Current nPOD projects

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.

The prevalence of type 1 diabetes (T1D) is on the rise, posing a significant burden on healthcare systems. T1D results from the immune system mistakenly attacking insulin–producing pancreatic β–cells, necessitating lifelong insulin treatment and continuous monitoring of blood sugar levels. The complex inflammatory nature of T1D, coupled with substantial gaps in our understanding of the molecular drivers behind β–cell destruction, presents challenges in developing effective treatment strategies.

The primary challenge this project tackles is the need for more effective approaches to treat T1D by addressing the underlying cause–the destruction of pancreatic β–cells. This requires a thorough understanding of the molecular mechanisms responsible for β–cell damage. This project centers around the role of the Hippo signalling pathway, known for its involvement in regulating organ size and tissue hemostasis. We were the first to demonstrate the pivotal role of the Hippo signaling pathway in β–cell apoptosis, regeneration, and function. Pilot data indicate that the terminal effector of the Hippo pathway, Yes–Associated Protein (YAP), typically repressed in mature β–cells, is upregulated in both exocrine and endocrine compartments of individuals with T1D, acting as a potential regulator of β–cell apoptosis and islet inflammation. Therefore, elevated expression of YAP is a novel metabolic aspect of T1D. We hypothesize that elucidating the molecular mechanisms linked to YAP–induced damage is crucial for the development of targeted therapies for T1D.

The project’s primary objectives are to unravel the role of YAP and its molecular partners in T1D. These objectives include: (1) Identifying how YAP expression is activated in T1D and characterizing the cell types responsible for YAP upregulation. (2) Investigating the expression and cellular localization of YAP partners. (3) Determining spatial transcriptome profiling of YAP–expressing pancreatic cells in T1D to identify and characterize gene programs and signaling events shaping their niche identities. Utilizing the nPOD collection of healthy and diseased human pancreatic tissue, the project aims to unravel the molecular aspects of islet autoimmunity and β–cell destruction in T1D. This knowledge will serve as the foundation for new therapeutic strategies targeting the Hippo pathway to protect β–cells in T1D.

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.

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.

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.

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. 

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.

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.

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.

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.

     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.

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  eIF5A^Hyp) in the progression of diabetes in mice.  In previously published studies, we showed that eIF5A^Hyp 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 eIF5A^Hyp 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 eIF5A^Hyp in the human pancreas and spleen from persons with T1D, T2D and controls.  The results of this study will uncover whether the accumulation of eIF5A^Hyp-expressing cells are a marker for diabetic disease in the pancreas.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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 .

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.

The human pancreas is a vital organ responsible for both endocrine and exocrine functions, playing a crucial role in glucose metabolism and digestion. It originates from the foregut endoderm, with the dorsal and ventral buds forming and eventually fusing to develop into a mature pancreas. Key stages of pancreas development include specification, branching morphogenesis, and differentiation into endocrine and exocrine cell types (Jennings et al., 2015). Transcription factors play pivotal roles in pancreas development. Some of the key transcription factors include:

PDX1 (Pancreatic and Duodenal Homeobox 1): essential for early pancreas development and beta–cell differentiation (Offield et al., 1996); maintains progenitor cell populations and is involved in ductal and endocrine differentiation (Seymour & Sander, 2011),

NGN3 (Neurogenin 3): NGN3 marks endocrine progenitors and is crucial for islet cell development (Rukstalis & Habener, 2009).

NKX6.1: NKX6.1 Plays a key role in the maturation and function of beta cells (Sander & German, 1997)

RFX3: RFX3 is a critical transcription factor involved in the development and function of the endocrine pancreas. It plays a significant role in regulating gene expression necessary for the growth of cilia in pancreatic endocrine cells in mice, which is essential for maintaining the cellular composition of islets of Langerhans, insulin production, and glucose tolerance. RFX3 is also crucial for the differentiation and function of mature β–cells and directly regulates the β–cell promoter of the glucokinase gene, a key enzyme in glucose metabolism in the mouse pancreas ([Ait–Lounis et al., 2007] [Ait–Lounis et al., 2010]). While its role in mouse pancreatic development is less defined, its role in human pancreas development has never been explored before.

RFX6: RFX6 is a crucial transcription factor involved in the development and function of the pancreas, particularly in the differentiation of insulin–producing β–cells and regulation of insulin production. It is essential for the proper differentiation of endocrine cells, as seen in conditions like Mitchell–Riley Syndrome, where mutations in RFX6 lead to impaired pancreatic development (Trott et al., 2020).RFX6 also influences insulin synthesis by regulating genes involved in mRNA translation (Cheng et al., 2019)and mutations in this gene are linked to neonatal diabetes and other pancreatic dysfunctions (Concepcion et al., 2014), however, its role in human exocrine and endocrine pancreas has never been explored before.

