Current nPOD projects

  • Category

Alpha Cell Physiology & Dysfunction

  • Nicolai Doliba & Doris Stoffers

    University of Pennsylvania
    Role of Alpha Cells in Pathogenesis of Type 1 Diabetes

    Multiple islet autoantibodies (AAb+) predict type 1 diabetes (T1D) and hyperglycemia within 10 years. By contrast, T1D develops in just ~15% of single AAb+ (generally against glutamic acid decarboxylase, GADA+) individuals; hence the single AAb+ state may represent an early stage of T1D. We previously discovered the functional defects in suppression of glucagon secretion in non-diabetic, normoglycemic GADA+ donors which precedes the insulin secretory defect seen in T1D. This GADA+ donor alpha cell phenotype is accompanied by altered cAMP signaling and alpha cell gene expression, particularly in glycolysis and oxidative phosphorylation. The mechanism of this glucagon secretion defect in GADA+ alpha cells remains poorly understood. Therefore, the goal of the current project is to test whether the reduced or lost suppression of glucagon secretion by glucose in alpha cells during the progression of T1D is causally related to alteration of the cAMP pathway and/or bioenergetics.

    This study will help to identify possible protective or pathogenic factors which may play a role in alpha cell dysfunction during development of T1D. These factors may then be used in the future for drug development to prevent or delay progression to T1D by targeting the early single AAb+ stage.

Beta Cell Physiology and Dysfunction

  • Andisheh Abedini & Ann Marie Schmidt

    NYU School of Medicine
    Uncovering pathological factors and mechanisms leading to β-cell death in type 1 diabetes

    We hypothesize that β-cell toxicity in T1D can be induced via different pathways by distinct pathological ligands (AGEs, S100-proteins, amyloidogenic amylin) of the receptor for advanced glycation endproducts (RAGE) that become upregulated in the diabetic pancreas. We also hypothesize that C-peptide (+) T1D subjects may be more susceptible to amylin-induced β-cell toxicity. Amylin is co-produced /co-secreted with insulin by β-cells; subjects that retain the ability to produce/secrete insulin may also produce/secrete amylin. We will test out hypotheses in three successive stages: 1) Method validation, 2) Hypothesis-driven proof-of-concept testing, and 3) Mechanistic studies guided by findings in Stage-2. Having completed Stage-1 using nPOD control pancreas sections (T2D and nondiabetic), we are ready to test our hypothesis in 4 phenotypes of human subjects (C-peptide(+) T1D, C-peptide(-) T1D, T2D and non-diabetic controls) according to two synergistic specific aims. Aim-1: We will determine whether RAGE lignads (AGEs, S100-proteins, amyloidogenic amylin) become upregulated in T1D islets. Aim-2: We will determine whether islet amyloidosis correlates with C-peptide levels in T1D. IHC-IF will be used to quantify RAGE ligands/signaling molecules (amylin/amyloid, AGEs, RAGE, mDia1) and loss in β-cell/islet area and apoptosis (insulin/synaptophysin, cleaved capase3) in pancreas sections. RAGE ligands and signaling molecules (S100A12, RAGE, mDia1) will also be measured in frozen pancreas specimens by qRT-PCR, and in plasma (S100B) by ELISA. Upregulated ligands/mediators identified in Stage-2 will be the focus of mechanistic studies in Stage-3; which aim to identify mediators of oxidative stress (NOX1, NOX4, NOS), inflammation (CCL2, TNFα, ILβ, IL6, IL10) and apoptosis (bax) in frozen pancreas and spleen (control) specimens viz qRT-PCR. Results will be biostatically compared/contrasted/correlated. Successful completion of the proposed studies will identify key pathological factors and associated mechanisms by which RAGE contributes to β-cell/islet pathology in T1D, providing important insight into new drug targets for β-cell preservation and treatment/prevention of this disease.

  • Ronadip Banerjee

    Johns Hopkins University School of Medicine
    Funded by: NIH/NIDDK
    Immediate-early gene transcription factor (IEG-TF) expression within human pancreatic islets during pregnancy and gestational diabetes

    Pregnancy is a unique physiologic state in adult females that occurs and resolves over a defined time period. The acute metabolic stress it causes on the mother triggers adaptations with maternal pancreatic islets. Failure of normal islet adaptation is a major contributor to gestational diabetes (GDM).In rodents, insulin-producing beta-cells proliferate causing expansion of beta-cell mass. This expansion is defective in rodent models of GDM. Normally, the gestationally-expanded mass regresses in the early postpartum period. We and others have begun to identify gene expression changes that occur during pregnancy and postpartum in this model system, including how defects in specific genes or signaling pathways contribute to GDM. We recently identified a number genes within specific islet cell types whose expression changes in late pregnancy or postpartum. Amongst these were “immediate early genes” (IEGs) including several transcription factors (TFs) that may regulate survival (or cell death) of beta-cells in late pregnancy. In this proposed project, we will test the hypothesis that IEG-TFs identified in our rodent studies regulate beta-cell mass and function in human pregnancy, and may be dysregulated in GDM. To do so, we will perform immunohistochemical staining for specific IEG-TFs. We will co-stain with hormone markers (insulin, glucagon) to confirm cell type of expression, as well as with a marker of apoptosis (TUNEL) to see if IEG-TF expression correlates with apoptosis or survival of beta-cells in pregnancy or GDM.

  • Richard Benninger

    University of Colorado Denver
    Funded by: Breakthrough T1D
    The role of protein kinase C delta in beta-cell death and dysfunction in type 1 diabetes

    The goal of this study is identify novel mechanisms of immune-mediated β-cell death
    and dysfunction such that these mechanisms can be exploited to protect against the
    onset and progression of T1D. To achieve this goal, we propose the following aims: 1)
    Determine if reduced electrical coupling, altered calcium signaling, and activation of
    protein kinase C delta (PKCδ) occur in islets from newly diagnosed and autoantibody
    positive donors compared to healthy donors 2) Determine if increased activation of
    PKCδ correlates with β-cell apoptosis in islets from newly diagnosed and autoantibody
    positive donors compared to healthy donors. The results of this study will identify a
    novel role for decreased electrical coupling, altered calcium signaling, and activation of
    PKCδ in mediating β-cell death and dysfunction in the pathogenesis of T1D.

  • Susan Bonner-Weir & Cristina Aguayo-Mazzucato

    Joslin Diabetes Center
    Funded by: NIH
    Aging Beta Cell

    Type 2 diabetes (T2D) increases with age with the majority of patients being above the fifth decade of life. These data underline the importance of studying how aging contributes to the reduced beta cell mass and dysfunctional insulin secretion found in T2D. However, the specific contribution of beta cell aging and senescence to diabetes has not been studied. Beta cells progress through different life stages, and, with age, the older subpopulation of cells becomes enriched. This enrichment has led us to identify specific markers of beta cell aging whose expression we have validated in mouse and human pancreas. We would like to further understand this process and its contribution to the development of T2D by exploring the expression of these markers in the pancreas of patients with T2D and lean and obese non-diabetic age-matched controls.

  • Lynne Coluccio & Hiroshi Tokuo

    Boston University
    Role of myosin-I in insulin secretion

    In pancreatic beta cells, insulin is produced as proinsulin and packaged into secretory granules at a region of the cell called the trans-Golgi network (TGN). After maturation which includes the conversion of proinsulin to insulin, insulin granules are trafficked to the cell membrane where their contents are released through a process known as exocytosis. Actin filaments participate in different phases of insulin granule production and release. Our laboratory studies myosin motor proteins, which associate with actin filaments and modulate their activity. We have found that loss of the unconventional myosin Myo1b reduces insulin and proinsulin content. This results in less insulin secretion from Myo1b-depletedinsulinoma cells. Importantly, using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, we found that loss of Myo1b impaired the early-stage trafficking of (pro)insulin granules. Our working model is that Myo1b is involved in early-stage insulin granule formation in beta cells. As diabetes is characterized by a reduction in insulin synthesis, insulin granule formation, and insulin secretion, our goal is to determine whether Myo1b loss is associated with the impaired insulin secretion in diabetic beta cells.

  • Yingfeng Ding

    City of Hope
    Uridine in the pathogenesis of T1D

    Blood uridine levels are elevated in T1D. However, its significance to the disease pathogenesis remains largely unexplored. Our pilot studies indicate uridine regulates glucosestimulated insulin secretion (GSIS) in mouse and human islets. We will use human pancreas tissue sections and live pancreas slices to study how chronic elevation in blood uridine might affect beta cell function and survival in T1D.

  • 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.

  • Yuval Dor & Elad Horwitz

    Hebrew University, Jerusalem, Israel
    Beta cell DNA double strand breaks in Type 1 diabetes

    Emerging evidence suggests beta cell dysfunction and failure precede autoimmune destruction during the development of type 1 diabetes, but the underlying mechanisms are not known. Defining and targeting the changes leading to impaired beta cell function may lead to clinical improvement and prolonged honeymoon period. We have recently discovered that in type 2 diabetes, beta cells suffer major damage to their DNA, and have partly delineated the pathway leading from high glucose levels to breaks in the DNA (Tornovsky, Cell Metabolism, 2014). While the consequences of DNA breaks in beta cells remain unknown, extensive information about the biology of DNA damage in other fields of biology strongly suggests that this is an important pathogenic process that may contribute to beta cell failure via multiple pathways. In unpublished preliminary experiments that form the basis for this proposal, we found increased DNA breaks in beta cells of human patients shortly after diagnosis of type 1 diabetes. We hypothesize that DNA damage precedes, and may contribute to, the development of autoimmune destruction of beta cells, and are seeking evidence for or against this hypothesis using nPOD material. Consistent with this hypothesis, previous studies have shown that DNA breaks can trigger an immune response, and that defects in the DNA repair machinery may lead to autoinflammatory disease.

    We request funds to support experiments that will solidify the observation of DNA damage in beta cells shortly after diagnosis with type 1 diabetes, and will test whether DNA damage occurs in beta cells prior to clinical diagnosis. In addition, we will characterize the beta cells and islets that show DNA damage to understand what may control this phenomenon. To so, we will stain histological material of the pancreas from people at risk for type 1 diabetes (autoantibody positive donors) and shortly after diagnosis with type 1 diabetes (as well as controls) for markers of DNA damage, oxidative stress, replicative stress and immune attack. Together, these may point towards DNA damage as a new potential modulator of the development and progression of type 1 diabetes.

  • Decio Eizirik, Ihsane Marhfour & Anne Op de beeck

    Universite Libre De Bruxelles, Belgium
    Funded by: JDRFI
    Characterization of beta cell ER stress associated to enteroviral infection in type 1 diabetes

    Enteroviral infections are associated with an increased risk to develop type 1 diabetes. Several stains of enteroviruses are able to infect beta cells leading to both inflammation and cell death, two major factors for the onset of type 1 diabetes (T1D).  The cellular factors that modulate cellular permissiveness to enteroviral infection and beta cell death remain largely unknown. Since ER stress is involved in beta cell death and viral infections induce ER stress in other cell types, the aim of this project is to address the role of ER stress in virally induced beta cell death and local inflammation.

    For this purpose, we will first determine whether components of the unfolded protein response (UPR) are induced during beta cell infection with a diabetogenic coxsackievirus (CVB5). Our preliminary data indicate an increase in CHOP, Bip and XBP1s mRNA expression in infected INS-1E cells. These results must be confirmed at the protein level and the activated UPR pathways further detailed.

    Second, the role of the putative viral-induced ER stress in beta cell death and release of inflammatory mediators will be addressed by modulation of their expression through a silencing/overexpression approach and further evaluation of cell viability after CVB5 infection. These experiments, and the above described experiments, to be initially made in INS-1E cells, will be confirmed in primary rat and human beta cells. The accumulation of the involved ER stress markers in CVB5-infected beta cells, as well as co-detection of the capsid viral protein, will be evaluated by immunohistochemistry. To confirm the in vivo relevance of these observations, we will evaluate the co-localisation of the involved ER stress markers with viral capsid proteins in sections of pancreas from controls and coxsackievirus infected- and non infected-T1D donors. Altogether these results should shed light on the role of ER stress in beta cell death during coxsackievirus infection. 

  • Feyza Engin

    University of Wisconsin-Madison
    Examination of ER stress markers in type 1 diabetes samples in humans

    The endoplasmic reticulum (ER) is an organelle that is responsible for the proper folding of proteins and biosynthesis of lipids and steroids. Disruption of ER homeostasis leads to ER stress and activates a highly conserved adaptive network called the unfolded protein response (UPR). ER stress has been implicated in several diseases associated with protein folding defects and low grade chronic inflammation including neurodegenerative diseases, type2 diabetes, and atherosclerosis. The ability of UPR mediators to activate inflammatory and apoptotic signaling suggest ER stress might also be critical in development or progression of type1 diabetes. Despite increasing evidence that points to a critical role for ER stress in the pathogenesis of type1 diabetes, to date alterations in this pathway in human diabetic patients has not been demonstrated leading to a great scientific gap in understanding how organelle stress and related stress responses function in the pathogenesis of this disease. Our specific aim is to investigate the ER stress marker expression in the pancreata of human type1 diabetes and control samples by using immunofluorescence (IF) technique.

  • Carmella Evans-Molina, Tatsuyoshi Kono & Emily Sims

    Indiana University School of Medicine
    Funded by: NIH
    Regulation of Beta Cell Secretory Pathway Calcium Homeostasis in Type 1 Diabetes

    Calcium plays a vital role in many processes that govern beta cell function, including the production, maturation, and regulated secretion of insulin. The fidelity of these processes depends on the maintenance of calcium subcompartments and their respective transmembrane gradients, which are organized at both the cellular and organelle level. Then endoplasmic reticulum, Golgi apparatus, and secretory granules comprise a functional module referred to as the beta cell secretory pathway, through which insulin mRNA is translated and preproinsulin protein is processed to mature insulin and packaged for exocytosis. In aggregate, these organelles also represent the largest intracellular depots of calcium. Calcium dyshomeostasis arising within the integrated secretory unit of the β cell is a characteristic of Type 1 diabetes and contributes to altered insulin production and processing and impaired β cell function and survival. The goal of this work is to define changes in the expression patterns of key molecular regulators of secretory pathway calcium and proinsulin processing.

  • Ji-Ming Feng

    Louisiana State University
    The cellular distribution of Golli protein in human pancreatic islets

    The objective of this application is to determine the cellular distribution of Golli protein in human pancreatic islets. Golli (gene expressed in oligodendrocyte lineage) is an alternatively spliced product of myelin basic protein (MBP) gene featured by the presence of a unique Golli domain which contains a 133-amino-acid sequence fused in the N-terminal of classic MBP. The Golli peptide share 79% homology between the human and the mouse. Our previous works in mouse have identified that Golli protein serves as an important regulator of Ca2+ release –activated Ca2+ (CRAC) channel in T lymphocytes and voltage-dependent calcium channel (VDCC) in oligodendrocytes and neurons. This application will start from our original findings in mice that: Golli protein is expressed in β cells of mouse Langerhans islet and plays a critical role in insulin secretion. We propose here to investigate cellular distribution of Golli protein in human pancreas (both normal and type-1 and -2 diabetic) tissues, aim to build up a direct correlation of our findings of Golli in rodents to the type-1 and type-2 diabetes in human.

  • Herbert Gaisano

    University of Toronto
    Funded by: Canadian Institutes of Health Research and Breakthrough T1D
    Spatio-temporal visualization of immune and non-immune islet injury in Type 1 Diabetes

    Type 1 Diabetes (T1D) generally results from a poorly understood autoimmune process that leads to selective destruction of pancreatic beta cells. Interestingly, beta cell function is often not correlated with mass and declines very early in the disease before symptom onset, but the mechanisms driving this are not known. Islet beta cells existing in a functional hierarchy, with a small proportion of key beta cells exerting a disproportional effect on overall islet insulin release. These key beta cells, termed ‘hub’ and ‘leader’ cells are the sources of calcium oscillations and coordinate connectivity between other ‘follower’ beta cells, thereby governing the amplitude and duration of insulin secretory granule release. Previous work has also shown that hub and leader beta cells are a stable beta cell subtypes that are sensitive to metabolic and inflammatory stress in vitro, but whether loss or dysfunction of these beta cell subtypes are causally linked to overall beta cell functional decline and inter-individual heterogeneity in T1D has not been explored. In this study, we propose to test the hypothesis that hub and leader beta cells are lost early on in T1D, which would lead to extensive heterogeneity in islet insulitis and functional beta cell mass both between individuals at the same disease stage and between different disease stages in the same individual. To test these ideas, we will carry outlive cell imaging and functional studies on live human donor pancreas slices from individuals at different disease stages (no diabetes/control, early-late autoimmunity, recent onset T1D) using genetically targeted probes to track calcium dynamics. We will apply computational approaches to characterize beta cell functional hierarchy in these slices, as well as use of optical probes to photo-label hub and leader populations in situ for analysis of their behavior in the autoimmune slice milieu. We propose to further characterize the inter-relationships between hub and leader beta cells, beta cell stress pathways and islet insulitis using fixed pancreas sections. Finally, using the live slice in situ model, we will explore how small molecules to that selectively beta cell stress pathways (including senescnece and unfolded protein response)impacts on islet functional hierarchy and connectivity. Together these studies will take us closer to understanding the contributions of beta cell heterogeneity to T1D heterogeneity.

  • Ivan Gerling

    University of Tennessee Health Science Center
    Funded by: Breakthrough T1D
    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.

  • David Harlan

    University of Massachusetts Medical School
    Transcriptome analysis of highly purified islet cell populations

    Therapies focused on sustaining or promoting endogenous insulin production for individuals at risk for or with T1D will require a thorough understanding of the gene expression patterns in islet α, β, and δ cells from control subjects and from individuals with T1D. Toward that end, we have developed methods to essentially purify α, β, and δ cells from human pancreata by intracellular staining for specified intracellular hormone content, sorting the populations by flow cytometry, then obtaining detailed transcriptomes from each population using next generation RNA sequencing. We propose to define the transcriptomes of isolated α, β, and δ cells from those with and without T1D and from β cells with varying HLA Class I expression (dim or bright) from subjects with T1D. We will define the changes in genes expressed by islet cells under the pathological conditions of T1D and specifically, from β and α cells hyper-expressing Class I proteins as a potential marker o a pro-inflammatory microenvironment with islets. These studies elucidate the β and α cell dysfunction in T1D and may provide targets for future therapeutic intervention(s) in T1D.

  • Pamela Itkin-Ansari, William Balch & Randal Kaufman

    UCSD
    Funded by: NIDDK HIRN
    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.

  • Pamela Itkin-Ansari

    Sanford-Burnham-Prebys Medical Discovery Institute
    The role inflammatory signaling crosstalk on human exocrine and beta cells

    There is increasing evidence for cross-talk between the islet and exocrine compartments of the pancreas. Diabetes is a risk factor for exocrine atrophy, pancreatitis, and pancreatic cancer. Conversely, pancreatitis is a risk factor for diabetes. Until now our ability to study human endocrine and exocrine cells has been limited to isolated islets and isolated exocrine tissue. The tissue slice program provides a unique opportunity to simultaneously examine the immediate effects of exposure to inflammatory associated with diabetogenic or pancreatitis on both human beta cell and exocrine cell physiology. Here we will treat pancreas slices with the exocrine inflammatory molecule caerulein or the diabetogenic cytokines IL1beta and IFNgamma, measuring cell function and stress in both compartments. We previously showed that short term treatment of isolated exocrine cultures with caerulein induced BMP signaling, the transcriptional repressors ID1 and ID3 that promoted a potent proliferative response, as well as expression of inflammatory cytokines that may affect islets (Lee et. al. 2011a). In contrast to exocrine cells, human islets responded to ID3 with replication stress and DNA damage (Lee et. al 2011b). We also recently profiled gene expression in human islets exposed to a cytokine cocktail of IL1beta and IFNgamma, finding that the islets underwent ER stress and were induced to express chemokines that may well impact neighboring exocrine cells. In summary, the pancreas slice program enables the study of human endocrine/exocrine cross-talk in tissue for the first time.

  • Michael Kalwat & Travis Johnson

    Indiana Biosciences Research Institute
    Funded by: Helmsley Charitable Trust George S. Eisenbarth nPOD Award for Team Science
    Leveraging heterogeneity to identify markers of resilient β-cells in T1D

    We aim to identify factors contributing to the destruction ofβcells in diabetes. In this project, we took advantage of publicly available bulk and single cell gene expression datasets from human islets of healthy and diabetic donors(T1D and T2D). We used deep transfer learning platform DEGAS1 combinedwith this data to identify subsets of βcells within human islet single cell RNA seq data whose transcriptomic signature correlates with that of diabetic islets. We determined the transcriptomic differences between highand lowscoring βcells within the human islet scRNAseq data. As part of this project, we identified the geneDLK1asa candidate marker of βcell resilience under diabetic stress. Our project will take two major approaches. 1)We will use antibodies to stain for the candidate protein DLK1 that we predict is expressed in βcells that persist in the face of autoimmunity. 2) We will use spatial transcriptomics in nondiabetic, AAB+, and T1Dhuman pancreas to measure the expression of candidate genes identified by DEGAS analysis and the spatial location of cells expressing these genes. By analyzing the gene expression patterns of these cells in we anticipate defining genes associated with different βcell states, spanning from resilient to vulnerable. We anticipate that this knowledge will help uncover new mechanisms to improve βcell survival, escape autoimmunity, and explain why some βcells persist but others do not.

  • Heiko Lickert & Teresa Rodriguez-Calvo

    Helmholtz Zentrum München
    IGF receptor-like 1 and 2, new therapeutic targets for increasing insulin sensitivity and beta cell health

    Diabetes mellitus is a complex and multifactorial disease characterized by progressive loss or dysfunction of the insulin-producing beta cells in the pancreas. This results in chronic hyperglycemia and systemic metabolic complications and, in the long-term, in multi-organ damages; together, these complications create enormous medical and social burdens as well as causing premature deaths. Today, over 422 million people worldwide have been diagnosed with diabetes and the number is expected to rise in the next 20 years. Currently no treatment can stop or revert diabetes progression, except bariatric surgery and islet transplantation. However, donor shortage and risks associated with life-long immunosuppression demand the development of alternative therapies. Increasing beta-cell health, proliferation and regeneration is disease modifying and has the potential to cure diabetes. In a screen for novel regulators of endocrinogenesis, we identified a previously uncharacterized regulator of insulin action, beta cell proliferation and blood glucose homeostasis. Experiments are ongoing to validate its usage as target to improve insulin sensitivity and to preserve β cell mass. Since the diabetic beta cells will be the target of the intervention, the nPOD samples will help us to discover changes in the receptor expression during the disease progression.

  • Amelia Linnemann

    Indiana University
    Regulation of Islet Autophagy in Type 1 Diabetes

    Oxidative stress occurs when increased production of cellular oxidants (e.g. Reactive Oxygen Species, or ROS) is not compensated  for  by  an  increase  in  cellular  antioxidants/antioxidant  enzymes.   A  considerable body  of  evidence  supports  the  conclusion  that  oxidative  stress  is  a common  feature  of type  1 diabetes (T1D), and that the process of autophagy plays a key role in mitigation of ROS to reduce oxidative stress and promote -cell survival. Indeed, we recently found that coupling of autophagy to the antioxidant response could reduce-cell ROS, and protect against oxidative damage and apoptosis in an STZ model of diabetes in mice. Autophagy has long been known to play a critical role in -cell homeostasis and survival in the context of type 2 diabetes, however much less is known about the role that autophagy plays in the pathogenesis of type 1 diabetes, particularly in humans. Therefore,  in  this  project  we  propose  to  analyze autophagy in  islets  from  cadaveric donors with type 1 diabetes. Number, spatial organization of autophagosomes and lysosomes, and  colocalization  of  these  vesicles  will  be  quantified.  Further,  since  a  specialized  form  of autophagy  known  as  crinophagy  is  important  for insulin  homeostasis,  we  will also determine  if lysosome changes are associated with the changes in proinsulin that have been recently reported in islets from type 1 diabetic individuals.

  • Clayton Mathews

    University of Florida
    Funded by: Breakthrough T1D
    Islet resistance to T1D

         Our proposed studies will address a major gap in the understanding of T1D, namely, to identify the mechanisms of beta cell failure from a metabolic as well as an immunological perspective. This specific project has high potential to reveal significant, novel, and translatable information regarding beta cell dysfunction that will be of therapeutic benefit for individuals with both established T1D as well as persons with pre-diabetes.  The goal is to identify components of the glucose sensing and insulin secretory machinery pathways that are altered during the natural history of disease progression in type 1 diabetes (T1D), and to characterize the metabolic and immunological signals responsible for the functional loss in beta cell activity that are specifically associated with this disease.  This aim will be achieved by performing two lines of experimentation.  First, gene expression arrays will be utilized to analyze islet RNA isolated from pancreatic sections of nPOD cases (representative from the natural history of T1D, alongside of appropriate matched controls) following laser capture microdissection (LCM). These analyses will be undertaken to determine the specific gene expression changes that result in beta cell dysfunction, as well as to identify the immunological signals which are responsible for beta cell failure in T1D.  In the second we will use primary human islets obtained from cadaveric donors with T1D as well as non-diabetic subjects, to confirm and extend our associations of phenotypes reflective of beta cell dysfunction. Also, using extended in vitro culture systems to allow for a recovery of beta cell insulin secretory function, followed by expression analysis, we will identify changes in signaling that are reflective of both cellular damage and recovery.  These experiments are posed to test the overall hypothesis that that following the appearance of autoantibodies, inflammatory signals act to increase islet inflammation and negatively impact beta cells through downregulation of genes essential for glucose sensing and insulin secretion. Based on previously published work, we expect that the effects of these signals are reversible.  Elucidating the immune signals that induce beta cell dysfunction would have a major impact on future treatment strategies as they should provide a means to revive the endogenous beta cell mass.

  • Clayton Matthews & Martha Campbell-Thompson

    University of Florida
    Funded by: NIH-NIAID
    Investigating the role of iron metabolism in beta cell function and the immunopathology of type 1 diabetes

    Insulin release from beta cells depends on ATP energy generated by mitochondrial. The electron transport chain is the primary group of proteins that make ATP energy and they require iron to do so. Consequently, iron is essential for insulin release, and iron deficiency inhibits beta cell function. Conversely, excess cellular iron leads to an increase in reactive oxygen species (ROS), which can damage cells and cause dysfunction. Evidence indicates that both ROS and mitochondrial dysfunction occur during diabetes progression. Beta cells have low antioxidant capacity making them susceptible to ROS from iron overload. Iron overload can cause mitochondrial fragmentation and dysfunction. Indeed, diseases of iron overload such as hereditary hemochromatosis and Friedreich’s ataxia have an associated risk of diabetes in the absence of autoimmunity. Inflammation alters iron metabolism by promoting sequestration within cells. Prolonged inflammation can cause anemia of inflammation, which has been reported in patients with T1D, where serum iron levels are too low to promote the differentiation of red blood cells. An inflammatory signaling molecule called interferon gamma (IFNy) is increased in individuals who develop diabetes long before the onset of disease. IFNy has been shown to increase cellular iron. Taken together, these data demonstrate the importance of understanding iron metabolism under basal conditions and during T1D disease progression in beta cells. Importantly, iron metabolism pathways may serve as targets for intervention for the treatment of diabetes.

  • Raghavendra Mirmira & Teresa Mastracci

    Indiana University School of Medicine
    Funded by: Breakthrough T1D
    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.

  • Feroz Papa & Mark Atkinson

    UCSF
    Validating KIRAs as β-cell-sparing drugs in islets from human patients with T1D

    Our lab has been focusing for many years on the intrinsic stress experienced by pancreatic islet β-cells, which (even in healthy states) live under a strict metabolic mandate to continuously produce and secrete insulin at intensely high rates that predispose these professional secretory cells to secretory exhaustion. β-cells evolved a high-capacity endoplasmic reticulum (ER) organelle in which the insulin precursor, proinsulin, undergoes initial oxidative protein folding and structural maturation. However, whenever secretory demand outpaces folding capacity, unfolded proteins begin to accumulate in the ER—a condition called “ER stress”.

    ER stress activates an intracellular signaling pathway called the unfolded protein response (UPR) that augments ER chaperones and protein-modifying activities by triggering adaptive (‘A’)-UPR transcriptional and translational programs. But if ER stress remains unresolved, the adaptive UPR outputs wane and are replaced by “terminal” (‘T’)-UPR programs that actively promote loss of differentiated cell identity, cause sterile inflammation and senescence, and ultimately trigger apoptosis. Mounting evidence shows that high ER stress/and consequent A>T-UPR conversion causes β-cell apoptosis, leading to diabetes in mouse models of T1D. However, it remains unclear whether human islets also demonstrate an A- to T-UPR switch during progression to T1D.

