| Pseudomonas aerueinosa is one of the dominant pathogens in hospital acquired infections, and the dominant bronchial pathogen in the majority of cystic fibrosis (CF) . Although P. Aeruginosa is only conditionally pathogenic in healthy human hosts, it is an important cause of chronic progressive lung disease in CF sufferers. The reason which the colonization persists and causes inflammatory damage in the CF patients remains unclear. One mechanism by which P. Aeruginosa could contribute to the pathophysiology of CF is to nonspecifically stimulate macrophages and T lymphocytes to release TNF and related cytokines. These cytokines are important for host to limit bacteria growth and clear pathogens. P.aeruginosa extoxin A is a virulence factor secreted by almost P.aeruginosa, and causes cytoxicity by inhibition of protein synthesis. Some studies have demonstrated that PEA influences the immune system at non-cytoxic doses. The studies about influence of PEA on immune system were often performed in murine models. There is not civil report about the effect of PEA on cytokines production of human perioheral blood mononuclear cells(PBMC) . We observed the level of TNF- a in bronchoalveolar lavage fluid(BALF) infected with P.aeruginosa, TNF- a production induced by heat killed P.aeruginosa(HKPA) in PBMC, inhibition of PEA on the production ofTNF- a , neutralization of anti-PE serum and try to determine the influence of PEA on host defense mechanism.13 BALF samples were from patients infected with P.aeruginosa and other 6 sample controls were from patients without bacterial infections. Two different assay, trypan blue dye exclusion method and MTT assay, were used to determine the uncytoxic range of PEA. P.aeruginosa ATCC 27853 was used as inducements, and killed by heating at 80癈 for 30 min. Nucleic acid and protein were destroyed. The concentration of bacteria was l10CFU/ml and diluted to0~10CFU/ml before using. PBMC were prepared from blood of 12 healthy volunteers by centrifugation on hypaque-ficoll. PBMC were adjusted to 210 /ml with 10% PCS RPMI-1640. Samples were divided to six groups according to the doses of PEA. 100 1 HKPA and 500 1 PBMC added with 100 1 PEA(concentration according to Result) were incubated at 37癈 in the presence of 5% CO2 and high humidity for 24 hour. Anti-PE serum and PEA were preincubated at 37癈 for 2 hour, then added to incubation. The supernatants were collected for TNF- a measurements. TNF- a were measured by an enzyme-linked immunosorbent assay(ELISA).Result:1. The level of TNF- a of 13 BALF samples was 851 ?191pg/ml, the TNF- a in negative control samples were not detected(16pg/ml) . The data suggested that P.aeruginosa could induced TNF- a production in vivo.2. PEA exhibited cytoxicity on human PBMC at 1000 ng/ml (P<0.01) , cell viability was not influenced apparently at 0-800 ng/ml.3. Stimulation of TNF- a production was 1273 ?190 pg/ml at 1 10CFU/ml bacteria. Production of TNF was respectively 720+126, 387?5, 157?5 pg/ml after incubation with PEA at 50. 100.200, 400 ng/ml. These data showed that PEA ( 100 ng/ml) inhibited the production of TNF- a (0.01) . The inhibition was correlated with the dose of PEA (r 0.82, P< 0.01) .4. The production of TNF was 1195 + 187 pg/ml in Anti-PE serum group and was not significant defferent versus value in the controll group (P>0.05). Anti-PE serum could completely neutralize the inhibiting effect of PEA. TNF was not detected in 10% PCS RPMI-1640 and PEA incubation with PBMC.Conclusion:This study proved that Pseudomonas aeruginosa could induce production of 7NF- a in HALF ?HKPA could effectively induce TNF- a in human PBMC, end uncytoxic dose of PEA can inhibit TNF- a production. The inhibitory effect was not due to direct cytotoxicity of PEA because PEA at 0-800 ng/ml did not influenced cell viability. TNF is one of pro-inflammatory cytokines, local TNF limits bacterial growth in lung,and play a. defensive role in the course of pneumonia. Therefore the inhibitory effect of PEA on TNF- a production may offer a d...
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