Kathleen Waugh

Dysregulated Toll-Like Receptor–Induced Interleukin-1β and Interleukin-6 Responses in Subjects at Risk for the Development of Type 1 Diabetes

We tested the hypothesis that altered Toll-like receptor (TLR) signaling may be involved in early stages of type 1 diabetes (T1D). To do so, we analyzed TLR-induced interleukin (IL)-1β and IL-6 responses in freshly isolated peripheral blood mononuclear cells (PBMNCs) from seropositive compared with seronegative subjects. Similar frequencies of myeloid dendritic cells (mDCs), plasmacytoid DCs (pDCs), and monocytes were observed in seropositive and seronegative subjects. Subjects with autoantibodies had increased proportions of monocytes expressing IL-1β ex vivo. Activating PBMNCs with TLR3, TLR4, or TLR7/8 agonists in vitro led to increased percentages of IL-1β–expressing monocytes and mDCs from seropositive versus seronegative subjects. TLR ligation also resulted in a diminished IL-6 response in seropositive individuals as lower frequencies of IL-6–expressing monocytes and mDCs were induced. The dysregulated TLR-induced IL-1β and IL-6 pathways were more readily detectable in children aged <11 years and from 11 to <21 years, respectively, and did not involve altered HbA1c or the presence of one or more autoantibodies. Finally, subjects with autoantibodies had lower amounts of serum chemokine (C-X-C motif) ligand 10 compared with autoantibody-negative subjects. Our data may imply that alterations in innate immune pathways are detectable in genetically susceptible individuals and could be linked with the early course of T1D.

Feasibility of screening for T1D and celiac disease in a pediatric clinic setting

Type 1 diabetes (T1D) or celiac disease (CD) develops in at least 2% of the general population. Early detection of disease‐specific autoimmunity and subsequent monitoring would be possible if screening tests were more widely available. Currently, screening for islet autoimmunity is available only in a research setting, and CD‐specific autoimmunity screening is limited to those in high‐risk groups. This study assessed the feasibility of incorporating T1D and CD autoantibody screening into a pediatric practice.

Increased inflammation is associated with islet autoimmunity and type 1 diabetes in the Diabetes Autoimmunity Study in the Young (DAISY)

Background Type 1 diabetes (TID) is characterized by a loss of pancreatic islet beta cell function resulting in loss of insulin production. Genetic and environmental factors may trigger immune responses targeting beta cells thus generating islet antibodies (IA). Immune response pathways involve a cascade of events, initiated by cytokines and chemokines, producing inflammation which can result in tissue damage. Methods A nested case-control study was performed to identify temporal changes in cytokine levels in 75 DAISY subjects: 25 diagnosed T1D, 25 persistent IA, and 25 controls. Serum samples were selected at four time points: (T1) earliest, (T2) just prior to IA, (T3) just after IA, and (T4) prior to T1D diagnosis or most recent. Cytokines (IFN-α2a, IL-6, IL-17, IL-1β, IP-10, MCP-1, IFN-γ, IL-1α, and IL-1ra) were measured using the Meso Scale Discovery system Human Custom Cytokine 9-Plex assay. Results Multivariate mixed models adjusting for HLA risk, first-degree relative status, age, and gender, showed MCP-1 and IFN-үto be significantly higher at T3 in T1D compared to IA subjects. At T4, IP-10 was significantly higher in IA subjects than controls. Conclusions This repeated measures nested case-control study identified increased inflammatory markers in IA children who developed T1D compared to IA children who had not progressed to clinical disease. It also showed increased inflammation in both T1D and IA children when compared to controls. Results suggest inflammation may be related to both the development of IA and progression to T1D.

Temporal profiles of plasma proteome during childhood development

Human blood plasma proteome reflects physiological changes associated with a childs development as well as development of disease states. While age-specific normative values are available for proteins routinely measured in clinical practice, there is paucity of comprehensive longitudinal data regarding changes in human plasma proteome during childhood. We applied TMT-10plex isobaric labeling-based quantitative proteomics to longitudinally profile the plasma proteome in 10 healthy children during their development, each with 9 serial time points from 9months to 15years of age. In total, 1828 protein groups were identified at peptide and protein level false discovery rate of 1% and with at least two razor and unique peptides. The longitudinal expression profiles of 1747 protein groups were statistically modeled and their temporal changes were categorized into 7 different patterns. The patterns and relative abundance of proteins obtained by LC-MS were also verified with ELISA. To our knowledge, this study represents the most comprehensive longitudinal profiling of human plasma proteome to date. The temporal profiles of plasma proteome obtained in this study provide a comprehensive resource and reference for biomarker studies in childhood diseases. Biological significance: A pediatric plasma proteome database with longitudinal expression patterns of 1747 proteins from neonate to adolescence was provided to the research community. 970 plasma proteins had age-dependent expression trends, which demonstrated the importance of longitudinal profiling study to identify the potential biomarkers specific to childhood diseases, and the requirement of strictly age-matched clinical samples in a cross-sectional study in pediatric population.

