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Background: Human rhinoviruses are known to predispose infants to asthma development during childhood and are often associated with exacerbations in asthma patients. MYADM epithelial expression has been shown to associate with asthma severity. The goal of this study was to determine if MYADM expression patterns were altered in asthma and/or rhinovirus infection and if increased MYADM expression is associated with increased asthma-associated factors., Methods: Utilizing H1HeLa cells and differentiated primary human airway epithelial cells (AECs), we measured the expression of MYADM and inflammatory genes by qRT-PCR in the presence or absence of RV-1B infection or poly I:C treatment and with siRNA knockdown of MYADM. Expression of MYADM in the asthmatic lung was determined in the ovalbumin (ova)-challenged murine model., Results: MYADM expression was upregulated in the lungs from ova-treated mice and in particular on the subsurface vesicle membrane in airway epithelial cells. Upon infection with RV-1B, human AECs grown at an air-liquid interface had increased the MYADM expression predominantly detected in ciliated cells. We found that the presence of MYADM was required for expression of several inflammatory genes both in a resting state and after RV-1B or poly I:C treatments., Conclusions: Our studies show that in a mouse model of asthma and during RV-1B infection of primary human AECs, increased MYADM expression is observed. In the mouse model of asthma, MYADM expression was predominantly on the luminal side of airway epithelial cells. Additionally, MYADM expression was strongly associated with increases in inflammatory genes, which may contribute to more severe asthma and RV-linked asthma exacerbations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Tanyaratsrisakul, Dy, Polverino, Numata and Ledford.)

Surfactant protein A (SP-A) is well-known for its protective role in pulmonary immunity. Previous studies from our group have shown that SP-A mediates eosinophil activities, including degranulation and apoptosis. In order to identify potential binding partners on eosinophils for SP-A, eosinophil lysates were subjected to SP-A pull-down and tandem mass spectrometry (MS/MS) analysis. We identified one membrane-bound protein, myeloid-associated differentiation marker (MYADM), as a candidate SP-A binding partner. Blocking MYADM on mouse and human eosinophils ex vivo prevented SP-A from inducing apoptosis; blocking MYADM in vivo led to increased persistence of eosinophilia and airway hyper-responsiveness in an ovalbumin (OVA) allergy model and increased airways resistance and mucus production in a house dust mite (HDM) asthma model. Examination of a subset of participants in the Severe Asthma Research Program (SARP) cohort revealed a significant association between epithelial expression of MYADM in asthma patients and parameters of airway inflammation, including: peripheral blood eosinophilia, exhaled nitric oxide (FeNO) and the number of exacerbations in the past 12 months. Taken together, our studies provide the first evidence of MYADM as a novel SP-A-associated protein that is necessary for SP-A to induce eosinophil apoptosis and we bring to light the potential importance of this previously unrecognized transmembrane protein in patients with asthma., (© 2021. The Author(s).)

The peptide hormone, angiotensin (Ang-(1-7)), produces anti-inflammatory and protective effects by inhibiting production and expression of many cytokines and adhesion molecules that are associated with a cytokine storm. While Ang-(1-7) has been shown to reduce inflammation and airway hyperreactivity in models of asthma, little is known about the effects of Ang-(1-7) during live respiratory infections. Our studies were developed to test if Ang-(1-7) is protective in the lung against overzealous immune responses during an infection with Mycoplasma pneumonia (Mp), a common respiratory pathogen known to provoke exacerbations in asthma and COPD patients. Wild type mice were treated with infectious Mp and a subset of was given either Ang-(1-7) or peptide-free vehicle via oropharyngeal delivery within 2 h of infection. Markers of inflammation in the lung were assessed within 24 h for each set of animals. During Mycoplasma infection, one high dose of Ang-(1-7) delivered to the lungs reduced neutrophilia and Muc5ac , as well as Tnf- α and chemokines ( Cxcl1 ) associated with acute respiratory distress syndrome (ARDS). Despite decreased inflammation, Ang-(1-7)-treated mice also had significantly lower Mp burden in their lung tissue, indicating decreased airway colonization. Ang-(1-7) also had an impact on RAW 264.7 cells, a commonly used macrophage cell line, by dose-dependently inhibiting TNF-α production while promoting Mp killing. These new findings provide additional support to the protective role(s) of Ang1-7 in controlling inflammation, which we found to be highly protective against live Mp-induced lung inflammation.

