Objective To investigate the role of Staphylococcus aureus enterotoxin B (SEB) in non-IgE mediated activation of mast cells (MCs) by in vitro co-culture of laboratory of allergic disease 2 (LAD2) cells and SEB.Methods The LAD2 cells were incubated with SEB at different concentrations of 0.01,0.1,1,10 and 100 μg/ml,A23187 positive control and negative control separately for 30 minutes.Then,effects of SEB on the morphology of MCs were observed by using a light microscope,and culture supernatants of the above incubation systems were collected.The concentration of tryptase released from MCs was analyzed by enzymatic activity assay,and the level of histamine was detected by enzyme-linked immunosorbent assay (ELISA).Results After 30-minute co-culture of LAD2 cells and SEB,MCs showed larger size,obscure boundaries,increased number of protuberances on the cell surface and decreased refractivity,with a radial burr fin-like appearance.After 30-minute co-culture of LAD2 cells and SEB at different concentrations of 0.01,0.1,1,10 and 100 μg/ml,the concentrations of tryptase in the culture supematants were 4.116 ± 0.651,5.344 ± 0.874,3.806 ± 0.459,1.309 ± 0.247,0.310 ± 0.199 ng/ml respectively.Additionally,the tryptase levels were significantly higher in the 0.01-,0.1-,1-μg/ml SEB groups than in the negative control group(1.538 ± 0.490,all P ＜ 0.05),and gradually decreased along with the increase of SEB concentrations.The histamine levels in the 0.01-,0.1-,1-,10-and 100-μg/ml SEB groups were 242.409 ± 63.915,522.491 ± 73.466,550.926 ± 84.466,334.397 ± 33.640,226.527 ± 5.678 ng/ml respectively.In the 0.01-,0.1-,1-μg/ml SEB groups,the levels of histamine released from MCs were gradually increased along with the increase of SEB concentrations,and were significantly higher than those in the negative control group (146.436 ± 3.100,all P ＜ 0.05).However,with the continued increase of SEB concentrations,the histamine levels gradually decreased.Conclusion SEB can directly activate MCs by a non-IgE mediated mechanism,followed by morphologic changes of MCs and release of tryptase and histamine.

Objective To detect the expression of proteinase activated receptor 2 (PAR-2) in the skin of patients with atopic dermatitis (AD) and to evaluate the effects of tacrolimus on the expression.Methods Six patients with acute moderate or severe AD were enrolled in this study and topically treated with tacrolimus 0.1％ ointment twice daily for 3 weeks.Tissue samples were obtained from the lesions and non-lesional skin at least 10 cm away from the lesions before and after the 3-week treatment.Skin specimens from 6 normal human controls served as the control.Patients were evaluated at the baseline,1 and 3 weeks after the beginning of treatment for clinical symptoms and signs by visual analogue scale (VAS),eczema area and severity index (EASI) and investigator's global assessment (IGA).The expression of PAR-2 in tissue specimens were determined by immunohistochemistry.Results PAR-2 was expressed throughout the whole epidermis,especially in the granular layer,hair follicles,sweat glands,endothelial cells and nerve fiber-like structures.Before treatment,the expression level (mean optical density) of PAR-2 in keratinocytes was 4339.6 ± 115.8 in lesional skin of AD patients,significantly higher than that in non-lesional skin (4189.0 t 228.9,t =2.85,P ＜0.05) and in normal skin (3864.0 ± 237.3,t =4.31,P ＜ 0.05).After the 3-week treatment with tacrolimus ointment,the expression level of PAR-2 significant decreased to 3942.4 ± 176.6 in keratinocytes from lesional skin of patients with AD (t =4.55,P ＜ 0.05).The expression level of PAR-2 was positively correlated with VAS score for itch,EASI and IGA score in the patients.Conclusions The expression of PAR-2 is enhanced in keratinocytes of lesions from AD patients,and is positively correlated with itch and lesion severity.Topical tacrolimus may suppress the overexpression of PAR-2 in keratinocytes in lesional skin of AD.

Objective:To establish a method to detect food allergen based on Latex-enhanced immune turbidimetry ( PETIA) and apply to clinic.Methods: The PETIA method was used for evaluating the detection system including standard curve , detection-limit,stability, intra-assay and inter-assay precision and cross-reactivity,clinical normal serum specimens and clinical allergic diseases serum specimens were measured to evaluated the perspective of the clinical application .Results: The standard curve :Y=837.1 x2-125.2x+10.036, linear range: 0.0-400 U/ml, the correlation coefficient of the standard curve was 0.996, the intrabatch and interbatch precision was<10%.The normal reference range was≤33.5 U/ml,the AUC of ROC curve was 0.957,the sensitivity was 89.61%,The specificity was 65.22%, the accuracy was 84.00%, and the positive predicted value was 89.61%, the negative predicted value was 65.22%.the test results have strong correlation with ELISA ( r=0.890 2 ) .Conclusion: The PETIA method for detecting food allergen achieved corresponding clinical application standards and may be used for the diagnosis of food allergies .

