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 observe the effects of Qingkailing injection on RBL-2H3 cell degranulation and histamine release, and discuss the possible mechanism of anaphylactoid reaction induced by Qingkailing injection.

METHODRBL-2H3 cells were incubated with Qingkailing injection for 30 min. Then the morphological changes of cells were observed by transmission electron microscopy. Cell degranulation rate was detected by Alcian blue dye assay, Annexin V binding assay and beta-hexosaminidase assay, and cell histamine release rate was detected by ELISA.

RESULTDifferent concentration of Qingkailing injection can induce the typical morphological changes in RBL-2H3 cell with degranulation. The rates of degranulation and histamine release in Qingkailing injection treated cells were significantly increased and dose-dependent.

CONCLUSIONRBL-2H3 cell degranulation and histamine release can be induced by single administration of Qingkailing injection, and then induced anaphylactoid reaction, which may be one of the possible mechanisms of serious adverse induced by Qingkailing injection for the first administration in clinic.

Animals ; Basophils ; drug effects ; immunology ; physiology ; Cell Degranulation ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Histamine ; metabolism ; Rats

Flow cytometry was used to identify and characterize monoclonal antibodies (mAbs) that react with rabbit leukocyte differentiation molecules (LDM). Screening sets of mAbs, developed against LDM in other species, for reactivity with rabbit LDM yielded 11 mAbs that recognize conserved epitopes on rabbit LDM orthologues and multiple mAbs that recognize epitopes expressed on the major histocompatibility class I or class II molecules. Screening of mAbs submitted to the Animal Homologues Section of the Eighth Human Leukocyte Differentiation Workshop yielded 7 additional mAbs. Screening of mAbs generated from mice immunized with leukocytes from rabbit thymus or spleen or concanavalin A activated peripheral blood and/or spleen lymphocytes has yielded 42 mAbs that recognize species restricted epitopes expressed on one or more lineages of leukocytes. Screening of the anti-rabbit mAbs against leukocytes from other species yielded one additional mAb. The studies show that screening of existing sets of mAbs for reactivity with rabbit LDM will not be productive and that a direct approach will be needed to develop mAbs for research in rabbits. The flow cytometric approach we developed to screen for mAbs of interest offers a way for individual laboratories to identify and characterize mAbs to LDM in rabbits and other species. A web-based program we developed provides a source of information that will facilitate analysis. It contains a searchable data base on known CD molecules and a data base on mAbs, known to react with LDM in one or more species of artiodactyla, equidae, carnivora, and or lagomorpha.
Animals ; Antibodies, Monoclonal/*immunology ; Antigens, Differentiation/*metabolism ; B-Lymphocytes/cytology/metabolism ; Basophils/cytology/metabolism ; Epitopes/genetics/metabolism ; *Flow Cytometry ; Gene Expression Regulation ; Granulocytes/cytology/metabolism ; Leukocytes/immunology/*metabolism ; Mice ; Monocytes/cytology/metabolism ; Rabbits ; T-Lymphocytes/cytology/metabolism

No abstract available.
Basophils/cytology/*metabolism ; Flow Cytometry ; HLA-DR Antigens/metabolism ; Humans ; Interleukin-3 Receptor alpha Subunit/metabolism ; Leukocyte Count ; Phosphoric Diester Hydrolases/metabolism ; Pyrophosphatases/metabolism ; Receptors, CCR3/metabolism ; Urticaria/*diagnosis/immunology/metabolism

OBJECTIVE: To explore the value of flow cytometry in anaphylactic shock diagnosis by CD63 expression being detected using flow cytometry to conform the activation of basophils. METHODS: Sixteen rats were randomly divided into two groups: control group and anaphylactic shock group. The model of anaphylactic shock rat with ovalbumin injection was established. CD63, CD45 and CD203c antibody combination, flow cytometry was employed to detected blood basophil CD63 expression. Immunofluorescence method was employed to observe the CD63 immunofluorescence staining in the rat lung tissue. RESULTS: (1) Pure basophils were obtained by CD45 and CD203c gating. (2) The percentages of basophils CD63 were (17.34 +/- 2.04)% and (1.52 +/- 0.35)% in the experimental and control group, respectively. The differences between two groups were statistically significant (P < 0.01). (3) Compared with the control group, the expression of CD63 in basophils increased in anaphylactic shock lung tissue. CONCLUSION The detection of CD63 by flow cytometry could be the supplement of vivo allergic reactions and have good clinical value.
Anaphylaxis/metabolism* ; Animals ; Basophil Degranulation Test/methods* ; Basophils/metabolism* ; Biomarkers/analysis* ; Disease Models, Animal ; Female ; Flow Cytometry ; Lung/pathology* ; Male ; Ovalbumin/administration & dosage* ; Phosphoric Diester Hydrolases/immunology* ; Pyrophosphatases/immunology* ; Random Allocation ; Rats ; Rats, Wistar ; Tetraspanin 30/metabolism*

