1746-P: Differential Depletion and Expansion of Autoreactive CD8+ T Cell Subsets after Alefacept Treatment in Type 1 Diabetes (T1D)

Alterations in RNA splicing may influence protein isoform diversity that contributes to or reflects the pathophysiology of certain diseases. Whereas specific RNA splicing events in pancreatic islets have been investigated in models of inflammation in vitro, how RNA splicing in the circulation correlates with or is reflective of type 1 diabetes (T1D) disease pathophysiology in humans remains unexplored. To use machine learning to investigate if alternative RNA splicing events differ between individuals with and without new-onset T1D and to determine if these splicing events provide insight into T1D pathophysiology. RNA deep sequencing was performed on whole blood samples from 2 independent cohorts: a training cohort consisting of 12 individuals with new-onset T1D and 12 age- and sex-matched nondiabetic controls and a validation cohort of the same size and demographics. Machine learning analysis was used to identify specific isoforms that could distinguish individuals with T1D from controls. Distinct patterns of RNA splicing differentiated participants with T1D from unaffected controls. Notably, certain splicing events, particularly involving retained introns, showed significant association with T1D. Machine learning analysis using these splicing events as features from the training cohort demonstrated high accuracy in distinguishing between T1D subjects and controls in the validation cohort. Gene Ontology pathway enrichment analysis of the retained intron category showed evidence for a systemic viral response in T1D subjects. Alternative RNA splicing events in whole blood are significantly enriched in individuals with new-onset T1D and can effectively distinguish these individuals from unaffected controls. Our findings also suggest that RNA splicing profiles offer the potential to provide insights into disease pathogenesis.

IntroductionAim of this study was to investigate the pancreatic exocrine function in patients with type 1 diabetes (T1D) by multiple non-invasive tests.Research design and methodsThe study is a single-center, cross-sectional...

Background: There are limited contemporaneous data about outcomes in pregnancies with pre-existing type 1 diabetes (T1D) in the U.S. Objective: To determine maternal and neonatal outcomes in pregnancies with preexisting T1D and pregnancies in age, parity and BMI matched healthy subjects. Methods: We retrospectively studied 32 pregnancies in patients with preexisting T1D matched for age (mean 25.83 years), gravidity/parity {G1P1 (66), G2P1 (19), G3P1 (9) and G4P1 (6) % respectively} and BMI with controls (n=32) during a 5 year period from 01/01/2015 at Mayo Clinic, Rochester. Results: In 32 T1D pregnancies, 4 (12.5%) congenital defects (CD) were observed in neonates, 1-dysplastic ribs with thumb hypoplasia and 3-congentinal heart defects leading to death in one in comparison to no congenital defect or death in controls. CD pregnancies were complicated by severe pre-eclampsia in all 4 and poor glycemic control (HbA1c >8) in 3. There was a significant difference in Apgar score at 1 min between T1D pregnancies and controls (p=<0.001). Significant respiratory distress was observed in neonates with T1D pregnancies (p=0.011) compared to control group. Significant difference was observed in gestational age b/w two groups (35.9 ± 3.1 vs. 39.4 ± 1.4, p=<0.001). More severe eclampsia was observed in T1D subjects (11 vs. 2, p=0.005) along with pre-term deliveries (12 vs. 1, p=<0.001) compared to controls. Frequency of C-sections was increased in T1D subjects (19 vs.3, p=<0.001) compared to controls. Smoking had a significant effect on Apgar score at 5 min (p=0.014) when compared with current smokers or former smokers. Hypothyroidism (being on T4) during pregnancy had an effect on Apgar score both at 1 min (p=0.003) as well as at 5 min (p=0.004). Conclusion: Pregnancies in T1D continue to be high risk pregnancies and need tight glycemic control with a more advanced system such as closed loop insulin delivery during pregnancy to improve maternal and neonatal outcomes. Disclosure R. Kaur: None. S. Rizvi: None. M. Trinidad: None. B.H. Smith: None. S. Batthula: None. S.K. McCrady-Spitzer: None. C. Reid: None. D. Desjardins: None. G. O’Malley: Research Support; Self; Abbott, Dexcom, Inc. K.N. Castorino: Research Support; Self; Abbott, Dexcom, Inc., Medtronic, Mylan, Novo Nordisk Inc. J.E. Pinsker: Advisory Panel; Self; Medtronic. Consultant; Self; Eli Lilly and Company, Tandem Diabetes Care. Research Support; Self; Dexcom, Inc., Eli Lilly and Company, Insulet Corporation, Medtronic, Tandem Diabetes Care. Speaker’s Bureau; Self; Tandem Diabetes Care. W.K. Kremers: Research Support; Self; AstraZeneca. E. Dassau: Consultant; Self; Eli Lilly and Company. Research Support; Self; Dexcom, Inc., DreaMed Diabetes, Tandem Diabetes Care, Xeris Pharmaceuticals, Inc. Speaker’s Bureau; Self; Roche Diabetes Care. Other Relationship; Self; Dexcom, Inc., Insulet Corporation, Roche Diabetes Care. C.J. Levy: Consultant; Self; Dexcom, Inc. Employee; Spouse/Partner; Allergan plc. Research Support; Self; Abbott, Dexcom, Inc., Insulet Corporation. Y.C. Kudva: Research Support; Self; Dexcom, Inc., Roche Diabetes Care. Other Relationship; Self; Abbott.

