Network For Pancreatic Organ Donors Research Articles

Enteroviral infections are not associated with type 2 diabetes.

For more than a century, enteroviral infections have been associated with autoimmunity and type 1 diabetes (T1D). Uncontrolled viral response pathways repeatedly presented during childhood highly correlate with autoimmunity and T1D. Virus responses evoke chemokines and cytokines, the "cytokine storm" circulating through the body and attack cells especially vulnerable to inflammatory destruction. Intra-islet inflammation is a major trigger of β-cell failure in both T1D and T2D. The genetic contribution of islet inflammation pathways is apparent in T1D, with several mutations in the interferon system. In contrast, in T2D, gene mutations are related to glucose homeostasis in β cells and insulin-target tissue and rarely within viral response pathways. Therefore, the current study evaluated whether enteroviral RNA can be found in the pancreas from organ donors with T2D and its association with disease progression. Pancreases from well-characterized 29 organ donors with T2D and 15 age- and BMI-matched controls were obtained from the network for pancreatic organ donors with diabetes and were analyzed in duplicates. Single-molecule fluorescence in-situ hybridization analyses were performed using three probe sets to detect positive-strand enteroviral RNA; pancreas sections were co-stained by classical immunostaining for insulin and CD45. There was no difference in the presence or localization of enteroviral RNA in control nondiabetic and T2D pancreases; viral infiltration showed large heterogeneity in both groups ranging from 0 to 94 virus+ cells scattered throughout the pancreas, most of them in the exocrine pancreas. Very rarely, a single virus+ cell was found within islets or co-stained with CD45+ immune cells. Only one single T2D donor presented an exceptionally high number of viruses, similarly as seen previously in T1D, which correlated with a highly reduced number of β cells. No association of enteroviral infection in the pancreas and T2D diabetes could be found. Despite great similarities in inflammatory markers in islets in T1D and T2D, long-term enteroviral infiltration is a distinct pathological feature of T1D-associated autoimmunity and in T1D pancreases.

A genomic data archive from the Network for Pancreatic Organ donors with Diabetes

The Network for Pancreatic Organ donors with Diabetes (nPOD) is the largest biorepository of human pancreata and associated immune organs from donors with type 1 diabetes (T1D), maturity-onset diabetes of the young (MODY), cystic fibrosis-related diabetes (CFRD), type 2 diabetes (T2D), gestational diabetes, islet autoantibody positivity (AAb+), and without diabetes. nPOD recovers, processes, analyzes, and distributes high-quality biospecimens, collected using optimized standard operating procedures, and associated de-identified data/metadata to researchers around the world. Herein describes the release of high-parameter genotyping data from this collection. 372 donors were genotyped using a custom precision medicine single nucleotide polymorphism (SNP) microarray. Data were technically validated using published algorithms to evaluate donor relatedness, ancestry, imputed HLA, and T1D genetic risk score. Additionally, 207 donors were assessed for rare known and novel coding region variants via whole exome sequencing (WES). These data are publicly-available to enable genotype-specific sample requests and the study of novel genotype:phenotype associations, aiding in the mission of nPOD to enhance understanding of diabetes pathogenesis to promote the development of novel therapies.

The extent and magnitude of islet T cell infiltration as powerful tools to define the progression to type 1 diabetes

