Dissertations / Theses on the topic 'Chronic rhinosinusitus'

Chronic Rhinosinusitis (CRS), an inflammation of the paranasal sinus cavities, is a common disorder of uncertain etiology that affects the upper airways and paranasal sinuses. Biopsy specimens had taken from CRS patients document disruption of the normal epithelial architecture, in addition to an intense infiltration of inflammatory cells, consisting mainly of eosinophils. Current clinical classification of CRS is based on the presence or absence of nasal polyposis; however, no consistent difference in histological aspect characterizes these two groups. Recently, we have identified distinct gene expression patterns in cultured epithelial cells obtained from surgical CRS subjects. These molecular signatures, which differ from clinical phenotype, may help better differentiate this disorder than clinical phenotype. In our current study we investigated the histological pattern associated with these two different molecular signatures in surgical biopsies obtained from CRS patients and control subjects. Cellular infiltrates were identified using immunohistochemistry (IHC) staining using three markers: neutrophil elastase (NE), CD68, Major basic protein (MBP). Macrophage activation status into classical and alternatively activated macrophages was verified by double-staining for CD68 and CD 206 markers. Results were reported both according to standard clinical criteria (CRSwNP and CRSsNP) and also according to their expression signature into two groups (CRS1, CRS2) and control subjects. Expression signatures were validated using immunohistochemical staining for the highest differentially expressed marker, CCL2. Results showed differences in the number of eosinophils, macrophages and neutrophils cells in CRS patients compared to the control subjects. Using conventional criterion, eosinophilia was higher in the CRSwNP group, but not greatly different for neutrophils or macrophages between the two groups. Using the molecular signatures to assign groups, eosinophilia was similar between both groups, however, there was a significant increase in the number of neutrophils and macrophages in CRS1 comparing to CRS2. The CRS2 group had a higher incidence of alternatively activated macrophages, supporting the concept of a less inflammatory, immunotolerant CRS2 phenotype. Validity of the molecular signature was supported by demonstration of increased levels of protein product of CCL2 expression in CRS1 compared to CRS2. Taken together, these results identify a molecular phenotype of CRS that is characterized by a marked neutrophilic infiltration, and a second one that is markedly less inflammatory, accompanied by alternative macrophage activation. This suggests that these expression signatures may identify novel mechanism-based phenotypes, which differ from the clinical phenotype, and can help in providing a better understanding of pathophysiologic mechanism and phenotypes of CRS.
La rhinosinusite chronique (RSC), une inflammation des sinus paranasaux, est un trouble commun avec une étiologie incertaine, qui affecte les voies respiratoires supérieures et les sinus paranasaux. Les biopsies des échantillons prélevés sur des patients atteints de RSC documentent la perturbation de l'architecture normale épithéliale, en plus d'une infiltration de cellules inflammatoires intense constituée principalement par des éosinophiles. La classification clinique actuelle de la RSC est basée sur la présence (CRSwNP) ou l'absence (CRSsNP) de polypose nasale, mais aucune différence consistente de l'aspect histologique caractérise ces deux groupes. Récemment, nous avons identifié des profils d'expression génique distincts dans des cultures de cellules épithéliales provenant de sujets atteints de la RSC ayant subis une chirurgie des sinus. Ces signatures moléculaires, qui diffèrent du phénotype clinique, peuvent aider à mieux différencier ce trouble que le phénotype clinique. Dans notre étude, nous avons étudié l'aspect histologique associé à ces deux différentes signatures moléculaires à partir de biopsies chirurgicales obtenus chez des patients atteints de la RSC et les sujets témoins. Les infiltrats cellulaires ont été identifiés par immunohistochimie (IHC), une coloration à l'aide de trois marqueurs: l'élastase de neutrophile (NE), le CD68 et la protéine basique majeure (MBP). L'état d'activation des macrophages dans les formes classiques et alternativement activés a été vérifié par une double-coloration pour les marqueurs CD68 et CD206. Les résultats ont été rapportés à la fois selon les critères cliniques habituels (CRSwNP et CRSsNP) et aussi en fonction de leur signature d'expression en deux groupes (CRS1, CRS2) et les sujets témoins. Les signatures d'expression ont été validées à l'aide de coloration immunohistochimique pour le marqueur le plus différentiellement exprimé, le CCL2.Les résultats ont montré des différences dans le nombre d'éosinophiles, macrophages et les cellules de neutrophiles chez les patients atteints de la RSC par rapport aux sujets témoins. Selon le critère classique, l'éosinophilie était plus élevée dans le groupe CRSwNP, mais pas très différent entre les deux groupes pour les neutrophiles ou les macrophages. En utilisant les signatures moléculaires pour assigner des groupes, l'éosinophilie était similaire entre les deux groupes, cependant, il y avait une augmentation significative du nombre de neutrophiles et de macrophages dans CRS1 comparativement à CRS2. Le groupe CRS2 avait une incidence plus élevée des macrophages alternativement activés, supportant le concept d'une inflammatoire basse, phénotype CRS2 immunotolérant. La validité de la signature moléculaire a été supportée par la démonstration du niveau accru de la protéine produite par l'expression de CCL2 dans CRS1 par rapport à CRS2.En somme, ces résultats mettent en évidence un phénotype moléculaire de la RSC qui se caractérise par une infiltration neutrophilique marquée, et une seconde qui est nettement moins inflammatoire, accompagnée par l'activation alternative des macrophages. Ceci suggère que ces signatures d'expression peuvent identifier de nouveaux mécanismes basés sur des phénotypes, qui diffèrent du phénotype clinique, et peuvent aider à fournir une meilleure compréhension du mécanisme physiopathologique et les phénotypes de la RSC.

