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Type 1 diabetes in children is heralded by a preclinical phase defined by circulating autoantibodies to pancreatic islet antigens. How islet autoimmunity is initiated and then progresses to clinical diabetes remains poorly understood. Only one study has reported gene expression in specific immune cells of at-risk children, associated with progression to islet autoimmunity. We analysed gene expression by RNAseq in CD4+ and CD8+ T cells, NK cells and B cells, and chromatin accessibility by ATACseq in CD4+ T cells, in five genetically at-risk children with islet autoantibodies who progressed to diabetes over a median of 3 years (‘Progressors’) compared to five children matched for sex, age and HLA-DR who had not progressed (‘Non-progressors). In Progressors, differentially expressed genes (DEGs) were largely confined to CD4+ T cells and enriched for cytotoxicity-related genes/pathways. Several top-ranked DEGs were validated in a semi-independent cohort of 13 Progressors and 11 Non-progressors. Flow cytometry confirmed progression was associated with expansion of CD4+ cells with a cytotoxic phenotype. By ATAC-seq, progression was associated with reconfiguration of regulatory chromatin regions in CD4+ cells, some linked to differentially expressed cytotoxicity-related genes. Our findings suggest that cytotoxic CD4+ T cells play a role in promoting progression to type 1 diabetes.

Background Short-chain fatty acids (SCFAs) produced by the gut microbiota have beneficial anti-inflammatory and gut homeostasis effects and prevent type 1 diabetes (T1D) in mice. Reduced SCFA production indicates a loss of beneficial bacteria, commonly associated with chronic autoimmune and inflammatory diseases, including T1D and type 2 diabetes. Here, we addressed whether a metabolite-based dietary supplement has an impact on humans with T1D. We conducted a single-arm pilot-and-feasibility trial with high-amylose maize-resistant starch modified with acetate and butyrate (HAMSAB) to assess safety, while monitoring changes in the gut microbiota in alignment with modulation of the immune system status. Results HAMSAB supplement was administered for 6 weeks with follow-up at 12 weeks in adults with long-standing T1D. Increased concentrations of SCFA acetate, propionate, and butyrate in stools and plasma were in concert with a shift in the composition and function of the gut microbiota. While glucose control and insulin requirements did not change, subjects with the highest SCFA concentrations exhibited the best glycemic control. Bifidobacterium longum, Bifidobacterium adolescentis, and vitamin B7 production correlated with lower HbA1c and basal insulin requirements. Circulating B and T cells developed a more regulatory phenotype post-intervention. Conclusion Changes in gut microbiota composition, function, and immune profile following 6 weeks of HAMSAB supplementation were associated with increased SCFAs in stools and plasma. The persistence of these effects suggests that targeting dietary SCFAs may be a mechanism to alter immune profiles, promote immune tolerance, and improve glycemic control for the treatment of T1D. Trial registration ACTRN12618001391268. Registered 20 August 2018,https://www.anzctr.org.au/Trial/Registration/TrialReview.aspx?id=375792

Background To determine the prevalence of enteric infections in Aboriginal children aged 0–2 years using conventional and molecular diagnostic techniques and to explore associations between the presence of pathogens and child growth. Methods Cross-sectional analysis of Aboriginal children (n = 62) residing in a remote community in Northern Australia, conducted from July 24th - October 30th 2017. Stool samples were analysed for organisms by microscopy (directly in the field and following fixation and storage in sodium-acetate formalin), and by qualitative PCR for viruses, bacteria and parasites and serology for Strongyloides-specific IgG. Child growth (height and weight) was measured and z scores calculated according to WHO growth standards. Results Nearly 60% of children had evidence for at least one enteric pathogen in their stool (37/62). The highest burden of infection was with adenovirus/sapovirus (22.9%), followed by astrovirus (9.8%) and Cryptosporidium hominis/parvum (8.2%). Non-pathogenic organisms were detected in 22.5% of children. Ten percent of children had diarrhea at the time of stool collection. Infection with two or more pathogens was negatively associated with height for age z scores (− 1.34, 95% CI − 2.61 to − 0.07), as was carriage of the non-pathogen Blastocystis hominis (− 2.05, 95% CI - 3.55 to − 0.54). Conclusions Infants and toddlers living in this remote Northern Australian Aboriginal community had a high burden of enteric pathogens and non-pathogens. The association between carriage of pathogens/non-pathogens with impaired child growth in the critical first 1000 days of life has implications for healthy child growth and development and warrants further investigation. These findings have relevance for many other First Nations Communities that face many of the same challenges with regard to poverty, infections, and malnutrition.

Children born to larger households have less allergic disease. T regulatory cell (Treg) development may be a relevant mechanism, but this has not been studied longitudinally.We aim to (i) describe how prenatal and postnatal environmental factors are associated with Treg development and (ii) investigate whether serial Treg measures predict allergic outcomes at 1 year of age.A birth cohort (n = 1074) with information on prenatal and postnatal early life factors. Both naïve Treg (nTreg) and activated Treg (aTreg) cell populations (as a proportion of CD4Infants born to larger households (3 or more residents) had higher longitudinal nTreg proportions over the first postnatal year with a mean difference (MD) of 0.67 (95% CI 0.30-1.04)%. Higher nTreg proportions at birth were associated with a reduced risk of infant allergic outcomes. Childcare attendance and breastfeeding were associated with higher longitudinal nTreg proportions (MD 0.48 (95% CI 0.08-0.80)%.Multiple prenatal and postnatal microbial factors are associated with nTreg and aTreg development. Larger household size was associated with higher nTreg at birth which in turn was associated with reduced allergic sensitization and disease at 12 months of age.

Loss of heterochromatin has been proposed as a universal mechanism of aging across different species and cell types. However, a comprehensive analysis of hematopoietic changes caused by heterochromatin loss is lacking. Moreover, there is conflict in the literature around the role of the major heterochromatic histone methyltransferase Suv39h1 in the aging process. Here, we use individual and dual deletion of Suv39h1 and Suv39h2 enzymes to examine the causal role of heterochromatin loss in hematopoietic cell development. Loss of neither Suv39h1 nor Suv39h2 individually had any effect on hematopoietic stem cell function or the development of mature lymphoid or myeloid lineages. However, deletion of both enzymes resulted in characteristic changes associated with aging such as reduced hematopoietic stem cell function, thymic involution and decreased lymphoid output with a skewing toward myeloid development, and increased memory T cells at the expense of naive T cells. These cellular changes were accompanied by molecular changes consistent with aging, including alterations in nuclear shape and increased nucleolar size. Together, our results indicate that the hematopoietic system has a remarkable tolerance for major disruptions in chromatin structure and reveal a role for Suv39h2 in depositing sufficient H3K9me3 to protect the entire hematopoietic system from changes associated with premature aging.