Objective:to investigate clinical significance of serum and urine NGAL (Neutrophil gelatinase-associated lipocalin) in the early kidney damage with primary hypertension.Methods:According to UAER ( urinary microalbumin excreting rate ) ,we divided 90 patients with primary hypertension into three groups (<30 mg/24 h,30-300 mg/24 h and >300 mg/24h),and selected 30 healthy people as the control group.Serum and urine NGAL and cystatin C ,serum creatinine,urea nitrogen,high sensitive C reactive protein , transferrin,and basis of blood pressure were detected and followed up one year.Results:Compared with healthy group ,GFR( glomerular filtration rate ) and serum NGAL were decreased significantly in medium and severe proteinuria groups , while urine NGAL was increased.Conclusion:Serum and urine NGAL have been a clear trend changes in kidney damage ,which could be used as a reliable indicator in monitoring renal function of patients with primary hypertension.

Objective To investigate the effect of sufentanil postconditioning on cardiomyocyte apoptosis during myocardial ischemia-reperfusion (I/R) in rats.Methods Thirty-six male Sprague-Dawley rats weighing 250-280 g were randomly divided into 3 groups ( n =12 each):sham operation group (group S),I/R group and sufentanil postconditioning group (group SP).Myocardial I/R was induced by occlusion of the left coronary artery for 30 min followed by 120 min reperfusion.Sufentanil 3.0μg/kg was injected iv at the beginning of reperfusion in group SP.HR and MAP were recorded during I/R.The rats were sacrificed at 120 min of reperfusion and their hearts were removed for determination of infarct size,number of apoptotic cardiomyocytes,and the expression of Bax mRNA and Bcl-2 mRNA,and apoptotic index was caculated.Results There was no significant difference in HR among the 3 groups ( P ＞ 0.05 ).Compared with group S,MAP and Bcl-2 mRNA expression were significantly decreased,apoptotic index and Bax mRNA expression increased in group I/R,and apoptotic index,and the expression of Bax mRNA and Bcl-2 mRNA were increased in group SP (P ＜ 0.05).Compared with group I/R,the infarct size,apoptotic index and Bax mRNA expression were decreased,and Bcl-2 mRNA expression was increased in group SP (P ＜ 0.05).Conclusion Sufentanil postconditioning can attenuate myocardial I/R injury in rats by up-regulating Bcl-2 expression,down-regulating Bax expression and inhibitting cardiomyocyte apoptosis.

Objective To investigate the effect of valsartan on vascular endothelial function and brachial-ankle pulse wave velocity(baPWV)in patients with essential hypertension(EH).Methods Blood pressure,NO value and brachial-ankle pulse wave velocity were determined and analyzed in 30 patients with essential hypertension before and after therapy of valsartan.Results Both blood pressure and brachial-ankle pulse wave velocity wefe re-duced greafly(P<0.05 and P<0.01),while serum NO increased significantly(P<0.01)following valsartan thera-Py,and brachial-ankle pulse wave velocity was well correlated to serum NO(r=-0.71,P<0.01).Conclusions Valsartan is not onlv effective in the control of blood pressure,but also effective in reveming the impaired endothelial function and artery elasticity in patients with essential hypertension,at the same time brachial-ankle pulse wave ve-locity is also good surrogate of endlothelial functional improvement induced by Valsartan therapy.

Objective To study the effect of bel-2 siRNA on apoptosis of HL-60 cells.Methods bcl-2 siRNA was synthesized in vitro transcription with silencer siRNA construction kit.The synthesized siRNA was transfected into HL-60 cells with Amine siPORT transfection.We used MTT flow cytometer and hoechst 33258 flourescence stainning t0 evaluate cell proliferation and apoptosis. Results.Bcl-2 siRNA could partially inhibit the growth of HL-60 cells.After incubated with bcl-2 siRNAl for 48 hours,HL-60 cells exhibited morphologic characteristic of apoptosis including chromatin condensation,crescents formation and nuclear fragmentation.Conclusion Effective bcl-2 siRNA can induce apoptosis and inhibit cell proliferation.

OBJECTIVETo compare skin sympathetic response(SSR) between patients with generalized anxiety disorder(GAD) and patients with major depression disorder(MDD).

METHODSThe latency and amplitude of SSR wave were measured in 30 GAD patients and 30 MDD patients, before and after 8-week treatment of anti-anxiety or anti-depression drugs. Thirty age and sex-matched healthy subjects served as healthy controls (HC).