Transcription factors belonging to the regulatory factor X (RFX) transcription factors are involved in regulating genes critical for ciliary development and function, playing roles in organogenesis and cellular differentiation across various tissues. The exact role of RFX3 in pancreas development is less understood but is thought to involve the regulation of gene expression patterns crucial for pancreatic differentiation (Chung et al., 2020). In this project, we are seeking to investigate the expression ofRFX3 and RFX6 in human fetal, neonatal, childhood and young adult pancreatic tissues to identify crucial novel factors of human pancreas development.

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.

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.

When a person is diagnosed with Type 1 diabetes (T1D), the amount of beta–cell 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[1–4]. These differ according to the degree of beta–cell loss, the composition of the islet–specific immune infiltrate, and how beta cells process insulin. Additionally, we now have preliminary data that suggests the beta–cells themselves may vary between the two forms), even hinting that successful beta–cell 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 beta–cell loss), construct a nuanced ‘pseudo’ timeline of disease progression in T1D to illustrate the life and loss of beta–cells in T1D and explore if this is the same in all people, randomly different between people– or 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.

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.

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.

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.

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.

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)

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

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.

Type 1 diabetes (T1D) is a pancreatic disease marked by the autoimmune destruction of insulin–secreting beta cells within the endocrine compartment, leading to chronic hyperglycemia and long–term 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 non–diseased control donors, have an elevated expression of a MHC class II molecule, HLA–DR. MHC class II molecules are known to be expressed by antigen presenting cells (APCs) that process and present extracellular antigens to CD4–expressing 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 HLA–DR 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 non–diseased donor tissues, using co–immunofluorescence 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.

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.

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. 

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.

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).

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.

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.

When a person is diagnosed with Type 1 diabetes (T1D), the amount of beta–cell 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[1–4]. These differ according to the degree of beta–cell loss, the composition of the islet–specific immune infiltrate, and how beta cells process insulin. Additionally, we now have preliminary data that suggests the beta–cells themselves may vary between the two forms), even hinting that successful beta–cell 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 beta–cell loss), construct a nuanced ‘pseudo’ timeline of disease progression in T1D to illustrate the life and loss of beta–cells in T1D and explore if this is the same in all people, randomly different between people– or 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.

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.  

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.

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. 

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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. 

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.

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 Co-Detection-by-InDEXing (CODEX), a multiplexed single-cell imaging technology that incorporates DNA barcoded antibodies to visualize 100 + cellular markers at the single-cell level to map islet stromal cells and TLS in nPOD samples to map tissue architecture and characterize stromal-immune 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 sub-types of stromal 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 have 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. 

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.

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.

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.

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. 

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.

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.

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. 

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.

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.

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.

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.

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.     

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.

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.

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.

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.

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.

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.  

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.

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.

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. 

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. 

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.

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.  

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.

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.

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. 

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.

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.

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.

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. 

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)

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.

Diabetes occurs when insulin producing pancreatic β-cells are impacted: type 1 diabetes due to destruction of β-cells, and type 2 diabetes due to a decrease in their number and/or potency. Consequently, therapy that aims to replace β-cells is a promising long term alternative to the currently prevalent insulin injections. However, the difficulty in finding viable sources to generate functional β-cells remains a crucial bottleneck. Herrera and colleagues recently discovered that under conditions of near-total depletion of β-cells in mice, only α-cells within pancreatic islets spontaneously convert into β-cells1. Encouragingly, there is in fact an overabundance of α-cells in the pancreas of diabetes patients. However, today we lack the knowledge to initiate and maintain α-to-β-cell conversion and are thus unable to exploit the availability of abundant α-cells to orchestrate this process reproducibly in humans.

Exciting preliminary results from our laboratory and others reveal that a subset of pancreatic α-cells in T1D juvenile donors lose α-cell gene expression and acquire the expression of several key β-cell genes including Insulin. These data suggest that α-to-β-cell conversion may have occurred in these T1D donors. Further, we identified genes that likely control α-cell fate by recapitulating our T1D findings in mice that were engineered to delete specific genes (Dnmt1 and Arx) in their α-cells. The work proposed here aims to extend our findings in multiple T1D donor samples with long or short disease durations. To our knowledge, the question of whether α-to-β-cell conversion occurs in T2D has not been previously studied.

We therefore request pancreas samples from T2D donors to analyze them for evidence towards α-to-β-cell conversion. We will use state-of-the-art technologies to enable us to isolate α-cells undergoing conversion and probe them for changes in their genetic and epigenetic landscape. These studies are expected to a) Elucidate the similarities and differences between the fates of pancreatic α-cells in early and late stage T1D, and between T1D and T2D, and b) Identify new molecules and pathways that can be targeted for developing drugs to enhance the generation of functional β-cells.