Temporal expression profiling of plasma proteins reveals oxidative stress in early stages of Type 1 Diabetes progression

Blood markers other than islet autoantibodies are greatly needed to indicate the pancreatic beta cell destruction process as early as possible, and more accurately reflect the progression of Type 1 Diabetes Mellitus (T1D). To this end, a longitudinal proteomic profiling of human plasma using TMT-10plex-based LC-MS/MS analysis was performed to track temporal proteomic changes of T1D patients (n=11) across 9 serial time points, spanning the period of T1D natural progression, in comparison with those of the matching healthy controls (n=10). To our knowledge, the current study represents the largest (>2000 proteins measured) longitudinal expression profiles of human plasma proteome in T1D research. By applying statistical trend analysis on the temporal expression patterns between T1D and controls, and Benjamini-Hochberg procedure for multiple-testing correction, 13 protein groups were regarded as having statistically significant differences during the entire follow-up period. Moreover, 16 protein groups, which play pivotal roles in response to oxidative stress, have consistently abnormal expression trend before seroconversion to islet autoimmunity. Importantly, the expression trends of two key reactive oxygen species-decomposing enzymes, Catalase and Superoxide dismutase were verified independently by ELISA. BIOLOGICAL SIGNIFICANCE The temporal changes of >2000 plasma proteins (at least quantified in two subjects), spanning the entire period of T1D natural progression were provided to the research community. Oxidative stress related proteins have consistently different dysregulated patterns in T1D group than in age-sex matched healthy controls, even prior to appearance of islet autoantibodies - the earliest sign of islet autoimmunity and pancreatic beta cell stress.

Deconvoluting Virome-Wide Antiviral Antibody Profiling Data

The ability to comprehensively characterize exposures and immune responses to viral infections will be critical to better understanding human health and disease. We previously described the VirScan system, a phage-display based technology for profiling antibody binding to a comprehensive library of peptides designed to represent the human virome. The previous VirScan analytical approach did not fully account for disproportionate representation of viruses in the library or for antibody cross-reactivity among sequences shared by related viruses. Here we present the ‘AntiViral Antibody Response Deconvolution Algorithm’ (‘AVARDA’), a multi-module software package for analyzing VirScan datasets. AVARDA provides a probabilistic assessment of infection at species-level resolution by considering alignment of all library peptides to each other and to all human viruses. We employed AVARDA to analyze VirScan data from a cohort of encephalitis patients with either known viral infections or undiagnosed etiologies. By comparing acute and convalescent sera, AVARDA successfully confirmed or detected antibody responses to human herpesviruses 1, 3, 4, 5, and 6, thereby improving the rate of diagnosing viral encephalitis in this cohort by 62.5%. We further assessed AVARDA’s utility in the setting of an epidemiological study, demonstrating its ability to determine infections acquired in a child followed prospectively from infancy. We consider ways in which AVARDA’s conceptual framework may be further developed in the future and describe how its analyses may be extended beyond investigations of viral infection. AVARDA, in combination with VirScan and other pan-pathogen serological techniques, is likely to find broad utility in the epidemiology and diagnosis of infectious diseases.

Higher Sensitivity and Earlier Identification of Celiac Disease Autoimmunity by a Nonradioactive Assay for Transglutaminase Autoantibodies.

Higher sensitive transglutaminase autoantibody (TGA) assay will detect the onset of celiac disease (CD) autoimmunity earlier. In developing a nonradioactive assay for TGA, we utilized electrochemiluminescence (ECL) technology and compared it to a high-performance radioimmunoassay (RIA) currently being used to screen patients with type 1 diabetes (T1D) and genetically at-risk individuals for CD. We selected 183 T1D patients with 60 patients having received biopsy and analyzed 396 sequential samples from 73 young children longitudinally followed up with TGA seroconversion, with 27 undergoing biopsy. In addition, 112 age-matched healthy control subjects were included in the study. With the 99th percentile of specificity, the ECL assay detected significantly more TGA positivity among patients with T1D (133/183) than RIA (114/183) and more of the sequential samples (34%) from 73 children than RIA (18%). The TGA assay performed by ECL was positive in all 59 subjects with villous atrophy. Among 73 longitudinally followed up children, ECL assay had earlier detection of TGA on 34 children by a mean of 2.5 years. In conclusion, the new TGA assay by ECL has a higher sensitivity than the current RIA assay and may better predict the onset of CD.