Pneumococcal Adherence to the Buccal Epithelial Cells of Cigarette Smokers: Results and Disscusion

smokersBackground adherence values were not significantly different in smokers (9.1 ±9.1), exsmokers (4.8±6.4), and nonsmokers (8.5±21.1). However, after incubation with S pneumoniae type 25, the pneumococcal adherence to ВЕС of smokers (12.3 ±6.9) was significantly greater (p<0.001) than that of nonsmokers (0.7±0.4), as shown in Figure 1. There was poor correlation between background adherence and pneumococcal adherence in nonsmokers (r = 0.5) and smokers (r=0.68). Smokers’ and nonsmokers’ ВЕС were indistinguishable in terms of maturation as determined microscopically. The correlation coefficient for the blinded adherence estimates between the two readers was.95,.90 and.85 for S, ES, and NS, respectively.

The reproducibility of the elevated pneumococcal adherence estimates is shown in Table 1. The mean pneumococcal adherence for the repeat adherence measurements is not significantly different from the original value, and the reproducibility for each individual subject also appears to be good.

There is no predictable relationship between either smokers age (r = 0.01) or pack years of smoking (r = 0.03) to pneumococcal adherence. Three years after cessation of smoking, all subjects have adherence values (0.5 ±0.4) which fall in the range of the nonsmokers, as shown in Figure 2. Prior to the three years, six of 13 had elevated values but the degree of elevation in these six (4.5 ±2.1) is significantly (p<0.05) less than that of current smokers (12.3 ±6.9). All exsmokers with elevated adherence had serum thiocyanate levels which were within the normal range. No pneumococci were isolated from any smoking or nonsmoking subjects.

The effect of smokers’ saliva on pneumococcal adherence to ВЕС of nonsmokers is shown in Table 2. Pneumococcal adherence to ВЕС of nonsmokers is similar when the cells are incubated with either the nonsmokers own saliva (1.1 ±0.9) or with saliva of another nonsmoker (0.9 ±0.2) but is significantly increased (p<0.001) to 8.2 ±4.4 by incubation of these cells in smokers saliva. The pneumococcal adherence to ВЕС of smokers, on the other hand, is not significantly changed by incubating their cells in their own saliva or in nonsmokers saliva (21.0± 13.8 to 16.2 ±6.2).

The salivary amylase in smokers was elevated compared to nonsmokers (135,800 vs 69,300 m|x/ml) and the SGOT of smokers was lower (10 vs 27 mp7ml). mucosal surface


Bacterial adherence to cells is tissue- as well as host-specific and it is an essentially irreversible molecular interaction between cell surfaces. It is believed to determine the ability of bacteria to colonize and to perhaps infect and invade many mucosal surfaces.

Several studies have shown a relationship between increased bacterial adherence to buccal or pharyngeal mucosal cells and the development of pneumonia. Johanson et al have shown that increased in-vitro adherence of Pseudomonas aeruginosa, Klebsiella pneumoniae, E coli, and Proteus mirabilis to buccal epithelial cells is associated with in-vivo colonization, which in turn has been correlated with increased risk of nosocomial pneumonia. Influenza infection has been shown to increase the adherence of Staphylococcus aureus, S pneumoniae, and Hemophilus influenza to pharyngeal epithelial cells. These organisms cause pneumonias which complicate influenzal respiratory illness.

This finding of increased adherence of type 25 S pneumoniae to smokers’ buccal epithelial cells is similar to the increased adherence demonstrated by Fainstein and Musher for type 1 and type 3 S pneumoniae. In both studies, there was no difference in background adherence values between smokers and nonsmokers. Ibis suggests that the increased adherence in smokers is mediated by some specific mechanism rather than a generalized increase in avidity of bacteria for smokers’ buccal epithelial cells. Pneumococcal adherence in smokers remained constantly elevated over a 14-week interval. Thus, day-to-day variations in cigarette consumption, oral hygiene, or other acute effects of cigarette smoking are probably not important factors in detemining adherence of S pneumoniae to buccal epithelial cells.

Adherence does not vary with age or duration of smoking. That is, it appears that pneumococcal adherence in smokers is fully expressed after a few years of smoking and does not intensify thereafter. Therefore, if adverse clinical consequences accrue from an increased adherence of Pneumococcus to oral epithelial cells, young smokers are at risk early in their smoking history. In this regard, the increased incidence of pneumonia in adolescent and young adult cigarette smokers is of interest.

The persistent adherence in exsmokers might be due to continued smoking, but the observation that these subjects had serum thiocyanate levels (half-life of 14 days) within the nonsmokers’ range gives us confidence that the history of smoking cessation given by these nonpaid volunteers was correct.

