David D. Lee

Recovery of Islet β-Cell Function in Streptozotocin- Induced Diabetic Mice An Indirect Role for the Spleen

Limitations in islet β-cell transplantation as a therapeutic option for type 1 diabetes have prompted renewed interest in islet regeneration as a source of new islets. In this study we tested whether severely diabetic adult C57BL/6 mice can regenerate β-cells. Diabetes was induced in C57BL/6 mice with high-dose streptozotocin (160−170 mg/kg). In the absence of islet transplantation, all diabetic mice remained diabetic (blood glucose >400 mg/dl), and no spontaneous reversal of diabetes was observed. When syngeneic islets (200/mouse) were transplanted into these diabetic mice under a single kidney capsule, stable restoration of euglycemia for ≥120 days was achieved. Removal of the kidney bearing the transplanted islets at 120 days posttransplantation revealed significant restoration of endogenous β-cell function. This restoration of islet function was associated with increased β-cell mass, as well as β-cell hypertrophy and proliferation. The restoration of islet cell function was facilitated by the presence of a spleen; however, the facilitation was not due to the direct differentiation of spleen-derived cells into β-cells. This study supports the possibility of restoring β-cell function in diabetic individuals and points to a role for the spleen in facilitating this process.

Glycemic Control Promotes Pancreatic Beta-Cell Regeneration in Streptozotocin-Induced Diabetic Mice

Background Pancreatic beta-cells proliferate following administration of the beta-cell toxin streptozotocin. Defining the conditions that promote beta-cell proliferation could benefit patients with diabetes. We have investigated the effect of insulin treatment on pancreatic beta-cell regeneration in streptozotocin-induced diabetic mice, and, in addition, report on a new approach to quantify beta-cell regeneration in vivo. Methodology/Principal Findings Streptozotocin-induced diabetic were treated with either syngeneic islets transplanted under the kidney capsule or subcutaneous insulin implants. After either 60 or 120 days of insulin treatment, the islet transplant or insulin implant were removed and blood glucose levels monitored for 30 days. The results showed that both islet transplants and insulin implants restored normoglycemia in the 60 and 120 day treated animals. However, only the 120-day islet and insulin implant groups maintained euglycemia (<200 mg/dl) following discontinuation of insulin treatment. The beta-cell was significantly increased in all the 120 day insulin-treated groups (insulin implant, 0.69±0.23 mg; and islet transplant, 0.91±0.23 mg) compared non-diabetic control mice (1.54±0.25 mg). We also show that we can use bioluminescent imaging to monitor beta-cell regeneration in living MIP-luc transgenic mice. Conclusions/Significance The results show that insulin treatment can promote beta-cell regeneration. Moreover, the extent of restoration of beta-cell function and mass depend on the length of treatment period and overall level of glycemic control with better control being associated with improved recovery. Finally, real-time bioluminescent imaging can be used to monitor beta-cell recovery in living MIP-luc transgenic mice.

Longitudinal confocal microscopy imaging of solid tumor destruction following adoptive T cell transfer

A fluorescence-based, high-resolution imaging approach was used to visualize longitudinally the cellular events unfolding during T cell-mediated tumor destruction. The dynamic interplay of T cells, cancer cells, cancer antigen loss variants, and stromal cells—all color-coded in vivo—was analyzed in established, solid tumors that had developed behind windows implanted on the backs of mice. Events could be followed repeatedly within precisely the same tumor region—before, during and after adoptive T cell therapy—thereby enabling for the first time a longitudinal in vivo evaluation of protracted events, an analysis not possible with terminal imaging of surgically exposed tumors. T cell infiltration, stromal interactions, and vessel destruction, as well as the functional consequences thereof, including the elimination of cancer cells and cancer cell variants were studied. Minimal perivascular T cell infiltrates initiated vascular destruction inside the tumor mass eventually leading to macroscopic central tumor necrosis. Prolonged engagement of T cells with tumor antigen-crosspresenting stromal cells correlated with high IFNγ cytokine release and bystander elimination of antigen-negative cancer cells. The high-resolution, longitudinal, in vivo imaging approach described here will help to further a better mechanistic understanding of tumor eradication by T cells and other anti-cancer therapies.

