[T-bet gene modified dendritic cells abrogate and reverse the airway inflammation in allergic asthma: experiment with mice].

OBJECTIVE: To explore the feasibility of the therapeutic strategy to use T-bet gene modified dendritic cells (DCs) to reverse the course of asthma. METHODS: (1) Mature DCs were derived from mononuclear cells obtained from femur of BALB/c mouse and divided into 3 groups, T-bet group transfected with recombinant adenovirus Ad-T-bet containing T-bet gene, LacZ group transfected with recombinant adenovirus Ad-LacZ containing LacZ gene, and control group. Seven days later ELISA was used to detect the interferon (IFN)-gamma level in the culture fluid. (2) Airway inflammation abrogating trial. Twenty-four BALB/c mice were sensitized with intraperitoneal injection of ovalbumin (OVA) (on day 1 and 15) to establish asthma models, and then randomly divided into 3 equal groups: T-bet group injected intravenously with T-bet-modified DCs on day 27, LacZ group injected with LacZ-modified DCs, and model control group without intravenous injection. Two days later the model mice began to undergo challenge by inhalation of OVA twice (on day 29 - 31). Eight mice were used as control group treated with PBS. On day 37 all mice were killed, ELISA was used to detect the blood interleukin (IL)-4 and IFN-gamma levels, and microscopy was conducted to observe the airway inflammation. (3) Airway inflammation reversing trial. Another 24 model mice were divided into 3 equal groups as well: re-challenged T-bet group injected intravenously with T-bet-modified DCs on day 27 and 42, re-challenged LacZ group injected intravenously with LacZ-modified DCs on day 27 and 42, and model control group. Since the day 45 OVA inhalation was given once a day for successive 3 days. On day 49 these mice were all killed to undergo the tests as mentioned above. RESULTS: The IFN-gamma level in the culture fluid of the T-bet gene modified DCs was (15.24 +/- 4.75) ng/ml, significantly higher than that of the LacZ gene modified DCs and control DCs [(3.08 +/- 0.61) and (2.35 +/- 0.41) ng/ml respectively, both P < 0.01]. The IFN-gamma in mice blood plasma of T-bet groups in abrogating and reversing trial were (130.2 +/- 10.5) and (145.7 +/- 16.7) pg/ml respectively, both significantly higher than those of the abrogating and reversing trial normal control groups [(25.0 +/- 6.5) and (24.6 +/- 5.9) pg/ml respectively], asthmatic model control groups [(20.7 +/- 4.5) and (16.5 +/- 7.0) pg/ml respectively] and LacZ groups [(17.6 +/- 7.0) and (24.2 +/- 9.0) pg/ml respectively] (all P < 0.01). However, the IL-4 levels in mice blood plasma of T-bet groups were both significantly lower than those of asthmatic model control groups and LacZ groups (all P < 0.01). The airway inflammation of T-bet groups were remarkable milder than those of the model control groups and LacZ groups. CONCLUSION: The asthma management strategy based on T-bet gene modified DCs is feasible with the plausible mechanism that the T-bet gene modified DCs regulate the T cells differentiation and polarization on the antigen presenting level.

[Vasoactive intestinal peptide expression and its clinical significance in gastric adenocarcinoma].

OBJECTIVE: To investigate the expression of vasoactive intestinal peptide (VIP) in gastric adenocarcinoma, and to evaluate the correlation of VIP level with clinical pathologic parameters. METHODS: The level of VIP in sera from gastric adenocarcinoma patients and healthy people was investigated by ELISA. Moreover, the differential gene expression between gastric adenocarcinoma, gastric dysplasia, and the corresponding normal gastric mucosa were determined by RT-PCR. Western Blot was also used to measure the expression of VIP in the gastric adenocarcinoma and the normal gastric mucosa. RESULTS: The serum level of VIP was (5.794 +/- 0.014) ng/ ml in normal control and was (14.437 +/- 0.825) ng/ml in gastric adenocarcinoma patients, showing significant difference (P < 0.05). Meanwhile,the V/B of gastric adenocarcinoma tissues was greater than that of gastric dysplasia and the corresponding normal gastric mucosa (P <0.01), the values of V/B were 1.5261 +/- 0.3028, 0.9334 +/- 0.2872,and 0.9051 +/- 0.2794, respectively. The values of V/B between normal gastric mucosa and gastric dysplasia were not different significantly (P > 0.05). There were significantly negative correlation between the VIP mRNA expression of the differentiation degree of tumor (P < 0.05). The VIP mRNA expression was higher in gastric adenocarcinoma with lymph node metastasis than that without lymph node matastsis (P < 0.05). The VIP protein expression of the gastric adenocarcinoma tissues was greater than that of normal control. CONCLUSION: This findings provide a direct evidence to support the possibility that VIP play a cofactor role in the pathogenesis of gastric adenocarcinoma.

