RESUMEN
Asthma is a heterogenous disease characterized by airway inflammation and variable expiratory airflow limitation resulting in variable respiratory symptoms. Characterization of airway inflammation is important to choose the optimal treatment for severe asthma patients eligible for biological treatment. However, counting cells in induced sputum samples are a time-consuming process, highly dependent on personal skills. Replacing eosinophil and neutrophil cell counting with qPCR for transcripts of selected mast cell, and basophil genes may provide more reproducible results. Aims: The objective of this study was to compare qPCR with microscopy in asthma endotyping. Methods: A qPCR method measuring five mast cell/basophil genes was applied on induced sputum samples from 30 severe asthma patients and compared with microscopy. Target gene Ct-values (CPA3, GATA2, HDC, MS4A2, TPSAB1/TPSB2) were referenced to household ß-actin Ct values as a measure of relative mRNA abundance of the target in each sample. Target/ß-actin-ratios in eosinophilic and non-eosinophilic groups determined by microscopy with an eosinophil threshold of 3% in 400 cells were compared using Mann-Whitney U Test. Spearman´s correlations were used to test for correlation between targets vs. FENO and targets vs. blood eosinophil counts. Results: The study demonstrated a statistical difference in relative mRNA abundance for four mast cell/basophil specific genes. CPA3, GATA2, HDC and MS4A2 were elevated in eosinophilic asthma versus non-eosinophilic asthma patients. The study found that GATA2, CPA3, MS4A2 and TPSAB1/TPSB2 transcripts are positively correlated with FENO. Neither the five mast cell genes nor the five-gene signature correlated with blood eosinophils. The five-gene signature with a target/ß-actin-ratio cut-off ≥2 generated sensitivity = 87%, specificity = 94%, NPV = 88% and PPV = 92% compared to microscopy. Conclusion: This study confirms the contribution of mast cells in the pathogenesis of EA and suggests that mast cell mRNA markers could be one of the biomarkers used to identify EA.
RESUMEN
BACKGROUND: Sequential allergen desensitization provides temporary tolerance for allergic patients. We adapted a clinical protocol to desensitize human blood basophils ex vivo and investigated the mechanism and allergen specificity. METHODS: We included 28 adult, grass allergic subjects. The optimal, activating allergen concentration was determined by measuring activated CD63(+) CD193(+) SS(Low) basophils in a basophil activation test with 8 log-dilutions of grass allergen. Basophils in whole blood were desensitized by incubation with twofold to 2.5-fold increasing allergen doses in 10 steps starting at 1 : 1000 of the optimal dose. Involvement of p38 mitogen-activated protein kinase (MAPK) was assessed after 3 min of allergen stimulation (n = 7). Allergen specificity was investigated by desensitizing cells from multi-allergic subjects with grass allergen and challenging with optimal doses of grass, birch, recombinant house dust mite (rDer p2) allergen or anti-IgE (n = 10). RESULTS: Desensitization reduced the fraction of blood basophils responding to challenge with an optimal allergen dose from a median (IQR) 81.0% (66.3-88.8) to 35.4% (19.8-47.1, P < 0.0001). CD63 MFI expression was reduced from 68 248 (29 336-92 001) to 30 496 (14 046-46 179, P < 0.0001). Basophils from multi-allergic subjects were desensitized with grass allergen. Challenge with grass allergen resulted in 39.6% activation (15.8-58.3). An unrelated challenge (birch, rDer p2 or anti-IgE) resulted in 53.4% activation (30.8-66.8, P = 0.16 compared with grass). Desensitization reduced p38 MAPK phosphorylation from a median 48.1% (15.6-92.8) to 26.1% (7.4-71.2, P = 0.047) and correlated with decrease in CD63 upregulation (n = 7, r > 0.79, P < 0.05). CONCLUSION: Desensitization attenuated basophil response rapidly and non-specifically at a stage before p38 MAPK phosphorylation.