Canadian Health&Care Mall: Research of Pneumococcal Adherence to the Buccal Epithelial Cells of Cigarette Smokers

bacterial colonizationAdherence to certain mucosal surfaces, a prerequisite for bacterial colonization, is believed to be a necessary initiating event in the pathogenesis of human diseases such as endocarditis, cystitis, pyelonephritis, gonococcal urethritis, Gram-negative bacterial pneumonia, shigellosis, and Escherichia coli enteritis. Treat all the mentioned above diseases including cystitis with Canadian Health&Care Mall – http://healthcaremall4you.com/cystitis-features-treatment.html.

Streptococcus pneumoniae is a common pathogen in cigarette smokers. To examine the relationship between cigarette smoking and adherence of S pneumoniae to buccal epithelial cells, the adherence of type 25 S pneumoniae was measured in smokers, nonsmokers and exsmokers. Findings were related to subject age, duration and intensity of smoking, and period of time after smoking cessation. Also investigated was the effect of saliva, a natural chemical and mechanical barrier, on pneumococcal adherence to these cells.

Methods

Study Groups

Three groups of volunteer, nonpaid subjects were studied: 15 nonsmokers (NS), ages 20 to 51, who had never smoked tobacco or other substances; 15 smokers (S), ages 25 to 65, who currently smoke >10 cigarettes a day and have smoked for at least five years; and 21 exsmokers (ES), ages 24 to 58, who stopped all smoking at least six months prior to testing. This study was approved by the Rochester General Hospital Human Investigations Committee. All volunteers gave informed consent.

No subject had respiratory symptoms or poor oral hygiene. Serum thiocyanate levels were measured in exsmokers to confirm abstinence from smoking. A cotton applicator was used to obtain cultures for S pneumoniae from the inner aspect of the cheek and pharynx in ten randomly chosen subjects from each group.

Assay

An in-vitro assay was performed concurrently on all experimental groups to determine the adherence of S pneumoniae type 25 to buccal epithelial cells. The buccal epithelial cells (ВЕС) were obtained by gently scraping the inner aspect of both cheeks with a blunt metal spatula, avoiding lingual and dental contamination. buccal epithelial cellsThe cells were collected in phosphate buffered saline (PBS), washed twice with PBS and harvested by differential centrifugation at 2.4G. Equal volumes of the bacterial (10е bacteria/ml) and buccal epithelial cell (104 cells/ml) suspensions were mixed and incubated in a shaking water both at 37°C for 35 minutes. After incubation, bacteria unattached to ВЕС were removed by four ten-minute differential centrifugations at 2.4G. The ВЕС were then stained with crystal violet to visualize adherent bacteria. Background adherence was measured simultaneously for each subject using ВЕС incubated with buffer solution alone. The number of bacteria adherent to 50 consecutive cells from both background and S pneumoniae incubated groups were counted. The total background count was divided by 50 to yield mean background adherence per cell. The total background count for each subject was subtracted from the total count of the cells that had been incubated with S pneumoniae and the difference divided by 50 to yield mean pneumococcal adherence per cell.

All slides were read by two independent observers in a blinded fashion. Five smoking and six nonsmoking subjects had adherence measurements repeated 14 weeks after the first determination. Buccal cell maturation was graded by accepted cytologic criteria for all subjects.

Effect of Saliva

Unstimulated fasting morning whole saliva was collected by pipette from the anterior lower gingival gutter of randomly chosen subjects in the smoking and nonsmoking groups. The saliva was centrifuged and the supernatant filtered to remove all cells and bacteria. The ВЕС were obtained from six randomly selected nonsmokers and four randomly selected smokers. The ВЕС from the nonsmoking subjects were then mixed with (A) the nonsmokers own saliva, (B) another nonsmoker s saliva, and (C) a smoker s saliva. The ВЕС from the smoking subjects were mixed with (A) the smokers own saliva, and (B) a nonsmoker s saliva. Saliva-BEC mixtures were incubated in a shaking water bath at 37°C for 30 minutes, then centrifuged. The supernatant was removed, and the cells were washed twice and resuspended in PBS. Equal volumes of ВЕС and bacteria were mixed, and adherence was determined.

The samples of saliva from three subjects in the S and NS groups were analyzed for pH, amylase, LDH, and SGOT. Results for all adherence measurements were expressed as a mean ± standard deviation. Statistical evaluation was performed by Wilcoxon rank analysis and by Kendalls coefficient of rank correlation.

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