Objective To investigate the expression of blood basophil activation markers in patients with allergic rhinitis (AR) and the effects of allergens on their expression. Methods The blood samples were collected from the following four groups: healthy control (HC), AR patients with negative skin prick test (nAR), seasonal AR patients (sAR) and perennial AR patients (pAR). Flow cytometry was employed to analyze the expression of basophil activation markers Immunoglobulin E receptor I alpha(FcepsilonRIα), CD63 and CD203c in AR patients. Plasma levels of interleukin 4 (IL-4) and IL-8 were measured by liquid-phase chip technology, and their correlations with the percentages of activated basophils were further analyzed. An ovalbumin-induced AR mouse model was established, and the expression levels of FcepsilonRIα and CD63 on blood basophils were detected. Results The expression of FcepsilonRIα, CD203c and CD63 on basophils were increased in nAR, sAR and pAR patients. Allergens enhanced the mean florescence intensity expression of CD63 and CD203c on basophils of sAR and pAR patients. The plasma levels of IL-4 and IL-8 were elevated in nAR, sAR and pAR patients, showing moderate to high correlations with the expression levels of basophil activation markers. The FcepsilonRIαand CD63 expression on basophils of AR mice were increased. Conclusion Allergens may contribute to AR pathogenesis by upregulating the expression of FcepsilonRIα, CD63 and CD203c, as well as promoting the secretion of IL-4 and IL-8.
Basophils/metabolism* ; Humans ; Allergens/immunology* ; Animals ; Rhinitis, Allergic/blood* ; Female ; Male ; Adult ; Mice ; Biomarkers/blood* ; Tetraspanin 30/blood* ; Interleukin-4/blood* ; Interleukin-8/blood* ; Receptors, IgE/blood* ; Phosphoric Diester Hydrolases ; Young Adult ; Pyrophosphatases ; Middle Aged ; Mice, Inbred BALB C

To prepare antibodies that specifically recognize the conserved domain in the large extracellular loop of the CD63 protein, we expressed anti-CD63 single-chain variable fragment (scFv) antibody in Pichia pastoris in a secreted form. The purified expression product was found to bind specifically with CD63 protein and recognize CD63 on the surface of SK-MEL-28 cells. The variable region of the anti-CD63 monoclonal antibody in an anti-CD63-positive cell line was sequenced. The anti-CD63 scFv consisted of a variable heavy chain and a variable light chain linked by a flexible peptide was then designed. After codon optimization, the gene was synthesized and cloned into the expression plasmid pPICZα-A. The SacI-linearized plasmid was electroporated into P. pastoris X33, and 1% methanol were used to induce the expression of scFv. The fermentation supernatant was purified by Ni column. Anti-CD63 scFv was identified by SDS-PAGE and Western blotting, and its biological activities were analyzed by immunoblotting, immunofluorescence, cell-based ELISA, and flow cytometry. A P. pastoris strain capable of expressing and secreting anti-CD63 scFv was successfully obtained. The antibody had a molecular weight of approximately 30 kDa and specifically recognized CD63 protein. The expression of anti-CD63 scFv in P. pastoris paves the way for the production of anti-CD63 antibodies on a large-scale, which is undoubtedly an economical and effective way of antibody acquisition.
Single-Chain Antibodies/immunology* ; Humans ; Tetraspanin 30/immunology* ; Recombinant Proteins/immunology* ; Pichia/genetics* ; Saccharomycetales/metabolism*

Objective To study the protein expression of cluster of differentiation 63 （CD63） in lung tissues of guinea pigs that died of anaphylactic shock and discuss the diagnostic value of CD63 for death from anaphylactic shock. Methods Twenty guinea pigs were randomly divided into control group, anaphylactic shock immediate death group, cold storage group （4 ℃ for 48 h） and frozen group （-20 ℃ for 7 d）. The animal model of guinea pigs that died of anaphylactic shock was established with human mixed serum injection. The expression changes of CD63 protein and CD63 mRNA in lung tissues were detected by hematoxylin-eosin （HE） staining, immunohistochemical staining, Western blotting, enzyme-linked immunosorbent assay （ELISA） and real-time RT-PCR. Results HE staining results showed congestion, and edema of lung tissues, and eosinophil infiltration in the anaphylactic shock groups. Western blotting analysis results showed that the expression of CD63 protein in the lung tissues of guinea pigs that died of anaphylactic shock was significantly higher than that in the control group （P<0.05）. Comparison between the anaphylactic shock groups was made, and the differences had no statistical significance. The results of immunohistochemical staining and real-time RT-PCR were consistent with that of Western blotting. ELISA results showed that CD63 protein expression in the immediate death group was higher than that in the control group （P<0.05）. Conclusion The expression of CD63 protein and CD63 mRNA in the lung tissues of guinea pigs that died of anaphylactic shock is significantly enhanced. Animal carcasses which were put in cold storage for 48 h and frozen for 7 d do not affect the examination of the above indicators. CD63 protein is expected to become an auxiliary diagnostic indicator of death from anaphylactic shock.
Anaphylaxis/mortality* ; Animals ; Disease Models, Animal ; Enzyme-Linked Immunosorbent Assay ; Guinea Pigs ; Humans ; Lung/metabolism* ; Real-Time Polymerase Chain Reaction ; Reverse Transcriptase Polymerase Chain Reaction ; Serum ; Tetraspanin 30/metabolism*