Lactate kinetics in individuals with type 1 diabetes (T1D) has not been assessed quantitatively. Defining lactate kinetics in T1D under steady state condition is vital for estimating non-steady state lactate turnover. We studied 5 T1D and 7 nondiabetic (ND) anthropometrically matched participants during [1-13C]-sodium-lactate bolus injection in the resting, overnight fasted state. A two-compartment model with loss only from the peripheral compartment reliably described [1-13C]-lactate kinetics. Volume of distribution of the accessible compartment, V1, was similar between T1D and ND groups (40.32±21.08 vs. 47.64±33.32 mL/kg, p=0.76) thus concordant to plasma volume (~40 ml/kg). The irreversible loss from the peripheral compartment was impaired in T1D vs ND (0.08±0.03 vs. 0.13±0.03 min−1, p=0.03); rate of lactate appearance (12.4±5.2 vs. 18.5±9.6 µmol/kg/min, p=0.34), disappearance (12.7±5.2 vs. 18.8±9.7 µmol/kg/min, p=0.34) and lactate clearance (15.9±4.7 vs. 21.9±7.8 mL/kg/min, p=0.20) were numerically but not statistically lower in T1D vs. ND individuals. To conclude, we have defined lactate kinetics under resting, overnight fasted state in T1D and ND subjects. While the volume of distribution was similar in both groups, irreversible loss from the peripheral compartment was lower in T1D subjects. Further studies are needed to better define lactate kinetics during exercise in individuals with and without T1D. Disclosure D.Romeres: None. F.Ruchi: None. Y.Yadav: None. C.Cobelli: None. A.Basu: None. R.Basu: Consultant; Sparrow Pharmaceuticals Inc, Research Support; Abbott Diabetes, AstraZeneca. Funding National Institutes of Health (DK29953 to R.B.), (DK85516 to A.B.)

