It has been demonstrated that smoking cessation alters the subgingival microbial profile; however, the response of individual bacteria within this ecosystem has not been well studied. The aim of this investigation, therefore, was to longitudinally examine the effect of smoking cessation on the prevalence and levels of selected subgingival bacteria using molecular approaches for bacterial identification and enumeration. Subgingival plaque was collected from 22 smokers at the baseline and 12 months following periodontal nonsurgical management and smoking cessation counseling. The prevalence and abundance of selected organisms were examined using nested PCR and multiplexed bead-based flow cytometry. Eleven subjects successfully quit smoking over 12 months (quitters), while 11 continued to smoke throughout (smokers). Smoking cessation led to a decrease in the prevalence of Porphyromonas endodontalis and Dialister pneumosintes at 12 months and in the levels of Parvimonas micra, Filifactor alocis, and Treponema denticola. Smoking cessation also led to an increase in the levels of Veillonella parvula. Following nonsurgical periodontal therapy and smoking cessation, the subgingival microbiome is recolonized by a greater number of health-associated species and there are a significantly lower prevalence and abundance of putative periodontal pathogens. The results indicate a critical role for smoking cessation counseling in periodontal therapy for smokers in order to effectively alter the subgingival microbiome.It is established that bacterial consortia within the subgingival microbiome play a critical role in the etiology of chronic periodontitis. Although tobacco smoking has been shown to preferentially enrich this microbiome for pathogenic species (26, 29), it is not known if smoking cessation is capable of reversing this pathogenic colonization, since current evidence is based only on cross-sectional comparisons of former and current smokers (10,25). A longitudinal examination of subgingival bacteria following smoking cessation would allow us to determine how the individual bacteria within this ecosystem respond to smoking cessation.We have previously shown, using a cultivation-independent, open-ended approach, that smoking cessation results in a shift in the subgingival microbial profile during recolonization following periodontal therapy (7). In order to identify the individual organisms that contributed to this compositional shift, it is necessary to use a targeted, sensitive, and quantitative approach to measure the levels of these species.Bead-based flow cytometry has recently been used to measure the abundance of target organisms in complex bacterial communities (24,28). This cultivation-independent approach is capable of simultaneously measuring the levels of several organisms, both cultivated and uncultivated. It also has a wide dynamic range; that is, it is capable of quantifying both the less numerous and the numerically dominant members of a microbial community.The goals of the present investigation were to ...

To understand the influence of oxidant stress on the barrier function of airway epithelium, we conducted studies to determine the effects of chemically generated reactive oxygen species on permeability, permselectivity, and active ion transport of ferret trachea. We examined the consequences of oxidant injury using ferret trachea mounted in Ussing-type chambers and bathed with a modified Krebs-Henseleit solution containing mannitol and xanthine. We added xanthine oxidase to the luminal bathing solution, which reacted with the xanthine to generate reactive oxygen species. Tissue electrical conductance and short-circuit current were significantly increased after the addition of xanthine oxidase. Simultaneous measurement of mannitol flux (as a marker of paracellular conductance) and the backflux of chloride (lumen to submucosa) demonstrated a significant oxidant-induced increase in mannitol flux and backflux of chloride. Mannitol flux and the backflux of sodium (submucosa to lumen) also increased after oxidant stress. Comparison of the diffusion of sodium relative to the diffusion of chloride in relation to predicted diffusion in free solution indicated that the paracellular pathway was cation selective after oxidant stress. Active ion transport, as reflected by the short-circuit current, was significantly increased transiently after oxidant stress. Studies with furosemide, amiloride, and diphenylamine-2-carboxylate are suggestive that oxidant stress transiently stimulates the Na-K-ATPase. These studies demonstrated that exposure to reactive oxygen species significantly altered the permeability of the tracheal epithelium as well as active ion transport.

Methotrexate (MTX) was injected intraperitoneally at a dose of 15 mg/kg into adult rats daily for three consecutive days. When compared with saline-injected, ad libitum-fed rats or pair-fed saline-injected rats, MTX decreased unstimulated parotid gland weight, RNA content, and amylase content. RNA content was also decreased in submandibular glands. The parotid and submandibular gland outputs of sodium and chloride, but not that of potassium, were decreased following MTX injection. Ductal perfusion demonstrated decreased net efflux of sodium and chloride from the excretory duct of the submandibular gland. The effects of MTX on unstimulated parotid gland weight, RNA content, and amylase content are consistent with its ability to inhibit protein synthesis through depletion of folate co-factors. However, the mechanism by which the alterations in ion transport occurred is not clear at this time.

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