Projects & Publications. Current nPOD Projects

Beta Cell Development, Differentiation & Regeneration

Domenico Accili & Chutima Talchai Columbia University Beta cell dedifferentiation in type 2 diabetes

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Justin Annes Stanford University Beta cell replication

Recent decades have seen an incredible growth in the prevalence of diabetes. While it is important to pursue multiple therapeutic strategies, harnessing the regenerative capacity of islet β-cells to increase an individual’s insulin secretion capacity is among the promising approaches.

Recently, discovery-oriented unbiased chemical screening led to the identification of the metabolic enzyme adenosine kinase (ADK) as a regulator of rodent and porcine β-cell replication. Importantly, small molecule ADK-inhibitors selectively stimulate replication of β-cells, but not alpha-cells, in vitro and in vivo. The identification of ADK as a regulator of β-cell regeneration has raised several important questions.

Does long-term inhibition of ADK in vivo promote β-cell mass expansion and protect against developing diabetes? How does a broadly expressed metabolic enzyme selectively control the growth of a specific cell-type?

Is human β-cell growth controlled by ADK? Using the nPOD resources, we are investigating the expression of ADK in normal and diabetic pancreatic tissues. We are interested in weather this molecule is expressed in human β-cells and whether its expression changes with diseased states.

Mark Atkinson University of Florida The ductal pancreatic progenitor microenvironment in type 1 diabetes

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Martha Campbell-Thompson, Alberto Pugliese, Clive Wasserfall & Mark Atkinson University of Florida Beta cell regeneration for Type 1 Diabetes

Understanding basic human beta cell physiology is crucial to targeted therapies and prevention strategies for type 1 diabetes. Clinical signs in type 1 diabetes start when beta cell function and/or numbers are unable to maintain adequate blood glucose levels. After onset of type 1 diabetes,  life-long insulin replacement therapy is needed. Factors regulating human beta cell regeneration are poorly understood. The purpose of these studies are to: 1) define pancreatic beta and alpha cell populations and how diabetes alters their function and numbers, 2) characterize potential autoimmune mediators of  islet inflammation (insulitis) and resultant beta cell death, and 3) correlate lymphocytic populations in pancreatic and nonpancreatic lymph nodes and spleen between  pre-diabetes and type 1 diabetic patients.   

Juan Dominguez-Bendala, Giacomo Lanzoni & Luca Inverardi University of Miami Progenitors of the beta cell lineage in the pancreas and biliary tree of diabetic patients

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Dieter Egli, Ruddy Leibel & Scott Noggle Columbia University Generation of pluripotent stem cells from diabetics

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Matthias Hebrok University of California, San Francisco Investigating the de-differentiated state of the β-cell in human diabetic patient tissues

It is well accepted that compromised ß-cell function is an integral part of diabetes development.  Several recent studies, mostly using complex genetic mouse models, have supported the notion that a change in the ß-cell state or identity due to varying insults causes diabetes in the absence of ß-cell death.  Our work has focused on the involvement of the von Hippel Lindau/Hypoxia Inducible Factor (VHL/HIF) pathway, a master regulator of cellular response to diminished oxygen (hypoxia).  In a mouse model that stabilizes the HIF pathway despite normal oxygen levels within the ß-cell, we observe the development of diabetes which correlates strongly with a) a loss of the ß-cell differentiation state as determined by markers such as Pdx-1, Nkx6.1, MafA, Glut-2, and Insulin, and b) inappropriate activation of embryonic signaling pathways and genes.  Immunofluorescence analyses on islets from diabetic animals displayed strikingly reduced level of insulin as compared to control, normoglycemic littermates, which indicate the cause of diabetes to be insulin insufficiency.  The broad disruption of gene expression observed in diabetic mice suggests a loss of ß-cell identity (or dedifferentiation) as the underlying cause.  We have also shown that ectopic expression of a key transcription factor Sox9 (normally expressed during embryogenesis) in the ß-cell causes dedifferentiation and diabetes.  Our goal is to translate our findings from the mouse models of diabetes into the human condition.  It is becoming apparent that diabetic patients can be stratified into distinct cohorts (based on onset of disease, age, weight, race) and it is critical to identify what sub-population of patients develops the disease due to ß-cell dedifferentiation.  To this end, samples available from the nPOD provide an invaluable resource to a) identify patients who have compromised ß-cell identity, and b) correlate the findings from mouse models into human patients by identifying what signaling pathways or genes are erroneously regulated in these samples.  Mouse tissue allows us to conduct genome wide expression analyses that can lead to identification of important regulators to be tested in the human samples.  Our focus is on the maintenance of identity of the ß-cell, and discovering important regulators of this process may enable the development of intervention therapies that could block or delay the onset of ß-cell damage.

Pedro Herrera University of Geneva Medical School Cell conversion in the human pancreas

The overall goal of my proposal is to identify means for improving β-cell regeneration in the adult pancreas. We previously developed a transgenic model of inducible total or partial β-cell ablation (termed RIP-DTR). We have reported that in these mice there is spontaneous reconstitution of new β-cells from heterologous (i.e. non-β) cells after near-total β-cell loss.

The RIP-DTR model has revealed an unsuspected degree of cellular plasticity in the pancreas of juvenile and adult mice, including aged individuals, regarding the spontaneous inherent capacity of islet a-cells to switch to insulin production upon β-cell loss.

During the next years we want to address the following fundamental questions:
1. What is (are) the signal(s) driving a-cell reprogramming upon near-total β-cell ablation?
2. Can the a-to-β-cell conversion be fostered? Why only a small fraction of a-cells engages into conversion? What is the nature of the epigenetic modifications in reprogrammed a-cells?
3. Can human a-cells reprogram to insulin production?
4. What is the influence of ageing on islet cell plasticity?
5. Can other islet cells, i.e. besides a-cells, reprogram to insulin production?
6. Can in vivo a-cell conversion be facilitated by means of compounds mimicking the effect of instructive signals?
7. Are human islets endowed with cell plasticity capabilities? How diabetic conditions influence i) the capacity of human/mouse a-cells to reprogram, and ii) the maintenance of the β-cell phenotype?
Fred Levine Sanford-Burnham Medical Research Institute Beta-cell regeneration by transdifferentiation

We have been interested in the mechanisms by which beta-cells regenerate in diabetes. Our laboratory is interested in how beta -cells regenerate under normal and pathophysiological conditions, with the goal of developing new therapies for diabetes that result in an increased number of those cells. There are two possible mechanisms for endogenous regeneration: beta-cell replication and beta-cell neogenesis. In 2013, we published that potent small molecule HNF4a antagonists induced beta-cell replication in mice and rabbits.

With respect to beta-cell neogenesis, we have developed a new model of pancreatic damage, combining pancreatic exocrine cell damage and chemical beta -cell ablation. In the model, high efficiency transdifferentiation of beta-cells to other islet cell types occurred. All of those experiments were done using mouse models. It is of interest to determine whether the findings in mice can be translated to humans. Thus, we propose to use samples from nPOD to examine for evidence of islet cell transdifferentiation.

Douglas Melton Harvard University Generation of tools to distinguish human pancreatic cell populations

Our work aims to understand the defects were deficiencies in the pancreatic beta cells of diabetics. We use a number of molecular methods, including but not limited to single cell RNA sequencing, to characterize the molecular phenotypes of islet cells.

Srinath Sanda Benaroya Research Institute Analysis of beta cell senescence in T1D subjects

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Maike Sander University of California, San Diego Preventing beta cell dedifferentiation in type 1 diabetes

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Andrew Stewart & Amy Cox University of Pittsburgh Medical Center Multidisciplinary approaches to driving human beta cell replication

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Beta Cell Physiology and Dysfunction

Graeme Bell & Manami Hara University of Chicago Beta-cell Death and Survival

Type 1 diabetes (T1D) is a progressive immune-mediated disease that is preceded by an asymptomatic preclinical period of highly variable duration. A better understanding of beta cell and islet function or dysfunction during this asymptomatic period and the nature of the “dialog” between islet endocrine cells and the immune system could lead to new therapeutic strategies to arrest the progression to autoimmunity. Here we propose to develop a framework for understanding the underlying biology of T1D by integrating histological and genomic (RNA-seq) analyses of the human pancreas, pancreatic islets and islet endocrine cells. We anticipate that these studies will lead to a new understanding of the pathophysiology of T1D and point to biomarkers of human beta-cell stress, death and survival.

Gary Cline & Richard Carson Yale University School of Medicine Imaging pancreatic beta-cells with PET neuroimaging agents

A clinically viable means to measure pancreatic beta-cell mass (BCM) is essential for evaluating the physiological basis of therapeutic approaches to restore deficient insulin secretory capacity. Major advances in imaging BCM have been made by taking advantage of receptor-specific imaging probes that have been successfully used for neuroimaging. Hence, Positron Emission Tomography (PET) imaging ligands that were originally developed to specifically bind to neurons may prove useful for imaging BCM.

The Yale PET Center has been at the forefront of imaging both brain receptors and BCM and proposes to evaluate its extensive library of human neuroimaging agents as in vivo probes to quantitatively determine BCM. To maximize the value of these studies, pancreatic imaging in humans will be obtained together with validation studies in healthy and type 1 and 2 diabetes mellitus pancreas. Imaging probes that show suitable in vivo specific uptake in pancreas and appropriate imaging properties in humans, and can distinguish between healthy and diabetic pancreas in vitro will then be tested in a limited clinical PET-imaging trial to assess whether there is a measurable decrease in radiotracer binding in the pancreas of T1DM patients.

In Specific Aim 1, we will evaluate whether the approved radiotracers currently in use at the Yale PET center for human neuroimaging can be used for imaging BCM in healthy individuals.

In Specific Aim 2, we will validate β-cell specificity of the radioligands in vitro in healthy, T1DM, and T2DM human pancreas tissue obtained from the network for Pancreatic Organ Donors with Diabetes (nPOD), and in human islets.

