Phl p 1-specific human monoclonal IgE and design of a hypoallergenic group 1 grass pollen allergen fragment.

Detailed understanding of how Abs of the IgE isotype interact with allergen at the onset of an allergic reaction is of great importance for deciphering mechanisms involved in the development of disease and may aid in the design of hypoallergenic variants. In this study, we have used a set of human monoclonal IgE Abs derived from the repertoires of allergic individuals, specific for the major timothy grass pollen allergen Phl p 1, to gain detailed information on the interaction between Abs and allergen. These allergen-specific IgE are to varying degrees cross-reactive toward both different allergen isoforms and various group 1 allergens originating from other grass species. The usage of human monoclonal IgE, as an alternative to polyclonal preparations or mouse Abs, allowed us to locate several important IgE-binding epitopes on the C-terminal domain of Phl p 1, all clustered to an IgE-binding "hot spot." By introducing three mutations in the IgE-binding area of the C-terminal domain we were able to significantly reduce its reactivity with serum IgE. In conclusion, our study shows the great potential of using human monoclonal IgE as a tool for studies of the molecular interactions taking place during allergic responses. Furthermore, we present a novel IgE-hyporeactive fragment with the potential to be used as a safer hypoallergenic alternative in specific immunotherapy than the pollen extracts used today.

Gene expression differences in nitric oxide and reactive oxygen species regulation point to an altered innate immune response in chronic rhinosinusitis.

BACKGROUND: 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. METHODS: Sinonasal tissue samples 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-). Samples 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. RESULTS: Twenty-three samples 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. CONCLUSION: 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.

Fungicidal activity of lysozyme is inhibited in vitro by commercial sinus irrigation solutions.

BACKGROUND: Lysozyme is an innate immune peptide with bactericidal and fungicidal activity (FA). Despite increased expression of lysozyme protein in chronic rhinosinusitis (CRS) sinus mucosa, CRS patients experience repeated bacterial and/or fungal infections. Commercial sinus irrigation solutions are often used to provide symptomatic relief. However, one of the mechanisms of action of lysozyme involves ionic interactions with the microbial cell wall, which may be inhibited by ionic solutions such as commercial sinus irrigation solutions. OBJECTIVE: Determine if the FA of lysozyme is reduced in the presence of solutions with increasing ionic strength and inhibited in the presence of commercial sinus irrigation solutions. METHODS: Using an in vitro colony-forming unit (CFU) assay, the FA of lysozyme (5 µM) was tested against a fungi commonly isolated from CRS patients, Aspergillus fumigatus, in solutions of increasing ionic strength or commercial sinus irrigation solutions. FA was presented as percent of control. RESULTS: FA of lysozyme against A. fumigatus was 95% in a 21-mM ionic strength solution. However, with increasing ionic strength, FA decreased and was abolished in a 46-mM ionic strength solution. Commercial sinus irrigation solutions abolished the FA of lysozyme against A. fumigatus. CONCLUSION: The in vitro FA of lysozyme is dependent on the ionic strength of the solution. The use of sinus irrigation solutions should be further evaluated with regard to maintaining functional activity of cationic antimicrobial peptides involved in sinonasal innate immunity.

Immunolocalization of NLRP3 Inflammasome in Normal Murine Airway Epithelium and Changes following Induction of Ovalbumin-Induced Airway Inflammation.

Little is known about innate immunity and components of inflammasomes in airway epithelium. This study evaluated immunohistological evidence for NLRP3 inflammasomes in normal and inflamed murine (Balb/c) airway epithelium in a model of ovalbumin (OVA) induced allergic airway inflammation. The airway epithelium of control mice exhibited strong cytoplasmic staining for total caspase-1, ASC, and NLRP3, whereas the OVA mice exhibited strong staining for active caspase-1, with redistribution of caspase-1, IL-1ß and IL-18, indicating possible activation of the NLRP3 inflammasome. Active caspase-1, NLRP3, and other inflammasome components were also detected in tissue eosinophils from OVA mice, and may potentially contribute to IL-1ß and IL-18 production. In whole lung, inRNA expression of NAIP and procaspase-1 was increased in OVA mice, whereas NLRP3, IL-1ß and IL-18 decreased. Some OVA-treated mice also had significantly elevated and tightly correlated serum levels of IL-1ß and TNFα. In cultured normal human bronchial epithelial cells, LPS priming resulted in a significant increase in NLRP3 and II-lp protein expression. This study is the first to demonstrate NLRP3 inflammasome components in normal airway epithelium and changes with inflammation. We propose activation and/or luminal release of the inflammasome is a feature of allergic airway inflammation which may contribute to disease pathogenesis.

