Limits...
Ag85B DNA vaccine suppresses airway inflammation in a murine model of asthma.

Wu J, Xu J, Cai C, Gao X, Li L, Zhong N - Respir. Res. (2009)

Bottom Line: IL-4 production was significantly decreased in the BAL fluid (32.0 +/- 7.6 pg/ml vs. 130.8 +/- 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 +/- 1.6 pg/ml vs. 10.1 +/- 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-gamma production was increased in the BAL fluid (137.9 +/- 25.6 pg/ml vs. 68.4 +/- 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 +/- 5.4 pg/ml vs. 11.3 +/- 3.2 pg/ml, p < 0.05).In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration.This protective effect was associated with decreased IL-4 and increased IFN-gamma production in the BAL fluid and in the supernatant of cultured splenocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiratory Disease, Peking University First Hospital, Beijing, PR China. wjxst@hotmail.com

ABSTRACT

Background: In allergic asthma, Th2 lymphocytes are believed to play important roles in orchestrating airway eosinophilia and inflammation. Resetting the Th1/Th2 imbalance may have a therapeutic role in asthma. The mycobacterium tuberculosis 30-kilodalton major secretory protein (antigen 85B, Ag85B) can protect animals from M. tuberculosis infection by inducing a Th1-dominant response.

Methods: In this study, the Ag85B gene was cloned into pMG plasmids to yield the pMG-Ag85B plasmid. The expression of Ag85B gene in murine bronchial epithelia cells was detected by Western blotting and immunohistochemical staining after intranasal immunization with reconstructed pMG-Ag85B plasmids. The protective effect of pMG-Ag85B plasmids immunization in airway inflammation was evaluated by histological examination and bronchoalveolar lavage (BAL). IL-4 and IFN-gamma levels in the BAL and supernatant from splenocyte culture were determined using ELISA kits.

Results: The Ag85B gene was successfully expressed in murine bronchial epithelia cells by intranasal immunization with reconstructed pMG-Ag85B plasmids. Using a murine model of asthma induced by ovalbumin (OVA), pMG-Ag85B immunization significantly inhibited cellular infiltration across the airway epithelium with a 37% decrease in the total number of cells (9.6 +/- 2.6 x 10(5)/ml vs. 15.2 +/- 3.0 x 10(5)/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 +/- 0.2 x 10(5)/ml vs. 5.4 +/- 1.1 x 10(5)/ml, p < 0.01) compared with the OVA-sensitized control group. There was no difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group. IL-4 production was significantly decreased in the BAL fluid (32.0 +/- 7.6 pg/ml vs. 130.8 +/- 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 +/- 1.6 pg/ml vs. 10.1 +/- 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-gamma production was increased in the BAL fluid (137.9 +/- 25.6 pg/ml vs. 68.4 +/- 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 +/- 5.4 pg/ml vs. 11.3 +/- 3.2 pg/ml, p < 0.05).

Conclusion: In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration. This protective effect was associated with decreased IL-4 and increased IFN-gamma production in the BAL fluid and in the supernatant of cultured splenocytes.

Show MeSH

Related in: MedlinePlus

Ag85B expression in murine bronchial epithelial cells. A. Murine cells were transfected with pMG plasmids (Lane 1) or pMG-Ag85B plasmids (Lane 2). Ag85B mRNA (992 bp) expression was tested 36 hours after transfection by RT-PCR. B. As described in A, Western blotting was used to determine Ag85B protein expression in the supernatant of pMG-Ag85B transfected murine bronchial epithelial cells (Lane 1) and pMG transfected cells (Lane 2). The positive control was 100 ng purified Ag85B protein (Lane 3). C, D: Mice were intranasally immunized with 100 μg pMG (C) or pMG-Ag85B plasmids (D), with a booster dose 7 days after the initial immunization. Immunohistochemistry staining shows Ag85B protein expression in the lung 48 hours after the booster dose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2713210&req=5