The aim of this thesis is to evaluate and optimise current pharmacotherapeutic options in rhinosinusitis. There is often a marked variation in treatment response in those afflicted with chronic rhinosinusitis, both within and between patients, attributable in part to different disease phenotypes/endotypes, poor awareness of treatment optimization options, and trivialization of symptoms by patients and physicians. Characteristically, these factors contribute to a typical remitting and relapsing disease course. The objectives of this work are to improve the therapeutic index and reach of commonly used medications by boosting efficacy whilst reducing concomitant side effects. The third chapter explores the use of initial oral steroids in patients with chronic rhinosinusitis and nasal polyposis, focusing on the role of the ostiomeatal complex in the perpetuation of disease symptoms. Often a short course of oral steroids is used in patients with moderate to severe disease to achieve initial control before maintenance with intranasal steroids. This is termed as a ‘medical polypectomy’ and anecdotally is commonly used in patients with chronic rhinosinusitis with nasal polyposis (CRSwNP). However, the evidence for its efficacy is tenuous and there are no data to evaluate if it indeed re-establishes ostiomeatal sinus complex drainage which is a condicio sine qua non of ensuring long-term symptom resolution. Further, it is known that monotherapy with nasal steroids may result in loss of symptom control. We have therefore in a double-blind placebo controlled trial (Chapter 4) evaluated the effect of this initial induction with oral steroids on subsequent sequential intranasal therapy. Perhaps, however, more crucially we have for the first time comprehensively addressed the safety of both oral and topical steroids in patients with CRSwNP who have other concomitant steroid-dependent illnesses like asthma and COPD. A particularly refractory subset of those with CRSwNP also have aspirin intolerance and asthma. While recent guidelines have recommended more aspirin challenge testing in these patients, it is unclear what the significance of a positive test is in the absence of overt clinical symptoms or in patients with only moderate disease. This is addressed in Chapter 5, as this significant phenotype of aspirin intolerant rhinosinusitis need close monitoring, dose optimization, polytherapy, and in selected cases may be suitable for aspirin desensitization. Penultimately, we evaluate in a double-blind placebo controlled trial (Chapter 6) the tachyphylaxis and rebound congestion that blights the medium to long-term use of sympathomimetic nasal decongestant sprays like oxymetazoline and if this can be reversed by the concomitant use of nasal steroids. We also characterized nasal blood flow as an outcome to evaluate in these patients and its relation to other rhinological outcome measures (Chapter 7).

Chronic rhinosinusitis (CRS) is a common and debilitating disorder. There is a deficit of knowledge about the epidemiology of CRS or the experience of sufferers. The aims of the study were to identify differences in socio-economic variables and quality of life between patients with chronic rhinosinusitis and healthy controls, to identify any significant associations between CRS and other medical co-morbidities, psychiatric disease or environmental exposures and to explore the experience of CRS from the perspective of CRS sufferers. This study consisted of a self-reported questionnaire distributed from 30 ENT clinics across the UK, and qualitative interviews with 21 patients with CRS. Additional studies were undertaken to support this work including further qualitative interviews with patients who have disturbed olfaction, and studies to assess new or unproven treatment regimens including a feasibility study for Clarithromycin for CRS and a trial of sodium citrate for hyposmia. No clear differences in socioeconomic variables were identified between cases and controls. CRS was found to be strongly associated with asthma and inhaled allergies as well as significantly impairing quality of life. Quality of life issues were very important to sufferers, and had been poorly addressed, particularly with regards to sense of smell. Further research is needed to better understand and manage CRS although better adherence to current guidelines would improve care in the interim.

Chronic rhinosinusitis is a common disorder of chronic inflammation of the upper respiratory tract. It is associated with significant symptoms and impairment of the quality of life of sufferers. Despite recent advances in the medical and surgical management of chronic rhinosinusitis, there remains a population of patients who fail to obtain relief from their symptoms. Chronic inflammation of the mucosa of the nasal cavity and paranasal sinuses is one of the hallmarks of chronic rhinosinusitis. This inflammation is demonstrated by an increased number of chronic inflammatory cells, elevated levels of pro-inflammatory cytokines, increased expression of adhesion molecules and metaplastic changes in the epithelium. The current medical treatments for chronic sinusitis aim to reduce this inflammation and consequently improve symptoms. In recent years, evidence has emerged that macrolide antibiotics have an anti-inflammatory effect that is separate from their anti-bacterial effect. This effect was first described in the treatment of diffuse panbronchiolitis, a disorder of chronic inflammation of the lower respiratory tract. Following the success of macrolides in treating this condition it was trialed in chronic rhinosinusitis. Several open-label trials have subsequently demonstrated a beneficial effect. Laboratory studies have investigated the mechanism of the anti-inflammatory effect of macrolides. These have shown that macrolides effect cytokine production, inflammatory cell apoptosis, expression of adhesion molecules, neutrophil oxidative burst, bacterial virulence and mucociliary function. In this thesis we report a series of experiments designed to further investigate the mechanism of action and clinical effect of macrolides. In vitro studies using whole sections of chronic rhinosinusitis mucosa cultured for 24 hours in macrolide, prednisolone or control showed that macrolide and prednisolone produced significant reductions in the production of interleukin-5, interleukin-8 and granulocyte-macrophage colony stimulating factor. The same cultured specimens also showed a reduction in expression of transforming growth factor-?. No reduction was seen in the expression of the key pro-inflammatory nuclear transcription factor Nuclear factor-?B. In our in vivo experiments, biopsies were taken from chronic rhinosinusitis patients who had received a 3-month course of macrolide. These biopsies showed a reduction in the number of neutrophils present following treatment. There was no reduction in the number of other inflammatory cells or in the expression of TGF-? and NK-?B. We have performed the first ever double-blinded, randomized, placebo-controlled trial of macrolide in the treatment of chronic rhinosinusitis. Patients receiving macrolide showed significant improvements in saccharine transit time, nasal endoscopic scoring and symptom scores following a 12 week course. Patients with low levels of serum immunoglobulin E showed significantly improved outcomes compared to those with high levels. Interleukin-8 levels in nasal lavage fluid were significantly reduced in the patients with low levels of IgE following macrolide treatment. No improvements in any of the objective or subjective outcome measures were seen in the placebo-treated patients. We have performed a series of experiments investigating the anti-inflammatory effect of macrolide antibiotics from 'the bench to the bedside'. These experiments have provided insight into the mechanism of action of macrolides in the laboratory setting and evidence of a beneficial effect in the treatment of chronic rhinosinusitis patients.