In Specific Aim 3, we will perform a clinical evaluation in healthy and T1DM volunteers of those agents that meet the criteria of 1) promising pancreas imaging characteristics determined in Aim 1 and 2) favorable β-cell specificity as determined in Aim 2. A limited clinical evaluation is necessary to establish 

Yuval Dor & Benjamin Glaser Hebrew University of Jerusalem Markers of stress in diabetic islets

Our project seeks to understand the molecular basis for beta cell failure in human diabetes. We have discovered that excessive glucose metabolism in beta cells may lead to double strand breaks in the DNA and activation of the tumor suppressor gene p53. With the help of nPOD, we are studying the significance if this response in human type 2 diabetes patients. In our experiments we utilize archived material from human pancreata to examine the expression of multiple markers of the DNA damage response, to understand the nature of this response.

Dieter Egli, Ruddy Leibel Columbia University Beta cell defects in cystic fibrosis related diabetes

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Decio Eizirik, Ihsane Marhfour & Anne Op de beeck Universite Libre De Bruxelles, Belgium Characterization of beta cell ER stress associated to enteroviral infection in type 1 diabetes

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Ivan Gerling University of Tennessee Health Science Center Defining islet heterogeneity using single islet transcriptomics

The goal of this project is to obtain transcriptome data from human islets using either hybridization arrays or RNA sequencing technology.  We plan to obtain complete information about relative expression of all genes in islets from a number of different organ donor groups with an emphasis on: 1) donors with no autoantibodies or diabetes, 2) donors with autoantibodies but no diabetes, 3) donors with newly diagnosed type 1 diabetes, 4) donors with long-standing (over 5 years) type 1 diabetes, and, 5) donors with type 2 diabetes.  The dataset will be analyzed to identify genes whose expression differs significantly between one or more of these donor groups.  The goal is to define what pathways and processes uniquely define islets in each donor group, in order to gain new insights into the pathophysiology of diabetes and discover possible new drug targets.  To further that goal each list of differentially expressed genes (distinguishing islets in one group from one or more of the other groups) will be subjected to a data mining process designed to define if the list is significantly enriched in genes that: 1) belong to specific pathways or gene ontologies; 2) are regulated by specific transcription factors; 3) are known to be influenced by specific drugs, proteins or other molecules.  This will provide a global (systems level) definition of gene expression patterns in islets from each of these donor groups.  However, we also intend to make this dataset available to other investigators who may want to examine the data for expression of genes relevant to their specific studies.

Gokhan Hotamisligil & Feyza Engin Harvard University Examination of ER stress markers in type 1 diabetes samples in humans

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Pamela Itkin-Ansari, William Balch & Randal Kaufman UCSD Insulin interacting proteins and stress markers in T1D

We have developed novel methods for proteomic study of beta-cells within native islets. Using
antibodies to proinsulin or insulin we reported the first Biosynthetic Interaction Network for insulin
(Pottekat, Cell Reports, 2013). Based upon our finding that many proteins which interact directly with
insulin are T1D GWAS candidates or proteins required for ER homeostasis, we propose to determine
how these proteins and others in their class are altered during progression to T1D. Moreover, based
upon recent evidence of ER stress in T1D islets and our identification of compounds which stabilize
the ER protein folding environment in vivo, we propose to test new strategies for preserving or
restoring beta-cell mass in T1D.

Timothy Kowalski & Shu-Cheng Chen Merck Confirmation of target gene expression in T1D and T2D pancreas

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Rudolph Leibel & Streamson Chua Columbia University Identification of a Gene Regulating Pancreatic Beta Cell Replication

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Rudolph Leibel, Wakae, Nao Columbia University E-cadherin mediates developmental effects on the proliferation and the function of β-cells in the islet of Langerhans

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Clayton Mathews University of Florida Islet resistance to T1D

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Raghavendra Mirmira & Teresa Mastracci Indiana University School of Medicine Deoxyhypusine synthase: a novel target for beta cell protection

The cellular processes giving rise to type 1 diabetes (T1D) and type 2 diabetes (T2D) involve the activation of inflammation, which leads to the eventual death of islet β cells.  An urgent priority in diabetes research is the discovery of biomarkers (simple blood tests) that can assist in the identification of persons at-risk for disease for the purposes of early therapy.  Recently, our laboratory has been investigating the involvement of a specifically altered form of the protein eIF5A (known as  eIF5AHyp) in the progression of diabetes in mice.  In previously published studies, we showed that eIF5AHyp is responsible for the production of a subset of inflammatory proteins in β cells and immune cells of the mouse.  However, the specific types of cells that exhibit eIF5AHyp in human diabetes have never been examined, largely because specific reagents for tissue analysis have heretofore been unavailable.  The purpose of this study is to determine the cell-type distribution of eIF5AHyp in the human pancreas and spleen from persons with T1D, T2D and controls.  The results of this study will uncover whether the accumulation of eIF5AHyp-expressing cells are a marker for diabetic disease in the pancreas.

Jerry Nadler, Margaret Morris & Kaiwen Ma Eastern Virginia Medical School 12/15 Lipoxygenase expression in type 1 diabetes

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John Andrew Pospisilik & Tess Tsai-Hsiu Lu Max-Planck Institute of Immunbiology and Epigenetics Epigenetic regulation of beta-cell function and identity

Diabetes is a disorder of complex interaction between genetic susceptibility and environmental perturbation. Various epigenetic models, such as intra-uterine growth retardation, have been shown to contribute to pancreatic beta-cell dysfunction. More recently, beta-cell dedifferentiation has been discovered as a mechanism for beta-cell dysfunction in diabetic mouse model. The project aim to understand the role of classic epigenetic modules in regulating beta-cell identity and function in human context. We’ll survey changes in histone modifications across human pancreas samples over various age groups and diabetic conditions. Understanding how histone modification changes with age and disease state in human will enable the research community to identity factors that underlie aging related decline in beta-cell function and identity.

Al Powers Vanderbilt University Pancreatic islet biology and vascularization

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Sasanka Ramanadham University of Alabama-Birmingham Role of Calcium-Independent Phospholipase A2β (iPLA2β) on β-Cell Apoptosis

Beta-cell apoptosis contributes to loss of β-cells and decreases in β-cell function in both types 1 and 2diabetes mellitus. It is therefore important to understand the mechanisms underlying β -cell apoptosis if this process is to be prevented or delayed. Our hypothesis is that the group VIA Ca2+-independent phospholipase A2 (iPLA2 β) participates in β -cell apoptosis. We observed that (a) ER stress, proinflammatory cytokines, and prolonged hyperglycemia promote expression and activity of iPLA2 β  inislets, (b) iPLA2 β  activation increases ceramide generation via neutral sphingomyelinase (NSMase)- catalyzed hydrolysis of sphingomyelins triggering the mitochondrial apoptotic pathway and β –cell apoptosis, (c) these outcomes are suppressed by inhibition of iPLA2 β  or NSMase, (d) iPLA2 β –null islets are less and iPLA2 β -transgenic (Tg) islets more sensitive to ER stress, (e) NSMase expression is unaffected in iPLA2 β -null islets and amplified in iPLA2 β -Tg islets, and (f) β -cell iPLA2 β  and NSMase messages are higher in the Akita mouse model of spontaneous β -cell ER stress that leads to diabetes. We also find that iPLA2 β  participates in human islet β -cell apoptosis induced by hyperglycemia and cytokines and that islet iPLA2 β  and NSMase messages are elevated in prediabetic NOD mice. Further, we recently found evidence for inhibition of iPLA2 β  leading to decreased incidence of T1D in diabetes-prone NOD mice. These findings strengthen our hypothesis thatiPLA2 β  participates in the onset and progression of T1D. We propose to validate our hypothesis in human T1D by correlating expression levels of iPLA2 β  with indices of beta-cell apoptosis in pancreas samples obtained from control and diabetic donor subjects.

Shiva Reddy University of Auckland, New Zealand Immunohistochemical identification of molecular markers of oxidative and nitrosative stress in β-cells during the early stages of human type 1 diabetes mellitus

This research seeks immunohistochemical evidence of beta cell oxidative and nitrosative stress during various stages of T1D and whether such stressors are also present preceding and during the early onset of the disease. Antibodies to nitrotyrosine, a marker of peroxynitrite-mediated cell damage and to 8-hydroxy-2-deoxyguanosine (a marker of DNA oxidation) are being employed in triple-label immunohistochemistry to determine if such processes are activated within the beta cell. Combined immunohistochemistry and histochemistry are being applied to correlate the immunolabelling of such deleterious markers with the extent and spatial distribution of leukocytic infiltrates within the islets and with beta cell co-localization. Studies to date have shown weak to moderate immunolabelling of nitrotyrosine restricted to a majority of the remaining beta cells from 5 T1D cases, being also present in islet beta cells with minimum insulitis. Nitrotyrosine was absent in glucagon cells and the islet leukocytes. It was absent or weakly expressed in islet cells of non-diabetic donors (2 cases). DNA oxidation was observed within islet endocrine cells but was not restricted to beta cells from T1D cases.

The expression of nitrotyrosine and DNA oxidation are being extended to non-diabetic cases with single or two autoantibodies.  In line with previous in vitro studies with human islets in culture, the current research is also exploring the possible activation of the pro-inflammatory cytokine-nitric oxide axis within the islets of T1D cases, likely under-expression of anti-oxidant enzymes and whether stressors within the pre-diabetic beta cell lead to beta cell apoptosis and attract the early immune response.

Protocol development, and search for suitable primary antibodies and optimization studies are in progress for the immunohistochemical localization of interleukin-1β, inducible nitric oxide synthase and interferon-α.

Charmaine Simeonovic, Christopher Parish, J. Dennis Wilson & Andrew Ziolowski Australian National University Heparan sulfate levels mark the health status of human islet β-cells

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Roland Stein Vanderbilt University Medical Center Analysis of Islet Enriched Transcription Factor Expression and Activity in Normal & Type 2 Diabetic Islets

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Gerald Taborsky, Jr. University of Washington Glucagon secretion and islet neuropathy

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Emil Unanue Washington University School of Medicine The immunogenicity of abnormal catabolites of insulin metabolism

We are searching for abnormal products of insulin metabolism that appear in beta cell as a result of the increased demands for insulin. These abnormal catabolites in NOD mice select for very unique CD4 T cells that initiate this chronic autoimmune reaction. The plans are to search for these abnormal products in islets from early diabetic subjects. Their presence in human islets may point to key antigens that may be the target of future immunotherapy.

