Inhibition of neutrophil inflammatory mediator expression by azithromycin.

BACKGROUND AND OBJECTIVE: Peri-implant tissues appear to exhibit a more vigorous inflammatory response during post-operative healing than periodontal tissues. There is evidence that a single dose of amoxicillin (AMX) prior to implant surgery reduces the risk of early peri-implant healing complications. This study compared the effects of AZM and AMX on neutrophil expression of mRNA for mediators involved in peri-implant healing. MATERIALS AND METHODS: Neutrophils were isolated from healthy human donors and pre-incubated with AZM (4 or 8 µg/ml) or AMX (2 or 4 µg/ml). Cells were then incubated with LPS (1 µg/ml), TNF-α (10 ng/ml), or medium alone (control) for 1, 2, and 4 h. Total RNA was analyzed with qPCR to quantify changes in expression of the six inflammatory mediators. RESULTS: LPS and TNF-α induced a similar pattern of IL-1ß mRNA expression, with peak expression at 1 h. For most mediators, gene expression in neutrophils activated by LPS was markedly reduced in a dose-dependent manner by AZM. Therapeutic concentrations of AZM (8 µg/ml) consistently reduced expression of mediators tested in this study. AMX was effective only in a few cases and under certain conditions. Therefore, AZM was more effective in its direct anti-inflammatory action. CONCLUSION: AZM is a consistent and effective inhibitor of neutrophil inflammatory mediator mRNA expression. CLINICAL RELEVANCE: Given that a single dose of AZM produces higher and more sustained concentrations of this agent in periodontal tissues than AMX when used as a pre-operative prophylactic antibiotic, AZM has greater potential to inhibit inflammatory mediator expression at peri-implant wound sites than AMX.

Azithromycin kills invasive Aggregatibacter actinomycetemcomitans in gingival epithelial cells.

Aggregatibacter actinomycetemcomitans invades periodontal pocket epithelium and is therefore difficult to eliminate by periodontal scaling and root planing. It is susceptible to azithromycin, which is taken up by many types of mammalian cells. This led us to hypothesize that azithromycin accumulation by gingival epithelium could enhance the killing of intraepithelial A. actinomycetemcomitans. [(3)H]azithromycin transport by Smulow-Glickman gingival epithelial cells and SCC-25 oral epithelial cells was characterized. To test our hypothesis, we infected cultured Smulow-Glickman cell monolayers with A. actinomycetemcomitans (Y4 or SUNY 465 strain) for 2 h, treated them with gentamicin to eliminate extracellular bacteria, and then incubated them with azithromycin for 1 to 4 h. Viable intracellular bacteria were released, plated, and enumerated. Azithromycin transport by both cell lines exhibited Michaelis-Menten kinetics and was competitively inhibited by l-carnitine and several other organic cations. Cell incubation in medium containing 5 µg/ml azithromycin yielded steady-state intracellular concentrations of 144 µg/ml in SCC-25 cells and 118 µg/ml in Smulow-Glickman cells. Azithromycin induced dose- and time-dependent intraepithelial killing of both A. actinomycetemcomitans strains. Treatment of infected Smulow-Glickman cells with 0.125 µg/ml azithromycin killed approximately 29% of the intraepithelial CFU of both strains within 4 h, while treatment with 8 µg/ml azithromycin killed ≥82% of the CFU of both strains (P < 0.05). Addition of carnitine inhibited the killing of intracellular bacteria by azithromycin (P < 0.05). Thus, human gingival epithelial cells actively accumulate azithromycin through a transport system that facilitates the killing of intraepithelial A. actinomycetemcomitans and is shared with organic cations.

An in vitro model for studies of attenuation of antibiotic-inhibited growth of Aggregatibacter actinomycetemcomitans Y4 by polyamines.

