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.

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.

Computational requirements for real-time ptychographic image reconstruction.

Ptychographic imaging techniques can be coupled with tomographic image reconstruction techniques to obtain cross-sectional 3D images with resolution on the nanometer scale. However, such ptychographic x-ray computed tomography (PXCT) techniques require the collection of a large number of diffraction patterns. This work derives a set of equations that can be used to calculate the rate at which data can be collected given an experimental setup. It also determines the computational system requirements needed to process ptychographic data in real time as soon as it has been collected. This will expedite the ptychography step of PXCT. These theoretical results are then applied to performance data collected from reconstructing simulated diffraction patterns in order to determine the computational resources needed for real-time ptychographic processing for representative experimental setups. All of our results are independent of any specific ptychographic reconstruction algorithm.

Impact of Induction Therapy on Circulating T Follicular Helper Cells and Subsequent Donor-Specific Antibody Formation After Kidney Transplant.

INTRODUCTION: The cellular events that contribute to generation of donor-specific anti-HLA antibodies (DSA) post-kidney transplantation (KTx) are not well understood. Characterization of such mechanisms could allow tailoring of immunosuppression to benefit sensitized patients. METHODS: We prospectively monitored circulating T follicular helper (cTFH) cells in KTx recipients who received T-cell depleting (thymoglobulin, n = 54) or T-cell nondepleting (basiliximab, n = 20) induction therapy from pre-KTx to 1 year post-KTx and assessed their phenotypic changes due to induction and DSA occurrence, in addition to healthy controls (n = 13), for a total of 307 blood samples. RESULTS: Before KTx, patients displayed comparable levels of resting, central memory cTFH cells with similar polarization to those of healthy controls. Unlike basiliximab induction, thymoglobulin induction significantly depleted cTFH cells, triggered lymphopenia-induced proliferation that skewed cTFH cells toward increased Th1 polarization, effector memory, and elevated programmed cell death protein 1 (PD-1)int/hi expression, resembling activated phenotypes. Regardless of induction, patients who developed DSA post-KTx, harbored pre-KTx donor-reactive memory interleukin (IL)-21+ cTFH cells and showed higher % cTFH and lower % of T regulatory (TREG) cells post-KTx resulting in elevated cTFH:TREG ratio at DSA occurrence. CONCLUSION: Induction therapy distinctly shapes cTFH cell phenotype post-KTx. Monitoring cTFH cells before and after KTx may help detect those patients prone to DSA generation post-KTx.

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.

Phenotype, function, and differentiation potential of human monocyte subsets.

Human monocytes have been grouped into classical (CD14++CD16-), non-classical (CD14dimCD16++), and intermediate (CD14++CD16+) subsets. Documentation of normal function and variation in this complement of subtypes, particularly their differentiation potential to dendritic cells (DC) or macrophages, remains incomplete. We therefore phenotyped monocytes from peripheral blood of healthy subjects and performed functional studies on high-speed sorted subsets. Subset frequencies were found to be tightly controlled over time and across individuals. Subsets were distinct in their secretion of TNFα, IL-6, and IL-1ß in response to TLR agonists, with classical monocytes being the most producers and non-classical monocytes the least. Monocytes, particularly those of the non-classical subtype, secreted interferon-α (IFN-α) in response to intracellular TLR3 stimulation. After incubation with IL-4 and GM-CSF, classical monocytes acquired monocyte-derived DC (mo-DC) markers and morphology and stimulated allogeneic T cell proliferation in MLR; intermediate and non-classical monocytes did not. After incubation with IL-3 and Flt3 ligand, no subset differentiated to plasmacytoid DC. After incubation with GM-CSF (M1 induction) or macrophage colony-stimulating factor (M-CSF) (M2 induction), all subsets acquired macrophage morphology, secreted macrophage-associated cytokines, and displayed enhanced phagocytosis. From these studies we conclude that classical monocytes are the principal source of mo-DCs, but all subsets can differentiate to macrophages. We also found that monocytes, in particular the non-classical subset, represent an alternate source of type I IFN secretion in response to virus-associated TLR agonists.