Hongju Wu & Vivian Fonseca Tulane University Pancreatic GLP-1 vs glucagon production in healthy and diabetic patients

Glucagon-like Peptide 1 (GLP-1) and glucagon shares the same precursor molecule proglucagon, but each arises from a distinct posttranslational process in a tissue-specific manner. Recently GLP-1 has been shown to be co-expressed with glucagon in pancreatic islet cells. Our preliminary data showed GLP-1 was progressively up-regulated in pancreatic islets during type 2 diabetes development. These data suggest intra-islet GLP-1 production may have an impact on the clinical and pathophysiological processes of these diseases and may be a target for therapeutic approaches. This project is thus designed to investigate this matter in human type 1 and type 2 diabetic patients. In addition, since GLP-1 based drugs, incretins, have been developed and approved for clinical use, it is thus essential to investigate their impact on intra-islet GLP-1 production. The main objectives of this project are thus 1) To investigate whether GLP-1 vs glucagon production in pancreas of human has changed with the development of type 1 and type 2 diabetes; and 2) To investigate whether the GLP-1 based therapies (i.e., incretin therapy) had any impact on the relative production of GLP-1 vs glucagon. To accomplish the goals, pancreatic tissues from normal, type 1 diabetic, type 2 diabetic without incretin treatment and type 2 diabetic with incretin treatment will be analyzed by immunofluorescence staining. The GLP-1 vs glucagon expression will be quantified and analyzed for statistical significance. In addition, the matching blood samples will be used to obtain physiological data related to glucose regulation. The results will help us understand the complete effect of incretin drugs, and identify potential safety issues. Therefore, the study will not only advance our knowledge on the regulation of intra-islet GLP-1 production in diabetes, but also have significant impact on the clinical practice regarding current use of incretins.

Bone Marrow Studies

Reza Abdi & Marwan Mounayar Brigham and Women’s Hospital Intrinsic immunoregulatory defect of bone marrow mesenchymal stem cells (BM-MSC) from T1D patients

Type 1 diabetes (T1D) represents a major public health burden demanding innovative treatment strategies. Mesenchymal stem cells (MSC) have profound immunomodulatory effects. Numerous clinical trials have focused on the immunomodulatory capacity of MSC to treat various immune-mediated diseases.  We were amongst the first to show the protective effects of allogeneic MSC in autoimmune diabetes in NOD mice. More studied need to be carried out in particular on human MSC to unravel the mechanisms of immunomodulation of MSC to help in the design of MSC therapy in T1D.  We are fortunate to collaborate with nPOD to receive samples from T1D patients to isolate and grown MSC for our studies to examine their immunomodulatory capacity. Data to be generated using these samples are crucial in better understanding as how MSC suppress autoimmunity which would support the bases for These data provide important preclinical data supporting the basis for further development of MSC-based therapies for T1D and potentially, for other autoimmune disorders.

Maria Grant University of Florida Bone marrow progenitor cell (BMPCs) dysfunction in diabetes is mediated by reduced bioavailability of NO

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Paolo Madeddu & Costanza Emanueli University of Bristol, UK Microangiopathy in diabetic bone marrow

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Core Lab

Mark Atkinson University of Florida Pancreatic immunologic and metabolic parameters

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Graeme Bell University of Chicago MODY mutation testing

Perform Maturity Onset Diabetes of the Young (MODY) testing for select nPOD samples.

Santica Marcovina University of Washington Northwest Lipid Research

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Janelle Noble Children’s Hospital Oakland Research Institute High resolution HLA typing

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ProImmune Ltd Oxford, UK HLA Testing

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Stephen Rich University of Virginia Immunochip Assays

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Clive Wasserfall University of Florida UF AAb Core

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Bill Winters University of Florida UF Clinical Pathology Lab/ICA lab

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Liping Yu University of Colorado-Denver Denver AAb Core

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Mark Stuart Anderson University of California, San Francisco Identification of novel Tissue-Specific Antigens expressed by human Extrathymic Aire-Expressing Cells, and determination of their potential contribution to the prevention of type 1 diabetes

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Manuela Battaglia San Raffaele Scientific Institute, Italy Dissecting the effector/regulatory compartments in the target tissues of T1D

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Jeffrey Bluestone & Armando Villalta University of California, San Francisco Vascular endothelial growth factor receptor signaling regulates the pathogenesis of type 1 diabetes

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Todd Brusko University of Florida Molecular Signature of Autoimmune T cells in Type 1 Diabetes and Treg TCR deep sequencing. Detect Cd226+ Treg and Teff ininsulitis patient samples

The Brusko Lab is generally interested in the cellular immune response involved in the disease process leading to type 1 diabetes (T1D). A strong genetic association between T1D and the MHC class II gene region of the adaptive immune system has implicated coordinated T and B cell responses as a central mediator of the disease process. To date, the majority of studies in humans have been limited to investigations using peripheral blood samples. While these studies have identified a number of T cells reactive to autoantigen targets (e.g., insulin, glutamic acid decarboxylase (GAD), islet antigen-2 (IA-2), zinc transporter-8 (ZnT8), among others), they have been unable to identify clonal signatures unique to T1D patients. In an effort to generate a disease specific signature, our lab initiated studies to isolate CD19+ B cells, CD8+ T cells, CD4+ T conventional cells (Tconv), and CD4+CD25+CD127-/lo Tregs from nPOD-donor tissues including spleen, pancreatic draining lymph nodes, irrelevant draining lymph nodes, and peripheral blood. From this donor material, we will generate a molecular profile of T1D including a characterization of the T cell receptor (TCR) and B cell receptor (BCR) repertoire, global DNA methylation profile, and transcriptional signature.  We believe that this effort will lead to novel insights into the specificity and functional properties of immune cells that lead to autoreactive beta cell destruction. Of particular interest, we have been focused on a T cell costimulatory pathway involving CD226 or TIGIT interacting with CD155. We believe the molecular signatures identified in cell subsets isolated from the site of autoimmune attack (e.g., the islets and draining lymph nodes) will provide insight into the cause of immunoregulatory defects leading to T1D. 

John Cambier & Peter Gottlieb University of Colorado, Denver Tracking the loss of anergic B cells in pre-diabetic and new onset T1DHLA Testing

Insulin-specific B lymphocytes play an important role in development of autoimmunity in Type 1 Diabetes (T1D). Since potentially offensive autoreactive cells are silenced by mechanisms of immune tolerance, participation of insulin-binding B cells (IBCs) in T1D must reflect escape from this silencing. Examining this question, we found that those IBCs bearing antigen receptors with high affinity for insulin occur only in the anergic BND (naive IgD(+)IgM(-) B cells [Duty, 2009 J Exp Med. 16: 139-51]) B cell compartment in peripheral blood of healthy subjects. Importantly, these cells are absent from this compartment in some “at risk” first degree relatives and all pre-diabetic and new-onset (<1yr) T1D patients. BND cells were found at normal levels in individuals diabetic for >1 year. Interestingly, these changes were associated with loss of the entire BND compartment. By extrapolation from parallel studies of in diabetic NOD mice, we believe that under these circumstances B cells relocalize in tissues rich in their autoantigen, where they become activated and can present antigen to T cells. These findings suggest that environmental events such as infection or injury may, by disrupting B cell anergy, dispose individuals to autoimmunity, the precise tissue target of which is specified by genetic risk factors.

Raphael Clynes Columbia University TCR signal transduction in diabetogenic T cells

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Howard Davidson University of Colorado Health Science Center Characterization of autoantigen-specific T cell receptors from PLNs

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Mark Davis & Ruth Taniguchi Stanford University Analysis of the antigen-specific T cell repertoire in T1D patients

The majority of human T cell antigens relevant to T1D have been defined using algorithms which predict high affinity peptide binding to MHC.   However, direct evidence showing that these antigens are presented to T cells by human islets and APCs in T1D patients has not been established for the vast majority of these peptides.  A major goal of our project is to identify T cell peptide antigens relevant to T1D which meet the following criteria: 1) the peptides are naturally processed and presented by human islets/APCs and 2) there are T cells specific for these antigens in the PLN and/or islets of T1D patients.   To achieve this goal, we are eluting peptides from class I MHC on HLA-A2+ human islets and identifying them by mass spectrometry to define naturally presented antigens.  We are also verifying natural antigen presentation by using antigen-specific T cell lines as “biosensors” of antigen presentation on islets and APCs.  Islet-specific peptides that are shown to be naturally presented will be used to make MHC-peptide tetramers and analyzed by CyTOF technology to measure the frequency, phenotype, and function of T cells specific for these antigens in T1D pancreatic draining lymph nodes obtained from the nPOD tissue bank.  By verifying the natural antigen presentation of islet-derived peptides and the presence of activated T cells specific for these peptides in T1D patients, we will be able to differentiate between peptide antigens with bonafide relevance to T1D from islet peptides that are irrelevant to T1D.  CyTOF analysis of the frequency, phenotype, and function of pathogenic T cells specific for disease-relevant islet antigens in T1D patients should be informative with respect this disease, potentially enabling sophisticated and early diagnoses and the ability to assess the efficacy of therapeutic intervention in human patients.

