Nichole Reisdorph

Chromogranin A is an autoantigen in type 1 diabetes

Autoreactive CD4+ T cells are involved in the pathogenesis of many autoimmune diseases, but the antigens that stimulate their responses have been difficult to identify and in most cases are not well defined. In the nonobese diabetic (NOD) mouse model of type 1 diabetes, we have identified the peptide WE14 from chromogranin A (ChgA) as the antigen for highly diabetogenic CD4+ T cell clones. Peptide truncation and extension analysis shows that WE14 bound to the NOD mouse major histocompatibility complex class II molecule I-Ag7 in an atypical manner, occupying only the carboxy-terminal half of the I-Ag7 peptide-binding groove. This finding extends the list of T cell antigens in type 1 diabetes and supports the idea that autoreactive T cells respond to unusually presented self peptides.

Pathogenic CD4 T cells in type 1 diabetes recognize epitopes formed by peptide fusion

T cells target peptide combos One of the enduring mysteries of autoimmunity is the identity of the specific proteins targeted by autoimmune T cells. Delong et al. used mass spectrometry to elucidate the peptide targets of autoimmune T cells isolated from a mouse model of type 1 diabetes. T cells targeted hybrid peptides formed by the covalent linking of a peptide derived from pro-insulin to other peptides derived from proteins found in pancreatic beta cells. T cells isolated from the pancreatic islets of two individuals with type 1 diabetes also recognized such hybrid peptides, suggesting that they may play an important role in driving disease. Science, this issue p. 711 Autoimmune T cells recognize covalently linked peptides derived from two distinct proteins. T cell–mediated destruction of insulin-producing β cells in the pancreas causes type 1 diabetes (T1D). CD4 T cell responses play a central role in β cell destruction, but the identity of the epitopes recognized by pathogenic CD4 T cells remains unknown. We found that diabetes-inducing CD4 T cell clones isolated from nonobese diabetic mice recognize epitopes formed by covalent cross-linking of proinsulin peptides to other peptides present in β cell secretory granules. These hybrid insulin peptides (HIPs) are antigenic for CD4 T cells and can be detected by mass spectrometry in β cells. CD4 T cells from the residual pancreatic islets of two organ donors who had T1D also recognize HIPs. Autoreactive T cells targeting hybrid peptides may explain how immune tolerance is broken in T1D.

Plasma sphingolipids associated with chronic obstructive pulmonary disease phenotypes

RATIONALEnChronic obstructive pulmonary disease (COPD) occurs in a minority of smokers and is characterized by intermittent exacerbations and clinical subphenotypes such as emphysema and chronic bronchitis. Although sphingolipids as a class are implicated in the pathogenesis of COPD, the particular sphingolipid species associated with COPD subphenotypes remain unknown.nnnOBJECTIVESnTo use mass spectrometry to determine which plasma sphingolipids are associated with subphenotypes of COPD.nnnMETHODSnOne hundred twenty-nine current and former smokers from the COPDGene cohort had 69 distinct sphingolipid species detected in plasma by targeted mass spectrometry. Of these, 23 were also measured in 131 plasma samples (117 independent subjects) using an untargeted platform in an independent laboratory. Regression analysis with adjustment for clinical covariates, correction for false discovery rate, and metaanalysis were used to test associations between COPD subphenotypes and sphingolipids. Peripheral blood mononuclear cells were used to test associations between sphingolipid gene expression and plasma sphingolipids.nnnMEASUREMENTS AND MAIN RESULTSnOf the measured plasma sphingolipids, five sphingomyelins were associated with emphysema; four trihexosylceramides and three dihexosylceramides were associated with COPD exacerbations. Three sphingolipids were strongly associated with sphingolipid gene expression, and 15 sphingolipid gene/metabolite pairs were differentially regulated between COPD cases and control subjects.nnnCONCLUSIONSnThere is evidence of systemic dysregulation of sphingolipid metabolism in patients with COPD. Subphenotyping suggests that sphingomyelins are strongly associated with emphysema and glycosphingolipids are associated with COPD exacerbations.

Urinary leukotriene E4 levels identify children with tobacco smoke exposure at risk for asthma exacerbation

BACKGROUNDnChildren with asthma exposed to secondhand smoke (SHS) might be at higher risk for severe exacerbations, but biomarkers of susceptibility to SHS exposure have not been previously reported.nnnOBJECTIVESnWe sought to assess the usefulness of urinary leukotriene E(4) (uLTE₄) levels in the prediction of increased risk of severe asthma exacerbations requiring emergency department (ED) or urgent care (UC) visits.nnnMETHODSnForty-four schoolchildren with moderate-to-severe asthma receiving inhaled corticosteroids were followed for 5 months with repeated measurements of uLTE₄ and monitoring of ED and UC visits. SHS exposure status was determined by using prestudy questionnaires and repeated measurements of urinary cotinine during the study.nnnRESULTSnNine (45%) of 20 children with SHS exposure experienced a severe exacerbation requiring an ED or UC visit compared with 3 (12.5%) of 24 children without significant SHS exposure (relative risk, 3.6; 95% CI, 1.1-11.5; P = .02). The uLTE₄ level was a significant predictor of exacerbation risk in children exposed to SHS (area under the curve, 0.85; P = .003). Other predictors, such as nighttime symptom frequency, prebronchodilator and postbronchodilator lung function, and exhaled nitric oxide levels, were not related to exacerbations in this group. uLTE₄ levels at or greater than 106 pg/mg achieved 67% (6/9) sensitivity and 100% (11/11) specificity for predicting children with SHS exposure who required an ED or UC visit.nnnCONCLUSIONSnChildren exposed to SHS are at increased risk for severe asthma exacerbations, despite use of inhaled corticosteroids. uLTE₄ levels identify children exposed to SHS at high risk for asthma exacerbations.