The progression of type 2 diabetes is mediated to a large extent by the deaths of β-cells, the cells in the pancreas that synthesize and secrete insulin.  We discovered that ARC (Apoptosis Repressor with CARD), an endogenous inhibitor of cell death, is expressed at high levels in β-cells of the mouse pancreas.  Using cell culture studies and genetic mouse models, we have further demonstrated that ARC functions as a critical β-cell survival factor during type 2 diabetes. However, little is known about the expression patterns of ARC in human β-cells and other cells within the endocrine pancreas.  This information is critical for understanding the role of ARC in the pathogenesis of diabetes in humans.  We have assessed whether ARC levels in islets decrease during diabetes, and found no significant changes in ARC abundance in old versus young ob/ob mice.  Unexpectedly, however, we observed that the subcellular localization of ARC differs in diabetic versus non-diabetic mice. In this study, we propose to characterize ARC expression in islet cells of human pancreatic tissue from non-diabetic individuals and those with type 2 diabetes.  

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

We cloned a genetic modifier of T2D in obese mice. The gene, Ildr2, encodes a molecule that plays a role in both lipid metabolism and cellular responses to stress.  Animals with lower levels of expression of this gene has reduced number of insulin producing cells in the pancreas, and, ultimately went on to develop mild diabetes.  How the apparent functions of Ildr2 account for these effects is currently under study by us using genetically manipulated mice.

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

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

We hypothesize that the alterations seen in diabetic BM are a major cause of impaired homeostasis in other vascular beds. Main objectives (1) to obtain a deeper insight into the cellular and vascular composition of diabetic BM using IHC, flow cytometry, and expressional studies (2) to determine whether BM microangiopathy is associated to poor metabolic control and increased susceptibility of cardiovascular complications. (adopted by nPOD staff)

Recent evidence derived from animal models of type 1 diabetes indicates that interaction of immune cells expressing the protein known as NKG2D with cells expressing other proteins, termed NKG2D ligands, plays an essential role in initiating diabetes development. In an effort to define more precisely the role these proteins play in type 1 diabetes progression, we developed novel mouse models. Based on results generated with these experimental models, we believe that expression of NKG2D ligands expressed on infiltrating immune cells within pancreatic islets is critical to the development of type 1 diabetes. Here we propose to confirm there is NKG2D ligand expression in the pancreas of type 1 diabetic donors.

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

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

The downregulation of cyclin D3 due to infiltration is causally related to beta cell dysfunction and beta cell-apoptosis in beta cells from NOD mice. We would like to confirm whether this is true in human beta cells. In order to validate cyclin D3 as a target for T1D therapy in humans, we need to evaluate the impact of inflammation on the expression levels of cyclin D3 in pancreatic beta cells from individuals at risk of developing T1D, newly diagnosed T1D patients and, non-medalist overtly diabetic patients, in comparison to control individuals, as an approach to study the disease in humans in relationship to cyclin D3 expression levels, as we have observed in NOD mice.

There is conflicting information regarding the role of natural killer (NK) cells in the development of Type 1 Diabetes (T1D).  NK cells represent an important effector arm against viral infection, and mounting evidence suggests that viral infection plays a role in the development of T1D in at least a portion of patients.  NK cells of diabetes prone NOD mice express high levels of the pro-inflammatory mediator 12-lipoxygenase (12-LO), and decreased levels of stimulatory receptors.  NK cells of 12-LO deficient NOD mice, conversely, express significantly higher levels of stimulatory receptors.  We previously found that 12-LO was upregulated in the pancreas of both T1D and T2D donors with insulin-containing islets.  It is not clear how this 12-LO becomes upregulated, and whether or not additional sources of 12-LO may contribute to diabetes pathogenesis.

Therefore, we propose to study NK cells that reside in the duodenum of non-diabetic, T1D, and AAb+ donor samples.  These cells will be characterized for their mRNA expression levels of anti-viral responses, as well as the expression of 12-LO and various key NK cell receptors.  Further studies will assess the functionality of NK cells from different donors with respect to their ability to produce IFN-gamma and lyse target cells.  Given the direct connection of the duodenum to the pancreas via the bile duct, there is a great potential for the immune cells in this area of the gut to influence the development of autoimmunity.

We have previously used Imaging Mass spectrometry (IMS) to identify Insulin within β-cells in the islets of Langerhans. The analysis utilized specially prepared pancreatic sections from the Network of Pancreatic Organ Donors with Diabetes (nPOD). In these studies, a comparative analysis of T1D MS spectra to those of Non-T1D revealed several uniquely expressed proteins between the two groups.  In the current study, we propose to perform inter- and intra-sample qualitative and quantitative analyses to verify protein expression between normal and T1D tissue samples in an expanded subset of samples.  We have developed methods that can distinguish proteins that are differentially present in T1D and non-diabetic samples. To this end, we have recently acquired a new instrument with a higher mass accuracy, resolution and sensitivity (Q-Exactive mass spectrometer, Thermo Fisher Scientific) on which we will perform LC-MS-MS analysis of additional samples in order to verify and validate the previously defined panel.  Therefore, we propose to identify and develop quantitative assays to determine the utility of these molecules as potential diagnostic biomarkers for T1D by continuing our studies in additional pancreas samples. 

