Craig James

Surfactant Protein D Expression in Chronic Rhinosinusitis Patients and Immune Responses In Vitro to Aspergillus and Alternaria in a Nasal Explant Model

Objectives/Hypothesis: Common fungi have been implicated in the pathogenesis of chronic rhinosinusitis (CRS) with eosinophilic mucus (EMCRS). Surfactant protein (SP)‐D plays an important role in the immune response to Aspergillus fumigatus in the lungs. We sought to determine whether SP‐D is expressed in nasal mucosa and investigated the response of SP‐D in vitro to fungal allergens.

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.

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.

Safety and efficacy of topical bacteriophage and ethylenediaminetetraacetic acid treatment of Staphylococcus aureus infection in a sheep model of sinusitis

Treatment of sinonasal bacterial biofilms continues to be a challenge in modern rhinology. This studys 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.

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.

Do nasal polyps and inverted papilloma have similar disorders in cell cycle regulation

Background The aim of this study was to determine the expression of cell cycle regulation genes in patients with inverted papilloma (IP) and compare this with expression in patients with nasal polyps (NPs). Methods Tissue from 18 patients with IP and 5 patients with NPs were stained by immunohistochemistry techniques for p53 and p27. Measurement of the gene expression was performed by three assessors, who we blinded with respect to the specimens. Results The mean score for p53 expression (3.33) was significantly higher in the IP group than the NP group (1.46). The mean difference between IP and NPs was 1.80 (CI, 1.15–2.46; p = 0.003). Additionally, we showed in a number of individuals variation in the p53 expression within the same specimen. There was no difference in the mean scores for p27, with the mean difference 0.79 (CI, 0.30–1.89; p = 0.147). Conclusion Our study established a significantly increased expression of p53 in IP when compared with NPs. Additionally, there appear to be two different cell populations identified within the same specimens, which exhibited variation in their p53 expression.

Topical colloidal silver as an anti-biofilm agent in a Staphylococcus aureus chronic rhinosinusitis sheep model.

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.

An in vivo safety and efficacy demonstration of a topical liposomal nitric oxide donor treatment for Staphylococcus aureus biofilm-associated rhinosinusitis

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.

Long-Term Safety of Topical Bacteriophage Application to the Frontal Sinus Region

Background: Staphylococcus aureus biofilms contribute negatively to a number of chronic conditions, including chronic rhinosinusitis (CRS). With the inherent tolerance of biofilm-bound bacteria to antibiotics and the global problem of bacterial antibiotic resistance, the need to develop novel therapeutics is paramount. Phage therapy has previously shown promise in treating sinonasal S. aureus biofilms. Methods: This study investigates the long term (20 days) safety of topical sinonasal flushes with bacteriophage suspensions. The bacteriophage cocktail NOV012 against S. aureus selected for this work contains two highly characterized and different phages, P68 and K710. Host range was assessed against S. aureus strains isolated from CRS patients using agar spot tests. NOV012 was applied topically to the frontal sinus region of sheep, twice daily for 20 days. General sheep wellbeing, mucosal structural changes and inflammatory load were assessed to determine safety of NOV012 application. Results: NOV012 could lyse 52/61 (85%) of a panel of locally derived CRS clinical isolates. Application of NOV012 to the frontal sinuses of sheep for 20 days was found to be safe, with no observed inflammatory infiltration or tissue damage within the sinus mucosa. Conclusion: NOV012 cocktail appears safe to apply for extended periods to sheep sinuses and it could infect and lyse a wide range of S. aureus CRS clinical isolates. This indicates that phage therapy has strong potential as a treatment for chronic bacterial rhinosinusitis.

Chitosan Dextran gel as an anti adhesion agent in a postlaminectomy spinal sheep model

INTRODUCTION Post-operative peridural adhesions increase morbidity after neurosurgical procedures. Aim of this study is to assess safety and efficacy of Chitosan-Dextran (CD) gel as an anti-adhesion agent in a spinal laminectomy sheep model. METHODS Eighteen sheep were used in this study with 6 animals in each treatment arm (namely, CD gel, Gelfoam paste and normal saline control). Posterior lumbar laminectomy was performed in all animals and the dura was exposed intact. Test agents were applied over the exposed dura and the wound was closed in layers. Sheep were euthanized at the end of three months. MRI spine was performed after euthanasia to assess epidural fibrosis. Adhesion in the spinal specimen was assessed by Peel test and histopathology was used to assess safety of the agents. RESULTS Average scores for the Peel test for CD gel, Gelfoam and normal saline control groups were 1.16 (95% CI, 0.5-1.7), 1.5 (95% CI, 0.6-2.3) and 3 (95% CI, 2.1-3.8) respectively. There was significant reduction in adhesions between treatment and normal saline treated groups (p=0.0292), with no difference between Gelfoam and CD gel groups (p=0.56). Average scores on MRI for CD gel, Gelfoam and normal saline groups were 1.4 (95% CI, 0.9-1.8), 1.5 (95% CI, 1.2-1.8) and 1.6 (95% CI, 1.3-1.8) respectively, with no significant difference in fibrosis amongst (p=0.2992). Histopathology did not show any adverse effects. CONCLUSION CD gel is an effective agent to reduce epidural adhesions with a good safety profile in neural tissue.