Background: Preclinical studies have shown that maternal gut microbiota during pregnancy play a key role in prenatal immune development but the relevance of these findings to humans is unknown. The aim of this prebirth cohort study was to investigate the association between the maternal gut microbiota in pregnancy and the composition of the infant’s cord and peripheral blood immune cells over the first year of life.Methods: The Barwon Infant Study cohort (n=1074 infants) was recruited using an unselected sampling frame. Maternal fecal samples were collected at 36 weeks of pregnancy and flow cytometry was conducted on cord/peripheral blood collected at birth, 6 and 12 months of age. Among a randomly selected sub-cohort with available samples (n=293), maternal gut microbiota was characterized by sequencing the 16S rRNA V4 region. Operational taxonomic units (OTUs) were clustered based on their abundance. Associations between maternal fecal microbiota clusters and infant granulocyte, monocyte and lymphocyte subsets were explored using compositional data analysis. Partial least squares (PLS) and regression models were used to investigate the relationships/associations between environmental, maternal and infant factors, and OTU clusters.Results: We identified six clusters of co-occurring OTUs. The first two components in the PLS regression explained 39% and 33% of the covariance between the maternal prenatal OTU clusters and immune cell populations in offspring at birth. A cluster in which Dialister, Escherichia, and Ruminococcus were predominant was associated with a lower proportion of granulocytes (p=0.002), and higher proportions of both central naïve CD4+ T cells (CD4+/CD45RA+/CD31−) (p+/CD45RA+/FoxP3low) (p=0.02) in cord blood. The association with central naïve CD4+ T cells persisted to 12 months of age.Conclusion: This birth cohort study provides evidence consistent with past preclinical models that the maternal gut microbiota during pregnancy plays a role in shaping the composition of innate and adaptive elements of the infant’s immune system following birth. BackgroundPreclinical studies have shown that maternal gut microbiota during pregnancy play a key role in prenatal immune development but the relevance of these findings to humans is unknown. The aim of this prebirth cohort study was to investigate the association between the maternal gut microbiota in pregnancy and the composition of the infant's cord and peripheral blood immune cells over the first year of life. MethodsThe Barwon Infant Study cohort (n=1074 infants) was recruited using an unselected sampling frame. Maternal fecal samples were collected at 36 weeks of pregnancy and flow cytometry was conducted on cord/peripheral blood collected at birth, 6 and 12 months of age. Among a randomly selected sub-cohort with available samples (n=293), maternal gut microbiota was characterized by sequencing the 16S rRNA V4 region. Operational taxonomic units (OTUs) were clustered based on their abundance. Associations between maternal fecal microbiota clusters and infant granulocyte, monocyte and lymphocyte subsets were explored using compositional data analysis. Partial least squares (PLS) and regression models were used to investigate the relationships/associations between environmental, maternal and infant factors, and OTU clusters. ResultsWe identified six clusters of co-occurring OTUs. The first two components in the PLS regression explained 39% and 33% of the covariance between the maternal prenatal OTU clusters and immune cell populations in offspring at birth. A cluster in which Dialister, Escherichia, and Ruminococcus were predominant was associated with a lower proportion of granulocytes (p=0.002), and higher proportions of both central naive CD4(+) T cells (CD4(+)/CD45RA(+)/CD31(-)) (p

Studies of the gut microbiome have focused on its bacterial composition. We aimed to characterize the gut fungal microbiome (mycobiome) across pregnancy in women with and without type 1 diabetes.Faecal samples (n = 162) were collected from 70 pregnant women (45 with and 25 without type 1 diabetes) across all trimesters. Fungi were analysed by internal transcribed spacer 1 amplicon sequencing. Markers of intestinal inflammation (faecal calprotectin) and intestinal epithelial integrity (serum intestinal fatty acid binding protein; I-FABP), and serum antibodies to Saccharomyces cerevisiae (ASCA) were measured.Women with type 1 diabetes had decreased fungal alpha diversity by the third trimester, associated with an increased abundance of Saccharomyces cerevisiae that was inversely related to the abundance of the anti-inflammatory butyrate-producing bacterium Faecalibacterium prausnitzii. Women with type 1 diabetes had higher concentrations of calprotectin, I-FABP and ASCA.Women with type 1 diabetes exhibit a shift in the gut mycobiome across pregnancy associated with evidence of gut inflammation and impaired intestinal barrier function. The relevance of these findings to the higher rate of pregnancy complications in type 1 diabetes warrants further study.

Background The gut microbiome changes in response to a range of environmental conditions, life events and disease states. Pregnancy is a natural life event that involves major physiological adaptation yet studies of the microbiome in pregnancy are limited and their findings inconsistent. Pregnancy with type 1 diabetes (T1D) is associated with increased maternal and fetal risks but the gut microbiome in this context has not been characterized. By whole metagenome sequencing (WMS), we defined the taxonomic composition and function of the gut bacterial microbiome across 70 pregnancies, 36 in women with T1D. Results Women with and without T1D exhibited compositional and functional changes in the gut microbiome across pregnancy. Profiles in women with T1D were distinct, with an increase in bacteria that produce lipopolysaccharides and a decrease in those that produce short-chain fatty acids, especially in the third trimester. In addition, women with T1D had elevated concentrations of fecal calprotectin, a marker of intestinal inflammation, and serum intestinal fatty acid-binding protein (I-FABP), a marker of intestinal epithelial damage. Conclusions Women with T1D exhibit a shift towards a more pro-inflammatory gut microbiome during pregnancy, associated with evidence of intestinal inflammation. These changes could contribute to the increased risk of pregnancy complications in women with T1D and are potentially modifiable by dietary means.

The Environmental Determinants of Islet Autoimmunity (ENDIA) Study is an Australia-wide observational pregnancy-birth cohort of children at genetic risk on account of a first-degree relative with type 1 diabetes (1). 1511 participants were recruited from all Australian States and Territories from 2013-2019 with 1473 live-born infants in follow-up. The standard protocol involves 3-monthly face-to-face visits from pregnancy until the child is 2 years of age, then 6-monthly visits. Study staff across nine centres in five States collect biospecimens (blood, urine, stool, swabs) and administer lifestyle and dietary questionnaires. Approximately 10% of the cohort are engaged in a Regional Participant Program (2) that requires self-collection of sample types except for venepuncture performed at local pathology services.

Background Except for bacteria, the taxonomic diversity of the human fecal metagenome has not been widely studied, despite the potential importance of viruses and eukaryotes. Widely used bioinformatic tools contain limited numbers of non-bacterial species in their databases compared to available genomic sequences and their methodologies do not favour classification of rare sequences which may represent only a small fraction of their parent genome. In seeking to optimise identification of non-bacterial species, we evaluated five widely-used metagenome classifier programs (BURST, Kraken2, Centrifuge, MetaPhlAn2 and CCMetagen) for their ability to correctly assign and count simulations of bacterial, viral and eukaryotic DNA sequence reads, including the effect of taxonomic order of analysis of bacteria, viruses and eukaryotes and the effect of sequencing depth. Results We found that the precision of metagenome classifiers varied significantly between programs and between taxonomic groups. When classifying viruses and eukaryotes, ordering the analysis such that bacteria were classified first significantly improved classification precision. Increasing sequencing depth decreased classification precision and did not improve recall of rare species. Conclusions Choice of metagenome classifier program can have a marked effect on results with respect to precision of species assignment in different taxonomic groups. The order of taxonomic classification can markedly improve precision. Increasing sequencing depth can decrease classification precision and yields diminishing returns in probability of species detection.

