Ling-Jia Wang

Coating Human Pancreatic Islets With Cd4+cd25highcd127− Regulatory T Cells as a Novel Approach for the Local Immunoprotection

Objectives:To develop a novel approach for local immunoprotection using CD4+CD25highCD127– T regulatory cells (Tregs) attached to the surface of the islets before transplantation. Background:Tregs expanded ex vivo can control allo and autoreactivity, therefore, Treg-based therapy may offer more effective protection for transplanted islets from immunologic attack than currently used immunosuppression. Local application of Tregs can make such therapy more clinically feasible and efficient. Methods:Human islets were isolated and coated with allogeneic ex vivo expanded Tregs using biotin-poly(ethylene glycol)-N-hydroxysuccinimide ester (biotin-PEG-NHS) and streptavidin as binding molecules. Results:Coating pancreatic islets with Tregs did not affect islet viability (>90% fluorescein diacetate/propidium iodide) or the insulin secretion profile in dynamic islet perifusion assays. After in vitro incubation with allogeneic T effector cells, Treg-coated islets revealed preserved function with higher insulin secretion compared with controls-native islets, coated islets with T effector cells or when Tregs were added to the culture, but not attached to islets (P < 0.05). In addition, the Enzyme-linked immunosorbent spot (ELISPOT) assay revealed suppression of interferon (IFN)-&ggr; secretion, when T effector cells were challenged with Treg-coated islets comparing to controls (99 ± 7 vs 151 ± 8 dots, respectively; P < 0.01). Conclusions:We demonstrated, for the first time, the ability to bind immune regulatory cells to target cells with preservation of their viability and function and protective activity against immune attack. If successfully tested in an animal model, local delivery of immunoprotective Tregs on the surface of transplanted pancreatic islets may be an alternative or improvement to the currently used immunosuppression.

Evidence of non-pancreatic beta cell-dependent roles of Tcf7l2 in the regulation of glucose metabolism in mice

Non-coding variation within TCF7L2 remains the strongest genetic determinant of type 2 diabetes risk in humans. A considerable effort has been placed in understanding the functional roles of TCF7L2 in pancreatic beta cells, despite evidence of TCF7L2 expression in various peripheral tissues important in glucose homeostasis. Here, we use a humanized mouse model overexpressing Tcf7l2, resulting in glucose intolerance, to infer the contribution of Tcf7l2 overexpression in beta cells and in other tissues to the metabolic phenotypes displayed by these mice. Restoring Tcf7l2 expression specifically in beta cells to endogenous levels, in face of its overexpression elsewhere, results in impaired insulin secretion, reduced beta cell number and islet area, corroborating data obtained in humans showing similar phenotypes as a result of manipulations leading to Tcf7l2 loss of function. Interestingly, the persistent overexpression of Tcf7l2 in non-pancreatic tissues results in a significant worsening in glucose tolerance in vivo, indicating that Tcf7l2 overexpression in beta cells does not account for the glucose intolerance in the Tcf7l2 overexpression mouse model. Collectively, these data posit that Tcf7l2 plays key roles in glucose metabolism through actions beyond pancreatic beta cells, and further points to functionally opposing cell-type specific effects for Tcf7l2 on the maintenance of balanced glucose metabolism, thereby urging a careful examination of its role in non-pancreatic tissues as well as its composite metabolic effects across distinct tissues. Uncovering these roles may lead to new therapeutic targets for type 2 diabetes.

Improved coating of pancreatic islets with regulatory T cells to create local immunosuppression by using the biotin-polyethylene glycol-succinimidyl valeric acid ester molecule

