Korean Red Ginseng aqueous extract improves markers of mucociliary clearance by stimulating chloride secretion.

BACKGROUND: Abnormal chloride (Cl-) transport has a detrimental impact on mucociliary clearance in both cystic fibrosis (CF) and non-CF chronic rhinosinusitis. Ginseng is a medicinal plant noted to have anti-inflammatory and antimicrobial properties. The present study aims to assess the capability of red ginseng aqueous extract (RGAE) to promote transepithelial Cl- secretion in nasal epithelium. METHODS: Primary murine nasal septal epithelial (MNSE) [wild-type (WT) and transgenic CFTR-/-], fisher-rat-thyroid (FRT) cells expressing human WT CFTR, and TMEM16A-expressing human embryonic kidney cultures were utilized for the present experiments. Ciliary beat frequency (CBF) and airway surface liquid (ASL) depth measurements were performed using micro-optical coherence tomography (µOCT). Mechanisms underlying transepithelial Cl- transport were determined using pharmacologic manipulation in Ussing chambers and whole-cell patch clamp analysis. RESULTS: RGAE (at 30µg/mL of ginsenosides) significantly increased Cl- transport [measured as change in short-circuit current (ΔISC = µA/cm2)] when compared with control in WT and CFTR-/- MNSE (WT vs control = 49.8±2.6 vs 0.1+/-0.2, CFTR-/- = 33.5±1.5 vs 0.2±0.3, p < 0.0001). In FRT cells, the CFTR-mediated ΔISC attributed to RGAE was small (6.8 ± 2.5 vs control, 0.03 ± 0.01, p < 0.05). In patch clamp, TMEM16A-mediated currents were markedly improved with co-administration of RGAE and uridine 5-triphosphate (8406.3 +/- 807.7 pA) over uridine 5-triphosphate (3524.1 +/- 292.4 pA) or RGAE alone (465.2 +/- 90.7 pA) (p < 0.0001). ASL and CBF were significantly greater with RGAE (6.2+/-0.3 µm vs control, 3.9+/-0.09 µm; 10.4+/-0.3 Hz vs control, 7.3 ± 0.2 Hz; p < 0.0001) in MNSE. CONCLUSION: RGAE augments ASL depth and CBF by stimulating Cl- secretion through CaCC, which suggests therapeutic potential in both CF and non-CF chronic rhinosinusitis.

Contribution of Short Chain Fatty Acids to the Growth of Pseudomonas aeruginosa in Rhinosinusitis.

Background: Chronic rhinosinusitis (CRS) is characterized by complex bacterial infections with persistent inflammation. Based on our rabbit model of sinusitis, blockage of sinus ostia generated a shift in microbiota to a predominance of mucin degrading microbes (MDM) with acute inflammation at 2 weeks. This was followed by conversion to chronic sinus inflammation at 3 months with a robust increase in pathogenic bacteria (e.g., Pseudomonas). MDMs are known to produce acid metabolites [short chain fatty acids (SCFA)] that have the potential to stimulate pathogen growth by offering a carbon source to non-fermenting sinus pathogens (e.g., Pseudomonas). The objective of this study is to evaluate the concentrations of SCFA within the mucus and its contribution to the growth of P. aeruginosa. Methods: Healthy and sinusitis mucus from the rabbit model were collected and co-cultured with the PAO1 strain of P. aeruginosa for 72 h and colony forming units (CFUs) were determined with the targeted quantification of three SCFAs (acetate, propionate, butyrate). Quantification of SCFAs in healthy and sinusitis mucus from patients with P. aeruginosa was also performed via high performance liquid chromatography. Results: To provide evidence of fermentative activity, SCFAs were quantified within the mucus samples from rabbits with and without sinusitis. Acetate concentrations were significantly greater in sinusitis mucus compared to controls (4.13 ± 0.53 vs. 1.94 ± 0.44 mM, p < 0.01). After 72 h of co-culturing mucus samples with PAO1 in the presence of mucin medium, the blue-green pigment characteristic of Pseudomonas was observed throughout tubes containing sinusitis mucus. CFUs were higher in cultures containing mucus samples from sinusitis (8.4 × 109 ± 4.8 × 107) compared to control (1.4 × 109 ± 2.0 × 107) or no mucus (1.5 × 109 ± 2.1 × 107) (p < 0.0001). To provide evidence of fermentative activity in human CRS with P. aeruginosa, the presence of SCFAs in human mucus was analyzed and all SCFAs were significantly higher in CRS with P. aeruginosa compared to controls (p < 0.05). Conclusion: Given that SCFAs are solely derived from bacterial fermentation, our evidence suggests a critical role for mucin-degrading bacteria in generating carbon-source nutrients for pathogens. MDM may contribute to the development of recalcitrant CRS by degrading mucins, thus providing nutrients for potential pathogens like P. aeruginosa.

