ORCID Profile
0000-0003-4719-9785
Current Organisation
University of Adelaide
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Publisher: Wiley
Date: 30-01-2015
DOI: 10.1002/ALR.21459
Abstract: Treatment of recalcitrant chronic rhinosinusitis (CRS) is a challenge with increasing antibiotic resistance, leading to re-emergence of topical therapies. The aim of this study was to assess safety and efficacy of topical colloidal silver solution for the treatment of Staphylococcus aureus biofilms in a sheep model. In the safety study, normal saline (control) and 30-ppm colloidal silver solution (test) was used to flush the frontal sinuses for 14 days in 8 sheep (4 sheep each). In the efficacy study, following frontal sinus infection with Staphylococcus aureus, sheep were treated with either control saline or topical silver solution of varying concentrations (30 ppm/20 ppm/10 ppm/5 ppm) for 5 days, with 4 sheep in each group. Blood silver level, full blood counts, and biochemical parameters were analyzed in both safety and efficacy studies. Sinus tissue was harvested for histological examination and ciliary structure analysis in safety and for biofilm biomass quantification by fluorescence in situ hybridization (FISH) technique and COMSTAT 2 software in the efficacy study. Results were analyzed using appropriate statistical tests. Sheep treated with silver showed a significant decrease in biofilm biomass (0.004, 0.004, 0.004, and 0.007, in the 4 silver-treated groups, respectively) compared to saline control (0.175), p < 0.001. Although average blood silver levels were higher in the treated groups compared to controls (p < 0.05), blood counts and biochemical parameters were normal. Histology and ciliary structure analysis did not show any difference between control and treatment groups. Topical colloidal silver solution has effective antibiofilm activity in Staphylococcus aureus CRS in a sheep model and appears safe.
Publisher: Wiley
Date: 15-01-2014
DOI: 10.1002/ALR.21259
Abstract: Colloidal silver is an alternative medicine consisting of silver particles suspended in water. After using this solution as a nasal spray, the symptoms of a previously recalcitrant Staphylococcus aureus (S. aureus)-infected chronic rhinosinusitis patient were observed to have improved markedly. The aim of this study was to determine whether colloidal silver has any direct bactericidal effects on these biofilms in vitro. S. aureus biofilms were grown from the ATCC 25923 reference strain on Minimum Biofilm Eradication Concentration (MBEC) device pegs, and treated with colloidal silver. Concentrations tested ranged from 10 to 150 μL colloidal silver diluted to 200 μL with sterile water in 50 μL cerebrospinal fluid (CSF) broth. Control pegs were exposed to equivalent volumes of CSF broth and sterile water. The s le size was 4 biomass values per treatment or control group. Confocal scanning laser microscopy and COMSTAT software were used to quantify biofilms 24 hours after treatment. Significant differences from control were found for all concentrations tested bar the lowest of 10 μL colloidal silver in 200 μL. At 20 μL colloidal silver, the reduction in biomass was 98.9% (mean difference between control and treatment = -4.0317 μm(3) /μm(2) , p < 0.0001). A maximum biomass reduction of 99.8% was reached at both 100 and 150 μL colloidal silver (mean differences = -4.0681 and -4.0675μm(3) /μm(2) , respectively, p < 0.0001). Colloidal silver directly attenuates in vitro S. aureus biofilms.
Publisher: Wiley
Date: 25-02-2016
DOI: 10.1002/ALR.21735
Abstract: Biofilms are clusters of bacteria embedded in a protective matrix that frequently cause failure of medical treatments and increase the risk of recurrent infections. In particular, Staphylococcus aureus biofilms are associated with a series of chronic and nosocomial infections that are increasingly resistant to antibiotics. This study proposes a novel intervention strategy targeting the essential iron metabolism for bacterial growth, survival and pathogenesis using the compounds deferiprone (Def) and gallium-protoporphyrin (GaPP). S. aureus biofilms were challenged with Def/GaPP as single and dual treatments. In vitro anti-biofilm efficacy was assessed by the AlamarBlue viability assay and confocal microscopy. In vitro cytotoxicity of the treatments was examined by the lactate dehydrogenase assay on mouse fibroblast (L929) and human bronchial epithelial cells (Nuli-1). Def (20 mM) and GaPP (200 μg/mL) monotherapy for 2 hours showed 35% and 74% biofilm removal, respectively, whereas simultaneous Def/GaPP administration showed 55% biofilm removal. In contrast, the consecutive treatment (2 hours Def followed by 2 hours GaPP) achieved 95% biofilm removal. Cytotoxicity studies indicated no cell hazard in all treatments. This study demonstrated the in vitro efficacy of a novel treatment combination against S. aureus biofilms targeting the bacterial iron metabolism. The consecutive Def/GaPP treatment showed significantly enhanced biofilm efficacy than the in idual compounds, while being not toxic to 2 cell lines. This novel treatment combination is a promising approach to combat S. aureus-associated biofilm infections having high potential for future clinical application.
Publisher: SAGE Publications
Date: 11-2012
DOI: 10.2500/AJRA.2012.26.3822
Abstract: The emerging concept of intracellular pathogens such as Staphylococcus aureus playing a role in chronic rhinosinusitis (CRS) has led to the development of numerous imaging techniques for their identification. Traditional methods of bacterial culture are not effective at localizing bacteria to the surface or within tissue s les. The aim of this study was to develop and validate a novel imaging technique using confocal scanning laser microscopy (CSLM) coupled with a fluorescence in situ hybridization (FISH) probe and nucleic acid counterstain (propidium iodide [PI]) that allows for simultaneous analysis of S. aureus intracellular status and surface biofilm within whole mucosal s les. A prospective study was performed including 17 patients undergoing endoscopic sinus surgery for CRS. Tissue s les were analyzed with both CSLM-FISH/PI and immunohistochemistry (IHC) for intracellular S. aureus status. Using CSLM-FISH/PI intracellular S. aureus was identified in 9/17 (47%) patients and in 7/17 (39%) using IHC. Surface biofilm can be identified with CSLM-FISH/PI in the same piece of tissue however, deeper imaging to the submucosa is impossible. IHC showed submucosal bacteria in three patients. Both CSLM-FISH/PI and IHC are complementary techniques that can be used to identify intracellular S. aureus. CSLM-FISH/PI allows for the simultaneous detection of intracellular status and surface biofilm within the tissue analyzed. IHC has a role in the identification of intracellular and submucosal S. aureus within these tissues. Additional investigation is required to identify the true pathogenic nature of intracellular organisms as well as any relationship to surface biofilm status.
Publisher: Wiley
Date: 21-08-2018
DOI: 10.1111/ALL.13580
Publisher: Public Library of Science (PLoS)
Date: 30-06-2015
Publisher: Elsevier BV
Date: 04-2017
Publisher: Wiley
Date: 13-06-2014
DOI: 10.1111/ALL.12440
Abstract: Group 2 innate lymphoid cells (ILC2s) were shown to be involved in the initiation and coordination of Th2-type immune responses in allergic disease animal models. Recently, ILC2s enrichment was noted in chronic rhinosinusitis (CRS) patients however, the role of ILC2s in coordinating the Th2 response in CRS remains to be elucidated. Here, we characterize the ILC2 compartment in CRS by investigating the correlations between ILC2s, Th2 cells and Th2 cytokines expression in CRS patients. We used flow cytometric analysis of sinonasal mucosal tissues of 29 CRS patients and 5 controls to quantify ILC2s and Th2 cells. Messenger RNA expression levels of IL-5, IL-13, IL-25, IL-33, TSLP and GATA3 were determined using qRT-PCR. ILC2s were significantly enriched in nasal polyps (CRSwNP) patients. Multivariate linear regression showed a significant positive association of ILC2 numbers with CRSwNP and allergic CRS and a negative association with the number of previous endoscopic sinus surgeries. Group 2 innate lymphoid cell numbers significantly correlated with Th2 cell frequencies. Messenger RNA expression levels of IL-5 and IL-13 were increased in CRSwNP compared with controls, while mRNA levels of IL-25 and GATA3 were significantly reduced. Our results characterize the complex interactions between ILC2s and other Th2 response elements in the context of CRS and suggest that ILC2 enrichment occurs in CRSwNP and in allergic CRS patients.
Publisher: Wiley
Date: 02-04-2019
DOI: 10.1111/ALL.13772
Publisher: The Company of Biologists
Date: 15-08-2022
DOI: 10.1242/BIO.059267
Abstract: Patient-derived organoids grown in three-dimensional cultures provide an excellent platform for phenotypic high-throughput screening and drug-response research. Organoid technology has been applied to study stem cell biology and various human pathologies. This study investigates the characteristics and cellular morphology of organoids derived from primary human nasal epithelial cells (HNECs) of chronic rhinosinusitis (CRS) patients. Nasal organoids were cultured up to 20 days and morphological, cell composition and functional parameters were measured by immunofluorescence, RT-qPCR, western blot and FACS analysis. The results showed that nasal organoids expressed the stem cell marker leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5), and markers for apical junction genes, goblet cells and ciliated cells. Moreover, we were able to regrow and expand the nasal organoids well after freezing and thawing. This study provides an effective and feasible method for development of human nasal organoids, suitable for the phenotypic high-throughput screening and drug response research.