  • Edward Phelps

    University of Florida
    Funded by: Breakthrough T1D
    Mechanisms and dynamics of GAD and GABA in human beta cells

    In humans, GAD65 is one of the first targets of autoimmunity and 70-80% of human diabetics have antibodies against this protein. In late-onset patients, circulating GAD65 autoantibodies may even be more predicative of future disease onset than antibodies to insulin. The early appearance of GAD65 antibodies may hold a clue that GAD65 is implicated during the earliest phases of the disease, which makes it an attractive target for interventional therapies to prevent clinical progression. One of the peculiarities of GAD is that this protein is expressed in two isoforms, and only the lighter weight form, GAD65 is immunogenic, while the heavier one, GAD67, is not. Mice predominantly express GAD67 in their beta cells, while rats express both GAD65 and GAD67 and humans only express GAD65. Because the majority of diabetes research is conducted on NOD mice where GAD65 is not well expressed in beta cells, the central importance of GAD65 in human diabetes may be overlooked in NOD mouse research. We are studying GAD65 in human tissues to better understand how this protein contributes to T1D. In addition, we have an interest in the role of gama-aminobutyric acid (GABA), the enzymatic product of GAD. GABA is a potent neurotransmitter that is strongly and selectively synthesized in neurons and pancreatic islets. Beta cells secrete GABA as a paracrine and autocrine signal to help regulate hormone secretion. GABA is also a negative regulator of immunity and helps shape the islet local immune microenvironment.

  • Al Powers, Marcela Brissova and Rachana Haliyur

    Vanderbilt University
    Funded by: Breakthrough T1D and NIH
    Pancreatic islet biology and vascularization

    Our group is interested in pancreatic islet development and function and how beta cells are affected in diabetes. Critical discoveries from nPOD samples and resources are changing our understanding of type 1 diabetes. We are using or hope to use nPOD samples for the following studies:

     1) Define and describe the normal human pancreas and islet over the human lifespan. In terms of normal pancreatic islet development, we are studying the fetal human pancreas, the juvenile human pancreas (less than age 18), and the adult human pancreas. We are interested in how islet morphology, gene expression, and function change as humans age and adapt to different physiologic conditions such as puberty and insulin resistance. In addition to studying islet gene expression (hormones, key islet-enriched transcription factors, and differentiation markers), we are also studying the innervation and vascularization of islets. Islets are richly innervated and vascularized and we are interested in interactions between these processes and islet cell function.

     2) Describe the features of insulin+ cells remaining in the pancreas of individuals with Type 1 diabetes (T1D). We seek to understand the functional characteristics, and molecular signatures of the insulin+ cells remaining in the pancreas of individuals with T1D. Little is known about the remaining insulin+ cells and their functional status, or how those cells have survived in the inhospitable metabolic and immunologic environment. It is not known whether these insulin+ cells are ‘normal’ β cells.

     3) Describe the features of islets and beta cells in the pancreas of individuals with cystic fibrosis-related diabetes (CFRD). This form of diabetes is becoming increasingly common, but the pathogenesis is incompletely understood. By studying samples in the nPOD repository, we hope to determine how these beta cells compare to normal beta cells.

     We are using or hope to use nPOD samples from both the control and diabetic pancreas. We will combine information from nPOD samples with our results from samples from human pancreatic tissues that our group has recently collected (fetal and juvenile). We will examine these tissues by immunohistochemistry and other methods such as tissue imaging mass spectrometry. We will use antibodies that are widely available and also new antibodies that are being developed by investigators in the Beta Cell Biology Consortium.

    July 11, 2014

  • Boone Prentice & Clive Wasserfall

    University of Florida
    Evaluation of Fatty Acid Levels in Human Pancreatic Tissue using Imaging Mass Spectrometry

    Alterations in metabolism are known to play profound roles inthe health ofindividuals with type 1 diabetes (T1D). These alterations lead to disruptions in cell structure, signaling, and energy homeostasis. While most studies focus on identifying genetic or protein signatures of disease, the metabolic profile is a far more dynamic, sensitive, and rapidly-responding molecular measure of phenotype. Free fatty acids (FFAs) in particular represent a class of metabolites known to play key roles in protective activities such as preventing beta-cell apoptosis, regulating plasma glucose levels, and enhancing insulin sensitivity as well as destructive activities such as exacerbating insulin resistance and producing lipotoxic conditions conducive to apoptosis. The nature of these roles is in part determined by the concentration of the FFAs as well as their molecular identity. Unambiguous detection and identification of these metabolites by conventional, antibody-based fluorescent imaging approaches is not feasible. Instead, we will leverage chromatography-tandem mass spectrometry (LC-MS/MS) to analyze homogenized tissues. However, LC-MS/MS lacksa spatial component to the measurement and does not provide for the direct in situmeasurement of metabolites in intact tissue sections. Detecting the spatial distributionand diversity of FFAs directly in pancreatic tissue, before these metabolites have been diluted in the bloodstream, would provide an unprecedented view of T1D metabolism. Herein, we propose to use label-free imaging mass spectrometry to enable the multiplexed and facile discrimination of metabolites in human pancreas tissue sections. We will analyze and quantify differences in the expression of free fatty acids between unaffected and T1D pancreases. Our preliminary data suggest that multiple genes involved in fatty acid biosynthesis are significantly upregulated in T1D tissue compared to unaffected pancreases. The generation of molecular maps by imaging mass spectrometry will determine whether the metabolic phenotypes agree with the genetic profiles of these individuals.

  • Sarah Richardson, Irina Stefana, John Todd & Noel Morgan

    University of Exeter Medical School
    Funded by: Breakthrough T1D, MRC and Wellcome
    Investigating the role of aggregation-prone proteins in pancreatic beta cell function and health

    Almost 20% of the chromosomal regions associated with the risk of developing of Type 1 diabetes (T1D) identified to date,have no identified candidate causal gene. Often, this is because the genes in those regions have no known roles in the immune system, reflecting the classical view that T1D is caused by the autoimmune destruction of pancreatic islet β cells.One possibility is that the causal gene(s) in these regions are not acting in the immune system but rather on the target tissue (the βcells)or in both. Our ongoing analyses show that >150 genes in over 60 T1D risk regions are expressed in β cells and that several T1D-candidate variants colocalize to regulatory motifs active specifically in human islets and not in immune cells.One region that may act via βcells, rather than the immune system,to predispose to T1D is the chromosomal region 17q21.31. The same genetic signal in region 17q21.31 is also associated with increased 2-hr glucose levels following an oral glucose tolerance test and, importantly, with increased plasma proinsulin levels, thus strengthening the rationale that the association in this region might act via β cells. To date, this region has been extensively studied for its association with neurodegenerative diseases, where highly disease-penetrant mutations have provided evidence that MAPT, encoding for the microtubule-associated protein Tau, is the causal gene. Classically thought of as a neuronal protein, Tau is also expressed in several peripheral tissues, including pancreatic islet βcells.We are currently investigating the role of Tau in β-cellfunction and survival under diabetes-relevant stresses.Using nPOD samples, we aim to examine the expression and subcellular localization of Tau and its pathological, hyperphosphorylated formin human pancreatic β cells from donors of different ages and diabetes status

  • Teresa Rodriguez-Calvo

    Helmholtz Zentrum München
    ORIGINAL: Proinsulin processing and presentation in human type 1 diabetes: a beta cell perspective ADDENDUM: Proinsulin processing and presentation in human type 1 diabetes: a beta cell perspective –T cell perspective

    ORIGINAL: Type 1 diabetes (T1D) is an autoimmune disease in which clinical symptoms arise as a result of insulin deficiency. While genetic and environmental factors contribute to the disease, in recent years it has become increasingly evident that beta cells might be contributing to their own destruction and might have an active role in type 1 diabetes development. Insulin production occurs thanks to processing enzymes, which transform the precursor of insulin (proinsulin) into insulin. Our hypothesis is that there might be a defect in the activity of these enzymes that would cause the accumulation of large amounts of proinsulin (which is not being transformed to insulin) and might also introduce insulin processing errors. These errors would lead to protein modifications that would be recognized by the immune system. Therefore, our objective is to investigate the mechanisms involved in proinsulin and insulin synthesis and the enzymes that process them. Consequently, we propose to study the biological processes that accompany insulin synthesis from a beta cell perspective, providing a more comprehensive understanding of the pathogenesis of type 1 diabetes. Ultimately, this work should help to develop novel therapeutic approaches targeting beta cell dysfunction possibly in combination with antigen-specific therapies.

    ADDENDUM: Type 1 diabetes is an autoimmune disease in which clinical symptoms arise as a result of insulin deficiency. While genetic and environmental factors contribute to the disease, in recent years it has become increasingly evident that beta cells might be contributing to their own destruction and might have an active role in type 1 diabetes development. Insulin production occurs thanks to processing enzymes, which transform the precursor of insulin (proinsulin) into insulin. Our hypothesis is that there might be a defect in the activity of these enzymes that would cause the accumulation of large amounts of proinsulin (which is not being transformed to insulin) and might also introduce insulin processing errors. These errors would lead to protein modifications that would be recognized by the immune system. Therefore, our objective is to investigate the mechanisms involved in proinsulin and insulin synthesis, the enzymes that process them and which protein fragments (modified or not) are recognized by lymphocytes, causing their activation and leading to beta cell destruction. Consequently, we propose to study the biological processes that accompany insulin synthesis not only from the beta cell perspective but also from the side of the immune system, providing a more comprehensive understanding of the pathogenesis of type 1 diabetes. Ultimately, this work should help to develop novel therapeutic approaches targeting beta cell dysfunction possibly in combination with antigen specific therapies.

  • Teresa Rodriguez-Calvo

    Helmholtz Zentrum München
    The insulin secretory granule components and its immune recognition in type 1 diabetes

    Our parent project studies the intracellular accumulation and molecular interaction of proinsulin, insulin and the proinsulin processing enzymes PC1/3, PC2 and CPE, which may be dysregulated during the development of the disease and lead to processing errors. Our interest was focused on proinsulin and insulin, as these are the most abundant molecules of the insulin granule, and their role in disease progression has been highlighted in several studies but has not been systematically investigated. However, other molecules inside the insulin granule, such as chromogranins, secretogranins, and islet amyloid polypeptide (IAPP) might undergo similar processing errors. These may contribute to beta cell demise, thereby leading to their recognition by the immune system. Here, we propose to study the expression and accumulation of insulin granule proteins other than insulin and proinsulin. By approaching this from the perspective of the beta cell, we will strive to provide a more comprehensive understanding of the pathogenesis of type 1 diabetes. Ultimately, this work should help to develop novel therapeutic approaches targeting beta cell dysfunction possibly in combination with antigen specific therapies.

  • Holger Russ

    University of Colorado
    Evaluating senescence induction in human beta cells upon transplantation

    We are interested in cell replacement therapy as a potential practical cure for patients suffering from diabetes. Using stem cell derived beta cell clusters and cadaveric human islets transplantation in preclinical animal models we find distinct changes to the beta cell phenotype. This project will test if our observations hold true in islet recipients. We will evaluate if the changes observed using model systems are also present in tissue sections from donors that previously received islet transplants.

    We find a small subpopulation of stem cell derived beta cells (sBC) marked by CD9. We found previously that CD9 marks beta cell subpopulations that are less functional in cadaveric islets preps (Dorrell et al. Nature com, 2016). CD9 cell % is up in T2D. When looking closely, part of the CD9 population in both sBC and cadaveric islets exhibits a senescent and senescence associated secretory phenotype. When we look at transplanted sBC we see a huge increase in CD9 cells (and senescence markers). We are currently testing if CD9 cells exhibit greater immunogenicity. We believe our findings have important implications to current efforts aimed at providing a practical cure via cell replacement therapy to diabetes patients.

  • Albert Salehi

    Lund University
    VDAC1 a Common Denominator of Pancreatic β-cell and Vascular Endothelial Cell Apoptosis in T1D and its Organ Complications

    Impaired insulin secretion from the β-cells characterizes both type 2 and the initial phase of type 1 diabetes (T1D). Although the two sub-forms have different causes, we postulate that the rapidly developing β-cell defect and death in T1D as well as the slowly progressing β-cell dysfunction associated with T2D share a common cellular mechanism.

    Long-term elevated plasma glucose (glucotoxicity) as well as presence of inflammatory cytokines in the pancreatic islets contribute to β-cell demise in both forms of diabetes. In the initial phase of T1D, islet cell antibodies are detected, while insulin secretion is attenuated but not abolished. Existing therapies are not preventing the progression of either T1D or T2D. We have recently shown that long-term exposure of pancreatic β-cells to high glucose (glucotoxicity) as well as β-cells isolated from T2D cadaveric organ donors show increased expression of the mitochondrial voltage-dependent anion channel-1 (VDAC1). This leads toVDAC1 miss-targeting (translocation) to the cell surface. Our preliminary data showed that inflammatory cytokines also increases VDAC1, which might contribute to the loss of β-cells in T1D. VDAC1 overexpression leads to its oligomerization, which participates in cell death. In preliminary results, we also observe upregulation of VDAC1 in a rat model of T1D. Since VDAC1 is a mitochondrial ATP transporter, its translocation to the cell surface is associated with ATP loss from the β-cells, leading to energy depletion. Prevention of ATP loss withVDAC1 inhibitors restores β-cell ATP levels and insulin secretion in T2D islets. Daily injections of a VDAC1 inhibitor prevent the onset of hyperglycemia in db/db mice, a T2D model. Based on the effects of cytokines, we want to extend our investigations to include T1D. To this end, we will studyVDAC1 protein expression and co-localization with other β-cell markers on pancreatic sections from T1D organ donors. We thus envision that novel therapeutics based on VDAC1 inhibitors i.e. newly developed small molecules or a specific VDAC1 antibody, may act either as complements to existing therapies, or as novel stand-alone treatments for T2D as well as prevention of the β-cell loss in T1D.

  • Charmaine Simeonovic, Christopher Parish, J. Dennis Wilson & Andrew Ziolowski

    Australian National University
    Funded by: Breakthrough T1D
    Heparan sulfate levels mark the health status of human islet β-cells

    Heparan sulfate (HS) is a sulfated glycosaminoglycan that consists of repeating disaccharides (composed of uronic acid and glucosamine), forming a linear polysaccharide. The HS chains are assembled onto the core proteins of heparan sulfate proteoglycans (HSPGs). We recently reported that HS is localized in the peri-islet basement membrane (BM) in mice and is intensely expressed and widely distributed throughout the intra-islet cell mass, being localized selectively inside insulin-producing beta cells. This intracellular distribution of HS is unique for beta cells, since normally HS is localized extracellularly in BMs and the extracellular matrix (ECM). Using in vitro studies, we have discovered that isolated mouse beta cells need intracellular HS for their survival and protection against oxidative damage. During T1D in NOD/Lt mice, HS is progressively lost from islet beta cells, due to the local production of heparanase (a HS-degrading enzyme) by insulitis leukocytes, resulting in beta cell death. Using nPOD human pancreas specimens, we have demonstrated that normal human islets also express high levels of intracellular HS in their beta cells. In parallel, our immunohistochemical analyses of the distribution of HSPG core proteins revealed strong intra-islet staining for collagen type XVIII and syndecan-1 HSPG core proteins. These findings provide support for the high levels of HS in human beta cells in situ. Like T1D in NOD/Lt mice and in contrast to normal human pancreas, nPOD pancreas specimens from donors early after T1D-onset (≤ 8 years post-onset) have shown selective loss of HS but not HSPG core proteins in remaining insulin-positive islet beta cells. In fact the HS content of T1D islets was only 10% of normal controls. In addition, peri-islet insulitis mononuclear cells in the T1D pancreas specimens strongly expressed heparanase. Our findings strongly support the testing of dual activity heparanase inhibitors/ HS replacers as new therapeutics for preventing T1D progression in new onset T1D individuals.

  • Emily Sims

    Indiana University School of Medicine
    Funded by: NIDDK
    Defining the link between Islet prohormone accumulation and secretion in type 1 diabetes

    An improved understanding of the pathophysiologic mechanisms contributing to insulin deficiency in type 1 diabetes (T1D) is crucial to optimize approaches to disease prevention and treatment. Multiple groups have described increases in beta cell prohormones in individuals at different stages of T1D development, both at the level of the islet and in circulation. These findings have highlighted the concept that dysfunctional beta cell prohormone processing may contribute to insulin deficiency and T1D progression. Because of historical limitations of access to human pancreatic tissue, prior human analyses have been unable to combine analysis of prohormone secretory dynamics with tissuelevel phenotyping of prohormone expression and processing. Our preliminary analysis identified heterogeneity in proinsulin secretion among the donors with a history of T1D. Extracellular vesicles (EVs) and their cargo contribute to islet intercellular communication. However, the relationship between the beta cell EV secretome and beta cell prohomone secretion has never been described. For this addendum to the proposal, our overarching hypothesis is that heterogeneity in islet proinsulin release could be associated with differences in islet EV content. We will specifically test EV proinsulin, EV programmed death ligand1 (PDL1), and EVs linked to autophagy (LC3+EVs). To test this, we propose analysis of EVs using the remaining perifusate samples already at our possession at IU, using the Exoview platform, with quantification of PDL1+ and LC3+ positive EVs (absolute and as a percentage of total EVs) as well as beadbased pulldown and permeablization to identify proinsulin+ EVs. These results will determine if heterogeneity islet proinsulin secretion is linked to altered EV composition, yielding critical insight into association of altered prohormone processing and release with insulin deficiency in T1D.

  • Jason Spaeth

    Indiana University School of Medicine
    Funded by: NIH/NIDDK and Indiana University School of Medicine
    Transcription factor:coregulator interactions are compromised in islet cells under diabetogenic conditions

    Beta cell dysfunction occurs in the early stages of type 1 (T1D) and type 2 diabetes (T2D) development. Strategies to improve outcomes for the mounting number of diabetic patients requires understanding the complex programs that coordinate a proper insulin release in response to changing blood glucose levels and how these mechanisms falter in diabetogenic settings. It has been proposed that expression and activity of islet enriched transcription factors (TFs) are altered in beta cells from diabetic individuals. We propose that interactions between TFs and their recruited transcriptional coregulators are negatively impacted during diabetes development and detrimentally alter gene expression programs essential for normal beta cell function. The Spaeth lab and others have demonstrated that Pdx1 interacting coregulators such as the Swi/Snf chromatin remodeling complex and the Nucleosome Remodeling and Deacetylase (NuRD) complex positively contribute to normal beta cell function in vitro and in vivo. Preliminary data suggests that beta cells from animals under high fat diet(which mimic human metabolic syndrome) and non-obese diabetic (T1D animal model)have reduced interactions between Pdx1:coregulators. We will utilize nPOD samples to evaluate if interactions between Pdx1 and these important coregulators are altered in primary human tissue from donors with multiple autoantibodies, overt T1D as well as donors that diagnosed as T2D.

  • Roland Stein

    Vanderbilt University
    Funded by: Breakthrough T1D and NIH
    Lipid droplet distribution study in human pancreas

    In collaboration with Dr. Alvin Powers’ at Vanderbilt University, we showed by electron microscopy (EM) that transplanted human islets have a much higher level of lipid droplets (LDs) than transplanted mouse islets (1). Moreover, we found that the number of LDs in transplanted human islets was increased in high fat diet (HFD) fed mouse recipients. We were intrigued by this observation, since we also observed that the high LD accumulation in human islets correlated with the unresponsiveness of the transplanted human islet β cells to the proliferation and insulin secretion signals observed in mouse islets (e.g. HFD & hyperglycemia (1)). Consequently, we extended this analysis by using both EM and the BODIPY 493/503 dye (i.e. a green fluorescent dye with hydrophobic properties that specifically stains neutral lipids and other lipophilic compounds) to analyze the LD content in intact human primary pancreas tissue sections. BODIPY 493/503 has the advantage over EM of allowing visualization of LD content/redistribution within a larger pancreatic area. BODIPY 493/503+ LD signals were shown to be: 1) randomly distributed between the acinar and endocrine compartment in healthy human samples; 2) in the α- and β-like cells generated from human embryonic stem cells; and strikingly, 3) enriched within T2D islets and longstanding T1D residual α cells in relation to the acinar space. These findings suggested the pancreatic LD accumulation patterns could be a marker of dysfunctional endocrine cells, and were published in Diabetes with Dr. Powers’ group (2). Dr. Xin Tong, the lead investigator on this project, also received a postdoctoral fellowship supporting this project through Breakthrough T1D in 2019. We now propose in Aim 1 to use frozen OCT embedded slides to obtain insight into the effect of gender and BMI on pancreatic LD distribution. While low LD levels were previously found in healthy juvenile/young samples, we never tested T1D donors within this age range, which could have a novel LD enriched endotype. Consequently, we propose in Aim 2 to perform a pilot study exploring the pancreatic LD accumulation patterns in juvenile – young adult T1D donors (i.e. 10-20 years). Successful assessment of the LD accumulation pattern in these conditions will help determine the relationship of LDs to islet function, and guide the human islet selection process for transplantation therapy.

  • Ian Sweet & Christiane Hampe

    University of Washington
    Funded by: Helmsley Foundation
    Pathogenic Action of Autoantibodies on Islet Function and Viability in Type 1 Diabetes

    Type 1 Diabetes (T1D) is known to develop over time when autoimmune processes attack and destroy beta cells thereby reducing the capacity to secrete adequate amounts of insulin. The relatively gradual progression of the disease offers an opportunity for therapeutic intervention to slow or prevent destruction of beta cells before the disease has reached symptomatic stages. Systematic development of drugs will be greatly accelerated with identification of steps that mediate the progression of the disease. An intrinsic hallmark of T1D is the appearance of several autoantibodies directed against islet cell antigens, including insulin and the 65kDa isoform of glutamate decarboxylase (GAD65). We recently revealed pathogenic effects of autoantibodies on isolated rat islets (that like human islets do express GAD65). The paradigm-shifting results of our now published study demonstrated the ability of GAD65mAb to

    i. penetrate and accumulate in beta cells,

    ii. localize to various and distinct subcellular structures and

    iii. affect insulin secretory function.

    Accordingly, our overall goal is to establish that our exciting in vitro results showing an impact of GAD65 autoantibodies (GAD65Ab) on intracellular processes in pancreatic beta cells support a mechanism for mediating Type 1 Diabetes (T1D). As a first step, we will endeavor to show the presence and effects of GAD65Ab in human islets and in particular in human islets from patients with T1D. Therefore we are requesting pancreatic slides to search for the presence of antibodies in GAD-positive vs. control patients.

  • Gladys Teitelman

    SUNY-Downstate Medical Center
    Preventive Insulin Therapy for Type 2 Diabetes (Addendum: Pancreatic beta cell heterogeneity; Addendum: Heterogeneity of human beta cells)

    Two major pathophysiologic abnormalities underlie most cases of type 2 diabetes (T2D): insulin resistance and defects in pancreatic beta-cell function. These defects are initially compensated by an increase in insulin secretion and in the number of insulin secreting cells. However, with time, beta cell dysfunction and T2D develops, requiring intervention therapies. Eventually, there is significant beta cell loss and increased difficulty in the management of hyperglycemia.

    It has been postulated that the increased rate of insulin synthesis induced by hyperglycemia could lead to beta cell failure. Insulin, initially synthesized as a larger polypeptide, is modified in the endoplasmic reticulum (ER) and post-translationally converted into insulin within the secretory/storage vesicles prior to secretion. Two proteases, the prohormone convertases (PC) PC2 and PC3/1, are responsible for this conversion. Deficiencies in insulin production could be due to defects in the processing of precursor insulin molecules in the ER and/or the secretory granules. Defects in the ER function unleash the unfolded protein response (UPR), which can be adaptive and recover normal cell function. Alternatively if the stress is chronic and severe, the UPR initiates cell death.

    In pilot studies, using immunocytochemical techniques, we discovered that chronic pharmacological induction of insulin synthesis in normoglycemic white outbred CD-1 mice induced the expression, in all islets of 30% of mice treated (N=10), of a transcription factor that indicates severe ER stress. Significantly, islets of CHOP+ mice had a dramatic decrease in the levels of PC3/1 and PC2. Similar defects were found in beta cells of all diabetic C57BI6/Ks db/db mice examined.

    The objective of this proposal is to ascertain the clinical relevance of our findings. We wish to determine whether increased demand for insulin synthesis in diabetic patients induce CHOP expression and/or abnormal low levels of the proprotein convertases PC3/1 and PC2 in pancreatic islets.

    Addendum 1 Summary:

    In this extension of the proposal we will test whether the heterogeneity of PC1/3 expression in human beta cells correlates with differences in the level of expression of mature insulin, proinsulin and of transcription factors involved in insulin synthesis, Second, whether there is a parallel between expression of proinsulin/mature insulin and that of proteins involved in insulin secretion. The third specific aim will evaluate the level of expression of proinsulin and of molecules involved in its processing in T2D islets. This analysis will allow ascertaining if the phenotypic variability in T2D reflects the presence, in each pancreas, of normal islets and of islets with different stages of beta cell dysfunction.

    Addendum 2 Summary: 

    This proposal will test the hypothesis that beta cell death in Type 1 Diabetes occurs sequentiallyand is initiated with the immune mediated -ablation of the ProIN+PC1/3-cells. Comparison of celltype in pancreatic islets from autoantibody positive, newly diagnosed and long term T1D donors may reveal heterogeneity in response of the three cell types to the immunological attack. 

  • Peter Thompson

    University of Manitoba
    Therapeutic targeting of senescent β cells in Type 1 Diabetes

    A growing body of evidence has implicated various forms of beta cell dysfunction in the pathogenesis of Type 1 Diabetes (T1D). Recently we discovered that during the natural history of T1D, a subpopulation of β cells become senescent. Senescence is a cell type-specific form of permanent growth arrest, which occurs during aging, tissue damage or stress and involves progressively acquired phenotypes including activation of a DNA damage response, upregulation of cyclin-dependent kinase inhibitors, an apoptosis resistance phenotype and a senescence-associated secretory phenotype (SASP). Using nPOD pancreas specimens from a small cohort of donors, we previously found that β cells expressing senescence markers indicating the DNA damage response, cyclin-dependent kinase activation and SASP accumulated in islets of autoantibody-positive (1 or 2 autoantibodies) and recent onset T1D donors (diagnosed ≥11-12 years of age, 0-6 years post-diagnosis) relative to control donors without T1D. Studies in the nonobese diabetic (NOD) model demonstrated that senescent β cells are pathologic and accelerate T1D development, as small molecule targeting of the apoptosis resistance phenotype of these cells prevents T1D. Therefore,ourprevious studies suggest that the apoptosis resistance phenotype of senescent β cells is a target for therapeutic intervention to delay or prevent T1D. However, the proteins that confer the apoptosis resistance phenotype in senescent human β cells are not known, therefore it remains to be determined whether senescent human β cells can be selectively targeted by small molecule therapy. The goals of this project are to define the apoptosis resistance phenotype in senescent human β cells that accumulate in autoantibody-positive and recent onset T1D donors and to evaluate the efficacy of small molecule inhibitors of apoptosis resistance in targeting senescent β cell in islets ex vivo. These studies will take us closer towards the clinical translation of anti-senescence therapies for T1D.

  • Peter Thorn

    University of Queensland
    Funded by: NHMRC and Diabetes Australia
    Beta cell polarity

    Insulin secretion is principally regulated by the beta cell response to glucose. This secretory response is multiphasic with an initial burst of insulin secretion followed by sustained or increasing secretion. This complex timing of secretion is important for body function and is lost in type 2 diabetes. Beta cells normally function, and are normally regulated, within the islets of Langerhans which are composed of many hundreds of cells. However, due to technical limitations, beta cell function within islets is not well understood. To this end we have developed new methods that study single beta cells within an islet. Our new evidence shows that beta cells are structurally polarised and secretion of insulin is targeted to the vasculature.

  • John Todd

    University of Oxford
    Funded by: Helmsley Trust and Wellcome Trust
    Quantitative proteomic profiling of human islets in healthy and diabetes associated pregnancy

    Normal pregnancy is associated with a rise in insulin secretion, which begins at placental implantation and increases progressively in parallel with placental growth. Following placenta delivery there is a sharp postpartum decline in insulin secretion. Gestational diabetes (GDM),the development of diabetes for the first time during pregnancy, is associated with pancreatic beta-cell dysfunction. Rodent models have been used to elucidate the biological alterations in beta-cells during normal pregnancy. However, these findings have not been investigated in human pregnancy and little is understood about the mechanisms that contribute to beta-cell dysfunction in women with GDM.

    From mouse models, it is proposed that lactogenic hormones (prolactin [PRL] and human placental lactogen [hPL]) secreted by the placenta drive beta-cell expansion in normal pregnancy. Lactogenic hormones activate the beta-cell membrane prolactin receptor (PRLR) to increase downstream serotonin production. Serotonin may either increase or decrease beta-cell proliferation, in a receptor-dependent manner: rodent models have shown that activation of the5-hydroxytryptophan receptor 2B (5-HT2B)increases beta-cell proliferation,whilst5-hydroxytryptophan receptor 1d (5-HT1D)receptor activation decreases proliferation. At midgestation,Htr2bmRNA levels are increased in mice, which normalise at the end of gestation, whilstHtr1dexpression is increased at the end of gestation. It is proposed that it is this switch in serotonin receptor expression that contributes to the change in beta-cell mass during and following pregnancy. Preliminary data from our existing nPOD project illustrates that PRLRand5-HT1D receptor expression is increased in3rdtrimesterhuman beta cells in women with normal pregnancy but not in GDM. However, we have further found thatthe5-HT2Breceptor is not detected in normal human pregnancy or GDM at any stage during in gestation. Hence, much is still to be understood regarding pancreatic pathogenic mechanisms in GDM whilst, even in normal pregnancy, mouse models may not fully clarify pregnancy-associated changes in pancreatic biology.