Hepatic extraction of insulin is associated with insulin sensitivity in humans. It is unknown if a diurnal pattern of HE exists and if this differs between nondiabetic (ND) and type 2 diabetes (T2D) subjects. Here are data presented for 10 ND (BMI 29.4±1.3 kg/m2, age 57±3 yrs) and 9 T2D subjects (BMI 33±2 kg/m2, age 58±4 yrs, HbA1c 57±4 mM/mol) studied thus far. They ingested identical mixed meals (75 g carbs; 8 kcal/kg) at either breakfast (B), lunch (L) or dinner (D) in randomized Latin square order on three consecutive days with frequent sampling of plasma glucose (G), insulin (I) and C-peptide (C) concentrations. Fasting and post-meal glucose concentrations were higher while fasting and post-meal insulin, C-peptide concentrations were lower during meals in T2D as compared to ND subjects. Total and basal hepatic insulin extraction (HE and HEb, respectively) were estimated using the Oral Insulin and C-peptide Minimal Models. Differences with B, L and D and between ND and T2D were assessed by N-way ANOVA (a p-value<0.05 was considered statistically significant). Results show that both HE and HEb were significantly lower (p=0.009, p=0.011, respectively) throughout the day in T2D than ND subjects (Fig 1). Our initial results suggest an emerging diurnal pattern of HE in T2D subjects with D being lower than other meals. These observations provide novel insights into the diurnal pattern of hepatic extraction throughout the day in T2D subjects. Disclosure D. Romeres: None. Y.R. Yadav: None. F. Ruchi: None. C. Cobelli: None. C. Dalla Man: Research Support; Self; Sanofi-Aventis Deutschland GmbH. A. Basu: Consultant; Spouse/Partner; GENFIT. Research Support; Spouse/Partner; AstraZeneca. R. Basu: Consultant; Self; GENFIT. Research Support; Self; AstraZeneca. Funding National Institutes of Health (R01DK029953 to R.B.), (R01DK085516 to A.B.), (DK059637, DK020593)

Metabolic dysfunction in Emirati subjects in Abu Dhabi: Relationship to levels of soluble RAGEs

BackgroundType 1 diabetes (T1D) is a T-cell mediated autoimmune disease targeting the insulin-producing pancreatic β cells. Naturally occurring FOXP3+CD4+CD25high regulatory T cells (Tregs) play an important role in dominant tolerance, suppressing autoreactive CD4+ effector T cell activity. Previously, in both recent-onset T1D patients and β cell antibody-positive at-risk individuals, we observed increased apoptosis and decreased function of polyclonal Tregs in the periphery. Our objective here was to elucidate the genes and signaling pathways triggering apoptosis in Tregs from T1D subjects.Principal FindingsGene expression profiles of unstimulated Tregs from recent-onset T1D (n = 12) and healthy control subjects (n = 15) were generated. Statistical analysis was performed using a Bayesian approach that is highly efficient in determining differentially expressed genes with low number of replicate samples in each of the two phenotypic groups. Microarray analysis showed that several cytokine/chemokine receptor genes, HLA genes, GIMAP family genes and cell adhesion genes were downregulated in Tregs from T1D subjects, relative to control subjects. Several downstream target genes of the AKT and p53 pathways were also upregulated in T1D subjects, relative to controls. Further, expression signatures and increased apoptosis in Tregs from T1D subjects partially mirrored the response of healthy Tregs under conditions of IL-2 deprivation. CD4+ effector T-cells from T1D subjects showed a marked reduction in IL-2 secretion. This could indicate that prior to and during the onset of disease, Tregs in T1D may be caught up in a relatively deficient cytokine milieu.ConclusionsIn summary, expression signatures in Tregs from T1D subjects reflect a cellular response that leads to increased sensitivity to apoptosis, partially due to cytokine deprivation. Further characterization of these signaling cascades should enable the detection of genes that can be targeted for restoring Treg function in subjects predisposed to T1D.