[Continuous inhalation of allergen induces immunotolerance in a mouse asthma model].

OBJECTIVE: To investigate the mechanism of immunotolerance caused by allergen immunotherapy in allergen-induced asthmatic airway inflammation. METHODS: Sixty ovalbumin (OVA)-sensitized BALB/c mice were randomly divided into two groups, 50 in the experimental group and 10 in the control group. The mice in the experimental group were treated with 3 injections of ovalbumin intraperitoneally (1 mg for each separated for two weeks) and challenged by ovalbumin inhalation 1 h/day for 10 successive days. Then the mice were divided further into group A, group B, group C, group D and E with 10 mice in each group. The mice in group A were sacrificed after 10 day challenge. The mice in group B and D were continuously exposed to inhaled OVA for 4 and 8 weeks (1 h/day, 5 days a week), respectively, then to inhaled OVA 1 h/day for 10 successive days. The inhalation was interrupted for 4 weeks in group C after initial challenge and restarted for another 10 days (1 h/day) afterwards. The mice in group E were exposed continuously to inhaled OVA for 4 weeks (1 h/day, 5 days a week) after initial challenge, which was interrupted for 4 weeks, and restarted for 10 days (1 h/day). Bronchoalveolar lavage (BAL) was performed, and total cells, eosinophils, lymphocytes were assessed; CD4+, CD8+, CD4+ IL-10+ cells were determined using flow cytometry, and IL-4, IFN-gamma and IL-10 in the BAL fluid were measured by ELISA. Serum ovalbumin-specific IgE, IL-4, IFN-gamma and IL-10 were also determined. Pathologic manifestation of the lung was analyzed. RESULTS: The percentage of EOS, B lymphocytes, CD4+ IL-10+ cells in BAL were 0.010 +/- 0.000, 2.1 +/- 1.9 and 4.9 +/- 1.5, respectively in the control group; 0.480 +/- 0.110, 5.1 +/- 2.6 and 5.1 +/- 2.3, respectively, in group A; 0.120 +/- 0.020, 8.9 +/- 3.6, and 10.4 +/- 3.6, respective, in group B; 0.560 +/- 0.050, 4.7 +/- 1.7 and 6.3 +/- 3.1, respectively, in group C; 0.070 +/- 0.030, 10.1 +/- 2.9 and 12.7 +/- 4.5, respectively, in group D; 0.680 +/- 0.030, 5.6 +/- 3.2 and 6.1 +/- 3.4, respectively, in group E. The difference was significant among different groups (F = 36.46, 31.89, 167.89 respectively; all P < 0.01). The percentage of CD4+ IL-10+ cells in BAL was increased in group B and group D, which were significantly higher than those in group A (q = 5.8, 6.4, P < 0.05). The levels of IL-4 and IL-10 in BAL fluid were (21 +/- 3) pg/ml and (44 +/- 12) pg/ml, respectively, in the control group; (128 +/- 23) pg/ml and (68 +/- 18) pg/ml, respectively, in group A; (54 +/- 12) pg/ml and (127 +/- 27) pg/ml, respectively, in group B; (133 +/- 21) pg/ml and (78 +/- 17) pg/ml, respectively, in group C; (8 +/- 18) pg/ml and (135 +/- 34) pg/ml, respectively, in group D; (143 +/- 26) pg/ml and (76 +/- 15) pg/ml, respectively, in group E. The difference was statistically significant among different groups (F = 37.20, 143.78 respectively; all P < 0.01). The levels of IL-10 in BAL fluid were increased in group B and group D, which were significantly higher than that in group A (q = 7.8, 9.6, all P < 0.05). CONCLUSION: The results show that continuous allergen inhalation suppresses allergen-induced airway inflammation and produces immunotolerance, in which IL-10 may play an important role.

