The success of human kidney allotransplantation was realized over six decades ago. First described 50 years ago, renal autotransplantation has been utilized sparingly as a salvage procedure for patients at risk of losing renal function, either from a benign or malignant condition. While classically associated with colorectal malignancies, Lynch syndrome also carries a small yet significant risk for the development of ureteral carcinoma. For these patients who develop chronic kidney disease, allotransplantation may not be an option due to the lifelong risk of several malignancies. We report the first known case of renal autotransplantation in a patient with metachronous ureteral cancer due to Lynch syndrome.

AIMS/HYPOTHESIS:Beta cell death triggered by pro-inflammatory cytokines plays a central role in the pathogenesis of type 1 diabetes and loss of transplanted islets. The nuclear factor κB (NF-κB) signalling pathway is a key regulator of beta cell stress response, survival and apoptosis. Withaferin A (WA), a steroidal lactone derived from Withania somnifera, has been demonstrated to be a potent, safe, anti-inflammatory molecule that can inhibit NF-κB signalling. Therefore, we evaluated the ability of WA to protect mouse and human islets from the damaging effects of pro-inflammatory cytokines in vitro and following intraportal transplantation.METHODS:Mouse and human islets were treated with a cytokine cocktail, and NF-κB activation was measured by immunoblots, p65 nuclear translocation and chromatin immunoprecipitation of p65-bound DNA. Intraportal transplantation of a marginal mass of syngeneic mouse islets was performed to evaluate the in vivo protective effect of WA.RESULTS:Treatment with WA substantially improved islet engraftment of syngeneic islets (83% for infusion with 200 islets + WA; 0% for 200 islets + vehicle) in a mouse model of diabetes, compared with marginal graft controls with superior islet function in WA-treated mice confirmed by glucose tolerance test. Treatment of human and mouse islets with WA prevented cytokine-induced cell death, inhibited inflammatory cytokine secretion and protected islet potency.CONCLUSIONS:WA was shown to be a strong inhibitor of the inflammatory response in islets, protecting against cytokine-induced cell damage while improving survival of transplanted islets. These results suggest that WA could be incorporated as an adjunctive treatment to improve islet transplant outcome.

Introduction: Prevention of early graft loss is a major issue in improving clinical outcomes of allogeneic as well as autologous islet transplantation. The instant blood mediated inflammatory reaction (IBMIR) has been well investigated in allogeneic islet transplant setting; however, inflammatory or coagulatory responses in autologous islet transplantation (AIT) have not been clearly documented. We focused on systemic inflammatory and coagulatory responses in early phase of clinical AIT. Methods: A total of ten consecutive patients with refractory chronic pancreatitis who underwent AIT at our institute were included in this study. Serum C-peptide, inflammatory and coagulation factors were measured at the following time points: prior, middle and end of islet infusion as well as 15 min, 1 hour, 3 hours, 6 hours and 24 hours after the IAT. Serum C-peptide response during islet infusion was considered as a surrogate maker of early islet destruction and the patients with the early C-peptide response over or less than 1000 pmol/L were grouped as high- or low-damage groups, respectively. Peak values of cytokine and coagulation factors within 24 hours after AIT were compared between the two groups using Mann-Whitney U test non-parametrically. Results: No significant differences were found in patient characteristics and transplant condition except for body mass index (p=0.04, Table 1)[Table 1. Patient and transplant characteristics]Tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-8, interferon γ-induced protein (IP)-10/CXCL10, monocyte chemotactic protein (MCP)-1/CCL2, interferon (IFN)-γ, thrombin-antithrombin complex (TAT), complement 3a (C3a) were significantly elevated within 24 hours after AIT compared to the prior infusion levels (p with nonparametric matched Wilcoxon signed-rank test < 0.01). Among these factors, significant differences were found in TNF-α and C3a levels between the two group (p < 0.05) while IP-10 had marginally significant difference (p = 0.06).[Fig. 1. Elevated inflammatory/coagulatory factors]Conclusions: Systemic inflammatory and coagulatory responses were observed in in early phase of AIT for the patients with chronic pancreatitis. Greater islet graft damage may be associated with higher release of inflammatory/coagulatory factors, suggesting that inhibition of these factors could enhance islet engraftment in AIT. The impact of IBMIR should be validated in large scale clinical studies on AIT.