Type 1 diabetes (T1D) is caused by immune-mediated destruction of insulin-producing β-cells. Insufficient control of autoreactive T cells by regulatory T cells (Tregs) is believed to contribute to disease pathogenesis, but changes in Treg function are difficult to quantify because of the lack of Treg-exclusive markers in humans and the complexity of functional experiments. We established a new way to track Tregs by using a gene signature that discriminates between Tregs and conventional T cells regardless of their activation states. The resulting 31-gene panel was validated with the NanoString nCounter platform and then measured in sorted CD4(+)CD25(hi)CD127(lo) Tregs from children with T1D and age-matched control subjects. By using biomarker discovery analysis, we found that expression of a combination of six genes, including TNFRSF1B (CD120b) and FOXP3, was significantly different between Tregs from subjects with new-onset T1D and control subjects, resulting in a sensitive (mean ± SD 0.86 ± 0.14) and specific (0.78 ± 0.18) biomarker algorithm. Thus, although the proportion of Tregs in peripheral blood is similar between children with T1D and control subjects, significant changes in gene expression can be detected early in disease process. These findings provide new insight into the mechanisms underlying the failure to control autoimmunity in T1D and might lead to a biomarker test to monitor Tregs throughout disease progression.

OBJECTIVES/GOALS: We assessed the relationship between C-peptide preservation and a serum exocrine pancreatic enzyme (trypsin) in a recently concluded clinical trial. We hypothesized that immunomodulatory treatment resulting in improved beta-cell function would be associated with improved trypsin levels in subjects with recent-onset type 1 diabetes (T1D). METHODS/STUDY POPULATION: In a three-arm, randomized, double-masked, placebo-controlled trial 'Antithymocyte Globulin (ATG) and pegylated granulocyte colony stimulating factor (GCSF) in New Onset Type 1 Diabetes’ 89 subjects with recent-onset T1D (duration <100 days) were enrolled and randomized to 3 groups: low-dose ATG (2.5 mg/kg IV) followed by pegylated GCSF (6 mg subcutaneously every 2 weeks for 6 doses), low-dose ATG alone, and placebo. We compared longitudinal serum levels of an exocrine enzyme (trypsin) in a subset of responders to therapy (defined as subjects with at least 60% of baseline area under the curve (AUC) C-peptide levels at 96 weeks, n=4) versus placebo 'responders’ (n=2) and non-responders (n=25), and treated (n=19) versus placebo (n=12) subjects at baseline, 2 weeks, and 6 months after treatment. RESULTS/ANTICIPATED RESULTS: There was no observed difference in treated (n=20) versus placebo (n=12) longitudinal trends in trypsin levels when compared to baseline levels. However, responders to immunotherapy (n=4) had 6 month trypsin levels that were 114% of baseline whereas placebo subject 'responders’ (n=2), placebo subjects (n=10), and non-responders to immunotherapy (n=15) had trypsin levels that were 81-93% of baseline (unpaired T test p=0.05). Overall, we found that serum trypsin, a marker of exocrine pancreatic function, had a normal upward trend in new-onset T1D subjects who responded clinically to immunotherapy but declined in subjects who did not respond or who were not treated. These results were bordering on statistical significance but did not reach significance, likely due to the small sample size. DISCUSSION/SIGNIFICANCE: An improvement in trypsin, a marker of exocrine function, after response to immunotherapy in new-onset T1D may be due to a direct impact on exocrine function versus an indirect effect from improved beta cell function. Future studies will be needed to confirm our findings in a larger sample and evaluate the mechanism for improved exocrine function.

Background/Objective: Diabetic ketoacidosis (DKA) is a life-threatening complication of type 1 diabetes (T1D) resulting from ketone body production and metabolic acidosis occurring due to insulin deficiency. We sought to define the occurrence of DKA amongst pediatric patients presenting with new-onset T1D in Indiana and to determine whether patterns of DKA were affected by the COVID-19 pandemic. Methods: This was a retrospective chart review for patients <18 years admitted to Riley Children’s Hospital with a clinical diagnosis of new onset T1D who had available chemistry values. Patients diagnosed from March 23- June 30, 2020 and over the same period in 2019 were included. DKA was classified as mild (bicarbonate 10-15 mmol/L) or severe (bicarbonate <10 mmol/L). Results: Ninety-four patients met inclusion criteria. The total number of incident T1D cases in 2019 and 2020 were similar (48 vs. 46, respectively). Similarly, there was no significant difference in rates of DKA (21 in 2019 vs. 25 in 2020; p>0.05). Of the 94 patients, 49% met criteria for DKA; 79% of cases were classified as severe and 21% as mild. More males were diagnosed with DKA in both 2019 and 2020 (61% of DKA cases). Non-Hispanic whites comprised 75% of all new onset T1D patients and no differences in race or ethnicity were present amongst those presenting in DKA. Conclusion: DKA was present in nearly half of all new onset pediatric T1D cases in Indiana in 2019 and 2020. There was no observed impact of the COVID-19 pandemic on T1D or DKA. Impact and Implications: DKA is common amongst pediatric patients with new onset T1D in Indiana. Prompt recognition of symptoms is needed to prevent this life-threatening complication of T1D.