Aims/hypothesisInsulitis is not present in all islets, and it is elusive in humans. Although earlier studies focused on islets that fulfilled certain criteria (e.g. ≥15 CD45+ cells or ≥6 CD3+ cells), there is a fundamental lack of understanding of the infiltration dynamics in terms of its magnitude (i.e. how much) and extent (i.e. where). Here, we aimed to perform an in-depth characterisation of T cell infiltration by investigating islets with moderate (1–5 CD3+ cells) and high (≥6 CD3+ cells) infiltration in individuals with and without type 1 diabetes.MethodsPancreatic tissue sections from 15 non-diabetic, eight double autoantibody-positive and ten type 1 diabetic (0–2 years of disease duration) organ donors were obtained from the Network for Pancreatic Organ Donors with Diabetes, and stained for insulin, glucagon, CD3 and CD8 by immunofluorescence. T cell infiltration was quantified in a total of 8661 islets using the software QuPath. The percentage of infiltrated islets and islet T cell density were calculated. To help standardise the analysis of T cell infiltration, we used cell density data to develop a new T cell density threshold capable of differentiating non-diabetic and type 1 diabetic donors.ResultsOur analysis revealed that 17.1% of islets in non-diabetic donors, 33% of islets in autoantibody-positive and 32.5% of islets in type 1 diabetic donors were infiltrated by 1 to 5 CD3+ cells. Islets infiltrated by ≥6 CD3+ cells were rare in non-diabetic donors (0.4%) but could be found in autoantibody-positive (4.5%) and type 1 diabetic donors (8.2%). CD8+ and CD8− populations followed similar patterns. Likewise, T cell density was significantly higher in the islets of autoantibody-positive donors (55.4 CD3+ cells/mm2) and type 1 diabetic donors (74.8 CD3+ cells/mm2) compared with non-diabetic individuals (17.3 CD3+ cells/mm2), which was accompanied by higher exocrine T cell density in type 1 diabetic individuals. Furthermore, we showed that the analysis of a minimum of 30 islets and the use of a reference mean value for T cell density of 30 CD3+ cells/mm2 (the 30–30 rule) can differentiate between non-diabetic and type 1 diabetic donors with high specificity and sensitivity. In addition, it can classify autoantibody-positive individuals as non-diabetic or type 1 diabetic-like.Conclusions/interpretationOur data indicates that the proportion of infiltrated islets and T cell density change dramatically during the course of type 1 diabetes, and these changes can be already observed in double autoantibody-positive individuals. This suggests that, as disease progresses, T cell infiltration extends throughout the pancreas, reaching the islets and exocrine compartment. While it predominantly targets insulin-containing islets, large accumulations of cells are rare. Our study fulfils the need to further understand T cell infiltration, not only after diagnosis but also in individuals with diabetes-related autoantibodies. Furthermore, the development and application of new analytical tools based on T cell infiltration, like the 30–30 rule, will allow us to correlate islet infiltration with demographic and clinical variables with the aim of identifying individuals at the very early stages of the disease.Graphical abstract

1467-P: Chemical Profiles of Acinar Tissue and Pancreatic Islets from Healthy and T2D-Affected Pancreata

Both the endocrine and exocrine pancreas demonstrate morphological and chemical changes associated with T2D development. Despite significant progress, chemical profiles of the islets and acinar tissue following disease progression are not well characterized. In this work we investigated isolated populations of pancreatic islets (n=18) and acinar tissues (n=7) collected from healthy and T2D affected pancreata using several mass spectrometry (MS) approaches. Samples are provided by the ADI IsletCore and the Network for Pancreatic Organ Donors with Diabetes (nPOD) . Both peptide and metabolite profiles were determined and compared via chemical profiling with matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance MS. Not surprisingly, the peptide profiles from T2D islets demonstrated lower signal intensities compared to healthy islets (ROC analysis, AUC=0.9) . Somatostatin, pancreatic icosapeptide, and insulin-related signals are most decreased. Preliminary data for acinar tissues indicate lower peptide levels in samples collected from T2D-affected (n=2) vs. healthy (n=5) pancreata. Several unique peptides are detected specifically in acinar tissue obtained from T2D-affected pancreata. Liquid chromatography-MS reveals a complex endogenous peptide complement of acinar tissue. Analysis of extracellular media for both islet types demonstrated similar differences (AUC=0.7) indicating higher levels of observed peptides in preparations from healthy islets, perhaps suggesting that peptides are released from the islets during shipment. Integrity of received islets was verified using dithizone staining prior to measurement. In contrast, almost no peptide markers of acinar tissue are found in corresponding extracellular media. Our results enhance knowledge on biochemical signature of T2D in the pancreas. Disclosure S.Rubakhin: None. E.V.Romanova: None. J.V.Sweedler: None. Funding American Diabetes Association (1-18-VSN-19)

Circulating C-Peptide Levels in Living Children and Young People and Pancreatic β-Cell Loss in Pancreas Donors Across Type 1 Diabetes Disease Duration.

