Inhibitory Effects Of Clarithromycin Research Articles

Clarithromycin suppresses IL-13-induced goblet cell metaplasia via the TMEM16A-dependent pathway in guinea pig airway epithelial cells.

Transmembrane protein 16A (TMEM16A) is associated with mucus secretion and ion transport in asthma. Clarithromycin (CAM) is reported to inhibit IL-13-induced goblet cell metaplasia. However, the effect of CAM on TMEM16A function and expression remains unclear. Tracheal epithelial cells from guinea pigs were cultured for ~14 days at an air-liquid interface in medium containing IL-13 (10 ng/ml) in the absence or presence of CAM (20 µg/ml) or a TMEM16A inhibitor, T16Ainh-A01 (10 µg/ml). Electrophysiological studies were performed by Ussing׳s short-circuit technique. The cells were used for immunofluorescence staining with antibodies against TMEM16A, MUC5AC, and α-tubulin. The cells were also examined by transmission electron microscopy. TMEM16A protein levels in the cell lysates were determined by ELISA. For the in vivo study, guinea pigs were treated intratracheally with IL-13 in the absence or presence of CAM or T16Ainh-A01. CAM decreased the MUC5AC-positive cells and reduced TMEM16A expression in them and increased the α-tubulin-positive cells. CAM inhibited TMEM16A protein levels in a dose-dependent manner, and decreased UTP-induced Cl ion transport. In cells treated with IL-13 for 24 h, TMEM16A appeared prior to MUC5AC protein expression, and was inhibited by CAM. In the in vivo study, CAM inhibited IL-13-induced goblet cell metaplasia and TMEM16A expression. The inhibitory effects of CAM were similar to those of T16Ainh-A01. CAM inhibited IL-13-induced TMEM16A expression, Cl ion transport and goblet cell metaplasia both in vitro and in vivo. CAM may thus improve airway mucociliary differentiation by attenuating TMEM16A expression in IL-13-related asthma.

Clarithromycin attenuates IL-13\u2013induced periostin production in human lung fibroblasts

BackgroundPeriostin is a biomarker indicating the presence of type 2 inflammation and submucosal fibrosis; serum periostin levels have been associated with asthma severity. Macrolides have immunomodulatory effects and are considered a potential therapy for patients with severe asthma. Therefore, we investigated whether macrolides can also modulate pulmonary periostin production.MethodsUsing quantitative PCR and ELISA, we measured periostin production in human lung fibroblasts stimulated by interleukin-13 (IL-13) in the presence of two 14-member–ring macrolides—clarithromycin or erythromycin—or a 16-member–ring macrolide, josamycin. Phosphorylation of signal transducers and activators of transcription 6 (STAT6), downstream of IL-13 signaling, was evaluated by Western blotting. Changes in global gene expression profile induced by IL-13 and/or clarithromycin were assessed by DNA microarray analysis.ResultsClarithromycin and erythromycin, but not josamycin, inhibited IL-13–stimulated periostin production. The inhibitory effects of clarithromycin were stronger than those of erythromycin. Clarithromycin significantly attenuated STAT6 phosphorylation induced by IL-13. Global gene expression analyses demonstrated that IL-13 increased mRNA expression of 454 genes more than 4-fold, while decreasing its expression in 390 of these genes (85.9%), mainly “extracellular,” “plasma membrane,” or “defense response” genes. On the other hand, clarithromycin suppressed 9.8% of the genes in the absence of IL-13. Clarithromycin primarily attenuated the gene expression of extracellular matrix protein, including periostin, especially after IL-13.ConclusionsClarithromycin suppressed IL-13–induced periostin production in human lung fibroblasts, in part by inhibiting STAT6 phosphorylation. This suggests a novel mechanism of the immunomodulatory effect of clarithromycin in asthmatic airway inflammation and fibrosis.

Does systemic clarithromycin therapy have an inhibitory effect on tympanosclerosis? An experimental animal study.