Chronic rhinosinusitis without nasal polyps (CRSsNP) is a heterogeneous condition with common symptoms, clinical and radiological findings. CRSsNP is typified by inflammation of the sinonasal epithelium and development of fibrosis, yet its precise pathophysiology remains elusive. Recently stromal cells have been shown to act like immune effector cells in orchestrating chronic inflammation. Histological analysis of tissue biopsies from patients with CRSsNP demonstrates recruitment of circulating inflammatory cells, though the precise role of structural cells such as epithelial and fibroblast cells in CRSsNP remains to be discovered. Aims 1. (a) Recruit phenotyped cohorts of control & CRSsNP participants. (b) Characterise recruited CRSsNP participants’ tissue samples and isolated epithelial & fibroblast cells. 2. Assay the sinonasal environment to determine any association between, infection, inflammation and remodelling. 3. Identify clusters of genes differentially expressed in CRSsNP & control participants. Methods Cohorts of healthy control and CRSsNP participants were recruited. Matched tissue biopsy, epithelial and fibroblast cells were harvested together with clinical, radiological, microbiological and mucosal swab data. Tissue and cellular samples were characterised to confirm their identity and disease status. The sinonasal environment was characterised from mucosal swabs and analysed for a range of 40 human disease biomarkers. Transcriptome analysis was performed using microarrays and RNA sequencing with downstream bioinformatics investigation of the data. Results 47 age and sex matched CRSsNP and control participants were recruited, differing significantly in symptom and radiological scores. Histological analysis of tissue biopsy specimens was consistent with CRSsNP and control samples. Matched epithelial and fibroblast cells were generated. Assay of the sinonasal microenvironment identified 13 discriminant mediators separating CRSsNP samples from controls using a novel, non-invasive technique. Transcriptomics identified 239 differentially expressed genes in CRSsNP tissue biopsy samples. Cellular samples differed significantly from their matched tissue biopsies. Conclusions This thesis characterises a cohort of tightly defined CRSsNP patients and healthy controls to investigate the potential role of epithelial and fibroblast cells in CRSsNP. Transcriptomics has demonstrated clusters of genes upregulated in CRSsNP, however changes were not consistent in matched cellular samples questioning the validity of cellular models in CRSsNP. Additionally, a straightforward, non-invasive measure of the CRSsNP cytokine profile has been demonstrated. The mediators identified in these assays could potentially be developed as biomarkers of sinonasal inflammation as an adjunct in patient management.