Publisher: Wiley
Date: 09-07-2018
DOI: 10.1111/ALL.13532
Publisher: Public Library of Science (PLoS)
Date: 21-03-2014
Publisher: Springer Science and Business Media LLC
Date: 09-07-2006
DOI: 10.1038/NCB1440
Abstract: Homologues of signal peptide peptidase (SPPLs) are putative aspartic proteases that may catalyse regulated intramembrane proteolysis of type II membrane-anchored signalling factors. Here, we show that four human SPPLs are each sorted to a different compartment of the secretory pathway. We demonstrate that SPPL2a and SPPL2b, which are sorted to endosomes and the plasma membrane, respectively, are functional proteases that catalyse intramembrane cleavage of tumour necrosis factor alpha (TNFalpha). The two proteases promoted the release of the TNFalpha intracellular domain, which in turn triggers expression of the pro-inflammatory cytokine interleukin-12 by activated human dendritic cells. Our study reveals a critical function for SPPL2a and SPPL2b in the regulation of innate and adaptive immunity.
Publisher: Wiley
Date: 05-02-2014
DOI: 10.1002/ALR.21297
Abstract: Our understanding of fungi in chronic rhinosinusitis (CRS) has been limited by previously employed detection techniques. This study examines the fungal component of the microbiome in CRS patients and controls using a highly sensitive culture-independent molecular technique. The aims of this study include the characterization of fungal richness, prevalence, abundance, temporal changes, and their relationship with patient outcomes. Swabs were collected from the sinuses of 23 CRS patients and 11 controls. Collection occurred intraoperatively, and at 6 and 12 weeks postoperatively. DNA was extracted from the swabs and fungal outcomes were determined through 18S ribosomal DNA (rDNA) fungal tag-encoded FLX licon pyrosequencing. Fungi were ubiquitous to all patients. A total of 207 fungal genera were detected, with a mean s le richness of 8.18 and 12.14 in the control and CRS groups, respectively. Malassezia was detected in all patients at surgery and was also the most abundant. Postoperatively, fungal richness decreased (p < 0.05) and was associated with declines in the prevalence of Fusarium and Neocosmospora (p < 0.05). Neocosmospora was also less abundant postoperatively (p < 0.05). No correlations were found with quality of life. This is the first study to use a highly sensitive pyrosequencing technique to reveal the true ersity of fungi in the sinuses of CRS patients and postoperative changes in richness. The presence of Malassezia, a genus not previously described in the sinuses, is of great interest, and its potential as a disease modifier should see further investigation given its association with atopic disease.
Publisher: Wiley
Date: 26-08-2013
DOI: 10.1002/ALR.21213
Abstract: The presence of Staphylococcus aureus biofilms on sinonasal mucosal surfaces is associated with recalcitrant chronic rhinosinusitis (CRS), but little is known about the innate immune response they trigger. We aimed to study the human pattern recognition receptor (PRR) nucleotide-binding oligomerization domain containing 2 (Nod2) receptor and downstream pathway in response to initial S. aureus biofilm infection. Using a validated protocol, sinonasal mucosae from 4 non-CRS donors were cultured with and without S. aureus biofilms and planktonic cells. After 24 hours, RNA was extracted and gene expression was analyzed using a human antibacterial response polymerase chain reaction (PCR) array. Immunohistochemistry was performed to confirm the presence and determine the immunolocalization of selected proteins. C-X-C motif (CXC) chemokine ligands 1 and 2, interleukin-6 (IL-6), and genes related to the Nod2 pathway were significantly upregulated in biofilm-treated tissues compared with control s les. Nod2 pathway-specific gene expression was increased in biofilm-treated tissues compared with planktonic S. aureus-treated explants. Enhanced expression of Nod2 and nuclear factor kappa B1 (NF-κB1) was also detected with immunohistochemistry in control and biofilm-treated tissues. S. aureus biofilms exerted a proinflammatory response in the mucosa and activation of the Nod2 pathway, indicating this receptor to be involved in the innate immune response to S. aureus biofilms. Further studies are required to elucidate the role of this pathway in CRS.
Publisher: Wiley
Date: 23-06-2016
DOI: 10.1002/LARY.26128
Abstract: The adenoid pad has been considered a reservoir for bacteria in the pathogenesis of otitis media with effusion. This study aimed to characterize the middle ear microbiota in children with otitis media with effusion and establish whether a correlation exists between the middle ear and adenoid microbiota. Prospective, controlled study. Middle ear aspirates adenoid pad swabs were collected from 23 children undergoing ventilation tube insertion. Adenoid swabs from patients without ear disease were controls. S les were analyzed using 16S rRNA sequencing on the Illumina MiSeq platform. Thirty-five middle ear s les were collected. The middle ear effusion microbiota was dominated by Alloiococcus otitidis (23% mean relative abundance), Haemophilus (22%), Moraxella (5%), and Streptococcus (5%). Alloiococcus shared an inverse correlation with Haemophilus (P = .049) and was found in greater relative abundance in unilateral effusion (P = .004). The microbiota of bilateral effusions from the same patient were similar (P .05) (permutational multivariate analysis of the variance). Dissimilarities between the local microbiota of the adenoid and the middle ear question the theory that the adenoid pad is a significant reservoir to the middle ear in children with otitis media with effusion. A otitidis had the greatest cumulative relative abundance, particularly in unilateral effusions, and shares an inverse correlation with the relative abundance of Haemophilus. NA Laryngoscope, 126:2844-2851, 2016.
Publisher: Wiley
Date: 25-03-2016
DOI: 10.1002/ALR.21742
Abstract: Chronic rhinosinusitis (CRS) is categorized into 2 types based on the absence (CRSsNP) and presence of nasal polyps (CRSwNP). Although CRSsNP patients lack nasal polyps, the mucosa may show variable degrees of polypoid change. This raises the question of whether or not the classification system is an over simplification and that CRSsNP and CRSwNP only represent 2 phenotypic extremes along a broader spectrum of immunologically different disease processes. To investigate this, adaptive and innate immune cells were compared in the different tissue types within CRSsNP and CRSwNP patients. Tissue from 15 CRSwNP, 6 CRSsNP, and 8 healthy control patients was obtained prospectively. Nonpolypoid mucosa, polypoid tissues, and polyps were obtained at the time of endoscopic sinus surgery and analyzed using flow cytometry for various adaptive and innate immune cell subsets. In the polyps from CRSwNP patients there were significantly more T regulatory (Treg) cells (12.86 ± 12.60 vs 2.83 ± 4.68) and Th17 cells (16.12 ± 11.75 vs 2.31 ± 2.13) compared to the polypoid tissue from CRSsNP patients. Cellular infiltrates in the nonpolypoid or polypoid mucosa of the different patient categories showed no difference in CRSwNP, CRSsNP and control groups. This observational study identified an increase in Treg and Th17 cells in CRSwNP patients implying that these cells may be implicated in polyp development. Importantly it also identified a similar inflammatory infiltrate in nonpolyp or polypoid mucosa across control, CRSsNP, and CRSwNP groups inferring that polyps should be s led when studying CRSwNP.
Publisher: Wiley
Date: 10-2014
DOI: 10.1002/ALR.21423
Abstract: Staphylococcus aureus infection is known to play a role in recalcitrant chronic rhinosinusitis (CRS). However, it is unknown if recurrent S. aureus infections are caused by the same strain or are due to independent acquisitions of different strains. S les were collected from patients with CRS from July 2011 to August 2012. S. aureus was isolated from mucosal swabs and tissue specimens from patients who underwent surgery during the study period, or from swabs of areas of purulence taken in the postoperative period under endoscopic guidance. Pulsed-field gel electrophoresis was used to characterize S. aureus isolates. Thirty-four patients were included in the study 79% showed persistence of the same S. aureus strain in their paranasal sinuses (p = 0.001 H1 ≠ 50%). Furthermore, a significantly high frequency of patients with known biofilm status were positive for S. aureus biofilm (p = 0.002 H1 ≠ 50%). When patients were stratified according to disease evolution postsurgery, certain strains appeared to be more commonly associated with symptom persistence. The same S. aureus strain appears to persist in the paranasal sinuses of CRS patients despite multiple courses of culture-directed antibiotics. This suggests that conventional antimicrobial therapies in patients with CRS may not eliminate the organism. This may be partly explained by the formation of biofilms in the paranasal sinus region.
Publisher: Springer Science and Business Media LLC
Date: 19-02-2002
DOI: 10.1038/NG848
Publisher: Elsevier BV
Date: 12-2015
DOI: 10.1016/J.MOLIMM.2015.09.024
Abstract: The present study describes and compares functional properties of Nuli-1 cells and primary human nasal epithelial cells (HNEC) including TLR expression and function. Differences in gene expression were identified for non-TLR genes that play a role in TLR response pathways. However, experiments comparing TLR gene expression for both Nuli-1 cells and HNECs indicated conserved expression in both cell types. Stimulation of the two cell types resulted in a conserved response to TLR3 agonists, but in differences in response to agonists for TLR5 and TLR6/2. HNECs were much more susceptible to infection with Staphylococcus aureus than NuLi-1 cells. Furthermore, when cultured at air-liquid interface (ALI), NuLi-1 cells possessed much lower trans-epithelial resistance than primary HNEC and did not exhibit maintenance of cell morphology or mucous production which was observed in HNECs. Nor did they produce the characteristic interconnecting pattern of tight junction complexes at the apicolateral margin of adjacent cells. Caution should therefore be exercised when selecting cell lines for immunological studies and a thorough screen of properties relevant to the study should always be carried out prior to commencement.