To demonstrate the inhibitory effects of clarithromycin on in vitro tympanosclerosis. Twenty-eight rats were divided into three groups: a clarithromycin group, a non-clarithromycin group and a negative control group. Those in the first two groups were injected with Streptococcus pneumoniae following a myringotomy, and tympanosclerosis was experimentally induced. Oral clarithromycin therapy was administered in the clarithromycin group. The other groups received no medical treatment. All eardrums in the clarithromycin and non-clarithromycin groups developed myringosclerosis, but there was only one eardrum, in the clarithromycin group, with very severe myringosclerosis. In the clarithromycin group, 11 ears showed no inflammation and there were no ears with severe inflammation. In the non-clarithromycin group, there were 11 ears with severe inflammation. The mean eardrum thickness in the clarithromycin group was 20.93 µm and in the non-clarithromycin group it was 42.71 µm. Acute otitis media and myringotomies induced tympanosclerosis, but clarithromycin reduced the severity of tympanosclerosis.

Effects of Clarithromycin at Sub-Minimum Inhibitory Concentrations on Early <i>erm</i>B Gene Expression, Metabolic Activity and Growth of an <i>erm</i>(B)-Expressing Macrolide-Resistant Strain of <i>Streptococcus pneumoniae</i>

Aim: To investigate the effects of exposure of a macrolide-resistant [erm (B)-expressing] strain of Streptococcus pneumoniae (strain 2507) to clarithromycin (0.5 and 5 mg/L) added at the outset and 6 hours after initiation of culture on early gene expression, energy metabolism, and growth. Methods: Bacterial growth was determined by turbidometric and colony counting procedures, energy metabolism by measurement of ATP, while analysis of gene expression was performed using reverse transcription-PCR and sequencing. Results: Addition of clarithromycin, at either concentration, at the outset of culture, caused transient suppression of growth of 10 - 12 hours duration, while delayed addition of antibiotic (during the logarithmic phase) resulted in an abrupt halt in growth followed by recovery. These inhibitory effects of clarithromycin on bacterial growth were associated with up-regulation of expression of erm(B), decreased ATP and protein synthesis, and were unaffected by inclusion of either catalase (500 and 1000 kunits/L), or competence-stimulating peptide (CSP-1, 0.5 mg/L). The inhibitory effects could, however, be overcome by pre-exposure of the bacteria to the antibiotic. Moreover, clarithromycin appeared to potentiate the antimicrobial actions of ceftriaxone, at sub-MIC concentrations, for strain 2507. Conclusions: Unlike several other common bacterial pathogens, the full expression of erm(B)-mediated macrolide resistance by the pneumococcus has a slow onset, which is associated with transient susceptibility to macrolides and inhibition of growth.

Macrolide antibiotics promote the LPS-induced upregulation of prostaglandin E receptor EP 2 and thus attenuate macrolide suppression of IL-6 production

We studied the influence of the inhibitory effect of clarithromycin (CAM) and erythromycin (EM) on the production of macrophage inflammatory protein (MIP)-2, interleukin-6 (IL-6), and prostaglandin E 2 (PGE 2), as well as PGE 2 receptor (EP 2) expression, by LPS-stimulated RAW264.7 cells. Production of IL-6 was significantly decreased by treatment with CAM or EM in a dose-dependent manner, but the inhibitory effect of CAM was significantly weaker than that of EM. In contrast, the production of MIP-2 and PGE 2 was inhibited to the same extent by CAM and EM. LPS induced the expression of EP 2 mRNA and its expression was promoted further by treatment with CAM or EM. In particular, CAM significantly upregulated EP 2 mRNA expression compared with that after stimulation by LPS alone. After treatment with a nonselective cyclooxygenase (COX) inhibitor (indomethacin), a selective COX-2 inhibitor (NS398), or an EP 2/EP 4 receptor antagonist (AH6809), the inhibitory effect of CAM and EM on LPS-induced IL-6 production was equalized. These results indicate that macrolide antibiotics upregulate the expression of EP 2, which then attenuates the suppressive effect on IL-6 production of these antibiotics, suggesting that these drugs have a variable anti-inflammatory effect that could influence host defenses.

Inhibitory effect of clarithromycin on costimulatory molecule expression and cytokine production by synovial fibroblast-like cells.