    In this study extension, we propose to study the proteomic profile of human islets from women with normal pregnancy and GDM. Towards this, we will isolate human pancreatic islets using laser capture microdissection (LCM) and perform proteomic analysis of the isolated tissue by quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS) to evaluate differences in islet protein expression and uncover pathways that are altered during normal and GDM pregnancy. This will improve our understanding of the changes in beta-cell biology in normal pregnancy, as well as GDM, and potentially provide valuable insights on whether they may be harnessed as beta-cell regenerative therapies for patients with diabetes.

  • Emil Unanue

    Washington University School of Medicine
    Funded by: Breakthrough T1D
    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.

  • Steven Zhang

    Rutgers Cancer Institute of New Jersey
    Role of Rab1A in Diabetes Mellitus

    We previously identified the Rab1A small GTPase as a conserved activator of mTORC1 in response to amino acid (AA) stimulation in yeast and mammalian cells. To better understand the physiological function of Rab1A- mTORC1 signaling in vivo, we genetically engineered conditional Rab1A knockout (Rab1A-KO) in mice. We found that tamoxifen-induced Rab1A knockout in young adult mice impairs insulin expression and beta cell proliferation, leading to hyperglycemia and glucose intolerance. Using beta-cell lines, we further found that Rab1A mediates AA signaling to regulate insulin transcription and beta cell proliferation through phosphorylation of the transcription factor Pdx1. Our results revealed a novel role of AA-Rab1A-mTORC1 signaling in controlling whole body’s glucose homeostasis in mammals. The objective of this study is to use human patient samples to obtain clinical evidence that Rab1A plays an important role in the progression of diabetes mellitus.

Beta Cell Development, Differentiation & Regeneration

  • Mark Atkinson

    University of Florida
    Funded by: NIH
    Quantitative and qualitative evaluation of the endocrine & exocrine pancreas in type 1 diabetes

    In the most widely recognized natural history model for type 1 diabetes, there is a presumption that all individuals begin life with the same number of beta cells. In combination with this, a consensus exists that the so-called “normal” adult pancreas possesses approximately one million islet cells. These concepts have drawn little attention or question over time as much of the research interest in terms of the disorder’s natural history has focused on genetic susceptibility and the trigger period, where the beta cells begin a process of linear loss.  However, a limited body of largely unnoticed data draws question to the notions of a normal pancreas as well as the number of islets and/or beta-cells that may be resident, both in early life through adulthood, in humans.

    We (in association with Dr. Martha Campbell Thompson and Dr. John Kaddis) recently published data (JAMA, 2012) involving nPOD tissues suggesting that the pancreas of individuals with type 1 diabetes is smaller, consistent with other studies over the previous two decades. But, until recently, the observation drew little investigator attention.  Moving away from pancreatic size, the situation becomes even more intellectually diffuse when one considers quantification of islet and/or beta-cell numbers.  In studies of pancreatic development in both humans and rodents, it appears that a wave of beta-cell death occurs as a normal physiological process following birth.  The implications for this process may influence T1D’s pathogenesis in at least two ways. First is how it might influence T1D relates to the initiation of autoimmunity. A second notion as being of equivalent if not an even greater potential relates to the aforementioned concept of “100%”.   We believe it conceivable that a major and yet highly unappreciated risk factor for T1D resides in the number of beta-cells present in early life.   Taken collectively, these findings support a hypothesis that immune and perhaps non-autoimmune mechanisms (e.g., developmental, environmental, genetic, etc.) influence a loss of beta-cell mass as well as loss of pancreatic mass in T1D, possibly even before full-blown disease; thus, forming clear objectives for our studies.

    Type 1 diabetes patients often exhibit exocrine insufficiency (REF), however, it remains unclear whether these alterations result from disrupted islet-acinar interactions secondary to the loss of functional β-cell mass or contribute directly to type 1 diabetes development. Thus, as a basis for a better understanding of pancreatic exocrine function, we propose to investigate the expression pattern of major digestive enzymes in the pancreas of nondiabetic, autoantibody positive without diabetes and T1 diabetes nPOD donors. This will be achieved by multiplex immunohistochemical staining for amylase, lipase and trypsinogen (amongst others) and by employing fresh tissue slicing technique to obtain functional readouts of pancreatic endocrine and exocrine cell function .

     

  • Anil Bhushan, Mark Atkinson, Todd Brusko & Bita Brodbar

    University of California, San Francisco
    Funded by: Breakthrough T1D
    Epigenetic analysis of nPOD islet cells

    Type 1 Diabetes (T1D) is a complex autoimmune disease affecting more than 30 million people worldwide. T1D is caused by a combination of genetic and epigenetic factors leading to immune destruction of insulin-secreting islet cells. Although significant progress has recently been made in elucidating the genetics of T1D, the non-genetics component has remained poorly defined. Multiple lines of evidence including studies of migrant populations and monozygotic twin pair discordance for T1D underline the role of epigenetic factors in this disease. Thus, the characterization of epigenome is critical for the development of new T1D therapeutics and for appropriate staging of disease. DNA methylation is one of the main epigenetic modifications involved in human disease. Using epigenome-wide analysis such as reduced representation bisulfite sequencing (RRBS), we aim to identify unique differentially methylated regions (DMRs) within the genome that accumulate in PBMCs isolated from circulating blood, that may reflect the immune insult that occurs with T1D onset. Then we are interested to study the role of these hypo or hyper methylated regions in T1D.

  • Susan Bonner-Weir & Anmar Khadra

    Joslin Diabetes Center
    Funded by: Rumsey Funds
    Origin of composite islet structure in humans

    In the adult human pancreas the majority of islets have a clear non-random organization of mantle of non beta cells around beta cell cores, with larger islets usually having multiple such subunits (composite) whereas smaller islets resembled the classic rodent pattern. There is far more variability in islet composition both within each human pancreas and among different human pancreas than in rodent pancreas. The intrapancreatic variability in human pancreas raises important questions about how islets evolve and function throughout life and how this might relate to the pathogenesis of diabetes. We will examine the origin of the composite structure of the human islets by histological analysis of pancreas of donors from newborn to 25 years old. Determining the islet organization pattern (composite vs simple) with the distribution of islet size over the age range of expansion of the beta cell mass will provide data for mathematical modeling, which will predict the mechanism of formation of the composite islets.

  • Susan Bonner-Weir & Jens Hoiriis Nielsen

    Joslin Diabetes Center
    Mechanisms Involved in Expansion of Beta Cells in Pregnancy

    Pregnancy is associated with a marked increase in the number of pancreatic beta cells in response to the increased insulin demand. In rats and mice the growth of the beta cell mass is primarily due to proliferation of the existing beta cells resulting in enlargement of the islets of Langerhans. However, in a study of islets in human pregnancy (Butler et al 2010) the beta cell mass was found to be increased by numerous small islets and no sign of proliferation, suggesting that the growth of the beta call mass is due to neogenesis from putative progenitor/stem cells. In the proposed project we want to test the hypothesis that neogenesis rather than proliferation of beta cells is involved for the expansion of the beta cell mass in human pregnancy. This will be performed by immunochemical staining for prolactin receptors, Ngn-3 and Pdx-1 as markers of beta cell regeneration in pancreatic sections from pregnant women.

  • Martha Campbell-Thompson, Alberto Pugliese, Clive Wasserfall & Mark Atkinson

    University of Florida
    ORIGINAL: Beta cell regeneration for Type 1 Diabetes ADDENDUM: Neuro-insular pathways regulating beta cell regeneration

    ORIGINAL: 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.

    ADDENDUM: The goal of this addendum is to utilize large scale 3D human pancreas imaging methods for quantification of islet neuroendocrine cells and intra-pancreatic neurons and fibers. The autonomic neuro-insular network may be an unrecognized co-factor in the loss of functional beta cells during the progression to type 1 diabetes and following an islet transplantation. This proposal is expected to provide key data regarding neuronal pathways regulating beta cell regeneration.

  • Juan Dominguez-Bendala, Giacomo Lanzoni & Luca Inverardi

    University of Miami
    Funded by: Diabetes Research Institute Foundation
    Progenitors of the beta cell lineage in the pancreas and biliary tree of diabetic patients

    Stemming from a collaboration with Lola Reid (University of North Carolina), we have described stem and progenitor cells in niches of the human biliary tree and pancreatic ducts. These cells can mature into pancreatic endocrine cells, including functional insulin-producing cells. Our data suggest that bile ducts, peribiliary glands, pancreatic ducts and pancreatic duct glands may comprise a continuous ramification of cells that are related in maturational lineages. We have identified an extended network of stem cell pockets that branch out from the more naïve stem cells in the biliary tree to more and more mature cells in the pancreas. The presence of these progenitors in adults strongly suggests that the human pancreas has the potential to replenish lost cells in normal homeostasis and disease. Preliminary data obtained through our nPOD collaboration with Ivan Gerling (University of Tennessee) suggest that islets at the early stages of Type 1 Diabetes (T1D) may be undergoing regeneration or neogenesis. We are: (1) investigating the presence of pancreatic endocrine stem/progenitor cells in T1D and T1D Medalist cases; (2) testing if the stem/progenitor cell pool is affected by (or responsive to) the pathology and (3) studying whether these progenitors are subject to autoreactive aggression or not. We are also studying progenitor cells in transplanted pancreata in the nPOD-T study group. The overall aim of the project is to understand if regenerative processes are ongoing in T1D, to clarify the grade of maturation of beta cells in T1D, and to determine the feasibility of autologous approaches aimed at beta cell regeneration in T1D patients. Our team is open to new nPOD collaborations; we offer expertise and technologies in the following fields: stem cell markers, pancreatic islet development, beta cell maturation, immunology, imaging, laser-capture microdissection.

  • Farzad Ensi

    University of Pittsburgh
    Funded by: NIH and Children’s Hospital of Pittsburgh of UPMC Research Advisory Committee
    Conversion of human acinar cells into insulin – producing cells

    Reprogramming of acinar cells toward functional β-like cells would offer an abundant and autologous source of insulin-producing cells. The current literature suggeststhat manipulation of genetic factors,in combination with cytokines or small molecules targeting specific pathways and the epigenetic machinery in humans,may eventually yield functional acinar-derived β-like cells. Our preliminary datashowthat treatment of adult mice with a compound that specifically inhibits the kinase activity of focal adhesion kinase (FAK) results in transdifferentiation of acinar cells into insulin-producing cells. Notably, the acinar-derived insulin producingcells infiltrate the pre-existing endocrine islets and are able to normalize the blood glucose levels.Here, we seek to determine whether FAK inhibition would convert human acinar cells into insulin-producing cells.

  • Kevan Herold & Mark Mamula

    Yale University
    Analysis of cellular proteins in β cells from patients with type 1 diabetes and healthy

    Our objective is to identify characteristics of β cells that survive immune attack. Our hypothesis is that
    these residual cells express immune inhibitory molecules and/or decrease expression of diabetes
    associated antigens. In addition, these cells may show changes consistent with dedifferentiated β cells.
    Our studies of protein modifications have also shown that there are post-translational changes in
    proteins produced by β cells during disease development. Finally, we have developed T cell library
    assays that will enable us to identify novel antigens that may be found in islets during progression of
    diabetes. Our objective is to determine whether β cells with these features can be found in the islets of
    patients with T1D.

  • Pedro Herrera

    University of Geneva Medical School
    Funded by: NIDDK and Breakthrough T1D
    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 acells 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?
  • Pia Leete & Joanna Boldison

    University of Exeter Medical School
    Funded by: RD Lawrence Fellowship Diabetes UK & Leona M. and Hary B. Helmsley Charitable Trust
    The Heterogeneity of Type 1 Diabetes: An Islets Story

    When a person is diagnosed with Type 1 diabetes (T1D), the amount of betacell mass each individual loses is highly variable despite their clinical need for insulin therapy. Attempts to detect robust markers in the blood that precisely describe disease activity in the islets are ongoing, but without a full understanding of the disease course in the pancreas, when attempting to target therapy to the organ, we are still operating partially blindfolded. We have previously reported that two forms, or endotypes, of T1D are evident in the pancreas[14]. These differ according to the degree of betacell loss, the composition of the isletspecific immune infiltrate, and how beta cells process insulin. Additionally, we now have preliminary data that suggests the betacells themselves may vary between the two forms), even hinting that successful betacell regeneration may be more likely in one form than the other. Our proposed study will expand on these investigations, and we will compare pancreatic protein expression profiles between people diagnosed with T1D at different ages, with different durations of disease and compare this to those without diabetes and with Type 2 diabetes (as a proxy for metabolic dysregulation). We will leverage advanced histological and imaging techniques to analyse the presence of multiple critical proteins in granular detail in whole sections of pancreas. We will then (based on several features of pancreatic T1D and betacell loss), construct a nuanced ‘pseudo’ timeline of disease progression in T1D to illustrate the life and loss of betacells in T1D and explore if this is the same in all people, randomly different between peopleor stratifiably different between groups of people living with T1D. With this information we will better understand if we should be looking for distinct forms of T1D, and this may help scientists and trialists understand why some people respond brilliantly in clinical trials, whilst other do not.

  • Heiko Lickert & Teresa Rodriguez-Calvo

    Helmholtz Zentrum München
    Identification of novel monoclonal antibodies directed against pancreatic endocrine cell types for in vitroand in vivo analysis and targeting

    Diabetes mellitus is a multifactorial disease characterized by progressive loss or dysfunction of the insulin-producing beta cells in the pancreas that leads to chronic hyperglycemia, systemic metabolic complications and multi-organ damages. Nowadays, no current treatments can stop or reverse the disease progression, apart bariatric surgery and human islet transplantation. Therefore, intensive efforts in the field of diabetes research are put into the development of novel therapeutic approaches.Regeneration of β-cells or replacement by β-like cells derived from stem cells hold great promise to stop or reverse disease progression. Unfortunately, the development of new treatment options is limited by our poor understanding of human pancreas organogenesis. The differentiation programs of all islet cells and the way in which islets are formed during pancreas development in their body niche, need to be fully understood1-3.Recently, many promising discoveries that could potentially lead to stem cell-derived functional human islets were made. However, the field currently still lacks of specific tools that can allow for enriching the endocrine progenitors during the differentiation process to improve efficiency and functionality. To date, there is only one beta cell marker available, which is already limited in that it doesn’t mark all beta cells and in early stages of life is not specific for beta cells4. To fill in those gaps, in a screen to identify novel monoclonal antibodies (mAbs) that can be used to isolate and/or target human pancreatic cells, we have identified several mAbs that specifically identify pancreatic endocrine, exocrine and endothelialcells. The mAbs produced have a great potential as: 1) tools to isolate pancreatic cells types and their different subtypes for in-depth characterization of pancreas organogenesis 2) markers to characterize pancreatic cell heterogeneity during diabetes progression 3) novel biomarkers of diabetes 4) as a method to enrich for endocrine progenitors during human stem cell differentiation for cell therapy 5) specific carriers for drug delivery or direct therapeutic tools.

  • Chloe Rackham & Sarah Richardson

    University of Exeter
    Funded by: Breakthrough T1D
    Exploring an islet-protective role for native pancreatic mesenchymal stromal cells in health and in type 1 diabetes

    Our research aims to investigate how a group of adult stem cells, known as mesenchymal stem /stromal cells(MSCs), located in the pancreas may be involved in helping to protect people from developing type 1 diabetes(T1D). MSCs are found in most parts (tissues) of the body, including the pancreas, bone marrow, fat and kidney. When samples of these tissues are taken, MSCs can be isolated in the lab and when fed with appropriate nutrients will expand in number, so that we have enough MSCs that may be used therapeutically to treat inflammatory and autoimmune conditions, including T1D. Once isolated and expanded in this way we refer to these cells as exogenous MSCs, as opposed to endogenous/native MSCs that are found naturally in the tissue from which they were isolated.

    Researchers in the diabetes field often refer to these exogenous MSCs as ‘Islet Helper’ cells. This is because experimental studies have shown that exogenous MSCs improve the ability of islet beta cells to produce insulin in response to increased sugar levels. Additionally, experimental studies have shown that exogenous MSCs can help to protect islet beta cells from immune attack and destruction. Clinical trials have shown that that exogenous bone marrow MSCs help to preserve the function of insulin producing islet beta cells in some individuals with newly diagnosed T1D, after infusion into the blood circulation.

    Whilst we have established a lot about the function of exogenous MSCs, our understanding of endogenous/native MSCs located in the pancreas, with regards to their role in health and T1D is limited. We aim to determine whether the number, distribution and function of native pancreas MSCs is altered in T1D.We will determine whether native pancreas MSCs have a role in health to protect pancreatic islets from immune attack and destruction. These studies will provide scientific information on MSC-related changes inT1D, that we can target therapeutically to help delay or prevent the progression of diabetes. We will further establish whether there are differences in native pancreas MSC number, distribution and function in different subgroups of individuals with T1D, thus determining which subgroups are most likely to benefit from MSC-based therapeutic intervention.

  • Sara Sackett & Jon Odorico

    University of Wisconsin-Madison
    Funded by: DHHS, PHS, NIH
    Probing the normal human pancreatic and islet matrisome throughout development

    Diabetes is a major world health problem, affecting millions of Americans, costing billions of dollars in treatment, and causing major late-stage health complications and death. Despite the promise of stem cell therapies for diabetes, the field of beta cell differentiation has not yet reached clinical application, mainly due to the lack of mature function and survival of the cells in vivo. A better understanding of the pancreatic and islet extracellular matrix (ECM), and what role ECM plays during beta cell development, function and maintenance may lead to a more translatable stem cell-derived beta cell product; as well as have important application in auto-and allo-islet transplantation.

  • Guido Sebastiani & Francesco Dotta

    Fondazione Umberto di Mario/ University of Siena
    Expression of Beta-Cell Dedifferentiation-Associated microRNAs in Type 2 Diabetes

    MicroRNAs are small endogenous RNAs which regulate gene expression through mRNA decay or translational inhibition. It has been previously demonstrated that microRNAs are pivotal regulators of cell phenotype and fate and are involved in many biological processes such as proliferation, differentiation or apoptosis in a wide type of cells and organisms. Given their prominent role in cell phenotype specification, our hypothesis is that microRNAs may contribute to the new recently described mechanism of beta-cell dysfunction, known as dedifferentiation, in type 2 diabetes (T2D). Indeed, in an in-vitro model of human pancreatic islets dedifferentiation, we demonstrated that microRNAs may be involved in the acquisition of islet cells undifferentiated phenotype, thus possibly playing a role in the dedifferentiation of human pancreatic islets in T2D also. Therefore, here we aim at characterizing microRNA signatures of T2D pancreatic islets in order to reveal whether microRNAs involved in in-vitro dedifferentiation of human pancreatic islets are also likely to be involved in the dedifferentiation of those islets deriving from T2D donors. In order to do this, we will specifically capture islets from pancreatic frozen sections of non-diabetic and T2D donors using Laser Capture Microdissection (LCM), and analyze microRNA profiles using high throughput technologies. The major outcome of this study will be to potentially highlight several microRNAs which may be involved in the dedifferentiation process of human pancreatic islets in T2D, thus shedding light on new possible mechanisms of beta-cell dysfunction and consequently discover new potential therapeutic target.

  • Andrew Stewart, Nathalie Fiaschi-Taesch

    Icahn School of Medicine at Mount Sinai
    Funded by: Breakthrough T1D and NIDDK
    Multidisciplinary approaches to driving human beta cell replication

    Understanding and controlling human beta cell replication and regeneration are major goals of the Breakthrough T1D specifically, and of the diabetes community in general. Whereas the field of beta cell replication is a relatively new one, and much new data has recently accumulated regarding the molecular control of rodent beta cells, very little is known regarding the molecular mechanisms that govern human beta cell replication. We have received support from the Breakthrough T1D and the NIDDK to specifically define the presence and subcellular location of all human cell cycle control molecules in the human beta cell. The major cell cycle control point in all cells is the “G1/S checkpoint”, or the “pRb pathway”, which contains the cell cycle proteins pRb, p107, p130, p15, p16, p18, p19, p21, p27, p57, p53, cyclins A,D,E, and cdks 1,2,4,6. Until recently, very little has been done to elucidate the presence, absence or role of these molecules in physiologic human beta cell regeneration, nor in attempts at therapeutic human beta cell replication. We are therefore trying to develop a “Human G1/S Proteome” Atlas, a task we have largely completed using human cadaveric islets provided by the Breakthrough T1D and by the IIDP. We have demonstrated that when we overexpress these cell cycle molecules using adenovirus, we can induce both robust human beta cell replication, as well as nuclear translocation of these molecules. Thus, this is an observation of great significance to beta cell regeneration investigators: it suggests that the proximate reason that human adult beta cells do not replicate and cannot be induced to replicate as that the requisite nuclear machinery does not, and apparently cannot, access the nucleus where it would drive beta cell replication. These are paradigm-changing observations, with major implications for therapeutic human beta cell replication and regeneration.\

    ADDENDUM: Type 1 Diabetes results from the loss of the insulin producing beta cells. It is now clear that beta cell replacement can reverse diabetes in humans. One way to replace beta cells is to induce the regeneration of the endogenous beta cells or to expend beta cells from human donors for transplant. However, we know little about human beta cell replication. Therefore, information on human beta cell replication is critical for the development of new therapies for diabetes. Adult human beta cells proliferate at an extremely slow pace and are mysteriously resistant to the induction of replication. Rare examples of physiological replication of human cells are during the first years after birth and during pregnancy. Recently, we have made the novel and unexpected observation that many key cell cycle accelerator proteins are excluded from the nucleus in adult human pancreatic beta cells. It is possible to induce some replication of adult human beta cells by manipulating the cell cycle. However, this induction of replication is dramatically reduced during diabetes, such as Type 2 Diabetes. We propose to define the expression of cell cycle molecules in neonatal human beta cells, or human beta cells from pregnant donors or Type 2 diabetic donors. The overall goal of our research is to identify therapeutic targets to promote human pancreatic beta cell proliferation.

  • Tamás Röszer

    Ulm University
    Distribution of neuropeptide FF in the human infant pancreas

    Improving adipose tissue (AT) insulin resistance and induce adipocyte browning are key therapeutic aims in the management of obesity associated insulin resistance (IR) and in type 2 diabetes (T2DM). Our laboratory has recently identified neuropeptide FF (NPFF) as a pancreatic hormone, which selectively improves IR in the AT and controls AT browning, especially in infancy (J Clin Invest 2017, 127(7):2842-2854). The selective effect on adipose tissue is due to the specific and high level of expression of an NPFF receptor (NPFFR2) by AT macrophages (ATMs). NPFF/NPFFR2 signaling leads to a metabolically healthy ATM quaility in the AT. We have shown that differentiation of AT in infancy is controlled by ATMs, and this protects the infant from obesity (J Clin Invest 2019, 129(6):2485-2499). We also have found that NPFF controls ATM function in infancy. Next, we aim to explore whether NPFF can protect from childhood obesity (J Clin Invest 2019, 129(6):2485-2499). We already have shown the presence of NPFF in adult human pancreatic islets (J Clin Invest 2017, 127(7):2842-2854) and in adult and infant mouse pancreatic islets. Now, we would like to test whether NPFF is expressed in the infant human pancreatic islets. This is important since our data suggest that the endocrine pancreas is the major source of NPFF in the adult human plasma.

     

  • Yue Wang

    Florida State University
    Harnessing the power of beta cell regeneration to treat diabetes

    Approximately 1 in 11 people worldwide is afflicted by diabetes, a disease caused by a shortage of beta cells. The current standard of care for patients with severe diabetes is insulin therapy or pancreatic islet transplantation. However, insulin therapy requires frequent insulin injection, detailed dose calibration and close monitoring of blood glucose; for islet transplantation, there is a global shortage of organ donors and the procedure requires lifelong immunosuppression. As an alternative way to treat diabetes, beta cell regeneration methods have been explored with the aim to compensate for the underlying beta cell deficiency of the disease.

    Human beta cells possess the potential to regenerate during normal cell turnover and under various physiological stresses. Therefore, to promote beta cell proliferation, one of the powerful approaches is to exploit the endogenous machinery of beta cell replication. However, research on human beta cell regeneration is hampered by the facts that in adult human, the basal rate of beta cell replication is less than 0.1% and bulk assays do not have enough sensitivity to distinguish signals from these rare proliferation events. To address this problem, we will develop and implement an experimental system to achieve targeted isolation of proliferating beta cells from primary human pancreas and characterize their global gene expression program.

    Our work will help to identify novel pathways to increase beta cell mass both in the islet transplantation context and directly in patients with diabetes who have residual beta cells.

Core Lab

  • Irina Kusmartseva

    University of Florida
    Funded by: Breakthrough T1D, NIH
    Pancreatic immunologic and metabolic parameters

    The Organ Processing and Pathology Core (OPPC), located at the University of Florida, receives nPOD donor tissue directly from OPOs, processes the tissue, ships samples directly to investigators and stores case samples and data.

  • Daniela Del Gaudio

    University of Chicago
    MODY mutation testing

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

  • Ben Giepmans

    University Medical Center Groningen
    Funded by: Breakthrough T1D and STW
    Nanotomy of human islets of Langerhans in type 1 diabetes

    Microscopy is of paramount importance in diabetes research: Paul Langerhans was the first to describe (1869) Islets based on a light microscopic (LM) evaluation of the pancreas. (Immuno-)EM-analysis of the Islets of Langerhans is an outstanding way to discriminate beta-cells from other endocrine cells: The crystalline appearance of insulin granules makes beta-cells readily distinguishable from other cells, allowing to assess total insulin content. In addition, analysis of environmental factors hypothesized to caused diabetes, e.g. viral infection, can be directly visualised. Ultrastructural analysis by EM reveals beta-cell condition, ER stress, abnormalities in mitochondria, fibrosis, as well as apoptosis, necrosis, macro- and micro-autophagy. EM can be used to follow the etiology of T1DM, the fate of transplanted Islets in both human as well as animal models and to assess the success of cellular trans differentiation to create insulin-producing cells. Moreover, EM allows other cell-types to be recognized in the Islet that affect the functioning of beta-cells, including immune cells attacking beta-cells, duct cells, endothelial cells and nerve cells. Our proposed approach enables identification of morphological and macromolecular characteristics in different cell-types within the Islets in a quantitative and high-throughput manner. A single dataset could resolve most of the features raised above. So why is EM-analysis not standard included? Traditionally, EM data-interpretation is mainly done by the publishing scientist(s). EM-examination is hampered by the laborious procedure that requires technical expertise and expensive equipment. In addition, data-sharing is limited, the field of view of the area inspected at high magnification is traditionally very small, the data is noisy making it difficult to objectively quantify and segment the greyscale EM-images. Without the knowledge of how Islets of Langerhans look like, it will remain cumbersome to establish the cause and solution to diabetes. Implementation of computational science has now revolutionized EM, paving the path for routine EM-analysis of complete Islets, as we have pioneered.

    We are creating and analyzing high-resolution EM-maps of cross-sections of Islets of Langerhans in health and diabetes, which we term Nanotomy of Islets. The datasets allow to determine many aspects, including leukocyte infiltration, insulin content and cellular stress. Importantly, small particles such as viruses will also be detectable. The data first will be shared with nPOD co-investigators and at a later stage will be made publically available. (adopted by nPOD staff)

  • Clive Wasserfall

    University of Florida
    UF AAb Core

    The Autoantibody Core at the University of Florida serves as the coordinating center for nPOD screening lab partners (LABS,Inc and Viracor-IBT Laboratories). Combi-kit manufacturing and quality control/assurance measures are managed here. Additionally, the UF Autoantibody Core tests all nPOD samples for autoantibodies to GAD, IA-2, and ZnT8 by ELISA.

  • William E. Winter

    University of Florida
    Northwest Lipid Research

    The Marcovina lab performs C-peptide testing for all nPOD samples.

  • Liping Yu

    University of Colorado-Denver
    Denver AAb Core

    The Autoantibody Core at the University of Colorado works with the Autoantibody Core at UF for quality control/assurance and tests all nPOD samples for autoantibodies to GAD, IA-2, Insulin, and ZnT8, by RIA.

Exocrine Pancreas

  • Brittany Bruggeman & Martha Campbell-Thompson

    University of Florida
    Congruence of lab versus histopathologic findings of acute pancreatitis in the nPOD consortium

    Acute pancreatitis is the cause of more than 200,000 hospital admissions in the United States each year (1). Acute pancreatitis presents very broadly, hence diagnosis can be difficult and requires confirmatory tests. Serum amylase and lipase levels have been established as markers for disease. However, they lack adequate specificity and sensitivity which might make their use insufficient in providing the most accurate diagnosis.

    This study aims to investigate whether varying degrees of elevation in lipase and amylase levels can serve as more accurate predictors of acute pancreatitis by histopathology. Additionally, the study seeks to identify other factors that can predict elevated amylase and lipase levels during the hospital course of deceased organ donors, as well as explore the clinical characteristics of donors who have acute pancreatitis based on histopathological examination.