C-peptide declines in type 1 diabetes, although many long-duration patients retain low, but detectable levels. Histological analyses confirm that β-cells can remain following type 1 diabetes onset. We explored the trends observed in C-peptide decline in the UK Genetic Resource Investigating Diabetes (UK GRID) cohort (N = 4,079), with β-cell loss in pancreas donors from the network for Pancreatic Organ donors with Diabetes (nPOD) biobank and the Exeter Archival Diabetes Biobank (EADB) (combined N = 235), stratified by recently reported age at diagnosis endotypes (<7, 7–12, ≥13 years) across increasing diabetes durations. The proportion of individuals with detectable C-peptide declined beyond the first year after diagnosis, but this was most marked in the youngest age group (<1-year duration: age <7 years: 18 of 20 [90%], 7–12 years: 107 of 110 [97%], ≥13 years: 58 of 61 [95%] vs. 1–5 years postdiagnosis: <7 years: 172 of 522 [33%], 7–12 years: 604 of 995 [61%], ≥13 years: 225 of 289 [78%]). A similar profile was observed in β-cell loss, with those diagnosed at younger ages experiencing more rapid loss of islets containing insulin-positive (insulin+) β-cells <1 year postdiagnosis: age <7 years: 23 of 26 (88%), 7–12 years: 32 of 33 (97%), ≥13 years: 22 of 25 (88%) vs. 1–5 years postdiagnosis: <7 years: 1 of 12 (8.3%), 7–12 years: 7 of 13 (54%), ≥13 years: 7 of 8 (88%). These data should be considered in the planning and interpretation of intervention trials designed to promote β-cell retention and function.

Substance Use Affects Type 1 Diabetes Pancreas Pathology: Implications for Future Studies.

Access to human pancreas samples from organ donors has greatly advanced our understanding of type 1 diabetes pathogenesis; however, previous studies have shown that donors have a high rate of substance use, and its impact on pancreatic histopathology in this disease is not well described. One-hundred-thirty-one type 1 diabetes and 111 control organ donor pancreata from persons 12-89 years of age (mean 29.8 ± 15.5 years) within the Network for Pancreatic Organ donors with Diabetes (nPOD) were examined for insulin positivity, insulitis, amyloid staining, acute and chronic pancreatitis, and chronic exocrine changes (acinar atrophy, fibrosis, fatty infiltration, or periductal fibrosis); findings were compared by history of substance use. A secondary analysis compared exocrine pancreatic histopathologic findings in type 1 diabetes versus control organ donors regardless of substance use history. We observed a high but congruent rate of substance use in type 1 diabetes and control organ donors (66.4% and 64% respectively). Among donors with type 1 diabetes (but not controls), islet amyloid (OR 9.96 [1.22, 81.29]) and acute pancreatitis (OR 3.2 [1.06, 9.63]) were more common in alcohol users while chronic exocrine changes (OR 8.86 [1.13, 69.31]) were more common in cocaine users. Substance use impacted the pancreata of donors with type 1 diabetes more than controls. Overall, despite similar rates of substance use, acute pancreatitis (15.3% versus 4.5%, p=0.0061), chronic pancreatitis (29.8% versus 9.9%, p=0.0001), and chronic exocrine changes (73.3% versus 36.9%, p<0.0001) were more common in type 1 diabetes donors than controls. Alcohol and/or cocaine use in type 1 diabetes organ donors increases exocrine pancreas pathology and islet amyloid deposition but does not affect insulitis or insulin positivity. Exocrine pathology in type 1 diabetes donors is common, and further study of the pathophysiology of these changes is needed.

Pancreatic beta cell autophagy is impaired in type 1 diabetes

Aims/hypothesisPancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development.MethodsPancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese diabetic (NOD) mice. Age- and BMI-matched pancreas tissue sections from human organ donors (N = 34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5).ResultsWe observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3–LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3–LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002).Conclusions/interpretationWe provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes.Graphical abstract

New Insights Into the Role of Autoreactive CD8 T Cells and Cytokines in Human Type 1 Diabetes.

Since the establishment of the network for pancreatic organ donors with diabetes (nPOD), we have gained unprecedented insight into the pathology of human type 1 diabetes. Many of the pre-existing “dogmas”, mostly derived from studies of animal models and sometimes limited human samples, have to be revised now. For example, we have learned that autoreactive CD8 T cells are present even in healthy individuals within the exocrine pancreas. Furthermore, their “attraction” to islets probably relies on beta-cell intrinsic events, such as the over-expression of MHC class I and resulting presentation of autoantigens such as (prepro)insulin. In addition, we are discovering other signs of beta-cell dysfunction, possibly at least in part due to stress, such as the over-expression of certain cytokines. This review summarizes the latest developments focusing on cytokines and autoreactive CD8 T cells in human type 1 diabetes pathogenesis.