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency, the most common enzymopathy in humans, is prevalent in tropical and subtropical areas where malaria is endemic. Anti-malarial drugs, such as primaquine and tafenoquine, can cause haemolysis in G6PD-deficient individuals. Hence, G6PD testing is recommended before radical treatment against vivax malaria. Phenotypic assays have been widely used for screening G6PD deficiency, but in heterozygous females, the random lyonization causes difficulty in interpreting the results. Over 200 G6PD variants have been identified, which form genotypes associated with differences in the degree of G6PD deficiency and vulnerability to haemolysis. This study aimed to assess the frequency of G6PD mutations using a newly developed molecular genotyping test., Methods: A multiplexed high-resolution melting (HRM) assay was developed to detect eight G6PD mutations, in which four mutations can be tested simultaneously. Validation of the method was performed using 70 G6PD-deficient samples. The test was then applied to screen 725 blood samples from people living along the Thai-Myanmar border. The enzyme activity of these samples was also determined using water-soluble tetrazolium salts (WST-8) assay. Then, the correlation between genotype and enzyme activity was analysed., Results: The sensitivity of the multiplexed HRM assay for detecting G6PD mutations was 100 % [95 % confidence interval (CI): 94.87-100 %] with specificity of 100 % (95 % CI: 87.66-100 %). The overall prevalence of G6PD deficiency in the studied population as revealed by phenotypic WST-8 assay was 20.55 % (149/725). In contrast, by the multiplexed HRM assay, 27.17 % (197/725) of subjects were shown to have G6PD mutations. The mutations detected in this study included four single variants, G6PD Mahidol (187/197), G6PD Canton (4/197), G6PD Viangchan (3/197) and G6PD Chinese-5 (1/197), and two double mutations, G6PD Mahidol + Canton (1/197) and G6PD Chinese-4 + Viangchan (1/197). A broad range of G6PD enzyme activities were observed in individuals carrying G6PD Mahidol, especially in females., Conclusions: The multiplexed HRM-based assay is sensitive and reliable for detecting G6PD mutations. This genotyping assay can facilitate the detection of heterozygotes, which could be useful as a supplementary approach for high-throughput screening of G6PD deficiency in malaria endemic areas before the administration of primaquine and tafenoquine.

Background: Specific IgE against Solenopsis invicta (imported fire ant) remains the current diagnostic tool for allergy to ants worldwide. However, S. invicta may not be the only cause of ant anaphylaxis in Thai patients., Objective: To characterize ant species causing anaphylaxis in Thai patients and to test allergenic reactivity to whole body extracts (WBE) of S. geminata (tropical fire ants) in patients with evidence of IgE-mediated ant anaphylaxis., Methods: Thirty-two patients with ant anaphylaxis were identified. The causative ants collected by the patients were subjected to species identification. Twelve patients with ant anaphylaxis and showed positive skin test or serum specific IgE to S. invicta and 14 control subjects were recruited. Whole body extraction from S. geminata was performed for protein characterization using SDS-PAGE and protein staining. IgE-immunoblotting and ELISA-specific IgE binding assays were performed on patients' sera and compared with controls., Results: Of 32 patients with ant anaphylaxis, the most common causative ant identified was S. geminata (37.5%). Western blot analysis of crude S. geminata revealed 13 refined protein components that bound to patients' serum IgE. Three major allergens with molecular masses of 26, 55 and 75 kDa were identified. All 12 patients gave positive results for specific IgE to S. geminata with statistically significant higher absorbance units of 0.390 ± 0.044, compared to healthy control group (0.121 ± 0.010), P < 0.01., Conclusions: S. geminata is identified as the most common causative ant anaphylaxis in Thai patients. Its WBE comprises of 13 IgE-binding components and 3 major allergens (26, 55 and 75 kDa), which supported possible IgE-mediated mechanism.

Asthma remains one of the most common respiratory diseases in both children and adults affecting up to 10% of the US population. Asthma is characterized by persistent symptoms, airway inflammation, airflow limitation and frequent exacerbations. Eosinophils are a key immune cell present in a large majority of asthmatics and their presence and dysregulation are clinically associated with more severe asthma. Surfactant protein A (SP-A) provides a first-line of defense in pulmonary innate immunity by virtue of its role in pathogen opsonization. SP-A is known to specifically bind to Mycoplasma pneumoniae (Mp), a pathogen associated with asthma exacerbations, and functions to attenuate Mp pathogenicity and abrogate lung inflammation. In addition, SP-A has been shown to inhibit Mp-induced eosinophil peroxidase (EPO) release, a toxic product that can compromise the integrity of the delicate airway epithelia. We have determined that genetic variation in SP-A2 at position 223 that results in a glutamine (Q) to a lysine (K) substitution alters the ability of SP-A to inhibit EPO release and may offer a mechanistic explanation as to why some SP-A extracted from subjects with asthma is unable to carry out normal immune regulatory functions.

