Muscarinic acetylcholine receptors regulating cell cycle progression are expressed in human gingival keratinocytes.

We have previously reported the presence in human gingival keratinocytes (GKC) of choline acetyltransferase, the acetylcholine (ACh) synthesizing enzyme, acetylcholinesterase, the ACh degrading enzyme, and alpha 3, alpha 5, alpha 7, beta 2 as well as alpha 9 nicotinic ACh receptor subunits. To expand the knowledge about the role of ACh in oral biology, we investigated the presence of the muscarinic ACh receptor (mAChR) subtypes in GKC. RT-PCR demonstrated the presence of m2, m3, m4, and m5 mRNA transcripts. Synthesis of the respective proteins was verified by immunoblotting with the subtype-specific antibodies that revealed receptor bands at the expected molecular weights. The antibodies mapped mAChR subtypes in the epithelium of human attached gingiva and also visualized them on the cell membrane of cultured GKC. The whole cell radioligand binding assay revealed that GKC have specific binding sites for the muscarinic ligand [3H]quinuclidinyl benzilate, Bmax = 222.9 fmol/106 cells with a Kd of 62.95 pM. The downstream coupling of the mAChRs to regulation of cell cycle progression in GKC was studied using quantitative RT-PCR and immunoblotting assays. Incubation of GKC for 24 h with 10 micro m muscarine increased relative amounts of Ki-67, PCNA and p53 mRNAs and PCNA, cyclin D1, p21 and p53 proteins. These effects were abolished in the presence of 50 micro m atropine. The finding in GKC of mAChRs coupled to regulation of the cell cycle progression demonstrate further the structure/function of the non-neuronal cholinergic system operating in human oral epithelium. The results obtained in this study help clarify the role for keratinocyte ACh axis in the physiologic control of oral gingival homeostasis.

A receptor-mediated mechanism of nicotine toxicity in oral keratinocytes.

Smoking and smokeless tobacco cause morbidity that originates from the epithelium lining of the skin and upper digestive tract. Oral keratinocytes (OKC) express nicotinic acetylcholine receptors (nAChRs) that bind nicotine (Nic). We studied the mechanism of the receptor-mediated toxicity of tobacco products on OKC. Preincubation of normal human OKC with Nic altered the ligand-binding kinetics of their nAChRs, suggesting that the nAChRs underwent structural changes. This hypothesis was confirmed by the finding that exposure of OKC to Nic causes transcriptional and translational changes. Through RT-PCR and immunoblotting, we found a 1.5- to 2.9-fold increase in the mRNA and protein levels of alpha3, alpha5, alpha7, beta2, and beta4 nAChR subunits. Exposure of OKC to Nic also changed the mRNA and protein levels of the cell cycle and cell differentiation markers Ki-67, PCNA, p21, cyclin D1, p53, filaggrin, loricrin, and cytokeratins 1 and 10. The nicotinic antagonist mecamylamine prevented these changes, which indicates that the Nic-induced changes in the expression of both the nAChR and the cell cycle and cell differentiation genes resulted from pharmacologic stimulation of nAChRs with Nic. To establish the relevance of these findings to the pathobiologic effects of tobacco products in vivo, we studied the above parameters in the oral tissue of rats and mice after their exposure for 3 weeks to environmental cigarette smoke or drinking water containing equivalent concentrations of Nic that are pathophysiologically relevant. The changes of the nAChRs and the cell cycle and cell differentiation genes were similar to those found in vitro. The results of indirect immunofluorescence assay of tissue specimens validated these findings. Thus, some pathobiologic effects of tobacco products in oral tissues may stem from Nic-induced alterations of the structure and function of keratinocyte nAChRs responsible for the physiologic regulation of the cell cycle by the cytotransmitter acetylcholine.

Choline acetyltransferase, acetylcholinesterase, and nicotinic acetylcholine receptors of human gingival and esophageal epithelia.

A non-neuronal cholinergic system that includes neuronal-like nicotinic acetylcholine receptors (nAChRs) has recently been described in epithelial cells that line the skin and the upper respiratory tract. Since the use of nicotine-containing products is associated with morbidity in the upper digestive tract, and since nicotine may alter cellular functions directly via nAChRs, we sought to identify and characterize a non-neuronal cholinergic system in the gingival and esophageal epithelia. mRNA transcripts for alpha3, alpha5, alpha7, and beta2 nAChR subunits, choline acetyltransferase, and the asymmetric and globular forms of acetylcholinesterase were amplified from gingival keratinocytes (KC) by means of polymerase chain-reactions. These proteins were visualized in the gingival and esophageal epithelia by means of specific antibodies. Variations in distribution and intensity of immunostaining were found, indicating that the repertoire of cholinergic enzymes and receptors expressed by the cells changes during epithelial maturation, and that an upward concentration gradient of free acetylcholine exists. Blocking of the nAChRs with mecamylamine resulted in reversible loss of cell-to-cell adhesion, and shrinking and rounding of cultured gingival KC. Activation of the receptors with acetylcholine or carbachol caused stretching and peripheral ruffling of the cytoplasmic aprons, and formation of new intercellular contacts. These results demonstrate that both the keratinizing epithelium of attached gingiva and the non-keratinizing epithelium lining the upper two-thirds of the esophageal mucosa possess a non-neuronal cholinergic system. The nAChRs expressed by these epithelia are coupled to regulation of cell adhesion and motility, and may provide a target for the deleterious effects of nicotine.