BACKGROUNDnWe showed that T regulatory (Treg) cells can be attached to the surface of pancreatic islets providing local immunoprotection. Further optimization of the method can improve coating efficiency, which may prolong graft survival. In this study, we compared the effectiveness of two different molecules used for binding of the Tregs to the surface of pancreatic islets. Our aim was to increase the number of Treg cells attached to islets without compromising islets viability and function.nnnMETHODSnThe cell surface of human Treg cells and pancreatic islets was modified using biotin-polyethylene glycol-N-hydroxylsuccinimide (biotin-PEG-NHS) or biotin-PEG-succinimidyl valeric acid ester (biotin-PEG-SVA). Then, islets were incubated with streptavidin as islet/Treg cells binding molecule. Treg cells were stained with CellTracker CM-DiL dye and visualized using a Laser Scanning Confocal Microscope. The number of Treg cells attached per islets surface area was analyzed by Imaris software. The effect of coating on islet functionality was determined using the glucose-stimulated insulin response (GSIR) assay.nnnRESULTSnThe coating procedure with biotin-PEG-SVA allowed for attaching 40% more Treg cells per 1 μm(2) of islet surface. Although viability was comparable, function of the islets after coating using the biotin-PEG-SVA molecule was better preserved than with NHS molecule. GSIR was 62% higher for islets coated with biotin-PEG-SVA compared to biotin-PEG-NHS.nnnCONCLUSIONnCoating of islets with Treg cells using biotin-PEG-SVA improves effectiveness with better preservation of the islet function. Improvement of the method of coating pancreatic islets with Treg cells could further facilitate the effectiveness of this novel immunoprotective approach and translation into clinical settings.

Influence of pharmacological immunomodulatory agents on CD4+CD25highFoxP3+ T regulatory cells in humans

T regulatory cells (Tregs) play a critical role in the immunologic tolerance to the graft in transplantation. Thus, due to their immunosuppressive capability, ex vivo expanded Tregs may be used as a cellular therapy and an attractive novel strategy to control chronic rejection and eliminate need for lifelong pharmacological immunosuppression. Since Treg therapy is still in its infancy, initially Tregs still need to be applied in combination with pharmacological agents to prevent rejection. Fortunately, some of the medications have been shown to enhance the function and number of Tregs. In the clinic, different immunosuppressive regimens are used for individual patients for different types of organ transplantation. In this review, we present the most commonly used pharmacological agents for immunosuppression and discuss how they affect the Treg population. It is extremely difficult to dissect the effect of single agent on Tregs population in clinical settings since usually the combination of several medications is applied at the same time for graft protection. Nevertheless, experimental and clinical data indicate that thymoglobulin as immunosuppressive induction and mTOR inhibitors as immunosuppressive maintenance agents have the most beneficial effect on Treg population in the blood. Among supplemental agents promoting Tregs, anti-TNFα preparations have been in clinical use (in autoimmune diseases) for many years, so they are optimal candidates for testing in transplant settings in combination with Treg based cellular therapy.

Application of Digital Image Analysis to Determine Pancreatic Islet Mass and Purity in Clinical Islet Isolation and Transplantation.

Pancreatic islet mass, represented by islet equivalent (IEQ), is the most important parameter in decision making for clinical islet transplantation. To obtain IEQ, the sample of islets is routinely counted under a microscope and discarded thereafter. Islet purity, another parameter in islet processing, is routinely assessed by estimation only. In this study, we validated our digital image analysis (DIA) system by using the software of Image Pro Plus and a custom-designed Excel template to assess islet mass and purity to better comply with current good manufacturing practice (cGMP) standards. Human islet samples (60 collected from a single isolation and 24 collected from 12 isolations) were captured as calibrated digital images for the permanent record. Seven trained technicians participated in determination of IEQ and purity by the manual counting method (manual image counting, Manual I) and DIA. IEQ count showed statistically significant correlations between the Manual I and DIA in all sample comparisons (r > 0.819 and p < 0.0001). A statistically significant difference in IEQ between Manual I and DIA was not found in all sample groups (p > 0.05). In terms of purity determination, statistically significant differences between assessment and DIA measurement were found in high-purity 100-μl samples (p < 0.005) and low-purity 100-μl samples (p < 0.001) of the single isolation. In addition, islet particle number (IPN) and the IEQ/IPN ratio did not differ statistically between Manual I and DIA. In conclusion, the DIA used in this study is a reliable technique to determine IEQ and purity. Islet sample preserved as a digital image and results produced by DIA can be permanently stored for verification, technical training, and information exchange among islet centers. Therefore, DIA complies better with cGMP requirements than the manual counting method. We propose DIA as a quality control tool to supplement the established standard manual method for islet counting and purity estimation.

