Methylglyoxal Adducts Are Prognostic Biomarkers for Diabetic Kidney Disease in Patients With Type 1 Diabetes.

More than 30% of patients with type 1 diabetes develop diabetic kidney disease (DKD), which significantly increases mortality risk. The Diabetes Control and Complications Trial (DCCT) and follow-up study, Epidemiology of Diabetes Interventions and Complications (EDIC), established that glycemic control measured by HbA1c predicts DKD risk. However, the continued high incidence of DKD reinforces the urgent need for additional biomarkers to supplement HbA1c. Here, we assessed biomarkers induced by methylglyoxal (MG), a metabolic by-product that forms covalent adducts on DNA, RNA, and proteins, called MG adducts. Urinary MG adducts were measured in samples from patients with type 1 diabetes enrolled in DCCT/EDIC who did (case patients; n = 90) or did not (control patients; n = 117) develop DKD. Univariate and multivariable analyses revealed that measurements of MG adducts independently predict DKD before established DKD biomarkers such as glomerular filtration rate and albumin excretion rate. Elevated levels of MG adducts bestowed the greatest risk of developing DKD in a multivariable model that included HbA1c and other clinical covariates. Our work establishes a novel class of biomarkers to predict DKD risk and suggests that inclusion of MG adducts may be a valuable tool to improve existing predictors of complications like DKD prior to overt disease, and to aid in identifying at-risk individuals and personalized risk management.

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.

Low risk for diabetic complications in type 1 diabetes patients carrying a protective insulin gene variant.

Type 1 diabetes patients carrying a 'protective' insulin gene (INS) variant present a disease endotype with reduced insulin antibody titers, preserved beta cell function and improved glycemic control. We tested whether this protective INS variant associated with lowered risk for development of proliferative diabetic retinopathy (PDR) and diabetic kidney disease (DKD) as long-term diabetic complications. Insulin gene polymorphisms were evaluated in 1,363 type 1 diabetes patients participating in the Diabetes Control and Complications Trial/Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC) study that compared intensive versus conventional insulin therapy in relation with development of PDR and DKD with a follow-up of over two decades. PDR and DKD were absent in type 1 diabetes patients carrying the protective INS variant and receiving intensive insulin therapy (the current standard of clinical care) 1-5 years from their diagnosis (n = 67; mean post-diagnosis follow up of 20.4 ± 1.6 years), versus 11 of 258 patients (4.3%) lacking this variant (20.4 ± 1.8 years follow up). In the secondary intervention group of the intensive therapy arm (1-15 years of disease), PDR was significantly less frequent in carriers of the protective INS variant than those without it (4 of 83 [4.8%] vs. 31 of 260 [11.9%]; p = 0.032; 26.1 ± 3.9 and 26.3 ± 4.1 years follow-up, respectively), whereas DKD frequencies were no different between those with or without this variant (5 of 83 [6.0%] vs. 11 of 260 [4.2%]). Carrying a copy of this protective INS variant further reduces the risk of diabetic complications achieved by intensive insulin therapy and marks a disease endotype with superior glycemic control, increased and extended beta cell function, and prevention of DKD and PDR.

Tolerogenic dendritic cells pulsed with islet antigen induce long-term reduction in T-cell autoreactivity in type 1 diabetes patients.

Introduction: Restoration of immune tolerance may halt progression of autoimmune diseases. Tolerogenic dendritic cells (tolDC) inhibit antigen-specific proinflammatory T-cells, generate antigen-specific regulatory T-cells and promote IL-10 production in-vitro, providing an appealing immunotherapy to intervene in autoimmune disease progression. Methods: A placebo-controlled, dose escalation phase 1 clinical trial in nine adult patients with long-standing type 1 diabetes (T1D) demonstrated the safety and feasibility of two (prime-boost) vaccinations with tolDC pulsed with a proinsulin peptide. Immunoregulatory effects were monitored by antigen-specific T-cell assays and flow and mass cytometry. Results: The tolDC vaccine induced a profound and durable decline in pre-existing autoimmune responses to the vaccine peptide up to 3 years after therapy and temporary decline in CD4 and CD8+ T-cell responses to other islet autoantigens. While major leukocyte subsets remained stable, ICOS+CCR4+TIGIT+ Tregs and CD103+ tissue-resident and CCR6+ effector memory CD4+ T-cells increased in response to the first tolDC injection, the latter declining thereafter below baseline levels. Discussion: Our data identify immune correlates of mechanistic efficacy of intradermally injected tolDC reducing proinsulin autoimmunity in T1D.