Publisher: Hindawi Limited
Date: 2016
DOI: 10.1155/2016/9798206
Abstract: Cytokine mediated changes in paracellular permeability contribute to a multitude of pathological conditions including chronic rhinosinusitis (CRS). The purpose of this study was to investigate the effect of interferons and of Th1, Th2, and Th17 cytokines on respiratory epithelium barrier function. Cytokines and interferons were applied to the basolateral side of air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs) from CRS with nasal polyp patients. Transepithelial electrical resistance (TEER) and permeability of FITC-conjugated dextrans were measured over time. Additionally, the expression of the tight junction protein Zona Occludens-1 (ZO-1) was examined via immunofluorescence. Data was analysed using ANOVA, followed by Tukey HSD post hoc test. Our results showed that application of interferons and of Th1 or Th2 cytokines did not affect the mucosal barrier function. In contrast, the Th17 cytokines IL-17, IL-22, and IL-26 showed a significant disruption of the epithelial barrier, evidenced by a loss of TEER, increased paracellular permeability of FITC-dextrans, and discontinuous ZO-1 immunolocalisation. These results indicate that Th17 cytokines may contribute to the development of CRSwNP by promoting a leaky mucosal barrier.
Publisher: SAGE Publications
Date: 2014
DOI: 10.2500/AJRA.2014.28.4001
Abstract: Staphylococcus aureus is the most common organism in recalcitrant chronic rhinosinusitis (CRS) and is often resistant to traditional antibiotic therapy. Bacteriophages (“phages”) are a potential candidate for a new, effective therapy. For phages to be useful in setting CRS, two minimum requirements must be presented: (1) phages must be effective against S. aureus biofilms and (2) phages must have a broad spectrum of activity. This study aimed to assess the in vitro activity of a phage cocktail (CockTail of Staphylococcus aureus specific bacteriophage [CT-SA]) against S. aureus biofilms and a broad panel of strains isolated from patients with CRS. The study examined 66 clinical isolates (CIs) of S. aureus. All isolates were tested for the susceptibility to phage lysis by spotting CT-SA onto bacterial lawns. To measure its effect on S. aureus biofilms, a minimum biofilm eradication concentration assay was used, using five S. aureus isolates. Biofilms of these isolates were grown, treated with CT-SA for 48 hours, fluorescently stained, and viewed using confocal scanning laser microscopy. CT-SA lysed 62 of 66 (94%) CIs of S. aureus. CT-SA treatment yielded significant reductions in biofilm mass for 4/5 CIs tested and for ATCC 25923. Challenge of S. aureus with a single phage resulted in the emergence of bacteriophage-insensitive mutants (BIM) with a frequency of 10 −7 , and challenge with CT-SA completely prevented their development. This study indicates that phage cocktail CT-SA can effectively eliminate S. aureus, in planktonic and biofilm forms, from the great majority of CIs from this hospital setting. In addition, its potential effect in preventing the emergence BIMs was a established. Thus, CT-SA has the potential to treat S. aureus infection and biofilm in CRS patients.
Publisher: Wiley
Date: 10-01-2014
DOI: 10.1002/ALR.21264
Abstract: Bacterial biofilms are thought to contribute to recalcitrance in chronic rhinosinusitis (CRS) patients. Manuka honey (MH) and its active component methylglyoxal (MGO) have demonstrated antibiofilm activity in vitro. This study evaluated the safety and efficacy of these agents in an in vivo model. To assess safety, ovine frontal sinuses were flushed twice daily for 14 days. In each sheep, 1 sinus was flushed with a panel of MGO concentrations ranging from 0.5 to 7.2 mg/mL alone and flushed with a panel of with 16.5% wt/vol MH enriched with MGO at the same range of concentrations (0.5-7.2 mg/mL designated MH/MGO). Contralateral sinuses were flushed with saline control. Tissue morphology was assessed histologically and with scanning electron microscopy. Efficacy was tested by developing Staphylococcus aureus biofilms in sheep sinuses. Twice-daily irrigation for 5 days was commenced with either saline, MGO (0.5-3.6 mg/mL) alone, or MH/MGO (with 0.5-3.6 mg/mL MGO). Biofilm biomass was compared between the groups (n = 4) using LIVE/DEAD BacLight staining and confocal scanning laser microscopy. The results of the safety assessment, for normal sinuses treated with MGO alone or with MH/MGO (≤1.8 mg/mL) showed normal pseudostratified epithelium and cilia structure however, higher concentrations caused cilia denudation and squamous metaplasia. As for efficacy, when compared to saline flush, treatment with MH/MGO at 0.9 mg/mL (0.608 ± 0.110 vs 0.316 ± 0.197 μm(3) /μm(2) , respectively p = 0.015) and 1.8 mg/mL (0.676 ± 0.079 vs 0.114 ± 0.033 μm(3) /μm(2) , respectively p = 0.001) significantly reduced biofilm biomass. Sinus irrigation with MH/MGO at MGO concentrations between 0.9 and 1.8 mg/mL is both safe to mucosa and efficacious against S. aureus biofilm. MH/MGO irrigation could represent a viable treatment option for recalcitrant CRS.
Publisher: Elsevier BV
Date: 10-2012
DOI: 10.1016/J.JIM.2012.07.015
Abstract: Immunofluorescence is a fundamental tool used to analyse tissue and cell s les with a wide variety of available antibodies targeting specific proteins or molecules. Staphylococcal surface protein A is used both in clinical, research and industrial settings for its ability to bind mammalian immunoglobulin G. Spurious binding between protein A and IgG antibodies can lead to false-positive fluorescence and misleading results. Here we demonstrate this occurring in formalin-fixed patient s les that harbour Staphylococcus aureus infection, and characterise methods to overcome this issue. Specifically the use of F(ab') fragment antibodies or blocking with human IgG is shown to prevent antibody-protein A interaction in formalin-fixed S. aureus smears, biopsies obtained from infected patients, and experimentally infected tissue s les.
Publisher: Wiley
Date: 24-07-2020
DOI: 10.1111/ALL.13964
Publisher: Microbiology Society
Date: 15-12-2021
Abstract: Prophages affect bacterial fitness on multiple levels. These include bacterial infectivity, toxin secretion, virulence regulation, surface modification, immune stimulation and evasion and microbiome competition. Lysogenic conversion arms bacteria with novel accessory functions thereby increasing bacterial fitness, host adaptation and persistence, and antibiotic resistance. These properties allow the bacteria to occupy a niche long term and can contribute to chronic infections and inflammation such as chronic rhinosinusitis (CRS). In this study, we aimed to identify and characterize prophages present in Staphylococcus aureus from patients suffering from CRS in relation to CRS disease phenotype and severity. Prophage regions were identified using PHASTER. Various in silico tools like ResFinder and VF Analyzer were used to detect virulence genes and antibiotic resistance genes respectively. Progressive MAUVE and maximum likelihood were used for multiple sequence alignment and phylogenetics of prophages respectively. Disease severity of CRS patients was measured using computed tomography Lund–Mackay scores. Fifty-eight S. aureus clinical isolates (CIs) were obtained from 28 CRS patients without nasal polyp (CRSsNP) and 30 CRS patients with nasal polyp (CRSwNP). All CIs carried at least one prophage (average=3.6) and prophages contributed up to 7.7 % of the bacterial genome. Phage integrase genes were found in 55/58 (~95 %) S. aureus strains and 97/211 (~46 %) prophages. Prophages belonging to Sa3int integrase group (phiNM3, JS01, phiN315) (39/97, 40%) and Sa2int (phi2958PVL) (14/97, 14%) were the most prevalent prophages and harboured multiple virulence genes such as sak, scn, chp, luk E/D, sea . Intact prophages were more frequently identified in CRSwNP than in CRSsNP ( P =0.0021). Intact prophages belonging to the Sa3int group were more frequent in CRSwNP than in CRSsNP ( P =0.0008) and intact phiNM3 were exclusively found in CRSwNP patients ( P =0.007). Our results expand the knowledge of prophages in S. aureus isolated from CRS patients and their possible role in disease development. These findings provide a platform for future investigations into potential tripartite associations between bacteria-prophage-human immune system, S. aureus evolution and CRS disease pathophysiology.
Publisher: Wiley
Date: 12-02-2013
DOI: 10.1002/ALR.21146
Abstract: Staphylococcus aureus (S. aureus) biofilm has been associated with severe and recalcitrant cases of chronic rhinosinusitis (CRS). However, its role in the pathophysiology of this condition is not completely understood. This study aims to develop a sinonasal tissue explant model to analyze the interaction of S. aureus biofilm with the mucosa in vitro. Sinonasal tissue s les from 5 control patients undergoing pituitary surgery were cultured with and without S. aureus biofilm in vitro. Confocal scanning laser microscopy (CSLM) using the Live/Dead BacLight stain and histology were performed on the tissue explants after 24 hours of biofilm challenge. Measurements of IL-6, at both the messenger RNA (mRNA) level (using quantitative reverse-transcriptase polymerase chain reaction [qRT-PCR]) and the protein level (using enzyme-linked immunosorbent assay [ELISA]), were undertaken to evaluate biofilm-mucosa interaction. Viability of the explants after 24 hours was confirmed by CSLM and histology. Although light microscopy failed to identify S. aureus biofilms, its presence was confirmed in the biofilm-challenged s les by CSLM. IL-6 mRNA transcript levels were 4.9-fold upregulated in biofilm-treated tissue compared to controls (p = 0.0485). A similar trend was observed at the protein level (p = 0.0313). The sinonasal tissue explant is a viable and functional model capable of analyzing direct biofilm-mucosal interactions and can advance our understanding of the role played by S. aureus biofilm in sinus inflammation. Our model suggests that S. aureus biofilms in the initial phase of growth are not inert bystanders but elicit an immune response in the sinonasal mucosa.