The Noble lab produces HLA genotyping for all nPOD samples.

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

References:

1.             Perron H, Germi R, Bernard C, et al. Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease. Mult Scler. 2012; 18: 1721-36.

2.             Dolei A and Perron H. The multiple sclerosis-associated retrovirus and its HERV-W endogenous family: a biological interface between virology, genetics, and immunology in human physiology and disease. J Neurovirol. 2009; 15: 4-13.

3.             Gaudin P, Ijaz S, Tuke PW, et al. Infrequency of detection of particle-associated MSRV/HERV-W RNA in the synovial fluid of patients with rheumatoid arthritis. Rheumatology (Oxford). 2000; 39: 950-4.

The rapid worldwide incidence increase suggests a major role for environmental factors in the aetiology of type 1 diabetes (T1D). According to cross-sectional and prospective studies on T1D patients and/or prediabetic individuals, virus infections may be one of these. Influenza viruses are known to cause severe pancreatic damage in birds and in some mammals, and we have preliminary strong evidence that these virus growth in primary pancreatic islet (human and mouse) cultures “in vitro”. The objective of our project is to demonstrate that influenza viruses may play a role in the etiopathogenesis of T1D. Due to the interdisciplinary nature of this study, two different Research Institutes will be involved: Istituto Zooprofilattico Sperimentale delle Venezie (I Capua, PI) for the influenza virus expertise and the San Raffaele Diabetes Research Institute (L Piemonti, CoPI) for the diabetes expertise.

The overall goal of this research is to demonstrate that influenza virus infection may be involved in the etiopathogenesis of diabetes.

Specifically we will aim to:

1)      Test “in vitro” the ability of influenza viruses to infect, replicate, and damage beta cell, pancreatic islet and endocrine cell

2)      Test “in vivo” (mouse) the ability of influenza viruses to induce beta cell damage and/or alterations of glucose metabolism

3)      Look for direct and/or  indirect  evidences of correlation between influenza viruses infection and autoimmunity/diabetes onset in humans

For the aim 3 we are testing the presence of influenza virus in pancreas at disease onset in T1D patients. For this purpose tissue samples are asked to the Network for Pancreatic Donors with Diabetes (nPOD). In situ hybridization will be performed to visualize viral RNA localized within cells using the Quantigene ViewRNA technique based on branched DNA signal amplification technology. Moreover the presence of influenza virus will be eventually confirmed by digital PCR and immunohystiochemistry

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing islet cells are targeted and destroyed by the immune system, especially by T cells. The events that lead to immune recognition of islet proteins in people genetically predisposed to autoimmunity are poorly understood. Many of the environmental triggers associated with T1D including viral infection, exposure to chemicals or free radicals, and dynamic glucose sensing, however, cause cellular stress within islets. In addition, the natural function of islet cells (to produce and release insulin) makes these cells especially susceptible to stress. The cells respond to stress by activating a program that protects them from death, but this results in mishandling of proteins leading to changes in there normal structure. In healthy individuals, this mishandling of proteins is not a problem; however, in individuals prone to autoimmunity, these proteins and the cells that make them may be targeted by the immune system. We hypothesize that the environmentally related events leading to this cellular stress in islet cells causes abnormal modification of proteins which in genetically predisposed patients, leads to abnormal immune system targeting causing the destruction of the islets. We have tested this hypothesis using a model system of T cell recognition of islets, in particular an islet protein called chromogranin A (CHgA) in a well established mouse model of T1D, demonstrating that T cells respond much more strongly to islets exposed to environmental triggers of cellular stress. These data support the hypothesis that cellular stress changes the proteins in the islet, and these changes facilitate the recognition of islets by T cells. We are now proposing to translate these findings further using human islet-reactive T cells, human islets and a humanized mouse model of T1D. The overarching goal of these studies is to determine in the humanized T1D animal model if the same modifications are demonstrated under conditions of cellular stress, and to determine how these modifications lead to the recognition of islets by the immune system. These studies are important because understanding how islet proteins come to be recognized by T cells will reveal targets for therapeutic intervention to prevent incorrect modification of islet proteins, protect islets from the immune system, and ultimately prevent T1D. 