ACE2 and SARS-CoV-2 Expression in the Normal and COVID-19 Pancreas

Diabetes is associated with increased mortality from Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Given literature suggesting a potential association between SARS-CoV-2 infection and diabetes induction, we examined pancreatic expression of the key molecule for SARS-CoV-2 infection of cells, angiotensin-converting enzyme-2 (ACE2). Specifically, we analyzed five public scRNAseq pancreas datasets and performed fluorescence in situ hybridization, Western blotting, and immunolocalization for ACE2 with extensive reagent validation on normal human pancreatic tissues across the lifespan, as well as those from coronavirus disease 2019 (COVID-19) patients. These in silico and ex vivo analyses demonstrated pancreatic expression of ACE2 is prominent in pancreatic ductal epithelium and the microvasculature, with rare endocrine cell expression of this molecule. Pancreata from COVID-19 patients demonstrated multiple thrombotic lesions with SARS-CoV-2 nucleocapsid protein expression primarily limited to ducts. SARS-CoV-2 infection of pancreatic endocrine cells, via ACE2, appears an unlikely central pathogenic feature of COVID-19 as it relates to diabetes.Funding: These efforts were supported by NIH P01 AI42288 and UC4 DK108132 (MAA), JDRF (MAA), NIH R01 DK122160 (MCT), NIH R01 AI134971 (DH), NIH P30 DK020541 (D.H.), JDRF 3-PDF-2018-575-A-N (VvdH), R01 DK093954 (CEM); VA Merit Award I01BX001733 (CEM), Imaging Core of NIH/NIDDK P30 DK097512 (CEM), gifts from the Sigma Beta Sorority, the Ball Brothers Foundation, and the George and Frances Ball Foundation (CEM), the Network for Pancreatic Organ donors with Diabetes (nPOD; RRID:SCR_014641) (5-SRA-2018-557-Q-R) and The Leona M. & Harry B. Helmsley Charitable Trust (2018PG-T1D053).Conflict of Interest: The authors declare no relevant conflicts of interest exist.Ethical Approval: Transplant-quality pancreas, duodenum, and kidney were recovered by JDRF nPOD (www.jdrfnpod.com) from 36 COVID-19 negative organ donors without diabetes (Table S2) according to established protocols and procedures (Campbell-Thompson et al., 2012), as approved by the University of Florida Institutional Review Board (201400486), the United Network for Organ Sharing (UNOS), and according to federal guidelines with informed consent obtained from each donor’s legal representative.

Pancreatic Alpha-Cells Contribute Together With Beta-Cells to CXCL10 Expression in Type 1 Diabetes.

C-X-C Motif Chemokine Ligand 10 (CXCL10) is a pro-inflammatory chemokine specifically recognized by the ligand receptor CXCR3 which is mostly expressed in T-lymphocytes. Although CXCL10 expression and secretion have been widely associated to pancreatic islets both in non-obese diabetic (NOD) mice and in human type 1 diabetic (T1D) donors, the specific expression pattern among pancreatic endocrine cell subtypes has not been clarified yet. Therefore, the purpose of this study was to shed light on the pancreatic islet expression of CXCL10 in NOD, in C57Bl/6J and in NOD-SCID mice as well as in human T1D pancreata from new-onset T1D patients (DiViD study) compared to non-diabetic multiorgan donors from the INNODIA European Network for Pancreatic Organ Donors with Diabetes (EUnPOD). CXCL10 was expressed in pancreatic islets of normoglycaemic and new-onset diabetic NOD mice but not in C57Bl/6J and NOD-SCID mice. CXCL10 expression was increased in pancreatic islets of new-onset diabetic NOD mice compared to normoglycaemic NOD mice. In NOD mice, CXCL10 colocalized both with insulin and glucagon. Interestingly, CXCL10-glucagon colocalization rate was significantly increased in diabetic vs. normoglycaemic NOD mouse islets, indicating an increased expression of CXCL10 also in alpha-cells. CXCL10 was expressed in pancreatic islets of T1D patients but not in non-diabetic donors. The analysis of the expression pattern of CXCL10 in human T1D pancreata from DiViD study, revealed an increased colocalization rate with glucagon compared to insulin. Of note, CXCL10 was also expressed in alpha-cells residing in insulin-deficient islets (IDI), suggesting that CXCL10 expression in alpha cells is not driven by residual beta-cells and therefore may represent an independent phenomenon. In conclusion, we show that in T1D CXCL10 is expressed by alpha-cells both in NOD mice and in T1D patients, thus pointing to an additional novel role for alpha-cells in T1D pathogenesis and progression.

