Definition of Naturally Processed Peptides Reveals Convergent Presentation of Autoantigenic Topoisomerase I Epitopes in Scleroderma.

ObjectiveAutoimmune responses to DNA topoisomerase I (topo I) are found in a subset of scleroderma patients who are at high risk for interstitial lung disease (ILD) and mortality. Anti-topo I antibodies (ATAs) are associated with specific HLA-DRB1 alleles, and the frequency of HLA-DR-restricted topo I-specific CD4+ T cells is associated with the presence, severity, and progression of ILD. Although this strongly implicates the presentation of topo I peptides by HLA-DR in scleroderma pathogenesis, the processing and presentation of topo I has not been studied.MethodsWe developed a natural antigen processing assay (NAPA) to identify putative CD4+ T cell epitopes of topo I presented by monocyte-derived dendritic cells (mo-DCs) from 6 ATA-positive patients with scleroderma. Mo-DCs were pulsed with topo I protein, HLA-DR-peptide complexes were isolated, and eluted peptides were analyzed by mass spectrometry. We then examined the ability of these naturally presented peptides to induce CD4+ T cell activation in 11 ATA-positive and 11 ATA-negative scleroderma patients.ResultsWe found that a common set of 10 topo I epitopes was presented by Mo-DCs from scleroderma patients with diverse HLA-DR variants. Sequence analysis revealed shared peptide-binding motifs within the HLA-DRβ chains of ATA-positive patients and a subset of topo I epitopes with distinct sets of anchor residues capable of binding to multiple different HLA-DR variants. The NAPA-derived epitopes elicited robust CD4+ T cell responses in 73% of ATA-positive patients (8 of 11), and the number of epitopes recognized correlated with ILD severity (P = 0.025).ConclusionThese findings mechanistically implicate the presentation of a convergent set of topo I epitopes in the development of scleroderma

A methodology for exploring biomarker – phenotype associations: application to flow cytometry data and systemic sclerosis clinical manifestations

BACKGROUND: This work seeks to develop a methodology for identifying reliable biomarkers of disease activity, progression and outcome through the identification of significant associations between high-throughput flow cytometry (FC) data and interstitial lung disease (ILD) - a systemic sclerosis (SSc, or scleroderma) clinical phenotype which is the leading cause of morbidity and mortality in SSc. A specific aim of the work involves developing a clinically useful screening tool that could yield accurate assessments of disease state such as the risk or presence of SSc-ILD, the activity of lung involvement and the likelihood to respond to therapeutic intervention. Ultimately this instrument could facilitate a refined stratification of SSc patients into clinically relevant subsets at the time of diagnosis and subsequently during the course of the disease and thus help in preventing bad outcomes from disease progression or unnecessary treatment side effects. The methods utilized in the work involve: (1) clinical and peripheral blood flow cytometry data (Immune Response In Scleroderma, IRIS) from consented patients followed at the Johns Hopkins Scleroderma Center. (2) machine learning (Conditional Random Forests - CRF) coupled with Gene Set Enrichment Analysis (GSEA) to identify subsets of FC variables that are highly effective in classifying ILD patients; and (3) stochastic simulation to design, train and validate ILD risk screening tools. RESULTS: Our hybrid analysis approach (CRF-GSEA) proved successful in predicting SSc patient ILD status with a high degree of success (>82 % correct classification in validation; 79 patients in the training data set, 40 patients in the validation data set). CONCLUSIONS: IRIS flow cytometry data provides useful information in assessing the ILD status of SSc patients. Our new approach combining Conditional Random Forests and Gene Set Enrichment Analysis was successful in identifying a subset of flow cytometry variables to create a screening tool that proved effective in correctly identifying ILD patients in the training and validation data sets. From a somewhat broader perspective, the identification of subsets of flow cytometry variables that exhibit coordinated movement (i.e., multi-variable up or down regulation) may lead to insights into possible effector pathways and thereby improve the state of knowledge of systemic sclerosis pathogenesis. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12859-015-0722-x) contains supplementary material, which is available to authorized users

Electronic Vaporization of Nicotine Salt or Freebase produces differential effects on metabolism, neuronal activity and behavior in male and female C57BL/6J mice

The use of Electronic Nicotine Delivery Systems (ENDS) is increasing in prevalence and popularity. ENDS are a rapidly evolving technology as devices and e-liquid formulations adapt to policy restrictions and market demand. To identify the impacts of nicotine formulation and concentration, we exposed female and male C57BL/6J mice to passive electronic vaporization of different nicotine formulations (freebase or salt) and concentrations (1% or 3%) and measured serum nicotine metabolite levels, brain activity by cFos expression, and anxiety-like and motivated behavior using the novelty suppressed feeding test. We found that the 3% freebase nicotine vapor group displayed significantly higher serum nicotine levels than either 1% or 3% nicotine salt formulations, and female mice displayed higher serum nicotine and cotinine levels compared to males. Central amygdala (CeA) activity was significantly elevated in male mice following nicotine vapor exposure, but the increase was not significantly different between nicotine vapor groups. CeA activity in female mice was unaffected. In contrast, increased activity in the ventral tegmental area (VTA) was only observed in female mice exposed to 3% nicotine freebase and specifically in the dopaminergic population. Anxiety-like behavior in female mice was relatively unaffected by nicotine vapor exposure, however male mice displayed increased anxiety-like behavior and reduced motivation to feed after vapor exposure, specifically in the 3% freebase group. These results identify important sex differences in the impact of nicotine formulation and concentration on nicotine metabolism, brain region-specific activity and anxiety-like behavior, which may have significant relevance for different consequences of vaping in men and women

Additional file 2: of A methodology for exploring biomarker – phenotype associations: application to flow cytometry data and systemic sclerosis clinical manifestations

IRIS Data Set and R code. (ZIP 291 KB

Additional file 1: of A methodology for exploring biomarker – phenotype associations: application to flow cytometry data and systemic sclerosis clinical manifestations

Flow Cytometry Details and Supplementary Results. (DOCX 1.93 MB