Donor Height in Combination With Islet Donor Score Improves Pancreas Donor Selection for Pancreatic Islet Isolation and Transplantation

To maximize the islet isolation yield for successful islet transplantation, the key task has been to identify an ideal pancreas donor. Since implementation of the islet donor score in donor selection, we have consistently obtained higher islet yields and transplantation rates. In this study, we tested whether assessing donor height as an independent variable in combination with the donor score could improve the pancreas donor selection. Donor and islet isolation information (nxa0= 22) were collected and studied between 2011 and 2012. Pearson correlation analysis was used in statistical analysis. Donor height as an independent variable was significantly correlated to the weight of the pancreas, pre-Islet Equivalents (pre-IEQ), post-IEQ, and IDS (Pxa0< .05). When donor with height of 179xa0cm ± 3 was selected in combination with IDS > 80, the clinical islet transplantation rate reached 80%.

Chronic pancreatitis and primary sclerosing cholangitis--first report of intrahepatic autologous islet transplantation.

BackgroundWe are reporting first successful intrahepatic autologous islet transplantation after total pancreatectomy in a patient with chronic pancreatitis and primary sclerosing cholangitis.MethodsTotal pancreatectomy and subsequent islet autotransplantation were performed in a 16-year-old boy with intractable pain due to chronic pancreatitis in the setting of ulcerative colitis and primary sclerosing cholangitis (PSC). Liver biopsy revealed PSC with focal bridging fibrosis. The pancreas was surgically removed and digested, and islets were isolated, highly purified, and infused intraportally.ResultsOver 18-month follow-up, the patient did not show progression of chronic liver disease or signs of portal hypertension. Magnetic resonance cholangiopancreatography revealed no new changes, and liver biopsy did not show progression of the periportal fibrosis. Pain medication was weaned over 12xa0months at which time glycemic control was excellent without exogenous insulin supplementation. HbgA1c was 5.9. Fifteen months after the procedure, stimulation with a mixed meal led to a fourfold increase of serum C-peptide and an eightfold increase of insulin level.ConclusionPancreatic autologous islets can be successfully transplanted into a liver affected by PSC without compromising hepatic or graft function. Durability of the procedure may be limited in the future by the natural course of the liver injury caused by PSC.

BETA-2 Score Allows for Convenient and Precise Monitoring of Islet Function and Early Detection of Islet Dysfunction

Introduction BETA-2 was developed to more conveniently and precisely assess islet graft function than commonly used beta-score, which requires a stimulation test. After validation of BETA-2 in our cohort of patients in relation to 90-min glucose in a mixed meal tolerance test and beta-score, we decided to assess the practical utility of BETA-2 in monitoring islet allograft function in individual patients. Methods We retrospectively analyzed BETA-2 calculations during clinical evaluations in islet allotransplantation (ITx) recipients with up to 3 islet infusions. We specifically looked in reference to previously established BETA-2 cut-offs for the detection of glucose intolerance <18 and insulin independence >13. Results We analyzed 298 BETA-2 scores calculated in 14 patients (7 women and 7 men) with an average age of 42.9 ± 9.5 years. In these patients, BETA-2 correlated well with islets function. Four patients, who experienced stable, long-term insulin independence after only one transplant, had a BETA-2 continuously over 18 (Fig 1). Figure. No caption available. Another 3 patients required a second transplant to reach the same outcome with the same BETA-2 characteristic (Fig 2). Figure. No caption available. In those patients, despite only discrete changes in fasting glucose level, the BETA-2 decreased to below 18 and continued gradually declining. This drop in BETA-2 always predicted islet dysfunction, the need for insulin support and subsequent transplant, which became clinically obvious as BETA-2 dropped below 13 (Fig 3 and 4). Figure. No caption available. Figure. No caption available. In the remaining patients, even when BETA-2 eventually reached peak above 18 after subsequent transplant, islet function gradually declined without obvious reason within a year or 2 with BETA-2 below 13 requiring re-introduction of insulin support. (Fig 4). One of these patients has not been able to stop insulin completely despite 3 subsequent transplants but BETA-2 has never increased above 18, which confirms utility of BETA-2. 12% (14/121) of BETA-2 measurements above 18 were made during the early period after iTx, when insulin was administered irrespectively of beta-cell function, in order to facilitate islet engraftment. In 80.2% of cases (142/177) where BETA-2 value was above the cut-off of 13, patients were off-insulin. The remaining 19.8% were on insulin due to failing islet function with dropping BETA-2 or supporting post infusion islet engraftment as described above. BETA-2 calculations below 13 were in 94.4% (113/121) in patients receiving insulin. Remaining 6.6% of BETA-2 calculations (8/121) were below the cut-off of 13, yet patients were still off insulin due to their non-compliance to the recommendation to resume insulin therapy based on clinically evident suboptimal glucose control. Conclusion BETA-2 score, based on only a single fasting blood sample is a very reliable tool for islet function assessment and allows early detection of islet graft decline before obvious clinical symptoms. This work was supported by the Illinois Department 370 of Public Health Grant “Pancreatic Islet Transplantation” US Public Health Service Grant DK-020595 to the University of Chicago Diabetes Research Training Center.