Prevalence of chronic rhinosinusitis in bronchiectasis patients suspected of ciliary dyskinesia.

BACKGROUND: Mucociliary clearance is a main defense mechanism of the airway and is impaired in ciliary dyskinesia. The objective of this study was to evaluate the prevalence of chronic rhinosinusitis (CRS) and its characteristics in bronchiectasis patients suspected of harboring ciliary dyskinesia. METHODS: Bronchiectasis patients referred to a rhinology clinic for nasal brush biopsy (NBB) were included in this study. NBB was performed using a curettage technique whereby ciliated epithelial cells were obtained from the surface of the inferior nasal turbinate. Results of transmission electron microscopy findings, primary ciliary dyskinesia (PCD) gene (35 genes) analyses (Invitae), and sinus computed tomography (CT) scans were reviewed. RESULTS: Twenty-three patients (age, 54 ± 2.9 years) were referred for NBB between 2015 and 2018. Thirteen patients (56.5%) met the criteria for diagnosis of CRS. Nineteen patients had ciliary ultrastructural defects. The most common finding was compound cilia (n = 11, 47.8%). Five patients (21.7%) had central microtubule defects (CMD) with higher forced expiratory volume in 1 second (FEV1 ) at the time of referral than those without CMD (CMD+ , 91 ± 3.7%; CMD- , 73.5 ± 5.7%; p = 0.023). Of 15 subjects with a PCD gene panel, 67% (9 of 15) carried at least 1 gene associated with PCD. Only 1 patient reached diagnosis of PCD. Approximately 50% of non-PCD carriers had a smoking history (p < 0.05). Lund-Mackay scores did not significantly differ between PCD and non-PCD carriers (p = 0.72). CONCLUSION: Nearly half of bronchiectasis patients referred for NBB had concurrent CRS. The presence of ciliary abnormalities was not amplified in bronchiectasis patients with CRS compared to those without CRS. Extrinsic factors may be related to ciliary structural abnormalities in non-PCD gene carriers.

In-vitro evaluation of a ciprofloxacin- and ivacaftor-coated sinus stent against Pseudomonas aeruginosa biofilms.

BACKGROUND: We recently developed a novel ciprofloxacin-coated sinus stent capable of releasing antibiotics over a sustained period of time. Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator that has synergistic bactericidal activity with ciprofloxacin and also enhances sinus mucociliary clearance. The objective of this study was to optimize and evaluate the efficacy of a ciprofloxacin- and ivacaftor-releasing biodegradable sinus stent (CISS) in vitro. METHODS: A CISS was created by coating ciprofloxacin/ivacaftor-embedded nanoparticles with an acrylate and ammonium methacrylate copolymer onto a biodegradable poly-L-lactic acid stent. In-vitro evaluation of the CISS included: (1) assessment of drug stability in nanoparticles by zeta potential, and drug-coating stability within the CISS using scanning electron microscopy (SEM); (2) determination of ciprofloxacin- and ivacaftor-release kinetics; and (3) assessment of anti-Pseudomonas aeruginosa biofilm formation by calculating relative optical density units (RODUs) compared with control stents at 590-nm optical density. RESULTS: The presence of drugs and a uniform coating on the stent were confirmed by zeta potential and SEM. Sustained drug release was observed through 21 days without an initial burst release. Anti-biofilm formation was observed after placing the CISS for 3 days onto a preformed 1-day P aeruginosa biofilm. The CISS significantly reduced biofilm mass compared with bare stents and controls (RODUs at 590-nm optical density; CISS, 0.31 ± 0.01; bare stent, 0.78 ± 0.12; control, 1.0 ± 0.00; p = 0.001; n = 3). CONCLUSION: The CISS maintains a uniform coating and sustained delivery of drugs providing a marked reduction in P aeruginosa biofilm formation. Further studies evaluating the efficacy of CISS in a preclinical model are planned.

l-Methionine anti-biofilm activity against Pseudomonas aeruginosa is enhanced by the cystic fibrosis transmembrane conductance regulator potentiator, ivacaftor.