Casting and Splinting Management for Hand Injuries in the In-Season Contact Sport Athlete.

CONTEXT: Upper extremity injuries are extremely common in contact sports such as football, soccer, and lacrosse. The culture of competitive athletics provides an environment where hand injuries are frequently downplayed in an effort to prevent loss of game time. However, studies have shown that many sport-induced hand injuries do not actually require immediate surgical attention and can be safely treated through immobilization so that the athlete may complete the athletic season. In these cases, appropriate casting and splinting measures should be taken to ensure protection of the injured player and the other competitors without causing loss of game time. EVIDENCE ACQUISITION: Articles published between 1976 and 2015 were reviewed to capture historical and current views on the treatment of hand injuries in the in-season athlete. STUDY DESIGN: Clinical review. LEVEL OF EVIDENCE: Level 5. RESULTS: Although traditionally many sports-induced traumatic injuries to the hand held the potential to be season-ending injuries, experience has shown that in-season athletes do not necessarily need to lose game time to receive appropriate treatment. A thorough knowledge of converting everyday splints and casts into game day, sport-approved protective immobilization devices is key to safely allowing athletes with select injuries to play while injured. CONCLUSION: Protective techniques allow for maximum functionality during gameplay while safely and effectively protecting the injury from further trauma while bony healing takes place.

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.

Should Antibiotics Be Prescribed to Treat Chronic Periodontitis?

Although scaling and root planing is a cost-effective approach for initial treatment of chronic periodontitis, it fails to eliminate subgingival pathogens and halt progressive attachment loss in some patients. Adjunctive use of systemic antibiotics immediately after completion of scaling and root planing can enhance the degree of clinical attachment gain and probing depth reduction provided by nonsurgical periodontal treatment. This article discusses the rationale for prescribing adjunctive antibiotics, reviews the evidence for their effectiveness, and outlines practical issues that should be considered before prescribing antibiotics to treat chronic periodontitis.

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.

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.

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.

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.

Long-term effects of alemtuzumab on regulatory and memory T-cell subsets in kidney transplantation.

BACKGROUND: Induction with lymphocyte-depleting antibodies is routinely used to prevent rejection but often skews T cells toward memory. It is not fully understood which memory and regulatory T-cell subsets are most affected and how they relate to clinical outcomes. METHODS: We analyzed T cells from 57 living-donor renal transplant recipients (12 reactive and 45 quiescent) 2.8±1.4 years after alemtuzumab induction. Thirty-four healthy subjects and nine patients with acute cellular rejection (ACR) were also studied. RESULTS: We found that alemtuzumab caused protracted CD4 more than CD8 T-lymphocyte deficiency, increased proportion of CD4 memory T cells, and decreased proportion of CD4 regulatory T cells. Reactive patients exhibited higher proportions of CD4 effector memory T cells (TEM) and CD8 terminally differentiated TEM (TEMRA), with greater CD4 TEM and CD8 TEMRA to regulatory T cell ratios, than quiescent patients or healthy controls. Patients with ongoing ACR had profound reduction in circulating CD8 TEMRA. Mixed lymphocyte assays showed significantly lower T-cell proliferation to donor than third-party antigens in the quiescent group, while reactive and ACR patients exhibited increased effector molecules in CD8 T cells. CONCLUSIONS: Our findings provide evidence that T-cell skewing toward TEM may be associated with antigraft reactivity long after lymphodepletion. Further testing of TEM and TEMRA subsets as rejection predictors is warranted.

Innate immunity alone is not sufficient for chronic rejection but predisposes healed allografts to T cell-mediated pathology.