Background Since its discovery 100 years ago, insulin, as the ‘cure’ for type 1 diabetes, has rescued the lives of countless individuals. As the century unfolded and the autoimmune nature of type 1 diabetes was recognised, a darker side of insulin emerged. Autoimmunity to insulin was found to be an early marker of risk for type 1 diabetes in young children. In humans, it remains unclear if autoimmunity to insulin is primarily due to a defect in the beta cell itself or to dysregulated immune activation. Conversely, it may be secondary to beta-cell damage from an environmental agent (e.g., virus). Nevertheless, direct, interventional studies in non-obese diabetic (NOD) mouse models of type 1 diabetes point to a critical role for (pro)insulin as a primary autoantigen that drives beta cell pathology. Scope of review Modelled on Koch's postulates for the pathogenicity of an infectious agent, evidence for a pathogenic role of (pro)insulin as an autoantigen in type 1 diabetes, particularly applicable to the NOD mouse model, is reviewed. Evidence in humans remains circumstantial. Additionally, as (pro)insulin is a target of autoimmunity in type 1 diabetes, its application as a therapeutic tool to elicit antigen-specific immune tolerance is assessed. Major conclusions Paradoxically, insulin is both a ‘cure’ and a potential ‘cause’ of type 1 diabetes, actively participating as an autoantigen to drive autoimmune destruction of beta cells - the instrument of its own destruction., Graphical abstract Image 1

Significance Sepsis is a condition wherein a microbial infection leads to life-threatening systemic hyperactivation of innate immunity. Blood pH is normally maintained tightly between 7.35 and 7.45, and lactic acidosis with a pH, Blood pH is tightly maintained between 7.35 and 7.45, and acidosis (pH

Background: Murine studies demonstrate that maternal prenatal gut microbiota influences brain development and behaviour of offspring. No human study has related maternal gut microbiota to behavioural outcomes during early life. This study aimed to evaluate relationships between the prenatal faecal microbiota, prenatal diet and childhood behaviour. Methods: A sub-cohort of 213 mothers and 215 children were selected from a longitudinal pre-birth cohort. Maternal prenatal exposure measures collected during the third trimester included the faecal microbiota (generated using 16S rRNA amplicon sequencing), and dietary intake. The behavioural outcome used the Childhood Behaviour Checklist at age two. Models were adjusted for prenatal diet, smoking, perceived stress, maternal age and sample batch. Findings: We found evidence that the alpha diversity of the maternal faecal microbiota during the third trimester of pregnancy predicts child internalising behaviour at two years of age (−2·74, (−4·71, −0·78), p = 0·01 (Wald test), R2=0·07). Taxa from butyrate-producing families, Lachnospiraceae and Ruminococcaceae, were more abundant in mothers of children with normative behaviour. A healthy prenatal diet indirectly related to decreased child internalising behaviours via higher alpha diversity of maternal faecal microbiota. Interpretation: These findings support animal studies showing that the composition of maternal prenatal gut microbiota is related to offspring brain development and behaviour. Our findings highlight the need to evaluate potential impacts of the prenatal gut microbiota on early life brain development. Funding: This study was funded by the National Health and Medical Research Council of Australia (1082307, 1147980), Murdoch Children's Research Institute, Barwon Health and Deakin University.

Aims/hypothesis Accurate prediction of disease progression in individuals with pre-symptomatic type 1 diabetes has potential to prevent ketoacidosis and accelerate development of disease-modifying therapies. Current tools for predicting risk require multiple blood samples taken during an OGTT. Our aim was to develop and validate a simpler tool based on a single blood draw. Methods Models to predict disease progression using a single OGTT time point (0, 30, 60, 90 or 120 min) were developed using TrialNet data collected from relatives with type 1 diabetes and validated in independent populations at high genetic risk of type 1 diabetes (TrialNet, Diabetes Prevention Trial–Type 1, The Environmental Determinants of Diabetes in the Young [1]) and in a general population of Bavarian children who participated in Fr1da. Results Cox proportional hazards models combining plasma glucose, C-peptide, sex, age, BMI, HbA1c and insulinoma antigen-2 autoantibody status predicted disease progression in all populations. In TrialNet, the AUC for receiver operating characteristic curves for models named M60, M90 and M120, based on sampling at 60, 90 and 120 min, was 0.760, 0.761 and 0.745, respectively. These were not significantly different from the AUC of 0.760 for the gold standard Diabetes Prevention Trial Risk Score, which requires five OGTT blood samples. In TEDDY, where only 120 min blood sampling had been performed, the M120 AUC was 0.865. In Fr1da, the M120 AUC of 0.742 was significantly greater than the M60 AUC of 0.615. Conclusions/interpretation Prediction models based on a single OGTT blood draw accurately predict disease progression from stage 1 or 2 to stage 3 type 1 diabetes. The operational simplicity of M120, its validity across different at-risk populations and the requirement for 120 min sampling to stage type 1 diabetes suggest M120 could be readily applied to decrease the cost and complexity of risk stratification. Graphical abstract

AIM: We aimed to characterize associations between diet and the gut microbiome and short chain fatty acid (SCFA) products in youth with islet autoimmunity or type 1 diabetes (IA/T1D) in comparison with controls. RESEARCH DESIGN AND METHODS: Eighty participants (25 diagnosed with T1D, 17 with confirmed IA, 38 sibling or unrelated controls) from the Australian T1D Gut Study cohort were studied (median [IQR] age 11.7 [8.9, 14.0] years, 43% female). A Food Frequency Questionnaire characterized daily macronutrient intake over the preceding 6 months. Plasma and fecal SCFA were measured by gas chromatography; gut microbiome composition and diversity by 16S rRNA gene sequencing. RESULTS: A 10 g increase in daily carbohydrate intake associated with higher plasma acetate in IA/T1D (adjusted estimate +5.2 (95% CI 1.1, 9.2) μmol/L p = 0.01) and controls (adjusted estimate +4.1 [95% CI 1.7, 8.5] μmol/L p = 0.04). A 5 g increase in total fat intake associated with lower plasma acetate in IA/T1D and controls. A 5% increase in noncore (junk) food intake associated with reduced richness (adjusted estimate -4.09 [95%CI -7.83, -0.35] p = .03) and evenness (-1.25 [95% CI -2.00, -0.49] p

In mice, the maternal microbiome influences fetal immune development and postnatal allergic outcomes. Westernized populations have high rates of allergic disease and low rates of gastrointestinal carriage of Prevotella, a commensal bacterial genus that produces short chain fatty acids and endotoxins, each of which may promote the development of fetal immune tolerance. In this study, we use a prebirth cohort (n = 1064 mothers) to conduct a nested case-cohort study comparing 58 mothers of babies with clinically proven food IgE mediated food allergy with 258 randomly selected mothers. Analysis of the V4 region of the 16S rRNA gene in fecal samples shows maternal carriage of Prevotella copri during pregnancy strongly predicts the absence of food allergy in the offspring. This association was confirmed using targeted qPCR and was independent of infant carriage of P. copri. Larger household size, which is a well-established protective factor for allergic disease, strongly predicts maternal carriage of P. copri., Incidence of food allergy in westernized populations is associated with low abundance of Prevotella. Here, the authors analyse the microbiome of a mother-infant prebirth cohort and find that maternal carriage, but not infant carriage, of P. copri during pregnancy predicts the absence of food allergy in the offspring.