Publisher: American Thoracic Society
Date: 10-2018
Publisher: Wiley
Date: 17-11-2015
DOI: 10.1002/ALR.21661
Abstract: A subgroup of chronic rhinosinusitis with nasal polyps (CRSwNP) patients is refractory to optimal surgical therapy and requires multiple revision sinus operations. Studies have shown that mucosal eosinophilia correlates with disease severity. We hypothesized that a high-grade tissue inflammatory load is associated with these refractory patients. A single-surgeon, retrospective case-control study comparing 20 CRSwNP patients requiring a second surgery during follow-up (refractory group) vs a matched cohort of 20 CRSwNP patients without needing further revision surgery (control group). Hematoxylin and eosin (H&E)-stained tissue harvested intraoperatively (×2 for the refractory group) were recalled for histopathological examination of subepithelial inflammation and basement membrane (BM) thickness. The refractory group had a significantly higher average eosinophil count (49 vs 18), relative eosinophilia (55% vs 32%) as well as total inflammatory cell count (86 vs 49) than the control group (p < 0.05). However within the refractory group, the eosinophil-lymphocyte ratio was reduced from their first to their second (revision) surgeries whereas the total averaged inflammatory cell count remained unchanged. No significant difference in BM thickness was found between the groups. These findings suggest that a higher inflammatory and eosinophilic load is associated with refractory disease and thus may be useful in predicting need for future revision surgery in CRSwNP.
Publisher: Wiley
Date: 26-03-2015
DOI: 10.1002/ALR.21517
Abstract: Chronic rhinosinusitis (CRS) is a cluster of disorders that result in sinonasal mucosal inflammation. Staphylococcus aureus (S. aureus) is associated with severe and recalcitrant CRS. The purpose of our study was to investigate the effect of S. aureus on respiratory epithelial barrier structure and function. Conditioned media from S. aureus reference strains (American Type Culture Collection [ATCC] 13565, 14458, and 25923) was applied to air-liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs) and transepithelial electrical resistance (TEER) was measured to assess cell-to-cell integrity. Electron microscopy was used to gauge the ciliated area and tight junctions (TJs). Additionally, the expression of the TJ protein zona occludens-1 (ZO-1) was examined via immunofluorescence. Statistical analysis was performed using analysis of variance (ANOVA) with pairwise Bonferroni-adjusted t tests. Secreted products applied to ALI cultures from S. aureus strain 13565 caused a concentration-dependent decline in electrical impedance compared to controls and reference strains 14458 and 25923 (p < 0.001). Electron microscopy showed a distinct separation between adjacent cells apically, in the region of TJs. The ciliated area was not affected however, ZO-1 expression became discontinuous in HNECs exposed to the 13565 strain's conditioned media. Conditioned media of the S. aureus strain 13565 damages the airway epithelium by disrupting the TJs between primary HNECs grown at an ALI. These findings suggest that strain-specific S. aureus-secreted product(s) compromise epithelial barrier function, which may constitute 1 of the roles played by S. aureus in the pathophysiology of recalcitrant CRS. Further research is required to uncover the relevant molecular mechanisms.
Publisher: Wiley
Date: 15-04-2016
DOI: 10.1002/ALR.21758
Abstract: Staphylococcus aureus (S. aureus) has been shown to exist within nasal epithelial cells in chronic rhinosinusitis (CRS) patients. This study investigates the localization of intracellular S. aureus (ICSA) in CRS patients, the associated histopathology changes, and their effect on long-term postoperative outcomes. A prospective study of patients with CRS with and without polyps and control patients (n = 25, 15, and 8, respectively) undergoing endoscopic sinus surgery was performed. Validated patient reported symptom scores and objective endoscopic scores were collected preoperatively and 12 months postoperatively. Mucosal tissue s les were collected and examined for the presence of ICSA using immunohistochemical analysis. Tissue also underwent routine hematoxylin and eosin and Sirius Red staining to evaluate the inflammatory cell load and extent of fibrosis. ICSA appeared to localize to the perinuclear region of the pseudostratified columnar respiratory epithelium. ICSA was more prevalent in CRS without nasal polyps (CRSsNP) than in CRS with nasal polyps (CRSwNP) or controls (80% vs 56% vs 38%, respectively). ICSA positive status did not appear to influence symptom or endoscopic scores at the time of surgery nor 12 months postoperatively. Lymphocytes and total inflammatory cells were significantly increased in ICSA(+) group than ICSA(-) groups (36.4 vs 22.4 cells/area and 53.8 vs 29.1 cells/area, respectively). There was no difference found in fibrosis. This study indicated that ICSA was most prevalent in CRSsNP patients and was associated with increased lymphocytia and total inflammatory cells but not with worse symptomatology, endoscopy results, or basement membrane (BM) thickening.
Publisher: Elsevier BV
Date: 12-2015
DOI: 10.1016/J.TRSL.2015.06.009
Abstract: The burden of drug resistance emerges in the wake of chronic and repeated antibiotic use. This underpins the importance of discovering alternatives to current antibiotic regimens. In chronic rhinosinusitis (CRS), topical therapy such as nasal douches and steroid sprays is the mainstay of treatment. However, bacterial sinusitis such as those with Staphylococcus aureus biofilm infection point to more recalcitrant CRS subtypes, focusing research efforts into topical antimicrobial therapies. In the sinuses, both local mucosal and systemic effects must be considered in designing any new topical medication. Nitric oxide (NO), an endogenous antimicrobial agent, is found at extremely low levels in CRS sinuses and high levels in healthy sinuses. As a novel treatment modality, we have designed a liposomal formulation of an NO donor (LFNO) using isosorbide mononitrate, as a topical sinus wash in a sheep model of S. aureus biofilm rhinosinusitis. Heart rate (HR), blood pressure, mean arterial pressure (MAP), and histologic and ciliary analyses were assessed in the safety component. Efficacy was assessed by quantifying biofilm biomass post-treatment. LFNO-treated sheep had lesser inflammation (P = 0.02), and comparable ciliary preservation (P = 0.86) than the control group. A transient increase in HR and decrease in MAP were observed in the LFNO group (P < 0.05), but this was not accompanied by observable side effects. LFNO sheep had significantly lower biofilm biomass vs controls (P = 0.044). Our findings demonstrate the localized and systemic safety of LFNO in an animal model despite using high NO concentrations, thus warranting further investigation for its possible therapeutic role in CRS.
Publisher: Elsevier BV
Date: 09-2006
DOI: 10.1016/J.MOLCEL.2006.07.005
Abstract: Myosin VI is the only myosin that moves toward the minus end of actin filaments, suggesting a unique biological function. Here, we show that myosin VI is present in the nucleus of mammalian cells where it colocalizes with newly transcribed mRNA and with RNA polymerase II (RNAPII) and is detected in the RNAPII complex. The colocalization and interaction of myosin VI with RNAPII require transcriptional activity. Chromatin immunoprecipitation (ChIP) demonstrates that myosin VI is recruited to the promoter and intragenic regions of active genes, encoding urokinase plasminogen activator (uPA), eukaryotic initiation factor 6 (p27/eIF6), and low-density lipoprotein receptor (LDLR), but not to noncoding, nonregulatory intergenic regions. Downregulation of myosin VI reduces steady-state mRNA levels of these genes in vivo, and antibodies to myosin VI reduce transcription in vitro. We suggest that myosin VI modulates RNAPII-dependent transcription of active genes, implicating the possibility of an actin-myosin based mechanism of transcription.
Publisher: Proceedings of the National Academy of Sciences
Date: 28-05-2002
Abstract: Normal vision in Drosophila requires NINAC, a class III myosin. Class III myosins are hybrid motor-signaling molecules, with an N-terminal kinase domain, highly conserved head and neck domains, and a class III-specific tail domain. In Drosophila rhabdomeres, NINAC interacts with actin filaments and with a PDZ scaffolding protein to organize the phototransduction machinery into a signaling complex. Recessive null mutations in Drosophila NINAC delay termination of the photoreceptor response and lead to progressive retinal degeneration. Here, we show that normal hearing in humans requires myosin IIIA, the human homolog of NINAC. In an extended Israeli family, nonsyndromic progressive hearing loss is caused by three different recessive, loss-of-function mutations in myosin IIIA. Of 18 affected relatives in Family N, 7 are homozygous and 11 are compound heterozygous for pairs of mutant alleles. Expression of mammalian myosin IIIA is highly restricted, with the strongest expression in retina and cochlea. The involvement of homologous class III myosins in both Drosophila vision and human hearing is an evolutionary link between these sensory systems.
Publisher: Frontiers Media SA
Date: 24-02-2017
Publisher: Royal Society of Chemistry (RSC)
Date: 2015
DOI: 10.1039/C4TB01953A
Abstract: The anti-biofilm effect of drug delivery systems composed of the antiseptic quaternary ammonium compound cetylpyridinium chloride (CPC) and cholesterol was evaluated in Staphylococcus aureus biofilm.
Publisher: SAGE Publications
Date: 2015
DOI: 10.2500/AJRA.2015.29.4130
Abstract: Staphylococcus aureus is one of the most common bacteria associated with chronic rhinosinusitis (CRS). Although S. aureus biofilms have been correlated with disease severity in CRS, little is known about the initial immune response that biofilms induce in the sinonasal mucosa. The aim of this study was to evaluate the innate immune response (in terms of cytokines) of nondiseased human sinonasal tissue to S. aureus biofilms. Full-thickness sinonasal explant cultures (n = 7 donors) were challenged with established S. aureus biofilms for 24 hours. The expression profiles of 17 cytokines were measured using multiplex analysis, real-time quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Differences in expression were evaluated using Student's t-test. Interleukin (IL)-1β, IL-10, TNF, IL-17A, and interferon (IFN)-γ were up-regulated at the RNA and protein levels in biofilm-treated tissues compared with controls. Elevation of caspase-3 in biofilm-treated s les indicates S. aureus biofilms induce apoptosis on the sinonasal mucosa. S. aureus biofilms induced apoptosis and a predominant proinflammatory immune response on normal sinonasal mucosal explants. This immune response appeared to be triggered by intrinsic bacterial elements but also by components of the biofilm matrix. Live biofilms were present on the mucosa at the end of the challenge, suggesting an inability of the induced immune response to eliminate the S. aureus biofilms.