There is growing evidence that memory T cells are associated with a variety of autoimmune conditions, including type 1 diabetes (T1D). While autoreactive T cells are detected in both patients and healthy control subjects, CD45RO+ memory autoreactive T cells are usually found in patients but not in controls. Our own studies in several pancreas transplant recipients with T1D recurrence have shown that autoreactive memory T cells can be found in the circulation and pancreas transplant lymph nodes. Moreover, the examination of pancreas transplant biopsies from two of such patients shows that most (~80%) of the islet infiltrating T cells have a memory phenotype. Based on this findings we hypothesize that memory cells should be present in the islet infiltrates of nPOD patients. Specifically, we plan to study pancreatic tissue obtained from patients with proven insulits for the presence of the T cells bearing the memory marker CD45RO. It is plausible that the frequency of the memory cells may be related to disease duration, and perhaps this could be higher in the lesions of patients with longer disease duration who still have insulitis. It is also possible that there will be a difference compared to pancreas transplant recipients with recurrent disease. Understanding whether memory T cells represent a significant component of islet infiltrating T cells is relevant to a more complete characterization of the insulitis. A recent clinical trial in new onset patients has tested Alefacept, an anti-memory T cell drug. Demonstrating memory T cells in the lesion would provide further rationale for regimens that target memory T cells, with Alefacept or with new drugs that have similar effects. Furthermore, if memory cells are demonstrated, we can propose additional work to better define the memory phenotype by staining for additional markers.

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

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

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

ADDENDUM: Despite considerable efforts directed at finding a cure for type 1 diabetes (T1D), the molecular processes that initiate and perpetuate beta cell destruction and how and why beta cells become targets of immune destruction are poorly understood.This knowledge gap and the realization that the underlying processes of T1D and the age of onset of T1D are heterogeneous have compounded progress in finding a cure.In addition, evidence of beta cell dysfunction is present well before clinical onset.

Beta cells have high rates of metabolism, protein synthesis and secrete insulin continuously in a pulsatile fashion and with increased output in response to meals. We propose that the highly metabolic beta cells are more susceptible to environmentally-induced insults, that oxidant-induced beta cell stress, leading to generation of abnormal cellular proteins due to their intrinsic inability to clear excess free radicals. The resulting abnormal proteins compromise beta cell function and act as antigenic targets of immune-mediated destruction.

Environmental factors such as certain viruses or toxins can also lead to excess and uncontrolled generation of reactive oxygen species (ROS) in beta cells, resulting in stress.Thus, sustained beta cell stress may be a key determinant of beta cell damage. In this research, we wish to demonstrate if the remaining beta cells from cases with new-onset T1D and those with longer duration of disease have low levels of three key ROS clearing enzymes, and express detectable levels of interferon-α (IFN-α), a marker of virally-infected beta cells and which my contribute to oxidative stress.The endoplasmic reticulum (ER), a vital subcellular organelle, is responsible for correctly processing and folding of nascent proteins and is exquisitely sensitive to imbalances in oxidative stress. We also wish to examine if oxidative stress disturbs the redox balance and promotes stress within the ER.

Our studies will provide key insights on why beta cells become targets of self-immunity and may pave the way for the development of future therapies aimed at alleviating beta cell stress in the early stages of type 1 diabetes and even prior to its onset and thus, prevent this serious disorder.

nPOD participated in the ImmunoChip consortium, that was established to design a cost effective genotyping array in order to fine map well established GWAS reported risk loci in immunologically related human diseases, including type 1 diabetes.

To determine the specificities and frequencies of islet autoreactive CD8 T-cells in pancreas draining lymph nodes, control lymph nodes, spleen and/or peripheral blood from diabetic and non-diabetic organ donors, we used the Diab-Q-kit, a nanotechnology that allows the direct detection of circulating autoreactive CD8 T-cells against an array of islet-epitopes simultaneously, reducing the needs of patient resources. The detection of these T-cells is based on the T-cell receptor (TCR) specificity for its peptide-HLA ligand. We produced HLA-monomers for an array of islet epitopes presented in the context of HLA -A2, -A3 and -B7. This allowed expanding the number of patients that could be analyzed considerably. Moreover, to characterize the memory phenotype of the autoreactive T-cells, we validated and upgraded the Diab-Q-kit through the inclusion of cell surface markers to distinguish naïve, effector memory and central memory T-cells. From the nPOD repository we selected a total of 29 cases to be analysed for this study from which we could obtain cell suspensions from blood and pancreas draining lymph nodes, control lymph nodes and/ or spleen.

Upon thawing of the samples we unexpectedly observed that far fewer viable cells could be recovered from the majority of the samples than the number reported to be frozen on the vial. For some cases, we also counted the number of dead cells in each vial. We found that the percentage of dead cells was very high in some cases, but not all. As an internal control, we always included samples from healthy controls collected and cryopreserved in our laboratory, which showed very high viability after thawing (>90%), indicating that the thawing procedure was correctly performed.

Similar issues on viability and recovery have since been reported by other partners in nPOD. Importantly, the quality control to detect any issues was critical to identify these problems and potential causes. For instance, issues with viability and recovery were determined immediately after thawing, while FACS analyses performed hours after thawing of the samples would have suggested excellent viability outcome, even in cases that QC earlier on had identified poor recovery and viability.