Backgrounds: We aimed to monitor pancreatic exocrine function longitudinally in relation to the development of islet autoimmunity (IA) and type 1 diabetes (T1D) in at-risk children with a first-degree relative with T1D, who were followed prospectively in the Environmental Determinants of Islet Autoimmunity (ENDIA) study. Methods: Fecal elastase-1 (FE-1) concentration was measured longitudinally in 85 ENDIA children from median age 1.0 (IQR 0.7,1.3) year. Twenty-eight of 85 children (progressors) developed persistent islet autoantibodies at median age of 1.5 (IQR 1.1,2.5) years, of whom 11 went on to develop clinical diabetes. The other 57 islet autoantibody-negative children (non-progressors) followed similarly were age and gender-matched with the progressors. An adjusted linear mixed model compared FE-1 concentrations in progressors and non-progressors. Results: Baseline FE-1 did not differ between progressors and non-progressors, or by HLA DR type or proband status. FE-1 decreased over time in progressors in comparison to non-progressors (Wald statistic 5.46, P =.02); in some progressors the fall in FE-1 preceded the onset of IA. Conclusions: Pancreatic exocrine function decreases in the majority of young at-risk children who progress to IA and T1D.

Inflammatory factors derived from adipose tissue have been implicated in mediating insulin resistance in obesity. We sought to identify these using explanted human adipose tissue exposed to innate and adaptive immune stimuli.Subcutaneous and omental adipose tissue from obese, insulin-resistant donors was cultured in the presence of macrophage and T-cell stimuli, and the conditioned medium tested for its ability to inhibit insulin-stimulated glucose uptake into human Simpson-Golabi-Behmel Syndrome (SGBS) adipocytes. The nature of the inhibitory factor in conditioned medium was characterized physicochemically, inferred by gene microarray analysis and confirmed by antibody neutralization.Conditioned medium from omental adipose tissue exposed to a combination of macrophage- and T-cell stimuli inhibited insulin action and adiponectin secretion in SGBS adipocytes. This effect was associated with a pronounced change in adipocyte morphology, characterized by a decreased number of lipid droplets of increased size. The bioactivity of conditioned medium was abolished by trypsin treatment and had a molecular weight of 46 kDa by gel filtration. SGBS adipocytes exposed to a bioactive medium expressed multiple gene transcripts regulated by interferon-gamma (IFN-γ). Recombinant human IFN-γ recapitulated the effects of the bioactive medium and neutralizing antibody against IFN-γ but not other candidate factors abrogated medium bioactivity.IFN-γ released from inflamed omental adipose tissue may contribute to the metabolic abnormalities seen in human obesity.

Remodelling of chromatin architecture is known to regulate gene expression and has been well characterized in cell lineage development but less so in response to cell perturbation. Activation of T cells, which triggers extensive changes in transcriptional programs, serves as an instructive model to elucidate how changes in chromatin architecture orchestrate gene expression in response to cell perturbation. To characterize coordinate changes at different levels of chromatin architecture, we analyzed chromatin accessibility, chromosome conformation and gene expression in activated human T cells. T cell activation was characterized by widespread changes in chromatin accessibility and interactions that were shared between activated CD4+ and CD8+ T cells, and with the formation of active regulatory regions associated with transcription factors relevant to T cell biology. Chromatin interactions that increased and decreased were coupled, respectively, with up- and down-regulation of corresponding target genes. Furthermore, activation was associated with disruption of long-range chromatin interactions and with partitioning of topologically associating domains (TADs) and remodelling of their TAD boundaries. Newly formed/strengthened TAD boundaries were associated with higher nucleosome occupancy and lower accessibility, linking changes in lower and higher order chromatin architecture. T cell activation exemplifies coordinate multi-level remodelling of chromatin underlying gene transcription.

Rotavirus (RV) remains the major cause of infantile gastroenteritis worldwide, although the advent of vaccination has substantially decreased associated mortality [1]. Recently, we observed a 15% decrease in the incidence of type 1 diabetes (T1D) in Australian 0–4-year-old children following the introduction of RV vaccination [2, 3], suggesting that RV vaccination could contribute to the primary prevention of this autoimmune disease. This finding builds on our human and animal studies implicating RV in the development of T1D in genetically susceptible children.

Background Microbial exposures in utero and early life shape the infant microbiome, which can profoundly impact on health. Compared to the bacterial microbiome, very little is known about the virome. We set out to characterize longitudinal changes in the gut virome of healthy infants born to mothers with or without type 1 diabetes using comprehensive virome capture sequencing. Methods Healthy infants were selected from Environmental Determinants of Islet Autoimmunity (ENDIA), a prospective cohort of Australian children with a first-degree relative with type 1 diabetes, followed from pregnancy. Fecal specimens were collected three-monthly in the first year of life. Results Among 25 infants (44% born to mothers with type 1 diabetes) at least one virus was detected in 65% (65/100) of samples and 96% (24/25) of infants during the first year of life. In total, 26 genera of viruses were identified and >150 viruses were differentially abundant between the gut of infants with a mother with type 1 diabetes vs without. Positivity for any virus was associated with maternal type 1 diabetes and older infant age. Enterovirus was associated with older infant age and maternal smoking. Conclusions We demonstrate a distinct gut virome profile in infants of mothers with type 1 diabetes, which may influence health outcomes later in life. Higher prevalence and greater number of viruses observed compared to previous studies suggests significant underrepresentation in existing virome datasets, arising most likely from less sensitive techniques used in data acquisition.

usc Aims/hypothesis: To investigate the longitudinal relationship between the gut microbiome, circulating short chain fatty acids (SCFAs) and intestinal permeability in children with islet autoimmunity or type 1 diabetes and controls. Methods: We analyzed the gut bacterial microbiome, plasma SCFAs, small intestinal permeability and dietary intake in 47 children with islet autoimmunity or recent-onset type 1 diabetes and in 41 unrelated or sibling controls over a median (range) of 13 (2-34) months follow-up. Results: Children with multiple islet autoantibodies (≥2 IA) or type 1 diabetes had gut microbiome dysbiosis. Anti-inflammatory Prevotella and Butyricimonas genera were less abundant and these changes were not explained by differences in diet. Small intestinal permeability measured by blood lactulose:rhamnose ratio was higher in type 1 diabetes. Children with ≥2 IA who progressed to type 1 diabetes (progressors), compared to those who did not progress, had higher intestinal permeability (mean [SE] difference +5.14 [2.0], 95% confidence interval [CI] 1.21, 9.07, P = .006), lower within-sample (alpha) microbial diversity (31.3 [11.2], 95% CI 9.3, 53.3, P = .005), and lower abundance of SCFA-producing bacteria. Alpha diversity (observed richness) correlated with plasma acetate levels in all groups combined (regression coefficient [SE] 0.57 [0.21], 95% CI 0.15, 0.99 P = .008). Conclusions/interpretation: Children with ≥2 IA who progress to diabetes, like those with recent-onset diabetes, have gut microbiome dysbiosis associated with increased intestinal permeability. Interventions that expand gut microbial diversity, in particular SCFA-producing bacteria, may have a role to decrease progression to diabetes in children at-risk. Refereed/Peer-reviewed