Histological validation of a type 1 diabetes clinical diagnostic model for classification of diabetes.

Misclassification of diabetes is common due to an overlap in the clinical features of type 1 and type 2 diabetes. Combined diagnostic models incorporating clinical and biomarker information have recently been developed that can aid classification, but they have not been validated using pancreatic pathology. We evaluated a clinical diagnostic model against histologically defined type 1 diabetes. We classified cases from the Network for Pancreatic Organ donors with Diabetes (nPOD) biobank as type 1 (n=111) or non-type 1 (n=42) diabetes using histopathology. Type 1 diabetes was defined by lobular loss of insulin-containing islets along with multiple insulin-deficient islets. We assessed the discriminative performance of previously described type 1 diabetes diagnostic models, based on clinical features (age at diagnosis, BMI) and biomarker data [autoantibodies, type 1 diabetes genetic risk score (T1D-GRS)], and singular features for identifying type 1 diabetes by the area under the curve of the receiver operator characteristic (AUC-ROC). Diagnostic models validated well against histologically defined type 1 diabetes. The model combining clinical features, islet autoantibodies and T1D-GRS was strongly discriminative of type 1 diabetes, and performed better than clinical features alone (AUC-ROC 0.97 vs. 0.95; P=0.03). Histological classification of type 1 diabetes was concordant with serum C-peptide [median <17pmol/l (limit of detection) vs. 1037pmol/l in non-type 1 diabetes; P<0.0001]. Our study provides robust histological evidence that a clinical diagnostic model, combining clinical features and biomarkers, could improve diabetes classification. Our study also provides reassurance that a C-peptide-based definition of type 1 diabetes is an appropriate surrogate outcome that can be used in large clinical studies where histological definition is impossible. Parts of this study were presented in abstract form at the Network for Pancreatic Organ Donors Conference, Florida, USA, 19-22 February 2019 and Diabetes UK Professional Conference, Liverpool, UK, 6-8 March 2019.

2115-P: Beta-Cell Autophagy Is Reduced in Type 1 Diabetes

Autophagy is a dynamic recycling mechanism that maintains beta cell homeostasis, whereas the related process of crinophagy also plays a specific role in insulin granule regulation. Impairment of autophagy and crinophagy have been implicated in beta cell death and dysfunction in the context of type 2 diabetes; however, there is currently no literature describing beta cell autophagy in type 1 diabetes pathogenesis. Therefore, we set out to determine if beta cell autophagy and crinophagy are impaired in the context of type 1 diabetes. We utilized immunofluorescent staining of LC3 (autophagosome marker), Lamp1 (lysosome marker), and proinsulin to analyze pancreatic islet autophagy and crinophagy in the NOD (non-obese diabetic) mouse model of type 1 diabetes as well as in human pancreas tissue obtained from the network for Pancreatic Organ donors with Diabetes (nPOD). We observed evidence of reduced macroautophagy in the residual proinsulin-positive islet cells of human donors with type 1 diabetescompared to controls, as measured by significantly reduced autophagosome:lysosome colocalization (p=0.034). Similarly, there was a trend towards reduced autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to nondiabetic NOR mice (p=0.08). We also observed significantly reduced colocalization of proinsulin with lysosomes in the proinsulin positive cells from human donors with type 1 diabetes in comparison to both controls without diabetes (p=0.0014) and autoantibody positive donors (p= 0.0278), as well as in diabetic NOD vs. nondiabetic NOR mice (p=0.0121), suggesting that crinophagy is also reduced as a function of type 1 diabetes. Collectively, we provide evidence that macroautophagy and crinophagy are reduced in the context of type 1 diabetes. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development. Disclosure C. Muralidharan: None. M. Marasco: Employee; Spouse/Partner; Eli Lilly and Company. J. Crowder: Employee; Spouse/Partner; Eli Lilly and Company. A.M. Conteh: None. A.K. Linnemann: None.

