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

To confirm local production of IgE, and evaluate role of immunoglobulins on eosinophil activation in nasal polyp (NP) tissue, we measured IgG, IgA, secretory IgA(sIgA), total (tIgE) and specific IgE (sIgE) to Dermatophagoides pteronyssinus(DP) by ELISA in NP tissue homogenates from 51 subjects. They were classified according to skin reactivity to DP: group I, 15 highly atopic subjects; group II, 18 weakly atopic subjects; and group III, 18 non-atopic subjects. Eosinophil cationic protein (ECP) level was measured by CAP system. Highest level of DP-sIgE was noted in group I, followed by group II and III (p<0.05). Nine (60%) of group I and 4 (22%) of group II subjects had detectable level of DP-sIgE with no significant differences in IgA, sIgA, and IgG. All of NP tissue had eosinophilic infiltration with no significant difference in activated eosinophil count or ECP level among 3 groups. A significant correlation was noted between EG2+ cell count and tIgE (r=0.55, p<0.05), and DP-sIgE level (r=0.60, p<0.05). A significant correlation was also noted between ECP and IgG (r=0.51, p<0.05) and DP-sIgE level (r=0.47, p<0.05) with no significant correlation with IgA or sIgA. These results suggest that DP-sIgE was detectable in NP tissue from weakly atopic subjects as well as highly atopic subjects. IgG and sIgE may have potential roles in eosinophil degranulation in NP tissue.
Blood Proteins/analysis ; Cell Degranulation/immunology ; Dermatophagoides pteronyssinus/immunology ; Eosinophil Granule Proteins ; Eosinophils/immunology ; Humans ; Immunoglobulin A/analysis/immunology ; Immunoglobulin E/analysis/immunology ; Immunoglobulin G/analysis/immunology ; Immunoglobulins/analysis/*immunology ; Nasal Polyps/*immunology/pathology ; *Ribonucleases

Eosinophil degranulation plays a crucial role in tissue inflammatory reactions associated with helminth parasitic nfections and allergic diseases. Paragonimus westermani, a lung fluke causing human paragonimiasis, secretes a large amount of cysteine proteases, which are involved in nutrient uptake, tissue invasion, and modulation of hos's immune responses. There is, however, limited information about the response of eosinophils to direct stimulation by cysteine proteases (CP) secreted by P. westermani. In the present study, we tested whether degranulation and superoxide production from human eosinophils can be induced by stimulation of the 2 CP (27 kDa and 28 kDa) purified from excretory-secretory products (ESP) of P. westermani newly excysted metacercariae (PwNEM). A large quantity of eosinophil-derived neurotoxin (EDN) was detected in the culture supernatant when human eosinophils isolated from the peripheral blood were incubated with the purified 27 kDa CP. Furthermore, the 27 kDa CP induced superoxide anion production by eosinophils in time- and dose-dependent manners. In contrast, the purified 28 kDa CP did not induce superoxide production and degranulation. These findings suggest that the 27 kDa CP secreted by PwNEM induces superoxide production and degranulation of human eosinophils, which may be involved in eosinophil-mediated tissue inflammatory responses during the larval migration in human paragonimiasis.
Animals ; Astacoidea/parasitology ; *Cell Degranulation ; Cysteine Endopeptidases/*immunology/isolation & purification ; Eosinophils/*immunology/metabolism ; Helminth Proteins/*immunology/isolation & purification ; Humans ; Paragonimiasis/*immunology/metabolism ; Paragonimus westermani/*enzymology/isolation & purification ; Superoxides/*immunology

