Peripheral blood biomarkers of cell-specific autoimmunity. Studies in children at increased risk for type 1 diabetes.

Objective While the incidence of children suffering from autoimmune type 1 diabetes (T1D) is increasing in Sweden and worldwide, the underlying etiology and cellular mechanisms behind this remain unknown. The predisposition of the high-risk HLA DR-DQ genotype and as yet unknown environmental triggers lead to autoimmunity and the onset of T1D, which is preceded by islet beta-cell autoantibodies acting as markers for ongoing autoimmunity. This study aims to identify peripheral blood biomarkers to predict and explain cellular autoimmune processes leading to beta-cell loss before and after seroconversion. We also investigate whether immune tolerance treatment with GAD-alum affects T-cells in nondiabetic children at increased genetic risk of T1D prospectively followed in longitudinal studies. Methods Children participating in the Swedish TEDDY cohort with or without islet beta-cell autoantibodies were studied. Complete blood count in these children was analyzed and related to autoantibody status, gender, HLA genotype, and glucose metabolism measures. HbA1c, a predictive biomarker for a subsequent autoantibody or T1D, was analyzed in the TEDDY cohort from Finland, Germany, Sweden, and the US. HbA1c trajectories were also studied in the progression from developing a single autoantibody to diagnosing T1D. Children aged 4–17.99 years at enrollment participating in the DiAPREV-IT2 clinical trial were studied and different T-cells were immunophenotyped to investigate the immune tolerance treatment with GAD-alum. Results A reduction in neutrophil counts primarily in boys and children with the HLA-DR3-DQ2/DR4-DQ8 genotype, and a reduction of red blood cell counts, hemoglobin, and hematocrit primarily in girls and in children with HLA-DR3-DQ2/DR4-DQ8 were inversely associated with autoimmunity and the number of beta-cell autoantibodies. A reduction in red blood cell indices (MCH and MCV) was associated with increased HbA1c, by increased number of beta-cell autoantibodies. Reduction in red blood cell count, hemoglobin, and hematocrit levels were associated with increased fasting blood glucose. Increased red blood cell counts and hemoglobin, hematocrit, and MCH were associated with increased fasting insulin. Increased HbA1c was associated with an increased risk of T1D regardless of the number and type of autoantibodies. The development of IA-2A as a second or fourth autoantibody was associated with decreased HbA1c levels. The HbA1c trajectories presented a more rapid increase of HbA1c as the number of autoantibodies increased from one to three. GAD-alum-treated children had lower T-helper cell (CD3+ CD4+ T-cells ) and cytotoxic T-cell (CD3+ CD8+ T-cells) levels 18–24 months after two immunizations with GAD-alum. Conclusion Reductions in neutrophil levels, red blood cells, and red blood cell parameters and increased levels of HbA1c are all associated with multiple autoantibodies, reflecting a prominent islet autoimmune burden. The reduction in different complete blood counts with increasing numbers of beta-cell autoantibodies may suggest an unknown effect of impaired beta-cell function on hematopoiesis. Predicted trajectories of HbA1c could be used to further develop a model to predict the time to T1D diagnosis in children with multiple autoantibodies. The decrease in HbA1c associated with the appearance of IA-2A may be a consequence of aggressive autoimmune destruction of beta-cells leading to insulin leakage into the bloodstream. These results should prove helpful for understanding the pathogenesis of T1D and better predicting the onset of T1D in seroconverted children. Immunization with GAD-alum has a long-term effect on T-cells 18–24 months after treatment

Beta cell function in participants with single or multiple islet autoantibodies at baseline in the TEDDY Family Prevention Study: TEFA

AimThe aim of the present study was to assess beta cell function based on an oral glucose tolerance test (OGTT) in participants with single islet autoantibody or an intravenous glucose tolerance test (IvGTT) in participants with multiple islet autoantibodies.Materials and methodsHealthy participants in Sweden and Finland, between 2 and 49.99 years of age previously identified as positive for a single (n = 30) autoantibody to either insulin, glutamic acid decarboxylase, islet antigen-2, zinc transporter 8 or islet cell antibodies or multiple autoantibodies (n = 46), were included. Participants positive for a single autoantibody underwent a 6-point OGTT while participants positive for multiple autoantibodies underwent an IvGTT. Glucose, insulin and C-peptide were measured from OGTT and IvGTT samples.ResultsAll participants positive for a single autoantibody had a normal glucose tolerance test with 120 minutes glucose below 7.70 mmol/L and HbA1c values within the normal range (ConclusionParticipants positive for a single autoantibody appeared to have a normal beta cell function. Participants positive for three or more autoantibodies had a lower FPIR as compared to participants with two autoantibodies, supporting the view that their beta cell function had deteriorated.</p