Elevated ratios of circulating unmethylated to methylated preproinsulin (INS) DNA have been suggested to reflect β-cell death in type 1 diabetes (T1D). We tested the hypothesis that absolute levels (rather than ratios) of unmethylated and methylated INS DNA differ between subjects with new-onset T1D and control subjects and assessed longitudinal changes in these parameters. We used droplet digital PCR to measure levels of unmethylated and methylated INS DNA in serum from subjects at T1D onset and at 8 weeks and 1 year post-onset. Compared with control subjects, levels of both unmethylated and methylated INS DNA were elevated at T1D onset. At 8 weeks post-onset, methylated INS DNA remained elevated, but unmethylated INS DNA fell. At 1 year postonset, both unmethylated and methylated INS DNA returned to control levels. Subjects with obesity, type 2 diabetes, and autoimmune hepatitis exhibited lower levels of unmethylated and methylated INS compared with subjects with T1D at onset and no differences compared with control subjects. Our study shows that elevations in both unmethylated and methylated INS DNA occurs in new-onset T1D and that levels of these DNA species change during T1D evolution. Our work emphasizes the need to consider absolute levels of differentially methylated DNA species as potential biomarkers of disease.

Multiple therapeutic strategies to restore immune regulation and slow type 1 diabetes (T1D) progression are in development and testing. A major challenge has been defining biomarkers to prospectively identify subjects likely to benefit from immunotherapy and/or measure intervention effects. We previously found that, compared with healthy controls, Tregs from children with new-onset T1D have an altered Treg gene signature (TGS), suggesting that this could be an immunoregulatory biomarker. nanoString was used to assess the TGS in sorted Tregs (CD4+CD25hiCD127lo) or peripheral blood mononuclear cells (PBMCs) from individuals with T1D or type 2 diabetes, healthy controls, or T1D recipients of immunotherapy. Biomarker discovery pipelines were developed and applied to various sample group comparisons. Compared with controls, the TGS in isolated Tregs or PBMCs was altered in adult new-onset and cross-sectional T1D cohorts, with sensitivity or specificity of biomarkers increased by including T1D-associated SNPs in algorithms. The TGS was distinct in T1D versus type 2 diabetes, indicating disease-specific alterations. TGS measurement at the time of T1D onset revealed an algorithm that accurately predicted future rapid versus slow C-peptide decline, as determined by longitudinal analysis of placebo arms of START and T1DAL trials. The same algorithm stratified participants in a phase I/II clinical trial of ustekinumab (αIL-12/23p40) for future rapid versus slow C-peptide decline. These data suggest that biomarkers based on measuring TGSs could be a new approach to stratify patients and monitor autoimmune activity in T1D. JDRF (1-PNF-2015-113-Q-R, 2-PAR-2015-123-Q-R, 3-SRA-2016-209-Q-R, 3-PDF-2014-217-A-N), the JDRF Canadian Clinical Trials Network, the National Institute of Allergy and Infectious Diseases of the National Institutes of Health (UM1AI109565 and FY15ITN168), and BCCHRI.