Publisher: Wiley
Date: 16-05-2021
DOI: 10.1111/ALL.14883
Abstract: Staphylococcus aureus is a pathogen of major concern in both acute infections and chronic conditions such as chronic rhinosinusitis (CRS). Bacteriophage (phage) therapy has recently regained interest for its potential to treat infections caused by antibiotic resistant strains including Methicillin Resistant Staphylococcus aureus (MRSA). However, bacteria can adapt and become resistant to phages. The aim of this study is to determine the potential for antibiotics to overcome phage resistance. The susceptibility of S. aureus clinical isolates (CIs) to phages J‐Sa36, Sa83 and Sa87 alone or in combination with protein synthesis inhibitor (PSI) antibiotics clindamycin, azithromycin and erythromycin was assessed using plaque spot assays, minimum inhibitory concentration (MIC) assays, double layer spot assays and resazurin assays. The safety and efficacy of subinhibitory PSI antibiotics in combination with phage was tested in a Sprague Dawley rat model of sinusitis infected with a phage resistant S. aureus CI. All three antibiotics at subinhibitory concentrations showed synergy when combined with all 3 phages against S. aureus CIs in planktonic and biofilm form and could sensitize phage‐resistant S. aureus to promote phage infection. The combination of topical subinhibitory clindamycin or azithromycin and phage was safe and could eradicate S . aureus sinonasal biofilms in vivo . Subinhibitory concentrations of PSI antibiotics could sensitize phage‐resistant S . aureus and MRSA strains to phages in vitro and in vivo . This data supports the potential use of phage‐PSI antibiotic combination therapies, in particular for difficult‐to‐treat infections with phage‐resistant S . aureus and MRSA strains.
Publisher: Wiley
Date: 16-08-2023
DOI: 10.1111/IMM.13655
Abstract: Chronic rhinosinusitis (CRS) represents chronic inflammation of the sinus mucosa characterised by dysfunction of the sinuses' natural defence mechanisms and induction of different inflammatory pathways ranging from a Th1 to a Th2 predominant polarisation. Recalcitrant CRS is associated with Staphylococcus aureus dominant mucosal biofilms however, S. aureus colonisation of the sinonasal mucosa has also been observed in healthy in iduals challenging the significance of S. aureus in CRS pathogenesis. We aimed to investigate the relationship between CRS key inflammatory markers, S. aureus biofilm properties/virulence genes and the severity of the disease. Tissue s les were collected during endoscopic sinus surgery from the ethmoid sinuses of CRS patients with (CRSwNP) and without (CRSsNP) nasal polyps and controls ( n = 59). CD3+ T‐cell subset frequencies and key inflammatory markers of CD4+ helper T cells were determined using FACS analysis. Sinonasal S. aureus clinical isolates were isolated ( n = 26), sequenced and grown into biofilm in vitro, followed by determining their properties, including metabolic activity, biomass, colony‐forming units and exoprotein production. Disease severity was assessed using Lund–Mackay radiologic scores, Lund–Kennedy endoscopic scores and SNOT22 quality of life scores. Our results showed that S. aureus biofilm properties and CRS severity scores correlated positively with total CD4+ T‐cell frequencies but looking into CD4+ T‐cell subsets showed an inverse correlation with Th1 and Th17 cell frequencies. CD4+ T‐cell frequencies were higher in patients harbouring lukF.PV‐ positive S. aureus while its regulatory and Th17 cell subset frequencies were lower in patients carrying sea− and sarT/U‐ positive S. aureus. Recalcitrant CRS is characterised by increased S. aureus biofilm properties in relation to increased total CD4+ helper T‐cell frequencies and reduced frequencies of its Th1, Th17 and regulatory T‐cell subsets. These findings offer insights into the pathophysiology of CRS and could lead to the development of more targeted therapies.
Publisher: Wiley
Date: 21-01-2014
DOI: 10.1002/ALR.21270
Abstract: Treatment of sinonasal bacterial biofilms continues to be a challenge in modern rhinology. This study's objective was to assess the safety and efficacy of topically applied Cocktail of S. aureus specific phage (CTSA) alone and in combination with ethylenediaminetetraacetic acid (EDTA) for treatment of Staphylococcus aureus biofilms in vivo. Using a sheep model of sinusitis, frontal sinuses (n = 6 per treatment) were flushed once daily with a CTSA (2 × 10(6) plaque forming units [PFU]/mL), with or without EDTA (0.075 mg/mL), and compared to a control flush containing saline and heat-inactivated CTSA. Safety was assessed using histology and scanning electron microscopy (SEM) after treatment for 3 days. Efficacy was assessed by quantifying the generation of S. aureus biofilms in the frontal sinuses after 5 days of treatment. Biofilm mass was compared between treatment groups and controls using LIVE/DEAD BacLight staining and confocal scanning laser microscopy to visualize the tissue sections. COMSTAT2 software was used to compute the biofilm mass present on tissue sections. Tissue morphology was conserved, with no significant signs of inflammation, when comparing control and test treatments. Furthermore, SEM analysis indicated test treatments were not toxic or damaging to mucosal cilia. COMSTAT2 quantification of biofilm showed a significant reduction in biofilm levels when comparing the control with CTSA (p = 0.0043), EDTA (p = 0.0095), and CTSA-EDTA (p = 0.0022) treatments. Results indicate that CTSA and EDTA are safe and efficacious for short-term topical application against S. aureus infection in a sheep sinusitis model, and have the potential to be translated to a clinical setting.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 02-2016
Publisher: Wiley
Date: 19-02-2013
DOI: 10.1002/ALR.21154
Abstract: Despite recent evidence suggesting that Staphylococcus aureus exists within the sinonasal epithelium of chronic rhinosinusitis (CRS) patients, certain questions remain. The intracellular environment may provide a protective niche for pathogenic bacteria to evade host immunity and yet provide a reservoir for reinfection. To date, no studies have examined the impact of this bacterial phenotype therefore, this study was designed to evaluate the role of intracellular S. aureus on postsurgical outcomes. This study included 51 patients undergoing endoscopic sinus surgery (ESS) for medically-recalcitrant CRS. Sinonasal mucosa harvested at the time of surgery was dually stained with fluorescent molecular probes and imaged using confocal scanning laser microscopy for biofilm and intracellular status. Patients were followed in their early and late postoperative course for evidence of ongoing disease and signs of clinical relapse. Intracellular S. aureus was identified in 20 of 51 (39%) patients, and all were associated with surface biofilm. Biofilm alone was found in 16 of 51 (31%) patients and 15 of 51 (29%) patients had no evidence of S. aureus. Intracellular positive patients had a significantly higher risk of late clinical and microbiological relapse (p = 0.014). In this study, biofilm status without coexisting intracellular bacteria did not appear to impact on outcomes. Clinical and microbiological relapse of disease following ESS is significantly associated with intracellular S. aureus. Evidence suggests that this disease association is independent to surface biofilm status. Intracellular bacteria should be taken into consideration when designing novel treatment strategies to lessen the chance of reinfection.
Publisher: Wiley
Date: 07-11-2012
DOI: 10.1002/ALR.21114
Abstract: The complex interplay between host, environment, and microbe in the etiopathogenesis of chronic rhinosinusitis (CRS) remains unclear. This study focuses on the host-microbe interaction, specifically the regulation of nitric oxide (NO) and reactive oxygen species (ROS) against the pathogenic organism Staphylococcus aureus (S. aureus). NO and ROS play crucial roles in innate immunity and in the first-line defense against microbial invasion. Sinonasal tissue s les were harvested from CRS and control patients during surgery. CRS patients were classified S. aureus biofilm-positive (B+) or biofilm-negative (B-) using fluorescence in situ hybridization and clinically as polyp-positive (P+) or polyp-negative (P-). S les were assessed using an NO polymerase chain reaction (PCR) array containing 84 genes involved in NO and ROS regulation, and gene expression of all subgroups were compared to each other. Twenty-three s les were analyzed with 31 genes significantly changed, the greatest seen in the B+P+ CRS patients. Four genes consistently displayed differential expression between the groups including the cytoprotective oxidation resistance 1 (OXR1) and peroxiredoxin 6 (PRDX6), neutrophil cytosolic factor 2 (NCF2), and the prion protein (PRNP) genes. Alteration in gene expression points to impaired innate immune responses differing among CRS subgroups based on S. aureus biofilm and polyp status. The consistent alteration of 4 genes among distinct groups demonstrates that S. aureus biofilms and polyps are associated with specific changes in gene expression. Further studies are required to validate these findings in a wider cohort of patients and correlate this to protein expression and disease manifestation.