Polyamines are ubiquitous polycationic molecules that are present in all prokaryotic and eukaryotic cells, and they serve as important modulators of cell growth, stress, and cell proliferation. Polyamines are present at high concentrations in the periodontal pocket and could potentially affect the stress response of periodontal bacteria to antibiotics. The effects of polyamines on inhibition of growth by amoxicillin (AMX), azithromycin (AZM), and doxycycline (DOX) were investigated with the Y4 strain of Aggregatibacter actinomycetemcomitans (Aa). Bacteria were grown in brain heart infusion broth under the following conditions: (1) Aa only, (2) Aa + polyamine mix (1 mM putrescine, 0.4 mM spermidine, and 0.4 mM spermine), (3) Aa + antibiotic, and (4) Aa + antibiotic + polyamines. Growth curve analysis, minimal inhibitory concentration determination, and transcriptomic studies were conducted. The presence of exogenous polyamines produced a small, but significant increase in Aa growth, and polyamines attenuated the inhibitory effects of AMX, AZM, and DOX on growth. Transcriptomic analysis revealed that polyamines upregulate expression of ribosomal biogenesis proteins and small subunits, attenuate the bacterial stress response to antibiotics, and modulate bacterial nutritional pathways in a manner that could potentially increase the virulence of Aa. In summary, the polyamine-rich environment found in periodontal pockets appears to protect Aa and reduce its susceptibility to several antimicrobial agents in this in vitro model.

Antibiotic prophylaxis for implant placement: a systematic review of effects on reduction of implant failure.

Introduction Despite excellent reviews in the past several years, the use of antibiotics as prophylaxis for implant placement remains controversial.Aim To assess the literature on the efficacy of prophylactic antibiotics prescribed prior to and immediately following implant surgery (PIFS).Outcomes Whether administration of antibiotics reduced implant failure and post-operative complications.Design Databases searched were PubMed and Medline via Ovid (1946 to February 2018), Cochrane Library (Wiley) and Google Scholar.Materials and methods Quality assessment, meta-analysis with a forest plot and incorporated assessment of heterogeneity. A two-tailed paired t-test was performed, analysing differences in mean failure rates between groups.Results Fourteen publications were collected; 5,334 implants were placed with pre-operative antibiotics, 82 implants with antibiotics PIFS and 3,862 placed with no antibiotics. The overall risk ratio (RR) was 0.47 (95% CI 0.39-0.58), with the implant failure rates significantly affected by pre-operative intervention (Z = 7.00, P <0.00001). The number needed to treat (NNT) was 35 (95% CI 26.3-48.2). The difference between mean failure rates was statistically significant (P = 0.0335).Conclusion Administering prophylactic antibiotics reduced the risk of implant failures. Further investigations are recommended to establish a standardised protocol for the proper use of antibiotic regimen.

Formylpeptide receptor single nucleotide polymorphism 348T>C and its relationship to polymorphonuclear leukocyte chemotaxis in aggressive periodontitis.

BACKGROUND: Aggressive periodontitis (AgP) is associated with impaired polymorphonuclear leukocyte (PMN) chemotaxis toward bacterial N-formylpeptides. Formylpeptide receptors (FPRs) play a major role in guiding PMNs to infection sites. Previous work revealed a significant association between FPR1 single nucleotide polymorphism (SNP) 348T>C and AgP in African Americans. We tested the hypothesis that 348T impairs PMN chemotaxis by decreasing FPR mRNA expression, thereby increasing susceptibility to AgP. METHODS: Blood samples were obtained from African American subjects (37 AgP cases and 38 controls). Chemotaxis to N-formyl-methionine-leucine-phenylalanine by freshly isolated PMNs was assayed in a modified Boyden chamber. RNA was isolated from PMNs, and FPR1 gene expression was quantified by real-time polymerase chain reaction (PCR). To detect FPR1 5' SNPs, genomic DNA was isolated, and four fragments spanning the FPR1 5' region were PCR-amplified and sequenced. Haplotype associations between SNP 348T>C and 5' SNPs were analyzed. RESULTS: The homozygous 348T genotype was only found in AgP cases (P = 0.017; odds ratio, 18.9). Subjects with this genotype exhibited a significantly lower PMN chemotactic response relative to controls and to subjects with the 348C/C or 348T/C genotype (P <0.05). There were no significant differences in PMN FPR1 expression among subjects with the 348C/C, 348T/C, and 348T/T genotypes. Eleven FPR1 5' SNPs were detected, but none of the predicted haplotypes reflected associations with AgP or with 348T. CONCLUSIONS: Although the 348T/T genotype is relatively rare, it is associated with significantly impaired PMN chemotaxis and an increased risk for developing AgP in African Americans. These associations do not seem to be related to significant reductions in FPR1 transcripts in subjects expressing 348T.