Marc Donath, Thierry Nordmann & Elise Dalmas University Basel, Switzerland Identification of islet associated immune cells in type 2 diabetic patients and further exploration of their role in disease progression and severity

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Garry Fathman Stanford University Microarray analysis of PLN from autoantibody positive donors

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Georgia Fousteri San Raffaele Research Institute T follicular helper (TFH) and regulatory (TFR) cells in T1D

Type 1 Diabetes (T1D) is characterized by the gradual loss of insulin-producing beta cells, which are eliminated by autoreactive cells infiltrating the pancreas. In humans and mouse models of the disease, T1D susceptibility is determined by genetic factors and is greatly influenced by environmental triggers (1). Progression to T1D is usually preceded by a period of anti-islet autoantibody production, which often can last for years (2). Today, autoantibodies are most widely used as serum biomarker (3), but T-cell readouts and metabolome studies are underway as they may strengthen diagnosis, prognosis and most importantly bring forward effective protocols for prevention and cure. T follicular helper (TFH) cells are essential for the formation of germinal centers (GCs), specialized structures in secondary lymphoid organs where maturation of B cells into high-affinity plasma cells and long-lived memory B cells takes place. It has become clear that precise control of the GC response is important for the production of optimal antibody responses that are devoid of selfreactivity. The exact mechanism by which autoreactive B cells are eliminated during this process is not fully understood, however the number of TFH cells together with a specialized subset to FOXP3+ regulatory T cells, namely T follicular regulatory cells (TFR), finely control the survival of pathogen-specific high-affinity B cells. It is currently not known whether TFH or TFR cells are involved in the production of islet-reactive, autoantibody-producing B cells in patients with T1D. However, given that autoantibodies emerge prior to disease onset, the effect of TFH cells in T1D progression can be inferred. With this study we aim at identifying whether TFH and TFR cells are implicated in autoantibody development in T1D. Investigating this mechanism poses a cardinal opportunity to increase the pipeline of therapeutic targets and to improve the number of disease biomarkers.

Kathleen Gillespie University of Bristol Maternal microchimerism in T1D pancreas

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Kathleen Gillespie & Abby Willcox University of Bristol Molecular insights into the type 1 diabetes pancreas

Pancreatic lymph nodes (PLN) are the central location for the autoimmune response that leads to type 1 diabetes (T1D), yet no histological studies of this crucial step in pathogenesis of human diabetes exist. In this study, we request sections from T1D and matched control PLN to allow us to follow up our preliminary observations showing that replicating germinal centers are diminished in recent onset T1D. In particular, we will focus characterizing the structure and cellular content of PLN in T1D and will test the hypothesis that lack of germinal center replication reflects attempted regulation of the immune response.

Dale Greiner & Leonard Shultz University of Massachusetts Diabetogenic function of autoimmune donor splenocytes in humanized mice

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Abdel Hamad & Thomas Donner Johns Hopkins University Fas Ligand: Unorthodox target for prevention of type 1 diabetes

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Kevan Herold & Alfred Bothwell Yale University Analysis of Class I MHC expression in islets from patients with type 1 diabetes

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Kevan Herold & Alfred Bothwell Yale University Reconstitution of HHLS mice with bone marrow from patients with T1DM

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Dolores Jaraquemada, Mercè Martì & Carme Roura-Mir University of Barcelona, Spain Tracing effector and regulatory T cell populations in type 1 diabetes

More information to come…

Sally Kent University of Massachusetts Medical Center Investigation of β cells in human islets and PLN in T1

More information to come…

David Leslie University of London Risk of autoimmune disease and human self-antigen expression

We have identified type 1 diabetes (T1D)-associated differential DNA methylated variable positions (T1D-MVPs) in CD14+ from peripheral blood mononuclear cells (PBMCs) from monozygotic (MZ) twins discordant for T1D (Rakyan V et al.; Plos Genetics 2011). We aim to determine the role of T1D-associated MVPs in the mechanisms that underpin immune dysfunction in T1D. We have therefore studied whether the same disease signature is detectable in T1D tissue compared with normal tissues relevant to the disease, namely: pancreatic lymph nodes (site of self-reactive T cells priming) compared with the spleen (as control). In addition we are examining the methylation state of T1D-MVPs in selected cells from human thymus and we intend to make these cells available to nPOD in due course.

Sara Michie & Baohui Xu Stanford University Tissue-selective chemokines and adhesion molecules in human T1D

Migration of lymphocytes from blood vessels into pancreatic lymph nodes (PanLNs) and pancreas is critical for the development of islet inflammation and beta cell destruction in type 1 diabetes (T1D). Adhesion of lymphocytes to the luminal surface of blood vessel endothelia in PanLNs and pancreas is an important step in this migration. However, the adhesion molecules that control migration of lymphocytes into human PanLNs and pancreas during the development of T1D are not well defined.

The goals of our project are to determine which endothelia adhesion molecules, and their lymphocyte receptors, are highly expressed in human PanLNs and pancreas during development of T1D, as compared to pancreas and PanLNs of controls (Aims 1 and 2). The physiologic importance of adhesion molecules that are expressed on lymphocytes and endothelia of PanLNs and pancreas during development of T1D will be determined using Stamper-Woodruff in vitro binding assays (Aims 3 and 4). Successful completion of these studies will help define the immunological mechanisms that are important for the development of islet inflammation and beta cell destruction in human T1D.


Maki Nakayama & Joe Larkin, III University of Colorado Dynamic Survey of T Cell Repertoire Targeting Pancreatic Beta Cells

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Massimo Pietropaolo & Michael Morran The Brehm Center for Diabetes Research, University of Michigan Identification of T1D-specific Fab fragments of IA-2 dominant conformational epitope

One of the primary characteristics of Type 1 Diabetes (T1D) is the presence of autoantibodies directed to self-antigens expressed in the pancreatic islets, specifically those associated with the insulin secretory machinery. Although T1D is well characterized as a T-cell mediated disease, a mechanistic role of islet autoantibodies generated during the progression of T1D have not been elucidated. The tyrosine phosphatase-like protein (IA-2) is a major neuroendocrine autoantigen associated with the progression of T1D. We previously identified a new candidate biomarker within the extracellular domain of IA-2 (IA-2ec) in humans, which is associated to rapid acceleration of T1D onset compared to conventional IA-2 antibody biomarkers of T1D. Little is known about the clonality and the V region structures of IA-2 autoantibodies, an important consideration given their diagnostic and predictive value. Thus, our objective is to perform de novo sequencing to determine the clonality and V region structures of human autoantibodies directed against the islet autoantigen IA-2.  This method can be applied to multiple serum samples and has been validated by the discovery of a clonotypic autoantibody specific for an immunodominant determinant of target antigens of a number of autoimmune diseases including systemic lupus erythematosus (SLE) rheumatoid arthritis (RA). In particular, we aim to characterize the IA-2 autoantibody sequence in the sera of the well-characterized population of T1D patients of the nPOD network in an effort to determine if these autoantibodies express a highly restricted B-cell clonotype repertoire which may be shared (public) across different disease phenotypes. We will then identify unique V region peptides to interrogate human sera for IA-2 by a targeted mass spectrometry (MS) protocol. Mass spectrometry-based detection of specific autoantibody motifs may provide a powerful new tool for analysis of humoral autoimmunity in T1D.

Alberto Pugliese & Francesco Vendrame Diabetes Research Institute, University of Miami Assessment of memory T cells in the insulitis lesion

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Helena Reijonen Benaroya Research Institute at Virginia Mason Correlation of Islet Autoantigen-Specific T-Cell Repertoire in the Pancreatic Lymph Nodes and Peripheral Blood in T1D Autoimmunity

More information to come…

Bart Roep Leiden University, Netherlands Detection of islet autoreactive CD8 T-cells in insulitis versus periphery

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Suparna Sarkar & Dirk Homann University of Colorado, Denver Pancreatic expression of chemokines in human type 1 diabetes

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Nora Sarvetnick & Kamiya Mehla University of Nebraska Medical Center To identify the role of IL-18R+ CD8 T cell subsets in islet destruction

More information to come…

David Serreze & Kevin Mills The Jackson Laboratory Targeting AID expressing B-lymphocytes as possible clinically applicable T1D intervention

Diabetogenic B-lymphocyte activity may be enhanced by SHM induced increases in Ig antigen
-SHM requires AID induced double strand DNA breaks repaired by RAD51 complex proteins.
-AID is expressed at higher levels in B-lymphocytes from NOD mice than control strains
-A small RAD51 blocking molecule specifically induces apoptosis of AID expressing B-lymphocytes
from both NOD mice and humans.
-RAD51 blockade is being tested as a new B-lymphocyte directed late disease stage T1D intervention
approach in NOD mice.
-For possible future clinical translation purposes it will be important to assess if diabetogenic Blymphocytes
in humans are undergoingAID induced SHM, and thus may be susceptible to depletion
by RAD51 blockade.
-To address this need nPOD is being requested to supply five cryopreserved PLN samples each from
T1D patients and controls.
-We will use these nPOD samples to determine if B-lymphocytes within PLNs of T1D patients express
higher AID levels than those from controls and thus more susceptible to RAD51 blockade induced
apoptotic death.
-Previous published work by Jax investigators demonstrate they have an ability to assess AID
expression by human B-lymphocytes, and determine their sensitivity to RAD51 blockade induced

Hubert Tse University of Alabama-Birmingham Redox regulation of anti-viral responses in type 1 diabetes

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disease resulting in the destruction of pancreatic â-cells. Infiltrating leukocytes generate an inflammatory environment consisting of reactive oxygen species (ROS) and proinflammatory cytokines that collectively participate in â-cell destruction and enhance the adaptive immune effector response of islet-specific T cells. Environmental factors that include viral infections are hypothesized to directly lyse pancreatic â-cells and generate a pro-inflammatory milieu necessary to enhance T cell autoreactivity. Currently, a gap exists in our understanding of why the immune system attacks itself in Type 1 diabetics and the signals involved in initiating immune system activation in T1D. Our previous work has demonstrated the importance of oxidative stress and the generation of ROS as essential to the pathogenesis of T1D. ROS production is required for autoimmune responses during the development of T1D in Non-Obese Diabetic (NOD) mice, a mouse model that is widely used to study T1D. Mice deficient in superoxide synthesis (NOD.Ncf1m1J) are resistant to the development of T1D and our proposed studies to further define the synergy of ROS on immune responses have the potential to provide novel insight on the mechanisms associated with pancreatic â-cell destruction in T1D. Our proposed studies will test the hypothesis that ROS synthesis is necessary for efficient anti-viral responses in T1D. Specifically, we will define the role of redox-dependent signals involved in the activation of human innate immune receptors such as Toll-like receptor 3 (TLR3), melanoma differentiation-associated gene 5 (MDA5), and retinoic acid-inducible gene I (RIG-I) in response to Coxsackie virus infection with PBMCs from recent onset T1D and healthy patients. The studies in this proposal will further expand our knowledge of how innate immune-derived pro-inflammatory signals synergize to mediate efficient autoreactive T cell responses in Type 1 diabetes.

