Co-administration of Chenopodium album allergens and CpG oligodeoxy- nucleotides effects on peripheral blood mononuclear cells of patients with allergic rhinitis treated with intranasal corticosteroids and antihistamines

Allergic Rhinitis [AR] is one of the most common chronic diseases in the developed countries. This study was performed to investigate the effect of CpG-ODN in alteration of T-helper [Th]l/Th2 balance of patients with AR treated with intranasal corticosteroids [INCs] and antihistamines. Peripheral blood mononuclear cells [PBMCs] of 20 patients with AR were isolated before and after 45 days therapy. Cytokine production [IL-4, IL-10, IL-13, IFN-gamma] and specific Ch.a IgE in response to CpG co-administration of natural chenopodium album [CpG/Ch.a] or recombinant Ch.a [CpG/rCh.a] allergen were investigated in supernatants.of cultured PBMCs using ELISA Intracellular IL-10 was also assessed in CD4[+] cells using flow cytometry. Significant increase in production of IFN-y and IL-10 and decrease in production of IL-4 were found in supernatants of cultured PBMCs activated with CPG/ch.a and CPG/rch.a. of both CpG/Ch.a and CpG/rCh.a compared to allergens alone, before and after therapy. After therapy, IFN-gamma production with CpG/Ch.a was significantly increased in comparison with before [237 vs. 44 pg/ml, p=0.001]. IFN-gamma and IL-10 production with CpG/rCh.a was significantly increased after therapy compared to before [407.6 vs. 109 pg/ml, p=0.0l for IFN- gamma; 171.7 vs. 52.6 pg/ml, p=0.008 for IL-10], whilst IL-4 was significantly decreased [2.1 vs. 5.8 pg/ml, p=0.02]. Intracellular IL-10 expression was also significantly increased in response to either CpG/Ch.a or CpG/rCh.a that showed intracellular assay could be more sensitive than ELISA. Also, treatment with intranasal corticosteroids and antihistamines could enhance this CpG effect, in vitro

Comparison of two flow cytometric methods for detection of human invariant natural killer T cells [iNKT]

Invariant natural killer cells [iNKT] are an important immunoregulatory T cell subset. Currently several flow cytometry-based approaches exist for the identification of iNKT cells, which rely on using the 6B11 monoclonal antibody or a combination of anti-V alpha 24 and anti-V beta 11 antibodies. The aim of this study was to compare the ability of two flow cytometry-based methods for detecting the frequency of circulating iNKT cells. The frequency of iNKT cells was detected in the peripheral blood of 37 healthy adult donors by flow cytometry using the 6B11 antibody or a combination of anti-V alpha 24 and anti-V beta 11 antibodies. The frequency of iNKT cells detected by 6B11 antibody or by combination of anti-V alpha 24 and anti-V beta 11 antibodies was significantly different [0.54% vs. 0.31%, respectively, p<0.001] but the values were highly correlated [Spearman r=0.742, p<0.0001]. The results of this study indicate that different combinations of mAbs detect different frequencies of peripheral blood iNKT cells and a consensus in the field needs to be established to allow better assessment of iNKT-related studies and suggest using different methods for accurate identification of iNKT cells

Production and characterization of monoclonal antibody against saffron pollen profilin, Cro s 2

Allergy to Saffron [Crocus sativus] pollen has been described in people involved in processing of saffron flower stamens. Profilins have been identified as a pan-allergen in different plant pollens and foods. This molecule is an actin-binding protein with a molecular weight of 12-16 kDa found in eukaryotic species. The aim of this study was to generate monoclonal antibody against Cro s 2 in order to characterize this major allergen of saffron pollen. BALB/c mice were immunized to obtain adequate humoral response. Splenocytes were prepared from the immunized animals, mixed with the P3-X63-Ag8.653 myeloma cells and fused by means of PEG 1500. After two weeks of culturing in HAT-containing media, the supernatant from those wells growing hybridomas were screened by ELISA using plates coated with Cro s 2. Cells from positive wells were cloned at least 3 times by limiting dilution. Specificity and cross-reactivity of the mAbs were determined by Western blot analysis and sandwich ELISA. Two stable hybridoma clones secreting mAbs against Cro s 2 were obtained and expanded. The anti-Cro s 2 mAbs were also found to cross-react with other plant profilins. Isotype of this mAb was identified as micro heavy chain and k light chain. The anti-Cro s 2 mAb could be a useful tool for characterization and standardization of many pollen and fruit-derived profilins