Neutrophil formylpeptide receptor single nucleotide polymorphism 348T>C in aggressive periodontitis.

BACKGROUND: Neutrophil formylpeptide receptors (FPRs) play an important role in bacterial recognition and chemotaxis. Defective FPR1 expression and impaired polymorphonuclear leukocyte chemotaxis toward bacterial formylpeptides are associated with aggressive periodontitis (AgP). The objective of this study was to determine whether single nucleotide polymorphisms (SNPs) in FPR1 are associated with AgP. METHODS: Genomic DNA was isolated from blood samples obtained from African Americans (30 subjects with AgP and 33 healthy controls) and Turks (75 subjects with AgP and 63 healthy controls). A highly polymorphic fragment of the FPR1 gene was amplified by polymerase chain reaction and sequenced for analysis of SNPs. RESULTS: Five previously identified SNPs were detected in African Americans, whereas six were detected in Turkish subjects. The frequency of synonymous SNP c.348T>C was significantly higher in African Americans with AgP than in controls (P = 0.029). The 348T allele was significantly associated with AgP (P = 0.010). Seven of the subjects with AgP, but none of the controls, were homozygous for 348T. FPR1 haplotypes 348T.568A, 348T.576T, and 348T.568A.576T were significantly increased in African Americans with AgP (P <0.02). The Turkish population did not exhibit significant differences in FPR1 SNP frequencies between subjects with AgP and controls. CONCLUSIONS: FPR1 SNP c.348T>C is associated with AgP in African Americans. Individuals who are homozygous for 348T may have an increased risk for developing this disorder.

Distribution of systemic clarithromycin to gingiva.

BACKGROUND: Aggressive and recurrent forms of periodontitis are associated with infections by Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) and Porphyromonas gingivalis. Because these pathogens invade tissue, they are difficult to eliminate by root planing alone. The use of systemic antibiotics in conjunction with root planing significantly enhances clinical and microbiologic treatment outcomes. Although it is not widely prescribed by periodontists, clarithromycin is potentially useful because it is taken up by host cells and has favorable antimicrobial activity. METHODS: Experimental gingivitis was induced in eight healthy subjects at one randomly selected maxillary posterior site. The contralateral maxillary site served as the healthy control. Thereafter, subjects were administered six doses of clarithromycin, 500 mg, every 12 hours. Blood was then drawn, and samples of gingiva were harvested from both sites. The samples were extracted, and clarithromycin content was analyzed by liquid chromatography. RESULTS: Mean clarithromycin concentrations in healthy control and inflamed gingiva (2.4 and 3.0 microg/g, respectively) were significantly higher than in serum (0.5 microg/ml; P <0.05). Clarithromycin levels at control and gingivitis sites were higher than serum by 5.7- and 7.0-fold, respectively (difference between sites was significant; P = 0.02). At control sites, a significant decrease in gingival crevicular fluid flow rate was evident at the conclusion of the clarithromycin regimen (P = 0.018). CONCLUSIONS: Clarithromycin can attain higher levels in gingiva than serum and reach higher levels in inflamed gingiva than in healthy gingiva. Its distribution profile seems to be suitable for the treatment of periodontitis. The reduction in crevicular fluid flow at control sites suggested that clarithromycin may produce anti-inflammatory effects.

Early wound healing following one-stage dental implant placement with and without antibiotic prophylaxis: a pilot study.

