John S. Kaddis

Insulitis and β-Cell Mass in the Natural History of Type 1 Diabetes

Descriptions of insulitis in human islets throughout the natural history of type 1 diabetes are limited. We determined insulitis frequency (the percent of islets displaying insulitis to total islets), infiltrating leukocyte subtypes, and β-cell and α-cell mass in pancreata recovered from organ donors with type 1 diabetes (n = 80), as well as from donors without diabetes, both with islet autoantibodies (AAb+, n = 18) and without islet autoantibodies (AAb−, n = 61). Insulitis was observed in four of four donors (100%) with type 1 diabetes duration of ≤1 year and two AAb+ donors (2 of 18 donors, 11%). Insulitis frequency showed a significant but limited inverse correlation with diabetes duration (r = −0.58, P = 0.01) but not with age at disease onset. Residual β-cells were observed in all type 1 diabetes donors with insulitis, while β-cell area and mass were significantly higher in type 1 diabetes donors with insulitis compared with those without insulitis. Insulitis affected 33% of insulin+ islets compared with 2% of insulin− islets in donors with type 1 diabetes. A significant correlation was observed between insulitis frequency and CD45+, CD3+, CD4+, CD8+, and CD20+ cell numbers within the insulitis (r = 0.53–0.73, P = 0.004–0.04), but not CD68+ or CD11c+ cells. The presence of β-cells as well as insulitis several years after diagnosis in children and young adults suggests that the chronicity of islet autoimmunity extends well into the postdiagnosis period. This information should aid considerations of therapeutic strategies seeking type 1 diabetes prevention and reversal.

Network for Pancreatic Organ Donors with Diabetes (nPOD): developing a tissue biobank for type 1 diabetes.

The Network for Pancreatic Organ Donors with Diabetes (nPOD) was established to recover and characterize pancreata and related organs from cadaveric organ donors with various risk levels for type 1 diabetes (T1D). These biospecimens are available to investigators for collaborative studies aimed at addressing questions related to T1D natural history and pathogenesis.

The Juvenile Diabetes Research Foundation Network for Pancreatic Organ Donors with Diabetes (nPOD) Program: goals, operational model and emerging findings

nPOD actively promotes a multidisciplinary and unbiased approach toward a better understanding of T1D and identify novel therapeutic targets, through its focus on the study of human samples. Unique to this effort is the coordination of collaborative efforts and real-time data sharing. Studies supported by nPOD are providing direct evidence that human T1D isa complex and heterogeneous disease, in which a multitude of pathogenic factors may be operational and may contribute to the onset of the disease. Importantly, the concept that beta cell destruction is almost completed and that the autoimmune process is almost extinguished soon after diagnosis is being challenged. nPOD investigators are exploring the hypothesis that beta cell dysfunction may also be a significant cause of hyperglycemia, at least around the time of diagnosis, and are uncovering novel molecules and pathways that are linked to the pathogenesis and etiology of human T1D. The validation of therapeutic targets is also a key component of this effort, with recent and future findings providing new strategic direction for clinical trials.

Analysis of self-antigen specificity of islet-infiltrating T cells from human donors with type 1 diabetes

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood–derived T cells, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors and isolation and identification of DQ8 and DQ2–DQ8 heterodimer–restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.

Pancreas organ weight in individuals with disease-associated autoantibodies at risk for type 1 diabetes.

improve EMR note quality. In the absence of evidence, only expert opinion remains. It is my opinion as a medical educator, experienced if not expert, that training has minimal lasting impact. Mark Twain wrote, “I didn’t have time to write a short letter, so I wrote a long one instead.” With EMR features such as copy-and-paste and one-click data import, it takes less time and effort to write long random notes than it does to write short structured ones. Until EMR systems are modified to reverse this time incentive, long notes will remain the norm. A similar situation existed with regard to handwashing by health care workers. Decades of training and cajoling did little to improve handwashing compliance rates. Compliance improved only after it was made easier and faster to comply—by installation of conveniently located hand sanitizer dispensers. If short, cogent EMR notes are the goal, EMR system redesign is necessary. I am impressed that Cornell’s Department of Medicine has committed faculty time and money to these efforts. However, while I sincerely hope that Dr Siegler proves me wrong, I remain skeptical regarding the efficacy of training in improving EMR notes.

Multicenter analysis of novel and established variables associated with successful human islet isolation outcomes.

Islet transplantation is a promising therapy used to achieve glycometabolic control in a select subgroup of individuals with type I diabetes. However, features that characterize human islet isolation success prior to transplantation are not standardized and lack validation. We conducted a retrospective analysis of 806 isolation records from 14 pancreas‐processing laboratories, considering variables from relevant studies in the last 15 years. The outcome was defined as postpurification islet equivalent count, dichotomized into yields ≥315 000 or ≤220 000. Univariate analysis showed that donor cause of death and use of hormonal medications negatively influenced outcome. Conversely, pancreata from heavier donors and those containing elevated levels of surface fat positively influence outcome, as did heavier pancreata and donors with normal amylase levels. Multivariable logistic regression analysis identified the positive impact on outcome of surgically intact pancreata and donors with normal liver function, and confirmed that younger donors, increased body mass index, shorter cold ischemia times, no administration of fluid/electrolyte medications, absence of organ edema, use of University of Wisconsin preservation solution and a fatty pancreas improves outcome. In conclusion, this multicenter analysis highlights the importance of carefully reviewing all donor, pancreas and processing parameters prior to isolation and transplantation.

