Omid Savari

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

BACKGROUND We 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. METHODS The 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. RESULTS The 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. CONCLUSION Coating 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.

A multicenter study: North american islet donor score in donor pancreas selection for human islet isolation for transplantation

Selection of an optimal donor pancreas is the first key task for successful islet isolation. We conducted a retrospective multicenter study in 11 centers in North America to develop an islet donor scoring system using donor variables. The data set consisting of 1,056 deceased donors was used for development of a scoring system to predict islet isolation success (defined as postpurification islet yield >400,000 islet equivalents). With the aid of univariate logistic regression analyses, we developed the North American Islet Donor Score (NAIDS) ranging from 0 to 100 points. The c index in the development cohort was 0.73 (95% confidence interval 0.70–0.76). The success rate increased proportionally as the NAIDS increased, from 6.8% success in the NAIDS < 50 points to 53.7% success in the NAIDS ≥ 80 points. We further validated the NAIDS using a separate set of data consisting of 179 islet isolations. A comparable outcome of the NAIDS was observed in the validation cohort. The NAIDS may be a useful tool for donor pancreas selection in clinical practice. Apart from its utility in clinical decision making, the NAIDS may also be used in a research setting as a standardized measurement of pancreas quality.

Distinct function of the head region of human pancreas in the pathogenesis of diabetes

The large size of the human pancreas challenges unbiased quantitative analyses that require a practical stereological approach. While many histological studies of the pancreas in the past lacked regional information, we have shown marked heterogeneity within an individual, where islet distribution/density is relatively low in the head and gradually increases through the body toward the tail region by > 2-fold.1, 2 Studies focusing on the tail region may be prone to overestimation of β-cell/islet mass when normalizing measured values per person by using pancreas weight or volume. In this article, beyond technical issues, we discuss the pathophysiological importance of studying the head region of the human pancreas regarding its unique characteristics in early development, and the anatomical disposition that may lead to a preferential loss of β-cells in patients with type 2 diabetes and the development of pancreatic cancer.

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.

Total Pancreatectomy with Islet Autotransplantation for the Ampullary Cancer. A Case Report

Total pancreatectomy (TP) leads to pancreatic exocrine deficiency and poorly controlled diabetes despite insulin treatment [1]. Simultaneous islet autotransplantation (IATx) can prevent diabetes in 30–40% of patients and improve glycemic control with insulin supplementation in an additional 30% of individuals leading to improvement in quality of life in the majority of patients [2]. Total pancreatectomy with islet transplantation (TPIAT) has been offered mostly as a last resort procedure to highly selected patients with chronic or recurrent acute pancreatitis and intractable pain despite medical, endoscopic, and other conventional surgical interventions. Although patients with advanced benign tumors who require TP also may receive IATx, application of TPIAT in the setting of pancreatic malignancy remains controversial due to the risk of contamination of the islet prep with tumor cells and possible dissemination of malignancy [1]. Nevertheless, encouraging results in single cases of IATx have been reported in those patients with pancreatic cancer, who required TP due to complications after initial Whipple procedure [3–7]. Here we present for the first time, a case report of a patient with a small, early-stage ampullary cancer without lymph node involvement, who developed disseminated neoplastic disease 6 months after TPIAT. Method

Comparative Analysis of Islet Development

The pancreatic islet plays a critical role in glucose homeostasis. The islet is a highly vascularized micro-organ embedded in the exocrine pancreas, which mainly consists of endocrine hormone-secreting cells: beta cells (insulin), alpha cells (glucagon), delta cells (somatostatin), pancreatic polypeptide (PP) cells, and epsilon cells (ghrelin). In this chapter, we review fetal endocrine pancreatic cell development in various species, providing a glimpse of the evolutionary changes followed by a molecular hierarchy of genes involved in pancreas development and a model of islet formation that recent technological advances have made possible. Considering current concerns, species differences between humans and rodents will be discussed in detail.