Os modelos experimentais têm um papel importante no conhecimento dos mecanismos envolvidos na patogênese da rinossinusite crônica (RSC). Objetivos: comprovar que sem inoculação de bactérias seria possível induzir alterações histológicas crônicas nos seios maxilares de coelhos através da obstrução do óstio de drenagem dos mesmos, produzindo um modelo experimental consistente e reproduzível para RSC. Secundariamente, comparar achados inflamatórios entre duas técnicas de oclusão do óstio do seio maxilar com N-butil cianocrilato: via transmaxilar (VTM) e via teto de fossa nasal (VTFN). Métodos: estudo experimental randomizado cego em animais de laboratório realizado na Unidade de Experimentação Animal do Centro de Pesquisa do Hospital de Clínicas de Porto Alegre, no qual foram sorteados dezesseis coelhos Nova Zelândia entre oclusão do seio maxilar direito VTM ou VTFN. Após 12 semanas de seguimento, os animaisforam anestesiados e sacrificados para análise histopatológica cegada da mucosa do seio maxilar. Resultados: apresentavam alterações histopatológicas compatíveis com RSC os oito (100%) seios maxilares intervindos através da técnica VTM e três (37,5%) através da técnica VTFN, com p 0,008 e 0,250, respectivamente, quando comparados lado direito com o lado controle. Comparando-se as duas técnicas de oclusão, a técnica VTM mostrou-se mais consistente em provocar alterações crônicas nas mucosas dos seios maxilares ocluídos (p 0,026). Conclusões: O modelo do presente trabalho obteve sucesso em provocar alterações histológicas compatíveis com RSC nos animais submetidos à técnica de oclusão VTM com seguimento de 12 semanas, podendo ser facilmente replicável para futuros estudos celulares na mucosa sinusal.
Experimental models have an important role in understanding the mechanisms involved in the pathogenesis of chronic rhinosinusitis (CRS). Objectives: To demonstrate that, without the inoculation of pathogenic bacteria, it is possible to induce chronic histological changes in the maxillary sinuses of rabbits secondary to sinus ostium obstruction, producing a consistent and reproducible experimental model for CRS. Secondly, to compare inflammatory findings between two techniques of experimental occlusion of the maxillary sinus ostium with N-butyl cyanoacrylate: transmaxillary and through the roof of the nasal cavity. Methods: In a randomized, blinded, experimental study, 16 New Zealand rabbits were assigned for occlusion of the right maxillary sinus through a transmaxillary approach or through the roof of the nasal cavity. The contralateral sinus was left undisturbed to serve as a control. After 12 weeks of follow-up, the animals were anesthetized and sacrificed for blinded histopathological analysis of the maxillary sinus mucosa. Results: Histopathological changes consistent with CRS were found in eight (100%) of the maxillary sinuses approached transmaxillary and three of thoseapproached through the roof of the nasal cavity (37.5%), p 0.008 and 0.250, respectively, comparing the right to the left control sinus. Comparing the occlusion techniques, the transmaxillary approach was more consistent in causing chronic mucosal changes (p 0.026). Conclusions: The proposed model was successful in causing histological changes compatible with CRS in animals subjected to sinus occlusion with a transmaxillary approach followed-up for 12 weeks. This experimental model can be easily replicated for future cellular studies of the sinus mucosa.

Background The mechanisms of the rhinitic process are complex. Previous studies upon nasal epithelial cells have begun to investigate rhinitis. HNECs from turbinate explant tissue were taken from three patient groups (Normals, Chronic Rhinosinusitics and Rhinitics). Aims The study, firstly, aims to establish fundamental differences in cytokine activity between allergic rhinitis and chronic rhinosinusitis by analysing baseline levels of cytokines IL-6 and IL-8 and subsequent impact of bacterial endotoxin. Secondly the study analyses the affect of macrolides on activity in each subgroup. Methods HNECs were grown from the biopsy specimens as explant culture. Standardised exposures to LPS bacterial endotoxin and macrolide were carried out. The concentration of each mediator present in the medium at the end of incubation was assessed by ELISA). A final quantity of total cellular protein was obtained. 3 Results Baseline levels of IL-6 in unstimulated Allergic Rhinitics are significantly higher than in Normal patients. Baseline levels of IL-8, however, are lowest in Allergics. LPS significantly stimulates Allergics to increase production of both IL-6 and IL-8. Macrolides lower IL-6 and IL-8 in both stimulated and unstimulated AR cells. Baseline levels of IL-6 and IL-8 are higher in CRS than AR and Normals. LPS significantly raises IL-6 and IL-8 in CRS. Macrolides increase IL-6 and IL-8 in stimulated CRS cells however reduce levels of both in un-stimulated cells. Discussion Pre-existing neutrophilic and eosinophilic activity in CRS subjects may explain the increased baseline levels of both cytokines upon macrolide exposure. Whilst some studies have suggested macrolides act as antimicrobial, others have suggested that it is their anti-inflammatory effects that are more relevant. Treatment for Allergic Rhinitis needs to be effective long-term. The results here are novel and encourage further research to improve understanding of the effects of macrolides in a potentially pivotal role.

