection criteria prior to liver transplantation for hepatocellular carcinoma utilizing the scientific registry of transplant recipients database

The current model of liver graft allocation in place in the United States favors transplantation of patients with small hepatocellular carcinomas (HCCs) within the Milan criteria (a single tumor up to 5 cm in diameter or up to three lesions, none larger than 3 cm). Although several reports have suggested that these criteria could be extended, there is currently no agreement on new selection tools. In this study, we performed an overview of 6478 adult recipients of an isolated first liver transplant registered in the Scientific Registry of Transplant Recipients (SRTR) database. From March 2002 to January 2008, increasing numbers of patients outside Milan criteria (P ≤ 0.001) have been registered for a transplant, but they still represent less than 5% of the transplants performed for HCC. Of all the tested variables (tumor number, largest tumor size, and Milan and University of California San Francisco criteria), only total tumor volume (TTV; P ≤ 0.05) and alpha fetoprotein (AFP; P ≤ 0.001) could predict patient survival. While these two parameters demonstrated independent behaviors (no patient demonstrated an increase in both values), a composite score was defined, with patients with a TTV > 115 cm3 or an AFP > 400 ng/mL being outside criteria. The combined TTV/AFP score efficiently predicted posttransplant survival (hazard ratio = 2, 95% confidence interval = 1.7‐2.4, P ≤ 0.001); patients not meeting these criteria had a survival below 50% at 3 years. Conclusion: According to the present SRTR data, Milan criteria are too restrictive, and patients with larger TTV can enjoy satisfactory posttransplant survivals. A composite patient selection score combining TTV and AFP was the most effective of all tested staging criteria for the prediction of posttransplant patient survival for candidates with HCC. (HEPATOLOGY 2009.)

Sirolimus-based immunosuppression for liver transplantation in the presence of extended criteria for hepatocellular carcinoma.

An increasing number of patients with hepatocellular carcinoma (HCC) are undergoing evaluation for listing for liver transplantation. Criteria for selection require ongoing review for suitability. A consecutive series of 40 patients with HCC within the standard Milan criteria (single tumors n = 19 <5 cm, or up to 3 tumors <3 cm) and beyond (Extended Criteria; single tumors n = 21 <7.5 cm, multiple tumors <5 cm) underwent liver transplant with a sirolimus‐based immunosuppressive protocol designed to minimize exposure to calcineurin inhibitors and steroids. At 44.3 ± 19.3 months (mean ± standard deviation) follow‐up, 1‐ and 4‐year survivals (Kaplan‐Meier) are 94.1 ± 5.7% and 87.4 ± 9.3%, in the Milan group, respectively, and 90.5 ± 6.4% and 82.9 ± 9.3% in the Extended Criteria group, respectively. Five patients died during follow‐up, only 1 from recurrent HCC. Five tumor recurrences have occurred at median 17 (mean 22 ± 17) months posttransplant, 1 in the Milan group and 4 in the Extended Criteria group. Median survival in the patients with recurrent tumor is 42 months (mean 45 ± 25), and the median postrecurrence survival is 15.5 months (mean 23 ± 16). The rate of patients who were alive and free of tumor at 1 and 4 years is 94.1 ± 5.7% and 81.1 ± 9.9%, respectively, in the Milan group and is 90.5 ± 6.4% and 76.8 ± 10.5%, respectively, in the Extended Criteria group. Five patients had sirolimus discontinued for toxicity, while 24 of 35 surviving patients have sirolimus monotherapy immunosuppression. In conclusion, the Milan criteria for liver transplantation in the presence of HCC can be carefully extended without compromising outcomes. This sirolimus based immunosuppression protocol appears to have beneficial effects on tumor recurrence and survival with an acceptable rate of rejection and toxicity. (Liver Transpl 2004;10:1301–1311.)

Intraductal collagenase delivery into the human pancreas using syringe loading or controlled perfusion.

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°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°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°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 × 103 IE in the perfusion group compared with 391 ± 57 × 103 IE for the syringe-loaded group. Postpurification recovery was higher in the perfused group (379 ± 45 vs. 251 ± 28 × 103 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.

Long-term follow-up after transplantation of insulin-producing pancreatic islets into patients with Type 1 (insulin-dependent) diabetes mellitus

SummaryPurified human islets and a kidney from the same donor were transplanted into four patients with Type 1 (insulin-dependent) diabetes mellitus. Two of the patients received additional islets that were isolated from multiple donors, cryopreserved, and stored in a tissue bank. The islets were embolized into the liver via the portal vein. Immunosuppression was induced with antilymphocyte globulin and maintained with azathioprine, prednisone and cyclosporine. In the first two patients, fasting serum C-peptide rose to levels of 0.5–2.0 ng/ml during the first 4–8 weeks and mixed meal feeding elicited increases to 2–3 ng/ml. C-peptide secretion persisted for 8 months, but at progressively lower levels and insulin therapy could not be withdrawn. In the next two patients who received cryopreserved islets in addition to fresh islets, serum C-peptide levels (fasting/post-meal) rose to 4–7 ng/ml and serum glucose was more stable, allowing withdrawal of insulin therapy after 69 days in one patient, and reduced insulin doses in the other. The insulin-independent patient has maintained normal fasting glucose, glycosylated haemoglobin, and oral glucose tolerance at 1 year following cessation of daily insulin therapy. Episodes of renal graft rejection occurred in three patients, including the insulin-independent patient. High-dose steroid therapy reversed the rejection in all instances, with apparent preservation of C-peptide secretion. These data show that transplantation of purified freshly-prepared and cryopreserved islets into Type 1 diabetic patients results in prolonged insulin secretion, and that sufficient function could be provided in one patient to sustain euglycaemia in the absence of insulin therapy at 1 year of follow-up.