Cellular therapies for type 1 diabetes.

Type 1 diabetes mellitus (T1DM) is a disease that results from the selective autoimmune destruction of insulin-producing beta-cells. This disease process lends itself to cellular therapy because of the single cell nature of insulin production. Murine models have provided opportunities for the study of cellular therapies for the treatment of diabetes, including the investigation of islet transplantation, and also the possibility of stem cell therapies and islet regeneration. Studies in islet transplantation have included both allo- and xeno-transplantation and have allowed for the study of new approaches for the reversal of autoimmunity and achieving immune tolerance. Stem cells from hematopoietic sources such as bone marrow and fetal cord blood, as well as from the pancreas, intestine, liver, and spleen promise either new sources of islets or may function as stimulators of islet regeneration. This review will summarize the various cellular interventions investigated as potential treatments of T1DM.

Early allograft dysfunction after liver transplantation: An intermediate outcome measure for targeted improvements

BACKGROUND The term early allograft dysfunction (EAD) identifies liver transplant (LT) allografts with initial poor function and portends poor allograft and patient survival. Aims of this study are to use EAD as an intermediate outcome measure in a large single center cohort and identify donor, recipient and peri-operative risk factors. MATERIAL AND METHODS In 1950 consecutive primary LT, donor, recipient and peri-operative data were collected. EAD was defined by the presence of one or more of the following: total bilirubin ≥ 10 mg/dL (171 μmol/L) or, INR ≥ 1.6 on day 7, and ALT/AST > 2,000 IU/L within the first 7 days. RESULTS The incidence of EAD was 26.5%. 1-, 3-, and 5-year allograft and patient survival for patients who developed EAD were significantly inferior to those who did not (P < 0.01 at all time points). Multivariate analysis demonstrated associations in the development of EAD with recipient pre-operative ventilator status, donation after cardiac death allografts, donor age, allograft size, degree of steatosis, operative time and intra-operative transfusion requirements (all P < 0.01). Patients with EAD had a significantly longer hospitalization at 20.9 ± 38.9 days (median: 9; range: 4-446) compared with 10.7 ± 13.5 days (median: 7; range: 3-231) in patients with no EAD (P < 0.01). CONCLUSIONS This is the largest single center experience demonstrating incidence of EAD and identifying factors associated with development of EAD. EAD is a useful intermediate outcome measure for allograft and patient survival. Balancing recipient pretransplant conditions, donor risk factors and intra-operative conditions are necessary for avoiding EAD.BACKGROUND The term early allograft dysfunction (EAD) identifies liver transplant (LT) allografts with initial poor function and portends poor allograft and patient survival. Aims of this study are to use EAD as an intermediate outcome measure in a large single center cohort and identify donor, recipient and peri-operative risk factors. MATERIAL AND METHODS In 1950 consecutive primary LT, donor, recipient and peri-operative data were collected. EAD was defined by the presence of one or more of the following: total bilirubin ≥ 10 mg/dL (171 μmol/L) or, INR ≥ 1.6 on day 7, and ALT/AST > 2,000 IU/L within the first 7 days. RESULTS The incidence of EAD was 26.5%. 1-, 3-, and 5-year allograft and patient survival for patients who developed EAD were significantly inferior to those who did not (P < 0.01 at all time points). Multivariate analysis demonstrated associations in the development of EAD with recipient pre-operative ventilator status, donation after cardiac death allografts, donor age, allograft size, degree of steatosis, operative time and intra-operative transfusion requirements (all P < 0.01). Patients with EAD had a significantly longer hospitalization at 20.9 ± 38.9 days (median: 9; range: 4-446) compared with 10.7 ± 13.5 days (median: 7; range: 3-231) in patients with no EAD (P < 0.01). CONCLUSIONS This is the largest single center experience demonstrating incidence of EAD and identifying factors associated with development of EAD. EAD is a useful intermediate outcome measure for allograft and patient survival. Balancing recipient pretransplant conditions, donor risk factors and intra-operative conditions are necessary for avoiding EAD.

IMPROVING NATIONAL RESULTS IN LIVER TRANSPLANTATION USING GRAFTS FROM DONATION AFTER CARDIAC DEATH DONORS.

