Objective To establish an animal model for immunological contact urticaria in mice.Methods A total of 60 BALB/c mice were randomly and equally divided into 5 groups:anti-dinitrophenol IgE monoclonal antibody (anti-DNP IgE) + 2,4-dinitrofluorobenzene (DNFB) group and anti-DNP IgE + trimellitic anhydride (TMA) group both injected with anti-DNP IgE via tail veins firstly,followed by topical treatment with DNFB and TMA respectively on the ears at 24 hours after the injection,DNFB group,TMA group and normal saline (NS) group all injected with NS via the tail vein firstly,followed by topical treatment with DNFB,TMA and NS on the ears 24 hours after the injection.In the following 14 days,mice were observed daily for the appearance of wheals and for scratching behavior.All the mice were sacrificed at the end of the study followed by determination of the percentage of degranulated mast cells and spleen index as well as observation of pathological changes.Results Wheals were observed in all the mice (12/12) in the anti-DNP IgE + DNFB group,some mice (8/12) in the anti-DNP IgE + TMA group,but not observed in any mice in the other 3 groups.Compared with the NS group,both the anti-DNP IgE + DNFB group and anti-DNP IgE + TMA group showed a significant increase in the percentage of degranulated mast cells (70.21％ ± 26.01％ and 54.25％ ± 39.57％ vs.14.45％ ±6.79％,F=14.41,P=0.000),spleen index (7.54 ± 1.56 and 7.87 ± 1.18 vs.5.37 ± 1.16,F=4.29,P=0.004) and scratching frequency ((31.58 ± 3.58)/h and (22.17 ± 3.81)/h vs.(2.00 ± 0.85)/h at 30 minutes,F =437.86,P ＜ 0.01).Conclusion A stable mouse model for immunological contact urticaria can be established quickly by sensitization with anti-DNP IgE and challenge with DNFB.

Objective To study the relationship of T, B and NK lymphocytes with the pathogenesis of chronic urticaria. Methods Flow cytometry was applied to assess the proportion of T, B and NK lymphocyte subgroups in the peripheral blood of 51 patients with chronic urticaria and 30 sex and age-matched human controls. The CD4:CD8 ratio was calculated. Moreover, the symptoms, disease course and response to antihistamines of these patients were evaluated by one physician. Results The percentage of CD8+ T and NK cells, CD4:CD8 ratio were (27.20±8.22)%, (21.20±10.84)% and 1.48±0.62, respectively, in these patients,(29.9±3.74)%, (17.5±3.56)%, 1.24±0.27, respectively, in the controls; the differences were significant between the two groups (all P＜0.05). Decreased levels of CD3+ T cells, CD8+ T cells and B cells were noted in patients resistant to antihistamines compared with those responsive to antihistamines[(61.81±11.70)% vs (75.74±2.36)%, (24.00±7.79)% vs (34.22±9.30)%, (10.78±2.07)% vs (15.25±4.10)%, P＜0.05, 0.01, 0.05, respectively)], while the CD4:CD8 ratio and percentage of NK cells were increased in antihistamine-resistant patients compared to those in antihistamine-sensitive patients [1.67±0.76 vs 1.17±0.41, (28.61±12.62)% vs (12.78±6.02)%, both P＜0.01 ]. In these patients with chronic urticaria, the percentages of CD3+ T and CD8+ T cells were negatively correlated with the symptom scores (R = -0.31, -0.28, respectively, both P＜0.05 ), while the percentage of B cells was positively correlated with the symptom scores and disease course (R = 0.53, 0.55, respectively, both P＜0.01 ). Conclusions There is an abnormality in the proportion of T, B and NK lymphocyte subgroups in patients with chronic urticaria,which indicates that humoral immunity may be involved in the pathogenesis of chronic urticaria and the mechanism for responsiveness to antihistamine.