Publisher: Wiley
Date: 10-01-2014
DOI: 10.1002/ALR.21279
Abstract: Staphylococcus aureus (SA) is a key pathogenic component of the chronic rhinosinusitis (CRS) microbiome and is associated with increased disease severity and poor postoperative outcomes. Probiotic treatments potentially offer a novel approach to the management of pathogenic bacteria in these recalcitrant patients through supporting a healthy community of commensal species. This study aims to investigate the probiotic properties of Staphylococcus epidermidis (SE) against SA in a mouse model of sinusitis. Twenty C57/BL6 mice received intranasal inoculations of phosphate buffered saline (PBS), SE, SA, or a combination of SE and SA (SE+SA) for 3 days. Following euthanasia, the mouse snouts were harvested and prepared for histological analysis. Counts of periodic acid-Schiff (PAS)-positive goblet cells were the primary outcome measure. Goblet cell counts were significantly higher in both the SA and SE+SA groups compared to those receiving PBS or SE alone (p < 0.05). However, the SE+SA group demonstrated significantly lower goblet cell counts compared to the SA group (p 0.05). The presence of SA postinoculation was confirmed by culture in both the SA and SE+SA groups. This study confirms the probiotic potential of SE against SA in a mouse model of sinusitis. Although the interactions that occur between many probiotic species and pathogens are yet to be fully understood, studies such as this support further exploration of ecologically-based treatment paradigms for the management of CRS.
Publisher: Public Library of Science (PLoS)
Date: 14-04-2015
Publisher: Georg Thieme Verlag KG
Date: 12-12-2016
Abstract: Nano-hemostats are synthetic amino acid chains that self-assemble into a scaffold under certain conditions. These have been shown to be effective in stopping bleeding in small animal models of hemorrhage. Proposed mechanisms for their effect are that they form a mesh analogous to the fibrin plug in native hemostasis and that they may potentiate both platelet activation and the coagulation cascade. These may potentially become valuable adjuncts to endoscopic skull base surgery where there is the potential for both major vessel injury and smaller perforator injury to eloquent areas where bipolar cautery may not be suitable. We present a summary of the clinical studies to date and a small pilot study of nano-hemostat in an endoscopic sheep model of major vessel hemorrhage to determine its efficacy in stopping bleeding in this potentially catastrophic complication.
Publisher: Wiley
Date: 02-10-2013
DOI: 10.1002/LARY.24322
Abstract: Clinical improvement in patients with chronic rhinosinusitis (CRS) treated with steroids alone has previously been ascribed to the steroids' anti‐inflammatory properties rather than any direct effect on the bacteria. The aim of this study was to determine if commonly used intranasal steroids directly reduce bacterial biofilm production in vitro. In vitro comparative controlled trial. Staphylococcus aureus biofilms were grown on minimum biofilm eradication concentration device pegs and treated with the commonly prescribed CRS topical steroids fluticasone, mometasone, or budesonide. These were dissolved in vehicle solvents and added to cerebrospinal fluid (CSF) broth. Concentrations (including therapeutic doses) tested for fluticasone and mometasone ranged from 25 μg/200 μL to 400 μg/200 μL, and from 16 μg/200 μL to 2000 μg/200 μL for budesonide. Control pegs were exposed to equivalent volumes of the appropriate solvent/CSF broth. Confocal scanning laser microscopy and COMSTAT software were used to quantify biofilms at 24 hours after treatment. Significant differences from control were found for fluticasone at 400 μg/200 μL (difference = −0.3065 μm 3 /μm 2 , P = .007), mometasone at 300 μg/200 μL and 400 μg/200 μL (difference = −0.15 μm 3 /μm 2 , P = .006, and difference = −0.9193 μm 3 /μm 2 , P = .034, respectively), and budesonide at 750 μg/200 μL, 1000 μg/200 μL and 2000 μg/200 μL (difference = −1.0137 μm 3 /μm 2 , P = .038, difference = −0.6164, P = .009, and difference = −0.1906 μm 3 /μm 2 , P = .029, respectively). The concentrations of 400 μg/200 μL of fluticasone, 300 μg and 400 μg/200 μL of mometasone, and 750 μg, 1,000μg, and 2,000 μg/200 μL of budesonide directly reduce biofilm production in vitro, outside of the inflammatory milieu. NA. Laryngoscope , 124:602–607, 2014
Publisher: American Chemical Society (ACS)
Date: 23-06-2017
Abstract: Biofilms are aggregates of bacteria residing in a self-assembled matrix, which protects these sessile cells against external stress, including antibiotic therapies. In light of emerging multidrug-resistant bacteria, alternative strategies to antibiotics are emerging. The present study evaluated the activity of colloidal silver nanoparticles (AgNPs) of different shapes against biofilms formed by Staphylococcus aureus (SA), methicillin-resistant SA (MRSA), and Pseudomonas aeruginosa (PA). Colloidal quasi-spherical, cubic, and star-shaped AgNPs were synthesized, and their cytotoxicity on macrophages (THP-1) and bronchial epithelial cells (Nuli-1) was analyzed by the lactate dehydrogenase assay. The antibiofilm activity was assessed in vitro by the resazurin assay and in an in vivo infection model in Caenorhabditis elegans. Cubic and star-shaped AgNPs induced cytotoxicity, while quasi-spherical AgNPs were not toxic. Quasi-spherical AgNPs showed substantial antibiofilm activity in vitro with 96% (±2%), 97% (±1%), and 98% (±1%) biofilm killing of SA, MRSA, and PA, respectively, while significantly reducing mortality of infected nematodes. The in vivo antibiofilm activity was linked to the accumulation of AgNPs in the intestinal tract of C. elegans as observed by 3D X-ray tomography. Quasi-spherical AgNPs were physically stable in suspension for over 6 months with no observed loss in antibiofilm activity. While toxicity and stability limited the utilization of cubic and star-shaped AgNPs, quasi-spherical AgNPs could be rapidly synthesized, were stable and nontoxic, and showed substantial in vitro and in vivo activity against clinically relevant biofilms. Quasi-spherical AgNPs hold potential as pharmacotherapy, for ex le, as topical treatment for biofilm-related infections.
Publisher: Springer New York
Date: 2015
DOI: 10.1007/978-1-4939-2871-2_17
Abstract: In order to relate the structural architecture of the BAM complex to its function in outer membrane protein assembly, the arrangement of each component within the complex is vital. This chapter explores the structure and topology of BamC, using a range of biochemical techniques to probe the topology and surface exposure.
Publisher: Wiley
Date: 26-03-2014
DOI: 10.1002/ALR.21326
Abstract: The most dreaded hemorrhagic complication in endoscopic endonasal surgery is injury to the internal carotid artery (ICA). Although a number of treatment protocols are currently used, none have been formally investigated. This study aims to compare the efficacy of the muscle patch, bipolar diathermy, and aneurysm clip on hemostasis, pseudoaneurysm formation, and long-term vessel patency for different injury types in a sheep model of carotid bleeding. Twenty-seven sheep underwent ICA dissection/isolation followed by the artery placement within a modified "sinus model otorhino neuro trainer" (SIMONT) model. Standardized linear, punch, and stellate injuries were made. Randomization of sheep to receive 1 of 3 hemostatic techniques was performed (muscle, bipolar, clip). Specific outcome measures included attainment of primary hemostasis, time to hemostasis, blood loss, pseudoaneurysm formation, and carotid patency on follow-up magnetic resonance imaging (MRI). Bipolar achieved primary hemostasis in 7 of 9 cases and 2 cases of secondary hemorrhage. It had no associated pseudoaneurysm formation. Carotid patency was variable on follow-up MRI. Muscle patch achieved 100% primary hemostasis with 2 cases of secondary hemorrhage. There were 2 cases of pseudoaneurysm and 100% patency rate on follow-up MRI. Aneurysm clip achieved 100% primary hemostasis with 1 case of secondary hemorrhage. No pseudoaneurysm formation and a 50% rate of carotid insufficiency on MRI. This study shows that the crushed muscle patch and aneurysm clip can be viable options in the management of ICA injury with short-term and long-term benefits. Complications associated with these techniques were comparable if not reduced when compared to the published literature.
Publisher: Wiley
Date: 09-07-2021
DOI: 10.1002/ALR.22858
Publisher: Wiley
Date: 27-02-2015
DOI: 10.1002/ALR.21489
Abstract: Use of muscle grafts for hemostasis during surgery has re-emerged recent animal model studies have shown effective bleeding control with their use. However, the mechanism of action is unknown. The aim of this study is to evaluate the action of muscle extracts on the coagulation pathways and platelet aggregation. Muscle extracts were prepared by dissolving crushed snap-frozen muscle tissue (0.04 to 0.8 mg) in 1 mL saline. Saline was used as control. Prothrombin time, activated partial thromboplastin time (APTT), thrombin time, and platelet aggregation studies were performed on both muscle extract and saline. Prothrombin time and APTT were repeated using factor VII-deficient plasma, factor X-deficient plasma, lupus plasma, and contact pathway-inhibited plasma. Mean readings in the muscle group and control group were compared using nonparametric Mann-Whitney U test (Wilcoxon rank sum test with continuity correction). Among the various coagulation parameters, there was no significant difference between saline and muscle (p > 0.05), except in the APTT using factor X-deficient plasma (mean APTT 133.89 seconds and 185.10 seconds for muscle and saline, respectively p 0.5 mg/mL) increased platelet aggregation from 23.9% to 85.5% (p = 0.0001). Platelet aggregation plays a role in the hemostatic efficacy of muscle grafts. Even though action on the coagulation pathway via APTT is statistically significant, clinical significance may be low.