BACKGROUND: One-stage implant placement has clinically acceptable treatment outcomes. Among other advantages, it may allow investigation of early wound healing. The purpose of this pilot study was to determine whether peri-implant crevicular fluid (PICF) can be used to detect early changes around implants placed with one-stage surgical protocol following 1 week of healing. METHODS: Twenty subjects (11 males and nine females; aged 22 to 72 years; two smokers) were included. Exclusion criteria were allergies to amoxicillin and systemic conditions that may affect healing. Subjects had a healthy periodontium and needed a single implant; eight received antibiotic prophylaxis, and 12 served as controls. Clinical healing was evaluated with plaque and gingival indices (PI and GI, respectively). Gingival crevicular fluid (GCF) from the surgical site was obtained prior to the surgery, whereas PICF was collected at the 1-week visit. Enzyme-linked immunosorbent assay was used to determine GCF/PICF interleukin (IL)-1beta and -8 concentrations. Peripheral blood and GCF antibiotic levels were measured by high-performance liquid chromatography. RESULTS: Postoperative PI and GI were slightly increased. Total GCF and PICF volumes did not show a significant difference between appointments. There was an increase in PICF IL-1beta and -8 levels at 1 week postoperatively. Mean amoxicillin serum concentration was 5.1 +/- 2 microg/ml at 1 to 4 hours following the initial dose, whereas GCF amoxicillin levels were below the limit of detection. Antibiotic prophylaxis had a modest effect on clinical indices (PI and GI) and no appreciable effect on biomarkers. CONCLUSIONS: PICF content can be studied as early as 1 week following one-stage implant placement. The results raise doubts regarding the clinical usefulness of amoxicillin prophylaxis.

The making of a miscreant: tobacco smoke and the creation of pathogen-rich biofilms.

We have previously reported that oral biofilms in clinically healthy smokers are pathogen-rich, and that this enrichment occurs within 24 h of biofilm formation. The present investigation aimed to identify a mechanism by which smoking creates this altered community structure. By combining in vitro microbial-mucosal interface models of commensal (consisting of Streptococcus oralis, Streptococcus sanguis, Streptococcus mitis, Actinomyces naeslundii, Neisseria mucosa and Veillonella parvula) and pathogen-rich (comprising S.oralis, S.sanguis, S.mitis, A.naeslundii, N.mucosa and V.parvula, Fusobacterium nucleatum, Porphyromonas gingivalis, Filifactor alocis, Dialister pneumosintes, Selenonomas sputigena, Selenominas noxia, Catonella morbi, Parvimonas micra and Tannerella forsythia) communities with metatranscriptomics, targeted proteomics and fluorescent microscopy, we demonstrate that smoke exposure significantly downregulates essential metabolic functions within commensal biofilms, while significantly increasing expression of virulence genes, notably lipopolysaccharide (LPS), flagella and capsule synthesis. By contrast, in pathogen-rich biofilms several metabolic pathways were over-expressed in response to smoke exposure. Under smoke-rich conditions, epithelial cells mounted an early and amplified pro-inflammatory and oxidative stress response to these virulence-enhanced commensal biofilms, and a muted early response to pathogen-rich biofilms. Commensal biofilms also demonstrated early and widespread cell death. Similar results were observed when smoke-free epithelial cells were challenged with smoke-conditioned biofilms, but not vice versa. In conclusion, our data suggest that smoke-induced transcriptional shifts in commensal biofilms triggers a florid pro-inflammatory response, leading to early commensal death, which may preclude niche saturation by these beneficial organisms. The cytokine-rich, pro-oxidant, anaerobic environment sustains inflammophilic bacteria, and, in the absence of commensal antagonism, may promote the creation of pathogen-rich biofilms in smokers.

Characterization of minocycline transport by human neutrophils.

BACKGROUND: Tetracyclines are used in periodontal therapy as antimicrobial agents and as inhibitors of matrix metalloproteinases. Neutrophils appear to accumulate minocycline and other tetracyclines through a mechanism that has not been fully characterized. METHODS: The transport of minocycline and other tetracyclines by isolated human neutrophils was characterized by measuring the increase in cell-associated fluorescence. RESULTS: Quiescent neutrophils took up minocycline through a saturable, concentrative, sodium-dependent mechanism with a Michaelis constant (K(m)) of 153 micro g/ml (501 microM) and a maximal velocity of 240 ng/minute/10(6) cells. The efficiency of minocycline transport was not influenced significantly by a two-unit variation in extracellular pH and was not enhanced upon cell activation with phorbol myristate acetate. Neutrophil incubation in medium containing 10 micro g/ml minocycline, doxycycline, or tetracycline yielded steady-state intracellular/extracellular concentration ratios of approximately 64.0, 7.5, or 1.8, respectively. The dilution of extracellular minocycline or doxycycline triggered efflux from cells loaded with these antibiotics. Minocycline transport was competitively inhibited by the organic cations carnitine, diphenhydramine, and verapamil, but penicillin and other organic anions failed to produce inhibition. CONCLUSION: Transport of tetracyclines by neutrophils could potentially enhance the effectiveness of these agents in periodontal therapy by enhancing or sustaining their therapeutic levels at inflammatory sites and by enhancing the killing of phagocytosed bacterial pathogens.

