[The regulatory effect of the supernatant of human airway epithelial cells transfected with DNA vaccine of 30-kilodalton major secretory protein of mycobacterium tuberculosis on the Th1/Th2 balance in mite allergic asthmatics].
OBJECTIVE
To investigate the regulatory effect of the supernatant of human airway epithelial cells transfected with DNA vaccine of 30-kilodalton major secretory protein of Mycobacterium tuberculosis (Ag85B) on the Th1/Th2 balance in peripheral blood mononuclear cells (PBMCs) from mite allergic asthmatics.


METHODS
Eukaryocytes expressing vectors of pMG-Ag85B were constructed. After sequential analysis, the DNA vaccine was transfected into human airway epithelial cells (16HBE) with lipofectin. By the use of reverse transcription-polymerase chain reaction (RT-PCR), expression of Ag85B mRNA was detected in 16HBE cells. PBMCs were separated from 18 asthmatic patients who were allergic to house dust mites and from 13 non-allergic normal subjects. PBMCs were cocultured with mites, or the supernatant of 16HBE cells transfected with pMG-Ag85B or the combination of both. After 96 hours incubation, levels of IL-5 and IFN-gamma released in the culture medium were determined by enzyme-linked immunoabsorbent assay.


RESULTS
The sequential analysis showed that the gene fragment was homogenous as that of original Ag85B gene. The plasmids of pMG-Ag85B had been successfully transfected into 16HBE cells and there was expression of Ag85B mRNA in 16HBE cells. Without any stimulus, no significant difference in IL-5 or IFN-gamma levels in PBMC culture medium was found between the patients and normal individuals. In the asthmatic group, there was an significant increase of IL-5 level after stimulation with mites as compared with control (17.8 +/- 1.9) pg/ml vs (11.9 +/- 0.9) pg/ml, P < 0.05. Level of IFN-gamma showed no difference after the stimulation by either mites or Ag85B transfected culture supernatant alone, but increased significantly by co-stimulation with mites plus Ag85B supernatant in comparison with the mites alone (111.5 +/- 15.9) pg/ml vs (58.8 +/- 7.8) pg/ml, P < 0.05. There were no difference in IL-5 or IFN-gamma levels among the four groups after above stimulation in normal subjects.


CONCLUSIONS
In the presence of mites, PBMCs from mite allergic asthmatics demonstrated Th2 predominant reaction, and the culture medium from human airway epithelium cells transfected with Ag85B DNA vaccine upregulated Th1 response and the Th1/Th2 balance to some extent in asthmatics. As a subunit of DNA vaccine, Ag85B may play a role in the preventive management of allergic asthma in the future.
D000217:Acyltransferases / Q000235:genetics / Q000276:immunology*; D000328:Adult; D000942:Antigens, Bacterial / Q000235:genetics / Q000276:immunology*; D001249:Asthma / Q000235:genetics / Q000276:immunology*; D001426:Bacterial Proteins / Q000235:genetics / Q000276:immunology*; D002478:Cells, Cultured; D004847:Epithelial Cells / Q000276:immunology*; D005260:Female; D006801:Humans; D007371:Interferon-gamma / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D008297:Male; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000276:immunology*; D020545:Respiratory Mucosa / Q000166:cytology; D006377:T-Lymphocytes, Helper-Inducer / Q000276:immunology*; D014162:Transfection; D019444:Vaccines, DNA
D000942:Antigens, Bacterial; D001426:Bacterial Proteins; D015850:Interleukin-6; D019444:Vaccines, DNA; D007371:Interferon-gamma; D000217:Acyltransferases; C121433:antigen 85B, Mycobacterium tuberculosis