1637-P: Role of mTORC1 Regulation in the T1D Organ Donor Pancreas

Current evidence suggests that alteration in cellular metabolism leads to pancreatic β-cell dysfunction and loss in type 1 diabetes (T1D). The mammalian target of rapamycin, mTOR is a key regulator of cell survival and growth in response to intracellular energy levels, nutrients, and growth factor signaling. Dysregulated mTOR complex 1 (mTORC1) has been implicated in β-cell loss and dysfunction in T2D but has not been well studied in T1D. We hypothesized that genes associated with three pathways involved in the regulation of mTORC1 would display altered expression in T1D organ donor pancreata compared to nondiabetic controls. We isolated total RNA from 30 T1D and 30 unaffected control human organ donor pancreata obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD) program and performed real-time qPCR on 26 genes related to these pathways, all of which demonstrated significant (p&amp;lt;0.05) induction. In relation to cellular energy balance, we observed induction of the AMPK-related genes. Concerning nutrient availability and the GCN2/ATF4 pathway, genes associated with all Sestrins and the GATOR 1 and GATOR 2 complexes were induced. In relation to growth factor signaling, components of the PI3K/AKT pathway were induced (Table 1). Both AMPK and PI3K/AKT pathways converge on TSC2, which had the largest degree of induction observed (22.4x). To conclude, our results clearly indicate dysregulation of mTORC1 in the T1D donor pancreas. Disclosure B.S. Bruggeman: None. D. Beachy: None. L.M. Jacobsen: None. H.S. Nick: None. M.A. Atkinson: None. D. Schatz: None. C. Wasserfall: None.

252-LB: Insulin Secretion Pathway Genes Correlate with Markers Associated with Viral Infections in Human Islets

Type 1 diabetes (T1D) is an autoimmune disease hypothesized, to be enhanced or triggered by viral infections. In this study we used organ donor pancreas to isolate islets that expressed known signs of viral infections, and subsequently investigated pathways that may be secondarily affected through analysis of gene expression profiles. Using samples provided by the Network for Pancreatic Organ Donors with Diabetes, (nPOD), islets were collected, by laser-capture, from nondiabetic donors (Control), autoantibody positive nondiabetic donors (AB+) and donors with T1D (T1D). Islets were categorized based on the presence of markers associated with viral infection (VIMs), HLA, Mx1, dsRNA, and PKR, identified via immunohistochemical staining. From each donor islets were pooled based on the number of VIMs (0 VIMs, 1 VIMs or ≥2VIMs). RNA was extracted, and microarray used to assess transcriptomes. GeneSpring software was used to generate a list of genes with differential expression between donors/VIMs. Using WebGestalt we identified pathways enriched by this list. A total of 383 genes with a fold change of ≥1.1 and p-value=0.001 had differential expression between islets with 0 VIMs and ≥1 VIMs. Pathway analysis showed strongest enrichment for genes in the insulin secretion pathway (p&amp;lt; 5.7E-5). As an example; CACNA1C and ABCC8 (important for the secretion of insulin), had lower expression in the islets with ≥1VIMs compared to 0 VIMs (p&amp;lt;0.03). CACNA1C also had lower expression in islets with 0 VIM vs. ≥1VIMs when only comparing samples from control donors (p=0.02). Islets selected for high expression for genes associated with viral infection had decreased expression of genes important for insulin secretion. This may suggest a pattern of dedifferentiation and/or functional impairment of beta cells in the setting of viral infection. Disclosure G. Nelson: None. N.I. Lenchik: None. T. Rodriguez-Calvo: None. D. Balcacean: None. I.C. Gerling: None. Funding JDRF; National Institute of Diabetes and Digestive and Kidney Diseases; Lebonheur Children’s Hospital