    This project will consist of an nPOD chart review to evaluate amylase and lipase levels in critically ill patients as predictors of acute pancreatitis by histology, and other factors that may contribute to elevated amylase/lipase levels such as DKA, renal failure, and GI illness. Our main hypothesis is that if the lipase levels in a person’s blood during their hospital stay are three times higher than the upper normal limit, it could be a good indicator of acute pancreatitis by histopathology. However, if the amylase levels are generally high or if the lipase levels are less than three times the upper normal limit, it may not accurately predict the presence of acute pancreatitis.

  • Anna Casu

    AdventHealth
    Funded by: Breakthrough T1D
    Stellate cells in type 1 diabetes research (STELLAR)

    The loss of insulin-producing β-cells is central to the pathogenesis of type 1 diabetes (T1D). More recently, the contribution of the exocrine pancreatic cellular components is becoming evident in determining the β-cells well-being. Other pancreatic cell types could be acting by reducing β-cell resistance to the autoimmune attack or inducing a stressful condition that will lead to neo-antigen expression or modulate the autoimmune response favoring β-cell damage.

    The pancreatic stellate cells (PSCs) reside in the exocrine pancreas in the peri-acinar space, but also in perivascular and periductal locations. They are implicated in the pathobiology of major exocrine pancreatic disorders such as chronic pancreatitis and pancreatic cancer. Activated PSCs lose their vitamin A content and are responsible for organ fibrosis, promote extracellular matrix deposition, release factors that promote an inflammatory state, interfere with acinar cell function, and reduce the insulin secretory capacity. The PSC activity known in other diseases leads to hypothesize that they could have a role either in determining the exocrine pancreas alterations seen in T1D and favoring β-cell function decline. In spite of the multiple possible causative mechanisms, the role of PSCs in the pathogenesis of T1D has not been investigated, and this proposal aims to close this knowledge gap.

    A preliminary analysis of a subset of HPAP single-cell data showed that PSCs from organ donors with T1D have an immune-activated phenotype as described by Baron et al. in other conditions. They overexpress in fact multiple chemokines and IL-6. This evidence supports our hypothesis that a dysfunctional and proinflammatory PSC activation in T1D contributes to and maintains β-cell stress accelerating β-cell functional decline. It is hypothesized that this interaction is mediated by PSC-secreted factors directly acting on the islets which promote a pro-inflammatory gene expression. In this study, we will investigate the role of activated PSC-secreted factors on islet function.

  • Hsun Teresa Ku

    City of Hope
    Funded by: NIH/NIDDK
    The role of human ductal cells in propagating pathogenesis of type 1 diabetes

    Type 1 diabetes (T1D) is a pancreatic disease marked by the autoimmune destruction of insulinsecreting beta cells within the endocrine compartment, leading to chronic hyperglycemia and longterm complications. Despite extensive studies into the autoimmunity that targets beta cells, a complete understanding of the pathogenesis of T1D remains unclear. A recent study by Fasolino et al. demonstrated that the exocrine ductal cells in T1D donors, compared to nondiseased control donors, have an elevated expression of a MHC class II molecule, HLADR. MHC class II molecules are known to be expressed by antigen presenting cells (APCs) that process and present extracellular antigens to CD4expressing T helper cells. Fasolino et al. also found that CD4+ T cells, compared to CD8+ T effector cells, are located more frequently in the neighborhood of HLADR expressing ductal cells. These results raised the intriguing possibility that pancreatic ductal cells may function as APCs. In this study, we propose to conduct a more comprehensive assessment of proteins involved in antigen processing and presentation in pancreatic ductal cells of T1D donor tissues, compared to control nondiseased donor tissues, using coimmunofluorescence staining technique. Our proposed studies, if successful, will contribute to a new line of research addressing the cross talk between exocrine and endocrine pancreas in the pathogenesis of T1D, which may lead to new therapeutic targets for T1D.

  • Kathrin Maedler

    University of Bremen
    Funded by: DFG
    Do enteroviruses trigger diabetes progression?

    Identification of the driver of autoimmunity and early intervention before established disease is urgently needed. As in many autoimmune diseases, viral infections have been associated with T1D, recently confirmed by large and highly sensitive screenings using wellcharacterized material from autopsy and biopsy. We looked closer into the pancreases from organ donors with early T1Dassociated autoimmunity before T1D diagnosis and with established T1D and found the factor YAP, cotranscriptional activator of the Hippo cell and organ developmental pathway highly increased. Absent in mature functional betacells, YAP was especially found in areas of enterovirusinfected cells which remain deposited in the pancreas long after acute viral infection and drive inflammation and immune cell infiltration. Such changes in the exocrine pancreas have been completely overlooked. To our surprise, in a scenario of T1D, YAP did not balance the immune response but acted oppositely of what has previously been known; it potentiated the deleterious effect of the enterovirus and constituted a vicious cycle; YAP increased virus load, inflammation and cell death in both exocrine and endocrine cells. Eventually, even YAP+cells migrated into islets. Our project shifts two paradigms: we hypothesize that (1) autoimmune diabetes starts in the exocrine pancreas with areas of inflammation and immune infiltration and (2) YAP acts as early driver of autoimmunity and T1D by potentiating inflammatory signals towards immune cell attack and damage to betacells. Our analyses will explain the early pathology seen very early even in first degree relatives (FDR) of patients with T1D, who have already reduced pancreas mass. The objective of this exploratory project is to unravel how YAP expression in viral+ cells in the pancreas  fosters T1D initiation and progression and how this cycle of inflammation can be interrupted to establish an early pharmaceutical intervention against autoimmunity andT1D.

Immunology

  • Reza Abdi & JianJun Cheng

    Brigham and Wowen's hospital
    Use of T1D pancreastic and lymphoid tissue to assess the expression of adhesion molecules

    While immunoregulatory therapies hold great promise for the treatment of T1D, their inadequacy and serious toxicity have limited research efforts to find a lifelong immunosuppression approach. This has motivated investigators to search for alternative approaches such as the use of nanotechnology to deliver a wide variety of drugs and biomolecules. In recent years, there have been major strides to address various obstacles in order to bring nanotechnology closer to clinical practice. Nanotechnology has been moving forward on two fronts: 1) synthesis of a NP which efficiently loads, retains and releases drugs and 2) development of an innovative strategy to deliver NP to specific sites.   Much progress has been made on NP surface properties and size to reduce burst release, blood opsonization and clearance to enhance the circulation half-life of NP from several minutes to tens of hours.

    On the front of delivering NPs to specific tissue sites, we have witnessed less progress. To make our nano-delivery approach applicable to Type 1 diabetes (T1D), we (Abdi and Cheng) have made major strides as follows: (A) we have successfully designed nanomedicines with the desired surface properties and controlled particle sizes to improve their pharmacokinetics, (B) demonstrated that loading and controlled release of the drug is markedly improved by developing a polylactide-drug conjugated nanoparticle, or nanoconjugate, via drug-initiated polymerization of lactide followed by nanoprecipitation, (C) our published data indicate that our nanotechnology platform is highly efficient in delivering immunosuppressive drugs, and importantly..

    (D) Importantly, our preliminary data indicate selective PNAd dependent delivery of the nanoparticles to the pancreas and pancreatic lymph nodes in NOD mice. We are interested in examining the expression of PNAd in pancreatic lymph nodes (PLN), pancreas, non draining LN and spleen by immunhistolgoy to assess the feasibility and potential success of our targeted therapy in humans.

  • Anil Bhushan & Seung Kim

    University of California San Francisco & Stanford University
    Stromal sentinel cells are activated in T1D and organize tertiary lymphoid structures

    Preliminary data indicates that stromal cells within the vicinity of islets acquire properties that can organize ectopic lymph node like structures (known as tertiary lymphoid structures or TLS)in NOD mice. These properties include differentiating of stromal cells into specific cell types that can secrete chemokines to organize infiltration of T and B cells and secrete growth factors to organize endothelial cells into venules through which these lymphocytes traffic. These ectopic lymph nodes correlate with disease progression and mice that don’t develop disease do not show an evidence of these TLS. In this application, we utilize CoDetectionbyInDEXing (CODEX), a multiplexed singlecell imaging technology that incorporates DNA barcoded antibodies to visualize 100 + cellular markers at the singlecell level to map islet stromal cells and TLS in nPOD samples to map tissue architecture and characterize stromalimmune interactions in T1D. As TLS have been recently been shown to be a feature of T1D pancreas in humans, we would like to assess whether the stromal cells in nPOD donors acquire TLS organizing properties. We also assess the subtypes of stromal cells with lymphoid organizing properties and expression of cell type specific markers of stromal cell subtypes involved in TLS organization. We have built our CODEX panel to include markers of lymphoid organizing stromal cells that will enable us to identify the subtypes of cells. The CODEX panel includes markers for T cells, B cells and endothelial structures called high endothelial venules (HEVs).We will also be able to assess the activation state of lymphocytes. Finally we haver markers for endocrine cells. We envision that such analysis of T1D samples will allow us to establish whether stromal cells are strategically positioned in the islet to provide a scaffold and orchestrate TLS formation by modulating immune cell maturation, migration and activation.

  • Todd Brusko

    University of Florida
    Funded by: 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. 

  • Georgia Fousteri

    San Raffaele Research Institute
    Funded by: Ministero della Salute, Giovani Ricercatori
    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.

  • Rachel Friedman

    University of Colorado
    Funded by: Helmsley Charitable Trust George S. Eisenbarth nPOD Award for Team Science
    Mononuclear phagocyte populations in T1D islets

    Little is known about how T cell pathogenesis is modulated in the islets during T1D, yet this is a critical obstacle for preventing destruction of remaining islets or beta cell grafts. Therefore, we must elucidate mechanisms that promote and prevent the pathogenic autoimmune response in the islets. Our data in the NOD mouse model show that different populations of CD11c mononuclear phagocytes have countervailing roles in restimulating or inducing tolerance in islet-infiltrating T cells as islet infiltration progresses. While macrophages have been identified in human pancreatic islets, these likely represent a complex assortment of several mononuclear phagocytic cell populations that play a role in islet destruction. As a result, our goal is to identify the populations of mononuclear phagocytes within the islets of normal, autoantibody positive, and type 1 diabetes subjects.

    ADDENDUM: Type 1 diabetes (T1D) lacks therapeutic approaches that stop the autoimmune destruction of the beta cells. T cells may be the major mediator of destruction in T1D, but they do not act alone. T cells are influenced by myeloid cells in the islets. Our preliminary data show that human islets contain multiple populations of myeloid cells with some populations that are only present in the islets of T1D patients. As a key regulator of T cells in T1D, the islet myeloid cells are a potential target for therapeutic intervention for T1D. However, human islet myeloid cells are severely understudied. Thus, before we can develop myeloid therapies for T1D, we must understand the human islet myeloid populations and identify specific molecules on the myeloid cells for therapeutic targeting. Our goals are to identify the myeloid cell populations in the islets of non-diabetic, autoantibody positive, and type 1 diabetic donors. We will determine where these populations are localized in the islets, as well as how these populations correlate with islet health and interact with T cells in the islets. These analyses will create a picture of the myeloid landscape of human islets and begin to elucidate the roles of human myeloid populations in normal and type 1 diabetic islets. Because myeloid cells are present in all islets, the knowledge gained will be relevant to understanding how these cell populations function in T1D and T2D.

  • Kathryn Haskins, Sally Kent & Thomas Delong

    University of Colorado School of Medicine
    Hybrid peptides as target antigens for pathogenic T cells in human T1D patients

    We have recently discovered a new form of beta-cell autoantigen, which is recognized by CD4 T cells, and is formed through a novel post-translational modification of beta-cell peptides that involves the fusion between insulin peptide fragments and peptides of other secretory granule proteins. The hybrid insulin peptides (HIPs) are highly antigenic for autoreactive and pathogenic CD4 T cell clones derived from the nonobese diabetic (NOD) mouse. Importantly, three of these HIPs have been shown to exist in nature, as we detected them by mass spectrometry of antigenic fractions from beta cell extracts. Moreover, we have also identified two HIPs that are antigenic for T cells clones derived from the islets of T1D deceased donors. Thus, our data support the involvement of HIPs in human T1D. In this project, our goal is to extend our study of human T cell responses to HIPs by isolating T cells from spleen and pancreatic lymph nodes  (PLN) of nPOD donors and then testing these T cells on a selected panel of HIPs. We will initially test the T cells through a sensitive assay (called ELISpot) for the inflammatory cytokine, interferon-γ, produced when CD4 T cells encounter antigen. Long-term goals are to identify HIPs that stimulate autoreactive T cells from human patients and to develop reagents with these HIPs that can detect the autoreactive T cells in the blood (or other tissues).

  • Eddie James

    Benaroya Research Institute
    Characterization of T Cells that Recognize Post-Translationally Modified Beta Cell Epitopes from within the PLN

    We have recently demonstrated that enzymatically modified peptides derived from beta cell antigens are preferentially recognized by autoreactive CD4+ T cell clones isolated from the peripheral blood of patients with T1D. Furthermore, T cells that recognize these modified peptides are present in the peripheral blood of patients with T1D and have a high affinity, enabling their detection by flow cytometry. Importantly, the expression and activity of enzymes responsible for these modifications are upregulated in response to cellular stress and inflammation. Therefore, modified peptides are likely to form in stressed islets and to elicit pathogenic T cell responses. Consistent with this notion, T cells that recognize two or three of these modified peptides were shown to be present in islets and pancreatic lymph nodes (PLN) of two organ donors with T1D. The goal of this project is to corroborate and extend our study of T cell responses to modified beta cell antigens are relevant to the pathogenesis of T1D by identifying and studying T cells that recognize these peptides within PLN from nPOD donors. We will identify and isolate these T cells using HLA class II tetramers. We will then characterize these cells by examining their cytokine profiles and sequencing their T cell receptors. Finally, we will compare these attributes with those of T cells with the same peptide specificity that are found in peripheral blood to define unique attributes of T cells that are most relevant to disease.

  • Jenny Jiang & Todd Brusko

    The University of Texas at Austin
    Human T cell repertoire profiling in Type 1 Diabetes

    Current strategies of studying islet autoantigen-specific T cells rely heavily on fluorescence labeled tetramers and are limited in the throughput of antigens that can be analyzed simultaneously. The profile of islet autoantigen-specific T cells that can be obtained is limited. We recently developed a new technology, Tetramer associated TCR sequencing (TetTCR-Seq), that is capable of de novo generating a large amount of peptides and mapping antigen recognition with single cell TCR sequences by DNA barcoded tetramers. It enables us to quickly survey T cell repertoire at single cell level for a large number of peptides. Meanwhile, simultaneous profiling of gene/protein expression at single cell level helps to provide a comprehensive evaluation of antigen-specific T cell repertoire. We propose to apply this novel technology to study islet autoantigen-specific T cells in T1D. By investigating islet autoantigen-specific T cells in different tissues from nPOD, including spleen, pancreatic draining lymph node, and irrelevant draining lymph nodes, we will characterize the antigen-specific TCR repertoire and disease-specific transcriptional signatures. We believe this will help us to gain deeper understandings of the onset of T1D, the cause of immune dysregulation and potentially develop new markers for the diagnosis and the treatment of T1D.

  • John Kearney

    University of Alabama at Birmingham
    Funded by: Breakthrough T1D
    Modulation of autoimmune diabetes by natural antibodies specific for N-acetyl-D- glucosamine

    The focus of our research project is to improve the understanding of how antigenic stimulation of the B lymphocyte repertoire development during early life may protect from Type 1 Diabetes (T1D). Specifically, we are investigating how exposure to the common pathogen Group A Streptococcus promotes the formation of a T1D-suppressive natural antibody repertoire. Exposure to microorganism associated antigens during early life can protect diabetes-prone mice from developing T1D, and epidemiological studies in humans as well as studies in diabetes prone mice and rats have reported that that immune responses to Group A Streptococcus (GAS) are associated with protection from T1D. In humans and mice, infection or immunization with GAS results in a robust B cell response and the production of antibodies reactive with GAS cell wall-polysaccharide-associated N-acetyl-D- glucosamine (GlcNAc) epitopes, yet the role of these responses in protection from T1D has not been previously examined. We have shown that GlcNAc-reactive antibodies, produced following exposure to GAS cell wall antigens, bind GlcNAc-modified beta cell antigens. GlcNAc-reactive B cells elicited during neonatal immune responses to GAS traffic to the pancreas during T1D in non-obese diabetic (NOD) mice, and transfer protection upon adoptive transfer into naïve recipients. GlcNAc-reactive natural antibodies produced by these cells opsonize damage-associated molecular patterns on stressed beta cells and promotes clearance of their diabetogenic antigens, thereby attenuating the pathogenesis of T1D. Our goal is to evaluate the role of GlcNAc-reactive B lymphocyte development and antibody production in T1D in humans to identify novel biomarkers for improved prediction of T1D susceptibility, and the development of vaccination or probiotic strategies aimed at boosting natural antibody-mediated suppression of T1D progression.

  • Sally Kent

    University of Massachusetts Medical Center
    Funded by: Breakthrough T1D
    Investigation of B cells in human islets and PLN in T1

    Our goal is to understand the role of autoreactive B cells in tissues draining the pancreas and their role in the type 1 diabetes (T1D) autoimmune response. While B cells are responsible for secreting autoantibodies, B cells have other functions including secretion of effector cytokines, antigen presentation and costimulation of T cell responses. Using a multiplexed, single cell interrogation assay called microengraving, we saw, ex vivo, that there is a statistically more frequent number of immature (IgM secreting) CD20 B cells secreting antibodies reactive with islet antigen GAD65 in the spleen from 50% individuals examined with recent onset (0-7 years) T1D as compared to those from healthy control: these cells could also be seen on the spleen of some GADA+ individuals without disease and in some individuals with long-term T1D. We saw this reservoir of GADA-secreting B cells of a mature phenotype (secreting IgG1, IgG3 or IgA) in the spleen of two individuals with recent onset T1D. We saw a low frequency of immature CD20 B cells secreting antibodies reactive with proinsulin in all groups. We wished to interrogate the autoreactive B cells for their cytokine secretion, but due to cell demise in the single cell assay at time points greater than 24 hours, we developed a 7-day culture assay that allows for survival of B cells and identification of these cells by their autoantibody secretion and cytokine profile in a two-color ELISpot assay. We are interrogating spleen samples from control individuals, individuals with autoantibody, but without disease, individuals with recent onset T1D (0-7 years), and individuals with long-term T1D (>7 years) for co-secretion of GAD65 autoantibody and IL-6/IL-10 or LTa/TNFa. These studies will provide a profile of the function of autoreactive B cells from individuals with T1D and their role in the autoreactive disease process.

  • Sally Kent & Eddie James

    University of Massachusetts & Benaroya Research Institute
    Funded by: Cystic Fibrosis Foundation
    T cell autoreactivity in pancreatic draining lymph nodes (pLN) of donors with cystic fibrosis-related diabetes (CFRD) and type 1 diabetes (T1D)

    Many individuals with cystic fibrosis (CF) develop diabetes or CFrelated diabetes (CFRD).Some published data indicates that CFRD shares autoimmune phenotypes in common with type 1 diabetes (T1D). These include a higher prevalence of islet autoantibodies for CFRD patients than the general population and signs of broad immune dysregulation (including a Th17 signature). Notably, in autoantibodypositive CF patients, diabetes onset occurs earlier and is generally associated with a higher risk of acute complications. In a study funded by the Cystic Fibrosis Foundation, T cells from peripheral blood mononuclear cells from matched cohorts of individuals with CFRD, with CF, with T1D, and from controls are being examined for isletreactivity and phenotype. Here, we propose to examine cryopreserved single cell suspensions of pancreatic draining lymph nodes from matched donors with CFRD, withT1D, and from donors without diabetes with and without pancreatitis (the latter serving as an inflammation control)for these paprameters. Our overall goal is to increase the understanding of the autoimmune response to isletantigens in donors with CFRD and in T1Das compared to those from control and to those from donors with pancreatitis (inflammatory state)and from control donors and identify shared T cell specificities and immune mechanisms that might contribute to their decreased insulin secretion and beta cell damage in CFRD and T1D.

  • Pia Leete & Joanna Boldison

    University of Exeter Medical School
    Funded by: RD Lawrence Fellowship Diabetes UK & Leona M. and Hary B. Helmsley Charitable Trust
    The Heterogeneity of Type 1 Diabetes: An Islets Story

    When a person is diagnosed with Type 1 diabetes (T1D), the amount of betacell mass each individual loses is highly variable despite their clinical need for insulin therapy. Attempts to detect robust markers in the blood that precisely describe disease activity in the islets are ongoing, but without a full understanding of the disease course in the pancreas, when attempting to target therapy to the organ, we are still operating partially blindfolded. We have previously reported that two forms, or endotypes, of T1D are evident in the pancreas[14]. These differ according to the degree of betacell loss, the composition of the isletspecific immune infiltrate, and how beta cells process insulin. Additionally, we now have preliminary data that suggests the betacells themselves may vary between the two forms), even hinting that successful betacell regeneration may be more likely in one form than the other. Our proposed study will expand on these investigations, and we will compare pancreatic protein expression profiles between people diagnosed with T1D at different ages, with different durations of disease and compare this to those without diabetes and with Type 2 diabetes (as a proxy for metabolic dysregulation). We will leverage advanced histological and imaging techniques to analyse the presence of multiple critical proteins in granular detail in whole sections of pancreas. We will then (based on several features of pancreatic T1D and betacell loss), construct a nuanced ‘pseudo’ timeline of disease progression in T1D to illustrate the life and loss of betacells in T1D and explore if this is the same in all people, randomly different between peopleor stratifiably different between groups of people living with T1D. With this information we will better understand if we should be looking for distinct forms of T1D, and this may help scientists and trialists understand why some people respond brilliantly in clinical trials, whilst other do not.

  • Enrique Montero

    Arthur Riggs Diabetes & Metabolism Research Institute
    CD6 and its ligands in Type 1 Diabetes

    Type 1 diabetes mellitus (T1D) results from the autoimmune attack of pancreatic β-cells, resulting in a loss of functional β-cell mass. The CD6 molecule is a membrane glycoprotein predominantly expressed on T cells and is implicated in lymphocyte adhesion, activation, and proinflammatory commitment. The CD6 ligand CD166/ALCAM is upregulated in mature dendritic cells and the inflamed tissue, facilitating infiltrating lymphocytes into the target organs. Furthermore, CD166 is implicated as essential in pancreatic development, and it has been found in the membrane of islet endocrine cells.

    CD318/CDCP1 was recently identified as a second CD6 ligand, contributing to the autoimmune response in experimental models. We have discovered in healthy human donors that CD318 is expressed by a subset of myeloid Dendritic Cells and activated monocytes. Moreover, CD318 expression can be induced on peripheral monocytes by proinflammatory cytokines. CD318+ cells significantly reduced the proliferation of polyclonal T cells and antigen-specific CD4+ T cells. Our unpublished data also uncover CD318 expression in human pancreatic islets from non-diabetic donors, and its expression is increased by proinflammatory cytokines.

    These findings suggest a novel role for CD318 as a co-inhibitory ligand for T cell activation and effector function. Therefore, we propose CD6 as a novel immune checkpoint with dual function based on the ligand expression: CD6 binding to CD166 triggers T cell inflammatory response in the pancreas while CD318 acts as a co-inhibitory ligand down-regulating T cell responses and preventing the perpetuation of local autoimmunity. This project aims to characterize the expression of CD6 and its ligands CD318 and CD166 in human pancreatic islets from T1D donors. This study may describe a novel natural mechanism for human islet protection from autoimmunity and may aid developing novel therapeutic strategies to cure T1D by targeting CD6.

  • Maki Nakayama

    University of Colorado
    Dynamic Survey of T Cell Repertoire Targeting Pancreatic Beta Cells

    Our project aims to define islet-specific T cell receptor (TCR) sequence repertoires directly from pancreas to pursue two goals; 1) to identify antigens targeted by T cells contributing to the development of type 1 diabetes (T1D); and 2) to pursue the potential of TCR sequences to be used as biomarker.   We hypothesize that common antigens targeted by T cells negatively (Treg) and positively (Tpath) contributing to the development of T1D in multiple patients are present.  Identification of such antigens and TCR repertoires will lead us to develop antigen-specific immunotherapy and immunodiagnostic tools.  We directly sequence TCR alpha and beta chains of T cells in the pancreas as well as in different T cell subsets in the immune organs and peripheral blood of patients having T1D utilizing the high-throughput Illumina and 454 sequencing technologies.  TCRs detected in the pancreas are expressed on TCR-null T cell hybridoma cells using the retroviral expression system and are tested for the response to known antigens as well as the islet cells.  Once TCR pairs responding to the islet cells are determined, we will further assess pathogenicity in inducing diabetes by generating retrogenic animals.  In addition, presence and frequency of pancreas-derived TCR sequences in the peripheral organs of the same patients as well as those having the same HLA alleles will be evaluated to “trace” T cells detected in the pancreas.  Given evidence that a large number of public TCR sequences detected from multiple individuals are present, we believe that TCRs associated with the development of T1D exist and are able to be used for diagnosis in combination with other T cell assays (e.g. tetramer analysis).  Common antigens associated with the pathogenesis and regulation of T1D development will gain our insights to develop antigen-specific immunotherapy for T1D.  

  • Mark Peakman

    Sanofi
    Credentialing immunotherapy targets in islet tissues with insulitis

    Sanofi is developing strategies to control beta cell destruction in type 1 diabetes (T1D) through immunotherapy. Strategies could include targeting specific molecules on cells that may be of importance in disease pathogenesis.

    Such approaches are in progress and candidate molecular targets of potential interest have been identified. As part of the next steps, our goal is to obtain a better understanding of the relevance of candidate molecules in T1D development. For this reason, in this project we aim to study whether molecules of interest are expressed in the pancreas; the location and identity of positive cells; and the relationship to disease. This will assist in the next key stages of drug development for T1D

  • Peter Linsley & Karen Cerosaletti

    Benaroya Research Institute
    The role of shared germline-like TCR alpha chains of self-reactive TCRs in type 1 diabetes

    Human islet antigen reactive CD4+ memory T cells (IAR T cells) play a key role in the pathogenesis of autoimmune type 1 diabetes (T1D). Using single cell RNA-sequencing (scRNA-seq) to identify T cell receptors (TCRs) in IAR T cells, we identified a multi-specific class of T1D-associated T cell receptors (TCRs) that share TCR alpha chains between individuals (“public”). We isolated IAR T cells from blood of healthy, new onset and established T1D donors using multiplexed CD154 enrichment and identified paired TCR alpha/beta sequences from 2767 individual cells. More than a quarter of TCR junctions were shared between 2 or more cells (“expanded”) and 29/47 (~62%) of expanded TCRs tested showed specificity for islet antigen epitopes. Public TCRs sharing TCR alpha junctions were most prominent in new onset T1D. Public TCR sequences were more germline-like than unique or ‘private’ TCRs, and had shorter junction sequences, suggestive of fewer random nucleotide insertions. Public TCR alpha junctions were often paired with mismatched TCR beta junctions in TCRs; remarkably, a subset of these TCRs exhibited cross-reactivity towards distinct islet antigen peptides. Our findings demonstrate a prevalent population of IAR T cells with diverse specificities determined by TCRs with restricted TCR alpha junctions and germline-constrained antigen recognition properties. Since these “innate”-like TCRs differ from previously described immunodominant TCR beta chains in autoimmunity, they have implications for fundamental studies of disease drivers, as well as translational application to the identification of immune biomarkers and targets for TCR-based therapeutics. In the present proposal, we seek to extend these findings with IAR T cells in blood to spleen.

  • Alessandra Petrelli & Anna Giovenzana

    IRCCS Ospedale San Raffaele
    Unraveling the Role of CD45RA+CCR7+CD4 T Cells in the Pathogenesis of Type 1 Diabetes

    This study aims to investigate the role of CD45RA+CCR7+CD4 T cells in the pathogenesis of type 1 diabetes (T1D). Historically, naïve T cells were deemed irrelevant in T1D development due to their presumed lack of antigen experience. However, distinct subsets of naïve T cells with varying phenotypes and functions have been identified, with recent literature indicating that autoreactive T cells in T1D exhibit stem-like properties with phenotypes between naïve and effector/memory states.

    Our data show that the enrichment of CD45RA+CCR7+CD4 T cells in the peripheral blood of newly diagnosed T1D children. Moreover, a higher frequency of this cell subset at diagnosis correlates with a poorer outcome, regardless of age. The hypothesis posits that CD45RA+CCR7+CD4 T cells form a heterogeneous pool enriched with stem-like cells possessing effector functions, actively contributing to beta cell damage and T1D development.

    To investigate the heterogeneity of CD45RA+CCR7+CD4 T cells, the study will utilize single-cell RNA sequencing coupled with TCR sequencing on longitudinal blood samples from relatives of T1D patients. This approach aims to explore the phenotype and TCR repertoire, monitoring changes in plasticity and expansion of TCR clones as individuals transition through T1D stages, with special attention to stem cell memory subsets expressing CD95. Additionally, multi-color staining of pancreas sections will be performed to localize CD45RA+CCR7+CD4 T cells relative to beta cells and assess cell infiltration dynamics in different T1D stages. Pancreas tissues from non-diabetic and autoantibody-positive subjects, recent onset and long-standing T1D patients will be analyzed.