BackgroundThe main inhibitor of the fibrinolytic system, Plasminogen Activator Inhibitor -1 (PAI-1), irreversibly binds tissue-type Plasminogen Activator (t-PA) and thereby inhibits the protective action of tPA against thrombus formation. Elevated levels of plasma PAI-1 are associated with an increased risk of cardiovascular events and are observed in subjects with type 2 diabetes (T2D) and obesity. Platelets contain the majority of PAI-1 present in blood and exhibit the ability to synthesis active PAI-1. Diabetic platelets are known to be hyper-reactive and larger in size; however, whether these features affect their contribution to the elevated levels of plasma PAI-1 in T2D is not established.ObjectivesTo characterize the PAI-1 antigen content and the mRNA expression in platelets from T2D subjects compared to obese and lean control subjects, in order to elucidate the role of platelet PAI-1 in T2D.MethodsNine subjects with T2D and obesity were recruited from Primary Care Centers together with 15 healthy control subjects (8 lean subjects and 7 with obesity). PAI-1 antigen levels in plasma, serum and platelets were determined by ELISA, and PAI-1 mRNA expression was analyzed by qPCR.ResultsThere was no significant difference in PAI-1 mRNA expression or PAI-1 antigen in platelets in T2D subject in comparison to obese and lean control subjects. An elevated level of plasma PAI-1 was seen in both T2D and obese subjects. PAI-1 gene expression was significantly higher in both obese groups compared to lean.ConclusionSimilar levels of protein and mRNA expression of PAI-1 in platelets from T2D, obese and lean subjects indicate a limited role of platelets for the elevated plasma PAI-1 levels. However, an increased synthesis rate of mRNA transcripts in platelets from T2D and an increased release of PAI-1 could also result in similar mRNA and protein levels. Hence, synthesis and release rates of PAI-1 from platelets in T2D and obesity need to be investigated to further elucidate the role of platelets in obesity and T2D.

Circulating PANDER concentration is associated with increased HbA1c and fasting blood glucose in Type 2 diabetic subjects

Type 1 diabetes (T1D) is expected to cause significant changes in the serum proteome; however, few studies have systematically assessed the proteomic profile change associated with the disease. In this study, a semiquantitative spectral counting-based two dimensional liquid chromatography mass spectrometry platform was used to analyze serum samples from T1D patients and controls. In this discovery phase, significant differences were found for 21 serum proteins implicated in inflammation, oxidation, metabolic regulation, and autoimmunity. To assess the validity of these findings, six candidate proteins including adiponectin, insulin-like growth factor binding protein 2, serum amyloid protein A, C-reactive protein, myeloperoxidase, and transforming growth factor beta induced were selected for subsequent immune assays for 1139 T1D patients and 848 controls. A series of statistical analyses using cases and controls matched for age, sex, and genetic risk confirmed that T1D patients have significantly higher serum levels for four of the six proteins: adiponectin (odds ratio (OR) = 1.95, p = 10(-27)), insulin-like growth factor binding protein 2 (OR = 2.02, p < 10(-20)), C-reactive protein (OR = 1.13, p = 0.007), serum amyloid protein A (OR = 1.51, p < 10(-16)); whereas the serum levels were significantly lower in patients than controls for the two other proteins: transforming growth factor beta induced (OR = 0.74, p < 10(-5)) and myeloperoxidase (OR = 0.51, p < 10(-41)). Compared with subjects in the bottom quartile, subjects in the top quartile for adiponectin (OR = 6.29, p < 10(-37)), insulin-like growth factor binding protein 2 (OR = 7.95, p < 10(-46)), C-reactive protein (OR = 1.38, p = 0.025), serum amyloid protein A (OR = 3.36, p < 10(-16)) had the highest risk of T1D, whereas subjects in the top quartile of transforming growth factor beta induced (OR = 0.41, p < 10(-11)) and myeloperoxidase (OR = 0.10, p < 10(-43)) had the lowest risk of T1D. These findings provided valuable information on the proteomic changes in the sera of T1D patients.