Introduction: Total pancreatectomy followed by islet autotransplantation (TP/IAT) is a promising therapeutic option for refractory chronic pancreatitis. Multiple centers have shown the efficacy of TP/IAT in reduction of pain while retaining pancreatic endocrine function. We have isolated 100% pure islets without any purification process due to pancreatic acinar atrophy. Here, we compare the outcomes in patients transplanted with pure islets versus mixed islet preparations using purification at our center. Methods: Total four patients with refractory chronic pancreatitis were identified as those transplanted with 100% purely digested islets in our institute where the pancreata were procured with ductal injection, preserved using two-layer method with perfluorocarbon and digested by modified Ricordi method. Digested tissue was purified when the volume was over 10 mL. The final preparation was infused into the liver though portal vein system using bag technique. Patient characteristics, isolation results and clinical outcomes were compared between 100% purely digested cases (pure group) and the others performed using purification (non-pure group, n = 20). Liberase MTF was used for 3 isolations (75%) in pure group and 17 isolations (85%) for non-pure group while the other isolations were employed with Serva collagenase NB (not significant difference). The Mann-Whitney U and Fisher's exact tests were applied to compare continuous and categorical values, respectively. Results: There were no statistically significant differences between these two groups in pre-transplant characteristics except for history of pancreatic surgery. Significantly higher proportion was seen in the pure group (75% versus 10%, p = 0.02, Table 1).[Table 1. Patient characteristics at pre TP-IAT]No significant difference was found in the cold ischemia time. Significantly lower pancreas weight, islet yields, tissue volume and portal vein pressure were observed in pure group. The proportion of patients who achieved insulin independence were not significantly different between the two groups (Table 2).[Table 2. Islet isolation and clinical outcomes]Conclusions: The pure islet cases were characterized by previous pancreatic surgery and by lower islet yield; however, infusion of pure islets minimized tissue volume as well the elevation of portal vein pressure, and achieved similar insulin independence rate compared to the non-pure group. These results suggested that chronic pancreatitis leading to acinar cell atrophy does not significantly affect post-transplant islet function, and does not preclude considering TP/IAT.

Introduction: Allogeneic islet cell transplantation (ICT) is a promising treatment for patients with brittle type 1 diabetes. Recent reports have demonstrated that T cell depletiing immunosuppression protocol could improve islet engraftment as well as prolong graft function. We have implemented the T cell depletion plus antiinflammatory (TCD-AI) protocol using antithymocyte globulin (ATG) and double blockage of IL-1β and TNF-α. Global investigation of peripheral blood transcriptions is helpful for understanding systemic responses as well as discovering diagnostic biomarkers. Modular level analysis for cDNA microarray allows effective interpretation on the background determination with multiple immunologic disease populations (1). In this study, peripheral blood transcriptions were measured to explore systemic biological response for early phase of ICT with TCD-AI protocol. Methods: Five islet recipients received TCD-AI protocol with ATG for induction, along with anakinra and etanercept. Peripheral blood samples were collected on the screening day (baseline), post-transplant days 4, 7, 14, 28 and 42. Isolated RNA were hybridized on to Illumina HumanHT-12 v4 BeadChip. Probe-level differential gene expression analysis was conducted using mixed model adjusting for correlation for longitudinal data to compare between baseline and post-transplant samples. A false discovery rate of 0.1 was used to account for multiple testing. Modular analysis of gene expressions were employed with previously determined sets of genes (1). Results: Four patients (80%) achieved insulin independence after single islet infusion and the median duration of insulin independence is 17.5 months (range: 4-36 months) as of the analysis. The number of genes that were expressed with significant difference (p< 0.05) are 866, 465, 492, 160, 353 and 80 on day 4, 7, 14, 21, 28 and 42 after transplant when compare to baseline. Modular analysis of cDNA microarray on day 4 revealed upregulated modules annotated as interferon responses (M3.4), inflammation (M4.2), myeloid lineage (M4.6) and cell cycle (M6.13), and downregulated modules as coagulation cascade (M3.6), T-cells (M4.1 and M6.15), lymphoid lineage (M4.7 and M6.9) and cytotoxicity (M4.15) (Figure 1). Among these modules, T-cells (M4.1 and M6.19) and cytotoxicity (M4.15) modules were downregulated throughout the study period.[Figure 1]Figure 1. Blood transcriptional fingerprints with islet recipients receiving TCD-AI protocol. Relative changes in whole blood gene expressions with islet recipients for day 4 to 42, compared to baseline. Colored spots represent the percentage of significantly upregulated (red) or downregulated (blue) transcripts (p< 0.002). Conclusion: Gene expression modules annotated as T-cells and cytotoxicity were downregulated in early phase of ICT, suggesting that effective control of cellular immune response as the background mechanism of improved outcome of TCD-AI protocol. (1) Chaussabel D, et al. Immunity 2008;29:150-164.