Large-scale electron microscopy database for human type 1 diabetes

Autoimmune β-cell destruction leads to type 1 diabetes, but the pathophysiological mechanisms remain unclear. To help address this void, we created an open-access online repository, unprecedented in its size, composed of large-scale electron microscopy images (‘nanotomy’) of human pancreas tissue obtained from the Network for Pancreatic Organ donors with Diabetes (nPOD; www.nanotomy.org). Nanotomy allows analyses of complete donor islets with up to macromolecular resolution. Anomalies we found in type 1 diabetes included (i) an increase of ‘intermediate cells’ containing granules resembling those of exocrine zymogen and endocrine hormone secreting cells; and (ii) elevated presence of innate immune cells. These are our first results of mining the database and support recent findings that suggest that type 1 diabetes includes abnormalities in the exocrine pancreas that may induce endocrine cellular stress as a trigger for autoimmunity.

Abnormalities in proinsulin processing in islets from individuals with longstanding T1D

We recently described the persistence of detectable serum proinsulin in a large majority of individuals with longstanding type 1 diabetes (T1D), including individuals with undetectable serum C-peptide. Here, we sought to further explore the mechanistic etiologies of persistent proinsulin secretion in T1D at the level of the islet, using tissues obtained from human donors. Immunostaining for proinsulin and insulin was performed on human pancreatic sections from the Network for Pancreatic Organ Donors with Diabetes (nPOD) collection (n = 24). Differential proinsulin processing enzyme expression was analyzed using mass spectrometry analysis of human islets isolated from pancreatic sections with laser capture microdissection (n = 6). Proinsulin processing enzyme mRNA levels were assessed using quantitative real-time PCR in isolated human islets (n = 10) treated with or without inflammatory cytokines. Compared to nondiabetic controls, immunostaining among a subset (4/9) of insulin positive T1D donor islets revealed increased numbers of cells with proinsulin-enriched, insulin-poor staining. T1D donor islets also exhibited increased proinsulin fluorescence intensity relative to insulin fluorescence intensity. Laser capture microdissection followed by mass spectrometry revealed reductions in the proinsulin processing enzymes prohormone convertase 1/3 (PC1/3) and carboxypeptidase E (CPE) in T1D donors. Twenty-four hour treatment of human islets with inflammatory cytokines reduced mRNA expression of the processing enzymes PC1/3, PC2, and CPE. Taken together, these data provide new mechanistic insight into altered proinsulin processing in long-duration T1D and suggest that reduced β cell prohormone processing is associated with proinflammatory cytokine-induced reductions in proinsulin processing enzyme expression.

1366-P: Effect of Substance Use on Type 1 Diabetes (T1D) Pancreas Histopathology

Recent efforts addressing the pathogenesis of T1D have benefited greatly from access to transplant-quality human pancreas samples obtained from organ donors. While alcohol use is linked to pancreatitis clinically, the effect of alcohol and other illicit substances on pancreatic histopathology in T1D is not known. Hence, we determined the influence of alcohol, marijuana, tobacco, cocaine, and other controlled substances in T1D pancreata. In total, 140 T1D organ donor pancreata [median age at T1D onset=11 (range 0-36) years, duration=12 (0-84) years, 47% female] were recovered through the Network for Pancreatic Organ donors with Diabetes (nPOD) program. Paraffin sections were stained and reviewed for insulin positivity, insulitis (≥6 CD3+ cells adjacent/within the islet in ≥3 islets/section), amyloid staining, acute (acinar cell loss, mononuclear cell infiltrate, fat necrosis, and hemorrhage) and chronic pancreatitis (acinar cell loss, mononuclear cell infiltrate, and fibrosis) as well as chronic exocrine changes (acinar atrophy, fibrosis, fatty infiltration, or periductal fibrosis). Terminal hospital records were reviewed for a history of alcohol, tobacco, marijuana, and any illicit substance use. Of 140 T1D donors, 51% had a history of alcohol use, 40% tobacco use, 29% marijuana, and 20% other illicit substances. Urine drug screening on admission was available for 74 donors with 36 positive (49%). Alcohol use was associated with histopathological features of acute pancreatitis (p&amp;lt;0.05) and islet amyloid deposition (p&amp;lt;0.05). History of cocaine use (p&amp;lt;0.05) as well as a positive urine drug screen (p&amp;lt;0.05) were associated with chronic exocrine changes. To conclude, in T1D donor pancreata, alcohol use was associated with acute pancreatitis and islet amyloid deposition while a history of cocaine use and a positive urine drug screen were associated with chronic exocrine changes. These findings should be given consideration in studies aimed at interpreting histopathologic changes in T1D pancreata. Disclosure B.S. Bruggeman: None. M. Campbell-Thompson: None. A.L. Posgai: None. M.A. Atkinson: None. D. Schatz: None. L.M. Jacobsen: None. Funding JDRF

