BACKGROUND: The indirect basophil activation test using flow cytometry is a promising tool for autoimmune urticaria diagnosis. We aimed to identify better donor basophils (from atopic vs. non-atopic donors and interleukin-3 primed vs. unprimed basophils) and improve basophil identification and activation markers (eotaxin CC chemokine receptor-3 [CCR3] vs. CD123 and CD63 vs. CD203c). METHODS: Donor basophils were obtained from non-atopic and atopic group O donors. Positive control sera were artificially prepared to simulate autoimmune urticaria patients' sera. Patient sera were obtained from nine children with chronic urticaria. Assay sensitivity was compared among each variation by using positive control sera (n=21), applying cutoff values defined from negative control sera (n=20). RESULTS: For basophil identification, a combination of CCR3 and CD123 markers revealed a higher correlation with automated complete blood count (r=0.530) compared with that observed using CD123 (r=0.498) or CCR3 alone (r=0.195). Three activation markers on the atopic donor basophils attained 100% assay sensitivity: CD203c on unprimed basophils, CD63+CD203+ or CD63 alone on primed basophils; however, these markers on the non-atopic donor basophils attained lower assay sensitivity. CONCLUSIONS: For basophil identification markers, a combination of CD123 and CCR3 is recommended, while CD123 alone may be used as an alternative. Donor basophils should be obtained from an atopic donor. For basophil activation markers, either CD203c alone on unprimed basophils or CD203c and CD63 on primed basophils are recommended, while CD63 alone on primed basophils may be used as an alternative.
Autoimmune Diseases/blood/*diagnosis/immunology ; Basophils/*immunology/metabolism ; Biomarkers/blood ; Child ; Flow Cytometry ; Humans ; Interleukin-3 Receptor alpha Subunit/blood ; Male ; Receptors, CCR3/blood ; Urticaria/blood/*diagnosis/immunology

OBJECTIVETo explore the effect of Astragalus membranaceus (AM) on T-helper cell type 1 (Thl) specific transcription factor T-box expressed in T cells (T-bet) expression and Thl/Th2 equilibrium.

METHODSThe levels of T-bet mRNA in peripheral blood mononuclear cells (PBMCs) from 15 patients with asthma and 15 healthy subjects were determined by reverse transcription-polymerase chain reaction (RT-PCR). PBMCs in asthma patients were incubated with AM and then the concentration of interferon gamma (IFN-gamma) and interleukin-4 (IL-4) in the supernate before and after AM intervention were determined by ELISA. The numbers of CD4 + CCR3 + and CD4 + CCR5 + cells were counted by flow cytometry.

RESULTSThe expression of T-bet mRNA and the level of IFN-gamma were lower, but level of serum IL-4 was higher in asthma patients when compared with those in healthy subjects respectively. After AM (60 microg/ml) intervention, the former two parameters raised and showed a positive correlation between them, while the level of IL-4 was decreased. The mean percentage of CD4 + CCR3 + cells in asthma patients was significantly higher but that of CD4 + CCR5 + cells was lower when compared with those in healthy subjects respectively. After AM intervention, the abnormal change in the two indexes was improved to certain extent, showing a reversing status of Th2 polarization.

CONCLUSIONAM could increase the expression of T-bet mRNA and Thl cytokines such as IFN-Y, and might reverse the Th2 predominant status in asthma patients.

Adult ; Asthma ; drug therapy ; immunology ; Astragalus membranaceus ; Cell Polarity ; Cross-Sectional Studies ; Female ; Humans ; Interferon-gamma ; biosynthesis ; blood ; Interleukin-4 ; blood ; Male ; Middle Aged ; Phytotherapy ; RNA, Messenger ; analysis ; Receptors, CCR3 ; Receptors, CCR5 ; blood ; Receptors, Chemokine ; blood ; T-Box Domain Proteins ; genetics ; Th1 Cells ; drug effects ; immunology ; Up-Regulation

DNA methylation may regulate gene expression by restricting the access of transcription factors. We have previously demonstrated that GATA-1 regulates the transcription of the CCR3 gene by dynamically interacting with both positively and negatively acting GATA elements of high affinity binding in the proximal promoter region including exon 1. Exon 1 has three CpG sites, two of which are positioned at the negatively acting GATA elements. We hypothesized that the methylation of these two CpGs sites might preclude GATA-1 binding to the negatively acting GATA elements and, as a result, increase the availability of GATA-1 to the positively acting GATA element, thereby contributing to an increase in GATA-1-mediated transcription of the gene. To this end, we determined the methylation of the three CpG sites by bisulfate pyrosequencing in peripheral blood eosinophils, cord blood (CB)-derived eosinophils, PBMCs, and cell lines that vary in CCR3 mRNA expression. Our results demonstrated that methylation of CpG sites at the negatively acting GATA elements severely reduced GATA-1 binding and augmented transcription activity in vitro. In agreement, methylation of these CpG sites positively correlated with CCR3 mRNA expression in the primary cells and cell lines examined. Interestingly, methylation patterns of these three CpG sites in CB-derived eosinophils mostly resembled those in peripheral blood eosinophils. These results suggest that methylation of CpG sites at the GATA elements in the regulatory regions fine-tunes CCR3 transcription.
Binding Sites ; Cell Line ; *CpG Islands ; DNA Methylation ; Enhancer Elements, Genetic ; Eosinophils/cytology/*metabolism ; Exons ; Fetal Blood/cytology/metabolism ; GATA1 Transcription Factor/*genetics/metabolism ; Gene Expression Regulation ; Humans ; Promoter Regions, Genetic ; RNA, Messenger/metabolism ; Receptors, CCR3/*genetics/metabolism ; Sequence Analysis, DNA ; *Transcription, Genetic