Type 1 diabetes, a disease defined by absolute insulin deficiency, is considered a chronic autoimmune disorder resulting from the destruction of insulin-producing pancreatic β-cells. The incidence of childhood-onset type 1 diabetes has been increasing at a rate of 3%–5% per year globally. Despite the introduction of an impressive array of therapies aimed at improving disease management, no means for a practical “cure” exist. This said, hope remains high that any of a number of emerging technologies (e.g., continuous glucose monitoring, insulin pumps, smart algorithms), alongside advances in stem cell biology, cell encapsulation methodologies, and immunotherapy, will eventually impact the lives of those with recently diagnosed or established type 1 diabetes. However, efforts aimed at reversing insulin dependence do not address the obvious benefits of disease prevention. Hence, key “stretch goals” for type 1 diabetes research include identifying improved and increasingly practical means for diagnosing the disease at earlier stages in its natural history (i.e., early, presymptomatic diagnosis), undertaking such efforts in the population at large to optimally identify those with presymptomatic type 1 diabetes, and introducing safe and effective therapeutic options for prevention.

MicroRNAs (miRNAs) regulate T cell development and function and the disruption of miRNAs in natural regulatory CD4(+) FOXP3(+) T cells (nTreg) leads to autoimmune disease in mice. To investigate miRNA expression in relation to autoimmune disease risk in humans we sequenced them in purified CD4(+) T cell subsets from individuals at high risk of type 1 diabetes (pre-T1D), as well as other healthy individuals. Differences in miRNA expression patterns were observed between specific T cell subsets and, within subsets, between pre-T1D and healthy individuals. Compared to healthy, naive CD4(+) T cells in pre-T1D displayed 32 differentially expressed miRNAs, potentially a template for altered miRNA expression in effector memory T cells in T1D. Naive nTreg in pre-T1D displayed two differentially expressed miRNAs, Let-7c and miR-15a. In contrast, nTreg activated in vivo displayed a large number of differentially expressed miRNAs, revealing a pro-inflammatory and FOXP3-repressive signature. Differential expression of specific miRNAs was also a signpost to altered T cell function. For example, in pre-T1D, increased expression of miR-26a in nTreg activated in vivo or in vitro was associated with decreased expression of its target, the histone methyltransferase EZH2. Chemical inhibition of EZH2 decreased the number of activated naïve nTreg and their expression of nTreg signature genes FOXP3 and TIGIT. Our findings demonstrate that miRNAs differentially expressed in CD4(+) T cell subsets are markers of risk and T cell dysfunction in T1D.

The HLA-DRB1*15:01-DQA1*01:02-DQB1*06:02 haplotype is linked to protection from the development of type 1 diabetes (T1D). However, it is not known at which stages in the natural history of T1D development this haplotype affords protection. We examined a cohort of 3,358 autoantibody-positive relatives of T1D patients in the Pathway to Prevention (PTP) Study of the Type 1 Diabetes TrialNet. The PTP study examines risk factors for T1D and disease progression in relatives. HLA typing revealed that 155 relatives carried this protective haplotype. A comparison with 60 autoantibody-negative relatives suggested protection from autoantibody development. Moreover, the relatives with DRB1*15:01-DQA1*01:02-DQB1*06:02 less frequently expressed autoantibodies associated with higher T1D risk, were less likely to have multiple autoantibodies at baseline, and rarely converted from single to multiple autoantibody positivity on follow-up. These relatives also had lower frequencies of metabolic abnormalities at baseline and exhibited no overall metabolic worsening on follow-up. Ultimately, they had a very low 5-year cumulative incidence of T1D. In conclusion, the protective influence of DRB1*15:01-DQA1*01:02-DQB1*06:02 spans from autoantibody development through all stages of progression, and relatives with this allele only rarely develop T1D.

Background: Despite intense interest in the relationship between gut microbiota and brain development, longitudinal data from human studies are lacking. This study aimed to investigate the relationship between the composition of gut microbiota during infancy and subsequent behavioural outcomes. Methods: A subcohort of 201 children with behavioural outcome measures was identified within a longitudinal, Australian birth-cohort study. The faecal microbiota were analysed at 1, 6, and 12 months of age. Behavioural outcomes were measured at 2 years of age. Findings: In an unselected birth cohort, we found a clear association between decreased normalised abundance of Prevotella in faecal samples collected at 12 months of age and increased behavioural problems at 2 years, in particular Internalizing Problem scores. This association appeared independent of multiple potentially confounding variables, including maternal mental health. Recent exposure to antibiotics was the best predictor of decreased Prevotella. Interpretation: Our findings demonstrate a strong association between the composition of the gut microbiota in infancy and subsequent behavioural outcomes; and support the importance of responsible use of antibiotics during early life. Funding: This study was funded by the National Health and Medical Research Council of Australia (1082307, 1147980, 1129813), The Murdoch Children's Research Institute, Barwon Health, Deakin University, Perpetual Trustees, and The Shepherd Foundation. The funders had no involvement in the data collection, analysis or interpretation, trial design, recruitment or any other aspect pertinent to the study. Keywords: Infant, Behaviour, Microbiota, Gut-brain axis

Human CD52 is a small glycopeptide (12 amino acid residues) with one N-linked glycosylation site at asparagine 3 (Asn3) and several potential O-glycosylation serine/threonine sites. Soluble CD52 is released from the surface of activated T cells and mediates immune suppression via its glycan moiety. In suppressing activated T cells, it first sequesters the pro-inflammatory high mobility group Box 1 (HMGB1) protein, which facilitates its binding to the inhibitory sialic acid-binding immunoglobulin-like lectin-10 (Siglec-10) receptor. We aimed to identify the features of CD52 glycan that underlie its bioactivity. Analysis of native CD52 purified from human spleen revealed extensive heterogeneity in N-glycosylation and multi-antennary sialylated N-glycans with abundant polyLacNAc extensions, together with mainly di-sialylated O-glycosylation type structures. Glycomic (porous graphitized carbon-ESI-MS/MS) and glycopeptide (C8-LC-ESI-MS) analysis of recombinant soluble human CD52-immunoglobulin Fc fusion proteins revealed that CD52 bioactivity was correlated with a high abundance of tetra-antennary α-2,3/6 sialylated N-glycans. Removal of α-2,3 sialylation abolished bioactivity, which was restored by re-sialylation with α-2,3 sialyltransferases. When glycoforms of CD52-Fc were fractionated by anion exchange MonoQ-GL chromatography, bioactive fractions displayed mainly tetra-antennary, α-2,3 sialylated N-glycan structures and a lower relative abundance of bisecting GlcNAc structures compared to non-bioactive fractions. In addition, O-glycan core type-2 di-sialylated structures at Ser12 were more abundant in bioactive CD52 fractions. Understanding the structural features of CD52 glycan required for its bioactivity will aid its development as an immunotherapeutic agent.