The consequences of low viability and recovery may differ depending on the type of analysis per project (functional assays vs. mRNA) and incentive (cloning T-cells may still be possible, but may not be representative of the total tissue sample). Such issues have been evaluated in depth and at length between the central facility and participants of nPOD. Indeed, the nPOD central facility was indispensable in identifying important correlations between recovery, viability and donor characteristics that may differ between tissue source of the leukocytes and that need not be independent co-variates ( PBMC: demographics, cause of death [anoxia vs > CV/stroke], disease [T2D/Ab+>T1D], donor age [o>y]; splenocytes: donor age [y>o], BMI [low>hi], transmission time [short>long]),  underscoring that the thawing procedure was not the primary parameter of poor outcome and implying that intrinsic features of the biosamples determines quality and reward.

Despite lower cell recovery than expected, we nevertheless proceeded performing the Diab-Q-kit analysis on all samples, to take maximal knowledge, benefit and reward from the precious nPOD material. Unfortunately, for more than half of the samples no reliable data could be obtained, due to the low number of CD8 T-cells present in each sample. This precluded reliable comparisons in frequencies and specificities of islet-autoreactive CD8 T-cells in the individual organs. The results from this study have been discussed at length with nPOD, and it has helped the nPOD consortium to decide on the feasibility of projects where viable cell suspensions are needed for analysis and functional assays. Measures have been undertaken to adjust allocations of samples on basis of this important new insight.

Cystic fibrosis related diabetes is a major morbidity for patients with cystic fibrosis and negatively impacts mortality.  The mechanistic basis for this form of diabetes is not well understood.  In this project we are analyzing the hypothesis that islet inflammation leads to islet amyloid deposition and beta cell failure in patients with CFRD.  Samples from the nPOD repository are being stained for both amyloid and interleukin 1 beta.   In the future we will look at additional inflammatory mediators in the islets.

Human chemokine superfamily of molecules mediate the recruitment of leukocytes to sites of inflammation. Many chemokines and chemokine receptors have emerged as key contributors and regulators of autoimmune disorders, including Type 1 Diabetes and may serve as potential surrogate biomarkers of inflammation or possible drug targets. More than one-half of all human and rodent chemokines have been implicated in the pathogenesis or complications of Type 1 diabetes. Most of the previously published work however is limited to the genetic association studies and chemokine and their receptor analyses in the peripheral blood. To delineate the complete spectrum of chemokine mRNA species transcribed by human islets in response to inflammatory stimuli, we used an established in vitro culture system in which islets purified from healthy organ donors were cultured in the absence versus the presence of IL-1β, TNFα, and IFNγ and subsequently subjected them to gene expression microarray analysis. Based on our microarray data and in collaboration with Homann laboratory, we study the expression and localization of chemokines in pancreatic tissue sections obtained through the Network for Pancreatic Organ Donors with Diabetes (nPOD) and included healthy control subjects.

The etiology of autoimmune diabetes is not known. However, the disease is accompanied by varying amounts of immune activation and inflammation. Although genetics clearly plays a role in determining who gets the disease, we do not know the instigating events. There has been a lot of investigation into the potential role of virus infections in triggering disease. However, there are no direct associations known between virus and disease in humans.

Recently, there has been increased interest in issues such as intestinal permeability and the microbiome in diabetes patients. One theory is that the leaky gut in T1D patients allows the penetration of bacteria and/or their products into the body, causing immune stimulation. When such products arrive into the pancreas after draining from the ducts, they cause changes in gene expression and inflammation. One set of factors that would get induced is bactericidal proteins, whose expression or subcellular localization is affected by exposure to bacterial products.

We have studied the expression of bactericidal proteins in circulation in new onset T1D patients. These studies indicated that there were coordinately high levels of expression in circulation in T1D patients less that five years after disease onset. These results suggest the upregulation of anti-bacterial responses in T1D patients. In this project, we will determine whether bactericidal factors are upregulated in the pancreas in T1D patients.

This project is important because it will tell us whether the disease may be mediated by the release of bacteria or their products into the pancreas, which would cause immune activation and inflammation. Several different bactericidal response elements will be investigated, each reflecting exposure to different bactericidal products including DNA, cell membrane constituents, and pigment molecules, an each providing an indication of the type of exposure the pancreas has encountered.