    Overall, this project seeks to shed light on the potential role of CD45RA+CCR7+CD4 T cells in beta cell damage, highlighting their potential significance as overlooked players in T1D pathogenesis and contributing to the decline of beta cell function. This project is under discussion for funding from Helmsley Charitable Trust.

  • Helena Reijonen

    City of Hope
    Correlation of Islet Autoantigen-Specific T-Cell Repertoire in the Pancreatic Lymph Nodes and Peripheral Blood in T1D Autoimmunity

    Islet autoantigen specific T cells can be found in both T1D patients and healthy individuals.  However, these T cells with the same antigen specificity are expected to differ in their phenotypic characteristics. We will investigate CD4 and CD8 T cells in nPOD samples from T1D patients, prediabetic and healthy subjects allowing us to compare T cell specificities and phenotypes at different stages of the disease. We have generated tetramers representing the most common HLA-DR types associated with T1D risk (DR401, 404 and 405, 301, DQ8) or protection (DR1501, DRB5 0101 and DQ 0602) containing immunodominant peptides from the major diabetes autoantigens GAD65, Proinsulin/insulin, IA2 and Zn-T8.  The approach is based on the utilization of tetramers to identify and isolate autoreactive T cells from pancreatic lymph node tissue, peripheral blood (and possibly bone marrow) offers an advantage that phenotypic analyses are performed on T cells responding to a single, known antigen mediated with a defined MHC restriction.  Tetramer-based technology also allows determination of the functional phenotype of autoantigen specific T cells, for example memory, avidity/affinity, cytokine profiles, homing properties, and these cells can be sorted for gene expression studies and TcR analysis. In the cases when both peripheral blood lymphocyte and pancreatic lymph node samples are available the proposed study would provide valuable information on the predictive value of blood samples as biomarker in the evaluation of the disease process.  Collaboration with other nPOD investigators allows our project to generate new knowledge on the key features and the population dynamics of CD4+ T cells directly associated with islet autoimmunity and beta-cell biology in human diabetes. 

  • Teresa Rodriguez-Calvo

    Helmholtz Zentrum München
    Targeting the innate immune system to discover new therapeutic pathways for the prevention of type 1 diabetes

    Type 1 diabetes (T1D) is an autoimmune disease, where immune cells (T cells) of the body attack and destroy the insulin-producing beta cells. T1D is, if left untreated, a devastating and life-threatening disease and despite intensive research in the field, there are still missing pieces in the pathogenetic puzzle of the disease. With this project, we plan to focus on the innate immune system and its role in the development of T1D. Innate immune cells, mainly represented here by macrophages (ΜΦ) and dendritic cells (DCs), are among the earliest responders to inflammation, infection and stress, yet their direct implication in the development of T1D remains unknown in humans. We propose to investigate the type, distribution, and main function of these innate immune cells in the human pancreas, by defining them based on their unique markers.

    By the end of this project, we expect to define the innate immune cell landscape and the changes that might take place as T1D progresses. Furthermore we will use specialized in vitro experiments, live cell- and 3D-imaging as well as super resolution microscopy, and transcriptomic and proteomic assays to study islet-immune cell interactions and beta cell function. Our goal is to better define the role of innate immune cells in the pathogenesis of T1D by acquiring quantitative data from human pancreata. In addition, we aim to obtain mechanistic information from in vitro experiments to understand how innate immune cells interact with beta cells. This will allow us to identify new “players” in the pathogenesis of T1D that will serve as targets for pharmacological intervention, bringing us closer to the prevention and cure of T1D.

  • Andrea Schietinger

    Memorial Sloan Kettering Cancer Center
    Funded by: NIDDK
    Decoding and Reprogramming Stem-Like Progenitor CD8 T cells Driving Type 1 Diabetes

    The pathogenesis of T1D is complex and involves many distinct immune populations. Autoreactive CD8 T cells play a major role as the ultimate mediators of beta cell destruction in the pancreas, leading to the loss of glucose homeostasis. However, many aspects of the programming and regulation of CD8 T cells mediating autoimmunity remain enigmatic. Utilizing the clinically relevant non-obese diabetic (NOD) mouse model of T1D, we identified two phenotypically and transcriptionally distinct beta cell-specific CD8 T cell populations in the pancreatic lymph node and pancreas with distinct functional roles in driving T1D in the NOD mouse. We propose to use T1D patient samples to investigate whether similar beta cell-specific CD8 T cell populations play a role in human disease using a multidimensional approach including multi-parameter flow cytometric phenotypic analyses, bulk and single-cell RNA-SEQ (and if enough material available also ATAC-SEQ). These studies will provide crucial insights into the little-understood programming and transcriptional regulation of CD8 T cells driving beta cell destruction and could identify novel targets for the treatment or prevention of T1D.

  • Mia Smith & Peter Gottlieb

    University of Colorado
    Mapping islet B cells in the pancreas on a single-cell level

    Recent evidence suggests a role for B cells in the pathogenesis of type 1 diabetes (T1D), particularly in individuals who develop T1D at a younger age and demonstrate rapid progression. However, little is known regarding the specificity, phenotype, and function of B cells in young-onset T1D. Recently we performed a cross-sectional analysis comparing insulin-reactive to tetanus-reactive B cells in the blood of newly-diagnosed T1D and controls using mass cytometry. Unsupervised clustering revealed the existence of a highly activated B cell subset, we term BND2, that falls within the previously defined anergic (unresponsive) BND subset. We found a specific increase in the frequency of insulin-reactive BND2 cells in the blood of young-onset T1D donors, which was further enriched in the pancreatic lymph nodes of T1D donors. We demonstrated BND2 cells are pre-plasma cells and can likely act as antigen-presenting cells to T cells. These findings suggest that activation of insulin-reactive anergic B cells likely plays a role in the rapid progression of young-onset T1D. Moreover, similar to previous findings by the Noel Morgan group, we have also identified an increase in number of B cells in the pancreas of young-onset T1D donors compared to older onset donors. However, it remains unknown what the phenotype and function of these B cells are within the pancreas. In this proposal, we will analyze the phenotype of total B cells, including whether BND2 cells are present in the pancreas, their activation status, location in relation to other immune cells, and islet health in the pancreas of young-onset versus older-onset T1D and non-diabetic organ donors using the high dimensional imaging platform, Multiplex Ion Beam Imaging (MIBI). The potential impact of these studies lies in identification of the pathogenic B cell(s) responsible for the rapid progression of disease, which will inform our understanding of the aggressiveness of young onset T1D and increase the precision of future age appropriate therapeutics.

  • Luc Teyton

    The Scripps Research Institute
    Funded by: NIH/NIAID, NIH/NIDDK
    Analysis of Immunocytes in Human Islets of Type 1 Diabetes Patients

    CD4+, CD8+ T cells and B cells will be isolated from islets of patients suffering from type 1 diabetes. Laser dissection will be used to localize and isolate single cells after tissue staining and confocal imaging at high resolution. T cell receptors and B cell receptors will be isolated from single cells and expressed to characterize antigen specificity. T cells will also be characterized for their expression profiles of cytokines, chemokines, co-receptors, and transcription factors. These studies are aimed at discovering important new antigenic targets in type 1 diabetes and understanding the pathogenic process leading to T1D.

  • Hubert Tse

    University of Alabama-Birmingham
    Funded by: NIH/NIDDK
    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 beta-cells. Infiltrating leukocytes generate an inflammatory environment consisting of reactive oxygen species (ROS) and proinflammatory cytokines that collectively participate in beta-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 beta-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.

  • Hubert Tse

    University of Alabama-Birmingham
    Funded by: NIH/NIDDK
    Analysis of innate immune response of myeloid cells in human Type 1 diabetes

    Type 1 diabetes (T1D) is a multifactorial autoimmune disease that requires genetic susceptibility as well as environmental triggers to induce disease onset. Genetic association studies have implicated the IFIH1 gene, encoding for melanoma differentiation-associated protein 5 (MDA5), a cytosolic sensor of dsRNA, to be involved in T1D susceptibility and resistance. Stimulation of the MDA5 signaling pathway activates the transcription factors IRF3 and NF-κB p65, which induce Type I IFNs synthesis, which through autocrine signaling initiate an antiviral transcriptional program. Single nucleotide polymorphisms (SNPs) in the IFIH1 gene such as rs1990760 and rs3747517, which results in a non-synonymous mutation that changes alanine at position 946 to a threonine and histidine at position 843 to arginine, respectively, is highly associated with increased risk for T1D. Studies in human PBMCs and mice have demonstrated that the A946T SNP results in an increased sensitivity to viral ligands and subsequently a stronger downstream IFN response. Our central hypothesis is that T1D-associated SNPs result in an exacerbated islet-resident macrophage and dendritic cell immune response that result in an increase in Type I IFNs which contributes to the initiation of autoimmunity. This will be tested by using spatial transcriptomics and proteomics of paraffin- and OCT- embedded section and pancreas slices from healthy donors, patients with autoantibody positivity, and patients with T1D that are genotyped for IFIH1 SNPs that contribute to T1D susceptibility.

  • Johnna Wesley, Matthias von Herrath

    Novo Nordisk Research Center
    Characterizing T-Cell Populations in Diabetes

    This study seeks to understand the immune cells important for beta cell loss by studying the function and type of cells present in lymphoid tissue.

  • Defu Zeng & Alberto Pugliese

    City of Hope
    Funded by: DMRI-COH
    Role of p21-activated secretory phenotype (PASP) in initiating type 1 diabetes pathogenesis

    Type 1 diabetes (T1D) results from autoimmune insulitis destruction of insulin-secreting β cells. β cell senescence with upregulated expression of P21 has been observed in the adult NOD mice and new-onset patients. However, it remains unclear whether the beta cell senescence is an outcome of persistent insulitis, and the factors trigger insulitis remains unclear.

    The initial infiltration in the islets of 3-week-old infant NOD mice was predominantly Mθ, but the mechanisms for the initial Mθ infiltration remain unclear. A recent report (Sturmlechner et al., Science2021) demonstrate that P21 activation not only results in cell-cycle arrest but also activate a large collection of genes that encoding chemokines (i.e., CXCL14) and cytokines (i.e., IL-6), called P21-activated secretory phenotype (PASP), which attract Mθ and other cell infiltration to exert immunosurveillance. It is well recognized that abnormal immune surveillance could lead to autoimmune diseases, although the mechanisms remain largely unknown. Our preliminary data with RNA-seq analysis and in vitro culture of CD45+ hematopoietic stem cell-derived immunocytes and CD45- parenchymal stem cell-derived fibroblast reticular cells (FRCs) and β cells from 3-week-old infant NOD, insulitis-resistant NOR, and non-autoimmune C57BL/6 (B6) mice indicate that exaggerated P21-activated PASP in fibroblast reticular cells (FRCs) and immunocytes (i.e., macrophages) intiates peri-insulitis and insulitis, leading to β cell senescence and destruction and full blown T1D.

    Imaging mass cytometry (IMC), a powerful novel technology, allows us to analyze in situ tissue cells with more than 30 markers on the cell surface, intracellularly, and in the nucleus, and can identify cell subsets with different intracellular cytokine and chemokine profiles as well as intracellular signaling pathways and spatial cell distribution. We have developed IMC techniques in our lab and have applied IMC to study of GVHD target tissues (Kong et al: Blood, 2013) and pancreatic islet cellular analysis of murine and human islets (see preliminary data). In the current studies, we will analyze murine and human islets with IMC in couple with other functional studies to test whether exaggerated P21-activated PASP exists in the islets of new-onset T1D patients. If successful, the proposed studies will help establish a paradigm-shift concept of T1D pathogenesis.

  • Li Zhang

    Indiana University
    To identify T1D epitope using highly specific autoantibodies

    DQ8/InsB:R3 was identified as an antigen by functional analysis of reactive T cells from peripheral blood, but direct evidence of antigenicity in islets is missing. The hypothesis is that the endogenous DQ8/InsB:R3 appear at the core organs of T1D in a disease stage-related timing. CODEX is a highly multiplexed tissue imaging platform enabling >50 markers to be simultaneously analyzed in single cells. We will use the CODEX platform to measure the expression of DQ8/InsB:R3 in different antigen-presenting cells, analyze the association of antigen with HLA-DQ and islet beta cells in the pancreas and pancreatic lymph nodes. Expression of DQ8/InsB:R3 only in patients and AAI+ individuals strongly support its pathogenic role in the development of T1D.

  • Todd Zion, Thomas Lancaster & Thillai Sathiyaseelan

    Akston Biosciences
    Targeting Islet-Antigen Specific B Cells for Deletion as a Means to Prevent Type 1 Diabetes

    Type 1 Diabetes (T1D) is an autoimmune disease in which the body attacks and destroys its own insulin-producing β-cells. It affects over 3 million people in the United States. For most T1D patients, it is extremely difficult to achieve normal glycemic control even with frequent blood glucose measurements, diet control, careful determination of insulin dosage, and multiple daily insulin injections. Considering the numerous approaches to reduce or eliminate the physical, emotional, psychological and economic hardships of T1D, arresting or reversing the disease before β-cell destruction is the most ideal but difficult solution to implement.

    A number of proposed therapeutics have focused on targeting and destroying the T-cells that are ultimately responsible for attacking and killing β-cells. However, in recent years a pathogenic role for B cells has also emerged. B cells can take up islet antigens, present them to helper T cells, and differentiate into antibody secreting plasma cells which enhance antigen uptake by antigen-presenting cells ultimately leading to the activation of cytotoxic T cells for β-cell destruction. A number of non-obese (NOD) diabetic mouse studies and at least one human clinical trial have demonstrated the ability to slow, arrest, or even reverse T1D through B-cell depletion. However, generalized B-cell depletion may lead to significant off-target safety issues thus restricting the allowable treatment duration and limiting its application to a small subpopulation of T1D patients for whom the risk is justified based on the anticipated benefits. An ideal product and treatment regimen would destroy only T1D specific Bcells while sparing the healthy B-cells. The development of just such a product, targeting insulin-specific B cells, is the focus of Akston Biosciences.

Novel Biomarkers

  • Mario Galgani

    Consiglio Nazionale delle Ricerche
    CD3+CD56+ T lymphocytes, as novel T1D biomarker

    Type 1 Diabetes (T1D) is a disease characterized by a lymphocyte-mediated destruction of insulin-producing beta-cells in genetically susceptible individuals. It has been demonstrated that lymphocytes with regulatory properties are essential for the maintenance of tolerance against self-tissues (pancreas) and alterations in the number and functions of these cells have been correlated with the break of immune tolerance.

    We have recently shown that the frequency, in the blood, of a lymphocyte population co-expressing CD56 and CD3 molecules (here defined as CD3+CD56+ cells), represents a valuable predictive marker of T1D progression (Galgani M et al. 2013). Preliminary data show that the absolute number and percentage of CD3+CD56+ cells are significantly reduced in the blood of a large cohort of new-onset T1D subjects compared with sex and age-related healthy individuals. In addition, the frequency of CD3+CD56+ lymphocytes in the blood positively correlated with good glycaemic control and fasting C- peptide in recent-onset T1D children (see later). Finally, additional studies have been showing that CD3+CD56+ cells, isolated from healthy controls, suppress the activity of triggered lymphocytes involved in pancreas damage; of note, this activity was impaired in CD3+CD56+ cells isolated from the blood of T1D subjects.

    We assume that number and function of CD3+CD56+ cells may serve as biological markers to monitor disease progression, in autoimmune diabetes. Functional defects of this “regulatory” cell subset could account for the exaggerate function of lymphocytes in T1D. Thus, the results of this proposal may pave the way to the better characterization of a novel cell population involved in the T1D pathogenesis, likely representing a useful biomarker potentially implicated in the control of the immune tolerance.

  • Emma Hamilton-Williams

    University of Queensland
    Funded by: Breakthrough T1D
    Meta – proteomic biomarkers in stool for prediction of islet autoimmunity progression

    Type 1 diabetes (T1D) is increasing around the world due to unknown changes in our lifestyle. One factor that may drive this change is the bacteria in our intestines. These bacteria interact potently with our immune system and metabolism. We have shown that changes in intestinal and pancreatic proteins found in stool are associated with risk of T1D development. By integrating these data with microbial community profiling, we have shown that host proteins linked to gut barrier function and exocrine pancreas function are associated with changes in specific gut microbes. Now we plan to investigate whether changes in proteins found in stoolare associated with inflammation present in either the intestine or pancreas of individuals with islet autoimmunity.

  • Mark Huising

    University of California, Davis
    Funded by: Breakthrough T1D
    Urocortin 3 as a sensitive marker for beta cell function

    Urocortin 3 (Ucn3) is a neuropeptide that was discovered by our group (Lewis et al., 2001). Ucn3 is specifically expressed in mouse beta cells, but is in human islets also expressed by alpha cells (Li et al., 2003; van der Meulen et al., 2012). Ucn3 is a member of the same neuropeptide family as corticotropin-releasing factor (CRF) and both peptides activate related receptors; CRF predominantly activates the type 1 CRF receptor, while Ucn3 exclusively activates the type 2 CRF receptor. We have in recent years discovered that CRF and Ucn 3 both contribute to the regulation of insulin secretion and the maintenance of functional beta cell mass by virtue of locally expressed CRF receptors in mouse and human islets (Huising et al., 2010, 2011). Interestingly, Ucn3 is not detectable in the developing mouse pancreas until embryonic day 17.5 and is not expressed by all beta cells until postnatal day 14 (van der Meulen et al., 2012). This is considerably later than the onset of expression of transcription factors that mark mature beta cells, such as Pdx1, Nkx6.1 and MafA as well as markers of functional beta cell maturity, including Pcks1, Znt8 (Scl30a8) or Glut2 (Scl2a2; in mouse).

    In line with findings in mouse pancreas, Ucn3 expression in human embryonic pancreas is not detectable until the end of the first trimester and trails the onset of expression of insulin and glucagon by several weeks (van der Meulen et al., 2013). However, in marked contrast to murine pancreas, where Ucn3 demonstrates exquisite selectivity for the beta cells, Ucn3 in both human and macaque (Macaca nemestrina) pancreas and in freshly isolated human donor islets is expressed in both beta and alpha cells (van der Meulen et al., 2012). Moreover, and perhaps to emphasize the species differences that exist regarding the local network of CRF-family neuropeptides, CRF expression is selective for human alpha cells and is not detected in mouse islets. In agreement to the above observations, Ucn3 in human embryonic stem cell-derived pancreatic endocrine cells is expressed relatively late and only after an extensive period of in vivo maturation in recipient mice and is then detectable in both mature beta and alpha cells (van der Meulen et al., 2012). These findings have established Ucn3 as a marker of beta cell maturity. We therefore hypothesize that Ucn3 is a biomarker of functional, mature beta cells, and is down regulated during diabetic conditions that are associated with beta cell dysfunction and dedifferentiation. Supported by nPOD, we will establish if Ucn3 expression is reduced in alpha or beta cells of T1D and T2D donors compared to non-diabetic controls.

  • Daniel Kaufman

    University of California, Los Angeles
    High-resolution mapping of the human pancreatic magnetic resonance landscape

    Magnetic resonance imaging (MRI) and magnetic resonance spectroscopy (MRS) both detect signals from atomic nuclei and can be performed together in conventional clinical MRI scanners. MRI provides the anatomical information, after which MRS scanning can provide biochemical information on the constituents within a region of interest. MRS has been used in clinical settings to study metabolic changes within breast and prostate tumors, as well as changes in small molecules in the brain associated with seizures, stroke, Alzheimer’s, and Parkinson’s disease. This technology has not yet been extended to the analysis of pancreatic small molecules in healthy and diabetic states. As an initial step in this direction, we request nPOD human pancreatic samples from healthy and T2D and T1D diabetic individuals in order to study them ex vivo by high-resolution MRS (HR-MRS). This will provide a new read-out of pancreatic small molecule constituents in healthy versus diabetic states. The results will provide insights into the disease processes and complement data gathered by other technologies. Importantly, the results will identify potential clinical MRS biomarkers of ß-cell health. Future clinical MRS studies can then focus on the candidate biomarkers using MRI pulse sequences or data editing programs that will enhance the signals from candidate biomarkers. Thus, our proposed studies will provide basic information on pancreatic constituents in health and disease states, and will lay the foundation for developing MRS as a noninvasive tool to monitor ß-cell health in the clinic.

  • Thomas Linn & Sebastian Petry

    Justus Liebig University
    Redoxins in the pathogenesis of type 1 diabetes

    We plan to quantify redoxin expression levels and the frequency and type of redoxin expressing pancreatic cells by imaging procedures established in our group. For this purpose we propose a pilot study with samples from #3 cases ideally with rapid (pre-type 1 or type 1 diabetes) or more chronic (type 2 diabetes) ongoing beta cell destruction process. To these samples we will apply well-established antibodies that were tested in our cell based models and non-diabetic human pancreas before. Thereby we will be able to identify the most obvious changes of expression of redoxins of interest with a focus on iron-sulphur containing clusters, such as glutaredoxins for the first time in human pathology. We will generate hypotheses from the collected results to perform power calculations and figure out if and how many samples are required for confirmation. Moreover, we will search for prominent associations of our findings to the medical history of the subjects, specifically to insulin reserve measured by C-peptide blood or urine concentrations whatever available.

    At our place of research Giessen, Germany a human islet transplantation program was performed for over 20 years, but it was halted due to changes of national transplantation laws with the result of reduced availability of pancreas organ donors. From this period we have continuously collected and restored pancreas samples both embedded in paraffin and frozen from non-diabetic donors.

  • Roberto Mallone

    INSERM Cochin Institute
    Identification and characterization of Coxsackievirus-reactive CD8+ T cells in pancreatic lymph nodes

    Despite accumulating epidemiological and histopathological evidence for an association between Coxsackievirus B (CVB) and type 1 diabetes (T1D), a causal link is missing. Very little is known about the T-cell response mounted against CVB infection and the epitopes targeted. Moreover, the ongoing CVB vaccination trials require: 1) the development of T-cell biomarkers to follow vaccination efficacy; and 2) the exploration of potential cross-reactivities with islet antigens that may precipitate rather than prevent T1D. We identified the HLA Class I viral peptidome of CVB-infected β cells, pinpointed a highly focused response toward very few immunodominant epitopes recognized by CD8+ T cells, and the functional features of these T cells, e.g. their capacity to destroy infected beta cells.

    We now aim to investigate whether these CD8+ T cells recognizing CVB peptides are also found in the pancreatic tissues targeted by islet autoimmunity, and whether they are enriched in these tissues as compared to peripheral ones. Such enrichment would lend support to the role of CVB infection in triggering islet autoimmunity.

Novel Technologies

  • Bernd Bodenmiller, Mark Atkinson, Harry Nick, & Clive Wasserfall

    University of Zurich
    Highly Multiplexed Imaging Mass Cytometry of the Human Pancreas Affected by Type 1 Diabetes

    Despite extensive efforts, understanding of the mechanisms of type 1 diabetes (T1D) development in human patients is collectively lacking, particularly with regard to contributions of islet endocrine cells, the immune system, and their interplay in the disorder’s development. Until recently, these questions have been particularly challenging to address due to technical limitations in the number of analytes that can be simultaneously measured from a single tissue section (i.e., typically four to six markers per section by immunohistochemistry or immunofluorescence). To address this, we have established Highly Multiplexed Imaging (HMI) methodology based on CyTOF mass cytometry to examine human pancreas samples from organ donors with or at varying levels of risk for T1D as well as control donors. We hypothesize that imaging mass cytometry (IMC) characterization of islet and immune cells in the human pancreas will provide novel clues regarding: 1) the impact of pre-T1D and T1D conditions on islet cell phenotypes and 2) the local intra-islet—immune interface throughout disease progression. Specifically, metal isotope-tagged antibodies serve as reporters allowing for unprecedented multi-parameter measurements of single cells, now in the three-dimensional (3D) tissue microenvironment. At the RNA, protein and protein modification level, 30-100 markers expressed by islet and immune cells can be simultaneously analyzed from human pancreas sections at single-cell resolution in 3D across the lifespan to identify “normal” islet development, islet plasticity, cell reprograming, and pathology throughout T1D disease staging. In this study, we are examining: 1) islet endocrine constituents (i.e., alpha, beta, and delta) to potentially identify reprograming cells or to discover novel subsets within these known cell types; 2) cellular signaling networks in endocrine, acinar, and immune cells; and 3) their interactions at various stages throughout the natural history of T1D. Indeed, we have developed the histoCAT 3D reconstruction toolbox to generate 3D data from serial tissue sections with subcellular (i.e., pixel) resolution. This work is revolutionizing our ability to examine the human pancreas in health and disease and has facilitated new avenues of research with new research questions that were previously impossible. We anticipate that we will identify novel pathways disrupted in human T1D, the cellular and molecular networks altered during disease pathogenesis, and new disease-defining/staging characteristics, each of which may represent a potential target for the development of novel therapies aimed at preventing or halting the disease progression.

  • Shalev Itzkovitz

    Weizmann Institute of Science, Israel
    Transcriptional heterogeneity in the intact human pancreas

    Pancreatic beta cells have been shown to be heterogeneous at multiple levels. However, spatially interrogating transcriptional heterogeneity in the intact tissue has been challenging. We developed an optimized protocol for single-molecule transcript imaging in the intact pancreas and used it to identify a sub-population of “extreme” beta cells with elevated mRNA levels of insulin and other secretory genes in mice. Extreme beta cells contain higher ribosomal and proinsulin content but lower levels of insulin protein in fasted states, suggesting they may be tuned for basal insulin secretion. They exhibit a distinctive intra-cellular polarization pattern, with elevated mRNA concentrations in an apical ER- enriched compartment, distinct from the localization of nascent and mature proteins. The proportion of extreme cells increases in type 2 diabetic db/db mice, potentially facilitating the required increase in basal insulin. Our results thus highlight a sub-population of beta cells that may carry distinct functional roles along physiological and pathological timescales. We would like to apply our new technology of smFISH in the intact human pancreas to assess whether similar heterogeneity of extreme and non-extreme beta cells exists in human.

  • Klaus Kaestner

    University of Pennsylvania
    Funded by: NIH/NIDDK
    Imaging Mass Cytometry for the Analysis of the Human Diabetic Pancreas

    By integrating systematic views of the transcription, translation machinery at single-cell level, we intend to gain insights into the molecular changes in diabetes and in aging and provide the research community with useful database resources.

  • Sally Kent

    University of Massachusetts Chan Medical School
    Funded by: NIDDK/NIAID
    De-differentiation of cryopreserved splenocytes to iPSC as an isogenic resource for differentiation to sc-islets

    In collaboration with the New York Stem Cell Foundation (NYSCF, see letter of collaboration), we will attempt de-differentiation of cryopreserved splenocytes to iPSC: this has not been attempted before to our knowledge. If successful, these iPSC may be differentiated into sc-islets and serve as an isogenic sources of sc-islets for interactions with the islet-derived T cell lines the Kent lab has generated and banked. If successful, we will submit a full addendum to this project for the study of the interaction of isogenic iPSC-derived sc-islets and autologous islet-derived T cells. This technology may be used by other investigators in their examination of the isogenic interaction of sc-islets and immune cells.

  • Sarah Kim & Martha Campbell-Thompson

    University of Florida
    Funded by: Breakthrough T1D & UF Informatics Institute
    Deep Learning-based Analyses of Pancreatic Islet Beta-Cell Heterogeneity and MRI Pancreas Volume as Biomarkers to Improve Understanding of Type 1 Diabetes Progression

    The overall objective of this project is to develop a deep learning based imaging analysis and informatics tool for better understanding of pathogenic mechanisms in T1D, using existing data which has been collected for 14 years by Network for Pancreatic Organ Donors with Diabetes (nPOD) study. The central hypothesis of this proposal is that modifications of the pancreatic islets due to loss of insulin-producing β-cells and infiltration of islets by CD3+T cells during the progression to T1D progression can be characterized and their heterogeneity can be quantified in pancreas whole-slide digital pathology images. Ultimately, our results will help in the rational design of T1D prevention and treatment strategies.

  • Chantal Mathieu

    KU Leuven
    Age-related heterogeneity of type 1 diabetes: a multi-omics, spatially resolved, exploration of the human pancreas at disease onset

    Type 1 diabetes (T1D) is a chronic autoimmune disease long thought to primarily affect children and adolescents. However, recent data suggest that nearly 70 percent of new T1D cases occur in adults. Striking differences in genetic, metabolic, and immune features as well as in diabetes management have been described between childhood- and adult-onset T1D, but many of these variances are poorly understood. An improved understanding of childhood- and adult-onset T1D heterogeneity is essential to provide optimal diabetes care but in addition, for designing novel disease-modifying therapies to prevent or arrest ongoing beta cell autoimmunity. I propose to implement the most advanced high-plex in situ assay platforms to study, at gene and protein level, the spatial context and interaction of cellular subtypes in pancreatic tissues obtained from childhood- and adult-onset T1D cases. Integrated data analyses will not only provide novel insights into precise cellular localizations but also identify cellular differentiation / activation states and neighborhoods. This will provide the best map yet of what is occurring in the pancreas at disease onset and how this differs between children and adults. Such knowledge is key to developing new treatments to cure T1D and to better define optimal treatment regimens.