Matthias von Herrath & Darius Schneider University of California, San Diego In situ detection of pro-inflammatory cytokines within pancreatic islets from diabetic subjects

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Thomas Waldmann & Jing Chen NCI/NIH The role of IL-15 and IL-15Ra in the pathogenesis of type 1 diabetes

Interleukin-15 (IL-15) is a pro-inflammatory cytokine that promotes the activation and maintenance of natural killer (NK) and CD8 (+) T-effector memory (T-EM) cells. In patients with type 1 diabetes, elevated serum levels of IL-15 have been reported. Using an assay developed in the lab, we demonstrated that the serum sIL-15Rα levels were significantly higher in patients with T1D compared with those of normal controls. To investigate whether islet overexpression of IL-15 and IL-15Rα could play a role in the pathogenesis of T1D, we generated double transgenic mice with β cell specific expression of both IL-15 and IL-15Rα under a rat insulin promoter. The mice developed hyperglycemia, marked mononuclear cell infiltration, β cell destruction and anti-insulin autoantibodies that mimic the early events of human T1D. Inhibiting IL-15 signaling with anti-IL2/IL15Rβ (anti-CD122), which blocks IL-15 transpresentation, or with the Janus kinase 2/3 inhibitor tofacitinib, reversed the diabetes in the transgenic mice. This suggested a causative role of IL-15 and IL-15Ra  in the induction of insulin dependent diabetes in mice.

To further investigate whether IL-15 and IL-15Ra play a role in the pathogenesis of human type 1 diabetes, we investigated the expression of IL-15 and IL-15Ra in the islets of patients with type 1 diabetes, anti-insulin autoantibodies and normal donors.  Immunohistochemistry demonstrated IL-15 expression in the islets in all T1D patients examined whereas IL-15 was detected only in the pancreatic islets in one out of four normal donors and two out of five autoantibody positive donors. Interestingly, in two cases where IL-15 was positive, we also detected islet expression of MxA, a GTPase induced by type 1 IFN (α/β) that has been reported to interfere with virus multiplication and spread. Furthermore, IL-15 and IL-15Rα mRNA levels were also higher in the islets from T1D patients compared to that from autoantibody positive donors or normal donors. The islets were microdissected based on insulin positivity using laser capture microscopy (LCM). Taken together, our data suggest that disordered IL-15 and IL-15Rα may be involved in the pathogenesis of human T1D.

Shannon Wallet University of Florida Role of mucosal epithelium in autoreactivity

One of the major questions we still don’t have an answer to is why some people who are genetically predisposed for development of T1D don’t progress and why others do. In addition, why there are different timings for the progression of this disease (i.e. why are some faster than others).  Our thoughts are that these differences are due to differences in environmental experiences.  It is important to note that we feel that these different environmental experiences can be big or small (i.e. living in different areas, eating different foods, or infection with different organisms). Thus, my laboratory studies the communication of the immune system with the environment at the largest environmental interface within our body, the gastrointestinal track.  We feel that if we can understand the difference in this communication that occurs in T1D and how it shapes the immune system, we can not only figure out the initiating events of this disease process, but design better therapies and even preventive treatments to curb the disease process.

Clive Wasserfall University of Florida Humoral immunity in type 1 diabetes

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Novel Biomarkers

Vitaly Ablamunits, Kevan Herold & Jasmin Lebastchi Yale University Comparison of total pancreatic insulin content and peripheral markers of beta cell function and desctruction

More information to come…

Ken Coppieters, Ditte Tornehave & Charles Pyke Novo Nordisk A/S IL-21/IL-21R and GLP-1R staining on pancreas tissues from (pre-)T1D, T2D and control donors

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Nika Danial & Ulupi Jhala Harvard Medical School Molecular signatures of islet inflammation in type 1 diabetes
The process of β-cell decline in the lead-up to the overt diabetes could provide critical clues for timely
therapeutic intervention. However the complexity, cell-type specificity, rapidly changing dynamics and
the cumulative effects of inflammation all inform the outcome of the autoimmune attack on beta cell.
This proposal is designed to use multiple in vitro models systems of beta cell inflammation, along with
human tissue samples as well as human serum samples from T1D children, to arrive at a temporal
proteomic and metabolomics profile of human islet inflammation and death.
These data are likely to contribute towards our understanding of biomarkers that would assist in
staging the progression of human type 1 diabetes. The ultimately goal is to be able to use the
information gleaned form the grant to identify windows of opportunity when therapeutic intervention
could preserve beta cell mass and function to delay the progression towards full-blown diabetes.
Zhiguang Guo, Alex Rabinovitch & Romano Regazzi Sanford Research/Sanford Health Identifying and Validating Noncoding RNAs as Human Beta Cell-Specific Biomarkers

Direct identification and validation of human β cell-specific biomakers is critical for monitoring the change of human β cell mass at prediabetes and the new-onset diabetes stage and during the therapeutic intervention. Due to technical and ethical constraints, our ability to prospective studies of human β cell biomarkers in response to β cell stress/apoptosis and its correlation with early β cell loss in human is limited. The objective of our study is to develop biomarkers of human β cell stress/apoptosis correlating with β cell loss that can be used to non-invasively monitor the development of diabetes pathogenesis and the outcome of treatments aimed at preserving or restoring functional β cell mass. The goal of this study is to identify potential biomarkers including a panel of noncoding RNAs in human islets that are correlated with β cell stress and apoptosis and to determine whether novel β cell biomarkers in circulation are correlated with early human β cell loss. We hypothesize that β cell ER stress and apoptosis induction increases a panel of noncoding RNAs in human islets in the pancreas of patients with diabetes and dead or dying human β cells can release them into circulation and the levels of these noncoding RNA in circulation are associated with β cell loss. We plan to collect islet cells for miRNA expression profiles by laser capture microdissection (LCM) and determine whether a panel of β-cell specific noncoding RNAs in islets of human pancreas specimens from patients with type 1 and type 2 diabetes are up-regulated by real-time PCR array. We will confirm some up-regulated noncoding RNAs in human islets are β-cell specific by miRNA fluorescence based in situ hybridization (FISH). We will validate this panel of noncoding RNAs, as β cell-specific biomarkers, in plasma of patients with type 1 and type 2 diabetes. Success in our proposed experiments would help us to further develop a panel of β cell biomarkers, which are correlated with early β cell loss, for clinical evaluation of disease stages including pre-diabetes and of therapeutic interventions to promote β cell health and survival.

Paul Harris, Masanori Ichise & Matthew Freeby Columbia University Determination of specific and non-specific binding of 18F-FP-DTBZ in whole pancreas homogenates obtained from controls and patients with longstanding type 1 diabetes

In the past we identified Vesicular Monoamine Transporter Type 2 (VMAT2), as a biomarker of beta cell mass that is quantifiable in vivo by Positron Emission Tomography (PET). PET is a tomographic imaging technique providing accurate non-invasive dynamic measurements of regional PET tracer uptake and clearance. These measurements are used to calculate VMAT2 density in the pancreas as a surrogate measure of beta cell mass.  Immunohistochemical staining of both control and long term T1D pancreata show excellent correlation between VMAT2 and insulin staining.  When PET and the radiopharmaceutical 18F-FP-DTBZ are used to non-invasively measure VMAT2 in vivo, we observe that healthy subjects have a significantly greater pancreatic VMAT2 signal than long term T1D patients with documented inability to secret normal amounts of insulin.  However there is a significant residual VMAT2 signal in the pancreata of the T1D patients.

The goals of our project are to understand the nature of this background signal.  There are three not mutually exclusive explanations of the background signal observed in pancreata believe to be beta cell ablated due T1D; 1) The radioligand 18F-FP-DTBZ is binding specifically to VMAT2 or some other receptors, not present in beta cells (specific and displaceable off-target binding).  Specific off target binding is being measured in a clinical PET study currently in progress. We have also quantified, using immunohistochemistry, the expression of VMAT2 laying outside the beta cell subset in human pancreata (e.g. PP cells).  The specific off target bind does not seem to account for the background,  2) 18F-FP-DTBZ may also bind non-specifically and non-displaceably to pancreas tissue (off target non-specific binding).  The in vivo non-specific binding is currently being measured in clinical PET study in progress.  Our in vitro estimates of non-specific binding using cadaveric pancreas tissue supplied by NPOD suggest that a significant portion of the in vivo background signal is due to non-specific binding.  However, these in vitro methods may overestimate in vivo binding of the tracer and must be interpreted with caution, 3) The last formal possibility, that our measurements of VMAT2 in the long term T1D pancreas indeed are on target and specific, representing residual VMAT2 possibly associated with beta linage cells is now under consideration.  Such a possibility seems to conflict with preexisting immunohistochemistry data, but immunohistochemistry is not the most sensitive technique available for detecting low levels of expressed proteins.  The next phase of our project will be to study long term diabetes pancreata for VMAT2 gene expression using in situ hybridization and RT-PCR techniques.  While the pancreas is a difficult organ to obtain RNA suitable for such studies, the generally excellent quality of NPOD tissues will improve our chances for success. 

Mark Huising The Salk Institute Urocortin 3 as a sensitive marker for beta cell function

More information to come…

Richard Kitsis Albert Einstein College of Medicine ARC, a novel beta cell survival factor in type 2 diabetes

more information to come…

Richard Lloyd, Heikki Hyoty, Joseph Petrosino & Ivan Gerling Baylor College of Medicine Beta-cell stress signatures and infection in T1D islets

This project will be the first to examine stress granule responses and the expression of long non coding RNAs in donor islet cells, a model beta cell line and the role of enterovirus infection in modulating these stress responses. This information will be used as a benchmark to examine nPOD tissues for these same stress responses, mobilization of innate immunity markers. The above experiments will also be performed in conjunction with analysis of the replication of terminally-deleted defective enteroviruses in the cell lines, and the stress responses they produce. This will inform secondary studies of actual nPOD tissues for the level of stress responses, key lncRNA expression profiles present. The nPOD tissues will also receive the most stringent analysis for virus capsid proteins and particularly viral RNA with single molecule sensitivity on a single cell basis that has yet been attempted.