Increased Cell Surface Fas Expression Is Necessary and Sufficient To Sensitize Lung Fibroblasts to Fas Ligation-Induced Apoptosis: Implications for Fibroblast Accumulation in Idiopathic Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is associated with the accumulation of collagen-secreting fibroblasts and myofibroblasts in the lung parenchyma. Many mechanisms contribute to their accumulation, including resistance to apoptosis. In previous work, we showed that exposure to the proinflammatory cytokines TNF-α and IFN-γ reverses the resistance of lung fibroblasts to apoptosis. In this study, we investigate the underlying mechanisms. Based on an interrogation of the transcriptomes of unstimulated and TNF-α– and IFN-γ–stimulated primary lung fibroblasts and the lung fibroblast cell line MRC5, we show that among Fas-signaling pathway molecules, Fas expression was increased ∼6-fold in an NF-κB– and p38mapk-dependent fashion. Prevention of the increase in Fas expression using Fas small interfering RNAs blocked the ability of TNF-α and IFN-γ to sensitize fibroblasts to Fas ligation-induced apoptosis, whereas enforced adenovirus-mediated Fas overexpression was sufficient to overcome basal resistance to Fas-induced apoptosis. Examination of lung tissues from IPF patients revealed low to absent staining of Fas in fibroblastic cells of fibroblast foci. Collectively, these findings suggest that increased expression of Fas is necessary and sufficient to overcome the resistance of lung fibroblasts to Fas-induced apoptosis. Our findings also suggest that approaches aimed at increasing Fas expression by lung fibroblasts and myofibroblasts may be therapeutically relevant in IPF.

A novel, in-solution separation of endogenous cardiac sarcomeric proteins and identification of distinct charged variants of regulatory light chain

The molecular conformation of the cardiac myosin motor is modulated by intermolecular interactions among the heavy chain, the light chains, myosin binding protein-C, and titin and is governed by post-translational modifications (PTMs). In-gel digestion followed by LC/MS/MS has classically been applied to identify cardiac sarcomeric PTMs; however, this approach is limited by protein size, pI, and difficulties in peptide extraction. We report a solution-based work flow for global separation of endogenous cardiac sarcomeric proteins with a focus on the regulatory light chain (RLC) in which specific sites of phosphorylation have been unclear. Subcellular fractionation followed by OFFGEL electrophoresis resulted in isolation of endogenous charge variants of sarcomeric proteins, including regulatory and essential light chains, myosin heavy chain, and myosin-binding protein-C of the thick filament. Further purification of RLC using reverse-phase HPLC separation and UV detection enriched for RLC PTMs at the intact protein level and provided a stoichiometric and quantitative assessment of endogenous RLC charge variants. Digestion and subsequent LC/MS/MS unequivocally identified that the endogenous charge variants of cardiac RLC focused in unique OFFGEL electrophoresis fractions were unphosphorylated (78.8%), singly phosphorylated (18.1%), and doubly phosphorylated (3.1%) RLC. The novel aspects of this study are that 1) milligram amounts of endogenous cardiac sarcomeric subproteome were focused with resolution comparable with two-dimensional electrophoresis, 2) separation and quantification of post-translationally modified variants were achieved at the intact protein level, 3) separation of intact high molecular weight thick filament proteins was achieved in solution, and 4) endogenous charge variants of RLC were separated; a novel doubly phosphorylated form was identified in mouse, and singly phosphorylated, singly deamidated, and deamidated/phosphorylated forms were identified and quantified in human non-failing and failing heart samples, thus demonstrating the clinical utility of the method.

An insulin-IAPP hybrid peptide is an endogenous antigen for CD4 T cells in the non-obese diabetic mouse.