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells, found within the islets of Langerhans in the pancreas, are destroyed by islet-infiltrating T cells. Identifying the antigenic targets of beta-cell reactive T cells is critical to gain insight into the pathogenesis of T1D and develop antigen-specific immunotherapies. Several lines of evidence indicate that insulin is an important target of T cells in T1D. Because many human islet-infiltrating CD4(+) T cells recognize C-peptide–derived epitopes, we hypothesized that full-length C-peptide (PI(33–63)), the peptide excised from proinsulin as it is converted to insulin, is a target of CD4(+) T cells in people with T1D. CD4(+) T cell responses to full-length C-peptide were detected in the blood of: 14 of 23 (>60%) people with recent-onset T1D, 2 of 15 (>13%) people with long-standing T1D, and 1 of 13 (

BACKGROUND: Beta cell function in type 1 diabetes is commonly assessed as the average plasma C-peptide concentration (CP(AVE)) following a mixed meal. Monitoring of disease progression and response to disease-modifying therapy would benefit from a simpler, more convenient and less costly measure. Therefore, we determined if CP(AVE) could be reliably estimated from routine clinical parameters. METHOD: Clinical and fasting biochemical data from eight randomised therapy trials involving participants with recently-diagnosed type 1 diabetes were used to develop and validate linear models to estimate CP(AVE) and to test their accuracy in estimating loss of beta cell function and response to immune therapy. RESULTS: A model based on disease duration, body mass index, insulin dose, HbA(1c), fasting plasma C-peptide and fasting plasma glucose most accurately estimated loss of beta cell function (area under ROC 0.89; 95% CI 0.87, 0.92) and was superior to the commonly used insulin dose-adjusted HbA(1c) (IDAA1C) measure (area under ROC 0.72; 95% CI 0.68, 0.76). Model-estimated CP(AVE) (CP(EST)) reliably identified treatment effects in randomised trials. CP(EST), compared to CP(AVE), required only a modest (up to 17%) increase in sample size for equivalent statistical power. CONCLUSION: CP(EST), approximated from six parameters at a single time-point, accurately identifies loss of beta cell function in type 1 diabetes and is comparable to CP(AVE) for identifying treatment effects. CP(EST) could serve as a convenient and economical measure of beta cell function in the clinic and as a primary outcome measure in trials of disease-modifying therapy in type 1 diabetes.

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing. Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy. Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased. Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

It has been proposed that interactions between mammalian chromosomes, or transchromosomal interactions (also known as kissing chromosomes), regulate gene expression and cell fate determination. Here we aimed to identify novel transchromosomal interactions in immune cells by high-resolution genome-wide chromosome conformation capture. Although we readily identified stable interactions in cis, and also between centromeres and telomeres on different chromosomes, surprisingly we identified no gene regulatory transchromosomal interactions in either mouse or human cells, including previously described interactions. We suggest that advances in the chromosome conformation capture technique and the unbiased nature of this approach allow more reliable capture of interactions between chromosomes than previous methods. Overall our findings suggest that stable transchromosomal interactions that regulate gene expression are not present in mammalian immune cells and that lineage identity is governed by cis, not trans chromosomal interactions., Author summary It is a widely held belief that, in the darkness of the nucleus, strands of DNA that make up different chromosomes frequently meet to ‘kiss’. These kisses, or transchromosomal interactions, are thought to be important for the expression of genes and thus cell development. Here, we aimed to identify novel transchromosomal interactions in mouse and human immune cells by high-resolution genome-wide chromosome conformation capture methods. Although we readily identified stable interactions within chromosomes and also between centromeres and telomeres on different chromosomes, surprisingly we identified no gene regulatory transchromosomal interactions in either mouse or human cells, including those previously described. Overall our findings suggest that stable transchromosomal interactions that regulate gene expression are not present in mammalian immune cells and that chromosomes are doing far less kissing than was previously believed.

To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6–24 h, before transfer and storage at −80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4 °C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals.

How T cells differentiate in the neonate may critically determine the ability of the infant to cope with infections, respond to vaccines and avert allergies. Previously, we found that naïve cord blood CD4

Background Parathyroid glands of people with relapsing remitting multiple sclerosis (RRMS) fail to respond to low serum 25-hydroxyvitamin D (25OHD) and low serum calcium, which are stimuli for parathyroid hormone (PTH) secretion. This led us to hypothesise: that there is suppression of PTH in RRMS due to higher than normal serum concentrations of fibroblast growth factor 23 (FGF23). We therefore sought evidence for dysregulation of the PTH-FGF23-vitamin D axis in RRMS. Methods Longitudinal study (winter to summer) with fasting venepunctures. For RRMS subjects who recruited a healthy control (HC) friend, pairs analyses were performed. For each pair, the within-pair difference of the variable of interest was calculated (RRMS minus HC). Then, the median of the differences from all pairs was compared against a median of zero (Wilcoxon) and the 95% confidence interval of that median difference (CI) was calculated (Sign Test). Results RRMS had lower winter PTH than HC, P = 0.005, (CI -2.4 to 0.5 pmol/L, n = 28 pairs), and lower summer PTH, P = 0.04, (CI -1.8 to 0.5, n = 21 pairs). Lower PTH associates physiologically with lower intact FGF23 (iFGF23), yet RRMS had higher iFGF23 than HC in winter, P = 0.04, (CI -3 to 15 pg/mL, n = 28 pairs) and iFGF23 levels comparable to HC in summer, P = 0.14, (CI -5 to 13, n = 21 pairs). As PTH stimulates and FGF23 reduces, renal 1-alpha hydroxylase enzyme activity, which synthesises serum 1,25-dihyroxyvitamin D (1,25(OH)2D) from serum 25OHD, we examined the ratio of serum 1,25(OH)2D to serum 25OHD. In winter, this ratio was lower in RRMS versus HC, P = 0.013, (CI -1.2 to - 0.3, n = 28 pairs). Conclusions This study revealed a dysequilibrium of the PTH-FGF23-vitamin D axis in RRMS, with lower plasma PTH, higher plasma iFGF23 and a lower serum 1,25(OH)2D to 25OHD ratio in RRMS compared with HC subjects. This dysequilibrium is consistent with the study hypothesis that in RRMS there is suppression of the parathyroid glands by inappropriately high plasma concentrations of iFGF23. Studying the basis of this dysequilibrium may provide insight into the pathogenesis of RRMS.

The association between central obesity and insulin resistance reflects the properties of visceral adipose tissue. Our aim was to gain further insight into this association by analysing the lipid composition of subcutaneous and omental adipose tissue in obese women with and without insulin resistance. Subcutaneous and omental adipose tissue and serum were obtained from 29 obese non-diabetic women, 13 of whom were hyperinsulinemic. Histology, lipid and gene profiling were performed. In omental adipose tissue of obese, insulin-resistant women, adipocyte hypertrophy and macrophage infiltration were accompanied by an increase in GM3 ganglioside and its synthesis enzyme ST3GAL5; in addition, phosphatidylethanolamine (PE) lipids were increased and their degradation enzyme, phosphatidylethanolamine methyl transferase (PEMT), decreased. ST3GAL5 was expressed predominantly in adipose stromovascular cells and PEMT in adipocytes. Insulin resistance was also associated with an increase in PE lipids in serum. The relevance of these findings to insulin resistance in humans is supported by published mouse studies, in which adipocyte GM3 ganglioside, increased by the inflammatory cytokine tumour necrosis factor-α, impaired insulin action and PEMT was required for adipocyte lipid storage. Thus in visceral adipose tissue of obese humans, an increase in GM3 ganglioside secondary to inflammation may contribute to insulin resistance and a decrease in PEMT may be a compensatory response to adipocyte hypertrophy.