Anna Moore & Amol Kavishwar Massachusetts General Hospital In vivo imaging of islets

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Jerry Nadler, Margaret Morris & Julius Nyalwidhe Eastern Virginia Medical School Protein based biomarkers for type 1 diabetes

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Bruce Seligmann, Klaus Pechhold & Horacio Rilo BioSpyder Technologies, Inc. In situ gene profiling and identification of T1D disease mechanisms

This CBDS program will establish an in situ RASL-Seq Islet Study Facility that will validate a novel in situ RASL-Seq platform for diabetes research using FFPE tissue from normal and Type 1 diabetes (T1D) diseased pancreas, and then apply this platform to address the gap in available methodology to take full advantage of T1D organ repositories such as nPOD in the effort to derive molecular profiles of individual -cells and cells of other types associated within the islet that are involved in the onset of silent T1D and disease progression. We will measure gene expression profiles from individual cells, and from this data identify biomarkers and gene expression phenotypes of silent T1D compared to more advanced stages of disease. We will exploit “big data” to apply pathway analysis to the gene expression results in order to identify putative molecular mechanisms of silent T1D and disease progression, providing a basis for the development of novel therapeutic approaches. We will test matched serum to identify useful biomarkers of silent T1D from this accessible tissue, and potentially to provide the capability to divide patients into subgroups based on prognostic outcome of whether disease will progress or not.

Terry Strom, Thomas Thornley & Maria Koulmanda Harvard Medical School To identify mRNA and miRNA biomarkers associated with exosomes that predict the onset of T1D

There are currently no clinically precise biomarkers that predict the development of type 1 diabetes (T1D). This limits the ability to identify patients who will become diabetic at a time when they have a greater beta cell mass and thus may respond more favorably to interventional therapeutics aimed at stopping T1D progression. To this end, we propose to identify biomarkers that predict T1D progression by examining exosomes in at-risk and nondiabetic control individuals. Exosomes are small particles released by cells during inflammation that communicate “danger” to other cells nearby and at a distance. Exosomes contain numerous mRNA and miRNA messengers that alter the biology of target cells, which include cells of the innate and adaptive immune systems. We propose to characterize the transcriptional (mRNA and miRNA) contents of exosomes from at-risk and non-diabetic control tissues in order to identify potential biomarkers that predict progression of T1D.

David Taylor-Fishwick Eastern Virginia Medical School The patterns of NADPH oxidase-1 expression in the course of human diabetes

Increased levels of reactive oxygen species (ROS) lead to dysfunction and subsequent loss of insulin-producing beta cells in pancreatic islets. Several lines of evidence support the importance of increased NADPH oxidase (NOX) activity as one of the contributing factors to elevated levels of ROS in beta cells. NOX-1 activity has been established as one of the sources of ROS in beta cells under experimental diabetic conditions. A contribution of NOX-1 to beta cell dysfunction in human diabetes is supported by preliminary studies. To further validate these observations as a first step we seek to establish the pattern of expression NOX-1 in normal and diabetic human islets. We hypothesize that NOX-1 will be elevated in diabetic islets. NOX-1 expression will be studied in relation to both the specific cells within pancreatic islets (beta cells, alpha cells) identified using well established specific protein markers for each phenotype as well as to the endocrine “sufficiency” status of such cells. In order to specifically examine qualitatively and quantitatively islets from 6 non-diabetic controls, 6 type 2 diabetes, 6 type 1 diabetes of short and 6 of long duration as well as 6 autoantibody positive would be employed; hence a total of 30 donors will be included in this study. Paraffin embedded 7 ìm thick sections of pancreas will be immunostained, using a protocol previously developed specifically for nPOD specimens. Commercially available antibodies raised against human NOX-1, insulin and glucagon will be used. Stained sections will be subjected to microscopic evaluation using both conventional fluorescent microscope and confocal microscope. A qualitative and quantitative analysis of the obtained images will be employed. We expect that an increase in expression NOX-1 will be observed in islets obtained diabetic and autoantibody positive donors.

Qibin Zhang & Mark Atkinson Pacific Northwest National Laboratory Human pancreatic tissue proteome of type 1 diabetes

The goal of this project is to comprehensively profile the proteins expressed in human pancreas within both the islets and their surrounding environments, and to obtain relative expression levels of pancreatic proteins from different organ donor groups using mass spectrometry based bottom-up and top-down proteomics technologies. Snap-frozen pancreatic tissue sections from organ donor groups with  1) autoantibody negative, no diabetes; 2) autoantibody positive, no diabetes; 3) autoantibody positive and T1D; and 4) autoantibody negative and T2D will be analyzed on both the peptide and intact protein levels for comprehensive identification and quantification of proteins and protein isoforms that differentially expressed among these subject groups. To complement with our study on pancreatic tissue sections, islets and beta cells will be isolated from pancreatic tissues using laser capture microdissection for their comprehensive proteomic characterization and comparison as well. The significantly changed proteins and the specific pathways and networks they are involved in may serve as candidate biomarkers for diagnosis and aid in elucidation of the pathogenesis of T1D.


Peter Butler, Alexandra Butler & Mark Atkinson University of California, Los Angeles Pancreatic duct glands in type 1 diabetes

The initial overall question to be tested by the collaborative of the Peter Butler laboratory at UCLA and the Mark Atkinson laboratory in Florida was to explore the possibility that the recently described pancreatic duct gland (PDG) compartment of the pancreas might serve as the elusive pancreatic stem cell niche, potentially offering an avenue to promote beta cell regeneration. Both laboratories had previously noted the presence of pancreatic beta cells in long standing type 1 diabetes mellitus (T1DM) and had been interested in the possibility that these were derived from beta cell regeneration rather than simply reflecting beta cell survivors.

The initial studies focused on quantification of the PDG compartment in T1DM with comparison to type 2 diabetes mellitus (T2DM) and non-diabetic controls. Since the primary interest in the PDGs was as a potential source of endocrine cells, we also examined the nPOD immunostained sections for insulin and glucagon to evaluate if the PDGs did indeed contain insulin or glucagon staining cells. During this process it was noted that there some of the nPOD sections had markedly expanded glucagon staining compared to normal controls. Further examination of the possible explanation of this led to the observation that the increased glucagon staining was most prominent in the group of individuals with T2DM treated with GLP-1 based therapy prior to their demise.

Following communication between the collaborating laboratories in Florida and Los Angeles, it became apparent that we had a responsibility to further delineate the unexpected differences between the pancreases of individuals with T2DM treated by GLP-1 based therapy and other therapies. The need for this was further heightened by the finding by the University of Florida group of neuroendocrine tumors expressing glucagon in GLP-1 treated T2DM patients. Further studies by an experienced pancreatic pathologist at UCLA raised concern that there was increased pancreatic dysplasia in the pancreases, and data from nPOD revealed increased overall pancreas replication (by Ki67) and expanded pancreas size. A question arose as to how best to make these observations known. It was decided that it would be preferable to include all the findings that seemed relevant to these observations in a peer-reviewed paper as well as to inform the domestic regulatory agency in charge of drug safety (i.e., the Food and Drug Administration – FDA). After months of rigorous peer review, the work saw publication in May of 2013 in the journal Diabetes.  In addition, following notification, the FDA posted a Drug Safety Communication in March 2013 noting that a group studying pancreatic pathology had suggested an increased risk of pancreatitis and pre-cancerous cellular changes in patients with type 2 diabetes treated with incretin mimetics.; however, the FDA could not reach any new conclusions about safety risks of incretin mimetic drugs. A vigorous response was seen from interested pharmaceutical interests including broad based subpoenas to multiple individuals at UCLA.  The current work in relation to the nPOD studies is now focused largely on repeating various aspects of the study (e.g., glucagon immunostaining originally preformed by nPOD at the University of Florida now being repeated at UCLA) to establish to what extent the criticism of the studies is valid (in which case the authors will acknowledge this) or if not, then respond with additional findings.  Since the initial FDA statement, they have subsequently published a follow up finding together with EMA (European Medicines Agency) in February 2014 in the New England Journal of Medicine. These agencies concluded that, based on current data, a causal association between incretin-based drugs and pancreatitis or pancreatic cancer could not be drawn.

In this regard the University of Florida group is now preparing a publication for peer review supporting the notion that the pancreas mass is indeed increased in individuals treated with GLP-1 based therapy. Moreover, the expansion is most apparent in the head of the pancreas, the region most vulnerable to pancreatic dysplasia.

The ongoing UCLA based studies to re-evaluate the alpha cell hyperplasia in GLP-1 treated individuals previously reported from the nPOD immune staining, has agreed with the conclusion of the prior analysis, that there is alpha cell hyperplasia in the incretin treated cases.

Once the issues of criticism in regards the prior study have been addressed the plan is to revert to the originally intended focus, specifically on the PDG compartment as a potential source of endocrine cell formation in humans.


Martha Campbell-Thompson, Dirk Homann & Richard Benninger University of Florida Mapping the histopathological landscape of human T1D: a pilot study (CLARITY)

The overall goal of this project is to extend studies examining human beta cells and immune cells in type 1 diabetes (T1D) to a new 3D fluorescent microscopy format for 500um or greater sized samples. These studies will adapt new tissue imaging methods (CLARITY) developed by  Dr. K. Deisseroth (Nat Methods 2013). The specific aims are: 1. Adapt tissue transparency methods for pancreas sample preparation and multiple immunofluorescence staining. 2. Create robust imaging and data collection methods to produce 3D islet topographical maps. 3. Examine islets in situ from donors with prediabetes and established diabetes for evidence of β‐cell regeneration and death. 4. Characterize immune cell subtypes infiltrating islets and their expression of cytokines and chemokines.

Roberto Gianani University of Colorado, Denver Characterization of type 1 diabetes subsets

More information to come…

Ben Giepmans University Medical Center Groningen Nanotomy of human islets of Langerhans in type 1 diabetes

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Akihisa Imagawa & Toshiaki Hanafusa Osaka University, Japan Histological differences between Japanese and Western type 1 diabetes

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George King & Susan Bonner-Weir Harvard University Joslin Medalist Study

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Jake Kushner Baylor College of Medicine Do de-granulated ß-cells persist in type 1 diabetes?