Assessment of simple indices based on a single fasting blood sample as a tool to estimate beta cell function after total pancreatectomy with islet autotransplantation - a prospective study

We investigated six indices based on a single fasting blood sample for evaluation of the beta‐cell function after total pancreatectomy with islet autotransplantation (TP‐IAT). The Secretory Unit of Islet Transplant Objects (SUITO), transplant estimated function (TEF), homeostasis model assessment (HOMA‐2B%), C‐peptide/glucose ratio (CP/G), C‐peptide/glucose creatinine ratio (CP/GCr) and BETA‐2 score were compared against a 90‐min serum glucose level, weighted mean C‐peptide in mixed meal tolerance test (MMTT), beta score and the Igls score adjusted for islet function in the setting of IAT. We analyzed values from 32 MMTTs in 15 patients after TP‐IAT with a follow‐up of up to 3 years. Four (27%) individuals had discontinued insulin completely prior to day 75, while 6 out of 12 patients (50%) did not require insulin support at 1‐year follow‐up with HbA1c 6.0% (5.5–6.8). BETA‐2 was the most consistent among indices strongly correlating with all reference measures of beta‐cell function (r = 0.62–0.68). In addition, it identified insulin independence (cut‐off = 16.2) and optimal/good versus marginal islet function in the Igls score well, with AUROC of 0.85 and 0.96, respectively. Based on a single fasting blood sample, BETA‐2 score has the most reliable discriminant value for the assessment of graft function in patients undergoing TP‐IAT.

Pain Control, Glucose Control, and Quality of Life in Patients With Chronic Pancreatitis After Total Pancreatectomy With Islet Autotransplantation: A Preliminary Report

BACKGROUNDnTotal pancreatectomy (TP) is offered as a last treatment option for pain relief in patients with chronic pancreatitis. Concurrent islets autotransplantation (TP-IAT) may improve glucose control.nnnMETHODSnWe analyzed results in 20 recent patients who underwent TP-IAT at The University of Chicago. The median observation period was 28 months (2-38). Data were collected prospectively then analyzed retrospectively.nnnRESULTSnThe number of patients requiring opioids daily for pain control decreased from 16 (80%) prior to surgery to 2 (13%) 1 year after, with only 1 (6.5%) patient experiencing persistent phantom pancreatic pain. Opioid requirements decreased from a median 56.3 (0-240) morphine equivalent dose to 5 (0-130) on day 75 and to 0 (0-30) at 1-year follow up. Five patients (25%) completely stopped insulin support prior to day 75 while maintaining hemoglobin A1c of 5.9% (5-6.3). Eight (53%) patients were insulin free at 1 year with A1c of 6% (5.5-6.8) and a similar rate persisted in next 2 years. For the remaining patients, the more islet function that was preserved, the less insulin they required and A1c was closer to optimal. Quality of Life (QoL) measured by SF36 Physical (PCS) and Mental (MCS) Component Score improved on day 75 (Pxa0< .001) and maintained improvement later on. Both PCS and MCS improved regardless of whether patient requires insulin support or not.nnnCONCLUSIONSnImprovements of QoL with pain resolution and good glucose control can be achieved after TP-IAT in properly selected patients with CP and intractable pain, regardless of patient insulin support status.