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

To investigate me material basis of Mahuang Fuzi Xixin decoction (MFXD) for anti-inflammation and immune-suppression based on the combined method of serum chemical and serum pharmacological. The LC-MS/MS fingerprints of MFXD, drug-containing serum and blank serum were compared to define the components in plasma. Histamine, β-hexosaminidase released from RBL-2H3 cell infulenced by drug-containing serum at different time points were measured by ELISA. The effect of drug-containing serum on lipopolysaccharide-induced splenocyte proliferation at different time points were determined by MTT. A correlation analysis was made on components of MFXD and pharmacological indexes based the stepwise regression method. After the intragastrical administration with MFXD, 32 components were discovered in rat serum, including 27 prototype components (10 from Mahuang, 13 from Fuzi and four from Xixin) and five unknown components. Compared with blank serum, drug-containing serum could reduce the release of histamine from RBL-2H3 induced by antigen at different time points (P < 0.05); except the 4-hour drug-containing serum, all of the remaining drug-containing serums could inhibit the RBL-2H3 mastocyte degranulation induced by antigen at different time points (P < 0.05). Drug-containing serum could significantly lipopolysaccharide-induced mouse splenocyte proliferation at 15 and 30 min (P < 0.05). A regression analysis was made on the chemical data of components absorbed into blood and pharmacological indexes, i. e. release rate of histamine, release rate of β-hexosaminidase and inhibition rate of splenocyte. This suggested the close correlations among methyl pseudo-ephedrine, pseudoephedrine and histamine released from RBL-2H3 induced by antigen; pseudoephedrine, hypaconine, methyl pseudoephedrine and β-hexosaminidase released from RBL-2H3 induced by antigen; as well as benzoyl hypaconine, benzoylaconine, 14-benzoyl-10-OH-mesaconine, mesaconine and lipopolysaccharide-induced mouse splenocyte proliferation. Methylpseudoephedrine, pseudoephedrine, benzoyl hypaconine, benzoylaconine and mesaconine may be part of material basis of MFXD on anti-inflammation and immune suppression.
Animals ; Anti-Inflammatory Agents ; chemistry ; pharmacology ; Cell Degranulation ; drug effects ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Female ; Histamine ; immunology ; Immunosuppressive Agents ; chemistry ; pharmacology ; Male ; Mass Spectrometry ; Mast Cells ; drug effects ; immunology ; Mice ; Rats ; Rats, Wistar ; Serum ; chemistry

OBJECTIVETo establish a novel flow cytometry-based assay for measuring the expression of lysosomal-associated membrane protein 1 (LAMP-1, CD107α) on the cell surface of natural killer (NK) cells and cytotoxic T lymphocyte (CTL) and evaluate the screening value of this assay for cytotoxic defects-related diseases such as familial hemophagocytic lymphopro-liferative (FHL) syndrome.

METHODThree suspected Chediak-Higashi Syndrome (CHS) patients, three suspected FHL patients and 10 healthy children were enrolled in the study from October 2010 to June 2011. Their PBMCs were separated and activated overnight with IL-2. After the granule release of NK cells activated by phytohemagglutinin (PHA) and CD8+T cells by anti-CD3, the CD107α expression were analyzed by flow cytometry. The peripheral blood DNA and RNA of the patients were extracted to analyze the pathogenic genes via DNA-PCR/RT-PCR and direct sequencing.

RESULTThe CD107α expression on CTL in the ten healthy children significantly increased after activation by anti-CD3 [(0.18 ± 0.07)% vs. (4.47 ± 2.36)%, P < 0.05] and NK cells after activation by PHA [(0.27 ± 0.07)% vs. (5.80 ± 2.83)%, P < 0.05]. The frequency of CD107α-expression NK cells in three suspected CHS after activation was significantly elevated when compared with the healthy control [0.5%, 0.6% vs. (5.80 ± 2.83)%] except patient 2. After the anti-CD3 activation, the frequency of CD107α expression on CTL cells also showed no significant difference [0.3%, 0.9%, 0.2% vs. (4.47 ± 2.36)%] in three patients. All of their mean fluorescence intensity (MFI) showed the same trend. Patient 1 and 3 were identified to have LYST mutations (Patient 1: c.5411-5414 del TTTC, L1741fsX1758 and c.7975 C > T, R2596X; Patient 3: c.4863G > A, R1563H and c.5392-5393delAA, E1739fsX1756). There was no mutation identified in the LYST gene for patient 2. CD107α expression of NK cells and CTL in the suspected FHL patients and in mirror of these findings, no underlying gene variation of PRF, MUNC13-4 and STX11 were identified.

CONCLUSIONWe developed a method to quantitatively assess cytotoxicity of the NK cells and CTL by measuring the expression of CD107α on the cell membrane, which appeared to be an effective and rapid screening test for cytotoxic defects-related diseases such as FHL and other HLH secondary to primary immunodeficiency.