RESULTSBefore the treatment, the latency of SSR in GAD patients was significantly shorter than that in HC group, while the amplitude was significantly higher than that in the HC (P<0.05). In MDD group, the latency before the treatment was significantly longer than that in the HC,while the amplitude was significantly lower than that in the HC (P <0.05). After treatment,the latency of SSR in GAD group was extended compared to the baseline level, and close to the level of the HC. The amplitude of SSR in GAD group became lower after treatment, but still higher than that of control group. The latency of SSR in MDD patients was significantly shorter after treatment compared to baseline level (P <0.05). In addition, the latency of SSR in MDD group was still longer than that in GAD group (P<0.05); meanwhile,the amplitude of SSR in MDD group was significantly lower that in GAD group (P<0.001). SSR parameters were positively correlated with HAMA and HAMD scores with a correlation coefficient of 0.57 and 0.73, respectively.

CONCLUSIONThere are significant differences in SSR parameters between patients with GAD and patients with MDD,indicating that SSR can be used as an objective index to distinguish anxiety from depression.

Adolescent ; Adult ; Anti-Anxiety Agents ; therapeutic use ; Antidepressive Agents ; therapeutic use ; Anxiety Disorders ; drug therapy ; physiopathology ; Case-Control Studies ; Depressive Disorder, Major ; drug therapy ; physiopathology ; Female ; Galvanic Skin Response ; Humans ; Male ; Middle Aged ; Skin ; physiopathology ; Sympathetic Nervous System ; physiopathology ; Young Adult

Objective Using hybridoma technique and screened hybridoma cell strains stably, efficiently secreted anti glycosylated hemoglobin monoclonal antibody to provide specific material for the development of glycosylated hemoglobin ELISA kit. Methods The immune antigen was prepared by maleimide method, multi-level immune mice by BALB/c,through cell culture fusion, screening of hybridoma cell culture medium HAT, ammonium sulfate salting out method and G protein chromatography. Monoclonal antibody subclasses were identified by monoclonal antibody subtype identi-fication Kit operation. Results Through cell fusion, screening and cloning culture, etc., the final selection screened 1 strain stably secreting specific antibody hybridoma cell line, named N5B4; cell culture supernatant liquid was 1:5000, ascites titer was 1:100 million; a standard curve to calculate the concentration in the sample human glycat ed hemoglobin was 99.2%. Conclusion After KET monoclonal antibody cell line to obtain a high specificity and high sen-sitivity of screening.

Objective To investigate the role of mast cells in Staphylococcus aureus enterotoxin B (SEB)-induced atopic dermatitis (AD)-like skin inflammation in BALB/c mice.Methods A total of 24 BALB/c mice were randomly and equally divided into 4 groups to be topically treated with ovalbumin (OVA group),SEB (SEB group),OVA + SEB (OVA + SEB group) and sodium chloride physiological solution (control group) respectively,so as to establish mouse models of epicutaneously induced AD-like skin inflammation.The AD-like skin lesions were evaluated by clinical observation and eczema area and severity index (EASI).Biopsy specimens were obtained from lesional skin of mice and then subjected to toluidine blue staining and immunohistochemical staining to count the mast cells,observe the morphology and distribution of mast cells,and calculate the percentage of degranulated mast cells.Results After 7-week treatment,the OVA group,SEB group and OVA + SEB group all showed severer local skin inflammation,higher EASI scores and denser infiltration of inflammatory cells compared with the control group.Moreover,the OVA + SEB group showed significantly severer local skin inflammation,skin lesions and degree of infiltration of inflammatory cells compared with the OVA group and SEB group (all P ＜ 0.05).The number of mast cells in the dermis of AD-like skin lesions per high-power field (× 400) was significantly higher in the OVA group (median [quartile range]:10.625 [3.675]),SEB group (11.000 [4.163]) and OVA + SEB group (13.875 [8.813]) than that in the control group (5.925 [2.088],all P ＜ 0.05).The SEB group (71.083％ ± 14.519％) and OVA + SEB group (58.767％ ±.16.978％) both showed significantly higher percentage of degranulated mast cells compared with the OVA group (24.050％ ± 11.161％,both P ＜ 0.05) and control group (23.617％ ± 8.132％,both P ＜ 0.05).Bivariate correlation analysis showed that the number of mast cells in the skin lesions was positively linearly correlated with the EASI scores (P ＜ 0.05).Conclusions Epicutaneous application of SEB can induce AD-like skin lesions in mice,and can exacerbate the severity of OVA-induced AD-like skin lesions.Mast cell proliferation,activation/degranulation and tryptase release may participate in the inflammation.