PURPOSE: Cefaclor is widely prescribed for various infectious diseases. As its consumption increases, the number of hypersensitivity reactions to cefaclor has increased. This study aimed to evaluate the immunologic findings of immediate hypersensitivity to cefaclor. MATERIALS AND METHODS: We enrolled 47 patients with immediate hypersensitivity to cefaclor from Ajou University Hospital and Asan Medical Center. Serum specific IgE, IgG1, and IgG4 antibodies to cefaclor-human serum albumin (HSA) conjugate were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The most common phenotype was anaphylaxis (Group I, 78.7%), followed by urticaria (Group II, 21.3%). The detection of specific IgE, IgG1, and IgG4 to cefaclor-HSA conjugate by ELISA tended to be higher in Group I (40.5%, 41.7%, 21.6%) than in Group II (20.0%, 20.0%, 0%) with no statistical significance. Significant associations were found between specific IgE and IgG1 or IgG4 (p<0.001, p=0.019). ELISA inhibition tests showed significant inhibitions by both free cefaclor and cefaclor-HSA conjugate. For basophil activation tests in patients having no specific IgE antibody, the CD63 expression level on basophils increased with incubations of free cefaclor. CONCLUSION: The most common manifestation of immediate hypersensitivity to cefaclor was anaphylaxis, most of which was mediated by IgE; however, a non-IgE mediated direct basophil activation mechanism was suggested in a subset of anaphylaxis patients.
Adolescent ; Adult ; Aged ; Anaphylaxis/*chemically induced/immunology ; Anti-Bacterial Agents/adverse effects/*immunology ; Antigens, CD63 ; Basophils/metabolism ; Cefaclor/*adverse effects/immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Hypersensitivity, Immediate/chemically induced/diagnosis/*immunology ; Immunoglobulin E/*blood ; Immunoglobulin G/immunology ; Male ; Middle Aged ; Retrospective Studies ; Skin Tests ; Urticaria/chemically induced/diagnosis/immunology ; Young Adult

OBJECTIVETo explore the protective effects and mechanism of ethanol extract of Inonotus obliquus (EEIO) injection on asthmatic mice.

METHODOVA was injected intraperitoneally and inhaled to produce the asthmatic model. Thirty two mice were randomly divided into four groups: control group, asthma group and I. obliquus groups of high and low dose. The concentrations of IL-4, IL-5, IL-13 and IFN-gamma in BALF, the phosphor-p38 MAPK in lung tissues were respectively measured by ELISA and Western blotting. The number of inflammatory cells in BALF and histopathology changes were observed.

RESULTIn asthmatic group, the number of inflammatory cells and the concentrations of IL-4, IL-5, IL-13 in BALF and phospho-p38 MAPK in lung tissue were higher, while IFN-gamma were lower than those in normal control mice (P < 0.05). In I. obliquus group, the number of inflammatory cells, the concentrations of IL-4, IL-5, IL-13 in BALF and phosphor-p38 MAPK in lung tissue were lower, but were higher than those in normal control mice (P < 0.05), and histropathology damage was alleviated significantly. There was no significant difference observed among the efficacies in the I. obliquus groups of high and low dose.

CONCLUSIONp38 MAPK may play a role in pathological process of asthma. I. obliquus effectively treats asthma by inhibiting the expression of phosphor-p38 MAPK, correcting the unbalance of IFN-gamma/IL-4 and decreasing the number of inflammatory cells.

Animals ; Anti-Asthmatic Agents ; isolation & purification ; pharmacology ; Asthma ; drug therapy ; metabolism ; pathology ; Basidiomycota ; chemistry ; Basophils ; drug effects ; metabolism ; Bronchoalveolar Lavage Fluid ; cytology ; immunology ; Disease Models, Animal ; Interferon-gamma ; drug effects ; metabolism ; Interleukin-13 ; metabolism ; Interleukin-4 ; metabolism ; Interleukin-5 ; metabolism ; Lung ; pathology ; Lymphocytes ; drug effects ; metabolism ; Mice ; Mice, Inbred BALB C ; Neutrophils ; drug effects ; metabolism ; Phytotherapy ; Plant Extracts ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; drug effects ; metabolism