Objective To determine the coagulation status as well as circulating levels of complement and inflammation markers in patients with chronic urticaria (CU) during acute attack and in remission,and to estimate the relationship of coagulant and anticoagulant factors as well as fibrinolytic markers with the development of chronic urticaira.Methods This study included 40 patients with CU (22 during acute attack and 18 in remission) and 40 healthy blood donors from the Guangzhou Blood Center.Venous blood samples were obtained from these subjects,and enzyme-linked immunosorbent assay (ELISA) was performed to measure the plasma levels of prothrombin fragrnent 1 +2 (F1 +2),tissue factor (TF),thrombomodulin (TM),high molecular weight kininogen (HMWK),tissue-type plasminogen activator (t-PA),C5a and serum levels of C3,C4,antistreptolysin O antibodies (ASO),rheumatoid factor (RF) and C-reactive protein (CRP).Erythrocyte sedimentation rate (ESR) was also determined in these patients.Comparisons of these parameters were carried out by using t test,and the correlation of these factors with CU was evaluated by using Spearman correlation coefficient.Results Compared with the healthy controls,the patients with CU showed significantly higher plasma levels of F1+2 and HMWK (both P ＜ 0.01),but lower levels of TF,TM and t-PA (all P ＜ 0.01).The plasma levels of F1 +2,HMWK,t-PA were significantly correlated with the symptom scores in patients with CU (r =0.81,P ＜ 0.01; r =-0.39,P ＜ 0.05; r =0.35,P ＜ 0.05).A significant increase was observed in the plasma concentration of F1 +2 in patients during acute attack compared with those in remission (P ＜ 0.01),whereas no significant differences were noted in the plasma levels of TF,TM,HMWK,t-PA,C5a,serum levels of C3,C4,ASO,RF and CRP or ESR between the two groups of patients (all P ＞ 0.05).Conclusions It seems that coagulation,anti-coagulation and fibrinolysis are all involved in the development of urticaria.There is an obvious difference in the plasma level of prothrombin F1 +2 between patients with CU during acute attack and in remission,suggesting that coagulation factors play a certain role in the initiation and progression of CU.

Objective To evaluate the relationship of clinical symptom to plasmic levels of D-dimer, activated factorⅦ (FⅦa) and tissue factor pathway inhibitor (TFPI)/X a in patients with urticaria. Methods A total of 27 patients with chronic urticaria (CU), 27 patients with acute urticaria (AU) and 26 normal human controls were included in this study. Symptom score was determined and disease course was surveyed in these patients. ELISA was used to detect the plasma levels of D-dimer, FⅦa and (TFPI)/Xa in patients and controls. The relation of clinical symptom and disease course to plasma levels of these parameters was assessed. Results In patients with AU and normal controls, the plasma level of D-dimer was 450.57± 242.13 ng/mL and 266.81±40.68 ng/mL, respectively, the level of FⅦa, 2.23± 0.74 ng/mL and 5.23±1.35 ng/mL, respectively, and the level of TFPI/Xa 0.87±0.13 nmol/L and 0.88 ~ 0.12 nmol/L, respectively. There was a significant difference in the level of both D-dimer and FⅦa (both P < 0.01 ), whereas no differ-ence was observed in that of TFPI/X a (P > 0.05) between patients with AU and normal controls. In addi-tion, increased level of D-dimer and decreased level of FⅦa were noticed in patients with CU compared with those in normal controls (593.80±294.04 ng/mL vs 266.81±40.68 ng/mL, 3.98±0.35 ng/mL vs 5.23± 1.35 ng/mL, both P < 0.01 ), but there was no significant difference in the plasma level of TFPI/Xa (0.87± 0.16 nmol/L vs 0.88±0.12 nmol/L, P > 0.05). Significant difference was observed in the plasma level of D-dimer and FⅦa between patients with AU and CU (450.57±242.13 ng/mL vs 593.80 ±294.04 ng/mL, P < 0.05; 2.23± 0.74 ng/mL vs 3.98± 0.35 ng/mL, P<0.01 ). The plasma level of D-dimer positively corre-lated to the symptom score of patients with CU and those with AU (r= 0.68, P< 0.01; r= 0.82, P< 0.01),but was independent of discase course (P> 0.05). Neither the level of FⅦa nor that of TFPI/Xa correlated to symptom score or disease course of patients (all P > 0.05). Conclusions There is an overactivation of coagulation cascade, consumption of blood coagulation factors and secondary fibrinolysis in patients with urticaria, suggesting that plasma D-dimer and FⅦa may be associated with the clinical symptoms of urticaria.

Objective To study the influence of sunscreens with different efficacy on delayed type hypersensitivity (DTH) and their immunoprotective effect in mice.Methods A cohort of mice were randomly divided into 5 groups with 10 mice in each group:group 1 as the positive control without irradiation,group 2 receiving solar-simulated radiation (SSR) only,group 3 receiving SSR and protected by sunscreen l with sun protection factor 15(SPF15)and persistent pigment darkening(PPD)12,group 4 receiving SSR and protected by sunscreen 2 with SPF 50 and PPD 28,and group 5 as the negative contml receiving SSR only.SSR was carried out on the back of mice with the UVA dose being 1.4 J/cm2 and UVB dose being 100 mJ/cm2 for 10 days.After a 5-day irradiation,the groups 1 to 4 were immunized by intraperitoneal injection with 100 μl(107 cells/ml) of Candida albicans suspension.On the 10th day both sides of the posterior foot pad were measured;then the foot pads were injected with additional 50 μl of the Candida albicans suspension.Twenty-four hours after the injection,the thickness of each foot pad was measured,and immunosuppression rate was calculated.Finally,the mice were sacrificed and skin samples were obtained from the back of these mice followed by the examination of CDla, CD80 and CD86 expression by Western blot.Resets The thickness of edema in foot pads was 0.41±0.38 mm,0.21±0.23 mm and 0.30 ± 0.25 mm in group 1,3 and 4,respectively,significantly higher than in group 5 and 2(0.04±0.03 mm,0.14±0.12 mm,respectively,all P<0.05),while no significant difference was observed between the group 3 and 4(P>0.05).Significant differences were observed in the immunosuppression rate between group 2,3 and 4(73.0%±11.3%,54.1%±6.4%,29.7%±7.5%,respectively,all P<0.01).Western blot revealed a significant increment in the expression of CDla protein in group 1 compared with group 2 as well as in the expression of CD86 protein in group 1 and group 3 compamd with group 2 and group 5(all P<0.05),but no statistical difference was observed between the other groups in the expression level of CDla,CD80 or CD86(P>0.05).Conclusions The exposure to sub-erythema dose of UV can induce DTH,and sunscreens have an immunoprotective effect in this process.Epidermal Langerhans cells are not essential for UV-induced immunosuppression.