Publisher: Wiley
Date: 25-02-2017
DOI: 10.1002/ALR.21924
Abstract: The unconventional toll-like receptor (TLR) CD180 is implicated in chronic inflammatory diseases however, its role in chronic rhinosinusitis (CRS) has yet to be investigated. Here we study the expression of CD180, its homologue TLR4 and myeloid differentiation factor 1 (MD1) on mucosal and systemic immune cell populations in relation to serum immunoglobulin G (IgG) levels. A total of 70 patients were recruited to the study. Mucosal and peripheral blood s les were prospectively collected from CRS patients and non-CRS controls without evidence of sinus disease. The expression of TLR4, MD1, and CD180 was investigated using qualitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, and flow cytometry. Serum IgG levels were determined using enzyme-linked immunosorbent assay (ELISA). CRS with nasal polyps (CRSwNP) patients had significantly increased messenger RNA (mRNA) expression of CD180 and MD1 compared to controls (5.54-fold and 2.1-fold, respectively, p < 0.01). B cells lacking CD180 were lower in CRSwNP tissue compared to CRS without nasal polyps (CRSsNP) and controls (21.07 ± 6.41 vs 41.61 ± 7.82 vs 40.06 ± 8.06 p < 0.01) but higher in blood (39.18 ± 8.3 vs 17.95 ± 7.82 and 12.49 ± 4.92 p ≤ 0.05). Changes in mucosal and peripheral CD180-expressing B cells were identified in CRSwNP patients compared to CRSsNP and controls. This suggests a role for these cells in the dysregulated immune response in these patients.
Publisher: Frontiers Media SA
Date: 26-12-2016
Publisher: Wiley
Date: 10-10-2017
DOI: 10.1002/LARY.26949
Abstract: Staphylococcus aureus (S. aureus) infection is known to contribute to the severity and recalcitrance of chronic rhinosinusitis (CRS), and its secreted products have been shown to alter the airway barrier. Extracellular proteases secreted by S. aureus are thought to be important in epithelial infection and immune evasion however, their effect on airway mucosal barrier function is not known. To investigate the impact of extracellular proteases on airway epithelial integrity, the purified S. aureus proteases V8 protease, Staphopain A, Staphopain B, Exfoliative toxin A, and serine protease-like A-F were applied to human nasal epithelial cell air-liquid interface (HNEC-ALI) cultures. Transepithelial electrical resistance (TEER), permeability (Papp) measurements, and immuno-localization of the tight junction proteins claudin-1 and ZO-1 were used to assess barrier integrity. Effects of the proteases on inflammation and cell viability were measured using interleukin-6 (IL-6) ELISA and a lactate dehydrogenase assay. Application of V8 protease to HNEC-ALI cultures caused a significant concentration and time-dependent decrease in TEER (22.67%, P < 0.0001), a reciprocal Papp increase (20.14-fold, P < 0.05), and a discontinuous ZO-1 immuno-localization compared to control. IL-6 production was significantly reduced in V8 protease-treated cells (153.5 pg/mL, P = 0.0069) compared to control (548.3 pg/mL), whereas no difference in cell viability was observed. S. aureus V8 protease causes dysfunction of mucosal barrier structure and function indicative of a leaky barrier. A reduction in IL-6 levels suggests that the mucosal immunity is impaired by this protease and thus has the potential to contribute to CRS recalcitrance. NA. Laryngoscope, 128:E8-E15, 2018.
Publisher: Elsevier BV
Date: 06-2021
Publisher: American Medical Association (AMA)
Date: 02-2017
DOI: 10.1001/JAMAOTO.2016.3105
Abstract: The adenoid pad has long been considered a reservoir for bacteria in the pathogenesis of otitis media with effusion (OME). However, bacteria more reminiscent of external auditory canal (EAC) commensals are often demonstrated within middle ear aspirates. To compare the microbiota of the EAC, the middle ear with OME, and the adenoid pad to further clarify the true source of middle ear bacteria. Middle ear fluid (MEF) aspirates and EAC lavages were collected from 18 children with OME undergoing ventilation tube insertion from June 1, 2014, to August 31, 2015, at Women and Children's Hospital, Adelaide, Australia. Adenoid pad and MEF s les were included from a previous study. S les were analyzed using sequencing of the 16S ribosomal RNA gene. Previously collected microbiota data from the adenoid pad were collated for analysis. Mean relative abundance of top bacterial genera for the MEF, EAC, and adenoid pad s les. Eighteen pediatric patients with chronic OME (6 female 12 male mean [SD] age, 48 [36] months) were recruited prospectively, with 34 paired MEF and EAC s les. The MEF microbiota (mean relative abundance [SD]) consisted of Alloiococcus otitidis (37.5% [40.0%]), Haemophilus (14.4% [29.1%]), Moraxella (10.0% [26.4%]), Staphylococcus (8.2% [21.9%]), and Streptococcus (3.8% [13.1%]). The mean relative abundance (SD) microbiota of the EAC demonstrated a sparsity of classic otopathogens, including Haemophilus (0.3% [0.8%]), Moraxella (0.3% [0.7%]), and Streptococcus (0.2% [0.6%]), but had a high abundance of Alloiococcus (58.0% [44.1%]), Staphylococcus (20.8% [34.0%]), and Pseudomonas (3.2% [17.1%]). In contrast, based on previously collected data, the microbiota of the adenoid pad showed a high abundance of the classic otopathogens with a sparsity of EAC genera for Alloiococcus (0.1% vs 28.9%, respectively P < .001), Haemophilus (25.2% vs 18.2%, respectively P = .002), Staphylococcus (0.2% vs 10.8%, respectively P = .02), Streptococcus (12.7% vs 4.2%, respectively P < .001), and Pseudomonas (0 vs 2.1% respectively P < .001). The microbiota of the MEF collected during 2 consecutive years were similar (Alloiococcus, 22.7% vs 37.5% Haemophilus, 22.5% vs 14.0% Staphylococcus, 10.9% vs 10.7% Moraxella, 5.0% vs 9.7% Corynebacteria, 6.2% vs 3.1% Streptococcus, 4.8% vs 3.7% and Pseudomonas, 1.1% vs 3.0% P ≥ .05). The EAC and the nasopharynx could serve as reservoirs for microbiota of the middle ear. Furthermore, the microbiota of the middle ear with effusion appear to be relatively stable over time and between populations with OME.
Publisher: Wiley
Date: 30-03-2020
DOI: 10.1111/ALL.14276
Abstract: The sinonasal microbiome remains poorly defined, with our current knowledge based on a few cohort studies whose findings are inconsistent. Furthermore, the variability of the sinus microbiome across geographical ides remains unexplored. We characterize the sinonasal microbiome and its geographical variations in both health and disease using 16S rRNA gene sequencing of 410 in iduals from across the world. Although the sinus microbial ecology is highly variable between in iduals, we identify a core microbiome comprised of Corynebacterium , Staphylococcus , Streptococcus , Haemophilus and Moraxella species in both healthy and chronic rhinosinusitis (CRS) cohorts. Corynebacterium (mean relative abundance = 44.02%) and Staphylococcus (mean relative abundance = 27.34%) appear particularly dominant in the majority of patients s led. Amongst patients suffering from CRS with nasal polyps, a statistically significant reduction in relative abundance of Corynebacterium (40.29% vs 50.43% P = .02) was identified. Despite some measured differences in microbiome composition and ersity between some of the participating centres in our cohort, these differences would not alter the general pattern of core organisms described. Nevertheless, atypical or unusual organisms reported in short‐read licon sequencing studies and that are not part of the core microbiome should be interpreted with caution. The delineation of the sinonasal microbiome and standardized methodology described within our study will enable further characterization and translational application of the sinus microbiota.
Publisher: Wiley
Date: 31-03-2022
DOI: 10.1111/ALL.15289
Publisher: Springer Science and Business Media LLC
Date: 31-01-2000
Abstract: Connexin 26 (GJB2) mutations lead to hearing loss in a significant proportion of all populations studied so far, despite the fact that at least 50 other genes are also associated with hearing loss. The entire coding region of connexin 26 was sequenced in 75 hearing impaired children and adults in Israel in order to determine the percentage of hearing loss attributed to connexin 26 and the types of mutations in this population. Age of onset in the screened population was both prelingual and postlingual, with hearing loss ranging from moderate to profound. Almost 39% of all persons tested harbored GJB2 mutations, the majority of which were 35delG and 167delT mutations. A novel mutation, involving both a deletion and insertion, 51del12insA, was identified in a family originating from Uzbekistan. Several parameters were examined to establish whether genotype-phenotype correlations exist, including age of onset, severity of hearing loss and audiological characteristics, including pure-tone audiometry, tympanometry, auditory brainstem response (ABR), and transient evoked otoacoustic emissions (TEOAE). All GJB2 mutations were associated with prelingual hearing loss, though severity ranged from moderate to profound, with variability even among hearing impaired siblings. We have not found a significant difference in hearing levels between in iduals with 35delG and 167delT mutations. Our results suggest that, in Israel, clinicians should first screen for the common 167delT and 35delG mutations by simple and inexpensive restriction enzyme analysis, although if these are not found, sequencing should be done to rule out additional mutations due to the ethnic ersity in this region.
Publisher: SAGE Publications
Date: 2016
DOI: 10.2500/AJRA.2016.30.4261
Abstract: The bacterial microbiome in chronic rhinosinusitis (CRS) remains poorly understood. Microorganisms are believed to be important contributors to the inflammatory response seen in these patients. To examine the bacterial CRS microbiome by using a pyrosequencing technique and determine the ersity, richness, prevalence, and abundance of bacterial species in these patients. Furthermore, the postoperative changes that occur in the microbiome and correlations with patient outcomes are assessed. Swabs were collected from 23 patients with CRS and 11 controls during surgery. Further postoperative swabs were collected in the CRS group. Bacterial DNA was extracted from the swabs and then sequenced by using 16S ribosomal DNA bacterial tag-encoded FLX licon pyrosequencing. A total of 456 unique bacterial species were detected. No difference was seen for richness or ersity between the study groups (p 0.05). Diversity declined after surgery in the CRS group (p = 0.01). Propionibacterium acnes and Staphylococcus epidermidis were the most prevalent species. Several significant differences were determined for prevalence and mean relative abundance (MRA) between the study groups. In particular, Acinetobacter johnsonii was more prevalent and had a higher MRA in the controls. Furthermore, the MRA of this species increased after surgery and was associated with improved quality of life. This study characterized the sinonasal microbiome in a group of controls and patients with CRS. Important differences in ersity, prevalence, abundance, and temporal changes were described. Of great interest is the potential association between A. johnsonii and health. These findings provide new insights into the interplay between the microbiome and health in the paranasal sinuses.