Generation of human islet cell type-specific identity genesets.

Generation of surrogate cells with stable functional identities is crucial for developing cell-based therapies. Efforts to produce insulin-secreting replacement cells to treat diabetes require reliable tools to assess islet cellular identity. Here, we conduct a thorough single-cell transcriptomics meta-analysis to identify robustly expressed markers used to build genesets describing the identity of human α-, ß-, γ- and δ-cells. These genesets define islet cellular identities better than previously published genesets. We show their efficacy to outline cell identity changes and unravel some of their underlying genetic mechanisms, whether during embryonic pancreas development or in experimental setups aiming at developing glucose-responsive insulin-secreting cells, such as pluripotent stem-cell differentiation or in adult islet cell reprogramming protocols. These islet cell type-specific genesets represent valuable tools that accurately benchmark gain and loss in islet cell identity traits.

Chronic marijuana usage by human pancreas donors is associated with impaired islet function.

We investigated the effect of chronic marijuana use, defined as 4 times weekly for more than 3 years, on human pancreatic islets. Pancreata from deceased donors who chronically used marijuana were compared to those from age, sex and ethnicity matched non-users. The islets from marijuana-users displayed reduced insulin secretion as compared to islets from non-users upon stimulation with high glucose (AUC, 3.41 ± 0.62 versus 5.14 ±0.47, p<0.05) and high glucose plus KCl (AUC, 4.48 ± 0.41 versus 7.69 ± 0.58, p<0.001). When human islets from chronic marijuana-users were transplanted into diabetic mice, the mean reversal rate of diabetes was 35% versus 77% in animals receiving islets from non-users (p<0.01). Immunofluorescent staining for cannabinoid receptor type 1 (CB1R) was shown to be colocalized with insulin and enhanced significantly in beta cells from marijuana-users vs. non-users (CB1R intensity/islet area, 14.95 ± 2.71 vs. 3.23 ± 0.87, p<0.001). In contrast, CB1R expression was not co-localized with glucagon or somatostatin. Furthermore, isolated islets from chronic marijuana-users appeared hypertrophic. In conclusion, excessive marijuana use affects islet endocrine phenotype and function in vitro and in vivo. Given the increasing use of marijuana, our results underline the importance of including lifestyle when evaluating human islets for transplantation or research.

Image-Based Machine Learning Algorithms for Disease Characterization in the Human Type 1 Diabetes Pancreas.

Emerging data suggest that type 1 diabetes affects not only the ß-cell-containing islets of Langerhans, but also the surrounding exocrine compartment. Using digital pathology, machine learning algorithms were applied to high-resolution, whole-slide images of human pancreata to determine whether the tissue composition in individuals with or at risk for type 1 diabetes differs from those without diabetes. Transplant-grade pancreata from organ donors were evaluated from 16 nondiabetic autoantibody-negative controls, 8 nondiabetic autoantibody-positive subjects with increased type 1 diabetes risk, and 19 persons with type 1 diabetes (0 to 12 years' duration). HALO image analysis algorithms were implemented to compare architecture of the main pancreatic duct as well as cell size, density, and area of acinar, endocrine, ductal, and other nonendocrine, nonexocrine tissues. Type 1 diabetes was found to affect exocrine area, acinar cell density, and size, whereas the type of difference correlated with the presence or absence of insulin-positive cells remaining in the pancreas. These changes were not observed before disease onset, as indicated by modeling cross-sectional data from pancreata of autoantibody-positive subjects and those diagnosed with type 1 diabetes. These data provide novel insights into anatomic differences in type 1 diabetes pancreata and demonstrate that machine learning can be adapted for the evaluation of disease processes from cross-sectional data sets.