  • Teresa Rodriguez-Calvo & Matthias Mann

    Helmholtz Zentrum München & Max-Planck-Institut of Biochemistry
    Unbiased single cell resolution spatial proteomics: defining the antigenic landscape in the context of HLA-I expression during type 1 diabetes progression

    Diabetes is a complex metabolic disease in which patients experience high blood glucose levels leading to a plethora of symptoms and long-term complications. In type 1 diabetes (T1D), this happens likely due to an autoimmune reaction targeting the insulin producing cell population, so-called beta cells, within the pancreatic islets. CD8+ T-cells are considered the main cell type implicated in beta cell destruction. However, their phenotype, recognized antigens, as well as how they interact with beta cells is still rather unknown. During disease progression, islets with a heightened expression of HLA class I (HLA-I) proteins can be observed. This feature is usually detected in all cells within one islet but not in all islets within one donor. The increased expression of HLA-I molecules on the surface of islet cells, and especially of beta cells, could be a precipitating factor and actively contribute to the presentation of self-antigens to the immune system. Based on this, we propose to investigate the differences at the proteomic level between islets with low, medium and high expression of HLA-I molecules on a cell-type resolved basis (in different cell types) using our recently published method Deep Visual Proteomics (DVP). This platform combines artificial-intelligence-driven image analysis of cellular phenotypes with automated single-cell or single-nucleus laser microdissection and ultrahigh-sensitivity mass spectrometry. Our goal is to analyze the proteomes of single-cell populations within their spatial context to provide answers regarding the significance of HLA-I upregulation with respect to disease etiology, as well as the proteomic profile of different islet cells during disease progression.

  • Maike Sander & Chun Zeng

    Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Germany
    Funded by: NIH-NIDDK (HIRN)
    Single cell analysis of the human pancreas in type 1 diabetes

    Type 1 diabetes (T1D) is an autoimmune disorder that affects 1.25 million individuals in the US and for which there is no known cause or cure. T1D is characterized by autoimmune destruction of insulin- producing beta cells in the pancreatic islets. The pancreas consists of multiple different cell-types relevant to diabetes pathogenesis including endocrine (beta, alpha, delta, gamma, and epsilon), exocrine (ductal and acinar), immune (macrophage, dendritic, T cells, and others), and endothelial cells. In addition, there is evidence for further cellular heterogeneity within individual cell types. Each cell- type has distinct functions in pancreas biology, yet the complete repertoire of pancreatic cell-types in non-diabetic and diabetic individuals, and the regulatory programs that define their identity and their roles in diabetes pathogenesis are unknown. Technological advances have enabled profiling chromatin and gene expression in individual cells which can reveal regulatory programs in specific cell types to facilitate understanding their role in disease. For this project we will adapt cutting edge single-cell methods on nPOD frozen samples from non-diabetic and diabetic donors to define cell-types and identify mechanisms of disease promoting cross-talk between cells.

Pathology

  • Joana Almaça

    University of Miami
    Funded by: NIH-NIDDK (HIRN)
    Changes in human islet microvasculature during the progression of type 1 diabetes

    Endocrine cells in pancreatic islets are embedded in a network of microvessels that regulate islet blood flow and hormone secretion. It is still unknown what happens to the islet microvasculature in type 1 diabetes (T1D), despite being the site of immune cell recruitment and infiltration. The microvasculature consists of capillary tubes made of a thin layer of endothelial cells covered by pericytes. Pericytes are the mural cells of the microcirculation. They are crucial for proper microvascular function, by giving structural stability, participating in angiogenesis, controlling vascular permeability and blood flow. While a wide body of literature exists on islet endothelial cells, the function of the islet pericyte is largely unknown. The aim of this research project is to look at pericytes in islets from non-diabetic donors as well as in individuals at different stages of type 1 diabetes. My hypothesis is that loss of pericyte coverage of capillaries leads to microvascular dysfunction and immune cell infiltration, contributing to the development of T1D. I also plan to assess whether pericytes control human islet capillary diameter, similarly to their role in mouse islets.

  • Joana Almaça

    University of Miami
    Changes in pericyte phenotype and function during type 1 diabetes

    Endocrine cells in pancreatic islets are embedded in a network of blood vessels that support adequate nutrient sensing, efficient release of hormones, and timely responses to changes in glycemia. This network of islet blood vessels consists of capillary tubes made of endothelial cells that are covered by pericytes. Compelling evidence indicates that significant structural alterations of islet blood vessels occur in individuals with T1D. These include not only changes in blood vessel diameter and density, altered expression of adhesion molecules by endothelial cells, but also perivascular alterations such as increased infiltration of immune cells, and altered composition of the extracellular matrix. Pericytes are part of the pancreatic islet vascular niche but it is not known what happens to them during T1D progression. The aim of this research project is to investigate pericytes and examine their phenotype and response profile in islets from non-diabetic donors as well as in individuals at different stages of T1D. Based on recent preliminary data obtained using nPOD material (under the parent project; see below), our hypothesis is that islet pericyte dysfunction early on in the disease impairs microvascular function, favoring immune cell infiltration and contributing to the development of T1D.

  • Alejandro Caicedo

    University of Miami
    Funded by: NIH/NIDDK
    The sensory innervation of the pancreatic islet in health and diabetes

    The role the nervous system plays in modulating islet inflammation and diabetes pathogenesis has barely been examined. Recent papers suggest that changes in sensory innervation initiate autoimmune diabetes in mice, which is in line with the notion that sensory neurons contribute to inflammation in several chronic disorders. Thus, the local interactions between the immune system and nerves in the islet may be crucial for the progression of diabetes. We propose that pathological activation of sensory nerves creates a chronic inflammatory milieu in the islet that ultimately leads to autoimmunity in susceptible individuals. Excessive activation of sensory nerves, as it may occur during hyperglycemic stress or infection with diabetogenic viruses, may lead to deranged secretion of neurotransmitters that alters vascular and macrophage functions, promoting inflammation. Studies, however, are limited by the difficulty to visualize and manipulate these processes in vivo and longitudinally in the hardly accessible pancreatic islets. Here we propose to identify the pattern of sensory innervation of the pancreatic islet in health and diabetes, using techniques our laboratory developed recently. At the successful completion, this project will provide fundamental knowledge about how the sensory innervation of the islet changes during diabetes progression. This in turn will provide the basis for functional and interventional studies to understand the role of sensory nerves in diabetes pathogenesis.

  • Nikki Farnsworth

    Colorado School of Mines
    Funded by: American Diabetes Association
    Changes in human islet microvasculature during the progression of type 1 diabetes

    The pancreatic islet is surrounded by an extracellular matrix (ECM) composed of laminin, collagen IV, and proteoglycans, that support islet function and viability. Type 1 diabetes (T1D) is characterized by the immunemediated loss of insulin-producing β-cells in the islet. Islet infiltration by activated immune cells results in the degradation of the peri-islet ECM eliminating critical signaling pathways for islet function and survival. The peri-islet ECM may provide a critical barrier to islet infiltration; however, the cellular and molecular contributions to loss of the peri-islet ECM are not well studied, limiting potential therapies targeted at this critical step in T1D. Furthermore, understanding the mechanisms underlying ECM regulation of islet function and how loss of these interactions affects β-cell survival in T1D is critical to understanding and halting T1D pathogenesis. The goal of this study is to determine the respective contributions of immune cells and β-cells to loss of the peri-islet ECM and determine how changes in the peri-islet ECM impact β-cell survival in the context of T1D. We propose 2 aims to achieve this goal: 1) Determine the specific contributions of CD4+ and CD8+ immune cells in degradation of the peri-islet ECM and loss of β-cell mass ECM in T1D, and 2) Determine the contributions of stressed β-cells in degradation of the peri-islet ECM and loss of β-cell mass ECM in T1D. Experiments for both aims will utilize human pancreas sections from the healthy controls, auto-antibody positive donors at risk of developing T1D, and donors with established T1D. The results from this project will define a new for changes in the peri-islet ECM in contributing to decreased islet survival in T1D and will provide novel therapeutic targets to halt immune infiltration of the islet and improve islet survival in T1D.

  • Rebecca Hull-Meichle & Sakeneh Zraika

    University of Alberta, Canada
    Funded by: Cystic Fibrosis Foundation, NIDDK Cystic Fibrosis Research Translation Center
    Islet Pathology in Cystic Fibrosis

    Insulin deficiency is a common feature of cystic fibrosis (CF), resulting in CF-related diabetes (CFRD) in ~20% of adolescents and ~50% of adults with the disease. The clinical consequences of insulin deficiency in CF are significant – CFRD is associated with worsened CF lung disease and increased morbidity and mortality. The loss of exocrine pancreatic tissue in CF is extensive, with its destruction being initiated around/before birth and resulting in pancreatic insufficiency in 85% of CF patients. In contrast, while most studies describe some loss of β cells, islets generally survive. This suggests that enhancing endogenous insulin secretion may be a viable therapeutic strategy in CF. However, the mechanisms underlying impaired insulin release in CF are poorly understood.

  • Marcia McInerney

    University of Toledo
    Insulin Receptor on T cells in T1D

    T cell movement into the islets in the pancreas, is a hallmark feature of Type 1 diabetes (T1D) that has been demonstrated on autopsy in human pancreas specimens and in mouse models of T1D such as the non-obese diabetic (NOD) mouse3-8. By determiningthe role that insulin receptor (IR)plays in this movement of the T cells may generate anew immunotherapeutic intervention for T1D, especially since chemotactic signaling and metabolic signaling are mediated by distinct parts of the IR9. Blocking IR mediated chemotaxis without affecting insulin binding and signaling would offer a new pharmacotherapeutic target, since it would effectively block T cell movement into the pancreas. Blocking T cell movement would stop or delay diabetes development in diabetes prone individuals and may allow for beta cell regeneration. Chemotaxis to the islets via IR expression is the hypothesis. To investigate this, we will examine T cells in human islets (control and T1D) for IR expression. 

  • Jonathan Sweedler

    University of Illinois at Urbana - Champaign
    Funded by: American Diabetes Association
    Understanding diabetes progression via tissue chemical imaging

    Type 1 diabetes (T1D) is a devastating multifactorial disorder manifested by the autoimmune destruction of insulin-producing beta cells leading to insulin deficiency. Our overarching aim is to investigate the spaciochemical changes in the human exocrine and endocrine pancreata during various stages of T1D with unmatched chemical specificity and multiplexity. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) will be used for multiplexed direct measurement of metabolites, lipids and peptides in control, no diabetes autoantibody positive and affected by clinically diagnosed T1D pancreata sections. Two groups of T1D affected donors will be investigated as well as no diabetes autoantibody positive group and compared to control group and group developed T2D. Donors will be similar to ones described in 1. The first will be those with less than one year since disorder diagnosis and the second with more than 7 years since diagnosis. C-peptide levels have to drop approximately 10 times for each group with most advanced T1D cases exhibiting lowest concentrations. We will perform a comparative analysis of the pancreas head and pancreas body regions which demonstrate different extent of damage during T1D progression.1 Also, for comparative analysis of malfunctioning pancreas biochemistry, we are requesting sections of late stage T2D-affected pancreata. This work will provide direct information on the multiplexed chemical heterogeneity and plasticity in the exocrine and endocrine tissues of the pancreatic head and body in health and disorder. This effort involves new and innovative measurement technologies including ones developed in collaboration with the nPOD2 to obtain information on metabolite, lipid, and peptides. Including their spatial distributions; these data cannot be easily obtained by other methods. This research will improve our understanding of the changes of tissue surrounding beta cell destruction in T1D by elucidating the chemical signals characteristic of T1D in comparison with control and T2D, and may help to identify novel factors and pathways for therapeutic intervention to treat this disease.

  • Bruce Verchere

    University of British Columbia
    Funded by: Breakthrough T1D/CIHR
    IAPP and innate immunity in T1D

    Islet amyloid polypeptide (IAPP) is a beta-cell peptide hormone that forms toxic and pro-inflammatory aggregates in pathological states, including T2D and islet transplants. Recent evidence suggests the presence of fibrillar aggregates of IAPP (islet amyloid) in some T1D pancreatic islets. In T2D and mouse
    models of islet amyloid formation, aggregates of human IAPP recruit and induce a pro-inflammatory phenotype in macrophages in pancreatic islets. Since islet macrophages may play a key role in triggering autoimmunity in T1D, it follows that IAPP aggregation in stressed islets could be a key initiating event in T1D, via induction of inflammation and islet macrophage activation. Here we will study nPOD pancreas sections for the presence of pre-fibrillar (oligomeric) IAPP aggregates and fibrillar amyloid, the association of IAPP aggregates/amyloid with islet macrophages, and the presence of markers of pro-inflammatory macrophage phenotype. The goal is to determine if induction of islet inflammation induced by IAPP aggregates may be an early event in T1D.

Type 1 Diabetes Etiology & Environment

  • Mark Atkinson, Irina Kusmartseva, Clive Wasserfall, Michael Haller, Martha Campbell-Thomspon, Bernd Bodenmiller, Stephan Speier & Alberto Pugliese

    University of Florida
    Analysis of major digestive enzymes expression in human pancreas

    Type I diabetes is characterized by significant loss of beta cells and decrease in pancreatic weight. It is widely accepted that decline of pancreatic weight is attributed to acinar atrophy which in turn could lead to pancreatic exocrine insufficiency frequently found in diabetic patients. However, the fact that the exocrine pancreas is affected in patients with T1D diabetes has often been overlooked. Thus, as a basis for a better understanding of pancreatic exocrine function, we propose to investigate the expression pattern of major digestive enzymes in the pancreas of nondiabetic, autoantibody positive without diabetes and T1 diabetes nPOD donors. This will be achieved by multiplex immunohistochemical staining for amylase, lipase and trypsinogen (amongst others) and by employing fresh tissue slicing technique to obtain functional readouts of pancreatic endocrine and exocrine cell function.

  • Clive Wasserfall, Mark Atkinson & Harry Nick

    University of Florida
    Role of Monogenic Diabetes, Integrated Stress Response, mTOR and NR4A genes in Type 1 Diabetes

    To date, over 60 loci have been identified, including the HLA and insulin regions and various single nucleotide polymorphisms (SNPs), conferring genetic susceptibility for type 1 diabetes (T1D); however, a number of these loci do not correspond to specific genes and as such, do not infer disease mechanisms or etiology. We have taken an innovative strategy to identify genes/biomarkers functionally and phenotypically associated with autoantibody positivity (AAB+, considered pre-T1D) and T1D, exploring early T1D pathogenic determinants in human pancreas. Our preliminary data demonstrate that genes associated with monogenic diabetes, which result from Mendelian inherited single gene mutations and are intrinsically non-redundant, display altered expression in pancreata from AAB+ and/or polygenic T1D. Using RTqPCR on total RNA from human pancreata from the Network for Pancreatic Organ donors with Diabetes (nPOD), we studied expression of 42 monogenic diabetes genes. We identified 19 genes induced in T1D versus control donor pancreata. 8 of the induced genes were functionally linked to the unfolded protein response (UPR): EIF2AK3 (PERK), head of one of three canonical UPR branches, exhibited the highest induction in T1D pancreata. Our data strongly argue that the T1D pancreas exhibits an integrated stress response (ISR), potentially in response to pathological challenges. Given that other stresses can activate the ISR, also regulating protein synthesis and cell viability, we will investigate pathways with logical overlap, namely those associated with mTOR (mammalian target of rapamycin) and NR4A (nuclear orphan receptor) and these test the hypothesis that differential gene expression in human pancreas and blood represent prognostic, diagnostic and therapeutic biomarkers/targets in T1D or pre-T1D.

     

  • Knut Dahl-Jorgensen & Lars Krogvold

    Oslo University Hospital & University of Oslo
    Funded by: European Union's Seventh Framework Programme
    Diabetes Virus Detection Study (DiViD)

    The Diabetes Virus Detection Study (DiViD) is the first to examine fresh pancreatic tissue at the diagnosis of type 1 diabetes (T1D) for the presence of viruses. Surgical minimal pancreatic tail resection was performed in general anesthesia 3-9 weeks after onset of type 1 diabetes in six adult patients (age 24-35 years). Pancreatic tissues were snap-frozen within three minutes after dissection, live tissues collected for functional studies, and tissues fixated with formalin and glutaraldehyde and stored. Duodenal biopsies were collected by gastroscopy. Serum, PBMCs and serum/plasma were also collected. All patients had HLA-risk for T1D and at least one positive autoantibody. All six cases fulfilled the criteria for insulitis (5-58% of the insulin-containing islets in the six patients were affected by ≥15 T cells). We demonstrated a low-grade, persistent enterovirus infection in the pancreatic islets of Langerhans. Both viral genome and viral capsid protein was detected and confirmed in different laboratories. We also observed a reserve reservoir of betacells in the islets that improved insulin secretion in a non-diabetogenic environment in vitro. This reservoir may restore the need for insulin if the disease process could be halted at the time of diagnosis. Recent gene expression studies of laser-captured pancreatic islets from the same DiViD patients show significant enrichment of gene ontologies for viral and reproduction/infectious cycle, and also significant mitochondrial dysfunction and increased cellular endoplasmatic reticulum (ER) stress. Pancreas samples from the nPOD program, recovered from organ donors, will be used as a control group together with the data generated from the biopsy of the patients from the DiViD study.

  • Dirk Homann & Mihaela Stefan

    Icahn School of Medicine at Mount Sinai
    Role of environmental chemicals in the development of type 1 diabetes

    There is a well-documented rise in the incidence of type 1 diabetes (T1D) in industrialized countries suggesting a role for environmental chemical exposures. Alarmingly, the increased incidence is seen mostly in children under age 5. However, research on environmental chemical exposures and T1D is very limited and there is an urgent need for research to fill this gap in our knowledge. Therefore, the goal of this proposal is to identify environmental pollutants that play a role in the increased incidence of T1D. We hypothesize that in the first years of life in T1D genetically susceptible children certain pollutants trigger damages specific to beta-cells making them more susceptible to the subsequent autoimmune attack. Of particular importance is the possibility that chemicals can directly affect beta cell physiology and thus may trigger initial insults precipitating the activation of self-reactive T-cells. Therefore, we have adapted a novel multi-chemical tissue mapping technology to study chemical quantitative distributions within the pancreatic tissues from T1D patients and healthy controls. Quantitative assessment of distribution of toxic and essential metals can reveal adverse (and protective) effects that occur at the cellular levels and may be overlooked in studies measuring average concentrations in homogenized pancreatic tissue. Laser ablation-based quantitative imaging provides a powerful tool to study the role of environmental toxicants and essential elements in T1D.

  • Heikki Hyoty

    University of Tampere, Finland
    Funded by: Breakthrough T1D
    Virus detection in pancreas and other tissues

    Enterovirus infections cause pancreatitis and damage endocrine cells in the pancreatic islets. Epidemiological studies have shown an association between enterovirus infections and type 1 diabetes, and viral structures have been detected in the pancreas of diabetic patients. Our recent epidemiological studies have suggested that among all different enterovirus types, the group of coxsackie B viruses is linked to the initiation of beta-cell autoimmunity and development of type 1 diabetes.  However, it is not known if this association is causal, and further studies are needed to address this important question. Current evidence suggest that enteroviruses can establish a slowly replicating persisting infection in the pancreas. In such case, the virus should be detectable by highly sensitive assays in the pancreas of diabetic and prediabetic patients. Our aim is to develop such assays, validate them in collaboration with other nPOD-V investigators and screen nPOD tissues for enteroviruses using the most optimal test panel.

    Our primary aims include the development of different histological methods, as well as PCR-based techniques, for enterovirus detection in various samples. These techniques are utilized in detecting enteroviruses in various tissue samples – such as pancreas, duodenum and spleen – of T1D, pre-diabetic and non-diabetic donors, and further in comparing the prevalence of EV infections between different donor groups, as well as in different tissues. We are applying three main methods to accomplish our goals:

    1. Immunohistochemistry (IHC) with a wide panel of both commercial and in-house antibodies suitable for FFPE and frozen samples.
    2. Standardized in situ hybridization (ISH) assay suitable for FFPE and frozen samples. We are using novel probes for both general and serotype-specific detection of enteroviruses.
    3. Highly sensitive RT-PCR method for the detection of viral RNA and for the sequencing of viral genome.

    Our studies have shown that these markers of enterovirus infection are more prevalent in type 1 diabetic and pre-diabetic subjects than in non-diabetic controls, and that the presence of enterovirus in pancreas correlates with that in other tissues.

  • Pia Leete & Joanna Boldison

    University of Exeter Medical School
    Funded by: RD Lawrence Fellowship Diabetes UK & Leona M. and Hary B. Helmsley Charitable Trust
    The Heterogeneity of Type 1 Diabetes: An Islets Story

    When a person is diagnosed with Type 1 diabetes (T1D), the amount of betacell mass each individual loses is highly variable despite their clinical need for insulin therapy. Attempts to detect robust markers in the blood that precisely describe disease activity in the islets are ongoing, but without a full understanding of the disease course in the pancreas, when attempting to target therapy to the organ, we are still operating partially blindfolded. We have previously reported that two forms, or endotypes, of T1D are evident in the pancreas[14]. These differ according to the degree of betacell loss, the composition of the isletspecific immune infiltrate, and how beta cells process insulin. Additionally, we now have preliminary data that suggests the betacells themselves may vary between the two forms), even hinting that successful betacell regeneration may be more likely in one form than the other. Our proposed study will expand on these investigations, and we will compare pancreatic protein expression profiles between people diagnosed with T1D at different ages, with different durations of disease and compare this to those without diabetes and with Type 2 diabetes (as a proxy for metabolic dysregulation). We will leverage advanced histological and imaging techniques to analyse the presence of multiple critical proteins in granular detail in whole sections of pancreas. We will then (based on several features of pancreatic T1D and betacell loss), construct a nuanced ‘pseudo’ timeline of disease progression in T1D to illustrate the life and loss of betacells in T1D and explore if this is the same in all people, randomly different between peopleor stratifiably different between groups of people living with T1D. With this information we will better understand if we should be looking for distinct forms of T1D, and this may help scientists and trialists understand why some people respond brilliantly in clinical trials, whilst other do not.

  • Kathrin Maedler & Federico Paroni

    University of Bremen
    Funded by: ERC
    Detection of coxsackievirus infection in β-cells in T1D by short fluorescently labeled oligonucleotide probes

    Type 1 diabetes (T1D) results from a complex interplay between genetic polymorphisms, immune system and environment. Viruses, mainly enteroviruses such as members of the Coxsackie B virus (CVB) family, have been suspected since long time to trigger diabetes and epidemiological studies support a causative role of viral infections during diabetes onset. Nonetheless, the presence of the enterovirus in diabetic tissue has not been fully confirmed. Here we present an adapted method to target single RNA molecules with short fluorescently labeled oligonucleotide probes in situ, that anneal to common regions of the coxsackievirus family. We successfully detect viral RNA in both cell culture and formalin fixed tissue sections bypassing the potential limitation of fragmented RNA. With the use of small contiguous RNA probes we were able to detect lower levels of strain specific infection compared to classical immunostaining. Specificity of detection was further confirmed without the well-known cross-reactivity with cellular proteins shown by antibodies. Furthermore the use of RNA probes allowed the discrimination between single RNA molecules and virus replication loci. Our data show a novel method to analyze human samples for the presence of enteroviral RNA and will be ideally suited to detect enterovirus infection with a very high sensitivity in paraffin embedded or frozen pancreatic sections.

  • Clayton Matthews & Martha Campbell-Thompson

    University of Florida
    Funded by: NIH-NIAID
    Investigating the role of iron metabolism in beta cell function and the immunopathology of type 1 diabetes

    Insulin release from beta cells depends on ATP energy generated by mitochondrial. The electron transport chain is the primary group of proteins that make ATP energy and they require iron to do so. Consequently, iron is essential for insulin release, and iron deficiency inhibits beta cell function. Conversely, excess cellular iron leads to an increase in reactive oxygen species (ROS), which can damage cells and cause dysfunction. Evidence indicates that both ROS and mitochondrial dysfunction occur during diabetes progression. Beta cells have low antioxidant capacity making them susceptible to ROS from iron overload. Iron overload can cause mitochondrial fragmentation and dysfunction. Indeed, diseases of iron overload such as hereditary hemochromatosis and Friedreich’s ataxia have an associated risk of diabetes in the absence of autoimmunity. Inflammation alters iron metabolism by promoting sequestration within cells. Prolonged inflammation can cause anemia of inflammation, which has been reported in patients with T1D, where serum iron levels are too low to promote the differentiation of red blood cells. An inflammatory signaling molecule called interferon gamma (IFNy) is increased in individuals who develop diabetes long before the onset of disease. IFNy has been shown to increase cellular iron. Taken together, these data demonstrate the importance of understanding iron metabolism under basal conditions and during T1D disease progression in beta cells. Importantly, iron metabolism pathways may serve as targets for intervention for the treatment of diabetes.

  • Noel Morgan & Sarah Richardson

    University of Exeter Medical School, UK
    Funded by: EU-FP7
    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 & Jainy Thomas

    University of Lyon - Geneuro
    Identification of HERV-W copies and insertion sites associated with T1D in human genomes

    T1D etiology is not clearly understood, but it lies at crossroads between genetic predisposition and environmental factors. Research on the human genome has so far identified numerous candidate sequences as genetic risk factors, most notably within the HLA (Human Leucocyte Antigen) region. However, an important part of the human genome has been neglected in the sequencing studies conducted so far, mostly including the so-called “mobile genetic elements”, which represent about half of the human genome. HERV (Human Endogenous Retroviruses) belong to this group, as retro-transposable elements. Moreover, the envelope protein from HERV-W family (HERV-W-Env) has recently been associated with T1D.

    Our goal is to identify T1D genetic risk factors belonging to mobile genetic elements, with an emphasis on HERV and HERV-W-env sequences. For this purpose, we propose to use novel sequencing technologies, which have not been previously used on the genome of T1D patients. These sequencing methods enable to detect genomic regions with repetitive or homologous sequences and to identify somatic DNA recombinations or rearrangements in affected tissue.

    HERV-W-env sequences and most of HERV sequences have so far been ignored by traditional sequencing methods. Sequencing technologies used in this project may allow us to identify individuals harboring HERV-W- env pathogenic DNA copies and to develop targeted therapies neutralizing the protein before the clinical onset of T1D.

  • Edward Phelps & Todd Brusko

    University of Florida/Immunocore
    Funded by: Immunocore Ltd.
    ImmTAAI technology and its potential use to treat autoimmunity

    We propose an industry collaboration between investigators in the University of Florida Diabetes Institute and an industry partner, Immunocore Ltd.

    Immunocore utilizes its T cell receptor (TCR) technology to generate tissue-targeted immune suppressant bispecifics called ImmTAAI (Immune modulating monoclonal TCR Against Autoimmune Disease) molecules. ImmTAAIs consist of a high affinity TCR-based targeting domain specific for a tissue-restricted peptide-HLA complex (pHLA), fused to an immune suppressive effector that inhibits autoreactive T cells (Fig 1). ImmTAAIs are designed to bind to target cells under autoimmune attack (e.g. pancreatic beta cells) and protect them from T cell driven destruction while not affecting the immune system elsewhere in the human body.

    Here, we propose to profile the ImmTAAIs in a disease-relevant system, a human pancreas tissue slice / T cell coculture that reproduces the autoreactive T cell attack towards beta cells in a meaningful environment. We will first test the specificity of a pre-proinsulin (PPI) ImmTAAI to bind beta cells in the complex native pancreatic environment using live tissue slices.

  • Matthew Poy

    Johns Hopkins All Children’s Hospital
    Functional Role of CADM1 during Type 1 Diabetes Pathogenesis

    Type 1 diabetes (T1D) is characterized by the hyperglycemia resulting from the autoimmune destruction of the insulin-expressing β-cells of the pancreas. As the prevalence of the disease continues to increase worldwide, it remains imperative to identify therapeutic strategies that can (1) preserve pancreatic beta-cell mass and function and (2) prevent the immune response that initiates the pathogenesis of T1D. Cell adhesion molecule 1 (referred to as human CADM1 and mouse Cadm1, and also known as SynCAM1, TSLC1, Igsf4a, and Necl2) is an immunoglobulin-domain-containing membrane protein shown to mediate homo-and heterophilic cell-to-cell contact with other Cadm family members. We previously performed Cadm1 loss of function studies in β-cell lines and transgenic mice and established endogenous Cadm1 as a negative regulator of exocytosis and proliferation in pancreatic β-cells. It has also been shown that Cadm1+ cells will bind CD8+ T-cells via the receptor Class 1-restricted T-Cell-associated Molecule (Crtam) present in the immune cells; however to date, Cadm1-mediated cellular interactions have not been studied in the context of T1D. Here we hypothesize that CADM1 expression within the human pancreatic islet mediates cell-to-cell contact with infiltrating immune cells via the interaction of CADM1 present in alpha and beta cells with CRTAM on CD8+ T cells.The goal of this work is to perform immunohistochemical analysis to visualize CADM1isletexpression (in both alpha and beta cells) with markers of immune cell populations (i.e. CD8) within the islet infiltrate. This work seeks to: (1)identify CADM1 as a mediator of cell contact between specific immune cell populations and islet endocrine cells (both alpha and beta)and (2) validate the co-localization of CADM1 in the alpha and beta cell and CRTAM in CD8+ T-cells or NK immune cell populations. Completion of the proposed studies will determine whether CADM1 may be a potential therapeutic target for preserving beta cell mass and function and for attenuating the inflammatory response during the pathogenesis of T1D.