Apart from its classical function in bone and calcium metabolism, vitamin D is also involved in immune regulation and has been linked to various cancers, immune disorders and allergic diseases. Within the innate and adaptive immune systems, the vitamin D receptor and enzymes in monocytes, dendritic cells, epithelial cells, T lymphocytes and B lymphocytes mediate the immune modulatory actions of vitamin D. Vitamin D insufficiency/deficiency early in life has been identified as one of the risk factors for food allergy. Several studies have observed an association between increasing latitude and food allergy prevalence, plausibly linked to lower ultraviolet radiation (UVR) exposure and vitamin D synthesis in the skin. Along with mounting epidemiological evidence of a link between vitamin D status and food allergy, mice and human studies have shed light on the modulatory properties of vitamin D on the innate and adaptive immune systems. This review will summarize the literature on the metabolism and immune modulatory properties of vitamin D, with particular reference to food allergy.

Soluble CD52 is a small glycoprotein that suppresses T-cell activation, but its effect on innate immune cell function is unknown. Here we demonstrate that soluble CD52 inhibits Toll-like receptor and tumor necrosis factor receptor signaling to limit activation of NF-κB and thereby suppress the production of inflammatory cytokines by macrophages, monocytes and dendritic cells. At higher concentrations, soluble CD52 depletes the short-lived pro-survival protein MCL-1, contributing to activation of the BH3-only proteins BAX and BAK to cause intrinsic apoptotic cell death. In vivo, administration of soluble CD52 suppresses lipopolysaccharide (LPS)-induced cytokine secretion and other features of endotoxic shock, whereas genetic deletion of CD52 exacerbates LPS responses. Thus, soluble CD52 exhibits broad immune suppressive effects that signify its potential as an immunotherapeutic agent.

OBJECTIVES: The side population (SP) contains cells with stem cell/progenitor properties. Previously, we observed that the mouse pancreas SP expanded after pancreatic injury. We aimed to characterize the SP in human pancreas as a potential source of stem cells. METHODS: Human organ donor pancreata were fractionated into islets and exocrine tissue, enriched by tissue culture and dispersed into single cells. Cells were phenotyped by flow cytometry, and the SP was defined by efflux of fluorescent dye Hoechst 33342 visualized by ultraviolet excitation. Cells were flow sorted, and their colony-forming potential measured on feeder cells in culture. RESULTS: An SP was identified in islet and exocrine cells from human organ donors: 2 with type 1 diabetes, 3 with type 2 diabetes, and 28 without diabetes. Phenotyping revealed that exocrine SP cells had an epithelial origin, were enriched for carbohydrate antigen 19-9 ductal cells expressing stem cell markers CD133 and CD26, and had greater colony-forming potential than non-SP cells. The exocrine SP was increased in a young adult with type 1 diabetes and ongoing islet autoimmunity. CONCLUSIONS: The pancreatic exocrine SP is a potential reservoir of adult stem/progenitor cells, consistent with previous evidence that such cells are duct-derived and express CD133.

The asymptomatic phase of type 1 diabetes is recognised by the presence of beta cell autoantibodies in the absence of hyperglycaemia. We propose that an accurate description of this stage is provided by the name 'Autoimmune Beta Cell Disorder' (ABCD). Specifically, we suggest that this nomenclature and diagnosis will, in a proactive manner, shift the paradigm towards type 1 diabetes being first and foremost an immune-mediated disease and only later a metabolic disease, presaging more active therapeutic intervention in the asymptomatic stage of disease, before end-stage beta cell failure. Furthermore, we argue that accepting ABCD as a diagnosis will be critical in order to accelerate pharmaceutical, academic and public activities leading to clinical trials that could reverse beta cell autoimmunity and halt progression to symptomatic insulin-requiring type 1 diabetes. We recognize that there are both opportunities and challenges in the implementation of the ABCD concept but hope that the notion of 'asymptomatic autoimmune disease' as a disorder will be widely discussed and eventually accepted. ispartof: Diabetologia vol:60 issue:1 pages:35-38 ispartof: location:Germany status: published

This study examines the association between rotavirus vaccination and declining rates of type 1 diabetes in children.

Vaccination against self-antigens to avert pathological immunity to self--'negative vaccination'--is the Holy Grail of autoimmune disease therapy. This approach depends on deletion or inactivation of pathogenic T cells, or induction of protective, 'regulatory' T cells. While effective in inbred rodent models, it is yet to be translated to humans. Reasons for this include its application only in end-stage disease, ignorance about antigen form, route of delivery and dose-schedule required for a bio-response, lack of meaningful and measurable bio-response markers, co-activation of pathogenic immunity and genetic heterogeneity.

Poor vitamin D nutrition is linked with dementia, but vitamin D has not been tested in a randomized controlled trial (RCT) in Alzheimer's disease (AD). Nasal insulin acutely improves cognition and vitamin D upregulates insulin receptor expression and enhances insulin action. In an RCT we examined the effect of high-dose vitamin D followed by nasal insulin on memory and disability in mild-moderate AD. 63 community-dwelling individuals aged > 60 were recruited; 32 with mild-moderate disease (Folstein Mini-Mental State Examination [MMSE] score 12-24) met entry criteria and were randomized. All took low-dose vitamin D (1000 IU/day) throughout. After run-in (8 weeks), they were randomized to additional high-dose D/placebo for 8 weeks, followed immediately by randomization to nasal insulin (60 IU qid)/placebo for 48 h. Primary outcome measures were Alzheimer's disease assessment scale-cognitive subscale (ADAS-cog) and Disability Assessment in Dementia (after high-dose D) and ADAS-cog and Wechsler Memory Scale-Revised Logical memory (WMS-R LM) for immediate and delayed recall (after nasal insulin). Baseline median (interquartile range, IR) age, MMSE, and ADAS-cog were 77.5 (69-80), 19.5 (17-22), and 25.5 (20-31), respectively. Median 25OHD increased from 49 to 60 nM (p < 0.01) after run-in and was 187 nM after high-dose vitamin D and 72 nM after placebo (p < 0.001). Neither cognition nor disability changed significantly after high-dose D. ADAS-cog improved by a median (IR) of 9 (1-11) with nasal insulin after placebo high-dose vitamin D (p = 0.02), but may represent regression to the mean as WLS-R LM did not change. We conclude that high-dose vitamin D provides no benefit for cognition or disability over low-dose vitamin D in mild-moderate AD.