We will select T2D patients, among them subjects with DNA variants, and profile gene expression, expressed mutants, and proteins from sub-areas down to the level of single islets and single cells within archived pancreas FFPE tissues using a novel in situ TO-Seq assay coupled with immunohistochemistry staining of protein biomarkers, retaining the morphologic context of the profiling data. Among the set of genes measured will be biomarkers of every known molecular pathway in addition to genes associated with the development, presentation, dysregulation, and pathogenesis of T2D. By obtaining in situ gene expression profiles defining for all the cell types involved in diseased vs normal tissue within both their morphologic context and the context of genetic variants we will be able to identify molecular phenotypes and mechanisms and correlate these to cell-cell interaction, tissue homeostasis, and genetic variants. Because TO-Seq is a targeted sequencing-based assay that can measure thousands of genes, and multiplex many samples within a single sequencing run it will enable profiling data to be obtained from many separate cells at an affordable cost/cell in order to achieve the statistical power required to produce quality data supporting reliable interpretation and bioinformatics analysis. No such data exists today, and there is no system capable of producing such data on a large scale at an affordable price. Laser capture microdissection (LCM) can produce single cell profiles within their morphological context, but at a high cost which severely limits the number of samples that can be profiled. We provide preliminary data demonstrating excellent correlation and sensitivity of TO-Seq measurement of gene expression from matched fixed and frozen tissue and successful implementation of the in situ TO-Seq protocol and its integration with immunohistochemical (IHC) staining of antibody biomarkers. We demonstrate the feasibility of performing single (prostate) gland profiling with this assay. Based on these preliminary data we will optimize the in situ TO-Seq/IHC assay, implement a single islet and cell sampling system benchmarked against LCM, implement the content for the assay, and then profile the cell types present in pancreas of normal vs T2D, and between areas of pancreas exhibiting a normal vs T2D histopathology. From these data we expect to gain an understanding of the impact of genetic variant(s) at the level of gene (and protein) expression, cell interactions, and upstream regulation, and molecular mechanisms and phenotypes as they potentially contribute to disease and its progression.

It was earlier reported the finding that the incidence of type 1 diabetes correlates with small rodent abundance in Sweden. This and other findings initiated an effort to isolate novel infectious agents from small rodents in an attempt to find an etiological agent for type 1 diabetes in humans. As a result of these efforts, a new picornavirus (Ljungan virus [LV]) was isolated from bank voles trapped in Sweden. Moreover, it has been shown that more than one-third of wild-caught bank voles and their offspring develop diabetes when kept under laboratory conditions. This was accompanied by changes in pancreatic islet structure, islet cells becoming immunohistochemically positive for LV and elevated levels of islet autoantibodies. The finding of LV in the pancreatic islets of the diabetic bank voles raised the general question whether or not LV can participate in the development of diabetes in rodents as well as in humans.

In pilot experiments using human pancreatic specimens from nPOD, we have found faint immunostaining for LV in two autoantibody-positive patients using a rabbit anti Ljungan virus protein 1 (LV VP1) antibody. Other type 1 diabetic patients, as well as non-diabetic controls, stained negative for LV in the pancreas. Due to the weak intensity of the immunostaining, results are inconclusive and not further pursued at this time.

Impairment and destruction of beta cells by an autoimmune attack leads to the onset of hyperglycemia in type 1 diabetes (T1D). However, knowledge on changes in islet cell physiology leading to dysfunction, failure and death of the cells is incomplete. This is partially due to technical limitations which do not allow the study of islet function within intact pancreatic tissue, but rely on the enzymatic isolation of islets. During isolation islets suffer from enzymatic and mechanical stress. After isolation the islets are separated from their surrounding tissue, including most of the infiltrating cells. In addition, enzymatic isolation of structurally damaged islets is challenging and hampers the study of islet physiology in T1D.

Our project aims to establish a pancreas tissue slice platform for the study of human islet physiology in the pathogenesis of T1D. Pancreas tissue slices allow the study of islet physiology in an intact environment in situ, avoiding isolation stress and separation from their surroundings. In rodents pancreas tissue slices have been successfully used to study rodent islet biology under various conditions. We recently established the pancreas tissue slice platform using human pancreas. We plan to establish this technique at the Diabetes Research Institute, University of Miami, and at the nPOD Organ Processing and Pathology Core, University of Florida. This will enable the use of pancreas tissue slices for the ex-vivo study and functional assessment of nPOD pancreata. Furthermore, we aim to develop an organotypic culture platform to allow investigating human islets within intact pancreatic tissue over several days. Finally, we will assess the feasibility of this culture platform by using exposure to viruses as a model system to explore effects on islet cell pathophysiology. We believe combination of this unique network with our novel approach will facilitate new insight into the pathogenesis of diabetes. Of note, this platform will empower additional studies to be conducted, beyond those described here. This effort is conceived as a collaborative working group effort which will seek support from the Helmsleüy Charitable Trust George Eisenbarth nPOD Award for Team Science.

The therapeutic treatment of type 1 and type 2 diabetes mellitus (T1DM, T2DM) has vastly improved over the past decades; however complete normalization of glucose remains elusive without proper functioning pancreatic β cells. Truly, sustained and effective treatment of T1DM and T2DM must involve retaining/replacing the function of pancreatic β cells. To this end, we must understand the molecular mechanisms that govern β cell development and function along with understanding the molecular progression of the disease.

The Stein lab and others have focused on the large Maf transcription factors, MafA and MafB, and their role in the β cell. Loss of MafB impedes normal β cell development in mouse models whereas loss of MafA restricts normal adult β cell function. In recent published data, we have shown that MafA and MafB are lowered in db/db mouse models and T2DM donor samples. Collectively, this data reveals the importance of MafA and MafB in normal β cell function and presents an area of research that needs to be further examined. 

