Sarah Vreugde

SPPL2a and SPPL2b promote intramembrane proteolysis of TNFα in activated dendritic cells to trigger IL-12 production

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 (TNFα). The two proteases promoted the release of the TNFα 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.

Association between Group 2 Innate Lymphoid Cells enrichment, nasal polyps and allergy in Chronic Rhinosinusitis

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.

Intracellular Staphylococcus aureus: the Trojan horse of recalcitrant chronic rhinosinusitis?

Background 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. Methods 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. Results 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. Conclusion 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.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.

Sinonasal Microbiome Sampling: A Comparison of Techniques

Background The role of the sino-nasal microbiome in CRS remains unclear. We hypothesized that the bacteria within mucosal-associated biofilms may be different from the more superficial-lying, free-floating bacteria in the sinuses and that this may impact on the microbiome results obtained. This study investigates whether there is a significant difference in the microbiota of a sinonasal mucosal tissue sample versus a swab sample. Methods Cross-sectional study with paired design. Mucosal biopsy and swab samples were obtained intra-operatively from the ethmoid sinuses of 6 patients with CRS. Extracted DNA was sequenced on a Roche-454 sequencer using 16S-rRNA gene targeted primers. Data were analyzed using QIIME 1.8 software package. Results At a maximum subsampling depth of 1,100 reads, the mean observed species richness was 33.3 species (30.6 for swab, versus 36 for mucosa; p > 0.05). There was no significant difference in phylogenetic and non-phylogenetic alpha diversity metrics (Faith’s PD_Whole_Tree and Shannon’s index) between the two sampling methods (p > 0.05). The type of sample also had no significant effect on phylogenetic and non-phylogenetic beta diversity metrics (Unifrac and Bray-Curtis; p > 0.05). Conclusion We observed no significant difference between the microbiota of mucosal tissue and swab samples. This suggests that less invasive swab samples are representative of the sinonasal mucosa microbiome and can be used for future sinonasal microbiome studies.

The fungal microbiome in chronic rhinosinusitis: richness, diversity, postoperative changes and patient outcomes.

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.

Liposome-encapsulated ISMN: a novel nitric oxide-based therapeutic agent against Staphylococcus aureus biofilms.

Background Staphylococcus aureus in its biofilm form has been associated with recalcitrant chronic rhinosinusitis with significant resistance to conventional therapies. This study aims to determine if liposomal-encapsulation of a precursor of the naturally occurring antimicrobial nitric oxide (NO) enhances its desired anti-biofilm effects against S. aureus, in the hope that improving its efficacy can provide an effective topical agent for future clinical use. Methodology S. aureus ATCC 25923 biofilms were grown in-vitro using the Minimum Biofilm Eradication Concentration (MBEC) device and exposed to 3 and 60 mg/mL of the NO donor isosorbide mononitrate (ISMN) encapsulated into different anionic liposomal formulations based on particle size (unilamellar ULV, multilamellar MLV) and lipid content (5 and 25 mM) at 24 h and 5 min exposure times. Biofilms were viewed using Live-Dead Baclight stain and confocal scanning laser microscopy and quantified using the software COMSTAT2. Results At 3 and 60 mg/mL, ISMN-ULV liposomes had comparable and significant anti-biofilm effects compared to untreated control at 24 h exposure (p = 0.012 and 0.02 respectively). ULV blanks also had significant anti-biofilm effects at both 24 h and 5 min exposure (p = 0.02 and 0.047 respectively). At 5 min exposure, 60 mg/mL ISMN-MLV liposomes appeared to have greater anti-biofilm effects compared to pure ISMN or ULV particles. Increasing liposomal lipid content improved the anti-biofilm efficacy of both MLV and ULVs at 5 min exposure. Conclusion Liposome-encapsulated “nitric oxide” is highly effective in eradicating S. aureus biofilms in-vitro, giving great promise for use in the clinical setting to treat this burdensome infection. Further studies however are needed to assess its safety and efficacy in-vivo before clinical translation is attempted.

Probiotic manipulation of the chronic rhinosinusitis microbiome

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.

Methylglyoxal-augmented manuka honey as a topical anti–Staphylococcus aureus biofilm agent: safety and efficacy in an in vivo model

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.

Bacteriophage reduces biofilm of Staphylococcus aureus ex vivo isolates from chronic rhinosinusitis patients.

Background 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. Methods 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. Results 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. Conclusion 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.

Small-colony variants and phenotype switching of intracellular Staphylococcus aureus in chronic rhinosinusitis.

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.