Publisher: Wiley
Date: 12-11-2020
DOI: 10.1002/LARY.29261
Abstract: Lymphoid neogenesis or the development of organised, de novo lymphoid structures has been described increasingly in chronically inflamed tissues. The presence of tertiary lymphoid organs (TLOs) has already been demonstrated to result in significant consequences for disease pathology, severity, prognosis and patient outcomes. Whilst the wider medical community has embraced TLOs as important markers of disease and potential therapeutic targets, the otolaryngology field has only begun turning to these entities in an academic capacity. This review aims to outline the role of tertiary lymphoid organs in disease and summarise key early findings in the ENT field. We also an overview of TLOs, their developmental process and clinicopathological implications. Literature review. A literature search for all relevant peer‐reviewed publications pertaining to TLOs and ENT diseases. Search was conducted using PubMed, Embase and CINAHL databases. A total of 24 studies were identified relevant to the topic. The majority of TLO research in ENT fell into the areas of oral squamous cell carcinoma (SCC) and chronic rhinosinusitis (CRS). Early research into both oral SCC and CRS suggests that TLOs have significant roles within ear, nose and throat (ENT) diseases. At this point in time, however, TLOs remain somewhat a mystery amongst otolaryngologists. As information in this field increases, we may develop a better understanding of how lymphoid neogenesis can influence disease outcomes amongst our patients and, ultimately, how they can be utilised in an immunotherapeutic manner. Laryngoscope , 131:1697–1703, 2021
Publisher: Wiley
Date: 29-07-2014
DOI: 10.1111/ALL.12457
Abstract: Chronic rhinosinusitis (CRS) has been linked to the gram-positive bacteria Staphylococcus aureus (S. aureus) in its biofilm or intracellular forms. Recent evidence suggests that S. aureus also exists in a small-colony variant (SCV) form as a mechanism of altering its virulence capabilities. The aim of this study was to investigate the presence of SCVs in sinonasal mucosa of CRS patients and whether the phenomenon of phenotype switching can be applied to intracellular epithelial infections. Sinonasal specimens were examined for the presence of intramucosal S. aureus and characterized to the strain level. An airway epithelial cell culture infection model was utilized to investigate whether bacteria were capable of alterations in virulence phenotype. Intramucosal organisms harvested from sinonasal biopsies demonstrate phenotypic growth patterns and lack of coagulase activity consistent with SCVs. Intracellular infection of airway epithelial cell cultures with S. aureus led to decreased secretion of enterotoxins and phenotypic growth alterations consistent with SCVs. Regulation of S. aureus virulence factors is a dynamic process, and exposure to the intracellular environment appears to provide the necessary conditions to enable these alterations in an attempt for the bacterium to survive and persist within host tissues. Further work is required to ascertain whether SCVs in CRS hold a clinically relevant pathogenic role in recalcitrant disease.
Publisher: Springer Science and Business Media LLC
Date: 06-2002
Abstract: Usher syndrome type 3 (USH3) is an autosomal recessive disorder characterised by the association of post-lingual progressive hearing loss, progressive visual loss due to retinitis pigmentosa and variable presence of vestibular dysfunction. Because the previously defined transcripts do not account for all USH3 cases, we performed further analysis and revealed the presence of additional exons embedded in longer human and mouse USH3A transcripts and three novel USH3A mutations. Expression of Ush3a transcripts was localised by whole mount in situ hybridisation to cochlear hair cells and spiral ganglion cells. The full length USH3A transcript encodes clarin-1, a four-transmembrane-domain protein, which defines a novel vertebrate-specific family of three paralogues. Limited sequence homology to stargazin, a cerebellar synapse four-transmembrane-domain protein, suggests a role for clarin-1 in hair cell and photoreceptor cell synapses, as well as a common pathophysiological pathway for different Usher syndromes.
Publisher: Wiley
Date: 17-12-2015
DOI: 10.1002/ALR.21680
Abstract: Staphylococcus aureus biofilms are a nidus for exacerbation of infectious conditions including chronic rhinosinusitis (CRS). Resistance of biofilms to current therapeutics stresses the need for the development of novel anti-biofilm strategies. The chimeric muralytic enzyme P128 was specifically engineered to target Staphylococcal sp. by combining the cell wall binding domain of lysostaphin and the peptidoglycan-degrading murein hydrolase derived from phage K. This study assessed the anti-biofilm activity of P128 against sinus-derived S. aureus. Biofilms from S. aureus ATCC 25923 and 3 sinus-derived methicillin-sensitive and methicillin-resistant CRS clinical isolates were grown for 48 hours and treated with various concentrations of P128 (0 to 100 μg/mL) for 2 and 24 hours, using the minimum biofilm eradication concentration (MBEC) assay and Alamar Blue (AB) assay. Biofilm present on the MBEC pegs was stained with LIVE/DEAD BacLight stain, imaged using confocal scanning laser microscopy and biomass determined by COMSTAT2 computation. In the AB assay, biofilm was measured by assessing the cell viability. Results were assessed using a Kruskal-Wallis test, with a Wilcoxon post hoc test and Bonferroni correction. Both the MBEC and AB assay indicated that P128 was effective against in vitro S. aureus biofilms with significant reductions in biofilm of up to 95.5% at concentrations ≥12.5 μg/mL for all tested strains. The engineered chimeric endolysin P128 was observed to be an effective anti-biofilm agent against S. aureus. Further study will proceed into the appropriate application of P128 to ensure both an economically and clinically feasible preparation.
Publisher: Wiley
Date: 09-07-2013
DOI: 10.1002/LARY.24255
Abstract: Biofilms are associated with clinical relapse following surgery for chronic rhinosinusitis. Encased bacteria are protected from innate immunity and antimicrobial therapy. Surfactants can disperse the biofilm into its planktonic phenotype so that traditional treatments may be effective. The aim of this study was to assess a surfactant for its cytotoxicity profile. In vitro explant-based cytotoxicity study. Sinonasal mucosa harvested from patients undergoing sinus surgery was tested using an air-liquid interface explant system. Surfactant at 1×, 2×, and 3× manufacturer's recommended concentrations were compared to control (saline) and Zinc Sulphate (ZnSO4 ), a known cytotoxic agent. Culture supernatant was analyzed for lactate dehydrogenase (LDH) as a marker of cellular toxicity. After 7 days, specimens were imaged using structured histopathology and scanning electron microscopy. Application of surfactant at 1× concentration did not elicit an elevation in LDH, whereas ZnSO4 caused a significant rise 1 day after application. Specimens tested with a 2× and 3× surfactant demonstrated LDH rises 4 days and 2 days after application, respectively. Mucosa tested with the 1× surfactant and control demonstrated intact cellular structures on histopathology and preserved cilial ultrastructure on SEM. In ZnSO4 -treated specimens, marked cellular degradation and ciliary denudation occurred. The surfactant does not appear to elicit cellular toxicity using an in vitro explant model at the manufacturer's recommended concentration. At higher concentrations, there may be dose-related toxicity that requires further investigation. In vivo testing is required to prove its efficacy in the treatment of recalcitrant chronic rhinosinusitis. N/A.
Publisher: Wiley
Date: 09-06-2021
DOI: 10.1111/ALL.14959
Abstract: Chronic rhinosinusitis (CRS) is a common chronic respiratory condition, frequently associated with asthma and affecting the majority of cystic fibrosis (CF) patients. Pseudomonas aeruginosa infections and biofilms have been implicated in recalcitrant CRS. One of the mechanisms of action for bacteria in CRS and CF is mucosal barrier disruption by secreted products that contribute to the inflammation. However, the role of biofilm and planktonic forms of P . aeruginosa in this process is not known. The aim is to determine the effect of P . aeruginosa exoproteins isolated from CF and non‐CF CRS patients on the mucosal barrier. Exoproteins from 40 P . aeruginosa isolates were collected in planktonic and biofilm forms and applied to air‐liquid interface (ALI) cultures of primary human nasal epithelial cells (HNECs). Mucosal barrier integrity was evaluated by transepithelial electrical resistance (TEER), passage of FITC‐dextrans and immunofluorescence of tight junction proteins. Cytotoxicity assays were performed to measure cell viability, and IL‐6 ELISA was carried out to evaluate pro‐inflammatory effects. Planktonic exoproteins from 20/40 (50%) clinical isolates had a significant detrimental effect on the barrier and significantly increased IL‐6 production. Barrier disruption was characterized by a reduced TEER, increased permeability of FITC‐dextrans and discontinuous immunolocalization of tight junction proteins and was significantly more prevalent in isolates harvested from patients with comorbid asthma ( P .05). Exoproteins from planktonic P . aeruginosa clinical isolates from asthmatic CRS patients have detrimental effects on the mucosal barrier and induce IL‐6 production potentially contributing to the mucosal inflammation in CRS patients.
No related grants have been discovered for Sarah Vreugde.