Using nPOD, we are currently characterizing transcription factor levels and investigating coregulator interactions in T1DM and T2DM donor samples. This data is crucial to the understanding of the human β cell situation and vital for β cell replacement therapy protocols. 

Though the pancreatic islets are known to play critical roles in both Type 1 diabetes (T1D) and Type 2 diabetes (T2D), the distinct mechanisms are poorly understood. Recent findings demonstrate that chemical heterogeneity exists within the classic islet endocrine cell types, even in the normal/healthy pancreas setting. For example, there are subtypes of beta cells with distinct transcriptomes that respond differently to chemical signals. One critical scientific question is how does this cellular heterogeneity influence chemical signaling in T1D and T2D? We hypothesize that characteristic changes in chemical heterogeneity within the various human islet cell types during diabetes progression alter the signaling pathways inside and outside of the pancreatic islets to potentiate disease. Determining the dynamics of these changes will provide insight into chemical mechanisms that can be exploited for the prevention and treatment of T1D and T2D. Hence, there is a need for high-throughput and highly sensitive technologies capable of probing a sufficient number of chemical constituents in single cells. We propose three unique measurement platforms that implement advanced single cell mass spectrometry and analyte separation techniques to characterize the peptide and small-molecule content in individual human islet cells affected by T1D and T2D: 1) high-throughput single cell matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS); 2) quantitative capillary electrophoresis (CE)-MS with a focus on small molecule metabolites; and 3) CE-laser induced fluorescence (LIF) and liquid chromatography (LC)-MS characterization of the levels of D-amino acids. We expect that this research will dramatically improve the understanding of the cellular mechanisms of both T1D and T2D pathogenesis, and elucidate new chemical parameters characteristic of each disease, helping to identify novel pathways for therapeutic intervention.

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

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

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

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.

In Type 1 Diabetes (T1D), the complex interplay of immune cells shifts the balance in favor of the development of lymphocytes, that attack structures of our own body: autoimmunity. These attacks resulting in an inflammation in the pancreas and, eventually, to the destruction of insulin producing beta cells.

Cytokines are small proteins that are released by cells and affect the behavior of other cells. They are oftentimes involved in promoting inflammation. We believe, that a network of “cytokines” determines the direction of the immune response towards the destruction of beta cells, and, as recent findings suggest, that this inflammatory milieu precedes the onset of the disease. However, it remains elusive so far, which cytokines are produced and when, and how they interact in such a complex network to sustain inflammation.

In this project, we use highly sophisticated chemical and bioinformational tools to detect important cytokines that are known to promote inflammation: Interleukin 1beta, Interleukin 6, and Tumor Necrosis Factor alpha in tissue pieces from diabetic patients, who have donated their pancreas for research.

This allows us to analyze both the presence of cytokines as well as their interactions during the very early days of diabetes at the single cell level and to create a so called “cytokine map” of the disease. Knowing which cytokine is released by which cell and when is a first step towards the development of very specific therapies for the treatment of diabetes and of other autoimmune diseases.

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

Type 1 diabetes results, at least in part, from invasion of pancreatic islets by T lymphocytes and antigen presenting cells (APCs) including macrophages and dendritic cells (DCs). What drives the migration of these immune cells into the islets remains unknown. A key step in the arrival of immune cells to a specific location is adhesion to the inner lining of blood vessels and then transit through the vessel wall into the surrounding tissue. Chemokines are important proteins in this process. Chemokines that are expressed by the cells making up blood vessels alert immune cells to nearby inflammation and assist the immune cells with leaving the vessel. One chemokine with this function is CCL21. Activated APCs and naïve T cells express the surface receptor CCR7 that binds CCL21 and directs the cells towards environments where CCL21 is present. We found through profiling of genes expressed in the islets of T1D subjects in the nPOD cohort that CCL21 levels are elevated in the islets that have been infiltrated by T cells but not islets lacking T cells. We hypothesize that endothelial cells of the islet are expressing CCL21 and that drugs that block CCL21 activity can prevent destructive immune invasion of the islets. This study is designed to determine where in the islet environment CCL21 protein is found. Since immune infiltrates are focal, we anticipate that CCL21 will co-localize with regions of the islet where APCs or naïve T cells have infiltrated.

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

Type 1 Diabetes (T1D) is a disease that results from the autoimmune destruction of insulin-producing beta cells in the pancreas. The current treatment for T1D does not cure the disease or prevent the possibility of other serious complications. The mechanism that leads to the loss of immune tolerance still remains unclear; scientists believe that both genetic and environmental triggers play a role, making it challenging to develop treatments that address both. Investigators at the Columbia University Center for Translational Immunology (CCTI) conduct many research studies aimed to determine factors involved in T1D. The purpose of our study is to investigate genetic variants limited to immune dysregulation in T1D through next generation sequencing of genetic material from tissues of T1D donors. Elucidating the genetic factors involved in T1D will contribute to our knowledge of this disease and autoimmunity.