BACKGROUND: Biofilms may contribute to refractory chronic rhinosinusitis (CRS), as they lead to antibiotic resistance and failure of effective clinical treatment. l-Methionine is an amino acid with reported biofilm-inhibiting properties. Ivacaftor is a cystic fibrosis transmembrane conductance regulator (CFTR) potentiator with mild antimicrobial activity via inhibition of bacterial DNA gyrase and topoisomerase IV. The objective of this study was to evaluate whether co-treatment with ivacaftor and l-methionine can reduce the formation of Pseudomonas aeruginosa biofilms. METHODS: P aeruginosa (PAO-1 strain) biofilms were studied in the presence of l-methionine and/or ivacaftor. For static biofilm assays, PAO-1 was cultured in a 48-well plate for 72 hours with stepwise combinations of these agents. Relative biofilm inhibitions were measured according to optical density of crystal violet stain at 590 nm. Live/dead assays (BacTiter-Glo™ assay, Promega) were imaged with laser scanning confocal microscopy. An agar diffusion test was used to confirm antibacterial effects of the drugs. RESULTS: l-Methionine (0.5 µM) significantly reduced PAO-1 biofilm mass (32.4 ± 18.0%; n = 4; p < 0.001) compared with controls. Low doses of ivacaftor alone (4, 8, and 12 µg/mL) had no effect on biofilm formation. When combined with ivacaftor (4 µg/mL), a synergistic anti-biofilm effect was noted at 0.05 µM and 0.5 µM of l-methionine (two-way analysis of variane, p = 0.0415) compared with corresponding concentrations of l-methionine alone. CONCLUSION: Ivacaftor enhanced the anti-biofilm activity of l-methionine against the PAO-1 strain of P aeruginosa. Further studies evaluating the efficacy of ivacaftor/l-methionine combinations for P aeruginosa sinusitis are planned.

Preclinical therapeutic efficacy of the ciprofloxacin-eluting sinus stent for Pseudomonas aeruginosa sinusitis.

BACKGROUND: The ciprofloxacin-coated sinus stent (CSS) has unique therapeutic potential to deliver antibiotics to the sinuses. The objective of this study is to evaluate the efficacy of the CSS stent in eliminating Pseudomonas aeruginosa infection in a rabbit model of sinusitis. METHODS: A ciprofloxacin-eluting sinus stent was created by coating ciprofloxacin/Eudragit RS100 on biodegradable poly-D/L-lactic acid (2 mg). After analyzing in-vitro inhibition of P aeruginosa (PAO-1 strain) biofilm formation, a total of 8 stents (4 shams, 4 CSSs) were placed unilaterally in rabbit maxillary sinuses via dorsal sinusotomy after inducing infection for 1 week with PAO-1. Animals were assessed 2 weeks after stent insertion with nasal endoscopy, sinus culture, computed tomography (CT) scan, histopathology, and scanning electron microscopy (SEM). RESULTS: PAO-1 biofilm formation was significantly reduced in vitro with exposure to the CSS (p < 0.0001). Insertion of the stent in PAO-1-infected rabbits for 2 weeks resulted in significant improvement in sinusitis according to endoscopy scoring (p < 0.0001) and CT scoring (p < 0.002). Histology and SEM revealed marked improvement in the structure of the mucosa and submucosa with no detection of biofilm structures in the CSS cohort. CONCLUSION: Although this study had a small sample size, we identified robust therapeutic efficacy of the CSS by reducing bacterial load and biofilm formation of P aeruginosa in a preclinical model of sinusitis after placement for 2 weeks.