Purpose: The sequence variations of the Der p 2 allergen of Dermatophagoides pteronyssinus diverge along 2 pathways with particular amino acid substitutions at positions 40,47,111, and 114. The environmental prevalence and IgE binding to Der p 2 variants differ among regions. To compare IgE binding to Der p 2 variants between sera from Bangkok, Thailand and Perth, Western Australia with different variants and to determine the variant-specificity of antibodies induced by vaccination with recombinant variants., Methods: The structures of recombinant variants produced in yeast were compared by circular dichroism and 1-anilinonaphthalene 8-sulfonic acid staining of their lipid-binding cavity. Sera from subjects in Bangkok and Perth where different variants are found were compared by the affinity (IC₅₀) of IgE cross-reactivity to different variants and by direct IgE binding. Mice were immunized with the variants Der p 2.0101 and Der p 2.0110, and their IgG binding to Der p 2.0103, 2.0104, and 2.0109 was measured., Results: The secondary structures of the recombinant variants resembled the natural allergen but with differences in ANS binding. The IC₅₀ of Der p 2.0101 required 7-fold higher concentrations to inhibit IgE binding to the high-IgE-binding Der p 2.0104 than for homologous inhibition in sera from Bangkok where it is absent, while in sera from Perth that have both variants the IC₅₀ was the same and low. Reciprocal results were obtained for Der p 2.0110 not found in Perth. Direct binding revealed that Der p 2.0104 was best for detecting IgE in both regions, followed by Der p 2.0101 with binding to other variants showing larger differences. Mouse anti-Der p 2.0101 antibodies had a high affinity of cross-reactivity but bound poorly to other variants., Conclusions: The affinity of IgE antibody cross-reactivity, the direct IgE binding, and the specificities of antibodies induced by vaccination show that measures of allergic sensitization and therapeutic strategies could be optimized with knowledge of Der p 2 variants.

Mycoplasma pneumoniae is an extracellular pathogen that colonizes mucosal surfaces of the respiratory tract and is associated with asthma exacerbations. Previous reports demonstrate that surfactant protein-A (SP-A) binds live M. pneumoniae and mycoplasma membrane fractions (MMF) with high affinity. Humans express a repertoire of single-amino acid genetic variants of SP-A that may be associated with lung disease, and our findings demonstrate that allelic differences in SP-A2 (Gln223Lys) affect the binding to MMF. We show that SP-A(-/-) mice are more susceptible to MMF exposure and have significant increases in mucin production and neutrophil recruitment. Novel humanized SP-A2-transgenic mice harboring the hSP-A2 223K allele exhibit reduced neutrophil influx and mucin production in the lungs when challenged with MMF compared with SP-A(-/-) mice. Conversely, mice expressing hSP-A2 223Q have increased neutrophil influx and mucin production that are similar to SP-A(-/-) mice. Using tracheal epithelial cell cultures, we show that enhanced mucin production to MMF occurs in the absence of SP-A and is not dependent upon neutrophil recruitment. Increased phosphorylation of the epidermal growth factor receptor (EGFR) was evident in the lungs of MMF-challenged mice when SP-A was absent. Pharmacologic inhibition of EGFR prior to MMF challenge dramatically reduced mucin production in SP-A(-/-) mice. These findings suggest a protective role for SP-A in limiting MMF-stimulated mucin production that occurs through interference with EGFR-mediated signaling. SP-A interaction with the EGFR signaling pathway appears to occur in an allele-specific manner that may have important implications for SP-A polymorphisms in human diseases., (Copyright © 2015 by The American Association of Immunologists, Inc.)

Pulmonary surfactant protein A (SP-A), a heterooligomer of SP-A1 and SP-A2, is an important regulator of innate immunity of the lung. Nonsynonymous single nucleotide variants of SP-A have been linked to respiratory diseases, but the expressed repertoire of SP-A protein in human airway has not been investigated. Here, we used parallel trypsin and Glu-C digestion, followed by LC-MS/MS, to obtain sequence coverage of common SP-A variants and isoform-determining peptides. We further developed a SDS-PAGE-based, multiple reaction monitoring (GeLC-MRM) assay for enrichment and targeted quantitation of total SP-A, the SP-A2 isoform, and the Gln223 and Lys223 variants of SP-A, from as little as one milliliter of bronchoalveolar lavage fluid. This assay identified individuals with the three genotypes at the 223 position of SP-A2: homozygous major (Gln223/Gln223), homozygous minor (Lys223/Lys223), or heterozygous (Gln223/Lys223). More generally, our studies demonstrate the challenges inherent in distinguishing highly homologous, copurifying protein isoforms by MS and show the applicability of MRM mass spectrometry for identification and quantitation of nonsynonymous single nucleotide variants and other proteoforms in airway lining fluid.