The goal of this proposal is to define the lineage mechanism of ß-cell persistence in type 1 diabetes (T1DM). A few ß-cells often persist in T1DM pancreata. But, the lineage mechanism of ß-cell persistence in T1DM remains poorly understood. Our overarching hypothesis is that ß-cell persistence in T1DM is due to ongoing ß-cell regeneration. Alternatively, ß-cell persistence in T1DM could be due regional differences in islet autoimmunity, or because ß-cells acquire a degranulated state that protects them from autoimmune mediated destruction. These hypotheses are readily testable with NPOD samples. However, by traditional techniques the analysis would require a huge number of slides and a many years to analyze. Our approach combines multi-labeling (5 antigens can be simultaneously detected) and high-throughput imaging technologies. This strategy will allow us to carry out this analysis using an absolute minimum of precious NPOD slides in a precise manner.

Alberto Pugliese Diabetes Research Institute, University of Miami Beta cells and autoimmunity correlations

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Christopher Rhodes University of Chicago “Empty Beta Cells” in type 1 diabetes

During the pathogenesis of Diabetes Mellitus, whether it be type 1 or 2 diabetes, islet cell heterogeneity becomes apparent. This is most commonly seen as a marked variability between the insulin content of some ß-cells versus others. Indeed, some ß-cells have such low insulin content that they appear to be empty. As such, in using insulin as a marker to estimate ß-cell numbers (as commonly done), one could be under-estimating the actual numbers of ß-cells presented since ‘empty ß-cells’ would not be accounted for. The concept of ‘empty ß-cells’ in the pancreas of diabetic patients could alter current thinking on ß-cell growth and regeneration. And, of course, if ß-cells are present in significant numbers, then it may become an issue of suitably refilling these cells with insulin rather than replacing/regenerating them.

We are interested in further examining the basis of ß-cell heterogeneity and ‘empty ß-cells’. Is this symptomatic of ß-cell dedifferentiation, a degranulated ‘hard working’ ß-cell and/or a stressed ß-cell approaching its end. We have used the transcription factor MafA as an alternative specific marker of adult pancreatic ß-cells, where a MafA+/Insulin- cell would indicate an ‘empty ß-cell’. But MafA is also susceptible to oxidation under oxidative stress conditions (e.g. hyperglycemia) and thus we are interested in examining other markers of ß-cell specificity to complement this analysis. We are also interested in examining adaptive plasticity of ß-cells and what mechanism guides the expansion of the rough endoplasmic reticulum, Golgi apparatus and trans-Golgi network reflective of upregulated insulin production to meet the increased demand in diabetes. Finally, we suspect that oxidative stress is a prime contributor to ß-cell destruction in the pathogenesis of diabetes and want to examine if an ‘empty ß-cell’ marks susceptibility to such stress and/or ß-cell very close to an apoptotic state.

Srinath Sanda & Rebecca Hull University of California, San Francisco IL-1beta expression in cystic fibrosis diabetes

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Lydia Sorokin & Eva Korpos University of Muenster, Germany Peri-capsular Basement Membrane Degradation During Leukocyte Penetration Into The Pancreatic Islet During Development of Human Type 1 Diabetes

Several steps are crucial for induction of type 1 diabetes (T1D); the first is extravasation of CD4+ T lymphocytes from blood vessels into the pancreatic tissue, the second is penetration of the peri-islet basement membrane (BM) surrounding the β-islets, and third the β-cell destruction which leads to appearance of disease symptoms.

BMs act to separate tissue compartments and represent barriers to the movement of both soluble molecules and cells. Hence, cells penetrating such protein barriers must employ specialized mechanisms (1,2). The main question addressed in our nPOD project is what is the mechanism used by leukocytes to penetrate the peri-islet BM and, thereby, reach the insulin producing  β-cells during the  development of T1D, in the non-obese diabetic (NOD) mouse model and in recently diagnosed T1D patients.

We recently published a comprehensive analysis of the extracellular matrix (ECM) composition of peri-islet capsules, composed of the peri-islet BM and subjacent interstitial matrix, in development of T1D in NOD mice and in human T1D (3), for which nPOD samples were crucial. Our data demonstrated global loss of peri-islet BM components only at sites of leukocyte infiltration into the islet in both mouse and man. Stereological analyses reveal a correlation between incidence of insulitis and the number of islets showing loss of peri-islet BM versus islets with intact BMs, suggesting that leukocyte penetration of the peri-islet BM is a critical step in disease development. We identified cathepsin S, W, and C activity at sites of leukocyte penetration of the peri-islet BM in association with a macrophage subpopulation in NOD mice and human T1D (3). Interestingly, the peri-islet BM is reconstituted once inflammation subsides, indicating that the peri-islet BM-producing cells are not lost due to the inflammation, which has important ramifications to islet transplantation studies (3).

We are now investigating how the cathepsins are involved in loss of the peri-islet BM: Studies are underway to detect and image cathepsin activity in mouse NOD and nPOD samples using cathepsin activity-based probes, which could lead to the development of novel diagnostic tools and therapeutic targets. In addition, we are examining the BM of the pancreas from different stages of human T1D and islet transplantation, with the aim of identifying ECM molecules that could promote islet survival.  

1) Korpos, E. et al, 2010. Cell Tissue Res. 339:47-57.
2) Wu, C. et al, 2009. Nat Med. 15:519-27.
3) Korpos et al, 2013. Diabetes 62:531-42.
Ranjeny Thomas & Katharine Irvine University of Queensland Pathological changes associated with chronic RelB activation in T1D

Dendritic cells (DCs) play a crucial role in establishing and maintaining the balance between immunity to pathogens and tolerance to self. Whether DCs evoke T cell activation or tolerance in response to antigen presentation is determined by the physiological context in which they differentiate and mature. DC differentiation and inflammatory signalling is abnormal in type 1 diabetes (T1D), but the relationship between this abnormality and disease pathogenesis requires clarification. We have shown constitutive activation and defective inflammatory responses in critical sub-units of the NF-kappaB transcription factor family, known as RelB and p65, which are required for myeloid DC differentiation, in T1D monocytes and DCs. Abnormal RelB activation may constitute a novel T1D risk phenotype. Our data indicate that peripheral blood cell RelB activation and associated inflammatory gene expression is increased in autoantibody-negative first-degree relatives of T1D patients. In autoantibody-positive and autoantibody-negative subjects, serum inflammatory cytokine expression is heterogeneous. We hypothesize that premature RelB activation and pro-inflammatory cytokine production during DC differentiation compromises the development of immunological tolerance and contributes to T1D pathogenesis. In this project we wish to investigate whether RelB activation and inflammatory cytokine expression in peripheral blood is associated with distinct pancreatic pathology in T1D and at-risk subjects. This information could enable us to stratify pancreatic pathology in living T1D patients and at-risk subjects, based on their peripheral blood RelB and inflammatory biomarker status. Moreover strategies for early therapeutic intervention to prevent or treat T1D may differ depending on pancreatic pathology.

Thomas Wight & Susan Perigo Benaroya Research Institute at Virginia Mason Extracellular matrix involvement in type 1 diabetes pancreatic islet destruction

The overall purpose of our research is to understand how changes in extracellular matrix affect insulitis initiation and progression and beta cell destruction in diabetes.

Our research focuses on a specific extracellular matrix molecule, hyaluronan, a long chain glycosaminoglycan distributed widely throughout different tissues. Hyaluronan is highly abundant in inflamed tissues and its synthesis is responsible for many of the physiologic changes associated with inflammation, including edema, vascular permeability changes, and leukocyte egress and activation at sites of injury, as well as the maturation of dendritic cells, antigen presentation, and the function and number of regulatory T cells. Hyaluronan exerts these effects through interactions and formation of macromolecular complexes with hyaluronan-binding molecules called hyaladherins, such as inter-α-inhibitor, versican, and tumor necrosis factor-stimulated gene-6. 

We have made the novel observations that hyaluronan and hyaladherins increase in pancreatic islets of younger donors and accumulate in regions of lymphocytic infiltrates in T1D, and that both the amount and distribution of these specific extracellular matrix molecules vary with time since diabetes onset. We have also shown that hyaluronan and hyaladherins are deposited in the follicular germinal centers and T-cell areas, regions of immune cell activation, in both human pancreatic lymph nodes and spleen in type 1 diabetes.

We are investigating (1) the regulation of hyaluronan accumulation in pancreatic islets and lymphoid tissue in T1D, (2) the functional significance of hyaluronan – hyaladherin complexes in initiation and progression of insulitis, (3) the effect of altered hyaluronan synthesis and hyaluronan interactions with hyaladherins on diabetes development, (4) the role of hyaluronan – hyaladherin matrices in modulation of immune cell interactions and their adhesive and migratory properties, and in promoting T-cell activation and proliferation.

Our novel findings were obtained from study of human samples provided by nPOD. Analysis of those tissues also forms the basis of the work in progress on changes in hyaluronan and hyaladherin matrices in human tissues in diabetes.

Type 1 Diabetes Etiology & Environment

William Bachovchin Tufts University Fibroblast Activation Protein in Type 1 Diabetes

More information to come…

David Carpenter, Miquel Porta & Sarah Howard University of Albany Persistent organic pollutants in the pancreatic tissue of people with and without diabetes

There is now strong evidence that persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) are major risk factors for type 2 diabetes.  While the mechanisms involved are not known with any certainty, the associations between development of type 2 diabetes and blood concentrations of POPs are stronger than those for almost any other factor, including obesity.  Some studies have suggested that obesity is in fact not a risk factor for type 2 diabetes, and that it is rather all of the POPs that are in the animal fats that are often consumed in excess that is really the risk factor. 

However almost nothing is known about any possible involvement of PCBs and other persistent organics as factors contributing to type 1 diabetes.  Type 1 diabetes is a quite different disease than type 2, but they do have some things in common.  Our proposal is to begin investigation of the possible role of POPs in type 1 diabetes by obtaining measurement of concentrations of PCBs in the pancreas of individuals with type 1 diabetes as compared to controls and those with type 2 diabetes.  PCBs will be used as an indicator POP.  All POPs are fat soluble and are found almost exclusively in the fat component of human tissues.  The pancreas, like every other organ, has some fat cells, and there are fats in the membranes of the beta cells that normally produce insulin.  One possibility is that there is excessive accumulation of POPs including PCBs in the pancreas in causes of type 1 diabetes.  This will be tested by direct determination of the PCB concentrations in tissues obtained through the nPOD project.  

