Stephen Hanley

Donor and Isolation Variables Predicting Human Islet Isolation Success

Background. Recent advances in the fields of islet transplantation and in vitro islet cell expansion place a renewed emphasis on the optimization of islet isolation from cadaveric human donor organs. We retrospectively analyzed 171 islet isolations to identify variables that predict islet yield and isolation success. Methods. Cadaveric human donor pancreata were procured and processed according to established protocols. Donor-, procurement-, and isolation-related variables were analyzed for correlation with islet yield and isolation success (≥250,000 islet equivalents). Results. Univariate analysis suggested correlations between islet yield and donor age (P<0.005), body surface area (P<0.005), duration of enzymatic digestion (P<0.001), and pancreatic β-cell volume (P<0.05). Donor sex (P<0.01), procurement team (P<0.05), and peridigestion serine protease inhibition (P<0.05) affected islet yield, whereas enzyme lot (P<0.01) and pancreatic fatty infiltration (P<0.05) influenced isolation success. By logistic regression, donor sex and age, and duration of enzymatic digestion could predict a successful isolation with 72% accuracy. The use of Liberase CI improved islet yield (P<0.05) in young donors (≤25 years). Conclusions. While donor-related variables are useful in predicting islet yield, these are likely surrogates for pancreatic β-cell volume. Enzyme lot, and the associated duration of enzymatic digestion (P<0.05), appears to be key determinants of isolation success.

Autocrine insulin action activates Akt and increases survival of isolated human islets

Aims/hypothesisThe phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays a critical role in promoting the survival of pancreatic beta cells. Akt becomes activated in isolated human islets following overnight culture despite significant levels of cell death. The aim of the current study was to identify the cause of the observed increase in Akt phosphorylation in isolated islets. We hypothesised that a factor secreted by the islets in culture was acting in an autocrine manner to activate Akt.MethodsIn order to identify the stimulus of the PI3K/Akt pathway in culture, we examined the effects of different culture conditions on Akt phosphorylation and islet survival during the immediate post-isolation period.ResultsWe demonstrated that islet-conditioned medium induced Akt phosphorylation in freshly isolated human islets, whereas frequent medium replacement decreased Akt phosphorylation. Following overnight culture, islet-conditioned medium contained significantly elevated levels of insulin, indicating that insulin may be responsible for the observed increase in Akt phosphorylation. Indeed, treatment with an anti-insulin antibody or with inhibitors of insulin receptor/IGF receptor 1 kinase activity suppressed Akt phosphorylation, leading to decreased islet survival. In addition, dispersion of islets into single cells also suppressed Akt phosphorylation and induced islet cell death, indicating that islet integrity is also required for maximal Akt phosphorylation.Conclusions/interpretationOur findings demonstrate that insulin acts in an autocrine manner to activate Akt and mediate the survival of isolated human islets. These findings provide new information on how culturing islets prior to transplantation may be beneficial to their survival by allowing for autocrine activation of the pro-survival Akt pathway.

The role of Islet Neogenesis-Associated Protein (INGAP) in islet neogenesis

Islet Neogenesis-Associated Protein (INGAP) is a member of the Reg family of proteins implicated in various settings of endogenous pancreatic regeneration. The expression of INGAP and other RegIII proteins has also been linked temporally and spatially with the induction of islet neogenesis in animal models of disease and regeneration. Furthermore, administration of a peptide fragment of INGAP (INGAP peptide) has been demonstrated to reverse chemically induced diabetes as well as improve glycemic control and survival in an animal model of type 1 diabetes. Cultured human pancreatic tissue has also been shown to be responsive to INGAP peptide, producing islet-like structures with function, architecture and gene expression matching that of freshly isolated islets. Likewise, studies in normoglycemic animals show evidence of islet neogenesis. Finally, recent clinical studies suggest an effect of INGAP peptide to improve insulin production in type 1 diabetes and glycemic control in type 2 diabetes.

Tumor necrosis factor-alpha production by human islets leads to postisolation cell death.

Background. Recent successes in islet transplantation highlight the importance of islet isolation by experienced centers and minimization of cell injury as crucial to the achievement of insulin independence. Islet injury may manifest as cell death by apoptosis, shorter graft survival, and the need for retransplantation. Although an inflammatory cytokine response at the graft site is known to inhibit engraftment, recent evidence indicates that islet cells may contribute to this response. Methods. Isolated human islets were cultured for up to one week in serum-free CMRL-1066 with 25 &mgr;M of tumor necrosis factor (TNF)&agr; inhibitor RDP58. Gene expression was measured by reverse transcriptase polymerase chain reaction, apoptosis and TNF&agr; secretion by enzyme-linked immunosorbent assay and enzyme-linked immunospot, and islet function by stimulated insulin secretion. Results. Isolation induced a twofold increase in TNF&agr; expression between days one and three (P<0.05), while TNF&agr; secretion peaked at day one. RDP58 reduced TNF&agr; secretion by 70.6% (P<0.02), though TNF&agr; gene expression was unaffected. RDP58 reduced the frequency of TNF&agr;-secreting islets by 64.4% (P<0.05) and reduced apoptotic levels by 26.4% within 24 hr postisolation (P<0.05). The reduction in apoptosis was maintained throughout the week (P<0.01), while apoptosis increased in control cultures. Finally, RDP58-treated islets displayed increased insulin secretion in response to both elevated glucose (1915.0±396.6 vs. 825.3±261.1 mU/L, P<0.01) and secretagogues (2294.3±529.5 vs. 939.8±333.7 mU/L, P<0.02). Conclusions. These data demonstrate that intraislet cytokine production should be considered as a factor leading to islet cell death postisolation and postengraftment, and strategies aimed at countering islet cytokine production represent a novel target for improving islet viability and function.