Background Published reports describing the national experience with liver grafts from donation after cardiac death (DCD) donors have resulted in reservations with their widespread utilization. The present study aimed to investigate if temporal improvements in outcomes have been observed on a national level and to determine if donor and recipient selection have been modified in a fashion consistent with published data on DCD use in liver transplantation (LT). Methods Patients undergoing DCD LT between 2003 and 2014 were obtained from the United Network of Organ Sharing Standard Transplant Analysis and Research file and divided into 3 equal eras based on the date of DCD LT: era 1 (2003-2006), era 2 (2007-2010), and era 3 (2011-2014). Results Improvement in graft survival was seen between era 1 and era 2 (P = 0.001) and between era 2 and era 3 (P < 0.001). Concurrently, an increase in the proportion of patients with hepatocellular carcinoma and a decrease in critically ill patients, retransplant recipients, donor age, warm ischemia time greater than 30 minutes and cold ischemic time also occurred over the same period. On multivariate analysis, significant predictors of graft survival included: recipient age, biologic MELD score, recipient on ventilator, recipient hepatitis C virus + serology, donor age and cold ischemic time. In addition, even after adjustment for all of the aforementioned variables, both era 2 (hazard ratio, 0.81; confidence interval, 0.69-0.94; P = 0.007), and era 3 (hazard ratio, 0.61; confidence interval, 0.5-0.73; P < 0.001) had a protective effect compared to era 1. Conclusions The national outcomes for DCD LT have improved over the last 12 years. This change was associated with modifications in both recipient and donor selection. Furthermore, an era effect was observed, even after adjustment for all recipient and donor variables on multivariate analysis.

Extracellular vesicles from bone marrow–derived mesenchymal stem cells protect against murine hepatic ischemia/reperfusion injury

Hepatic ischemia/reperfusion injury (IRI) and associated inflammation contributes to liver dysfunction and complications after liver surgery and transplantation. Mesenchymal stem cells (MSCs) have been reported to reduce hepatic IRI because of their reparative immunomodulatory effects in injured tissues. Recent studies have highlighted beneficial effects of extracellular vesicles from mesenchymal stem cells (MSC‐EV) on tissue injury. The effects of systemically administered mouse bone marrow–derived MSC‐EV were evaluated in an experimental murine model of hepatic IRI induced by cross‐clamping the hepatic artery and portal vein for 90 minutes followed by reperfusion for periods of up to 6 hours. Compared with controls, intravenous administration of MSC‐EV 30 minutes prior to IRI dramatically reduced the extent of tissue necrosis, decreased caspase 3–positive and apoptotic cells, and reduced serum aminotransferase levels. MSC‐EV increased hepatic messenger RNA (mRNA) expression of NACHT, LRR, and PYD domains‐containing protein 12, and the chemokine (C‐X‐C motif) ligand 1, and reduced mRNA expression of several inflammatory cytokines such as interleukin 6 during IRI. MSC‐EV increased cell viability and suppressed both oxidative injury and nuclear factor kappa B activity in murine hepatocytes in vitro. In conclusion, the administration of extracellular vesicles derived from bone marrow–derived MSCs may ameliorate hepatic IRI by reducing hepatic injury through modulation of the inflammatory response.Liver Transplantation 23 791–803 2017 AASLD.

Comparison of longterm outcomes and quality of life in recipients of donation after cardiac death liver grafts with a propensity‐matched cohort