Using routine clinical laboratory techniques, none of our subjects had positive pharyngeal cultures for S pneumoniae. Similarly, the enhanced in-vitro adherence of S pneumoniae following experimentally-induced influenza infection was not associated with pneumococcal colonization. This inability to culture S pneumoniae in asymptomatic cigarette smokers may be in part due to technical difficulties in identifying small numbers of colonized organisms. pneumoniae In any event, these observations do not refute the relation of adherence to colonization, but rather emphasize the multifactorial nature of the interaction of bacteria with their environment. Local defense mechanisms such as bacterial antagonism, surface immunoglobulins, salivary flow, surface fluids chemistry, and cell desquamation exist in the upper airway in-vivo and provide protection against surface colonization. If these are unfavorably altered in the host, the tendency for increased pneumococcal adherence may manifest itself in colonization and subsequent disease may then take place.

The mechanism by which smoking raises adherence and by which the increased adherence was maintained after smoking cessation in some subjects is not known. Hie elevation of nonsmokers adherence after incubation of their epithelial cells in a cell-free filtrate of smoker s saliva suggests that a constituent of smokers saliva alters the surface properties of buccal epithelial cells in a manner that enhances their avidity for Pneumococcus, perhaps by making binding sites on buccal epithelial cells available to type 25 S pneumoniae. Although this study was not designed to determine how this takes place, one possibility is the removal of an antiadherence factor from the epithelial cell. Such a mechanism exists in the urinary tract where adherence of Gram-negative organisms to bladder mucosal cells rises when surface glycosaminogly-cans are removed, and in the oral cavity where removal of fibronectin by trypsin enhances the adherence of Pseudomonas aeruginosa to the buccal cells. Whole saliva contains many enzymes including amylase and trypsin, which may act upon cell surfaces in an analogous fashion to increase pneumococcal adherence. Salivary amylase was elevated in the three of the smoking subjects tested.

It has been shown that in-vitro adherence of S pneumoniae to buccal epithelial cells of cigarette smokers is increased. This increased adherence remains constant over a period of several months in active cigarette smokers and may persist for up to three years after smoking cessation. Since increased adherence of other bacteria to surface cells is an established pathogenetic step in infection in both the lung and other organs, this observation may explain the increased risk of lower respiratory infection that exists in cigarette smokers.’’ This increased adherence is mediated by a factor found in the cell-free filtrate of smokers saliva.


Figure 1. Pneumococcal adherence of 15 nonsmokers and 15 smokers.


Figure 2. Pneumococcal adherence after smoking cessation in 21 exsmokers. Stippled area is the range of normal for smokers.

Table 1—Repeat Measurements of Pneumococcal Adherence in Smokers and Nonsmokers

OCTSmokers JAN
25.1 16.1
5.9 9.2
11.7 16.9
16.0 18.0
12.0 14.0
MEAN ± SD 14.1 ±7.1 14.8±3.5
0.4 0.1
0.2 0.6
1.2 1.7
0.4 0.6
0.4 0.5
0.6 0.3
MEAN ± SD 0.5 ±0.4 0.6±0.6

Table 2—Effect of Incubating Nonsmokers Cells with Own, Other ‘Nonsmokers у and Smokers Saliva

PneumiOwn xx>ccal Adherence (Bact< Other Nonsmokers jria/Cell)Smokers
0.4 0.9 13.3
2.7 0.5 8.6
1.0 0.8 5.5
0.4 1.0 13.5
0.6 0.9 4.4
1.5 1.2 3.8
MEAN±SD 1.1±0.9 0.9±0.2 8.2±4.4

Canadian Health&Care Mall: Outcomes of Niflumic Acid and AG-1478 Reduce Cigarette Smoke-Induced Mucin Synthesis

Treatment With Niflumic AcidIn Vivo Studies

Cigarette Smoke Up-regulates CLCA1 and EGFR mRNA and Protein Expressions and MUC5AC mRNA Expression in Rats: In the nonsmoking group, CLCA1, EGFR, and MUC5AC mRNA expressions were constitutively expressed in rat lung and trachea. The smoke-exposed group showed significant up-regulation of all the three mRNA expressions from day 21 onwards (p < 0.001) [Fig 1]. This effect was stronger in the trachea than in the lung. Similarly, the protein expressions of CLCA1 and both resting EGFR and activated pEGFR were significantly increased on day 28 (p < 0.001) [Fig 2].

Niflumic Acid, AG-1478, or Both Inhibit the Cigarette Smoke-Induced MUC5AC Gene Expression: Niflumic acid is a blocker of the CLCAs, and AG-1478 is an inhibitor of EGFR tyrosine kinase. In the smoke-exposed treatment group, niflumic acid, AG-1478, or both significantly inhibited the upreglation of MUC5AC mRNA expression (p < 0.001) without affecting the upregulation of CLCA1 and EGFR mRNAs. Combining both drugs did not show a significant synergistic effect on MUC5AC mRNA expression (Fig 3). On the protein level, neither niflumic acid, AG-1478, nor both could inhibit the upregulation of CLCA1 protein by cigarette smoke. However, EGFR upregulation was partially inhibited by AG-1478, while pEGFR expression was abolished by it (p < 0.001). Niflumic acid had no effect on the protein expressions of EGFR and pEGFR (Fig 2). Continue reading “Canadian Health&Care Mall: Outcomes of Niflumic Acid and AG-1478 Reduce Cigarette Smoke-Induced Mucin Synthesis”

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