Case-Control Studies ; Cell Degranulation ; immunology ; Cell Membrane ; metabolism ; Chediak-Higashi Syndrome ; diagnosis ; genetics ; immunology ; metabolism ; Child, Preschool ; Cytotoxicity, Immunologic ; Female ; Flow Cytometry ; methods ; Humans ; Infant ; Interleukin-2 ; metabolism ; Killer Cells, Natural ; immunology ; metabolism ; Lymphohistiocytosis, Hemophagocytic ; diagnosis ; genetics ; immunology ; metabolism ; Lysosomal-Associated Membrane Protein 1 ; metabolism ; Male ; Mutation ; Phytohemagglutinins ; metabolism ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism

OBJECTIVETo study the effects of polysaccharides of cultured Cryptoporus volvatus(CVPS) on airway hyperresponsiveness of ovalbumin-sensitized rats and to evaluate their mechanisms.

METHODSPolysaccharides A, B (5mg/kg, 20mg/kg) and ketotifen(5mg/kg) or vehicle(same volume of saline) were administrated orally for 10 days in ovalbumin -sensitized rats, methacholine bronchial provocation tests were performed to determine airway hyperresponsiveness. Bronchoalveolar lavage fluid (BALF) and peritoneal lavage fluid were prepared after the animals were challenged by nebulized antigen. The differential white cell count in BALF,and the degranulated mast cell count as well as differential white cell count in peritoneal lavage fluid were performed.

RESULTPolysaccharides markedly inhibited the increased lung resistance and the decreased lung compliance induced by antigen challenge,significantly reduced total cell counts and absolute eosinophil counts in BALF(P<0.05); polysaccharides B was more effective than polysaccharides A. They also inhibited recruitment of inflammatory cells in peritoneal lavage fluid and inhibited the allergen-induced mast cell degranulation.

CONCLUSIONPolysaccharides of CVPS inhibit airway hyperresponsiveness by stabilizing mast cell membranes and reducing infiltration and chemotaxis of eosinophils and may be developed as a potential anti-asthmatic drug.

Animals ; Anti-Asthmatic Agents ; pharmacology ; Bronchial Hyperreactivity ; drug therapy ; Bronchoalveolar Lavage Fluid ; cytology ; Cell Degranulation ; drug effects ; Male ; Mast Cells ; drug effects ; physiology ; Ovalbumin ; immunology ; Polyporaceae ; chemistry ; Polysaccharides ; pharmacology ; Rats ; Rats, Sprague-Dawley

This study is to investigate the anti-allergic effect of anthocyanidin and to explore its possible mechanism. The experiments of passive cutaneous anaphylaxis reaction (PCA) and colorimetry were used to determine the effect of anthocyanidin on degranulation of mast cells in vivo. For in vitro study, various concentrations of anthocyanidin (100, 50 and 25 micromol x L(-1)) were added to the culture medium of mast cells cultured with 100 microg x L(-1) of dinitrophenyl (DNP) specific IgE overnight. The azelastine (100 micromol x L(-1)) was selected as the positive control. The antigen (DNP-human serum albumin, DNP-HAS)-induced release of degranulation was measured by enzymatic assay, histamine was determined by EIA, and interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) were measured by Western blotting, separately. In addition, the effects of anthocyanidin on phosphorylation of NF-kappaB, p38MAPK and Akt were observed by Western blotting. The results showed that treatments with anthocyanidin (100 and 50 mg x kg(-1)) were followed by a decrease in PCA of rats. Anthocyanidin (100 and 50 micromol x L(-1)) obviously suppressed the degranulation from mast cells, whereas results from anthocyanidin (100 and 50 micromol x L(-1)) group indicated significant inhibitory effect on histamine, the calcium uptake, TNF-alpha, IL-6, phosphorylation of NF-kappaB, p38MAPK and Akt of mast cells induced by antigen. Anthocyanidin may suppress the anaphylactic reaction by inhibiting the action of mast cells. NF-kappaB, p38MAPK and Akt at least in part contribute to this event.
Animals ; Anthocyanins ; pharmacology ; Anti-Allergic Agents ; pharmacology ; Calcium ; metabolism ; Cell Degranulation ; drug effects ; Histamine Release ; drug effects ; Immunoglobulin E ; immunology ; Interleukin-6 ; metabolism ; Male ; Mast Cells ; immunology ; metabolism ; physiology ; Passive Cutaneous Anaphylaxis ; drug effects ; Proto-Oncogene Proteins c-akt ; metabolism ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism ; p38 Mitogen-Activated Protein Kinases ; metabolism