Oral clonidine pretreatment prior to venous cannulation.

Clonidine is a preferential alpha-2 agonist drug that has been used for over 35 years to treat hypertension. Recently, it has also been used as a preoperative medication and as a sedative/anxiolytic drug. This randomized, double-blind, placebo-controlled crossover clinical trial characterized the effects of oral clonidine pretreatment on intravenous catheter placement in 13 patients. Parameters measured included the bispectral index (BIS), Observer's Assessment of Alertness/Sedation Scale (OAA/S), frontal temporal electromyogram (EMG), 30-Second Blink Count (Blink), Digit Symbol Substitution Test (DSST), State Anxiety Inventory (SAI), fingertip versus forearm skin temperatures, and multiple questionnaires. Oral clonidine significantly decreased SAI scores, OAA/S, EMG, and Blink, but did not cause statistically significant BIS or DSST reductions. Subjects preferred oral clonidine pretreatment prior to venipuncture compared to placebo. Questionnaires also indicated that clonidine provided minimal sedation, considerable anxiolysis, and some analgesia. Fingertip versus forearm skin temperature differentials were decreased. Reduced fingertip versus forearm temperature differentials suggest increased peripheral cutaneous blood flow prior to venous cannulation. Oral clonidine pretreatment not only helped control patient anxiety and pain but also provided cardiovascular stability.

Relative effectiveness of azithromycin in killing intracellular Porphyromonas gingivalis.

Invasive infections by Porphyromonas gingivalis are associated with persistent periodontal attachment loss and can be difficult to eliminate by scaling and root planing. Azithromycin (AZM) inhibits P. gingivalis and is actively accumulated by most human cells. We used an in vitro infection model to compare the effectiveness of AZM in killing intracellular P. gingivalis to the combined regimen of amoxicillin (AMX) and metronidazole (MET). Transport of [3H]-AZM by human gingival fibroblasts was characterized. Monolayers of Smulow-Glickman gingival epithelial cells or gingival fibroblasts were infected with P. gingivalis (strain 33277 or W83). After extracellular bacteria were eliminated with teicoplanin, infected cells were treated with therapeutic concentrations of AZM, AMX, or AMX + MET. Viable intracellular bacteria were released by cell lysis and plated on blood agar for enumeration. Antimicrobial activity against planktonic P. gingivalis was also evaluated. While survival of intraepithelial P. gingivalis 33277 was not significantly different after treatment with the three regimens, survival in infected fibroblasts was significantly lower after AZM treatment (65.9 ± 5.5%) compared with AMX (92.2 ± 3.5%) or AMX + MET (79.8 ± 5.2%, P < 0.01). Carnitine, a competitive inhibitor of AZM transport, reduced killing by AZM by ~55% (P < 0.05). Survival of intrafibroblast P. gingivalis W83 was also significantly lower after AZM treatment compared with the other regimens (P < 0.05). At therapeutic concentrations, AZM was significantly more active against intracellular P. gingivalis than against planktonic P. gingivalis (P < 0.0083). Gingival epithelial cells and fibroblasts possess a transport system that accumulates AZM and enhances elimination of intracellular P. gingivalis. Compared with the combination of AMX and MET, AZM was equally effective against intraepithelial P. gingivalis 33277 and significantly more effective against both strains of P. gingivalis from infected gingival fibroblasts. The results suggest that AZM could be a reasonable alternative to the regimen of AMX and MET for periodontal patients who should not take these agents due to known side effects or compliance issues.

Effect of biologic mediators on ciprofloxacin accumulation by gingival fibroblasts.