With increased survival rates after liver transplantation (LTX), current posttransplant treatment aims at minimizing long term complications and side effects of immunosuppression. We analyzed whether donor or recipient age have impact in the incidence of acute cellular rejection (ACR) in the first 6 months after LTX. 1275 patients underwent primary LTX at one institution btween 2001-2010. Data collected prospectively was analyzed retrospectively. 230 patients (18%) were over the age 60 (P60), and 1145 were younger (82%). Patients aged > 60 received lighter immunosuppression by protocol. Their disease severity by MELD score was lower than of younger patients (median laboratory MELD 15 in patients >60 and 17 in younger patients, p=0.0002) Patients with ACR had a lower median age (51 vs. 53with no ACR, p=0.0006). Patients aged >60 had an ACR incidence of 26.4% when receiving organs from donors > 50 years old, 28.8% with organs from donors < 50. Their younger counterparts (< 60) had ACR rates of 39.2% with organs from donors >50 and 34.7% with younger donors (p=0.0381). Steroid resistant rejection was found in 3.5% of patients aged >60 and 5.9% in younger patients (p=0.0381). In contrast, patients transplanted with organs from donors >50 had more steroid-resistant ACR (p=0.026). Univariate logistic regression showed age>60 as the only factor associated with less ACR (OR 0.68, p=0.0159). Univariate regression in patients aged > 60 did not reveal risk factors of significance for ACR. Donor age >50 was associated with an increased risk for steroid resistant ACR (OR 1.78, p=0.0184) in stepwise logistic regression analysis, but not with steroid sensitive ACR. In conclusion, despite the use of less heavy immunosuppression and a lower pretransplant disease severity, patients aged > 60 have a lower incidence of ACR. This justifies minimization of immunosuppression in this patient age category to allow reduction of side effects and less complications from immunosuppressant medication.

Introduction: Instant blood-mediated inflammatory reaction (IBMIR) is known to cause the loss of a significant mass of transplanted islets. Major characteristics of IBMIR include activation of complement and coagulation cascades and infiltration of innate immune cells into the islets. We provide evidence using an in vitro model that blockade of transcription factor NF-κB results in significant inhibition of this inflammatory reaction. Methods: Purified human islets were mixed with heparinized autologous or ABO-matched allogeneic blood in an in vitro tube model. The in vitro model was validated by measurement of pO2, pH and glucose in the blood+islet mix which showed that the mixing of islets and blood retained normal physiological conditions up to 6 h. Low-molecular weight dextran sulfate (LMWDS) alone and in combination with Withaferin A (WA)or resveratrol (known NF-kB inhibitors) were added to the blood-islet mix. Islet viability was measured using Hoechst 33341/propidium iodide staining followed by image analysis. Plasma samples and islet clots were retrieved at regular time points to analyze cytokines and cellular infiltration respectively. Panel of pro-inflammatory cytokines including IFN-γ, IL-6, and IL-8 were measured using Luminex assay. Results: Islet viability increased by 26% over control in the presence of LMWDS; however, addition of WA or resveratrol significantly improved islet viability over control (107-112%; p< 0.02). Exposure of purified islets to both autologous and allogeneic blood increased production of inflammatory cytokines with significant release at 6 hour. Mixing of allogeneic blood invoked a stronger cytokine response (>10 fold) when compared to autologous combination. Presence of WA or resveratrol significantly inhibited release of IFN-γ (4.6±0.1 vs. 2.0±1.0 pg/mL; p< 0.04), IL-6 (6444±196 vs. 712±127 pg/mL; p< 0.001), IL-8 (6368±309 vs. 4257±84 pg/mL; p< 0.005) in allogeneic combination. Immunohistochemistry of islets showed significant reduction in infiltration of neutrophils in both allogeneic (8.2±2.9 vs. 0.5±0.7 cells/islet; p< 0.005) and autologous samples (8.1±2.7 vs. 1.7±0.8 cells/islet; p< 0.001). Conclusion: The results showed that combination of LMWDS and NF-κB blockers offered remarkable protection of islets from IBMIR during the initial phase of the exposure of islets to blood. In summary, control of NF-kB activation could be a key strategy to minimize damage to both autologous and allogeneic islet transplants due to IBMIR.