Doença respiratória exacerbada por aspirina, conhecida como AERD (Aspirin exacerbated respiratory disease) é caracterizada por rinossinusite crônica eosinofílica, polipose nasossinusal, asma e hipersensibilidade a aspirina e outros anti-inflamatórios não-esteroidais. Expressão aumentada do biomarcador periostina foi descrita em pacientes com AERD, em tecido nasossinusal, incluindo membrana basal, matriz extracelular e pólipo nasal. Avaliamos níveis de periostina sérica em pacientes com AERD e comparamos com níveis em pacientes com rinite alérgica perene (RAP) e indivíduos saudáveis. Foram selecionados 29 pacientes (20F/9M) com diagnóstico de AERD, dentre aqueles atendidos nos Ambulatórios de Alergia e de Otorrinolaringologia do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (HCFMRP-USP). Estes pacientes realizaram exames confirmatórios, incluindo teste de provocação oral com aspirina, e foram submetidos a biópsia de pólipos nasais por nasofibroscopia. Como controles, foram selecionados 12 pacientes com RAP (9F/3M) e 23 indivíduos saudáveis (14F/9M). Eosinófilos foram quantificados em sangue periférico e em tecido de pólipo ou mucosa nasal. IgE total foi determinada por ImmunoCAP, e periostina sérica foi medida por ELISA. Número de eosinófilos teciduais por campo de grande aumento (CGA), número de eosinófilos por milímetro cúbico em sangue periférico, níveis de IgE total e de periostina sérica em pacientes com AERD foram comparados aos de pacientes com RAP e indivíduos saudáveis. Pacientes com AERD tinham idade maior (mediana 54 anos, faixa 22-60) que pacientes com RAP (mediana 30 anos, faixa 19-57, p=0,0001) e indivíduos saudáveis (mediana 29 anos, faixa 19-53, p=0,0001), sem diferença entre os sexos. Números de eosinófilos em sangue periférico e em tecido foram mais elevados em pacientes com AERD que em pacientes com RAP e indivíduos saudáveis. A mediana do número de eosinófilos em sangue periférico foi 640eos/µL (faixa 100-5.100); 200eos/µL (faixa 100-500); e 100 eos/µL (faixa 100-400) em pacientes com AERD, RAP e indivíduos saudáveis, respectivamente (AERD versus RAP, p=0,0003; AERD versus indivíduos saudáveis, p=0,01). A média do número de eosinófilos teciduais foi de 113,3células/CGA; 2,5células/CGA; e 0,7células/CGA, respectivamente (AERD versus RAP, p=0,017; AERD versus indivíduos saudáveis p=0,003). A média geométrica da IgE total foi de 290,18kU/L (faixa 59,5-8.140); 69,96kU/mL (faixa 5,5-898); e 43,14kU/mL (faixa 4- 1.328) em pacientes com AERD, RAP e indivíduos saudáveis, respectivamente, sem diferença entre os grupos. Periostina sérica foi mais elevada em pacientes com AERD quando comparados a indivíduos saudáveis. A mediana de periostina sérica foi de 602ng/ml (faixa 290,7-1.055); 535,6ng/mL (faixa 209-733,2); e 496,7mg/mL (faixa 327,4-713,4), em pacientes com AERD, RAP e indivíduos saudáveis, respectivamente (AERD versus indivíduos saudáveis, p=0,01). Em subgrupo de pacientes brasileiros com AERD, observamos elevado número de eosinófilos em sangue periférico e em tecido, quando comparados a pacientes com RAP e indivíduos saudáveis. Níveis mais elevados de periostina sérica foram observados em pacientes com AERD, quando comparados a indivíduos saudáveis, indicando forte resposta do tipo 2 em pacientes com AERD em nosso meio
Aspirin exacerbated respiratory disease (also known as AERD), is characterized by eosinophilic chronic hypertrophic rhinosinusitis, nasosinusal polyps, asthma and hypersensitivity to Aspirin or other non-steroidal anti-inflammatory drugs. A higher expression of the biomarker periostin has been described in patients with AERD, in nasosinusal tissue, including basal membrane, extracellular matrix and nasal polyps. We evaluated the levels of serum periostin in patients with AERD, and compare those levels with patients with perennial allergic rhinitis (PAR), and with healthy subjects. Twenty-nine patients (20F/9M) with AERD were selected from the Allergy and Otolaryngology Clinics, from the Clinical Hospital of the Ribeirão Preto Medicine School, University of São Paulo (HCFMRP-USP). Those patients underwent confirmatory exams, such as Oral Provocation test with aspirin, and were submitted to polyp biopsy through nasofibroscopy. As a control group, 12 patients (9F/3M) with PAR and 23 healthy subjects (14F/9M) were selected. Eosinophils were quantified in peripheral blood and in polyp tissue or nasal mucosa. Total IgE was determined by ImmunoCAP, and serum periostin was measured by ELISA. The number of tissue eosinophils by high magnification field (HMF), number of eosinophils by cubic milliliter in peripheral blood, total IgE levels and serum periostin levels in patients with AERD were compared with those from patients with PAR and healthy subjects. Patients with AERD were older (median 54 years, and range 22-60) than patients with PAR (median 30 years, range 19-57, p=0,0001) and healthy subjects (median 29 years, range 19-53, p=0,0001), with no difference between genders. The numbers of eosinophils in peripheral blood and in tissue were higher in patients with AERD than patients with PAR or healthy subjects. The median of eosinophil number in peripheral blood was 640eos/µL (range 100-5.100); 200eos/µL (range 100-500); e 100eos/µL (range 100-400) in patients with AERD, PAR and healthy subjects respectively (AERD vs PAR, p=0,0003; AERD vs healthy subjects, p=0,01). The average number of tissue eosinophils was 113,3cels/HMF; 2,5cels/HMF; e 0,7cels/HMF, respectively (AERD vs PAR, p=0,017; AERD vs healthy subjects, p=0,003). The geometric mean for total IgE was 290,18kU/mL (range 59,5-8.140); 69,96kU/mL (range 5,5-898); and 43,14kU/mL (range 4-1.328) in patients with AERD, PAR and healthy subjects respectively, with no difference between the groups. Serum periostin was higher in patients with AERD when compared with healthy subjects. The median for serum periostin was 602ng/ml (range 290,7-1.055); 535,6ng/mL (range 209-733,2); e 496,7ng/mL (range 327,4-713,4), in patients with AERD, PAR and healthy subjects respectively (AERD vs healthy subjects, p=0,01). In a Brazilian subgroup of patients with AERD, we observed an elevated number of eosinophils in peripheral blood and tissue, when compared with patients with PAR and healthy subjects. Higher levels of serum periostin were observed in patients with AERD, when compared with healthy subjects, indicating a strong type 2 response in patients with AERD in our environment.