Objective To investigate the role of regulatory T (Treg) / T helper type 17 (Th17) cells in the pathogenesis of chronic spontaneous urticaria (CSU).Methods Eighty-nine patients with CSU were enrolled in this study,including 48 in active stage and 41 in remission stage.Forty-eight health check-up examinees,who were collected from the community hospitals in Guangzhou city,served as the healthy controls.Fluorescence-based realtime quantitative PCR was performed to determine the expression of transcription factors FOXP3 and RORγt in PBMCs from these subjects.Results Compared with the patients with CSU in remission stage and healthy controls,the patients in active stage showed a significantly higher level of FOXP3 mRNA (0.57 ± 0.19 vs.0.11 ± 0.21 and 0.13 ± 0.23,both P ＜ 0.05),but a significantly lower level of RORγt mRNA (0.43 ± 0.39 vs.0.89 ± 0.40 and 0.87 ± 0.43,both P ＜ 0.05).Conclusions The expression of Treg cell regulator FOXP3 increases,while the expression of Th17 cell regulator RORγt decreases in patients with CSU,suggesting that the imbalance between Treg and Th17 cells induced by the interaction between FOXP3 and RORγt may be involved in the pathogenesis of CSU.

Objective To evaluate effects of tea polyphenols on the mRNA and nucleoprotein expression of Nrf2/Bach1 in human skin fibroblasts (HSFs).Methods Some HSFs were incubated with tea polyphenols at different concentrations of 0,2.5,5,10,20 and 40 mg/L for 24 hours.Methyl thiazolyl tetrazolium (MTT) assay was conducted to evaluate the proliferative activity of HSFs to screen the optimal concentration of tea polyphenols.Then,some other HSFs were treated with tea polyphenols at this optimal concentration for 24 hours.Real-time quantitative PCR (RT-qPCR) was performed to determine mRNA expression of Nrf2 and Bach1,Western blot analysis to measure nuclear expression of Nrf2 and Bach1 proteins,and immunofluorescence assay to determine the distribution of Nrf2 and Bach1 protein in the cell nucleus.Results MTT assay showed that 5 mg/L tea polyphenols had no obvious effects on the proliferation of HSFs,so 5 mg/L was chosen as the optimal concentration of tea polyphenols for subsequent experiments.HSFs cultured without tea polyphenols served as control group.After the treatment,the 5-mg/L tea polyphenol group showed significantly decreased mRNA and nuclear protein expression of Bach 1 (mRNA:0.629 ± 0.077 vs.0.940 ± 0.033,t =6.397,P ＜ 0.05;protein:1.424 ± 0.171 vs.16.966 ± 1.702,t =15.730,P ＜ 0.05),but significantly increased mRNA and nuclear protein expression of Nrf2 (mRNA:1.467 ± 0.076 vs.0.977 ± 0.091,t =7.133,P ＜ 0.05;protein:6.929 ± 0.121 vs.3.537 ± 0.126,t =33.636,P ＜ 0.05) compared with the control group.Immunofluorescence assay showed increased accumulation of Nrf2 protein,but decreased accumulation of Bach1 protein in the nucleus.Conclusion Tea polyphenols can promote the mRNA and nuclear protein expression as well as nuclear distribution of Nrf2,but suppress the mRNA and nuclear protein expression as well as nuclear distribution of Bach 1,finally exerting antioxidative effects.

