ABSTRACT
Objective:To investigate characteristics and changes of skin microbiota in atopic dermatitis-like mouse models induced by different concentrations of 2,4-dinitrochlorobenzene (DNCB) .Methods:Totally, 30 male specific-pathogen-free BALB/c mice were randomly divided into 3 groups by using a random number table: negative control group topically treated with 200 μl of mixture of acetone and olive oil at a volume ratio of 3∶1 on the back twice a week for 6 consecutive weeks; high-and low-concentration DNCB groups both topically treated with 200 μl of 1% DNCB on the first and third day at the first week, followed by topical application of 200 μl of 0.5% and 0.1% DNCB, respectively, twice a week for 5 weeks from the second week. Twenty-four hours after the last treatment, the severity of skin lesions was evaluated, and the transepidermal water loss and stratum corneum hydration were measured. After the experiment, the mice were sacrificed, and skin tissues were resected from the back of the mice for histopathological examination. Full-thickness skin tissue samples were obtained from the back of 3 mice in each group. Illumina Miseq PE300 high-throughput sequencing was performed to sequence the V3-V4 variable region of 16S rRNA gene of skin microbiota on the back of the mice, and the composition and structure of the skin microbiota and changes in the relative abundance of different genera were analyzed. One-way analysis of variance was used to analyze differences in indices among the 3 groups, and the Games-Howell method was used for multiple comparisons.Results:The severity scores of skin lesions were significantly higher in the high-and low-concentration DNCB groups (9.83 ± 2.45 points, 2.71 ± 0.56 points, respectively) than in the negative control group (0.51 ± 0.12 points, t=-7.19,-2.85, respectively, both P < 0.05) . Compared with the negative control group, the high-and low-concentration DNCB groups showed significantly increased transepidermal water loss ( t=-7.72,-2.68, respectively, both P < 0.05) , but significantly decreased stratum corneum hydration ( t=6.77, 5.99, respectively, both P < 0.05) ; the transepidermal water loss was significantly higher in the high-concentration DNCB group than in the low-concentration DNCB group ( t=2.76, P < 0.05) , while no significant difference in the stratum corneum hydration was observed between the high-and low-concentration DNCB groups ( P > 0.05) . There was a significant difference in the relative abundance of Corynebacterium among the 3 groups ( F=249.85, P < 0.001) , which was highest in the high-concentration DNCB group. No significant differences in the observed species and Chao1 index of the skin samples were observed among the 3 groups (both P > 0.05) , and the Shannon index was significantly lower in the high-concentration DNCB group than in the low-concentration DNCB group and negative control group ( t=6.96,-6.37, respectively, both P < 0.05) . Conclusion:DNCB could induce atopic dermatitis-like dermatitis in mice, and the severity of skin lesions and degree of barrier function impairment were related to the concentration of DNCB; the species diversity of skin microbiota markedly decreased in the high-concentration DNCB group, indicating that high-concentration DNCB modeling has more advantages in studying microbiological changes associated with atopic dermatitis.
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Objective:To study the effects of agonistic anti gp130 monoclonal antibody B S12 on the differentiation, maturation and function of dendritic cells (DC).Methods:Monocytes isolated from human peripheral blood were cultured with GM CSF plus IL 4, and differentiated into immature DC. The phenotype of DC was analyzed by cytometry after the addition of B S12 antibody to the culture of immature DC. In addition, the abilities for DC to uptake antigen, secrete IL 12, initiate the mixed lymphocyte reaction and chemoattract T cells were tested. The effects of agonistic anti CD40 monoclonal antibody 5C11 on the differentiation, maturation and function of DC were simutaneously compared with those of B S12 antibody.Results:Agonistic anti gp130 monoclonal antibody B S12 had DC to up regulate the expression of CD1a, costimulatory molecules CD80 and CD86 as well as CD83, which is special marker for mature DC, down regulate the expression of CD14. Moreover, B S12 antibody decrease the up take of antigen by DC, enhance the abilities for DC to secrete IL 12, initiate the mixed lymphocyte reaction and chemoattract T cells. The comparison of roles of B S12 and 5C11 antibodies in DC showed that 5C11 was more effective than B S12.Conclusion:The direct stimulation of gp130 on immature DC by B S12 antibody could induce immature DC to differentiate into mature DC.
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objective : To prepare mouse anti-human CD40 antigen functional monoclonal antibody and to further study it's biological effects by triggering the Cd40 molecules on B cells and dendritic cells(DCs) Methods: Using cell fusion, McAb screening immunofluorescence analysis immunoblotting and competition test obtain mouse anti-human CD40 McAb;by the proliferation assay of B cells and DCsand the analysis of expression of differentiation antigens on DCs. Results: On the basis of phenotype analysis, Western blotting and competition test, it is verified that 5Cll recognizes human CD40 antigens specially; 5Cll can augement the proliferation of tonsil B cells in the LCD32cells and IL-4 culturing system; 5Cll can medicate DCs to get proliferation and maturatio. Conclusion: 5Cll+LCD32 +IL4 can make tonsil resting B cells survive and long-term proliferate in vitro, the setup of CD40 System furnish necessary tool for the study of B cells; McAb5Cll also triggered the generation, proliferation and maturation of dendritic cells from peripheral blood monocytes. Thus 5Cll is a McAb withspecial function and important application value.
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Objective:To study the course and mechanism of the immune response to SARS virus. Methods:The recombinant SARS virus S1 subunit was expressed in E. Coli according to the results of bioinformatics analysis. After purification, the recombinant S1 protein was identified by 6 serum samples of recovered SARS patients and 6 serum samples of health donors, which were collected before out-break of SARS. Results:Sequencing analysis confirmed that the recombinant protein has the same sequence of natural SARS virus S1 subunit. The recombinant S1 protein could react with all the samples from recovered SARS patients but not the control samples from healthy donors according to the results of Western blot. Conclusion:The recombinant SARS virus S1 subunit may provide a good tool for the research of immune response to SARS virus and the producing of recombinant vaccine to prevent people from SARS.