Objectives: In the past four decades, the median age of survival has nearly doubled for individuals with cystic fibrosis (CF), where over half the population is now adults. The prevalence of chronic diseases such as chronic rhinosinusitis (CRS) has increased with older age. In the non-CF population, CRS is associated with reduced health-related quality of life (HRQoL). Our objectives were to determine the prevalence of CRS among adults with CF and evaluate its impact on their HRQoL. Methods: One hundred sixty individuals from an academic teaching hospital in Vancouver, Canada were eligible to participate in this cross-sectional study. Included subjects were above the age of 18 years, had a confirmed diagnosis of CF and attended the CF clinic between September 2013 and April 2014. Participants completed a CF-specific HRQoL questionnaire (i.e. CFQ-R 14+), and underwent symptom and endoscopic assessment to diagnose CRS. Medical charts were reviewed for potential confounders that included socio-demographic (age, gender and body-mass index) and clinical factors (age of CF diagnosis, type of CF mutation, lung function and chronic Pseudomonas aeruginosa infection). Multivariable linear regression was used to model the relationship between CRS and HRQoL, adjusted for potential confounders. Results: One hundred twenty-one individuals were contacted prior to clinic visits of which, 113 (93.4%) consented to participate. The prevalence of CRS was found to be 64.2%. Socio-demographic and clinical factors were similarly distributed between CRS-positive and negative groups, except age of CF diagnosis. CRS-positive individuals were diagnosed with CF at younger age than non-CRS counterparts, although this finding was not significant (mean difference: 6.5 years, p=0.13). In unadjusted analysis, those with CRS reported worse HRQoL on 10 of 12 domains of the CFQ-R 14+. These findings remained despite adjustment for potential confounders. Individuals with CRS reported significantly worse HRQoL on Respiratory symptoms (adjusted regression coefficient: -13.33, p=0.001) and Digestion (adjusted regression coefficient: -8.71, p=0.03) domains, than non-CRS counterparts. Conclusion: The majority of adults with CF suffer from concomitant CRS. CRS is associated with worse HRQoL based on multiple domains of the CFQ-R 14+. CRS should be diagnosed and managed to optimize the HRQoL for adults with CF.
Medicine, Faculty of
Population and Public Health (SPPH), School of
Graduate

Chronic rhinosinusitis (CRS) is a common inflammatory condition of the sinonasal mucosa from its interaction with bacteria and/or fungi. The toll-like receptor (TLR) family plays an important role in innate immunity by recognizing distinct pathogen-associated molecular patterns. We evaluated single nucleotide polymorphisms (SNPs) in candidate genes from the TLR signaling pathway in 206 individuals with severe CRS and 200 controls. We also investigated the association between these SNPs and total serum IgE level. A total of 96 out of 104 SNPs were successfully genotyped. Although we could not confirm an association with CRS, 3 SNPs in the IRAK4 gene – rs1461567, rs4251513, and rs4251559 – were found to be associated with total serum IgE levels (p < 0.004). The finding was replicated in a second independent population with asthma, from the Saguenay-Lac-Saint-Jean region (p < 0.031). These results demonstrate a clear association between SNPs in the IRAK4 gene and serum IgE levels in patients with CRS and asthma.
La rhinosinusite chronique (RSC) est une maladie fréquente qui cause l'inflammation des sinus. Les récepteurs Toll-like (TLR) sont importants dans l'immunité innée, répondant aux microorganismes. Nous avons évalué les polymorphismes ponctuels de séquence (SNPs) dans les gènes codant les voies de signalisation TLR chez 206 patients atteints de RSC sévère et 200 témoins. Nous avons aussi investigué l'association entre ces SNPs et le niveau d'IgE sanguin. En tout, 96 sur 104 SNPs ont été génotypés avec succès. Bien que nous ne pouvions pas confirmer l'association avec la RSC, 3 SNPs dans le gène IRAK4 – rs1461567, rs4251513, and rs4251559 – étaient associés a un niveau d'IgE sanguin élevé (p < 0.004). Le résultat a été répliqué dans une seconde population indépendante d'individus souffrant d'asthme provenant du Saguenay-Lac-Saint-Jean (p < 0.031). Ces résultats suggèrent qu'une modification génétique dans le gène IRAK4 prédispose à un niveau élevé d'IgE dans les maladies respiratoires.

Background: Chronic rhinosinusitis (CRS) is a heterogeneous disease characterized by persistent sinonasal inflammation and sinus microbiome dysbiosis. The basis of this heterogeneity is poorly understood. We sought to address the hypothesis that a limited number of compositionally distinct pathogenic bacterial microbiota exist in CRS patients and invoke discrete immune responses and clinical phenotypes in CRS patients. Results: Sinus brushings from patients with CRS (n = 59) and healthy individuals (n = 10) collected during endoscopic sinus surgery were analyzed using 16S rRNA gene sequencing, predicted metagenomics, and RNA profiling of the mucosal immune response. We show that CRS patients cluster into distinct sub-groups (DSI-III), each defined by specific pattern of bacterial co-colonization (permutational multivariate analysis of variance (PERMANOVA); p = 0.001, r(2) = 0.318). Each sub-group was typically dominated by a pathogenic family: Streptococcaceae (DSI), Pseudomonadaceae (DSII), Corynebacteriaceae [DSIII(a)], or Staphylococcaceae [DSIII(b)]. Each pathogenic microbiota was predicted to be functionally distinct (PERMANOVA; p = 0.005, r(2) = 0.217) and encode uniquely enriched gene pathways including ansamycin biosynthesis (DSI), tryptophan metabolism (DSII), two-component response [DSIII(b)], and the PPAR-gamma signaling pathway [DSIII(a)]. Each is also associated with significantly distinct host immune responses; DSI, II, and III(b) invoked a variety of pro-inflammatory, T(H)1 responses, while DSIII(a), which exhibited significantly increased incidence of nasal polyps (Fisher's exact; p = 0.034, relative risk = 2.16), primarily induced IL-5 expression (Kruskal Wallis; q = 0.045). Conclusions: A large proportion of CRS patient heterogeneity may be explained by the composition of their sinus bacterial microbiota and related host immune response-features which may inform strategies for tailored therapy in this patient population.