[The roles of dendritic cells in antigen presentation and the pathogenesis of asthma].

OBJECTIVE: To investigate the roles of dendritic cells (DCs) in the pathogenesis of asthma. METHODS: (1) DCs were derived from peripheral blood monocytes of asthmatics with acute exacerbation (group A, n = 20), patients at remission (group B, n = 15) and healthy volunteers (group C, n = 10), and cultured using media supplemented with granulocyte-macrophage colony stimulation factors (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha). The antigen-uptaking function of the monocyte-derived DCs was evaluated by phagocytosis of fluorescence labeled ovalbumin (OVA), and their antigen-presenting function was determined by expression of membrane markers (MHC-II) and co-stimulatory molecules (CD(80) and CD(86)). The proliferation of T lymphocytes induced by DCs was assessed using autologous mixed T lymphocyte reaction. (2) The expression of MHC-II and CD(80) and CD(86) of DCs were determined from the bronchoalveolar lavage (BAL) of asthmatic rats. The effects of dexamethasone on the expression of these markers and molecules were studied. Twenty-four rats were divided into the experimental group (group D) and the control group (group E). In group D, rats were subdivided into two groups with 8 rats in each. One group (group D(2)) received pre-treatment with dexamethasone (10 mg/kg), and another group (group D(1)) did not received dexamethasone pre-treatment, while group E was matched with the experimental group with regard to both age and weight. The rats were sensitized and challenged with OVA, and BAL was obtained. (3) All the membrane markers and co-stimulatory molecules on the surface of DCs were determined using flow cytometric method. RESULTS: (1) The phagocytosis of fluorescence labeled OVA in group A was increased [(41 +/- 12)%] compared with those in group B [(29 +/- 10)%, P < 0.01] and group C [(29 +/- 10)%, P < 0.01]. The expression of MHC-II, CD(1alpha) and CD(80) on the surface of DCs was the highest in group A [(44 +/- 15)%, (32 +/- 11)% and (32 +/- 13)%, respectively] compared with those in group B [(22 +/- 10)%, (19 +/- 9)% and (19 +/- 10)%, all P < 0.01, respectively], as well as with those in group C [(18 +/- 12)%, (13 +/- 7)% and (14 +/- 7)%, all P < 0.01, respectively]. There was no statistical difference in the expression of CD(86) on the cell membrane in group A, group B and group C. In autologous mixed T lymphocyte reaction study, when DC/T ratio was 1/5, DCs had the highest ability in stimulating the proliferation of T lymphocytes in group A (stimulating index = 2.32 +/- 0.44) compared with those in group B (1.01 +/- 0.11, P < 0.01), and those in group C (1.62 +/- 0.27, P < 0.01). (2) The expression of MHC-II in BAL cells of asthmatic rats reached peak value at 6 h after challenge, (15.2 +/- 5.0)% in group D(1), and (2.0 +/- 1.0)% in group E, P < 0.01. The expression of CD(80) and CD(86) increased rapidly 2 h after challenge in group D(1) [(10.6 +/- 3.9)% and (7.5 +/- 3.8)%, respectively] compared with those in group E [(2.1 +/- 0.7)%, P < 0.01 and (1.7 +/- 0.7)%, P < 0.05, respectively]. The maximal inhibition effect of dexamethasone on the expression of MHC-II was at 10 h, (7.8 +/- 2.4)% in group D(1), and (2.8 +/- 1.5)% in group D(2), P < 0.01. The inhibition effect of dexamethasone on the expression of CD(80) and CD(86) was maximal at 24 h, (5.8 +/- 2.7)% and (5.5 +/- 1.5)% respectively in group D(1), and (2.8 +/- 1.1)% and (2.9 +/- 1.6)% respectively in group D(2), P < 0.05. CONCLUSIONS: The antigen up-taking and presenting function of DCs were significantly enhanced in asthmatics at exacerbation and in a rat asthmatic model. Pretreatment with dexamethasone inhibited the function of DCs significantly in the animal model. The results suggest that DCs play important roles in antigen presentation in the pathogenesis of asthma, and the inhibition of glucocorticiod on DCs might be a critical mechanism in treatment of bronchial asthma.