OBJECTIVE: To investigate the diagnostic significance of basophil activation test (BAT) in anaphylaxis to non-ionic contrast media through testing the content of CD63, mast cell-carboxypeptidase A3 (MC-CPA3), and terminal complement complex SC5b-9 of the individuals by testing their levels in the normal immune group and the anaphylaxis groups to β-lactam drugs and non -ionic contrast media. METHODS: The CD63 expression of basophilic granulocyte in blood was detected by flow cytometry. The levels of MC-CPA3 in blood serum and SC5b-9 in blood plasma were detected by ELISA. RESULTS: The CD63 expression of basophilic granulocyte in blood, the levels of MC-CPA3 and SC5b-9 of anaphylaxis to non-ionic contrast media and β-lactam drugs were significantly higher than that in normal immune group (P < 0.05). CONCLUSION There is activation of basophilic granulocytes, mast cells and complement system in anaphylaxis to non-ionic contrast media. BAT can be used to diagnose the anaphylaxis to non-ionic contrast media.
Anaphylaxis/diagnosis* ; Basophils/cytology* ; Carboxypeptidases A/metabolism* ; Complement Membrane Attack Complex/metabolism* ; Contrast Media ; Flow Cytometry ; Granulocytes/cytology* ; Humans ; Mast Cells/cytology* ; Tetraspanin 30/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*

OBJECTIVETo establish a new, real time, dynamic and direct optical detection method for mast cell degranulation caused by anaphylactoid reaction.

METHODA CD63-GFP plasmid was constructed and introduced steadily into rat basophilic leukemia (RBL-2H3) cells. The movements of CD63-GFP, which was located on both the granule membranes and the plasma membranes of RBL cells stimulated by Compound 48/80, were studied by confocal laser scanning microscope (CLSM) and total internal reflection fluorescence microscope (TIRFM) both inside and on the surface of living RBL-2H3 cells.

RESULTBefore antigen stimulation, most granules with CD63-GFP hardly moved in RBL cells. However, after antigen stimulation, the granules moved dramatically. They reached the plasma membranes in a few minutes and fused with them instantaneously. The velocity of the granule movement toward the plasma membranes on antigen stimulation was calculated to be 0.05 micron x s(-1).

CONCLUSIONAnalysis of the movement of each granule provided a new insight into the elementary process of degranulation. The method is rapid, sensitive and reliable, which could be used as a new detection method for anaphylactoid reaction in vitro.

Anaphylaxis ; diagnosis ; immunology ; metabolism ; Animals ; Antigens, CD ; genetics ; Cell Degranulation ; Cell Line, Tumor ; Cell Movement ; Mast Cells ; cytology ; immunology ; Microscopy, Confocal ; Microscopy, Fluorescence ; Platelet Membrane Glycoproteins ; genetics ; Rats ; Tetraspanin 30 ; Time Factors

OBJECTIVETo determine the serum levels of CD62p (alpha-granular membrane protein) and CD63 (lysosome intact membrane protein) in patients with head injury and to observe its relation to injury severity.

METHODSFifty-three patients with head injury were divided into 3 groups; Group A patients with mild head injury; Group B with moderate head injury; and Group C with severe head injury. The serum levels of CD62p, CD63 were measured on 12 h, d 1, 3, 5 and 7 after injury.

RESULTThe serum levels of CD62p and CD63 in Group B and Group C were higher than those in Group A and control (P<0.05). The serum level of CD62p in Group C was higher than that in Group B (P<0.05). The serum levels of CD62p in Group C on d 1, 3, 5 after injury were higher than those on 12 h (P<0.05). The serum level of CD63 in Group B on d 3 after injury were higher than that on 12 h (P<0.05). The serum levels of CD63 in Group C on d 1, 3, 5 after injury were higher than those on 12 h (P<0.05).

CONCLUSIONThe serum levels of CD62p and CD63 in patients with head injury may be helpful for identifying the severity of injury, and CD62p seems to be more sensitive.

Adolescent ; Adult ; Aged ; Antigens, CD ; blood ; Blood Platelets ; metabolism ; Brain Injuries ; blood ; Female ; Humans ; Male ; Middle Aged ; P-Selectin ; blood ; Platelet Activation ; Platelet Membrane Glycoproteins ; Tetraspanin 30 ; Trauma Severity Indices