Introdução: A rinossinusite crônica (RSC), doença multifatorial, na qual podem estar envolvidos fatores genéticos e ambientais, ainda tem muitos aspectos obscuros na sua patogênese. A genética tem se mostrado promissora na elucidação dessa complexa doença. Alguns estudos apontam que a expressão de interleucina (IL) 22 se apresenta reduzida em pacientes com RSC, podendo resultar também na redução da barreira epitelial e diminuição da produção de citocinas pró-inflamatórias Th1. Objetivos: Pesquisar a frequência dos polimorfismos no gene IL22RA1 (receptor subunidade alfa um da interleucina 22) em pacientes portadores de RSC com e sem pólipos nasais e em indivíduos normais, utilizando a técnica de sequenciamento, pelo método de Sanger, para análise de mutações; comparar as frequências dos polimorfismos encontrados no gene IL22RA1 entre os grupos e com a literatura médica e também comparar a técnica de Sanger com outras técnicas convencionais descritas na literatura. Casuística e Métodos: Foram avaliados 247 pacientes, no período de maio de 2011 a fevereiro de 2016, subdivididos em três grupos: 122 pacientes portadores de RSC com pólipos nasais (RSCcPN), 21 casos de RSC sem pólipos nasais (RSCsPN) e 104 voluntários sem sintomatologia nasal. Foram colhidas amostras de sangue venoso periférico de todos os casos e controles, e realizada a extração de DNA, com posterior análise das mesmas. Após a exclusão das perdas, restaram 70 casos de RSCcPN, 14 de RSCsPN e 68 controles. Resultados: O sequenciamento apontou 10 polimorfismos no gene IL22RA1, nos exon 2 (rs10903022, c.113_114insA/Q26Pfs*11, c.74T>A e c.141C>A), exon 4 (rs17852649), exon 5 (rs16829204), exon 6 (rs142356961) e exon 7 (rs17852648, rs34967816 e rs3795299). Os polimorfismos encontrados nos exons 2 (em homozigose), 5 e 6 foram exclusivos do grupo das patologias analisadas (RSC com e sem PN), sendo as duas últimas consideradas variáveis não sinônimas, ou seja, com capacidade de alterar a estrutura da proteína, podendo produzir impacto na patogênese da RSC. A alteração do exon 6 foi a única variante encontrada, com frequência do alelo menor (MAF) inferior a 0,01, exclusiva do grupo RSCcPN. Conclusões: Foram detectados três polimorfismos no gene IL22RA1, que até o momento não estão descritos na literatura, sendo a inserção c.113_114insA/Q26Pfs*11, possivelmente patogênica, com frequência maior nos grupos com RSC. O polimorfismo rs17852649 em heterozigose no exon 4, foi o único com diferença estatística, com predominância do alelo mutado no grupo controle, podendo conferir proteção contra o fenótipo. Também se destaca o polimorfismo rs142356961, no exon 6, do tipo não sinônimo, ou seja, capaz de alterar a estrutura final da proteína, com índice MAF<0,01, sendo exclusiva de pacientes negros portadores de RSCcPN. Estudos de replicação e com maiores coortes serão necessários para determinar se os achados do presente estudo se deram ao acaso.
Introduction: Chronic rhinosinusitis (CRS), a multifactorial disease, with genetic and environmental factors that may be involved, still have many aspects of its pathogenesis unknown. Genetics has shown itself promising in the elucidation of this complex disease. Some studies have indicated that the expression of IL-22 is reduced in patients with CRS, which may result in reduction of the epithelial barrier and decrease in the production of Th1 proinflammatory cytokines. Objectives: To investigate the frequency of polymorphisms in the IL22RA1 gene in patients with chronic rhinosinusitis with and without nasal polyps and in individuals without these pathologies, using the Sanger sequencing technique for mutation analysis; to compare the frequencies of the polymorphisms found in the IL22RA1 gene between the groups and the medical literature and also to compare the Sanger technique with other conventional techniques in the medical literature. Casuistic and Methods: From May 2011 to February 2016, 247 patients were evaluated, subdivided into three groups: 122 patients with chronic rhinosinusitis with nasal polyps (CRSwNP), 21 cases of chronic rhinosinusitis without nasal polyps (CRSsNP) and 104 volunteers without nasal symptoms. Samples of peripheral venous blood were collected from all cases and controls, and DNA extraction was performed, with subsequent analysis at the Molecular Genetics Laboratory - Ribeirão Preto Medical School Blood Center - USP. After the loss exclusion, there were 70 cases of CRSwNP, 14 CRSsNP and 68 controls. Results: Sequencing indicated 10 polymorphisms in the IL22RA1 gene, exon 2 (rs10903022, c.113_114insA / Q26Pfs * 11, c.74T> A and c.141C> A), exon 4 (rs17852649), exon 5 (rs16829204), exon 6 (rs142356961) and exon 7 (rs17852648, rs34967816 and rs3795299). Polymorphisms in exons 2 (in homozygosis), 5 and 6 were exclusive from the analyzed pathologies group (RSC with and without NP), the latter two being considered non-synonymous variables, that is, with capacity to alter the protein structure, being able to produce impact on the pathogenesis of CRS. The exon 6 alteration was the only variant found, with the minor allele frequency (MAF) under 0.01, exclusive of the RSCcPN group. Conclusions: Three polymorphisms were detected in the IL22RA1 gene, which until now are not described in the literature, and the possibly pathogenic insert c.113_114insA / Q26Pfs * 11, with a higher frequency in the groups with CRS. The polymorphism rs17852649 in heterozygosis in exon 4 was the only one with a statistical difference, with predominance of the mutated allele in the control group, which could confer protection against the phenotype. Also notable is the polymorphism rs142356961, in exon 6, of the non-synonymous type, that is, capable of altering the final structure of the protein, with MAF index <0.01, being exclusive in black patients with chronic rhinosinusitis with nasal polyps. Replication studies and larger cohorts are necessary to rule out the findings at random.