Figure 1: Ag85B expression in murine bronchial epithelial cells. A. Murine cells were transfected with pMG plasmids (Lane 1) or pMG-Ag85B plasmids (Lane 2). Ag85B mRNA (992 bp) expression was tested 36 hours after transfection by RT-PCR. B. As described in A, Western blotting was used to determine Ag85B protein expression in the supernatant of pMG-Ag85B transfected murine bronchial epithelial cells (Lane 1) and pMG transfected cells (Lane 2). The positive control was 100 ng purified Ag85B protein (Lane 3). C, D: Mice were intranasally immunized with 100 μg pMG (C) or pMG-Ag85B plasmids (D), with a booster dose 7 days after the initial immunization. Immunohistochemistry staining shows Ag85B protein expression in the lung 48 hours after the booster dose.

Mentions: The Ag85B expression vector, pMG-Ag85B was constructed by inserting a 992-bp Ag85B gene into the XBal I and BamHI sites of the pMG vector. Transfection was confirmed by restriction enzyme digestion, PCR and sequential analysis (data not shown). Ag85B mRNA was detected in murine bronchial epithelial cells 36 hours after transfection with endotoxin-free pMG-Ag85B plasmids, but not in pMG plasmid-transfected cells (Fig. 1A). Ag85B protein was also detected in the supernatant of the pMG-Ag85B-transfected cells using Western blotting (Fig. 1B). We then examined Ag85B gene expression in vivo. Ag85B mRNA was detected in lung tissue 36 hours after the second intranasal immunization with pMG-Ag85B. Immunohistochemical staining revealed that the Ag85B gene was mainly expressed in bronchial epithelial cells, bronchiolar submucosa and alveolar epithelial cells (Fig. 1D).


Ag85B DNA vaccine suppresses airway inflammation in a murine model of asthma.

Wu J, Xu J, Cai C, Gao X, Li L, Zhong N - Respir. Res. (2009)

Ag85B expression in murine bronchial epithelial cells. A. Murine cells were transfected with pMG plasmids (Lane 1) or pMG-Ag85B plasmids (Lane 2). Ag85B mRNA (992 bp) expression was tested 36 hours after transfection by RT-PCR. B. As described in A, Western blotting was used to determine Ag85B protein expression in the supernatant of pMG-Ag85B transfected murine bronchial epithelial cells (Lane 1) and pMG transfected cells (Lane 2). The positive control was 100 ng purified Ag85B protein (Lane 3). C, D: Mice were intranasally immunized with 100 μg pMG (C) or pMG-Ag85B plasmids (D), with a booster dose 7 days after the initial immunization. Immunohistochemistry staining shows Ag85B protein expression in the lung 48 hours after the booster dose.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2713210&req=5

Figure 1: Ag85B expression in murine bronchial epithelial cells. A. Murine cells were transfected with pMG plasmids (Lane 1) or pMG-Ag85B plasmids (Lane 2). Ag85B mRNA (992 bp) expression was tested 36 hours after transfection by RT-PCR. B. As described in A, Western blotting was used to determine Ag85B protein expression in the supernatant of pMG-Ag85B transfected murine bronchial epithelial cells (Lane 1) and pMG transfected cells (Lane 2). The positive control was 100 ng purified Ag85B protein (Lane 3). C, D: Mice were intranasally immunized with 100 μg pMG (C) or pMG-Ag85B plasmids (D), with a booster dose 7 days after the initial immunization. Immunohistochemistry staining shows Ag85B protein expression in the lung 48 hours after the booster dose.
Mentions: The Ag85B expression vector, pMG-Ag85B was constructed by inserting a 992-bp Ag85B gene into the XBal I and BamHI sites of the pMG vector. Transfection was confirmed by restriction enzyme digestion, PCR and sequential analysis (data not shown). Ag85B mRNA was detected in murine bronchial epithelial cells 36 hours after transfection with endotoxin-free pMG-Ag85B plasmids, but not in pMG plasmid-transfected cells (Fig. 1A). Ag85B protein was also detected in the supernatant of the pMG-Ag85B-transfected cells using Western blotting (Fig. 1B). We then examined Ag85B gene expression in vivo. Ag85B mRNA was detected in lung tissue 36 hours after the second intranasal immunization with pMG-Ag85B. Immunohistochemical staining revealed that the Ag85B gene was mainly expressed in bronchial epithelial cells, bronchiolar submucosa and alveolar epithelial cells (Fig. 1D).