The effects of nitric oxide on Staphylococcus aureus biofilm growth and its implications in chronic rhinosinusitis.

BACKGROUND: The relationship between sinonasal nitric oxide (NO) levels and the pathogenic organism Staphylococcus aureus is yet to be established. High NO levels measured in healthy sinuses likely contribute to maintenance of relative sterility. Lower concentrations such as is found in the sinuses of chronic rhinosinusitis (CRS) patients may decrease this effect. S. aureus in biofilm form has recently been implicated in recalcitrant CRS, its isolation predicting a higher risk of posttreatment reinfection. This in vitro study aims to characterize the changes in S. aureus biofilm formation when exposed to different NO levels mimicking the normal and diseased NO sinus concentrations reported in previous literature in an in vitro setting. METHODS: S. aureus ATCC 25923 and 7 clinical isolates were cultured in biofilm form using the MBEC device and the established biofilms exposed to 1 to 1000 µM NO concentrations. Biofilms were visualized using Live/Dead Baclight stain and confocal scanning laser microscopy, and quantified using Comstat2, a biofilm quantification software. RESULTS: Biofilm biomass decreases from an average of 0.105 to 0.057 µm(3) /µm(2) at higher NO concentrations (125-1000 µM), but is increased to 0.470 µm(3) /µm(2) at lower NO concentrations (0.9-2.0 µM). The average biomass at high vs low concentrations are statistically significant (p < 0.001). CONCLUSION: S. aureus biofilm formation varies across exposure to different NO levels, with antibiofilm effects at higher concentrations, and enhanced biofilm formation at lower or subphysiologic concentrations. These results coincide with the often dualistic function of NO, and have implications in its future use in the treatment of CRS.

Efficacy of using a hydrodebrider and of citric acid/zwitterionic surfactant on a Staphylococcus aureus bacterial biofilm in the sheep model of rhinosinusitis.

BACKGROUND: Biofilms in chronic rhinosinusitis (CRS) patients are associated with recalcitrant disease patterns. Citric acid/zwitterionic surfactant (CAZS) has been shown to have significant efficacy at in vitro biofilm eradication. Unique hydrodebrider systems have been shown to have effective antibiofilm efficacy. The aim of this study was to evaluate the efficacy of the CAZS/hydrodebrider treatment on Staphylococcus aureus biofilms in the sheep model of CRS. METHODS: Forty-two sheep frontal sinuses were inoculated with S. aureus, followed by 7 days of biofilm growth. Sinuses were randomized to one of nine treatment groups: control, hydrodebrider with saline or CAZS (killed on day 0 or day 7 posttreatment), CAZS, or saline flush (killed on day 0 or day 7 posttreatment). Confocal scanning laser microscopy (CSLM) was used to confirm extent of biofilm reduction. Samples of each sinus were taken, assessing cilia morphology using electron microscopy. RESULTS: A mean of 37.22 ± 9.65% of control (no treatment) CSLM images taken showed biofilms. Both CAZS flush and CAZS/hydrodebrider treatments showed initial improvements in biofilms but biofilm regrowth followed treatment, 14.74 ± 9.58% to 18.91 ± 12.14% and 15.60 ± 13.92% to 24.70 ± 3.66%, respectively (p > 0.05). Saline/hydrodebrider treatments showed a nonsignificant improvement in biofilm treatment, which was maintained, 18.34 ± 11.85% to 12.04 ± 10.28% (p > 0.05). Cilial morphology grade was significantly worse in the CAZS treatment groups (p < 0.05). CONCLUSION: CAZS solution (with and without a hydrodebrider system) can significantly adversely affect cilia morphology. The hydrodebrider system, when combined with saline, may be a useful treatment adjunct for mucosal biofilms.

Human lysozyme has fungicidal activity against nasal fungi.