The use of liver grafts from donation after cardiac death (DCD) has been limited due to the increased rate of graft failure, mostly related to ischemic cholangiopathy (IC). It is our hypothesis that longterm outcomes and quality of life (QOL) similar to patients undergoing liver transplantation (LT) with donation after brain death (DBD) can be achieved. Clinical outcomes of all patients undergoing DCD LT (n = 300) between 1998 and 2015 were compared with a propensity score–matched cohort of patients undergoing DBD LT (n = 300). Patients were contacted for a follow‐up questionnaire and short‐form (SF)–12 QOL Survey administration. Median follow‐up was >5 years. Graft survival at 1‐, 3‐, and 5‐years was 83.8%, 75.5%, and 70.1% in the DCD LT group and 88.4%, 80.3%, and 73.9% in the DBD LT group (P = 0.27). Patient survival at 1‐, 3‐, and 5‐years was 92.3%, 86.1%, and 80.3% in the DCD LT group and 92.3%, 85.1%, and 79.5% in the DBD LT group (P = 0.81). IC developed in 11.7% and 2% of patients in the DCD LT group and DBD LT group, respectively (P < 0.001). DCD LT recipients who developed IC had inferior graft survival compared with both the DCD non‐IC group (P < 0.001) and the DBD LT group (P < 0.001); no difference in graft survival was observed between the DCD non‐IC group and the DBD LT group (P = 0.50). Physical and Mental Composite Scores on the SF‐12 QOL questionnaire were similar between the DCD LT and DBD LT groups (44.0 versus 45.4; P = 0.34 and 51.9 versus 52.2; P = 0.83), respectively. Similar longterm survival and QOL scores can be achieved between DCD LT and DBD LT. Prevention of IC in DCD LT yields excellent graft and patient survival with virtually no difference compared with DBD LT. Liver Transplantation 23 342–351 2017 AASLD.

Effects of the Share 35 Rule on Waitlist and Liver Transplantation Outcomes for Patients with Hepatocellular Carcinoma.

Introduction Several studies have investigated the effects following the implementation of the “Share 35” policy; however none have investigated what effect this policy change has had on waitlist and liver transplantation (LT) outcomes for hepatocellular carcinoma(HCC). Methods Data were obtained from the UNOS database and a comparison of the 2 years post-Share 35 with data from the 2 years pre-Share 35 was performed. Results In the pre-Share35 era, 23% of LT were performed for HCC exceptions compared to 22% of LT in the post-Share35 era (p = 0.21). No difference in wait-time for HCC patients was seen in any of the UNOS regions between the 2 eras. Competing risk analysis demonstrated that HCC candidates in post-Share 35 era were more likely to die or be delisted for “too sick” while waiting (7.2% vs. 5.3%; p = 0.005) within 15 months. A higher proportion of ECD (p<0.001) and DCD (p<0.001) livers were used for patients transplanted for HCC, while lower DRI organs were used for those patients transplanted with a MELD≥35 between the 2 eras (p = 0.007). Conclusion No significant change to wait-time for patients listed for HCC was seen following implementation of “Share 35”. Transplant program behavior has changed resulting use of higher proportion of ECD and DCD liver grafts for patients with HCC. A higher rate of wait list mortality was observed in patients with HCC in the post-Share 35 era.

Intraregional model for end‐stage liver disease score variation in liver transplantation: Disparity in our own backyard

Variation in average Model for End‐Stage Liver Disease (MELD) score at liver transplantation (LT) by United Network for Organ Sharing (UNOS) regions is well documented. The present study aimed to investigate MELD variation at the interregional, intraregional, and intra–donation service area (DSA) levels. Patients undergoing LT between 2015 and 2016 were obtained from the UNOS standard analysis and research file. The distribution of allocation MELD score including median, skew, and kurtosis was examined for all transplant programs. Intraregional median allocation MELD varied significantly within all 11 UNOS regions. The largest variation between programs was seen in region 5 (MELD 24.0 versus 38.5) and region 3 (MELD 20.5 versus 32.0). Regions 1, 5, and 9 had the largest proportion of programs with a highly negative skewed MELD score (50%, 57%, and 57%, respectively), whereas regions 3, 6, 10, and 11 did not have any programs with a highly negative skew. MELD score distribution was also examined in programs located in the same DSA, where no barriers exist and theoretically no significant difference in allocation should be observed. The largest DSA variation in median allocation MELD score was seen in NYRT‐OP1 LiveOnNY (MELD score variation 11), AZOB‐OP1 Donor Network of Arizona (MELD score variation 11), MAOB‐OP1 New England Organ Bank (MELD score variation 9), and TXGC‐OP1 LifeGift Organ Donation Ctr (MELD score variation 9). In conclusion, the present study demonstrates that this MELD disparity is not only present at the interregional level but can be seen within regions and even within DSAs between programs located as close as several city blocks away. Although organ availability likely accounts for a component of this disparity, the present study suggests that transplant center behavior may also play a significant role. Liver Transplantation 24 488–496 2018 AASLD.