  • Alberto Pugliese

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

    nPOD has now assembled a “cloud” of investigators with diverse expertise, who are interested in collaborative studies, and is ready to take the nPOD research model to a higher level: the nPOD working groups. These groups are intended to collectively tackle key questions in diabetes research.

    The nPOD virus working group (nPOD-V) represents a self-assembled a collaborative effort with the goal of investigating the role of viruses in type 1 diabetes through the study of nPOD samples. An innovative concept implemented by the group is “real-time” data sharing, to help coordinate studies and inform strategy adjustments. This approach should accelerate the rate of discovery and maximize the potential for robust results. The premise underlying this collaboration, based on the nPOD research model and the concept of data sharing and collaboration promoted by the nPOD leadership, has been fully embraced by the nPOD-V investigators; we believe that this in itself represent a major step-forward and innovation in the study of complex questions in human disease.

    Many studies have linked enterovirus infection to islet autoimmunity and diabetes. Yet, many questions remain about which virus serotypes are linked to T1D, what type of infection they cause, and how this may contribute to the autoimmune process leading to diabetes. Earlier studies from individual laboratories suffered from limitations with samples and reagents, and did not have access to the latest methodological advances. This collaborative effort recognizes those limitations, and addresses them with an integrated and multidisciplinary approach that includes many innovative and powerful technologies that have not been used before for studying viruses in type 1 diabetes. The availability of shared tissues from the same patients and their coordinated analysis provide unprecedented opportunity that investigations can be exhaustive and the most informative.

    The precise identification of enterovirus serotypes associated with T1D and a better molecular characterization of the virus host interaction and relation to pancreas pathology could lead to the identification of novel, T1D-specific, biomarkers of infection and potentially novel therapeutic targets. If a virus plays a role in islet autoimmunity, a vaccine or drugs that target viral responses could perhaps become important therapeutic avenues for preventing the triggering of autoimmunity or its progression, reducing patient burden and potential exposure to immunosuppressive or immunomodulatory treatments which can be associated with significant side effects and long term concerns, especially when children are concerned.

  • Nadav Sharon

    Technion - Israel Institute of Technology
    Funded by: EFSD
    Identification of protective niches in T1D pancreata

    Type 1 diabetes (T1D) is caused by an autoimmune attack mediated by cytotoxic T cells. However as the attack unfolds, many other cell types participate–and some play an anti-inflammatory, regulatory, role. Interestingly, in the early stages of the disease, attacked islets often reside next to non-attacked islets. We hypothesized that the “tranquil” islets harbor elements which protect against the immune attack, and so ventured to compare attacked and non-attacked islets in pancreata at the pre-symptomatic stage of the disease. In this framework, we characterize the different cell populations which reside in either the attacked or non-attacked islet niche. Specifically, we focus on a population of macrophages with anti-inflammatory characteristics which reside exclusively in non-attacked islets. We now study the potential role of these cells in attenuating attack progression, and look for ways to activate them as means to delay–or even prevent–T1D.

  • John Todd, Rachael Bashford-Rogers & Sarah Richardson

    University of Oxford
    Funded by: Breakthrough T1D and Wellcome Trust
    Characterising the organisation of the gut associated lymphoid tissue in type 1 diabetes and tracking T and B cell receptor clonotype / isotype usage and islet antigen-specific lymphocytes across tissues in the nPOD resource.

    The intestinal microbiome plays an essential role in host health, where microbial communities have immune-modulating activity and impact on regulatory immune cell compartmentalisation in the gut associated lymphoid tissues. Increasing evidence points to a dysfunctional relationship between the microbiome and host tissues in individuals who develop type 1 diabetes (T1D). Alterations in gut microbial composition and diversity have been observed in cohorts of individuals who go on to develop diabetes and the amount of antibody binding to common microbes is altered in diabetics, controlled in part by T1D risk HLA haplotypes. Antibodies play a key role in the composition and healthy function of the gut microbiota.  Development of the most aggressive form of T1D, with the near complete destruction of insulin producing beta cells in children diagnosed under the age of 7, is associated with an increased number and proportion of B lymphocytes infiltrating the pancreas.  Using samples from tissues across affected and systemic tissues in T1D (duodenum, spleen, pancreatic lymph node and pancreas tissues) from nPOD, we will test the hypothesis that perturbations in microbial populations and intestinal barrier function promote cross-reactivity to pancreatic beta-cell antigens thus driving development of T1D in children. We will apply a sequencing approach to deeply characterise the mucosal-associated microbial community in intestinal tissues derived from control, autoantibody positive and T1D donors. In parallel, in our approved study, we will test whether T and B cells present in the gut are related to those found in the spleen, pancreatic lymph nodes and the pancreas using a sequencing-based approach to identify cells with a shared ancestry. We will trace related clones across sites through the B/T cell receptor sequences and determine their binding characteristics using cloning and expression approaches to test for microbial and pancreatic beta-cell specificity. 

  • Clive Wasserfall & Harry Nick

    University of Florida
    Persistence of Pancreatic Insulin mRNA Expression and Proinsulin Protein and the Autoantigens GAD, IA-2 and ZnT8 in Type 1 Diabetes Pancreata

    The canonical notion that type 1 diabetes (T1D) results following a complete destruction of β cells has recently been questioned as small amounts of C-peptide are detectable in patients with long-standing disease. Previously, we analyzed protein and gene expression levels for proinsulin, insulin, C-peptide, and islet amyloid polypeptide within pancreatic tissues from T1D, autoantibody positive (Ab+), and control organs. Insulin and C-peptide levels were low to undetectable in extracts from the T1D cohort; however, proinsulin and INS mRNA were detected in the majority of T1D pancreata. Interestingly, heterogeneous nuclear RNA (hnRNA) for insulin and INS-IGF2, both originating from the INS promoter, were essentially undetectable in T1D pancreata, arguing for a silent INS promoter. Expression of PCSK1, a convertase responsible for proinsulin processing, was reduced in T1D pancreata, supportive of persistent proinsulin. These data implicate the existence of β cells enriched for inefficient insulin/C-peptide production in T1D patients, potentially less susceptible to autoimmune destruction. Moving forward, we propose to expand these studies to address the molecular mechanisms, including those of genetics (at a single cell to whole pancreas level) that contribute to novel aspects of T1D development, including the stress responses, protein misfolding, and mitochondrial dysfunction. Follow up experiments will determine the relationship of autoantigens to autoantibody titer. It is well known that autoantibodies wane after the onset of type 1 diabetes. Herein we are investigating the rate of loss of autoantibodies post onset with loss of these target proteins in the beta cells.

Archived Projects

  • Reza Abdi & Marwan Mounayar

    Brigham and Women’s Hospital
    Funded by: Breakthrough T1D
    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.

  • Vitaly Ablamunits, Kevan Herold & Jasmin Lebastchi

    Yale University
    Funded by: Yale DERC
    Comparison of total pancreatic insulin content and peripheral markers of beta cell function and desctruction

    In both Type 1 and Type 2 diabetes, there is a gradual loss of pacreatic beta cells with time. The degree of beta cell destruction may be indirectly measured by the appearance of islet-specific components in circulation. For instance, insulin mRNA is increased in circulation in T1D patients before clinical loss of function of transplanted islets. We have recently demonstrated, that hypomethylated insulin gene characteristic for beta cells leaks into circulation during beta cell death and can predict hyperglycemia in both STZ-induced and spontaneous (NOD) mouse models. In pre-diabetic NOD mice, hypomethylated proinsulin DNA in circulation negatively correlated with total pancreatic insulin content measured in acid-ethanol extracts. However, in a natural history of prediabetes, the destruction may be slow at the very beginning due to low numbers of effector T cells, or be slow immediately before clinical precipitation of diabetes, because most of the target beta cell mass has been lost already. Thus the same level of destruction may have different prognostic value. The only accurate way to interpret the level of destruction would be to know residual beta cell mass. For that, in clinical setting, surrogate markets have been traditionally used, such as C-peptide, or, more recently, beta cell mass have been visualized by positron emission tomography. However, in humans, this data has never been directly correlated to total pancreatic insulin content. We hypothesize that circulating C-peptide (amylin, proinsulin) levels strongly correlate with total pancreatic insulin content, and that a ratio between the biomarker of beta cell death and C-peptide determines prognosis.

  • Domenico Accili & Chutima Talchai

    Columbia University
    Funded by: NIH
    Beta cell dedifferentiation in type 2 diabetes

    Diabetes is associated with β-cell dysfunction. But it remains unclear whether the latter results from reduced β-cell number or function. Purpose of this study is to investigate the hypothesis that β-cell failure is the result of β-cell dedifferentiation, rather than apoptosis or degranutaion. Transcription factor FoxO1 integrates β-cell proliferation with adaptive β-cell function. The rationale for the proposed project arises from the observation that mice lacking FoxO1 in β-cells develop hyperglycemia with reduced β-cell mass following physiologic stress, such as multiparity and aging. Surprisingly, lineage-tracing experiments demonstrate that loss of β-cell mass is due to β-cell dedifferentiation, not death. Dedifferentiated β-cells revert into progenitor-like cells expressing Neurogenin3, Hdac1,and L-Myc. They also partly convert into non-β-cells, resulting in hyperglucagonemia. Strikingly, we identify the same sequence of events as a feature of different models of murine diabetes. We intend to investigate whether humans with type 1 and 2 diabetes develop the same abnormalities observed in Foxo1 β-cell-specific knockouts.

  • Taro Akiyama

    Merck
    Confirmation of target gene expression in T1D and T2D pancreas

    Characterization of expression of novel anti-diabetes targets in pancreas of Type 1 and Type 2 diabetics.

  • Mark Stuart Anderson

    University of California, San Francisco
    Funded by: Breakthrough T1D
    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

    Aire+ cells were readily identified by immuno-staining in both PN and nPLN sections. Like in mouse, human eTACs were relatively rare but localized outside the B-cell follicles and stained with the hallmark “nuclear speckling” pattern. Furthermore, the eTACs were ubiquitously positive for MHC class II but lacked high expression of CD11c, calling into question their identification as a known resident DC population. Quantitative PCR confirmed the expression of Aire in whole PLN and in sorted CD45+, MHC class II+ populations from fresh human spleen. No difference was found in the relative expression of Aire between T1D, no diabetes, and pre-T1D PLN specimens. However, the expression of ladinin-1, a putative Aire-regulated TSA in mouse eTACS, was positively correlated with Aire expression, suggesting that it might be an Aire-regulated TSA in humans as well.

    Overall, our data confirm that Aire is indeed expressed outside the thymus in humans and that human eTACs appear analogous to the more fully characterized murine eTACs. Their potential for promoting peripheral tolerance in a therapeutic setting remains an exciting possibility.

  • Justin Annes

    Stanford University
    Funded by: NIDDK
    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, Irina Kusmartseva, Clive Wasserfall & Todd Golde

    University of Florida
    Microtubule-associated protein TAU and the Islets of Langerhans

    Tau is one of the most important known molecules for microtubular dynamic and trafficking within the cells. In neurogenerative disorders such as Alzheimer disease, the tau becomes hyperphosporylated and form insoluble intracellular protein aggregates leading to upregulation of unfolded protein response. At the same time, patients with Alzheimer’s disease show impaired glucose metabolism. Moreover, both insulin-dependent and non-insulin-dependent diabetes lead to cognitive deficits in diabetes patients. Recent studies demonstrated the presence of TAU and its hyperphosphorylated pathogenic forms in islets of Langerhans in Type 2 diabetes patients,suggesting an potential role of TAU in pancreatic endocrine cell function and insulin secretion. Thus, this project seeks to elucidate presence and distribution of total and hyperphosphorylated TAU protein in endocrine cells of pancreatic islets and establish whether accumulation of hyperphosphorylated TAU is associated with aberrant islet unfolded protein response in type 1 and type 2 diabetes.

  • Mark Atkinson & Anil Bhushan

    University of Florida & University of California, San Francisco
    Beta Cell Senescence and T1D

    Type 1 diabetes (T1D) results from an autoimmune disorder characterized by T-cell-mediated destruction of pancreatic beta cells. While the role of auto-reactive T cells in targeting beta cells has been well defined, the pathological mechanisms operating in beta cells remains poorly understood. To investigate beta cell stress responses during disease progression but before diabetes onset, we performed single-cell RNA-seq (scRNA-seq) on islet cells from 8 week and 14 nonobese diabetic(NOD) mice. This analysis revealed two transcriptionally-distinct sub-populations of beta cells in the 14 week beta cells, which had hallmarks of stress response senescence and the senescence-associated secretory phenotype (SASP). The SASP consists of proinflammatory cytokines, chemokines, extracellular matrix factors, metalloproteases and growth factors secreted from senescent cells that regulate the tissue microenvironment and provide signals for immune surveillance and senescent cell clearance. As the SASP has been previously implicated in the pathogenesis of a variety of age-related diseases, we hypothesized that accumulation of SASP beta cells could be a contributing factor to disease progression in T1D. Consistent with our hypothesis, further studies revealed that targeted elimination of SASP beta cells with small molecule inhibitors prevented T1D in the NOD mouse model.To determine whether beta cells undergo senescence and SASP during the natural history of T1D in humans, we have already performed two studies on a cohort of nPOD specimens from similarly age nondiabetic, autoantibody-positive and T1D donors. Xgal staining of cryosections was used to detect the activity of senescence-associated βgalactosidase (SA-βgal), a sensitive and widely used biomarker for senescent cells in vivo. This analysis showed dramatically elevated SA-βgal activity in the islets of T1D donors as compared with nondiabetic and autoantibody-positive donors, consistent with a higher number of senescent beta cells. We also performed immunohistochemistry for senescence (CDKN1A, CDKN2A, Ser139 phospho-H2A.X) and SASP (IL-6, IL-8, MMP9, SERPINE1) markers. While nondiabetic donors generally had no expression of these markers in beta cells, autoantibody-positive and T1D donors had significantly increased levels. These data extend and build upon our findings in the NOD mouse model and suggest involvement of beta cell SASP inthe progression of human T1D. In order to test the hypothesis that beta cell SASP plays a role in T1D pathogenesis in humans, as well as to investigate whether hallmarks of SASP could provide novel biomarkers for disease progression, the following experiments requiring nPOD specimens are planned:1) Evaluate exosome RNA biomarkers in nPOD donor serum/plasma. We will evaluate candidate exosome small noncoding RNA biomarkers identified from small RNA-seq analysis of SASP islet exosome RNA human serum from nondiabetic, autoantibody-positive and T1D donors; and 2) Evaluate therapy for targeted elimination of human SASP beta cells using nPOD donor islets. Using our current small molecule therapy for clearance of SASP beta cells in NOD mice and islets, we will perform similar studies on nPOD donor islets in culture to provide proof-of-concept of this therapy for ablating human SASP beta cells.

  • William Bachovchin

    Tufts University
    Funded by: NIH
    Fibroblast Activation Protein in Type 1 Diabetes

    Fibroblast activation protein is an extracellular membrane bound and soluble protease that is commonly used as a marker of activated fibroblasts. Expression of FAP in healthy adult tissues is limited, but up-regulation is observed in a number of inflammatory and tissue remodeling processes, including wound healing, liver fibrosis and epithelial cancer, where it is thought to participate in remodeling of the extracellular matrix via its protease activity.

    We hypothesized that FAP would also be up-regulated in the pancreatic remodeling and inflammatory process associated with type I diabetes. FAP has the potential to participate in this disease process in a number of ways. First, it has been suggested that immune cells cannot infiltrate pancreatic islets unless the membranous capsule is destabilized in some way. As a protease with collagenase activity, FAP may participate in degradation of the basement membrane allowing immune cell infiltration into the islets. Second, FAP may cleave hormones involved in glucose metabolism. The FAP KO mouse has been reported to have improved glucose tolerance, particularly after high fat feeding, suggesting as yet undiscovered substrates may exist for FAP. Preliminary work in our lab has identified FAP cleavage of a number of metabolically active hormones in vitro and FAP consensus sequences exist in many soluble factors including the hormone betatrophin. Thus FAP may have substrates of consequence related to beta cell regeneration or glucose control.  

    The goal of this project is to quantify FAP activity in the pancreas and serum of type 1 and type 2 diabetics compared to controls. This study will also be, to our knowledge, the first to examine FAP activity in the human pancreas.     

  • Manuela Battaglia

    San Raffaele Scientific Institute, Italy
    Funded by: Breakthrough T1D
    Dissecting the effector/regulatory compartments in the target tissues of T1D

    An altered balance between pathogenic and regulatory pathways in autoimmunity has been hypothesized and at times demonstrated in peripheral blood of patients with type 1 diabetes. However, our knowledge in humans at the sites of the autoimmune attack (i.e., the pancreas) is scanty. Studies generated in our lab. demonstrate that FOXP3+ T regulatory (Treg) cells, identified based on their epigenetic imprinting, are present within the pancreatic lymph-nodes of patients with type 1 diabetes but they lack a suppressive activity towards both polyclonal and insulin-specific effector responses. We have found that there is an important discrepancy in Treg cells of T1D patients between their epigenetic imprinting, FOXP3 expression and regulatory function when these cells reside in the draining lymph nodes but not when they circulate in the blood.

    Given the crucial role that microRNA (miRNA) genes have demonstrated in modulating immune responses, it is possible that dysregulation of miRNA-expression contributes to the pathogenesis of autoimmune diseases, type 1 diabetes included, as recently demonstrated in other autoimmune diseases (e.g. multiple sclerosis).

    Objectives of our work are: (i) to characterize the miRNA expression profile in Treg cells residing in different tissues of the same donor (healthy and with type 1 diabetes), and (ii) to understand whether Treg cells residing in the lymph-nodes of T1D patients are post-transcriptionally regulated in a different way than those isolated from healthy donors. Results from this study may be instrumental for our comprehension of type 1 diabetes and for the development of novel therapeutics to target miRNAs to modulate immune responses.

  • Manuela Battaglia

    San Raffaele Hospital
    Funded by: Breakthrough T1D
    Innate immunity and diabetes

    Although T1D is considered to be mainly a T-cell mediated and a beta-cell specific disease we have generated over the years the hypothesis (and some supporting data) that innate immunity is also key in disease initiation and that T1D might be a disease of the whole pancreas that manifests in autoimmunity to beta cells.

    Our published data indicate that neutrophils infiltrate the pancreas of patients with T1D [1] and our still unpublished results suggest that peripheral markers of Neutrophil Extracellular Traps (NETs: a networks of extracellular fibers composed of DNA extruded from neutrophils, which can bind pathogens but can also lead to tissue damage and dangerous exposure of self antigens) are found increased patients with T1D. In addition, we have evidence that neutrophils infiltrate the whole pancreas and not only the islets [1] (a phenomenon also reported for CD8+T cells [2]). Neutrophils can also exert interesting functions when invade tissues (e.g.,production of neutrophil extracellular traps -NETs, interaction with other innate immune cells, and source of post-translationally modified self antigens) –functions that overall we call the “innate network”.

    Thus, the objective of this project is to test the hypothesis that neutrophils and NETs are increased in AAb pos and T1D nPOD donors. We propose to use the POD cohort to identify innate immune cells and their markers of activity. These important samples will allow us to test our hypothesis and –if confirmed–to design new therapeutic approaches.

    ADDENDUM: Although T1D is considered to be mainly a T-cell mediated and a beta-cell specific disease we have generated over the years supporting data that innate immunity is also key in disease initiation and that T1D might be a disease of the whole pancreas that manifests in autoimmunity to beta cells.

    Our published data indicate that neutrophils infiltrate the pancreas of patients with T1D [1] and peripheral markers of Neutrophil Extracellular Traps (NETs: a networks of extracellular fibers composed of DNA extruded from neutrophils, which can bind pathogens but can also lead to tissue damage and dangerous exposure of self antigens) are found increased in patients with T1D. In addition, we have evidence that neutrophils infiltrate the whole pancreas and not only the islets(a phenomenon also reported for CD8+T cells2). Neutrophils can also exert interesting functions when they invade tissues (e.g.,production of neutrophil extracellular traps -NETs, interaction with other innate immune cells, and source of post-translationally modified self antigens) –functions that overall we call the “innate network”.

    We recently generated new data3demonstrating that: (i) purified circulating neutrophils have a specific gene-signature in pre-symptomatic subjects and in patients with T1D. This signature is already present at very early stages of the disease (i.e., in autoAb negative subjects); (ii) NETting neutrophils (cells that have protruded Neutrophil Extracellular Traps) are present in the pancreas of subjects with recent onset T1D and autoAb positive donors but not in non-diabetic controls.

    Thus, the objective of this “addendum” is to isolate pancreas infiltrating neutrophils for transcriptomic characterization and validate the peripheral correlate through the DepArray™ technology.We propose to use the nPOD cohort to delineate the transcriptomic signature of neutrophils attracted into the pancreas of pre-symptomatic and symptomatic T1D subjects that would differ from that of non-diabetic donors.

    These important samples will allow us to test our hypothesis and –if confirmed –to design new therapeutic approaches.

  • 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.

  • Jeffrey Bluestone & Armando Villalta

    University of California, San Francisco
    Vascular endothelial growth factor receptor signaling regulates the pathogenesis of type 1 diabetes

    1. To test the hypothesis that vascular endothelial cells in the pancreas of recent-onset diabetic patients have increased expression of VEGF receptors (VEGF-R).
    2. To examine the expression of VEGF or VEGF-R on islet-invading CD4+ T lymphocytes.
    3. To examine the utility of TCR repertoire between different T cell subsets as a marker of T1D disease initiation and progression by high throughput sequencing.

  • Bernhard Boehm & Prabhakar Shyam

    Nanyang Technological University
    Funded by: A*STAR Singapore and LKCMedicine
    Validation of blood – derived single cell gene expression signature in peri – pancreatic lymph nodes of Type 1 Diabetes subjects

    Type 1 diabetes (T1D) is an organ specific autoimmune disease resulting in an immune-mediated destruction of insulin-secreting beta cells. The etiology of T1Dis puzzled by a multifaceted interaction of environmental, genetic and epigenetic factors leading to variousendotypesof the disease. Fine mapping of T1D-associated genetic variants with cell-specific epigenetic markers or expression quantitative traits (eQTLs) indicates that the disease riskfactors most probably exert their function by changing the transcriptome profile of immune cells in a cell specific manner. This explains the heterogeneous nature of T1D in terms of both clinical representation and underlying pathogenic pathways. Therefore, measuring the cell-specific transcriptome profile and composition of peripheral immune cells is considered as the key to identify the exact cellular and molecular pathogenic mechanisms of the diseaseprogressand develop novelimmunesignatures for monitoring disease progression.In spite ofextensive research into T1D-associated immune signature, there is still no consensus about T1D-associated immune cell typesand their pathogenic signature.By taking advantage of the unbiased single cell RNA sequencing technology, we profiled transcriptome landscape of peripheral blood mononuclear cells (PBMCs) derived from 40 European T1D subjects and 35 healthy controls matched for age, gender and HLA alleles. We detectedsignificant alteration in relative frequency of several immune cell types(particularly subtypes of CD4+ T, CD8+T, and regulatory Tcells) inT1D subjects compared with healthy controls. Interestingly, we found that the aberrant transcriptome profile of these cell types issignificantly enriched in immune pathwaysassociated with enhanced antigen priming capacity(DAP12 signaling pathway)and transendothelial migratory potential(Rho GTPase activate ROCK pathway) of T cells. Since it is well known that peri-pancreatic lymph nodesare essential for the priming of auto-reactive T cellsin T1D, we hypothesized that the observed signatures in circulating blood cells root from the peri-pancreatic lymph nodes wherein enhanced antigen priming and trans-endothelial migration potential of activated cells lead to the pathogenesis of the disease. To test our hypothesis, the currentproposal aims tocharacterizeimmune subtype proportionand transcriptomic profilein the peri-pancreatic lymph nodes ofT1D cases and controls(aim 1), identify aberrantcell-specific genes/pathways (aim2) and cross-validate lymph node signature using single cell analysis of PBMCs from the same individuals as well as our single cellPBMC datasetfrom the European cohort(aim3). We envisage thatthe current research projectwill work as an essential step for the development of a blood-basedblueprint associated with pathogenesis and progression of the disease.

  • Marika Bogdani

    Benaroya Research Institute at Virginia Mason
    Funded by: Breakthrough T1D
    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.

  • 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.

  • Alexandra Butler, Sangeeta Dhawan

    University of California Los Angeles
    Funded by: National Institutes of Health/NIDDK and Helmsley Charitable Trust George S. Eisenbarth nPOD Award for Team Science
    ORIGINAL: Growth and Development of the Endocrine Pancreas in Humans: Extrapolating Normal Physiology to the Regenerative Capacity of the Endocrine Compartment in Type 1 and Type 2 Diabetes; ADDENDUM: The Role of Neuropeptide Y (NPY) in Human Beta Cell Maturation and Diabetes Pathogenesis

    ORIGINAL: Chromogranin A is a global marker of endocrine cells. Most endocrine cells in the pancreas, in addition to Chromogranin A, also express other hormones, Insulin and Glucagon being most frequent, with Pancreatic Polypeptide, Somatostatin and Ghrelin in smaller numbers of endocrine cells. We have found that in adult human pancreas a small number of pancreatic endocrine cells (<1% of the total number) express Chromogranin A but not any of the other known pancreatic hormones; we have termed these cells Chromogranin Positive Hormone Negative [CPHN] cells.

    We found that there is a very high percentage of CPHN cells in sections from human fetus and newborn pancreas, which suggests that these cells are a precursor of mature endocrine cells. We also found that the percentage of CPHN cells is higher in pancreas from both type 1 and type 2 diabetic subjects, suggesting that this may be an attempt at restoring the beta cells lost as a consequence of disease.

    Our initial observations are that these cells occur mostly in small clusters of 1-3 cells rather than in established islets, suggesting that these cells may be newly formed endocrine cells. The source of these CPHN cells is of great interest, as they could potentially occur de novo (neogenesis), by transformation of existing cells (transdifferentiation) such as from cells in the ducts or exocrine pancreas, or by replication of existing endocrine cells.

    Our goal is to characterize these cells by looking at the frequency of replication and apoptosis , and the transcription factors Nkx6.1 and Nkx2.2, in the CPHN cell population. Our findings will have significance with regard to the regenerative capacity of human endocrine cells in human pancreas.

    ADDENDUM: Both type 1 and type 2 diabetes result from loss of functional beta-cell mass. Functional defects in evolving T1D and T2D are marked by the uncoupling of ambient glucose and insulin secretion, reminiscent of functionally immature beta cells in early pancreatic development. To develop successful beta-cell replacement for reversal of beta-cell dysfunction in diabetes, it is therefore critical to understand mechanisms that regulate beta cell maturity. Beta cells are formed during embryonic life, but acquire function only after birth, in a process called beta cell maturation. We have identified Neuropeptide Y (NPY) as a marker of functionally immature, neonatal beta cells. NPY is known to inhibit insulin secretion, and we show its co-localization to the insulin secretory granules in neonatal human beta cells. Our preliminary data show that NPY is down-regulated in adult beta cells, and re-expressed in beta cell in mouse models of type 2 diabetes. Thus, we propose to examine NPY in human beta cell maturation and diabetes pathogenesis.

  • 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, et.al. 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.

  • Martha Campbell-Thompson, Dirk Homann & Richard Benninger

    University of Florida
    Funded by: Breakthrough T1D
    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.

  • Martha Campbell-Thompson, Lizette Duckworth-Vila, Ralph Hruban, Gunter Kloppel, Chen Liu, Nickolas Papadopoulos & Mark Atkinson

    University of Florida
    Diabetes and Pancreatic Cancers: Pilot study with molecular profiling

    Our study aims are the following:
    1. Histopathology review of pancreata from patients without diabetes (no diabetes and autoantibody+) and
    those within the diabetic and other groups (type 1, type 2, gestational, other (those with unclassified diabetes,
    excludes cystic fibrosis)).
    2. Molecular profiling of PanNET for neuroendocrine hormones and other genes (Isl1, p53, Ki67), FISH
    (telomerase shortening), MEN1 mutations (DNA), and gene expression (RNA). Assays will be conducted at the
    University of Florida and Johns Hopkins laboratories.

  • David Carpenter, Miquel Porta & Sarah Howard

    University at 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.  