BACKGROUND: The cationic antimicrobial peptide lysozyme is the most prevalent innate immune protein in nasal secretions but there is a paucity of research regarding its role in paranasal sinus disease. Lysozyme is generally regarded as an antibacterial agent; however, some data suggest activity toward yeast. This study was designed to determine if lysozyme displays fungicidal activity toward fungi commonly identified in patients with chronic rhinosinusitis (CRS) or fungal sinusitis. METHODS: Using a colony-forming unit assay the fungicidal activity of lysozyme (0, 0.5, 5, and 50 micromolar; 0- to 7-hour treatment) was tested against strains of Aspergillus fumigatus, the yeast Candida albicans, and other fungi commonly identified in mucin of patients with CRS. Fungi cultured directly from the mucin of two CRS patients were also tested to determine if they were resistant to the fungicidal activity of lysozyme. RESULTS: The fungicidal effect of lysozyme was both concentration and time dependent. After 7-hour treatment lysozyme (5 micromolar) had >80% fungicidal activity against A. fumigatus, Penicillium sp., Acremonium sp., C. albicans, and Candida parapsilosis. The fungicidal activity of lysozyme toward Alternaria alternata could not be determined. Lysozyme was also fungicidal toward the clinical isolates A. fumigatus and Aspergillus terreus cultured from the mucin of CRS patients. CONCLUSION: Lysozyme displays fungicidal activity toward many fungi commonly identified in patients with CRS, as well as clinical fungi isolates cultured from the mucin of CRS patients. Additional studies are required to determine the regulation of lysozyme in CRS.

A sheep model to investigate the role of fungal biofilms in sinusitis: fungal and bacterial synergy.

BACKGROUND: The role of fungi in the spectrum of chronic rhinosinusitis (CRS) is poorly understood. Fungal biofilms have recently been discovered in CRS patients. We have developed an animal model for the investigation of sinonasal fungal biofilms. The role of type I hypersensitivity and pathogenic bacteria is presented. METHODS: Thirty sheep were sensitized with fungal antigens-Aspergillus fumigatus and Alternaria alternata, or control. Endoscopic surgery was performed to expose both frontal sinus ostia-1 was occluded. Fungi with or without Staphylococcus aureus were inoculated into the sinus. Skin-prick tests assessed for fungal allergy. Fungal and S. aureus biofilms, histology, and culture rates were assessed. RESULTS: Forty-five percent of experimental sheep were sensitized to fungal antigen. Only 1 sinus inoculated with fungus developed minimal fungal biofilm. Eighty percent developed fungal biofilm when S. aureus was co-inoculated. The presence of hypersensitivity to fungus was not related to fungal biofilm development. CONCLUSION: Significant fungal biofilm only occurred when S. aureus was the co-inoculum. Hypersensitivity was not requisite. The relationship of S. aureus to fungal biofilms is of great clinical interest. Fungi may be opportunistic pathogens that simply require inflamed mucosa with weakened innate defenses; alternatively, a cross-kingdom synergy could be contributing to fungal proliferation.

Effects of simulated bleeding in an in vitro nasal fibroblast wound healing model.

BACKGROUND: We investigated the effect of simulated bleeding on plasminogen activity, matrix metalloproteinase (MMP) expression, and wound healing using a human fibroblast model. METHODS: Nasal fibroblasts from three chronic rhinosinusitis (CRS) patients with nasal polyps and three controls were grown in culture and a standardized injury was created using a punch. To mimic bleeding, fibroblasts were stimulated with plasminogen (100 microg/mL), plasminogen + tranexamic acid (TA; 100 microg/mL) or media only. At 24, 48, and 72 hours after injury, we measured urokinase plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activities and inactive and active MMP-2 and -9 expression. RESULTS: Injury stimulated the nasal fibroblasts to express uPA and tPA and active and inactive MMP-2 and -9. In CRS patients, plasminogen significantly decreased MMP-9 expression after 48 hours (p < 0.04). In untreated fibroblasts, we observed a decrease in active MMP-9 expression, whereas plasminogen increased active MMP-9 expression after 48 hours (p < 0.04). At 24 hours, active MMP-9 expression was reduced by plasminogen +/- TA (p < 0.02). Plasminogen also stimulated uPA expression in CRS patient fibroblasts after 48 hours (p < 0.04). Fibroblast proliferation occurred when exposed to plasminogen and was strongly modulated by uPA and inactive and active MMP-2. The quality of wound healing was affected by inactive MMP-2, uPA and tPA, simulation, and inhibition of bleeding. CONCLUSION: Activation of the plasminogen pathway and inactive MMP-2 expression tended to increase both proliferation of nasal fibroblasts and MMP-9 expression as a marker for deterioration of the quality of wound healing.