Gun Frisk & Olle Korsgren Uppsala University, Sweden Different expression of extracellular CAR in islets in pancreatic sections and isolated islets

The etiology of the type 1 diabetes (T1D) is unclear but it has been shown that genetic factors influence the pathogenesis. In addition, several studies have shown that environmental factors likely contribute to the disease development. One such factor is virus infections, particularly the Coxsackie B viruses (CBVs), are among the main candidates and there has been numerous of studies showing association between these gut viruses and T1D. To be able to infect a certain cell it has to express an appropriate receptor and the main receptor for CVBs is the tight junction protein Coxsackie-Adenovirus-Receptor (CAR). The expression of the CAR protein has been shown to be increased during healing and during inflammation. The primary aim of this study is to determine the expression of CAR in pancreas especially on b-cell, and to what extent this expression is affected by the disease progress in T1D. The expression on human isolated islets, infected and un-infected cultured for prolonged periods will be studied also be studied. CAR expression in pancreas will also be studied in human pancreatic tissue from recent onset T1D donors, long standing T1D donors, islet autoantibody positive donors, T2D donors and controls to see if this protein “normally” is expressed in pancreas, especially on the b-cells. Studies are also on-going to see if the expression is affected by the auto- immunity in the pre diabetic and T1D islets. Preliminary results indicate that the expression level of CAR is significantly higher in islets from diabetes related donors compared to controls.  It is also 10-100 fold higher expression in the endocrine tissue from pre diabetic and T1D donors compared to the exocrine tissue from the same donors.

Dale Greiner & Rita Bortell University of Massachusetts Medical School Investigate the molecular link between increased risk of diabetes among patients with psychiatric disorders

Compared to the general population, studies show 2-3 times increased risk of developing metabolic syndrome in drug naïve patients with severe mental illnesses like schizophrenia or bipolar disorder. Impaired glucose metabolism in non-obese never-treated patients and in first degree relatives of schizophrenic patients, suggests a possible genetic association between diabetes and schizophrenia. We are proposing to study the molecular cause of increased metabolic risk among patients with psychological disorders.

Heikki Hyoty University of Tampere, Finland Virus detection in pancreas and other tissues

More information to come…

Richard Lloyd & Joseph Petrosino Baylor College of Medicine Virome and microbiome in T1D onset

T1D is recognized to result from both genetic and environmental factors.  Chief among environmental factors that are strongly linked to T1D are Type B enteroviruses (HEV-B), yet the association of these viral triggers is not proven and many questions exist. Which of the 60 HEV-B serotypes trigger T1D, what type of infections do they cause in the pancreas, and how can they trigger autoimmunity are just a few. We are working within the nPOD-V, the virus working group, to attempt to amplify and isolate infectious viruses from nPOD samples, and to characterize the type of infections that occur in pancreatic cells.   Upon amplification, samples are subjected to next generation deep sequencing to identify all viruses and microbes present in the samples in an unbiased manner.  Samples are also examined using very sensitive qRT-PCR assays that detect single copies of enterovirus genomes.  This work will provide the most complete picture of what infectious agents may be harbored in the pancreas of T1D patients, both before and after disease onset.

Grant Morahan, Patrick Concannon & Stephen Rich University of Western Australia Genetics of nPOD

We have found that people with type 1 diabetes fall into one of six subtypes that can be defined on the basis of the genetic variants they have. These subtypes differ with respect to various clinical features, including immunological traits and risk of diabetic complications.

In this proposal, we plan to use genetic information from the nPOD donors to define which T1D subtype they belonged to. This will allow analyses of findings from nPOD researchers to see if any of the features they are testing differ between the subtypes. This could be very important information for understanding T1D and future treatment of people with T1D.

In addition, we will set up a user interface that will allow nPOD researchers to test whether any of the information they have found from nPOD samples vary according to any of the genetic variants that have been tested.

Noel Morgan & Sarah Richardson University of Exeter Medical School, UK Enteroviral infection as a causative factor in type 1 diabetes

Considerable evidence has accumulated implying that enteroviral infection is associated with the development of type 1 diabetes but the precise nature of this relationship remains unclear. Many studies have revealed that enteroviral RNA is detectable in patient serum at, or before, the onset of disease but it is unclear whether viral infection also occurs commonly in other organs and tissues at this time. We are investigating this possibility using pancreas (and other tissue) samples recovered from patients with recent-onset type 1 diabetes which are made available from within the nPOD collection. We study these samples in parallel with a larger collection available to us within the UK.

Our initial aims are to evaluate whether enteroviral infection can be detected at the level of the pancreas in patients with type 1 diabetes and to compare the prevalence with relevant age-matched controls. To achieve this, we are employing immunological approaches using antisera raised against enteroviral capsid proteins to detect infected cells and to provide unambiguous identification of these cells. Such studies are coupled to parallel experiments designed to define whether a “viral footprint” can also be found which would indicate that infected cells mount an active anti-viral response. Among the candidate molecules comprising such a “footprint” we are examining relevant pathogen recognition receptors, pro-and anti-apoptotic proteins, interferon response genes and downstream targets of interferon signalling.

A further goal is to establish whether the infection of islet cells by enteroviruses follows an atypical course in type 1 diabetes. In particular, we are developing methods to differentiate between more classical acute infections (which culminate in cell lysis) and those which persist for prolonged periods without causing the demise of the cells. We hypothesise that the development of a persistent infection within islet beta-cells might be critical to the initiation of islet cell autoimmunity and we are examining the molecular events that could underpin the development of such a response. 

Hervé Perron & Virginie Vidal Geneuro, Switzerland Evaluation of HERV-W Envelope antigen expression in Pancreas and serum from patients with Type 1 Diabetes.

Endogenous retroviruses are known to represent 8% of the Human genome.  HERV-W family retains elements expressing an envelope protein (Env), which activates a pro-inflammatory and autoimmune cascade through interaction with Toll-Like receptor 4 (TLR4). This Env protein was evidenced in brain lesions, sera and circulating mononuclear cells of patients with Multiple Sclerosis (MS)1. Thus, all the background science from the past two decades that has brought independent confirmations and reproductions of an involvement of HERV-W in MS 2 convinced us to further study its association with other “autoimmune diseases”. If no association was found when testing series of patients with rheumatoid arthritis3 or systemic lupus1,  about one-third of sera from patients with T1D revealed positive for the HERV-W Envelope antigen. We therefore further explored whether this immunopathogenic endogenous protein could be expressed in pancreas from T1D versus controls, which revealed positive in about 40% of first pilot series, and are now preparing to extend these analyses to larger number of serum, PBMC and pancreas samples. In parallel, we are developing an HERV-W-Env induce mouse model of diabetes for pre-clinical studies with an anti-Env neutralizing humanized antibody.

1.             Perron H, Germi R, Bernard C, et al. Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease. Mult Scler. 2012; 18: 1721-36.
2.             Dolei A and Perron H. The multiple sclerosis-associated retrovirus and its HERV-W endogenous family: a biological interface between virology, genetics, and immunology in human physiology and disease. J Neurovirol. 2009; 15: 4-13.
3.             Gaudin P, Ijaz S, Tuke PW, et al. Infrequency of detection of particle-associated MSRV/HERV-W RNA in the synovial fluid of patients with rheumatoid arthritis. Rheumatology (Oxford). 2000; 39: 950-4.


Lorenzo Piemonti & Ilaria Capua San Raffaele Scientific Institute, Italy Role of influenza viruses in the etiopathogenesis of diabetes

More information to come…

Alberto Pugliese Diabetes Research Institute, University of Miami The nPOD-Virus Group

More information to come…

John Todd, Vincent Plagnol & Herbert Virgin, IV University of Cambridge, UK Viral sequence discovery by high throughput DNA sequencing of the nPOD diabetic pancreatic samples

More information to come…

Antonio Toniolo, Andreina Baj & Roberto Accolla University of Insubria Medical School, Italy Detection of enteroviruses in lymphoid tissue of donors with T1D of short duration and attempts to identify the infected cell type(s)

If type 1 diabetes (T1D) is caused/triggered by a viral infection in select genetic backgrounds – and Enteroviruses (EVs) are felt as major culprits -  investigators are expecting to find EVs into pancreatic Langerhans islets and also in association with spleen, lymph nodes, peripheral blood leukocytes. Infection of lymphoid cells, in fact, is common in systemic infections. Using an original method that couples virus amplification in susceptible cells with RNA amplification assays capable of detecting persistent and mutated EV types, we showed that different EV species are present in blood leukocytes of two thirds of children/adolescents at the clinical onset of T1D. Initial study of living cells from nPOD cases (T1D, non-diabetics) demonstrated that EV genomes and infectivity are present also in spleen and lymph nodes of T1D cases (1 to 30 years post-diagnosis). Thus, EVs appear to produce chronic silent infections in diabetes as in the post-polio syndrome (that is being studied by our group). If the viral genome is present, then virus proteins should also be detectable. Initial work done with colleagues looking at the same nPOD samples by ISH, immunohistochemistry, proteomics showed a strong concordance between genome detection and protein expression. So far, we have been successful in sequencing short stretches of RNA encoding conserved EV enzymes. We failed, however, in detecting genes encoding EV capsid proteins. This may be due to the pronounced variability of these proteins, especially in chronic infections. An additional aim of our studies is to determine which leukocyte subsets are carrying EVs. Preliminary results suggest that T cell subsets may be infected. Evidence in this direction, would push the idea that virus-induced immune disturbances might contribute to autoimmunity in T1D. Proof that chronic EV infections are indeed associated with T1D has not been reached, however collaborative studies of the same nPOD cases using many different approaches will certainly reveal if the virus hypothesis is a viable one. Should EVs be implied, major implications for human health will ensue (antivirals, novel preventive measures, etc.).

Steven Tracy University of Nebraska Enteroviral infection and T1D

More information to come…