HbA1c as a time predictive biomarker for an additional islet autoantibody and type 1 diabetes in seroconverted TEDDY children

Objective Increased level of glycated hemoglobin (HbA1c) is associated with type 1 diabetes onset that in turn is preceded by one to several autoantibodies against the pancreatic islet beta cell autoantigens; insulin (IA), glutamic acid decarboxylase (GAD), islet antigen-2 (IA-2) and zinc transporter 8 (ZnT8). The risk for type 1 diabetes diagnosis increases by autoantibody number. Biomarkers predicting the development of a second or a subsequent autoantibody and type 1 diabetes are needed to predict disease stages and improve secondary prevention trials. This study aimed to investigate whether HbA1c possibly predicts the progression from first to a subsequent autoantibody or type 1 diabetes in healthy children participating in the Environmental Determinants of Diabetes in the Young (TEDDY) study. Research Design and Methods A joint model was designed to assess the association of longitudinal HbA1c levels with the development of first (insulin or GAD autoantibodies) to a second, second to third, third to fourth autoantibody or type 1 diabetes in healthy children prospectively followed from birth until 15 years of age. Results It was found that increased levels of HbA1c were associated with a higher risk of type 1 diabetes (HR 1.82, 95% CI [1.57-2.10], p Conclusion In conclusion, increased HbA1c is a reliable time predictive marker for type 1 diabetes onset. The increased rate of increase of HbA1c from first to third autoantibody and the decrease in HbA1c predicting the development of IA-2A are novel findings proving the link between HbA1c and the appearance of autoantibodies.</p

Complete blood counts with red blood cell determinants associate with reduced beta-cell function in seroconverted Swedish TEDDY children

Objectives: To investigate whether changes in complete blood count (CBC) in islet autoantibody positive children with increased genetic risk for type 1 diabetes are associated with oral glucose tolerance tests (OGTT) and HbA1c over time. Methods: The Environmental Determinants of Diabetes in the Young (TEDDY) study follows children with increased risk for type 1 diabetes in the United States, Germany, Sweden and Finland. In the current study, 89 Swedish TEDDY children (median age 8.8 years) positive for one or multiple islet autoantibodies were followed up to 5 (median 2.3) years for CBC, OGTT and HbA1c. A statistical mixed effect model was used to investigate the association between CBC and OGTT or HbA1c. Results: HbA1c over time increased by the number of autoantibodies (p <.001). Reduction in mean corpuscular haemoglobin (MCH) and mean cell volume (MCV) was both associated with an increase in HbA1c (p <.001). A reduction in red blood cell (RBC) counts (p =.003), haemoglobin (p =.002) and haematocrit (p =.006) levels was associated with increased fasting glucose. Increased red blood cells, haemoglobin, haematocrit and MCH but decreased levels of red blood cell distribution widths (RDW) were all associated with increased fasting insulin. Conclusions: The decrease in RBC indices with increasing HbA1c and the decrease in RBC and its parameters with increasing fasting glucose in seroconverted children may reflect an insidious deterioration in glucose metabolism associated with islet beta-cell autoimmunity

Immunocyte single cell analysis of vaccine-induced narcolepsy

n/aFunding Agencies|Alfred osterlunds Stiftelse; Anna och Edwin Bergers Stiftelse; Magnus Bergvalls Stiftelse; Crafoord Foundation; Filip Lundbergs Stiftelse; Fredrik och Ingrid Thurings Stiftelse; Royal Physiographic Society of Lund- Hedda; John Forssmans Foundation; Gunvor och Josef Aners Stiftelse; Gyllenstiernska Krapperupsstiftelsen; Jerringfonden; Kronprinsessan Lovisas Forening For Barnasjukvard/Stiftelsen Axel Tielmans Minnesfond; Linnea och Josef Carlssons Stiftelse; Neuro Sweden; Rune Ljungdahls Stiftelse; Stiftelsen Samariten; Stiftelsen till minne av Personalforeningarna i Holmia Forsakring AB; The Gun and Bertil Stohnes Foundation; Segerfalk Foundation; Svenska Lakaresallskapet; Tage Bluchers Stiftelse; Region Skane FoU; ALF grants; Swedish Foundation for Strategic ResearchSwedish Foundation for Strategic Research [IRC15-0067]; Swedish Research Council, Linnaeus grantSwedish Research Council [349-2006-237]</p

