Current nPOD projects

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

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.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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.