Beta2-adrenergic receptor gene polymorphism in Chinese Northern asthmatics.

OBJECTIVE: To investigate whether the polymorphisms of beta2-adrenergic receptor (beta2-AR) at position 16, 27, 164 are associated with asthma in Northern Chinese subjects. METHODS: Genomic DNA was collected from unrelated Northern Chinese population of Han ethnicity, including 125 unrelated asthmatic individuals and 96 healthy controls. Beta2-AR genes at position 16, 27, 164 were amplified by using restriction fragment length polymorphism (RFLP) and allelic specific polymerase chain reaction methods. All asthmatics had their serum IgE (total and specific) antibody or skin-prick test measured, bronchial reactivity to methacholine (Mch) and bronchial reversibility by beta2-agonist evaluated. RESULTS: (1) The frequency of Gly 16 homozygous was significantly higher in the asthmatic group than that in healthy controls (22.4% vs. 8.3%, P < 0.05), OR was 2.9 with 95% CI 1.26-6.78. The proportion of Gly 16 allele was also higher in asthmatics than that in control (0.46 vs. 0.36, P < 0.05); Gly16 homozygous was not independently associated with asthma pathogenesis (P = 0.21, OR 0.42 with 95% CI 0.11-1.61). (2) Of 51 night attack patients, 18 carrying Gly16 homozygosity, if compared with 10 of 74 nonnocturnal asthmatics carrying this genotype, there was significant difference between these two groups (35.3% vs. 13.5%, P < 0.01). (3) The average dose of PD20-Mch was significantly lower in patients carrying Gln 27 homozygous than those carrying homozygous Glu 27 and Gln/Glu 27 heterozygous (0.2 +/- 0.3, 1.6 +/- 0.8, and 2.1 +/- 3.0 micromol/L, P < 0.05). CONCLUSION: Beta2AR gene polymorphisms might confer susceptibility to asthma in Chinese Northern patients. Beta2-AR gene, coordinated with other candidate loci, plays a role in the development of asthma.

[The evolvement of Th1/Th2 imbalance accommodates to the progress of airway inflammation in asthmatic subjects and rat model].

OBJECTIVE: To observe the production of IFN-gamma and IL-4 released in peripheral T lymphocytes in asthmatic patients and rat models and to clarify the dynamic changes of proliferation and differentiation of T lymphocytes during the progress of airway inflammation. METHODS: Twenty patients with moderate to severe acute exacerbation and 15 patients with remission of asthma were included in this study. Ten normal volunteers were also enrolled as control group. T lymphocytes were obtained from their peripheral blood and the percentage of IFN-gamma(+)CD4(++), IFN-gamma + CD8(++) and IL-4(+)CD4(++) cells were determined by flow cytometric method. Asthmatic rat models were established by sensitizing and challenging by ovalbumin (OVA), and the blood samples were taken and the percentage of IFN-gamma(+)CD4(+), IFN-gamma + CD8(+) and IL-4 + CD4(+) cells were assessed 2, 4, 6, 8, 10, 12, 24, 48, and 72 h after OVA challenge. RESULTS: (1) The percentages of IFN-gamma(+)CD4(+), IFN-gamma(+)CD8(+) and IL-4(+)CD4(+) cells in the peripheral blood were significantly higher in the patients with acute exacerbation [(30% +/- 10%), (42% +/- 15%), and (4.2% +/- 1.6%) respectively] than those in the patients with remission asthma [(20% +/- 8%), (30% +/- 10%), and (2.0% +/- 0.8%) respectively, and P < 0.001;] and those in the normal subjects [(18% +/- 8%), and (24% +/- 9%), P < 0.01, and (1.9% +/- 0.9%), P < 0.001; respectively]. But all the parameters were not different significantly between the patients with remission asthma and the normal subjects. (2) In the asthmatic rat models, the IFN-gamma(+)CD4(+) cells in peripheral blood increased 2 h after OVA challenge (5.7% +/- 1.6%), and peaked 10 h after (9.9% +/- 4.4%), and decreased afterwards. The IFN-gamma(+)CD8(+) cells in peripheral blood increased 2 h after (11.5% +/- 5.1%) OVA challenge, and peaked 10 h after (38.7% +/- 6.3%), and then decreased afterwards. The IL-4(+)CD4(+) cells increased 2 h (1.7% +/- 1.0%) after OVA challenge, and peaked 36 h after (4.0% +/- 1.6%), and then decreased. CONCLUSION: Both Th1 (IFN-gamma) and Th2 (IL-4) cytokines generated by peripheral blood mononuclear cells in asthma subjects and asthmatic rat models play a role in the development of airway inflammation in bronchial asthma dynamically. Th1 cytokines dominate at the early stage of airway inflammation, whereas Th2 cytokines achieves the major effects at later stage of the inflammation. It suggests that using different strategy in treatment of asthmatics in early stage and late stage may be necessary to control asthma.