Human Pancreatic Islets and Diabetes Research

Human islet research is crucial to understanding the cellular biology of the pancreas in developing therapeutic options for diabetes patients and in attempting to prevent the development of this disease. The national Islet Cell Resource Center Consortium provides human pancreatic islets for diabetes research while simultaneously addressing the need to improve islet isolation and transplantation technologies. Since its inception in 2001, the consortium has supplied 297.6 million islet equivalents to 151 national and international scientists for use in clinical and laboratory projects. Data on the volume, quality, and frequency of shipments substantiate the importance of human islets for diabetes research, as do the number of funded grants for beta-cell projects and publications produced as a direct result of islets supplied by this resource. Limitations in using human islets are discussed, along with the future of islet distribution centers. The information presented here is instructive to clinicians, basic science investigators, and policy makers who determine the availability of funding for such work. Organ procurement coordinators also may find the information useful in explaining to donor families why research consent is so valuable.

A multicenter study to standardize reporting and analyses of fluorescence-activated cell-sorted murine intestinal epithelial cells

Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxons rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.

Validation of a Rapid Type 1 Diabetes Autoantibody Screening Assay for Community Based Screening of Organ Donors to Identify Subjects at Increased Risk for the Disease

The Network for Pancreatic Organ donors with Diabetes (nPOD) programme was developed in response to an unmet research need for human pancreatic tissue obtained from individuals with type 1 diabetes mellitus and people at increased risk [i.e. autoantibody (AAb)‐positive] for the disease. This necessitated the establishment of a type 1 diabetes‐specific AAb screening platform for organ procurement organizations (OPOs). Assay protocols for commercially available enzyme‐linked immunosorbent assays (elisas) determining AAb against glutamic acid decarboxylase (GADA), insulinoma‐associated protein‐2 (IA‐2A) and zinc transporter‐8 (ZnT8A) were modified to identify AAb‐positive donors within strict time requirements associated with organ donation programmes. These rapid elisas were evaluated by the international islet AAb standardization programme (IASP) and used by OPO laboratories as an adjunct to routine serological tests evaluating donors for organ transplantation. The rapid elisas performed well in three IASPs (2011, 2013, 2015) with 98‐100% specificity for all three assays, including sensitivities of 64–82% (GADA), 60–64% (IA‐2A) and 62–68% (ZnT8A). Since 2009, nPOD has screened 4442 organ donors by rapid elisa; 250 (5·6%) were identified as positive for one AAb and 14 (0.3%) for multiple AAb with 20 of these cases received by nPOD for follow‐up studies (14 GADA+, two IA‐2A+, four multiple AAb‐positive). Rapid screening for type 1 diabetes‐associated AAb in organ donors is feasible, allowing for identification of non‐diabetic, high‐risk individuals and procurement of valuable tissues for natural history studies of this disease.

Standardized Transportation of Human Islets: An Islet Cell Resource Center Study of More Than 2,000 Shipments

Preservation of cell quality during shipment of human pancreatic islets for use in laboratory research is a crucial, but neglected, topic. Mammalian cells, including islets, have been shown to be adversely affected by temperature changes in vitro and in vivo, yet protocols that control for thermal fluctuations during cell transport are lacking. To evaluate an optimal method of shipping human islets, an initial assessment of transportation conditions was conducted using standardized materials and operating procedures in 48 shipments sent to a central location by eight pancreas-processing laboratories using a single commercial airline transporter. Optimization of preliminary conditions was conducted, and human islet quality was then evaluated in 2,338 shipments pre- and postimplementation of a finalized transportation container and standard operating procedures. The initial assessment revealed that the outside temperature ranged from a mean of −4.6 ± 10.3°C to 20.9 ± 4.8°C. Within-container temperature drops to or below 15°C occurred in 16 shipments (36%), while the temperature was found to be stabilized between 15°C and 29°C in 29 shipments (64%). Implementation of an optimized transportation container and operating procedure reduced the number of within-container temperature drops (£15°C) to 13% (n = 37 of 289 winter shipments), improved the number desirably maintained between 15°C and 29°C to 86% (n = 250), but also increased the number reaching or exceeding 29°C to 1% (n = 2; overall p < 0.0001). Additionally, postreceipt quality ratings of excellent to good improved pre- versus postimplantation of the standardized protocol, adjusting for preshipment purity/viability levels (p < 0.0001). Our results show that extreme temperature fluctuations during transport of human islets, occurring when using a commercial airline transporter for long distance shipping, can be controlled using standardized containers, materials, and operating procedures. This cost-effective and pragmatic standardized protocol for the transportation of human islets can potentially be adapted for use with other mammalian cell systems and is available online at http://iidp.coh.org/sops.aspx.