Objective To evaluate the scavenging effect of crude polysaccharides extracted from Lycium barbarum (LBP) on reactive oxygen species in ultraviolet radiation-induced HaCaT cells,and to explore its possible mechanism.Methods Cultured immortalized human keratiuocyte HaCaT cells were divided into 6 groups:blank control group receiving no treatment,LBP group treated with crude LBP alone,ultraviolet A (UVA) group treated with UVA radiation alone,ultraviolet B (UVB) group treated with UVB radiation alone,UVA + LBP group treated with crude LBP for 24 hours followed by UVA radiation,and UVB + LBP group treated with crude LBP for 24 hours followed by UVB radiation.MTT colorimetry was performed to evaluate the cellular proliferative activity,UV spectrophotometric method to measure the UVA and UVB absorption of crude LBP,dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe assay to detect the level of ROS,enzymatic-biochemical method to estimate the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px),as well as to detect the leakage of lactate dehydrogenase (LDH).Results Crude LBP at different concentrations of 0,100,200,300,400,500,600,1 500,2 000 mg/L had no obvious effects on the proliferative activity of HaCaT cells.Crude LBP had a high transmittance of ultraviolet rays at 280-400 nm.Compared with the blank control group,the UVA group and UVB group both showed significantly higher LDH leakage and ROS level,lower activities of SOD and GSH-Px (P ＜ 0.001 or 0.05).Pretreatment with crude LBP before the ultraviolet radiation could significantly increase the activities of SOD and GSH-Px,decrease the LDH leakage and ROS level in the UVA + LBP group and UVB + LBP group compared with the UVA group or UVB group (P ＜ 0.05).Conclusion Crude LBP have no effect of sunscreening agents,but can effectively scavenge ROS,decrease LDH leakage,inhibit ultraviolet radiation-induced photodamage in HaCaT cells,which may be associated with the enhancement of antioxidant enzyme activity.

Objective To investigate the dynamic changes of three types of anti-poliovirus neutralizing antibodies and anti-hepatitis A virus (HAV) IgG antibody in children who were immunized with inactivated enterovirus 71 (EV71) vaccine (human diploid cell).Methods Serum samples were collected from the subjects immunized with inactivated EV71 vaccine.Neutralizing antibodies against EV71 and poliovirus were detected by micro-cytopathic effect neutralization test.Enzyme linked immunosorbent assay (ELISA) was used to detect IgG antibody against HAV.Results The geometric mean titers (GMTs) of anti-EV71 neutralizing antibody increased to 4.85 following the first-dose injection of inactivated EV71 vaccine.A significant increase of GMTs (up to 64.37) could be observed 28 days after the second-dose vaccination.Meanwhile, results of the dynamic monitor showed that there were slight fluctuations in the neutralizing antibodies against three types of poliovirus on day 28 (28 days after the first-dose vaccination) compared with those on day 0 (before vaccination) (P<0.05);types Ⅰ and Ⅲ anti-poliovirus neutralizing antibodies on day 56 (28 days after the second-dose vaccination) remained slightly different from those on day 0 (P<0.05), but type Ⅱ anti-poliovirus neutralizing antibody on day 56 had restored to normal level (P>0.05).The level of anti-HAV IgG antibody was stable and no significant difference was found during the observation period (P>0.05).Conclusion This study shows that inactivated EV71 vaccine has no impact on anti-HAV IgG antibody in Children during the two-dose vaccination and in anti-EV71 antibody-producing period, but has slight influence on the anti-poliovirus antibodies.In general, changes in antibody profile do not affect the clinical efficacy of immune response.

After sequencing the Asia 1 foot-and-mouth disease virus (FMDV) (As01 strain), we amplified the two fragments covering the whole genome by overlapping PCR and long PCR. The 5' fragment was 1.8 kb in length including 15Cs, and the 3' fragment was 6.7 kb in length. The two fragments were cloned into the pBluescript SK vector to construct recombinant plasmid pBSAs carrying the full-length cDNA of FMDV As01 strain. The RNA transcript was synthesized in vitro using T7 polymerase and transfected into BHK-21 cells. We observed the typical CPE caused by rescued FMDV. The harvested virus was confirmed to be Asia 1 FMDV by RT-PCR, indirect immunofluorescence assay (IFA) and electron microscope observation. The rescued virus showed a similar pathogenicity in suckling mouse (LD50) compared to its wild-type virus. The infectious cDNA clone of the FMDV As01 strain laid a new ground for further investigation of FMDV virulence determinants and development of novel vaccines against FMD.
3' Untranslated Regions ; 5' Untranslated Regions ; Animals ; Base Sequence ; Cattle ; Cell Line ; Cloning, Molecular ; DNA, Complementary ; genetics ; Foot-and-Mouth Disease ; metabolism ; virology ; Foot-and-Mouth Disease Virus ; classification ; genetics ; growth & development ; Genome, Viral ; Mice ; Molecular Sequence Data ; Plasmids ; RNA, Viral ; genetics ; Transfection ; Virulence ; Virus Replication