Epidermal growth factor induces adult human islet cell dedifferentiation

Given the inherent therapeutic potential of the morphogenetic plasticity of adult human islets, the identification of factors controlling their cellular differentiation is of interest. The epidermal growth factor (EGF) family has been identified previously in the context of pancreatic organogenesis. We examined the role of EGF in an in vitro model whereby adult human islets are embedded in a collagen gel and dedifferentiated into duct-like epithelial structures (DLS). We demonstrated that DLS formation was EGF dependent, while residual DLS formation in the absence of added EGF was abrogated by EGF receptor inhibitor treatment. With respect to signaling, EGF administration led to an increase in c-Jun NH2-terminal kinase (JNK) phosphorylation early in DLS formation and in AKT and extracellular signal-regulated kinase (ERK) phosphorylation late in the process of DLS formation, concomitant with the increased proliferation of dedifferentiated cells. In the absence of EGF, these phosphorylation changes are not seen and the typical increase in DLS epithelial cell proliferation seen after 10 days in culture is attenuated. Thus, in our model, EGF is necessary for islet cell dedifferentiation, playing an important role in both the onset of DLS formation (through JNK) and in the proliferation of these dedifferentiated cells (through AKT and ERK).

Estimating the Risk of Prolonged Air Leak after Pulmonary Resection Using a Simple Scoring System

BACKGROUND The high rate of prolonged air leak (PAL) after pulmonary resection has prompted interest in surgical adjuncts designed to prevent this complication. However, these adjuncts are costly and might not be beneficial if used routinely. Identification of patients at highest risk might allow for more effective use of these adjuncts. Therefore, we sought to develop a simple scoring system to predict PAL. STUDY DESIGN A derivation set of 580 patients was identified from a prospectively entered database of consecutive pulmonary resections at a single institution from 2002 to 2007. Patient and operative characteristics were compared using Students t-test and chi-square tests. Significant variables on univariate analysis were entered into a stepwise logistic regression to establish a simple predictive model to estimate the risk of PAL. This scoring system was then validated in a consecutive set of 381 patients operated at the same institution from 2007 to 2009. RESULTS The rate of PAL was 14% in the derivation set and 18% in the validation set. Poor pulmonary function (forced expiratory volume in 1 second and carbon monoxide diffusing capacity, percent predicted) and pleural adhesions were significantly associated with PAL in the derivation set. A weighted scoring system was devised using pleural adhesions (+2 points), forced expiratory volume in 1 second (+1 per 10% below 100%), and carbon monoxide diffusing capacity (+1 per 20% below 100%). Total number of points estimated the probability of PAL. Hosmer-Lemeshow goodness-of-fit test confirmed validity (p > 0.2) of this scoring system in the validation set. CONCLUSIONS We have devised and validated a simple scoring system to predict the probability of PAL after pulmonary resection.

Production and characterization of the recombinant Islet Neogenesis Associated Protein (rINGAP)

Islet Neogenesis Associated Protein (INGAP) is implicated in pancreatic islet neogenesis. INGAP peptide, a pentadecapeptide comprising amino acids 104-118, reverses diabetes in rodents and improves glucose homeostasis in patients with diabetes. The mechanism of INGAP action is unknown, but such studies would benefit from the availability of the full-length recombinant protein (rINGAP). Here we report the production of rINGAP from 293-SF cells following lentiviral transduction, and its characterization by MALDI-TOF and Q-TOF Mass Spectrometry, and HPLC. Importantly, we show that rINGAP exhibits 100x the bioactivity of INGAP peptide on a molar basis in an in vitro assay of human islet regeneration.

Safety and feasibility of endovascular aortic aneurysm repair as day surgery

Objective: The adoption of endovascular aneurysm repair (EVAR) during the past two decades has led to significantly shorter length of stay as well as lower hospital resource use. Currently, most patients are admitted to the hospital after EVAR; however, there are no standard observation periods, and timing of discharge is based on clinical judgment. The aim of this study was to confirm the safety and feasibility of performing EVAR as outpatient surgery. Methods: We developed criteria to identify patients for potential same‐day discharge (infrarenal aneurysm, low perioperative risk, to be accompanied for first 24 hours). We then implemented a prospective trial that observed patients planned for same‐day discharge and compared them with a historical control group (patients who had undergone EVAR during the previous 2 years and met same‐day discharge criteria). Basic demographic and operative data as well as length of stay, inpatient and perioperative complications, emergency department visits, readmissions, reinterventions, and deaths were collected. The primary outcome was the 30‐day complication rate, and the study was powered to assess noninferiority. Results: Prospectively, we assessed 266 patients and planned 110 (41%) for outpatient EVAR (62% of historical controls met outpatient criteria). Demographic characteristics were similar between planned outpatients and historical controls. In planned outpatients, hospital stay was significantly shorter (0.7 ± 2.6 days vs 2.5 ± 6.9 days; P < .01), and 79% were discharged the same day of surgery. The 30‐day follow‐up was available for all study patients and 94% of control patients; there were no differences in complication (11% vs 9%), readmission (2% vs 4%), reintervention (4% vs 4%), or mortality (1% vs 1%) rates, but study patients had significantly more emergency department visits (15% vs 6%; P < .05). Unsuccessful same‐day discharge was associated with longer operative times, increased blood loss, and use of general anesthesia. Conclusions: In selected patients undergoing elective EVAR, same‐day discharge is feasible without increasing complication rates. Health resource utilization remains a challenge in transitioning to an outpatient model.