Bacteremia following subgingival irrigation and scaling and root planing.

The purpose of this study was to determine the incidence of bacteremia after a single professional subgingival irrigation with a 0.12% chlorhexidine gluconate mouthrinse (CHX) as well as after a subsequent scaling and root planing (S/RP) during the same visit. Thirty subjects each with at least 1 site that probed 4 mm or more and bled on probing were randomly assigned to the following groups: 1) irrigation with 0.12% CHX; 2) irrigation with sterile water; and 3) non-irrigated controls. To begin the study blood was drawn just before and 2 minutes after irrigation. Thirty minutes later, blood was drawn again just before and 2 minutes after S/RP at the same site. Specimens were cultured for anaerobic and aerobic microorganisms using standard cultural techniques. Eighteen blood cultures from 15 subjects yielded positive cultures resulting in 23 isolates. Gram-positive rods comprised 34.8% of the total isolates; Gram-positive cocci 34.8%, Gram-negative rods 21.7%, and Gram-negative cocci 8.7%. In the CHX group, bacteremia was detected in 5 subjects after irrigation and in 2 other subjects after S/RP. In the water group, bacteremia was detected in one subject after irrigation and in 4 subjects after S/RP. The control group had 3 bacteremias after S/RP. There was no significant difference between the incidence of bacteremia associated with irrigation by CHX or sterile water (P = 0.141). There was also no significant difference in the incidence of bacteremia after S/RP between the CHX and sterile water irrigation groups and in patients who did not receive irrigation (control group) (P = 0.88).(ABSTRACT TRUNCATED AT 250 WORDS)

Current status of periodontal therapy for furcation involvements.

The treatment of furcation involvements is difficult because of the anatomic problems that interfere with the clinician's accessibility in treating the area and the patient's ability in maintaining adequate plaque control afterwards. The goal of traditional methods of treatment is to arrest the progression of bone loss; the rate of success with these methods has been poor, except for Grade I involvements. Advances in the last decade have resulted in the development of treatment techniques that attempt to reconstruct the lost periodontal structures. These techniques have greatly improved the prognosis of Grade II furcation involvement. The recommended technique combines the principles of guided tissue regeneration using polytetrafluoroethylene membranes with grafting with porous hydroxyapatite. The use of decalcified freeze-dried bone instead of hydroxyapatite also may be a successful method. Grade III and IV furcation involvements still have a poor long-term prognosis because predictable reconstructive techniques for their treatment have not been demonstrated. When possible, a root resection approach may be advisable.

Clinical and microbiological effects of subgingival and gingival marginal irrigation with chlorhexidine gluconate.

Recent interest in the local delivery of antimicrobial and anti-inflammatory agents has stimulated interest in the efficacy of various treatment regimens. Chlorhexidine gluconate (CHX) delivered daily by home-applied marginal irrigation as a 0.04% solution in combination with a single professional irrigation of 0.12% CHX was tested over a 3-month period. Sixty periodontal maintenance patients each having at least 2 pockets greater than or equal to 4 mm probing depth, and bleeding on probing were assigned to either Group 1: one professional subgingival 0.12% CHX (Peridex) irrigation (Perio Pik) followed by adjunctive daily home marginal 0.04% CHX irrigation (Pik Pocket); Group 2: one professional subgingival 0.12% CHX irrigation followed by adjunctive daily home marginal water irrigation; Group 3: one professional subgingival water irrigation followed by adjunctive daily home marginal water irrigation; or Group 4: control. At baseline and 3 month visits, subgingival plaque samples were taken from 2 sites per patient. Cultural microbiological analysis was performed using non-selective and selective media. Plaque Index, Gingival Index, pocket probing depths, and gingival recession were assessed. Scaling and root planing (supportive periodontal treatment) was provided for each patient followed by subgingival irrigation as outlined above. At 3 months the Gingival Index and pocket probing depths were both significantly reduced (P less than .05) in all irrigation groups compared to baseline. There were no significant changes in clinical parameters in the control group from baseline to 3 months. In Group 1 the GI was significantly reduced (P less than .05) compared to Group 4 at 3 months.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of subgingival irrigation on bacteremia following scaling and root planing.

The purpose of this study was to determine the effects of professional subgingival irrigation, together with subsequent patient administered home marginal irrigation, on the incidence of bacteremia after scaling and root planing (Sc/RP). A total of 60 periodontal maintenance patients were assigned to either Group 1: subgingival irrigation, with 0.12% CHX and daily marginal irrigation with 0.04% CHX; Group 2: subgingival irrigation with 0.12% CHX and daily marginal irrigation with water; Group 3: subgingival and daily marginal irrigation with water; Group 4: Non-irrigation (control). Patients entered the study after receiving a thorough periodontal maintenance appointment including a complete examination, Sc/RP, and standard oral hygiene instruction. Blood samples were taken at the 3-month visit before and after Sc/RP. Microbiological culturing was done using the Septi-Chek system, selective and non-selective media. Results from 54 patients showed that bacteremia was detected prior to Sc/RP in 2 patients and after Sc/RP in 10 patients. No significant effect by treatment regimens on post Sc/RP bacteremia could be detected. The organisms isolated included Eubacterium lentum, Propionibacterium acnes, Streptococcus species, Neisseria species, Candida albicans, Staphylococcus species, and un-identified Gram-negative rods.