DNA methylation mediates development of HbA1c-associated complications in type 1 diabetes.

Metabolic memory, the persistent benefits of early glycaemic control on preventing and/or delaying the development of diabetic complications, has been observed in the Diabetes Control and Complications Trial (DCCT) and in the Epidemiology of Diabetes Interventions and Complications (EDIC) follow-up study, but the underlying mechanisms remain unclear. Here, we show the involvement of epigenetic DNA methylation (DNAme) in metabolic memory by examining its associations with preceding glycaemic history, and with subsequent development of complications over an 18-yr period in the blood DNA of 499 randomly selected DCCT participants with type 1 diabetes who are also followed up in EDIC. We demonstrate the associations between DNAme near the closeout of DCCT and mean HbA1c during DCCT (mean-DCCT HbA1c) at 186 cytosine-guanine dinucleotides (CpGs) (FDR < 15%, including 43 at FDR < 5%), many of which were located in genes related to complications. Exploration studies into biological function reveal that these CpGs are enriched in binding sites for the C/EBP transcription factor, as well as enhancer/transcription regions in blood cells and haematopoietic stem cells, and open chromatin states in myeloid cells. Mediation analyses show that, remarkably, several CpGs in combination explain 68-97% of the association of mean-DCCT HbA1c with the risk of complications during EDIC. In summary, DNAme at key CpGs appears to mediate the association between hyperglycaemia and complications in metabolic memory, through modifying enhancer activity at myeloid and other cells.

Hospital time prior to death and pancreas histopathology: implications for future studies.

AIMS/HYPOTHESIS: Diabetes research studies routinely rely upon the use of tissue samples from human organ donors. It remains unclear whether the length of hospital stay prior to organ donation affects the presence of cells infiltrating the pancreas or the frequency of replicating beta cells. METHODS: To address this, 39 organ donors without diabetes were matched for age, sex, BMI and ethnicity in groups of three. Within each group, donors varied by length of hospital stay immediately prior to organ donation (<3 days, 3 to <6 days, or ≥6 days). Serial sections from tissue blocks in the pancreas head, body and tail regions were immunohistochemically double stained for insulin and CD45, CD68, or Ki67. Slides were electronically scanned and quantitatively analysed for cell positivity. RESULTS: No differences in CD45+, CD68+, insulin+, Ki67+ or Ki67+/insulin+ cell frequencies were found when donors were grouped according to duration of hospital stay. Likewise, no interactions were observed between hospitalisation group and pancreas region, age, or both; however, with Ki67 staining, cell frequencies were greater in the body vs the tail region of the pancreas (∆ 0.65 [unadjusted 95% CI 0.25, 1.04]; p = 0.002) from donors <12 year of age. Interestingly, frequencies were less in the body vs tail region of the pancreas for both CD45+ cells (∆ -0.91 [95% CI -1.71, -0.10]; p = 0.024) and insulin+ cells (∆ -0.72 [95% CI -1.10, -0.34]; p < 0.001). CONCLUSIONS/INTERPRETATION: This study suggests that immune or replicating beta cell frequencies are not affected by the length of hospital stay prior to donor death in pancreases used for research. DATA AVAILABILITY: All referenced macros (adopted and developed), calculations, programming code and numerical dataset files (including individual-level donor data) are freely available on GitHub through Zenodo at https://doi.org/10.5281/zenodo.1034422.

Intestinal Enteroendocrine Lineage Cells Possess Homeostatic and Injury-Inducible Stem Cell Activity.