Characterization of bacterial and fungal biofilms in chronic rhinosinusitis.

BACKGROUND: Conclusive evidence exists that biofilms are present on the mucosa of chronic rhinosinusitis (CRS) patients. Less is known about the species constituting these biofilms. This study developed a fluorescence in situ hybridization (FISH) protocol for characterization of bacterial and fungal biofilms in CRS. METHODS: Fifty CRS patients and 10 controls were recruited. Bacteria FISH probes for Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa and a universal probe for fungi were applied to sinus mucosal specimens and then analyzed using confocal scanning laser microscopy. RESULTS: Thirty-six of 50 CRS patients had biofilms present in contrast to 0/10 controls, suggesting a role for biofilms in the pathogenesis of this disease. S. aureus was the most common biofilm-forming organism. Eleven of 50 CRS patients had characteristic fungal biofilms present. CONCLUSION: This is the largest study of biofilms in CRS. It has validated mucosal tissue cryopreservation for delayed biofilm analysis. Fungal biofilms have been identified and the importance of S. aureus biofilms in the polymicrobial etiology of CRS is highlighted.

Innate immunity in the paranasal sinuses: a review of nasal host defenses.

BACKGROUND: Chronic rhinosinusitis (CRS) is a common inflammatory disorder of the paranasal sinuses. An abnormal host response to common bacterial or fungal pathogens is thought to be an important factor in the disease process. Host sinonasal epithelium plays an important role in initially recognizing the presence of microbes and responding by increasing production of antimicrobial peptides and cytokines, with recruitment of phagocytes and lymphocytes of the adaptive immune system, to eliminate the infection. Recently, the innate immune system and its complex interplay with the adaptive immune system are increasingly being recognized as important in the pathogenesis of chronic inflammatory diseases such as asthma and CRS. METHODS: Review of recent findings on innate immunity in the pathogenesis of CRS. RESULTS: New areas of research into potentially novel therapies for CRS are highlighted in this review, with emphasis on toll-like receptors, antimicrobial peptides (cathelicidins and defensins), and surfactant proteins. CONCLUSION: This review provides an overview of innate immunity in the sinonasal tract and discusses potential use of innate immune peptides as treatments against fungi, biofilms, and superantigens in CRS.

Reduced levels of lactoferrin in biofilm-associated chronic rhinosinusitis.

OBJECTIVE/HYPOTHESIS: The diverse antipathogenic action of lactoferrin has been well characterized. In addition, it is the human body's only known antimicrobial peptide with antibiofilm properties. The purpose of this study was to examine the nasal mucosal expression of lactoferrin in the biofilm-mediated disease, chronic rhinosinusitis (CRS). STUDY DESIGN/METHODS: Nasal biopsies from 41 CRS patients and 21 healthy controls were analyzed using confocal scanning laser microscopy (CSLM) and scanning electron microscopy (SEM) for the presence of biofilms. The messenger ribonucleic acid (mRNA) and protein level of lactoferrin in this tissue were also determined by quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbent assay (ELISA), respectively. RESULTS: Lactoferrin expression in chronic rhinosinusitis patients at both mRNA and protein level was downregulated relative to controls. Biofilm-positive CRS patients showed a much greater reduction in lactoferrin expression than biofilm-negative patients; mRNA median fold change biofilm positive = 0.03 (interquartile range 0.005-0.15) and biofilm-negative CRS median fold change = 0.49 (interquartile range 0.15-0.81) with median lactoferrin protein expression biofilm-positive patients' median lactoferrin protein expression = 32.58 ng/mL (interquartile range 8.67-59.9 ng/mL) and biofilm-negative patients' median lactoferrin expression = 114.40 ng/mL (interquartile range 75.41-163.1 ng/mL). CONCLUSIONS: Genetic, transcriptional, or translational deficiencies in lactoferrin synthesis may reduce the functional level of this important antimicrobial/antibiofilm peptide in the nasal secretions of CRS patients, predisposing certain individuals to bacterial colonization, biofilm development, and recalcitrant sinus disease.