INTRODUCTION:When patients do not become insulin independent after islet cell transplantation (ICT), another aim is to eliminate severe hypoglycemia. Previously we reported that a secretory unit of islet transplant objects (SUITO) index score >10 was associated with a reduction of severe hypoglycemia. In this study, we assessed patients' satisfaction with their insulin therapy based on the SUITO index. METHODS:The study involved 11 islet recipients with type 1 diabetes who underwent ICT but still used insulin. From those patients, 41 Insulin Therapy Satisfaction Questionnaires (ITSQ) were collected. The SUITO index (fasting C-peptide [ng/mL] × 1500/blood glucose [mg/dL] - 63) was calculated at the same outpatient visits that the survey was administered. ITSQ scores were summarized using subscales and compared among 3 groups: the pre-ICT group, the low-SUITO group (SUITO index score <10 post-ICT), and the high-SUITO group (SUITO index score ≥10). Higher survey scores indicated better satisfaction. RESULTS:Significant trend relationships across the 3 groups were observed in the ITSQ total score (P = .02 with Jonckheere-Terpstra test) and subscale scores of glycemic control (P < .001), hypoglycemic control (P = .01), and inconvenience of regimen (P = .004). The pairwise comparisons between the 3 groups found significant differences: high SUITO versus both pre-ICT and low SUITO for the total ITSQ score (P = .03 and .005, respectively) and glycemic control score (P = .008 and .001, respectively), and high SUITO versus low SUITO for hypoglycemic control score (P = .04) and inconvenience of regimen score (P = .008). CONCLUSION:Islet recipients with a SUITO index ≥10 experienced higher satisfaction with insulin injection therapy compared with the pre-ICT group, even though they were insulin dependent. A SUITO index ≥10 is a reasonable benchmark for successful ICT.

Introduction. Islet purification is mainly performed by the density gradient method. However, purification of the embedded islets that are surrounded by exocrine tissue should be difficult, because their density is similar to exocrine tissue. In this study, we performed chart review to assess the relationship between the ratio of embedded islets and efficacy of purification. Then, we tested several conditions of a new method to free the islets from surrounded exocrine tissues using high osmolality solution with gentle agitation.Materials and Methods. First, we performed chart review of our human islet isolation. Second, embedded islet-enriched human islet fractions (embedded islets >50%) were suspended in University of Wisconsin (UW) solution (UW group, 320 mOsm/kg/H(2)0) or osmolality-adjusted UW solution (400, 500, and 600 mOsm/kg/H20; 400 group, 500 group, and 600 group, respectively). Each tube was gently shaken at 4 degrees C. The tissue samples were taken before shaking and after 15, 30, and 60 minutes. Islet yield, percentage of embedded islets, and viabilities were assessed.Results. The chart review revealed that high ratio of embedded islets deteriorated the efficacy of islet purification. The islet yield in all groups except for the 600 group did not change at 15 minutes, but it decreased in all groups at 60 minutes. The average percentage of embedded islets before shaking was 62.6%. Although percentage of embedded islets were decreasing in all groups, it was < 20% at 15 minutes in the 500 and 600 groups whereas it was >44% in the UW group, which indicated that higher osmolality would have a greater effect. Viability was >95% in all groups at 30 minutes.Conclusions. The embedded islets deteriorated the efficacy of islet purification. Gentle agitation of embedded islets in high osmolality (500 mOsm/kg/H(2)0, 15 minutes) could release islets from surrounded exocrine tissue.