Several cell populations have been reported to possess intestinal stem cell (ISC) activity during homeostasis and injury-induced regeneration. Here, we explored inter-relationships between putative mouse ISC populations by comparative RNA-sequencing (RNA-seq). The transcriptomes of multiple cycling ISC populations closely resembled Lgr5+ ISCs, the most well-defined ISC pool, but Bmi1-GFP+ cells were distinct and enriched for enteroendocrine (EE) markers, including Prox1. Prox1-GFP+ cells exhibited sustained clonogenic growth in vitro, and lineage-tracing of Prox1+ cells revealed long-lived clones during homeostasis and after radiation-induced injury in vivo. Single-cell mRNA-seq revealed two subsets of Prox1-GFP+ cells, one of which resembled mature EE cells while the other displayed low-level EE gene expression but co-expressed tuft cell markers, Lgr5 and Ascl2, reminiscent of label-retaining secretory progenitors. Our data suggest that the EE lineage, including mature EE cells, comprises a reservoir of homeostatic and injury-inducible ISCs, extending our understanding of cellular plasticity and stemness.

Factors That Influence the Quality of RNA From the Pancreas of Organ Donors.

OBJECTIVES: Attaining high-quality RNA from the tissues or organs of deceased donors used for research can be challenging due to physiological and logistical considerations. In this investigation, METHODS: RNA Integrity Number (RIN) was determined in pancreatic samples from 236 organ donors and used to define high (≥6.5) and low (≤4.5) quality RNAs. Logistic regression was used to evaluate the potential effects of novel or established organ and donor factors on RIN. RESULTS: Univariate analysis revealed donor cause of death (odds ratio [OR], 0.35; 95% confidence interval [CI], 0.15-0.77; P = 0.01), prolonged tissue storage before RNA extraction (OR, 0.65; 95% CI, 0.52-0.79; P < 0.01), pancreas region sampled (multiple comparisons, P < 0.01), and sample type (OR, 0.32; 95% CI, 0.15-0.67; P < 0.01) negatively influenced outcome. Conversely, duration of final hospitalization (OR, 3.95; 95% CI, 1.59-10.37; P < 0.01) and sample collection protocol (OR, 8.48; 95% CI, 3.96-19.30; P < 0.01) positively impacted outcome. Islet RNA obtained via laser capture microdissection improved RIN when compared with total pancreatic RNA from the same donor (ΔRIN = 1.3; 95% CI, 0.6-2.0; P < 0.01). CONCLUSIONS: A multivariable model demonstrates that autopsy-free and biopsy-free human pancreata received, processed, and preserved at a single center, using optimized procedures, from organ donors dying of anoxia with normal lipase levels increase the odds of obtaining high-quality RNA.

Tissue distribution and clonal diversity of the T and B cell repertoire in type 1 diabetes.

The adaptive immune repertoire plays a critical role in type 1 diabetes (T1D) pathogenesis. However, efforts to characterize B cell and T cell receptor (TCR) profiles in T1D subjects have been largely limited to peripheral blood sampling and restricted to known antigens. To address this, we collected pancreatic draining lymph nodes (pLN), "irrelevant" nonpancreatic draining lymph nodes, peripheral blood mononuclear cells (PBMC), and splenocytes from T1D subjects (n = 18) and control donors (n = 9) as well as pancreatic islets from 1 T1D patient; from these tissues, we collected purified CD4+ conventional T cells (Tconv), CD4+ Treg, CD8+ T cells, and B cells. By conducting high-throughput immunosequencing of the TCR ß chain (TRB) and B cell receptor (BCR) immunoglobulin heavy chain (IGH) on these samples, we sought to analyze the molecular signature of the lymphocyte populations within these tissues and of T1D. Ultimately, we observed a highly tissue-restricted CD4+ repertoire, while up to 24% of CD8+ clones were shared among tissues. We surveyed our data set for previously described proinsulin- and glutamic acid decarboxylase 65-reactive (GAD65-reactive) receptors, and interestingly, we observed a TRB with homology to a known GAD65-reactive TCR (clone GAD4.13) present in 7 T1D donors (38.9%), representing >25% of all productive TRB within Tconv isolated from the pLN of 1 T1D subject. These data demonstrate diverse receptor signatures at the nucleotide level and enriched autoreactive clones at the amino acid level, supporting the utility of coupling immunosequencing data with knowledge of characterized autoreactive receptors.