93-OR: Expression of Genes Associated with Host Viral Responses in Insulitic Islets

Background: Type 1 diabetes (T1D) is an autoimmune condition long hypothesized to be enhanced or triggered by viral infections. Islet pathology in T1D is characterized by destruction and subsequent loss of insulin producing pancreatic beta cells. In this study we evaluated specific physiological pathways associated with the disorder’s pathogenesis by examining differential expression of host genes within islets, with special emphasis on identifying evidence suggestive of responses to a viral infection. Methods: Pancreatic tissue samples were obtained from nondiabetic donors, autoantibody positive donors, and donors with T1D through the Network for Pancreatic Organ donors with Diabetes (nPOD) program. Laser microdissection was used to isolate individual islets based on immunohistochemical documentation of presence or absence of insulin (INS+) and T-lymphocytes (CD3+). RNA was isolated and microarray used to assess transcriptomes. Results: Increased expression in insulitic islets was seen in a variety of genes including TLR pathway inhibitors: LAIR1 (p=2.77E-8), SIGLEC7 (p=8.33E-6), SIGLEC9 (p=1.59E-4), and C1q (p=6.11E-16); as well as genes downstream of the TLR pathway: CD163 (p=2.34E-11), CTSS (p=9.89E-7) and IFNγ (p=5.9E-8). We also observed variances in the frequency of viral response genes, including those known to be associated with T1D at a GWAS level: STAT1 (p=1.64E-7) and OAS3 (p=1.04E-8). Pathway analysis demonstrated increased representation of antigen processing and presentation genes (p=3.0E-14). Conclusions: In sum, these studies noted an increased expression of multiple host genes associated with anti-viral responses. The demonstration of specific protein involvement in host responses to infections are up-regulated in T1D islets, specifically in those with signs of immune activation and residual insulin positivity (INS+CD3+), supports the potential involvement of host viral response pathways in the development of T1D. Disclosure G. Nelson: None. N.I. Lenchik: None. M. Campbell-Thompson: None. M.A. Atkinson: None. I.C. Gerling: None. Funding National Institutes of Health (UC4DK104155)

The Official Journal of ATTD Advanced Technologies &amp; Treatments for Diabetes Conference Berlin, Germany—February 20–23, 2019

The Official Journal of ATTD Advanced Technologies &amp; Treatments for Diabetes Conference <i>Berlin, Germany—February 20–23, 2019</i>

Low-Level Insulin Content Within Abundant Non-β Islet Endocrine Cells in Long-standing Type 1 Diabetes.

Although most patients with type 1 diabetes (T1D) continue to produce small amounts of insulin decades after disease onset, very few β-cells persist within their pancreata. Consequently, the source of persistent insulin secretion within T1D remains unclear. We hypothesized that low-level insulin content within non-β-cells could underlie persistent T1D insulin secretion. We tested for low levels of insulin (insulinlow) within a large cohort of JDRF Network for Pancreatic Organ Donors With Diabetes (nPOD) human pancreata across a wide range of ages and T1D disease durations. Long exposures, high-throughput imaging, and blinded parallel examiners allowed precise quantification of insulinlow cells. Of note, abundant islet endocrine cells with low quantities of insulin were present in most T1D pancreata. Insulinlow islet abundance and composition were not influenced by age, duration of diabetes, or age of onset. Insulinlow islets also contained β-cell markers at variable levels, including Pdx1, Nkx6.1, GLUT1, and PC1/3. Most insulinlow cells contained abundant glucagon and other α-cell markers, suggesting that α-cells drive much of the insulinlow phenotype in T1D. However, pancreatic polypeptide, somatostatin, and ghrelin cells also contributed to the insulinlow cell population. Insulinlow cells represent a potential source of persistent insulin secretion in long-standing T1D and a possible target for regenerative therapies to expand β-cell function in disease.