This study was undertaken to investigate the immunomodulatory effect of clarithromycin against synovial fibroblast-like cells (synoviocytes). Synovial tissue obtained from rheumatoid arthritis (RA) or osteoarthritis (OA) patients was enzymatically digested to separate synoviocytes. The synoviocytes were cultured with or without cytokines in the presence of various concentrations of clarithromycin. The expression of costimulatory molecules was examined on the surface of the synoviocytes, using specific MoAbs and flow cytometry. The production of cytokines by synoviocytes was also measured using an immunoenzymatic assay. Finally, autologous T cells were stimulated by interferon-gamma (IFN-gamma)-treated synoviocytes in response to purified protein derivative (PPD). In some experiments, MoAbs specific for costimulatory molecules or clarithromycin were added and 3H-thymidine incorporation was counted. Intercellular adhesion molecule-1 (ICAM-1), LFA-3 and vascular cell adhesion molecule-1 (VCAM-1) were detected on the surface of both RA and OA synoviocytes. However, ICAM-2, B7-1 and B7-2 were not detected, and cytokines failed to induce these molecules. Both spontaneous and up-regulated expression of ICAM-1, LFA-3 and VCAM-1 by IFN-gamma, IL-1beta or 12-o-tetradecanoyl phorbol 13-acetate (TPA) were markedly suppressed by clarithromycin in a dose-dependent manner at concentrations between 0.1 and 10 microg/ml. The production of IL-1beta, IL-6, IL-8, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) but not IL-1alpha and tumour necrosis factor-alpha (TNF-alpha) by synoviocytes was detected. Clarithromycin significantly suppressed the production of these cytokines, but did not enhance IL-10 production. Finally, autologous T cells were stimulated by IFN-gamma-treated synoviocytes in response to PPD. As clarithromycin suppressed HLA-DR and costimulatory molecule expression was enhanced by IFN-gamma, autologous T cell proliferation was markedly inhibited by clarithromycin. Clarithromycin has a considerable immunosuppressive effect on synoviocytes by inhibiting costimulatory molecule expression, cytokine production and antigen-specific T cell proliferation induced by synoviocytes.

EVIDENCE FOR THE VALIDITY OF CORTISOL 6β-HYDROXYLATION CLEARANCE AS A NEW INDEX FOR IN VIVO CYTOCHROME P450 3A PHENOTYPING IN HUMANS

This study uses stable isotope methodology to evaluate the validity of 6beta-hydroxylation clearance of endogenous cortisol as a new index for in vivo CYP3A phenotyping in humans. Important factors contradictory to the use of a conventional index of urinary ratio of 6beta-hydroxycortisol to cortisol (6beta-OHF/F) to evaluate in vivo CYP3A activity are also discussed. Stable isotopically labeled cortisol (3-5 mg) was orally administered to three healthy adult subjects to accurately determine the fractional metabolic clearance specific for the 6beta-hydroxylation of cortisol. Plasma concentrations of labeled cortisol and urinary excreted amounts of labeled cortisol and 6beta-OHF were analyzed by gas chromatography-mass spectrometry simultaneously with their endogenous counterparts. There was a good correlation between endogenous and exogenous 6beta-hydroxylation clearances in the three subjects tested (r = 0.7733, 0.9112, and 0.9534 for 2-, 4-, and 6- to 8-h urine collection periods, respectively). This strongly suggests that the endogenous 6beta-hydroxylation clearance can be used as an appropriate index for phenotyping the in vivo CYP3A activity. Furthermore, observed intra- (2.1- to 4.6-fold) and interindividual variabilities (ca. 5-fold) in the labeled cortisol renal clearance suggest that the urinary ratio 6beta-OHF/F, a function of 6beta-hydroxylation clearance and renal clearance of cortisol, does not always reflect the in vivo CYP3A activity. When a macrolide antibiotic, clarithromycin, was administered to a healthy volunteer in a dose of 200 mg every 12 h for 6 days, the inhibitory effects of clarithromycin on the in vivo CYP3A activity were clearly seen by the 6beta-hydroxylation clearance of endogenous cortisol but not by the urinary ratio 6beta-OHF/F.

The role of nuclear factor-kappa B in interleukin-8 expression by human adenoidal fibroblasts.