[The evolvement of Th1/Th2 imbalance in the process of airway inflammation in a rat asthmatic model].

OBJECTIVE: T lymphocytes play an important role in the development of asthmatic airway inflammation. By observing the production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) released by peripheral T lymphocytes in asthmatic rat models, this study was designed to clarify the changes of T lymphocytes during the course of airway inflammation. The influence of dexamethasone pre-treatment on the production of IFN-gamma and IL-4 by T lymphocytes was also investigated in the study. METHODS: Twenty-four rats were divided into the study group (group A) and the control group (group B). In the study group, rats were subdivided into two groups with 8 rats in each group. one group (group A(2)) received pre-treatment with intraperitoneal injection with dexamethasone (10 mg/kg), and another group (group A(1)) did not receive dexamethasone pre-treatment, The control group was matched with the study group with regard to both age and weight. Rats were sensitized and challenged by ovalbumin (OVA), and blood samples were obtained and the percentage of IFN-gamma(+)CD(4)(+), IFN-gamma(+)CD(8)(+) and IL-4(+)CD(4)(+) cells were assessed by intracellular cytokine staining and flow cytometric analysis at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h, 72 h, 6 d, 9 d, 12 d, and 15 d after OVA challenge. RESULTS: In group A(1), the peripheral IFN-gamma(+)CD(4)(+) cells, IFN-gamma(+)CD(8)(+) cells, and IL-4 CD(4)(+) cells increased 2 h after OVA challenge [(5.7 +/- 1.6)%, [(11.5 +/- 5.1)%, (1.66 +/- 0.95)%] compared with group A(2) [(2. 6 +/- 1.3)%, (6.1 +/- 1.8)%, (0.77 +/- 0.37)%, P < 0.05 respectively] and group B [(1.8 +/- 0.7)%, (3.9 +/- 1.5)%, (0.65 +/- 0.12)%, P < 0.05 respectively]. The peripheral IFN-gamma(+)CD(4)(+) cells and IFN-gamma(+) CD(8)(+) cells in group A(1) reached their maximal level at 10 h [(9.9 +/- 4.4)%, (38.7 +/- 6.3)%] compared with group A(2) [(4.9 +/- 1.7)%, (15.7 +/- 8.7)%, P < 0.05 respectively and group B [(1.5 +/- 0.5)%, (6.4 +/- 1.5)%, P < 0.05 respectively], and then decreased afterward. The IL-4(+) CD(4)(+) cells in group A(1) at 10 h after challenge was (2.16 +/- 0.75)%. There was no statistical difference compared with group A(2) [(1.39 +/- 0.77)%, P > 0.05], but it was higher than group B [(0.68 +/- 0.15)%, P < 0.05]. The highest level of IL-4(+) CD(4)(+) cells of group A(1) was observed at 36 h [(4.0 +/- 1.6)%], and there was a statistical difference compared with group A(2) [(1.5 +/- 1.0)%, P < 0.05] and group B [(0.7 +/- 0.3)%, P < 0.05]. CONCLUSIONS: Both IFN-gamma and IL-4 generated by peripheral blood T cells in the rat asthmatic models played a role in the development of airway inflammation. INF-gamma dominated at early stage of the airway inflammation, whereas IL-4 took the major effect at the late stage of inflammation. The production of IFN-gamma and IL-4 by T lymphocytes in the asthmatic model was inhibited significantly by dexamethasone.