OBJECTIVE Excess accumulation of advanced glycation end products (AGEs) contributes to aging and chronic diseases. We aimed to obtain evidence that exposure to AGEs plays a role in the development of type 1 diabetes (T1D). RESEARCH DESIGN AND METHODS The effect of AGEs was examined on insulin secretion by MIN6N8 cells and mouse islets and in vivo in three separate rodent models: AGE-injected or high AGE–fed Sprague-Dawley rats and nonobese diabetic (NODLt) mice. Rodents were also treated with the AGE-lowering agent alagebrium. RESULTS β-Cells exposed to AGEs displayed acute glucose-stimulated insulin secretory defects, mitochondrial abnormalities including excess superoxide generation, a decline in ATP content, loss of MnSOD activity, reduced calcium flux, and increased glucose uptake, all of which were improved with alagebrium treatment or with MnSOD adenoviral overexpression. Isolated mouse islets exposed to AGEs had decreased glucose-stimulated insulin secretion, increased mitochondrial superoxide production, and depletion of ATP content, which were improved with alagebrium or with MnTBAP, an SOD mimetic. In rats, transient or chronic exposure to AGEs caused progressive insulin secretory defects, superoxide generation, and β-cell death, ameliorated with alagebrium. NODLt mice had increased circulating AGEs in association with an increase in islet mitochondrial superoxide generation, which was prevented by alagebrium, which also reduced the incidence of autoimmune diabetes. Finally, at-risk children who progressed to T1D had higher AGE concentrations than matched nonprogressors. CONCLUSIONS These findings demonstrate that AGEs directly cause insulin secretory defects, most likely by impairing mitochondrial function, which may contribute to the development of T1D.

Detection of human Ag-specific T cells is limited by sensitivity and blood requirements. As dendritic cells (DCs) can potently stimulate T cells, we hypothesized that their induction in PBMCs in situ could link Ag processing and presentation to Ag-specific T-cell activation. To this end, unfractionated PBMCs (fresh or frozen) or whole blood were incubated for 48 hours with protein or peptide Ag together with different DC-activating agents to rapidly and sequentially induce, pulse, and mature DCs. DC activation was therefore lined up with Ag recognition by neighboring T cells, thus telescoping the sequential steps of T-cell activation. Efficient processing of protein Ags made prior knowledge of epitopes and HLA restrictions dispensable. While reducing stimulation time, manipulation and blood requirements, in situ DC induction specifically amplified Ag-specific T-cell responses (cytokine secretion, proliferation, CD137/CD154 up-regulation, and binding of peptide-HLA multimers). IL-1β, although released by DCs, was also secreted in an Ag-specific fashion, thus providing an indirect biomarker of T-cell responses. These accelerated cocultured DC (acDC) assays offered a sensitive means with which to evaluate T-cell responses to viral and melanoma Ag vaccination, and may therefore find application for immune monitoring in viral, tumor, autoimmune, and transplantation settings.

Mouse dendritic cells (DC) have been extensively studied in various tissues, especially spleen, and they comprise subsets with distinct developmental origins, surface phenotypes, and functions. Considerably less is known about human DC due to their rarity in blood and inaccessibility of other human tissues. The study of DC in human blood has revealed four subsets distinct in phenotype and function. In this study, we describe four equivalent DC subsets in human spleen obtained from deceased organ donors. We identify three conventional DC subsets characterized by surface expression of CD1b/c, CD141, and CD16, and one plasmacytoid DC subset characterized by CD304 expression. Human DC subsets in spleen were very similar to those in human blood with respect to surface phenotype, TLR and transcription factor expression, capacity to stimulate T cells, cytokine secretion, and cross-presentation of exogenous Ag. However, organ donor health status, in particular treatment with corticosteroid methylprednisolone and brain death, may affect DC phenotype and function. DC T cell stimulatory capacity was reduced but DC were qualitatively unchanged in methylprednisolone-treated deceased organ donor spleen compared with healthy donor blood. Overall, our findings indicate that human blood DC closely resemble human spleen DC. Furthermore, we confirm parallels between human and mouse DC subsets in phenotype and function, but also identify differences in transcription factor and TLR expression as well as functional properties. In particular, the hallmark functions of mouse CD8α+ DC subsets, that is, IL-12p70 secretion and cross-presentation, are not confined to the equivalent human CD141+ DC but are shared by CD1b/c+ and CD16+ DC subsets.

OBJECTIVE Insulin in pancreatic β-cells is a target of autoimmunity in type 1 diabetes. In the NOD mouse model of type 1 diabetes, oral or nasal administration of insulin induces immune tolerance to insulin and protects against autoimmune diabetes. Evidence for tolerance to mucosally administered insulin or other autoantigens is poorly documented in humans. Adults with recent-onset type 1 diabetes in whom the disease process is subacute afford an opportunity to determine whether mucosal insulin induces tolerance to insulin subsequently injected for treatment. RESEARCH DESIGN AND METHODS We randomized 52 adults with recent-onset, noninsulin-requiring type 1 diabetes to nasal insulin or placebo for 12 months. Fasting blood glucose and serum C-peptide, glucagon-stimulated serum C-peptide, and serum antibodies to islet antigens were monitored three times monthly for 24 months. An enhanced ELISpot assay was used to measure the T-cell response to human proinsulin. RESULTS β-Cell function declined by 35% overall, and 23 of 52 participants (44%) progressed to insulin treatment. Metabolic parameters remained similar between nasal insulin and placebo groups, but the insulin antibody response to injected insulin was significantly blunted in a sustained manner in those who had received nasal insulin. In a small cohort, the interferon-γ response of blood T-cells to proinsulin was suppressed after nasal insulin. CONCLUSIONS Although nasal insulin did not retard loss of residual β-cell function in adults with established type 1 diabetes, evidence that it induced immune tolerance to insulin provides a rationale for its application to prevent diabetes in at-risk individuals.

OBJECTIVE Insulin resistance and other features of the metabolic syndrome have been causally linked to adipose tissue macrophages (ATMs) in mice with diet-induced obesity. We aimed to characterize macrophage phenotype and function in human subcutaneous and omental adipose tissue in relation to insulin resistance in obesity. RESEARCH DESIGN AND METHODS Adipose tissue was obtained from lean and obese women undergoing bariatric surgery. Metabolic markers were measured in fasting serum and ATMs characterized by immunohistology, flow cytometry, and tissue culture studies. RESULTS ATMs comprised CD11c+CD206+ cells in “crown” aggregates and solitary CD11c−CD206+ cells at adipocyte junctions. In obese women, CD11c+ ATM density was greater in subcutaneous than omental adipose tissue and correlated with markers of insulin resistance. CD11c+ ATMs were distinguished by high expression of integrins and antigen presentation molecules; interleukin (IL)-1β, -6, -8, and -10; tumor necrosis factor-α; and CC chemokine ligand-3, indicative of an activated, proinflammatory state. In addition, CD11c+ ATMs were enriched for mitochondria and for RNA transcripts encoding mitochondrial, proteasomal, and lysosomal proteins, fatty acid metabolism enzymes, and T-cell chemoattractants, whereas CD11c− ATMs were enriched for transcripts involved in tissue maintenance and repair. Tissue culture medium conditioned by CD11c+ ATMs, but not CD11c− ATMs or other stromovascular cells, impaired insulin-stimulated glucose uptake by human adipocytes. CONCLUSIONS These findings identify proinflammatory CD11c+ ATMs as markers of insulin resistance in human obesity. In addition, the machinery of CD11c+ ATMs indicates they metabolize lipid and may initiate adaptive immune responses.