Peak cyclosporine levels (Cmax) correlate with freedom from liver graft rejection: results of a prospective, randomized comparison of neoral and sandimmune for liver transplantation (NOF-8).

BACKGROUND Despite two decades of use, there are limited data on the best way to administer and monitor cyclosporine (CsA) for liver transplantation. The present study was undertaken (1) to determine whether treatment with a new formulation of CsA, Neoral, would improve the results of liver transplantation; and (2) to study the relationships between pharmacokinetic parameters and clinical outcomes after transplantation. METHODS A double-blind, randomized, comparison of Sandimmune (SIM) with Neoral (NEO) was conducted at five Canadian centers in 188 consecutive adults undergoing primary orthotopic liver transplantation. Patients were induced with intravenous CsA then switched to NEO or SIM. Dose adjustments were made daily, or as needed, to reach a target trough CsA level of 350 ng/ml in both groups. Pharmacokinetic studies were performed on days 5, 10, 15, and 16 weeks after transplantation. RESULTS The NEO group was slightly younger, with a median age of 50 years (range: 23-70) versus 55 years (range: 24-71) for SIM (P = 0.007); otherwise the two groups were well balanced. The NEO group stopped intravenous CsA earlier (5.8+/-2.6 days vs. 8.7+/-4.7 days, P<0.0001). This group required a lower median daily oral dose (7.5 mg/kg vs. 9.0 mg/kg, P<0.01) to maintain comparable trough CsA levels. Five SIM patients, but no NEO patients, discontinued the study due to 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 (93% NEO vs. 91% SIM), graft survival (90% NEO vs. 86% SIM), and rejection-free survival (54.1% NEO, 51.8% SIM). The incidence of serious adverse events was also similar and did not correlate with CsA pharmacokinetic profiles. The NEO group had a higher area under the drug concentration curve for the first 6 hr after the dosing interval (AUC0-6) and peak CsA levels (Cmax). There was a strong correlation between freedom from graft rejection during the first month after transplantation and (a) AUC0-6 and (b) Cmax at days 5 and 10 after transplantation, but only in the NEO group did this reach statistical significance. In contrast, there was a poor correlation between trough CsA and graft rejection. In patients on NEO, the concentration of CsA 2 hr after dosing (C2) closely reflected AUC0-6 (r2 = 0.93), whereas there was a poorer correlation in patients on SIM (r2 = 0.73) CONCLUSIONS Cmax and/or AUC0-6 may provide better markers than trough levels for monitoring CsA-based immune suppression after orthotopic liver transplantation. Prospective studies are underway to determine whether dosing to C2, which provides a good estimation of Cmax, can be used to take full advantage of NEOs improved absorption profile.

Normoglycaemia after transplantation of freshly isolated and cryopreserved pancreatic islets in Type 1 (insulin-dependent) diabetes mellitus

SummaryPurified islets of Langerhans and a kidney were transplanted into a 36-year-old patient who suffeded from renal failure secondary to a 25 year history of Type 1 (insulin-dependent) diabetes mellitus. The islet graft contained 243 000 fresh islets (mean islet diameter 150 μm) that were syngeneic with the kidney fraft and 368 000 cryopreserved islets that had been collected from four other donors. The total of 10 000 islets/kg body weight was infused into the liver via the umbilical vein. Immunosupperession was induced with antilymphocyte globulin and maintained with prednisone, cyclosporine and azathioprine. Serum C-peptide levels (ng/ml) during fasting and after standard mixed metal feeding (Sustacal) were <0.12 preoperatively. Postoperatively, insulin secretion was restored: fasting C-peptide rose during the first 4 weeks to levels of 4 to 5 and Sustacal elicited a further rise to 6 to 7. Transplant renal function was stable. Dialy fasting glucose (mmol/l, mean±SD) was 5.6±1 and 5.3±0.6 during the first and second months respectively and post-Sustacal glucose was 5.7+-0.8. Exogenous insulin therapy was progressively withdrawn and stopped duting the ninth week. Thereafter, fasting glucose was 4.7+-0.5, 24 h mean glucose was 6.6+-0.5, and normoglycaemia was maintained after Sustacal. These data show that this mass of freshly isolated and cryopreserved islets from multiple donors provided sustained function (3 months) that reversed insulin-dependence in an immunosuppressed Type 1 diabetic patient treated with simultaneous islet-kedney transplantation.