[A study on the active efflux mechanism in Staphylococcus aureus resistant to quinolone].

OBJECTIVE: To explore the effect, the characteristic, and potential approaches to the active efflux mechanism in straphylococcus aureus (S. a) resistant to quinolones. METHODS: S. a standard strain ATCC25923 and clinical isolates susceptible to quinolone were inoculated onto MH agar containing ofloxacin at a concentration of 4 x MIC (in the presence or absence of 20 micro g of reserpine/ml). Following incubation at 35 degrees C for 48 h, the inhibiting effect of reserpine on the occurrence of induced resistance was observed and the MIC of the induced resistant strain to ethidium bromide (EB), ofloxacin and ciprofloxacin (in the presence or absence of 20 micro g of reserpine/ml) was determined. The influence of reserpine to the MIC of induced resistant strains was also determined. The accumulation and loss of EB was determined based on the fact that EB's fluorescence can be strengthened when combined with DNA. Reserpine inhibition test was used to study the active efflux in clinical S. a resistant to quinolone. RESULTS: The active efflux mechanism in S. a resistant to second generation quinolones was confirmed by the reserpine's influence on the level of ethidium bromide (EB) in the cytoplasm of S. a. Reserpine reduced the 50 percent resistant rate to quinolone in induced resistant S. a, and decreased the MIC of induced resistant strains. Reserpine inhibited the active efflux of EB from the cytoplasm of S. a. CONCLUSIONS: Active efflux is an important mechanism in S. a resistant to quinolone. Reserpine can inhibit its active efflux mechanism, and has synergistic effect with quinolone, which hold therapeutic potential for S. a resistant to quinolones.

[Association of polymorphism of human beta 2-adrenergic receptor gene and bronchial asthma].

OBJECTIVE: To investigate whether beta 2-adrenergic receptor gene (beta 2AR) polymorphism at position 16, 27, 164 is in association with asthma susceptibility or asthmatic phenotype (including nocturnal asthma, serum IgE level, bronchial responsiveness, the status of asthmatics). METHODS: By using PCR-RFLP and allelic-specific PCR (ASP), the polymorphism of beta 2AR gene at position 16, 27, 164 in 125 Han origin asthmatics and 96 normal healthy controls with the same ethnic nearby Beijing region were genotyped. All patients had their serum total IgE (TIgE) measured by RAST, pulmonary ventilatory function assessed by FEV1% and FEV1/FVC, bronchial responsiveness challenged by methacholine (if FEV1% > 70%), and brocho-reversibity by inhaling beta 2-agonist. RESULTS: There was higher prevalence of Gly16 homozygous of beta 2AR in asthmatics than that in normal healthy controls (22.4% vs 8.3%, P < 0.05), with odd ratio (OR) 2.918 (95% CI: 1.256-6.781); Also there was higher frequency of Gly16 homozygous of beta 2AR in nocturnal asthmatics than that in nonnocturnal asthmatics (35.3% vs 13.5%, P < 0.01), but Gly16 homozygous of beta 2AR was low an independent risk factor for the pathogenesis of asthma. The dose of methacholine was low in asthmatics carrying Gln27 homozygous beta 2AR than Glu27 homozygous beta 2AR and Gln/Glu27 heterozygous beta 2AR in brocho-challenge test [(0.205 +/- 0.275) vs (2.11 +/- 3.00) vs (1.575 +/- 0.828) mumol, P < 0.05]. CONCLUSIONS: Gly16 homozygous beta 2AR was associated with asthma susceptibility in Chinese patients with Han ethnic nearby Beijing region, and Gly16 homozygous beta 2AR was associated significantly with nocturnal asthma. Glu27 homozygous beta 2AR was related to hyper-bronchial reactivity of asthmatics.