In vitro activity of mupirocin on clinical isolates of Staphylococcus aureus and its potential implications in chronic rhinosinusitis.

BACKGROUND: It has been postulated that bacterial biofilms are involved in the pathogenesis of chronic rhinosinusitis (CRS). Biofilms present on sinus mucosa are difficult to eradicate with conventional antibiotic therapy and are thought to provide a nidus for recurrent infection. Topical delivery of antibiotics via nasal irrigation may present a way of delivering high concentrations of antibiofilm agents with potentially low systemic absorption and side effects. This study investigates the effectiveness of mupirocin and two other antibiotics, ciprofloxacin and vancomycin, on established in vitro biofilms of Staphylococcus aureus isolated from patients with CRS. METHODS: S. aureus American Type Culture Collection 25923 and 12 clinical isolates were investigated for their ability to form biofilms in an in vitro setting using a 96 well microtiter crystal violet (CV) plate assay and confocal scanning laser microscopy (CSLM). Antimicrobial susceptibility tests to determine minimum inhibitory concentrations were performed on planktonic and biofilm forming strains. In addition, established biofilms were subjected to the antimicrobial agents at a twofold dilution series. A CV analysis of biofilm mass was performed after 1 and 24 hours of treatment, and minimum biofilm inhibition concentrations at 50% (MIB50) and 90% (MIB90) biofilm inhibition were recorded. RESULTS: With use of a 96-well microtiter plate CV assay, 8 of the 12 clinical isolates formed mature biofilms after 8 days of culture. These results correlated with findings from CSLM analysis of in vitro biofilms grown on Permanox chamber slides. Increased antimicrobial resistance was observed in the biofilm isolates when compared with planktonic counterparts. Mupirocin was capable of reducing biofilm mass by greater than 90% at concentrations of 125 mug/mL or less in all S. aureus isolates. Ciprofloxacin and vancomycin were largely ineffective in attaining MIB90 concentrations within safe dosage ranges. CONCLUSIONS: The topical application of mupirocin via nasal irrigation may be useful in eliminating S. aureus biofilms present on the sinus mucosa of patients with CRS and may offer an additional treatment to patients with recalcitrant sinusitis.

The efficacy of topical antibiofilm agents in a sheep model of rhinosinusitis.

BACKGROUND: Biofilms have been shown to be resistant to conventional antibiotic treatment. This study uses a sheep biofilm model developed by our department to investigate several novel topical anti-biofilm treatments. METHODS: Staphylococcal biofilms were grown in 54 sheep frontal sinuses over 8 days: Each sinus was randomized to (1) no intervention, (2) single mupirocin flush, (3) regular 12-hourly mupirocin flushes for 5 days, (4) Citric Acid Zwitterionic Surfactant (CAZS) via hydrodebrider, (5) gallium nitrate, (6) CAZS with gallium nitrate, (7) CAZS with mupirocin, and (8) saline regular flushes. Sheep were sacrificed and the sinus mucosa harvested 1 or 8 days after treatment to assess treatment and any biofilm regrowth. Confocal scanning laser microscopy was used to confirm the presence or absence of biofilms, and the extent of biofilm reduction was quantitated using fluorescent in situ hybridization and colony forming unit counts. RESULTS: In the control sheep biofilm coverage averaged 31.7%. Saline and mupirocin b.d. washes for 5 days had 23% and 0.84% coverage, respectively, when harvested on day 8. A single mupirocin and gallium wash had 7.7% and 16.2% on day 1 and 5.88% and 16.0% on day 8. CAZS with hydrodebrider had 6.66% on day 1 but 21.95% on day 8 whereas CAZS with hydodebrider and gallium had 13.3% on day 8. CONCLUSION: This study shows that regular treatment with mupirocin produced the most marked reduction in biofilm surface area coverage (0.84% and 1.25%) with sustained effects over the 8-day follow-up period.

Human cathelicidin antimicrobial peptide is up-regulated in the eosinophilic mucus subgroup of chronic rhinosinusitis patients.