The production of cytokines by adenoids is known to be associated with inflammation of nasopharynx and the pathogenesis of otitis media with effusion. However, the role of adenoids in producing inflammatory cytokines such as interleukin-8 (IL-8) is not yet clear. In the present study, expression of IL-8 in adenoidal fibroblasts was investigated at the level of transcription factors. Further, the effects of clarithromycin, a 14-member ring macrolide, on IL-8 gene expression and nuclear factor-kappa B (NF-kappa B) activation in adenoidal fibroblasts were evaluated. In vitro study for the production of inflammatory cytokine from human adenoidal fibroblasts. Adenoidal fibroblasts were incubated with nontypeable Haemophilus influenzae endotoxin or interleukin-1 beta. Then the expression of IL-8 and the influence of NF-kappa B inhibitor and clarithromycin were evaluated. Interleukin-8 protein production was assessed by ELISA, and IL-8 messenger RNA production was measured by Northern blot analysis and reverse transcriptase-polymerase chain reaction. Activation of NF-kappa B and inhibition of its activation were determined by electrophoretic mobility shift assay. The expression of both IL-8 protein and messenger RNA in adenoidal fibroblasts was enhanced by Haemophilus influenzae endotoxin and interleukin-1 beta and was positively correlated with increases in NF-kappa B activity. Treatment of cells with the NF-kappa B inhibitor N-tosyl-(L)-phenylalanine chloromethyl ketone, as well as with clarithromycin, reduced expression of IL-8 and NF-kappa B activity in a dose-dependent manner. Results suggest that adenoidal fibroblasts produce IL-8 in response to endotoxin through NF-kappa B activation. The inhibitory effects of clarithromycin on NF-kappa B activation and IL-8 production in adenoidal fibroblasts might explain, in part, the mechanism of this drug in improving otitis media with effusion.

Inhibitory effect of macrolide antibiotics on biofilm formation by Pseudomonas aeruginosa

In recent years, various medical indwelling devices have been developed and used. Bacteria adhering to these devices often cause refractory infection. In the field of otolaryngology, refractory infection accompanying these medical indwelling devices such as middle ear ventilation tubes and artificial auditory ossicles has been reported. The concept of bacterial biofilm infection has been suggested as an explanation for the refractory infection. Furthermore it has been reported that a bacterial biofilm is involved in refractory infection unrelated to medical indwelling devices. Topical biofilm formation was detected in patients with chronic sinusitis, chronic purulent otitis media or habitual tonsillitis. In this study, we morphologically and quantitatively examined the biofilm-forming capacity of a clinically isolated strain of mucoid type Psecudomonas aeruginosa on Teflon to investigate the effects of macrolide antibiotics on bacterial biofilm formation. In the morphological examination, P.aeruginosa was cultured together with a Teflon sheet in minimal medium containing various concentrations of the macrolide antibiotics clarithromycin (CAM), erythromycin (EM) and midecamycin (MDM), at 37 degrees C for 7 days. The surfaces of the Teflon sheets were examined by electron microscopy. The adherent bacteria and biofilm formation on Teflon sheets soaked in minimal medium containing CAM or EM were found to be decreased in a dose-dependent manner. However, in the Teflon sheets soaked in minimal medium containing MDM, there was no decrease in biofilm formation regardless of the MDM concentration. In the quantitative examination, P. aeruginosa was cultured in minimal medium containing various concentrations of the macrolide antibiotics at 37 degrees C for 7 days together with Teflon beads. The levels of hexose, protein and alginate adhering to the Telfon beads were quantified as an estimation of biofilm formation. On Teflon beads treated with CAM or EM, there were dose-dependent decreases in hexose, protein and alginate levels. In particular, marked decreases were noted when CAM and EM concentrations were 10 micrograms/ml or more. Furthermore, there was no significant difference between CAM and EM. However, in the presence of MDM, there was no decrease in hexose, protein or alginate levels regardless of the MDM concentration. The minimal inhibitory concentration (MIC100) of each macrolide against P. aeruginosa used in this experiment was 100 micrograms/ml or more. There may be no bactericidal effect on this strain at the macrolide concentrations used in this experiment. However, this experiment used 7-day treatment. The long-term bactericidal activity of macrolides was examined. In the presence of CAM or MDM, bacterial levels after culture were similar to preculture levels or slightly lower than the preculture levels. In the presence of EM, bacterial levels were similar to the preculture levels. These results demonstrated that CAM and EM, which are 14-membered macrolides inhibited biofilm formation, while MDM which is 16-membered macrolide, did not. These inhibitory effects of CAM and EM may be related to actions other than bactericidal activity. In our experiment, CAM and EM inhibited biofilm formation at 10 micrograms/ml or more. This concentration corresponded to 1/20 x MIC. This concentration can be achieved in tissues, nasal discharge and sputum with actual clinical doses. Therefore, these agents may be effective against biofilm disease caused by P. aeruginosa in the field of otolaryngology.