BACKGROUND: Human gingival fibroblasts actively accumulate fluoroquinolone antimicrobials. Because fibroblasts are prevalent in gingiva, they may help sustain therapeutic fluoroquinolone levels at that site. The purpose of this study was to determine whether mediators associated with infection or injury can enhance ciprofloxacin accumulation by gingival fibroblasts. METHODS: Quiescent fibroblast monolayers were treated for 1, 6, or 24 hours with several concentrations of tumor necrosis factor (TNF)-alpha, transforming growth factor (TGF)-beta1, platelet-derived growth factor (PDGF)-BB, fibroblast growth factor (FGF)-2, or insulin-like growth factor (IGF)-1. Transport was assayed by measuring cell-associated fluoroquinolone fluorescence. RESULTS: All mediators significantly enhanced ciprofloxacin transport in a dose dependent manner (P < 0.05; ANOVA). Except for TNF, this enhancement was associated with a decrease in the Km of ciprofloxacin transport. Maximal enhancement was observed with 10 ng/ml PDGF or FGF and 30 ng/ml TNF, TGF, or IGF. Brief (1 hour) treatment with TNF or FGF upregulated ciprofloxacin accumulation by a maximum of 13% to 14%, whereas TGF, PDGF, and IGF enhanced this process by 19% to 24%. All of the mediators enhanced ciprofloxacin accumulation by a maximum of 19% to 24% after 6 hours and 30% to 38% after 24 hours. The accumulation of other fluoroquinolones (e.g., gatifloxacin) was also slightly enhanced. CONCLUSIONS: Gingival fibroblasts treated with cytokines or growth factors accumulate significantly more ciprofloxacin than untreated controls. This provides a mechanism by which ciprofloxacin could be preferentially distributed to gingival wound or inflammatory sites, yielding local therapeutic levels that are more sustained than in serum.

Azithromycin enhances phagocytic killing of Aggregatibacter actinomycetemcomitans Y4 by human neutrophils.

BACKGROUND: Aggregatibacter actinomycetemcomitans resists killing by neutrophils and is inhibited by azithromycin (AZM) and amoxicillin (AMX). AZM actively concentrates inside host cells, whereas AMX enters by diffusion. The present study is conducted to determine whether AZM is more effective than AMX at enhancing phagocytic killing of A. actinomycetemcomitans by neutrophils. METHODS: Killing assays were conducted in the presence of either 2 µg/mL AZM or 16 µg/mL AMX (equipotent against A. actinomycetemcomitans). Neutrophils were loaded by incubation with the appropriate antibiotic. Opsonized A. actinomycetemcomitans strain Y4 was incubated with the indicated antibiotic alone, with loaded neutrophils and antibiotic, or with control neutrophils (without antibiotic) at multiplicities of infection (MOIs) of 30 and 90 bacteria per neutrophil. RESULTS: Neutrophil incubation with 2 µg/mL AZM yielded an intracellular concentration of 10 µg/mL. At an MOI of 30, neutrophils loaded with AZM failed to kill significantly more bacteria than control neutrophils during the 60- and 90-minute assay periods. At an MOI of 90, neutrophils loaded with AZM killed significantly more bacteria than either AZM alone or control neutrophils during 60- and 90-minute incubations (P < 0.05), and killed significantly more bacteria after 90 minutes than the sum of the killing produced by AZM alone or neutrophils alone. Neutrophils incubated with AMX under identical conditions also killed significantly more bacteria than either AMX alone or control neutrophils, but there was no evidence of synergism between AMX and neutrophils. CONCLUSIONS: Neutrophils possess a concentrative transport system for AZM that may enhance killing of A. actinomycetemcomitans. Its effects are most pronounced when neutrophils are greatly outnumbered by bacteria.

Comparison of Azithromycin and Amoxicillin Before Dental Implant Placement: An Exploratory Study of Bioavailability and Resolution of Postoperative Inflammation.