Immunocyte single cell analysis of vaccine-induced narcolepsy

Increased incidence of narcolepsy type 1 (NT1) was observed following Pandemrix®-vaccination, initiated as a preventive measure against the 2009 Influenza pandemic. Here, single cell analysis was conducted to suggest a lower number of CD8+CD27+ T cells among these patients. These findings provide understanding into the autoimmune pathogenesis of NT1

Reduction in White Blood Cell, Neutrophil, and Red Blood Cell Counts Related to Sex, HLA, and Islet Autoantibodies in Swedish TEDDY Children at Increased Risk for Type 1 Diabetes

Islet autoantibodies (IAs) precede the clinical onset of type 1 diabetes (T1D); however, the knowledge is limited about whether the prodrome affects complete blood counts (CBCs) in 4- to 12-year-old children with increased genetic risk for T1D. This study tested whether CBCs were altered in 4- to 12-year-old children without (n = 376) or with one or several IAs against insulin, GAD65, or IA-2 (n = 72). CBC was analyzed during longitudinal follow-up in 448 Swedish children enrolled in The Environmental Determinants of Diabetes in the Young (TEDDY) study. A linear mixed-effects model was used to assess potential association between IA and CBC measurements over time. The white blood cell and neutrophil counts were reduced in children with IAs, primarily in boys. In contrast, girls had lower levels of hemoglobin and hematocrit. Positivity for multiple IAs showed the lowest counts in white blood cells and neutrophils in boys and red blood cells, hemoglobin, and hematocrit in girls. These associations were primarily observed in children with the HLA-DR3-DQ2/DR4-DQ8 genotype. We conclude that the reduction in neutrophils and red blood cells in children with multiple IAs and HLA-DR3-DQ2/DR4-DQ8 genotype may signal a sex-dependent islet autoimmunity detected in longitudinal CBCs

Heterogeneity of beta-cell function in subjects with multiple islet autoantibodies in the TEDDY family prevention study - TEFA

Individuals with multiple islet autoantibodies are at increased risk for clinical type 1 diabetes and may proceed gradually from stage to stage complicating the recruitment to secondary prevention studies. We evaluated multiple islet autoantibody positive subjects before randomisation for a clinical trial 1 month apart for beta-cell function, glucose metabolism and continuous glucose monitoring (CGM). We hypothesized that the number and type of islet autoantibodies in combination with different measures of glucose metabolism including fasting glucose, HbA1c, oral glucose tolerance test (OGTT), intra venous glucose tolerance test (IvGTT) and CGM allows for more precise staging of autoimmune type 1 diabetes than the number of islet autoantibodies alone.Peer reviewe

Long-Term GAD-alum Treatment Effect on Different T-Cell Subpopulations in Healthy Children Positive for Multiple Beta Cell Autoantibodies

Objective. The objective of this study was to explore whether recombinant GAD65 conjugated hydroxide (GAD-alum) treatment affected peripheral blood T-cell subpopulations in healthy children with multiple beta cell autoantibodies. Method. The Diabetes Prevention–Immune Tolerance 2 (DiAPREV-IT 2) clinical trial enrolled 26 children between 4 and 13 years of age, positive for glutamic acid decarboxylase autoantibody (GADA) and at least one other autoantibody (insulin, insulinoma antigen-2, or zinc transporter 8 autoantibody (IAA, IA-2A, or ZnT8A)) at baseline. The children were randomized to two doses of subcutaneously administered GAD-alum treatment or placebo, 30 days apart. Complete blood count (CBC) and immunophenotyping of T-cell subpopulations by flow cytometry were performed regularly during the 24 months of follow-up posttreatment. Cross-sectional analyses were performed comparing lymphocyte and T-cell subpopulations between GAD-alum and placebo-treated subjects. Results. GAD-alum-treated children had lower levels of lymphocytes (109 cells/L) (), T-cells (103 cells/μL) (), T-helper cells (103 cells/μL) (), and cytotoxic T-cells (103 cells/μL) () compared to the placebo-treated children 18 months from first GAD-alum injection. This difference remained 24 months after the first treatment for lymphocytes (), T-cells (), T-helper cells (), and cytotoxic T-cells (). Conclusion. Our findings suggest that levels of total T-cells and T-cell subpopulations declined 18 and 24 months after GAD-alum treatment in healthy children with multiple beta-cell autoantibodies including GADA