BACKGROUND: Eosinophilic mucus chronic rhinosinusitis (EMCRS) patients are a subgroup of CRS with a poorer prognosis due to frequent recurrences of disease. The cathelicidin antimicrobial peptide (CAMP) is an important innate defense peptide but its role in CRS is not well characterized. The purpose of this study was to investigate CAMP mRNA and protein expression from EMCRS, CRS, and normal control patients. METHODS: Biopsy specimens of nasal mucosa and nasal polyps were taken from 59 CRS patients and 9 controls. CAMP mRNA and protein levels were analyzed by real-time quantitative reverse-transcriptase polymerase chain reaction, immunoassay, Western blot, and immunohistochemistry. RESULTS: The expression of CAMP mRNA was significantly increased in EMCRS patients compared with CRS patients (p = 0.0004). By immunohistochemistry, expression of CAMP was localized to nasal epithelial, submucosal glands, and inflammatory subepithelial cells. Western blotting confirmed the presence of CAMP in EMCRS, CRS, and control patients. However, we did not detect statistically significant differences in the protein levels in tissue homogenates between EMCRS, CRS, and control patients. CONCLUSION: This study shows expression of CAMP in nasal mucosa supporting its role in innate defenses against inhaled pathogens. Although CAMP mRNA was up-regulated in EMCRS patients, there were no statistically significant differences in protein levels in the nasal mucosa of EMCRS compared with CRS patients and controls.

Nasal mucosa expression of lactoferrin in patients with chronic rhinosinusitis.

OBJECTIVE/HYPOTHESIS: Antimicrobial peptides, such as lactoferrin, are an important component of the innate immune system. They offer the body a first line defense against a wide range of invading pathogens. The diverse antipathogenic action of lactoferrin has been well characterized; however, the role that this peptide plays in chronic conditions such as rhinosinusitis remains largely unknown. This study aims to examine the level of lactoferrin expression in the nasal mucosa of patients with chronic rhinosinusitis (CRS). STUDY DESIGN/METHODS: Nasal biopsies of 85 chronic rhinosinusitis patients, subclassified into allergic fungal sinusitis (AFS), nonallergic fungal eosinophilic sinusitis (NAFES), nonallergic, nonfungal eosinophilic sinusitis (NANFES), and CRS were studied by quantitative real-time reverse-transcriptase polymerase chain reaction and enzyme-linked immunosorbent assay for their expression of lactoferrin at an mRNA and protein level, respectively. RESULTS: All groups of patients with CRS showed a decrease in lactoferrin mRNA expression relative to controls (median fold-change of CRS relative to controls, 0.1550; AFS, 0.1800; NANFES, 0.1900; and NAFES, 0.2100). All groups also showed a decreased expression of lactoferrin protein (controls, 163.3 ng/mL; CRS, 82.19 ng/mL; AFS, 104.1 ng/mL; NANFES, 118.9 ng/mL; and NAFES, 74.33 ng/mL). The most significant reduction was evident in the CRS subgroup as well as in patients with nasal polyposis at the time of surgery. CONCLUSIONS: This is the first study of its kind to objectively examine lactoferrin expression in the nasal mucosa of CRS patients. We report a reduction in the expression of this important antimicrobial peptide at both the mRNA and protein level. Such a defect in the innate immune system may explain the predisposition of certain individuals to develop CRS and nasal polyposis, providing further insight into the pathogenesis of such conditions.

Fungal allergens induce cathelicidin LL-37 expression in chronic rhinosinusitis patients in a nasal explant model.