BACKGROUND: Acute allograft rejection is dependent on adaptive immunity, but it is unclear whether the same is true for chronic rejection. Here we asked whether innate immunity alone is sufficient for causing chronic rejection of mouse cardiac allografts. METHODS: We transplanted primarily vascularized cardiac grafts to recombinase activating gene-knockout (RAG(-/-)) mice that lack T and B cells but have an intact innate immune system. Recipients were left unmanipulated, received adjuvants that stimulate innate immunity, or were reconstituted with B-1 lymphocytes to generate natural IgM antibodies. In a second model, we transplanted cardiac allografts to mice that lack secondary lymphoid tissues (splenectomized aly/aly recipients) and studied the effect of NK cell inactivation on T cell-mediated chronic rejection. RESULTS: Acute cardiac allograft rejection was not observed in any of the recipients. Histological analysis of allografts harvested 50 to 90 days after transplantation to RAG(-/-) mice failed to identify chronic vascular or parenchymal changes beyond those observed in control syngeneic grafts. Chronic rejection of cardiac allografts parked in splenectomized aly/aly mice was observed only after the transfer of exogenously activated T cells. NK inactivation throughout the experiment, or during the parking period alone, reduced the severity of T cell-dependent chronic rejection. CONCLUSIONS: The innate immune system alone is not sufficient for causing chronic rejection. NK cells predispose healed allografts to T cell-dependent chronic rejection and may contribute to chronic allograft pathology.

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.

Azithromycin concentrations in blood and gingival crevicular fluid after systemic administration.

BACKGROUND: Azithromycin is a macrolide antibiotic that is active against several periodontal pathogens. Macrolides are taken up and concentrated inside gingival fibroblasts, which could influence their pharmacokinetics. This study tests the hypothesis that steady-state levels of azithromycin are higher and more sustained in gingival crevicular fluid (GCF) than in serum. METHODS: Four healthy patients received an initial dose of 500-mg azithromycin followed by 250-mg doses on each of the next 2 days. Serum and GCF samples were obtained 2 hours after the last dose on day 2, and on days 4 and 7. GCF samples were collected from maxillary posterior sites with paper strips. The strips were pooled and eluted with high-purity water. After extraction, the azithromycin content of the serum samples and GCF eluates was determined with an agar diffusion bioassay. RESULTS: On days 2, 4, and 7, the concentrations of azithromycin in blood serum were 0.22 ± 0.02, 0.08 ± 0.02, and 0.04 ± 0.01 µg/mL, respectively. The concentrations in GCF were 8.82 ± 1.25, 7.90 ± 1.72, and 7.38 ± 1.15 µg/mL, respectively. Mean GCF levels were significantly higher than mean serum levels (P ≤0.02; paired t test). CONCLUSIONS: The findings demonstrate that the pharmacokinetic profiles of azithromycin are different in GCF and serum. At steady state, azithromycin concentrations in GCF were higher and more sustained than those in serum. Based on previous studies, the levels observed in GCF were above the minimal inhibitory concentration for Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans), Porphyromonas gingivalis, and Prevotella intermedia.

Memory T cells migrate to and reject vascularized cardiac allografts independent of the chemokine receptor CXCR3.

BACKGROUND: Memory T cells migrate to and reject transplanted organs without the need for priming in secondary lymphoid tissues, but the mechanisms by which they do so are not known. Here, we tested whether CXCR3, implicated in the homing of effector T cells to sites of infection, is critical for memory T-cell migration to vascularized allografts. METHODS: CD4 and CD8 memory T cells were sorted from alloimmunized CXCR3 and wildtype B6 mice and cotransferred to congenic B6 recipients of BALB/c heart allografts. Graft-infiltrating T cells were quantitated 20 and 72 hr later by flow cytometry. Migration and allograft survival were also studied in splenectomized alymphoplastic (aly/aly) recipients, which lack secondary lymphoid tissues. RESULTS: We found that polyclonal and antigen-specific memory T cells express high levels of CXCR3. No difference in migration of wildtype versus CXCR3 CD4 and CD8 memory T cells to allografts could be detected in wildtype or aly/aly hosts. In the latter, wildtype and CXCR3 memory T cells precipitated acute rejection at similar rates. Blocking CCR5, a chemokine receptor also upregulated on memory T cells, did not delay graft rejection mediated by CXCR3 memory T cells. CONCLUSIONS: CXCR3 is not critical for the migration of memory T cells to vascularized organ allografts. Blocking CXCR3 or CXCR3 and CCR5 does not delay acute rejection mediated by memory T cells. These findings suggest that the mechanisms of memory T cell-homing to transplanted organs may be distinct from those required for their migration to sites of infection.