Bottom Line: IL-4 production was significantly decreased in the BAL fluid (32.0 +/- 7.6 pg/ml vs. 130.8 +/- 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 +/- 1.6 pg/ml vs. 10.1 +/- 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-gamma production was increased in the BAL fluid (137.9 +/- 25.6 pg/ml vs. 68.4 +/- 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 +/- 5.4 pg/ml vs. 11.3 +/- 3.2 pg/ml, p < 0.05).In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration.This protective effect was associated with decreased IL-4 and increased IFN-gamma production in the BAL fluid and in the supernatant of cultured splenocytes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Respiratory Disease, Peking University First Hospital, Beijing, PR China. wjxst@hotmail.com

ABSTRACT

Background: In allergic asthma, Th2 lymphocytes are believed to play important roles in orchestrating airway eosinophilia and inflammation. Resetting the Th1/Th2 imbalance may have a therapeutic role in asthma. The mycobacterium tuberculosis 30-kilodalton major secretory protein (antigen 85B, Ag85B) can protect animals from M. tuberculosis infection by inducing a Th1-dominant response.

Methods: In this study, the Ag85B gene was cloned into pMG plasmids to yield the pMG-Ag85B plasmid. The expression of Ag85B gene in murine bronchial epithelia cells was detected by Western blotting and immunohistochemical staining after intranasal immunization with reconstructed pMG-Ag85B plasmids. The protective effect of pMG-Ag85B plasmids immunization in airway inflammation was evaluated by histological examination and bronchoalveolar lavage (BAL). IL-4 and IFN-gamma levels in the BAL and supernatant from splenocyte culture were determined using ELISA kits.

Results: The Ag85B gene was successfully expressed in murine bronchial epithelia cells by intranasal immunization with reconstructed pMG-Ag85B plasmids. Using a murine model of asthma induced by ovalbumin (OVA), pMG-Ag85B immunization significantly inhibited cellular infiltration across the airway epithelium with a 37% decrease in the total number of cells (9.6 +/- 2.6 x 10(5)/ml vs. 15.2 +/- 3.0 x 10(5)/ml, p < 0.05) and a 74% decrease in the number of eosinophils (1.4 +/- 0.2 x 10(5)/ml vs. 5.4 +/- 1.1 x 10(5)/ml, p < 0.01) compared with the OVA-sensitized control group. There was no difference in the number of neutrophils in BAL fluid between the pMG-Ag85B group, the OVA-sensitized control group and the empty pMG group. IL-4 production was significantly decreased in the BAL fluid (32.0 +/- 7.6 pg/ml vs. 130.8 +/- 32.6 pg/ml, p < 0.01) and in the splenocyte supernatant (5.1 +/- 1.6 pg/ml vs. 10.1 +/- 2.3 pg/ml, p < 0.05) in the pMG-Ag85B group compared with the OVA-sensitized control group, while IFN-gamma production was increased in the BAL fluid (137.9 +/- 25.6 pg/ml vs. 68.4 +/- 15.3 pg/ml, p < 0.05) and in the splenocyte supernatant (20.1 +/- 5.4 pg/ml vs. 11.3 +/- 3.2 pg/ml, p < 0.05).

Conclusion: In a murine model of asthma induced by OVA, intranasal immunization with pMG-Ag85B significantly reduced allergic airway inflammation with less eosinophil infiltration. This protective effect was associated with decreased IL-4 and increased IFN-gamma production in the BAL fluid and in the supernatant of cultured splenocytes.

Show MeSH
Related in: MedlinePlus