BACKGROUND: Studies suggest that a single prophylactic dose of amoxicillin reduces early implant complications, but it is unclear whether other antibiotics are also effective. This study compared the local antimicrobial and anti-inflammatory effects resulting from a single dose of azithromycin or amoxicillin before surgical placement of one-stage dental implants. METHODS: Healthy adult patients requiring one-stage dental implant placement were allocated randomly to receive either 2 g amoxicillin (n = 7) or 500 mg azithromycin (n = 6) before surgery. Peri-implant crevicular fluid (PICF) samples from the new implant and gingival crevicular fluid (GCF) from adjacent teeth were sampled on postoperative days 6, 13, and 20. Inflammatory mediators in the samples were analyzed by immunoassay, and antibiotic levels were measured by bioassay. RESULTS: On day 6, azithromycin concentrations in GCF and PICF were 3.39 ± 0.73 and 2.77 ± 0.90 µg/mL, respectively, whereas amoxicillin was below the limit of detection. During early healing, patents in the azithromycin group exhibited a significantly greater decrease in GCF volume (P = 0.03, analysis of variance). At specific times during healing, the azithromycin group exhibited significantly lower levels of interleukin (IL)-6 and IL-8 in GCF than the amoxicillin group and exhibited significantly lower levels of granulocyte colony stimulating factor, IL-8, macrophage inflammatory protein-1ß, and interferon-gamma-inducible protein-10 in PICF. CONCLUSIONS: Azithromycin was available at the surgical site for a longer period of time than amoxicillin, and patients taking azithromycin exhibited lower levels of specific proinflammatory cytokines and chemokines in GCF and PICF. Thus, preoperative azithromycin may enhance resolution of postoperative inflammation to a greater extent than amoxicillin.

Distribution of systemic ciprofloxacin and doxycycline to gingiva and gingival crevicular fluid.

BACKGROUND: Systemic fluoroquinolones and tetracyclines reach steady-state levels in gingival crevicular fluid (GCF) that are several-fold higher than their levels in serum. The mechanism by which this occurs is unclear, but gingival fibroblasts are known to accumulate these agents. Uptake by fibroblasts could enhance their distribution to gingiva. To test this hypothesis, steady-state levels of these agents were assayed in serum, gingival connective tissue (GCT), and GCF. METHODS: Healthy subjects who needed resective periodontal surgery participated in the study. Approximately 78 hours prior to the surgical appointment, each subject began a 3-day regimen of ciprofloxacin or doxycycline. At the surgical appointment (scheduled approximately 6 hours after the last dose), samples of blood and GCT were collected. GCF samples were collected on paper strips and measured with an electronic device. Samples were extracted and analyzed by high performance liquid chromatography. RESULTS: Mean ciprofloxacin levels in serum, GCT, and GCF were 0.40 microg/ml, 1.38 microg/g, and 1.66 microg/ml, respectively (P<0.001, N=9). For doxycycline, these levels were 1.11 microg/ml, 2.03 microg/g, and 2.41 microg/ml, respectively (P=0.002, N=8). For both agents, the GCT and GCF levels were significantly higher than serum levels (P<0.05), but not significantly different from each other. CONCLUSIONS: Our findings suggest that fibroblasts could play an important role in the distribution of fluoroquinolones and tetracyclines to the gingiva. By accumulating these agents in GCT, fibroblasts could contribute to the relatively high levels they attain in GCF.

An in vitro model of ciprofloxacin and minocycline transport by oral epithelial cells.

BACKGROUND: Fluoroquinolones and tetracyclines can penetrate epithelial cells, but the mechanism by which they cross the plasma membrane is unclear. In this study, a cell line derived from oral epithelium was used as a model to demonstrate a role for active transport. METHODS: Transport of ciprofloxacin and minocycline by confluent cell monolayers was assayed by measuring the increase in cell-associated fluorescence. RESULTS: Uptake of both agents was saturable and was inhibited at low temperatures. At 37 degrees C, the cells transported ciprofloxacin and minocycline with Km values of 351 and 133 microg/ml, respectively, and maximum velocities of 5.11 and 13.4 ng/min/microg cell protein, respectively. When ciprofloxacin and minocycline were removed from the extracellular medium, the intracellular levels of both agents decreased. Ciprofloxacin efflux from loaded cells occurred more rapidly than with minocycline. Cells accumulated intracellular drug levels that were at least 8-fold higher than extracellular levels for ciprofloxacin and at least 40-fold higher for minocycline. Transport of ciprofloxacin and minocycline was significantly influenced by pH and was most favorable at pH 7.7 and 7.2, respectively. While ciprofloxacin transport was Na+ independent, minocycline transport was strongly inhibited when sodium in the medium was replaced with choline. Transport of both agents was inhibited by a variety of organic cations, but the pattern of inhibition was different. Papaverine, phenylephrine, and doxycycline competitively inhibited minocycline transport, but inhibited ciprofloxacin transport by a non-competitive mechanism. CONCLUSIONS: Epithelial cells take up ciprofloxacin and minocycline via different active transport systems. These transporters may play an important role in enhancing the effectiveness of these agents against invasive pathogens.