Islet cell hyperexpression of HLA class I antigens: a defining feature in type 1 diabetes.

AIMS/HYPOTHESIS: Human pancreatic beta cells may be complicit in their own demise in type 1 diabetes, but how this occurs remains unclear. One potentially contributing factor is hyperexpression of HLA class I antigens. This was first described approximately 30 years ago, but has never been fully characterised and was recently challenged as artefactual. Therefore, we investigated HLA class I expression at the protein and RNA levels in pancreases from three cohorts of patients with type 1 diabetes. The principal aims were to consider whether HLA class I hyperexpression is artefactual and, if not, to determine the factors driving it. METHODS: Pancreas samples from type 1 diabetes patients with residual insulin-containing islets (n = 26) from the Network for Pancreatic Organ donors with Diabetes (nPOD), Diabetes Virus Detection study (DiViD) and UK recent-onset type 1 diabetes collections were immunostained for HLA class I isoforms, signal transducer and activator of transcription 1 (STAT1), NLR family CARD domain containing 5 (NLRC5) and islet hormones. RNA was extracted from islets isolated by laser-capture microdissection from nPOD and DiViD samples and analysed using gene-expression arrays. RESULTS: Hyperexpression of HLA class I was observed in the insulin-containing islets of type 1 diabetes patients from all three tissue collections, and was confirmed at both the RNA and protein levels. The expression of ß2-microglobulin (a second component required for the generation of functional HLA class I complexes) was also elevated. Both 'classical' HLA class I isoforms (i.e. HLA-ABC) as well as a 'non-classical' HLA molecule, HLA-F, were hyperexpressed in insulin-containing islets. This hyperexpression did not correlate with detectable upregulation of the transcriptional regulator NLRC5. However, it was strongly associated with increased STAT1 expression in all three cohorts. Islet hyperexpression of HLA class I molecules occurred in the insulin-containing islets of patients with recent-onset type 1 diabetes and was also detectable in many patients with disease duration of up to 11 years, declining thereafter. CONCLUSIONS/INTERPRETATION: Islet cell HLA class I hyperexpression is not an artefact, but is a hallmark in the immunopathogenesis of type 1 diabetes. The response is closely associated with elevated expression of STAT1 and, together, these occur uniquely in patients with type 1 diabetes, thereby contributing to their selective susceptibility to autoimmune-mediated destruction.

Survival of Patients with Gastrointestinal Cancers Can Be Predicted by a Surrogate microRNA Signature for Cancer Stem-like Cells Marked by DCLK1 Kinase.

Doublecortin-like kinase 1 (DCLK1) is a gastrointestinal (GI) tuft cell kinase that has been investigated as a biomarker of cancer stem-like cells in colon and pancreatic cancers. However, its utility as a biomarker may be limited in principle by signal instability and dilution in heterogeneous tumors, where the proliferation of diverse tumor cell lineages obscures the direct measurement of DCLK1 activity. To address this issue, we explored the definition of a miRNA signature as a surrogate biomarker for DCLK1 in cancer stem-like cells. Utilizing RNA/miRNA-sequencing datasets from the Cancer Genome Atlas, we identified a surrogate 15-miRNA expression signature for DCLK1 activity across several GI cancers, including colon, pancreatic, and stomach cancers. Notably, Cox regression and Kaplan-Meier analysis demonstrated that this signature could predict the survival of patients with these cancers. Moreover, we identified patient subgroups that predicted the clinical utility of this DCLK1 surrogate biomarker. Our findings greatly strengthen the clinical significance for DCLK1 expression across GI cancers. Further, they provide an initial guidepost toward the development of improved prognostic biomarkers or companion biomarkers for DCLK1-targeted therapies to eradicate cancer stem-like cells in these malignancies. Cancer Res; 76(14); 4090-9. ©2016 AACR.