  • 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 

  • Raphael Clynes

    Columbia University
    Funded by: NIH, Breakthrough T1D, and NIDDK
    TCR signal transduction in diabetogenic T cells

    Tyrosine kinase inhibitors (TKis) effectively limit autoimmunity in animal models and are under clinical study in RA, MS and psoriasis. Recent studies in NOD mice have demonstrated proof-of-principal for promiscuous PTK blockade, using inhibitors initially developed to inhibit oncogenic pathways in cancer. Our recent work has shown that the selective Syk inhibitor R788 limits diabetes progression in NOD mice. Spleen tyrosine kinase (Syk) is most widely known as the PTK downstream of the B cell antigen receptor (BCR) and the IgG receptor (FcR) in myeloid cells and Syk PTKis have demonstrated clinical activity in RA and ITP. R788-treated NOD mice had decreased autoantibody levels presumably the result of BCR-inhibition and decreased T cell priming likely due to syk inhibition in DCs. A direct effect of Syk inhibtion on T cells was not reported, since syk expression in T cells was not expected. However, aberrant Syk expression may occur in autoimmune T cells. Indeed in our preliminary studies, discussed below, Syk is seen expressed in autoaggressive CD8 T cells infiltrating the skin and in activated murine and human T cells ex vivo.

    Targeting of T cell activation via small molecule interference with protein tyrosine kinases is a promising approach to autoimmunity and could be directed at TCR signaling or co-stimulatory pathways. In T1D, these efforts will require determination of which signal transduction targets are specifically expressed in diabetogenic T cells in the islet and pancreatic lymph node. Autoaggressive T cells responding to sustained autoantigen drive may express signal transducing molecules not ordinarily expressed in resting or transiently active normal T cells thus providing attractive therapeutic targets. Our preliminary data shows that the Syk kinase is expressed in persistently activated T cells in vitro and in autoimmune T cells in vivo.

  • 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

    Our aim is to assess the protein expression level in conjunction with histological and (sub)cellular localization of the cytokine IL-21, its receptor IL-21R and GLP-1R in normal controls, (pre-)type 1 and type 2 diabetics (with history of incretin treatment for GLP-1R). (adopted by nPOD staff)

  • Barbara Coulson

    The University of Melbourne at the Peter Doherty Institute for Infection and Immunity
    Funded by: Agency: Australian Government-National Health and Medical Research Council
    Analysis of type 1 interferon responses to rotavirus in patients with type 1 diabetes

    Rotavirus infection of children at-risk of type 1 diabetes is associated with disease progression. Similarly, infection of adult non-obese diabetic (NOD) mice with Rhesus monkey rotavirus RRV accelerates diabetes onset. This acceleration correlates with RRV spread to the mesenteric and pancreatic lymph nodes, where virus associates with and activates dendritic cells and induces B and T cell activation. We also have shown that RRV exposure ex vivo induces generalized B and T cell activation by triggering toll-like receptor 7 (TLR7) signaling and type I interferon production by plasmacytoid dendritic cells (pDC) from NOD mice. As this activation includes islet autoantigen specific T cells, we hypothesize that type I interferon-mediated bystander activation contributes to diabetes acceleration by RRV in NOD mice. Notably, RRV induces significantly more activation of NOD mouse than C57BL/6 mouse cells, providing evidence that these responses to RRV are heightened in mice at-risk of T1D. Influenza virus and coxsackievirus B, which also signal through TLR7, have been shown to induce heightened pDC-dependent type I interferon responses in PBMCs from T1D patients compared to controls. We hypothesize that rotavirus will also induce heightened pDC-dependent lymphocyte activation and type I interferon secretion in PBMCs and other immune cells from patients with T1D compared to controls. Therefore, the overall aim of our study is to evaluate activation and type I interferon expression following stimulation with rotavirus in immune cells from patients with T1D compared to controls.

  • Nika Danial & Ulupi Jhala

    Harvard Medical School
    Funded by: NIH-HIRN
    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 from 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.

  • Howard Davidson

    University of Colorado Health Science Center
    Funded by: Breakthrough T1D
    Characterization of autoantigen-specific T cell receptors from PLNs

    Type 1A Diabetes mellitus (T1D) is a predominantly T cell mediated autoimmune endocrine disease. Both CD4+ and CD8+ T cells reactive to diabetic autoantigens can be identified at low frequency in the peripheral blood of diabetic subjects and their at-risk relatives, but the extent to which these peripheral responses reflect those in the pancreatic draining lymph nodes (PLN) and insulitic lesions is currently unclear, mainly due to the relative inaccessibility of these tissues in living subjects. Answering this question is of central importance to the development of mechanistically relevant biomarker assays for staging disease and monitoring therapeutic efficacy, as well as for identifying appropriate targets for antigen specific immunotherapy. The goal of our project is to isolate and characterize T cells reactive to the major T1D autoantigens (with special focus on insulin, GAD65, IA-2, IGRP, and ZnT8) from the PLN and spleen of nPOD donors. The frequency, phenotype, epitope specificity and clonal diversity of islet-reactive T cells from these tissues will be compared with those present in islets and peripheral blood from the same donors (when available), and in PBMCs from HLA-matched living subjects at various stages of disease. 

  • Mark Davis & Yu Wong

    Stanford University
    Funded by: Breakthrough T1D and Howard Hughes Medical Institute
    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

    Growing evidence suggests inflammation as a major underlying mechanism in the pathogenesis of type 2 diabetes mellitus (T2DM). Supporting the involvement of the immune system in T2DM, cross-sectional and prospective studies have associated elevated circulating levels of acute-phase proteins. Furthermore, clinical studies have demonstrated that anti-inflammatory drugs may improve glycaemia. Morphological and therapeutic intervention studies have uncovered an inflammatory process in islets of patients with type 2 diabetes characterized by the presence of cytokines, macrophages, β cell apoptosis, amyloid deposits, and fibrosis. However, the immune cells involved in pancreatic islet inflammation during T2D are insufficiently described. In human tissue samples, only CD68 positive macrophages have been documented. The level of activity of these cells as well as the occurrence and role of other immune cell types remain to be investigated.

    Using the pancreatic tissue samples supplied by nPOD, we plan on investigating the presence of innate and adaptive immune cells within or around the islets of T2DM patients versus healthy donors. Defining the inflammatory milieu within the islets of T2DM patients will help us target further inflammatory treatments. 

  • Dieter Egli, Bjarki Johannesson & Ruddy Leibel

    Columbia University
    Funded by: Helmsley Foundation
    Generation of pluripotent stem cells from diabetics ADDENDUM: Generation of iPS cell lines from a majority of T1D cases

    ORIGINAL PROJECT: Established different skin fibroblast cell lines, both from control and T1D nPOD donors. Graciously offered this as a resource available to current nPOD investigators.

    ADDENDUM: The pancreata of nPOD cases are some of the best characterized type 1 diabetes cases. The availability of beta cells from stem cells would greatly enhance this resource by providing the ability to both study the primary pancreas, as well as living beta cells or other cell types in a defined environment with the genotype of the patient. This project aims to generate cell lines for as many of the nPOD cases as possible, and to convert selected cell lines to iPS cells, and stem cell derived beta cells to determine whether disease phenotypes are cell-autonomous effects of the genotype, or acquired through interactions with the environment or with other cell types.

  • Dieter Egli, Ruddy Leibel

    Columbia University
    Beta cell defects in cystic fibrosis related diabetes

    The aim of this study is to determine how CFTR mutations affect beta cell performance, and whether
    their effect is direct (cell-autonomous), or indirect, by affecting other cell types/physiological
    processes with secondary consequences for beta cell function and viability. It is likely that any
    mechanistic insights obtained from the study of this specific etiology of diabetes will be applicable to
    other types as well.

  • Garry Fathman

    Stanford University
    Microarray analysis of PLN from autoantibody positive donors

    As with many autoimmune diseases, T1D is initiated by some unknown inciting event (or events) that results in the appearance of disease-relevant biomarkers.  Serum autoantibodies to islet antigens or changes in whole blood gene expression that appear before the onset of glucose intolerance can serve as biomarkers to identify pre-diabetic individuals. As we enter the era of intervention trials to block progression of T1D before beta cell destruction, it is important and beneficial to identify relevant biomarkers that can be used to stage early disease progression in T1D. Recent studies from my lab have demonstrated that changes in gene expression of pancreatic lymph node (PLN) tissue samples from AA+ individuals provided by nPOD, can be seen before the onset of abnormal glucose tolerance.  These data also demonstrated that gene expression in the PLN is similar between T1D patients who have had hyperglycemia for two or more years and normal subjects, suggesting that the inflammation accompanying progression to hyperglycemia in AA+ subjects can be identified by changes in gene expression in AA+ subjects, and may be used to identify biomarkers, and/or suggest targets of, or response to therapy.  Because PLN tissues cannot be readily biopsied from AA+ individuals, our rationale is to examine gene expression in PBC samples provided by the TrialNet Natural History tissue repository to ask if there are distinct changes in gene expression seen in the PBCs (similar to those seen in the PLN) that occur with the progression of disease. As a second project arising from these data, we will ask if there are changes in gene expression that accompany intervention therapy with CTLA4-Ig in a TrialNet intervention trial being run by Dr. Bill Russell at Vanderbilt.  These approaches may help identify early biomarkers of disease that will allow sub-setting of AA+ patients based on stage of disease or rate of progression, and could identify prediction of, or response to, therapy.

  • Brian Fife & Marc Jenkins

    University of Minnesota
    Funded by: Minnesota Partnership for Biotechnology and Medical Genomics, Breakthrough T1D and NIH
    Tracking islet autoantigen specific CD4+ T Cells

    The goal of this research project is to identify self-reactive T cell clones and monitor the frequency and activation state during the progression of autoimmune diabetes.  Identification of T-cell reactivity to beta-cell antigen epitopes is important to study pathogenesis and monitor antigen specific interventions in T1D.  We have successfully generated and validated human tetramer reagents to diabetes relevant target antigens to determine frequency and activation state of autoreactive CD4+ T cells in patients with diabetes. Tetramer reagents could provide much needed biomarker reagents to predict T1D earlier in ‘at risk’ patients, stage clinical disease, and determine if therapeutic approaches have an effect on the CD4+ T cell level during T1D pathogenesis.  If successful, the tetramer biomarkers and enrichment approach we are testing could precede autoantibody production and provide an earlier diagnosis of T1D, before irreparable damage has occurred to the islets.  Therefore, these reagents would have great impact to provide a new in vivo diagnostic approach to identify T1D T cell responses as an early diagnosis for high risk patients.  With a better understanding of beta cell specific targets we will be able to develop novel therapeutic approaches to cure diabetes.  

  • Paolo Fiorina & Francesca D'Addio

    Boston Children's Hospital
    Role of TMEM219 Expression in Type 1 Diabetes

    The recent failure of immunotherapeutic strategies in the cure of T1D raised questions in considering autoimmunity the sole mechanism responsible for the pathogenesis of T1D. It has been recognized that activation of immune response against self-peptides is essential in the onset of T1D, but its role in the destruction of beta cells, which leads to reduced insulin secretion and glycometabolic control, is a matter of debate. The recent studies demonstrating a relevant role for the environmental factors in the pathogenesis of diabetes, confirmed that non-immunological determinants and beta cell regulating molecules might contribute in initiating and/or perpetrating this process. The increasing body of evidences that autocrine and paracrine signaling pathways control the homeostasis of several type of cells, such as stem cells and pancreatic beta cells, drew the attention of scientists on the possibility that even circulating hormones may be involved. More importantly, altered secretion of factors/hormones during T1D leads us to hypothesize that systemic factors may target pancreatic beta cells and modulate fate/function. Our previous studies showed that a circulating hepatic-released factor IGFBP3, exerts a detrimental effect on intestinal stem cells (ISCs) and induces their apoptosis/death by binding its receptor, TMEM219, expressed on ISCs surface. Interestingly, TMEM219, is expressed in human islets and in a beta cell line. We hypothesize that an overexpression of TMEM219 may favor beta cells destruction and affect beta cell mass, and the consequent hyperglycemia may perpetrate the process, given that TMEM219 expression is sensitive to high glucose exposure.

  • Gun Frisk & Olle Korsgren

    Uppsala University, Sweden
    Funded by: EU and Diabetesförbundet
    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.

  • Michael German & Chester Chamberlain

    University of California San Francisco
    Funded by: The Caring for Carcinoid Foundation
    Analysis of normal pancreas from PNET patients with and without diabetes

    Spontaneous recovery from established type 1 diabetes (T1D) is so rare that only one well-documented case has ever been described in an adult, and in that case recovery resulted from an insulin-secreting tumor, not regeneration of the patient’s normal beta-cells. Therefore, when we saw a 50 year old man at UCSF with T1D who got off insulin (Patient 36300562), we investigated the cause. He had a pancreatic neuroendocrine tumor (PNET), but it was not producing insulin. Instead, in the few islets in his pancreas that still contained beta-cells, those beta-cells were proliferating at rates far exceeding anything previously reported in humans, suggesting that PNETs may produce factors that can promote human beta-cell regeneration. We also found alpha-cells of Patient 36300562 were producing ghrelin, suggesting that gut hormone expression in islet cells may be a characteristic of the T1D pancreas. SPECIFIC AIM 1: Characterize the expression of ghrelin in the pancreatic islets of patients with and without diabetes. We will perform immunostaining assays for ghrelin in sections of normal and T1D pancreas samples from the nPOD. We will also examine the expression of PYY and somatostatin to gain a better understanding of how hormone gene expression may be different in the islet of T1D patients. These studies will help clarify whether the expression of ghrelin in the alpha-cells of Patient 363000562, who had T1D, is a consequence of the PNET or T1D. These studies will also add to our knowledge of the T1D pancreas phenotype. SPECIFIC AIM 2: Characterize the beta-cell phenotypes in patients with PNET. The high beta-cell proliferation rate and T1D recovery of Patient 36300562 suggests that PNETs may produce factors that can promote human beta-cell regeneration. We will therefore also examine markers of beta-cell proliferation (Ki67) and death (TUNEL). These studies will help confirm the beta cell phenotype of Patient 36300562, and may help justify additional studies to identify novel factors from PNETs that promote human beta-cell regeneration.

  • Roberto Gianani

    University of Colorado, Denver
    Characterization of type 1 diabetes subsets

    Type 1 diabetes is a clinically and pathologically heterogeneous disease. A large subset of individuals with Type 1 diabetes has an autoimmune form of the disease characterized by subtotal loss of beta cells and positivity for islet autoantibodies as well an increase prevalence of DR3 and DR4. We have recently characterized a pathological pattern of beta cell loss in Type 1 diabetes that does not appear to be associated with autoimmunity. We hypothesized that this represent a novel disease characterized by “degenerative” loss of beta cells possibly due to increased apoptosis (affecting also alpha and delta cells), pathogenetically distinct from the beta cell loss seen in Type 2 diabetes.

    The Specific Aims Are:

    1. Morphometric characterization of Pattern B type 1 diabetes in relation to the beta cell, alpha cell and delta cell relative volume as compared to controls and type 2 subjects.
    2. Investigation of surviving expression at the protein and RNA level (initially these studies will be limited to surviving).
    3. Comparison of apoptosis rate between different groups
    4. Investigation of amyloidosis in the different groups
    5. Collaborative efforts with Dr. Sechi Laboratory.
  • Nick Giannoukakis & Mark Atkinson

    University of Pittsburgh
    Role of bacterial biofilms in the onset of pancreas inflammation in T1D

    Even though the immunopathogenic triggering event(s) in type 1 diabetes (T1D) remain enigmatic, there is accumulating evidence, mainly in rodent models of the disorder, suggesting that pancreas-resident antigen-presenting cells (APC) are activated very early post-natally. This results in the recruitment of innate immune cells like neutrophils into the pancreas even before adaptive immune cells that are islet antigen-specific are detected. Activated neutrophil accumulation into new-onset T1D human pancreata has also been documented. While it is possible that this innate immune activation very early-on reflects a reparative response to some endogenous natural post-natal “remodeling” process gone-astray, or to the incursion of viruses, the direct associations have not been conclusively established. The kinetics of the activation of resident APC and the post-natal aggravation of innate immunity prior to any signs of T-cell incursion is better associated with pattern recognition receptor-mediated signals, implying a bacterial response. Indeed, the NOD/ShiLtJ mouse strain as well as human T1D patients, have been shown to exhibit a “leaky gut” phenomenon and treatment of NOD mice with antibiotics or an exchange of the gut microbiota has resulted in prevention of disease onset. Furthermore, microbiota from the “leaky gut” have been shown to localize inside the pancreas, triggering an inflammation. In spite of recent studies aimed at identifying specific microbes as triggers, there is no evidence pointing to a specific pathogen. Our hypothesis, instead, proposes that any bacterial pathogen can trigger the resident APC through the formation of biofilms. Bacterial biofilms are omnipresent in tissues in which bacteria reside and act not-too-unlike extracellular trap structures. These highly pro-inflammatory microenvironments compel tissue repair and remodeling. Biofilm constituent molecules not only protect and promote the survival of entrapped bacteria, but also act as “danger” signals to the pancreas-resident APC. Biofilm formation, consequent to post-natal “leaky gut”-facilitated bacterial deposition inside the pancreas is hypothesized to trigger the resident APC, to attract neutrophils and migratory macrophages and dendritic cells, provoking an inflammation that ultimately culminates in the activation of autoreactive lymphocytes inside the pancreas-draining lymph nodes. The objective of this study, using nPOD human pancreas tissue, is to first demonstrate the existence of bacterial biofilms inside the pancreas of T1D subjects. Then, to compare the incidence of biofilms in the T1D pancreata to non-diabetic pancreata and to determine any associations of biofilm presence, density, and “signature” with age-at-onset of disease. In parallel, we will determine if the presence of biofilms and/or specific signatures, is associated with the presence of particular innate immune cell populations and their state of activation in situ. To realise these objectives experimentally, we will deploy multiparameter immunofluorescence microscopy to identify specific immune cell populations; fluorescence in situ hybridization to identify biofilms, and sequencing technology to identify biofilm signatures in the pancreas tissues. On its own, we believe that this project is novel since it is the first-in-kind to explore the potential involvement of biofilms in organ-specific autoimmunity, and it can also inform a better understanding of the initiating trigger of T1D altogether.

  • Kathleen Gillespie

    University of Bristol
    Funded by: Diabetes UK
    Maternal microchimerism in T1D pancreas

    Maternal microchimerism (MMc) is acquired by an infant during pregnancy and these cells are maintained in some individuals for decades.  Encountering maternal antigens during pregnancy represents the first immunological challenge for the fetus although maternal cells are protected from detection by induction of suppressive fetal regulatory T cells.

    In type 1 diabetes increased levels of maternal microchimerism have been demonstrated in peripheral blood DNA and maternal cells have been identified in human pancreas. The aim of this study is to phenotype these maternal cells in well characterised male pancreases in order to help understand how the presence of maternal cells could contribute to risk of T1D. The methodology used will utilise using X/Y chromosome FISH with concomitant immunofluorescence.  Once the phenotype is established, we will attempt to confirm the maternal origin using single cell laser capture with DNA profiling. 

  • Kathleen Gillespie & Abby Willcox

    University of Bristol
    Funded by: Diabetes UK
    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.

  • Maria Grant

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

    Despite advances in understanding the pathogenesis of diabetic retinopathy (DR), the nature and time course of the molecular changes associated with DR remain incompletely understood. We propose to investigate the mechanism of bone marrow (BM) failure in diabetes and test diverse therapeutic approaches to correct BM and bone marrow progenitor cell (BMPC) dysfunction in order to restore retinal vascular health. Healthy BMPC participate in the process of re-endothelialization and also contribute indirectly to vascular repair by the secretion of paracrine factors. In health, distinct populations of BMPC work in concert to orchestrate vascular repair. The small pool of CD34+ cells has robust reparative function and modulates, via paracrine signaling, the function of the much larger pool of CD14+ cells which, depending on the local milieu, have the potential to also become efficient reparative cells. Freshly isolated BMPC can be expanded in vitro to generate two additional populations of reparative cells: early endothelial precursor cells (eEPC) or late outgrowth endothelial cells (OEC) that may have more therapeutic utility than freshly isolated cells. To date the optimal progenitor cell for vascular repair has not been identified.

    In diabetes, BMPC demonstrate deficient reparative function and may even have a deleterious effect on the repair process. We demonstrated that: 1) diabetic CD34+ cells do not repair acellular capillaries in the diabetic retina, whereas CD34+ cells from nondiabetic individuals do (a similar observation was also made using CD14+ cells); 2) compared to nondiabetic CD34+ cells, diabetic CD34+cells have reduced bioavailable nitric oxide (NO) which results in their reduced migratory ability that can be corrected by restoring NO levels to normal; and 3) diabetes is associated with the loss of circadian rhythmicity of BMPC release from the BM into the circulation. Decrease in blood flow through the vasa nervorum, the small arteries that provide blood supply to peripheral nerves, has been implicated in the development of diabetic neuropathy. We postulate that in diabetes reduced NO initiates changes in the vasa nervorum and is a mechanistic contributor to reduced sympathetic neurotransmission which ultimately results in BM dysfunction. Loss of this neurotrophic support leads to changes in the BM microenvironment which contribute to, not only reduced release of these cells, but also to their impaired function. This dysfunction is further exacerbated by elevated reactive oxygen species (ROS) in these cells. Based on our findings that diabetic retinopathy is linked to peripheral neuropathy, we put forth the following hypothesis:

    Loss of circadian rhythmicity of release of BMPC from the BM (due to dysregulated NO in the vasa nervorum and subsequent BM neuropathology) and increasing levels of intracellular ROS, increasing endogenous NOS inhibitors and inappropriate secretion of pro-inflammatory cytokines and proangiogenic growth factors compromise the reparative ability of BMPC leading to development of diabetic retinopathy.

  • Dale Greiner & Leonard Shultz

    University of Massachusetts
    Diabetogenic function of autoimmune donor splenocytes in humanized mice

    Our understanding of type 1 diabetes (T1D) has been advanced greatly by studies carried out using mice and rats. However, rodents are not humans and progress in the understanding of the pathogenesis of T1D in humans has been impeded by the a lack of assays that permit the direct in vivo analysis of diabetogenic human T cell populations without putting individuals at risk. The availability of spleen, bone marrow, and thymus samples from T1D and islet autoantibody-positive donors provides an opportunity to analyze the diabetogenic function of these cell populations following engraftment in newly developed humanized mouse model systems. This work is based on our recently developed immunodeficient mice that express human HLA molecules and are optimized for human immune cell engraftment. Our goal is to use unique tissues available from nPOD to recapitulate autoimmune T1D in humanized mice. We will engraft human hematopoietic stem cells and immune cells into HLA-matched NSG mice and determine the ability of cells from T1D donors to attack islets and induce the destruction of insulin-producing cells. The knowledge obtained from these studies will permit us to elucidate the pathogenesis of T1D and to develop new approaches for preventing or curing the disease.

  • 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.

  • Zhiguang Guo, Alex Rabinovitch & Romano Regazzi

    Sanford Research/Sanford Health
    Funded by: NIH
    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.

  • Abdel Hamad & Thomas Donner

    Johns Hopkins University
    Fas Ligand: Unorthodox target for prevention of type 1 diabetes

    Despite improvement in insulin delivery, maintaining tight control of glucose homeostasis continues to be a challenge that results in bouts of severe hypoglycemia and hyperglycemia and serious long term complications in many patients. Therefore, developing an immunotherapy for type 1 diabetes (T1D) remains a major goal. Reaching this goal requires detailed knowledge of the diabetogenic process. Particular knowledge of the molecules that play pivotal roles in driving the pathogenic mechanisms but are not required for mounting normal immune responses could lead to uncovering of safe therapeutic targets. Fas ligand (FasL) is a rare example of such molecules. It is an apoptosis-inducing member of the TNF family that is not required for immune responses. Yet it plays a critical role in driving the diabetogenic process as non-obese-diabetic (NOD) mice bearing spontaneous loss-of-function mutation of FasL (gld) of Fas (lpr) are completely protected from the disease. We hypothesize that aberrant FasL-mediated apoptosis of IL-10-producing regulatory B cells is critical for initiating the diabetogenic process. This hypothesis is supported by strong published and unpublished data in the NOD mouse model. By working with Breakthrough T1D/nPOD, we are investigating whether a dysregulated Fas pathway is compromising the role of IL-10-producing regulatory B cells in humans. We have collected strong evidence, so far, indicating that, as in NOD mice, aberrant FasL-mediated apoptosis may a pathogenic mechanism in T1D. Such results will not only provide new insights into the disease process, but also a platform for novel therapeutic modalities that do not impair the patient host defense.

  • Manami Hara

    University of Chicago
    Funded by: Breakthrough T1D
    Beta-cell destruction and preservation in early and late onset type 1 diabetes

    Type 1 diabetes (T1D) is one of the most common chronic diseases of childhood with onset peaking between 5-7 years of age and around puberty. Early childhood-onset T1D often exhibits severe beta-cell depletion compared to late adult onset T1D. We propose to test the hypothesis that immature beta-cells in early childhood are more susceptible to autoimmune destruction, whereas the cell death of mature beta-cells in adult onset T1D undergoes different kinetics and its process is substantially slower than previously considered. A better understanding of distinct beta-cell destruction and preservation during childhood could lead to new therapeutic strategies to treat young children with T1D.

  • Paul Harris, Masanori Ichise & Matthew Freeby

    Columbia University
    Funded by: NIDDK and Breakthrough T1D
    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. 

  • Matthias Hebrok & Long Cai

    University of California, San Francisco
    Funded by: Breakthrough T1D
    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.

  • Kevan Herold & Alfred Bothwell

    Yale University
    Funded by: Breakthrough T1D
    Reconstitution of HHLS mice with bone marrow from patients with T1DM

    The focus of our Breakthrough T1D Autoimmunity Center is to create and study a humanized mouse model of Type 1 diabetes. The approach that we are developing involves use of immune deficient (γc-/-) mice with “knock-in” of human cytokine and other genes that are needed for optimal reconstitution with human cells. In addition, we have created transgenic mice that express human proinsulin in beta cells and in addition, have human MHC+ mouse MHC- mice in which the presentation and responses to human antigens (e.g. proinsulin, insulin) can be studied. We hope to reconstitute the immune deficient mice with human CD34+ bone marrow cells from patients with diabetes. The duration of diabetes should not be an issue so the use of nPOD tissue would be appropriate. We plan to study the ability of the reconstituted mice to develop diabetes – spontaneously or with provocateurs such as low dose streptozotocin, immunization with antigens, or even transgenic expression of B7.1 on beta cells. (adopted by nPOD staff)

  • Akihisa Imagawa & Toshiaki Hanafusa

    Osaka University, Japan
    Funded by: Gift Fund
    Histological differences between Japanese and Western type 1 diabetes

    (1) To clarify the infiltration of mononuclear cells in exocrine pancreas of Western type 1A diabetes and compare the histological findings to those of Japanese patients

    (2) To clarify the expressions of the molecules related to innate immunity or viral infection in Western type 1A diabetes and compare the hitological findings to those of Japanese patients

  • Dolores Jaraquemada, Mercè Martì & Carme Roura-Mir

    University of Barcelona, Spain
    Tracing effector and regulatory T cell populations in type 1 diabetes

    Our group is studying T cell phenotype and antigen presentation and recognition by T cells in T1D, with three main objectives. (i) The characterization of role of T cells involved in immunoregulation, i.e. regulatory T cells (nTregs or iTregs) and NKT cells. We are studying the mechanisms of regulation by analyzing the role of soluble and membrane bound molecules on the regulatory effect of each cell type; and the cooperation between regulatory populations to achieve a higher degree of suppression of T effector cells. (ii) The characterization of the TCR repertoire both on the pancreas infiltrate and on PBMCs from T1D patients. TCR expressed by autoreactive T cells is important as a tracer of the different clonotypes involved in T1D pathogenesis. We are also working on the hypothesis that some of the T cells involved in the maintenance of the chronicity share the TRBV-CDR3 as public TCRs. If proven, TCR repertoires prone to autoimmunity could be defined when accompanied with the HLA-associated alleles. Expanded iNKT cells will be analyzed as a model of public TCR in those patients. (iii) The third aim addresses antigen recognition by autoreactive T cells by the characterization of peptides presented in situ by HLA molecules. We are now able to retrieve a good number of peptides from small samples (i.e. islets). We will compare the T-cell epitopes already identified in T1D with those naturally presented in the islets by HLA class I and class II molecules. Because of the low number of HLA-II positive cells in the islets, we expect results for class I and are working on alternative approches for class II.

    The first part of this project was in part funded by the Breakthrough T1D and we are now funded by the Spanish Department of Science.

  • Jonathan Katz

    Cincinnati Children’s Research Foundation
    Manipulating DNA damage-response signaling for the treatment of type 1 diabetes

    As T cell toggle through their various life cycle, certain distinct biological changes occur that can be specifically targeted with small molecule drugs to target these cells for cell death. This is particularly true of activated autoreactive T cells. We have developed a combination of small molecular drugs that can target activated T cells and push these cells into apoptosis during (re-)activation. We propose here to test if these drugs can specifically target islet reactive T cells from T1D patients and pre-diabetic individuals while specifically sparing Treg and T memory cell subsets.