Resolution of localized chronic periodontitis associated with longstanding calculus deposits.

This report, which is based on nonstandardized serial radiographs obtained over a period of 15 years, documents a case of localized chronic periodontitis associated with progressive deposition of calculus on the distal aspect of a mandibular second molar. The site was treated by scaling and root planing, followed by a course of adjunctive systemic azithromycin. Treatment yielded favorable reductions in probing depth and clinical inflammation, leaving only few isolated sites with pockets no deeper than 4 mm. Two years after completion of active treatment, there was radiographic evidence of increased bone density distal to the second molar.

Effect of gingivitis on azithromycin concentrations in gingival crevicular fluid.

BACKGROUND: Macrolide antibiotics yield high concentrations in inflamed tissue, suggesting that their levels in gingival crevicular fluid (GCF) could be increased at gingivitis sites. However, the increased volume of GCF associated with gingivitis could potentially dilute macrolides. To determine whether these assumptions are correct, the bioavailability of systemically administered azithromycin was compared in GCF from healthy and gingivitis sites. METHODS: Experimental gingivitis was induced in one maxillary posterior sextant in nine healthy individuals. Contralateral healthy sextants served as controls. Participants ingested 500 mg azithromycin, followed by a 250-mg dose 24 hours later. Four hours after the second dose, plaque was removed from experimental sites. GCF was collected from eight surfaces in both the experimental and control sextants and pooled separately. GCF samples were subsequently collected on days 2, 3, 8, and 15, and azithromycin content was determined by agar diffusion bioassay. RESULTS: On days 2 and 3, the pooled GCF volume at experimental sites was significantly higher than at control sites (P <0.01), and the total azithromycin mass in 30-second GCF samples pooled from experimental sites was significantly higher than at control sites (P <0.02). However, there were no significant differences in azithromycin concentration between the experimental and control pools at any point. Concentrations exceeded 7.3 µg/mL on day 2 and 2.5 µg/mL on day 15. CONCLUSION: Azithromycin concentrations are similar in GCF from gingivitis sites and healthy sites, suggesting that the processes that regulate GCF azithromycin concentration can compensate for local inflammatory changes.

Clarithromycin accumulation by phagocytes and its effect on killing of Aggregatibacter actinomycetemcomitans.

BACKGROUND: Clarithromycin inhibits several periodontal pathogens and is concentrated inside gingival fibroblasts and epithelial cells by an active transporter. We hypothesized that polymorphonuclear leukocytes (PMNs) and less mature myeloid cells possess a similar transporter for clarithromycin. It is feasible that clarithromycin accumulation inside PMNs could enhance their ability to kill Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans). METHODS: To test the first hypothesis, purified PMNs and cultured HL-60 cells were incubated with [(3)H]-clarithromycin. Clarithromycin transport was assayed by measuring changes in cell-associated radioactivity over time. The second hypothesis was examined with PMNs loaded by incubation with clarithromycin (5 µg/ml). Opsonized bacteria were incubated at 37°C with control and clarithromycin-loaded PMNs. RESULTS: Mature human PMNs, HL-60 cells differentiated into granulocytes, and undifferentiated HL-60 cells all took up clarithromycin in a saturable manner. The kinetics of uptake by all yielded linear Lineweaver-Burk plots. HL-60 granulocytes transported clarithromycin with a K(m) of ≈250 µg/ml and a V(max) of 473 ng/min/10(6) cells, which were not significantly different from the values obtained with PMNs. At steady state, clarithromycin levels inside HL-60 granulocytes and PMNs were 28- to 71-fold higher than extracellular levels. Clarithromycin-loaded PMNs killed significantly more A. actinomycetemcomitans and achieved shorter half-times for killing than control PMNs when assayed at a bacteria-to-PMN ratio of 100:1 (P <0.04). At a ratio of 30:1, these differences were not consistently significant. CONCLUSIONS: PMNs and less mature myeloid cells possess a transporter that takes up and concentrates clarithromycin. This system could help PMNs cope with an overwhelming infection by A. actinomycetemcomitans.