BACKGROUND: Fungus is thought to play an important role in some subgroups of chronic rhinosinusitis (CRS) patients with eosinophilic mucus (EMCRS). The cathelicidin LL-37 is an important innate defense peptide with antimicrobial activity but its responses in CRS and EMCRS patients have not been established. We investigated the innate immune responses of LL-37 in nasal tissue from CRS and EMCRS patients to fungal allergen challenge. METHODS: The levels of LL-37 produced by nasal tissue and secreted in response to fungal allergen challenge were determined by a nasal tissue explant in vitro model. LL-37 mRNA and protein levels were quantified by real-time reverse-transcriptase-polymerase chain reaction and immunoassay methods. RESULTS: LL-37 mRNA expression in CRS, but not EMCRS patients, is significantly upregulated by Aspergillus (mean fourfold increase) and Alternaria (mean sixfold increase) extracts in a dose-response manner (p < 0.001). LL-37 peptide levels in the nasal tissue from CRS patients are increased in response to Alternaria (p < 0.05). In contrast, with EMCRS patients, the expression of LL-37 peptide in nasal tissue is increased with Aspergillus (p < 0.001) but is reduced with Alternaria. We also observed a trend where levels of secreted LL-37 were decreased with higher doses of Alternaria and Aspergillus extracts. CONCLUSION: LL-37 is significantly up-regulated at the mRNA and protein level in CRS patients in response to fungal allergens. However, EMCRS patients do not show increased LL-37 at either the mRNA or the protein level in response to Alternaria.

Confocal scanning laser microscopy evidence of biofilms in patients with chronic rhinosinusitis.

OBJECTIVES: The recent detection of bacterial biofilms on the sinus mucosa of patients with chronic rhinosinusitis (CRS) has implicated biofilms in the pathogenesis of this condition. Electron microscopy has been the main modality used to document the presence of biofilms on sinus tissue, however, it has inherent problems associated with tissue preparation and sampling. Recently, Confocal Scanning Laser Micrsocopy (CSLM) has emerged as a noninvasive, nondestructive technique for the analysis of biofilms. This study used CSLM as the means of investigating biofilm presence in CRS patients. STUDY DESIGN AND METHODS: A prospective study comparing the presence of bacterial biofilms on the sinus mucosa of CRS and control patients was conducted using CSLM. Thirty eight CRS patients undergoing endoscopic sinus surgery and nine control patients were enrolled in this study. Demographic and clinical information was recorded from each patient and intraoperatively, sinus culture specimens and mucosal samples were obtained for microbiologic and microscopic examination. RESULTS: Using previously documented CSLM criteria, bacterial biofilms were found in 17 (44%) of the 38 CRS patients. No biofilm structures were evident in any of the controls. Patients having undergone previous sinus surgery seemed to have a higher incidence of biofilms compared with the incidence for those undergoing their first procedure. The difference however was not statistically significant. No correlation between positive bacterial cultures and biofilm presence was observed. CONCLUSIONS: The CSLM detection of biofilms in CRS patients and their absence in controls further supports the hypothesis that biofilms may play a role in the pathogenesis of CRS. This study's lower reported incidence of biofilms compared with that of previous studies might reflect the increased accuracy of biofilm detection with CSLM.

Response of peripheral blood lymphocytes to fungal extracts and staphylococcal superantigen B in chronic rhinosinusitis.

BACKGROUND: Previous studies have suggested that chronic rhinosinusitis may result from a hypersensitivity response of the nasal mucosa to the presence of fungal antigens or staphylococcal superantigens in the nasal mucus. Both of these groups of antigens are present so frequently in the nasal mucus of patients with chronic rhinosinusitis that their presence together is likely to be a common event. OBJECTIVE: The objective of this study was to determine whether the combined presence of fungal antigens and staphylococcal superantigens exert a synergistic proinflammatory effect on peripheral blood lymphocytes from patients with chronic rhinosinusitis. METHODS: Peripheral blood lymphocytes were extracted from patients with chronic rhinosinusitis with and without nasal polyposis (n = 7 for both groups) and normal controls (n = 7). These cells were cultured for 48 hours after the addition of fungal extracts (Aspergillus and Alternaria), staphylococcal superantigen type B (SEB), or a combination of these two antigens. Real-time polymerase chain reaction was used to determine the level transcription of interleukinL-5 and interferon-gamma genes. RESULTS: Fungal extracts alone resulted in minimal changes in the levels of cytokine expression in peripheral blood lymphocytes. SEB increased the expression of IFN-gamma, and this effect was magnified by the addition of SEB and fungal extracts together to the culture medium. There were no differences in the magnitude of responses seen in patients with and without polyps nor between patients with chronic rhinosinusitis and normal controls. CONCLUSION: SEB exerts a powerful proinflammatory effect on peripheral blood lymphocytes and fungal extracts may act synergistically to promote this action.