BDC-6.9, a diabetogenic CD4 T cell clone isolated from a non-obese diabetic (NOD) mouse, responds to pancreatic islet cells from NOD but not BALB/c mice. We recently reported that a hybrid insulin peptide (HIP), 6.9HIP, formed by linkage of an insulin C-peptide fragment and a fragment of islet amyloid polypeptide (IAPP), is the antigen for BDC-6.9. We report here that the core 12-mer peptide from 6.9HIP, centered on the hybrid peptide junction, is also highly antigenic for BDC-6.9. In agreement with the observation that BALB/c islet cells fail to stimulate the T cell clone, a single amino acid difference in the BALB/c IAPP sequence renders the BALB/c version of the HIP only weakly antigenic. Mutant peptide analysis indicates that each parent molecule-insulin C-peptide and IAPP-donates residues critical for antigenicity. Through mass spectrometric analysis, we determine the distribution of naturally occurring 6.9HIP across chromatographic fractions of proteins from pancreatic beta cells. This distribution closely matches the profile of the T cell response to the fractions, confirming that 6.9HIP is the endogenous islet antigen for the clone. Using a new MHC II tetramer reagent, 6.9HIP-tet, we show that T cells specific for the 6.9HIP peptide are prevalent in the pancreas of diabetic NOD mice. Further study of HIPs and HIP-reactive T cells could yield valuable insight into key factors driving progression to diabetes and thereby inform efforts to prevent or reverse this disease.

Predictors of asthma control and lung function responsiveness to step 3 therapy in children with uncontrolled asthma.

BACKGROUNDnPredictors of improvement in asthma control and lung function to step 3 therapy in children with persistent asthma have not been identified despite reported heterogeneity in responsiveness.nnnOBJECTIVEnWe sought to evaluate potential predictors of asthma control and lung function responsiveness to step 3 therapy.nnnMETHODSnA post hoc analysis from the Best Add-On Giving Effective Response (BADGER) study tested the association between baseline biological, asthma control, pulmonary function, and demographic markers and responsiveness to step-up to a higher dose of inhaled corticosteroid (ICS step-up therapy) or addition of leukotriene receptor antagonist (LTRA step-up therapy) or long-acting β₂-agonist (LABA step-up therapy).nnnRESULTSnIn multivariate analyses higher impulse oscillometry reactance area was associated (P = .048) with a differential FEV₁ response favoring LABA over ICS step-up therapy, whereas higher urinary leukotriene E₄ levels were marginally (P = .053) related to a differential FEV₁ response favoring LTRA over LABA step-up therapy. Predictors of differential responses comparing ICS with LTRA step-up therapy were not apparent, probably because of suppression of allergic markers with low-dose ICS treatment. Minimal overlap was seen across FEV₁ and asthma control day predictors, suggesting distinct mechanisms related to lung function and asthma control day responses.nnnCONCLUSIONnLevels of impulse oscillometry reactance area indicating peripheral airway obstruction and urinary leukotriene E₄ levels indicating cysteinyl leukotriene inflammation can differentiate LABA step-up responses from responses to LTRA or ICS step-up therapy. Further studies with physiologic, genetic, and biological markers related to these phenotypes will be needed to predict individual responses to LABA step-up therapy.

SPLUNC1 regulation in airway epithelial cells: role of Toll-like receptor 2 signaling.

BackgroundRespiratory infections including Mycoplasma pneumoniae (Mp) contribute to various chronic lung diseases. We have shown that mouse short palate, lung, and nasal epithelium clone 1 (SPLUNC1) protein was able to inhibit Mp growth. Further, airway epithelial cells increased SPLUNC1 expression upon Mp infection. However, the mechanisms underlying SPLUNC1 regulation remain unknown. In the current study, we investigated if SPLUNC1 production following Mp infection is regulated through Toll-like receptor 2 (TLR2) signaling.MethodsAirway epithelial cell cultures were utilized to reveal the contribution of TLR2 signaling including NF-κB to SPLUNC1 production upon bacterial infection and TLR2 agonist stimulation.ResultsMp and TLR2 agonist Pam3CSK4 increased SPLUNC1 expression in tracheal epithelial cells from wild type, but not TLR2-/- BALB/c mice. RNA interference (short-hairpin RNA) of TLR2 in normal human bronchial epithelial cells under air-liquid interface cultures significantly reduced SPLUNC1 levels in Mp-infected or Pam3CSK4-treated cells. Inhibition and activation of NF-κB pathway decreased and increased SPLUNC1 production in airway epithelial cells, respectively.ConclusionsOur data for the first time suggest that airway epithelial TLR2 signaling is pivotal in mycoplasma-induced SPLUNC1 production, thus improving our understanding of the aberrant SPLUNC1 expression in airways of patients suffering from chronic lung diseases with bacterial infections.

Leukotriene-E4 in human urine: Comparison of on-line purification and liquid chromatography-tandem mass spectrometry to affinity purification followed by enzyme immunoassay

A new analytical method suitable for high throughput measurements of LTE(4) in human urine is described. The methodology utilizes on-line enrichment and liquid chromatography/tandem mass spectrometry (LC/MS/MS). The novel LC/MS/MS method is rapid, linear from 5 to 500pg/ml in spiked urine samples of both healthy and asthmatic subjects and more accurate and precise than enzyme immunoassay (EIA) and previous LC/MS/MS methods. Results from sample integrity experiments and preliminary values of urinary LTE(4) from healthy adults and children are reported.