The influence of type 1 diabetes on pancreatic weight.

AIMS/HYPOTHESIS: Previous studies of pancreases obtained at autopsy or by radiography note reduced pancreas weight (PW) and size, respectively, in type 1 diabetes; this finding is widely considered to be the result of chronic insulinopenia. This literature is, however, limited with respect to the influence of age, sex, anthropometric factors and disease duration on these observations. Moreover, data are sparse for young children, a group of particular interest for type 1 diabetes. We hypothesised that the pancreas-to-body weight ratio would normalise confounding inter-subject factors, thereby permitting better characterisation of PW in type 1 diabetes. METHODS: Transplant-grade pancreases were recovered from 216 organ donors with type 1 diabetes (n = 90), type 2 diabetes (n = 40) and no diabetes (n = 86). Whole-organ and head, body and tail weights were determined. The relative PW (RPW; PW [g] / body weight [kg]) was calculated and tested for normalisation of potential differences due to age, sex and BMI. RESULTS: PW significantly correlated with body weight in control donors (R (2) = 0.76, p < 0.001) while RPW (1.03 ± 0.36, mean ± SD) did not significantly differ across ages (0-58 years). Donors with type 1 diabetes (0.57 ± 0.18, p < 0.001), but not those with type 2 diabetes (0.93 ± 0.30), had significantly lower RPW. The relative weights of each pancreatic region from donors with type 1 diabetes were significantly smaller than those of regions from control donors and donors with type 2 diabetes (p < 0.001). Perhaps most interestingly, the RPW was not significantly associated with duration of type 1 diabetes or type 2 diabetes. CONCLUSIONS/INTERPRETATION: RPW allows for comparisons across a wide range of donor ages by eliminating confounding variables. These data validate an interesting feature of the type 1 diabetes pancreas and underscore the need for additional studies to identify the mechanistic basis for this finding, including those beyond the chronic loss of endogenous insulin secretion.

Insulitis and ß-Cell Mass in the Natural History of Type 1 Diabetes.

Descriptions of insulitis in human islets throughout the natural history of type 1 diabetes are limited. We determined insulitis frequency (the percent of islets displaying insulitis to total islets), infiltrating leukocyte subtypes, and ß-cell and α-cell mass in pancreata recovered from organ donors with type 1 diabetes (n = 80), as well as from donors without diabetes, both with islet autoantibodies (AAb(+), n = 18) and without islet autoantibodies (AAb(-), n = 61). Insulitis was observed in four of four donors (100%) with type 1 diabetes duration of ≤1 year and two AAb(+) donors (2 of 18 donors, 11%). Insulitis frequency showed a significant but limited inverse correlation with diabetes duration (r = -0.58, P = 0.01) but not with age at disease onset. Residual ß-cells were observed in all type 1 diabetes donors with insulitis, while ß-cell area and mass were significantly higher in type 1 diabetes donors with insulitis compared with those without insulitis. Insulitis affected 33% of insulin(+) islets compared with 2% of insulin(-) islets in donors with type 1 diabetes. A significant correlation was observed between insulitis frequency and CD45(+), CD3(+), CD4(+), CD8(+), and CD20(+) cell numbers within the insulitis (r = 0.53-0.73, P = 0.004-0.04), but not CD68(+) or CD11c(+) cells. The presence of ß-cells as well as insulitis several years after diagnosis in children and young adults suggests that the chronicity of islet autoimmunity extends well into the postdiagnosis period. This information should aid considerations of therapeutic strategies seeking type 1 diabetes prevention and reversal.