Objective: 1) Systematic review on the accuracy of Chronic Rhinosinusitis (CRS) identification in administrative databases; 2) Develop an administrative data algorithm to identify CRS patients who have endoscopic sinus surgery (ESS). Methods: A chart review was performed for all ESS surgical encounters at The Ottawa Hospital from 2011-12. Cases were defined as encounters in which ESS for performed for Otolaryngologist-diagnosed CRS. An algorithm to identify patients who underwent ESS for CRS was developed using diagnostic and procedural codes within health administrative data. This algorithm was internally validated. Results: Only three studies meeting inclusion criteria were identified in the systematic review and showed inaccurate CRS identification. The final algorithm using administrative and chart review data found that encounters having at least one CRS diagnostic code and one ESS procedural code had excellent accuracy for identifying ESS: sensitivity 96.0% sensitivity, specificity 100%, and positive predictive value 95.4%. Internal validation showed similar accuracy. Conclusion: Most published AD studies examining CRS do not consider the accuracy of case identification. We identified a simple algorithm based on administrative database codes accurately identified ESS-CRS encounters.

In der vorliegenden Arbeit wurde Gewebe aus den Nasennebenhöhlen von Patienten mit Nasenpolypen untersucht. Außerdem wurden Zusammenhänge zwischen den Zellpopulationen und den Angaben zu allergischen Erkrankungen und wiederholtem Auftreten der Polypen analysiert. Es fand sich eine interindividuell unterschiedlich starke Infiltration mit eosinophilen Granulozyten. Es konnten keine Unterschiede in der prozentualen Verteilung von eosinophilen Granulozyten im Polypengewebe bei allergischen/ nichtallergischen Patienten oder Patienten mit/ ohne Rezidiv nachgewiesen werden. Die Untersuchungen erfolgten mit dem Laser Scanning Zytometer (LSC), das mit der Standardmethode, der Begutachtung mittels Lichtmikroskop, verglichen wurde. Mit der beschriebenen Methode erfolgte die Untersuchung von Polypengewebe nach einem speziell für diese Anwendung entwickelten Protokoll. Die Ergebnisse korrelierten gut mit den Ergebnissen der Lichtmikroskopie. Aufgrund der Weiterentwicklung des LSC und der ständig wachsenden Anzahl der Nachweismöglichkeiten der an der Polyposis nasi beteiligten Zytokine stellt das LSC eine ideale Methode für die Erforschung der Pathogenese von chronischen Entzündungen der Nasennebenhöhlen dar.

Introdução: Embora a fisiopatogenia da rinossinusite crônica (RSC) ainda não esteja totalmente elucidada, em virtude da sua heterogeneidade e multifatorialidade, existe um crescente corpo de evidências apontando que as bactérias exerçam um papel significativo na gênese ou perpetuação da inflamação crônica. Uma das possíveis formas de atuação são os biofilmes bacterianos, comumente encontrados em pacientes com RSC e que estão relacionados com má evolução clínica. Ainda, existem evidências de que algumas espécies bacterianas, especialmente o Staphylococcus aureus (S. aureus), são capazes de invadir as células epiteliais e permanecerem viáveis em seu interior. Por fim, tem se demonstrado que pacientes RSC com pólipo nasal (RSCcPN) revelam alta associação com a presença de superantígenos estafilocócicos na mucosa respiratória, responsáveis pela estimulação acentuada de respostas inflamatórias locais. Apesar de essas diferentes formas bacterianas estarem bem descritas na RSC, não se sabe ainda com clareza como elas estão associadas nesses indivíduos. Objetivos: Avaliar a associação entre a presença de biofilmes, bactérias intracelulares e superantígenos estafilocócicos em pacientes com RSC (com e sem pólipo nasal), comparados com o grupo controle. Casuística e Métodos: Avaliou-se a prevalência de biofilmes bacterianos, bactérias intracelulares e presença de superantígenos bacterianos em indivíduos com RSCcPN, sem pólipo nasal (RSCsPN) e controles, analisando a associação de distribuição de prevalência desses diferentes grupos (teste exato de Fisher, nível de significância quando p<0,05). Os biofilmes foram definidos por características morfológicas à microscopia eletrônica de varredura (MEV), as bactérias intracelulares foram analisadas por microscopia eletrônica de transmissão (MET) e hibridização fluorescente in situ (FISH) para S. aureus, e superantígenos de S. aureus A-E foram quantificados pela técnica de ELISA (Enzime Linked Imunosorbent Assay). Foram incluídos 90 indivíduos, divididos em três grupos: 1) 38 pacientes com RSCcPN, 2) 26 com RSCsPN e 3) 26 controles. Resultados: Quarenta e dois por cento dos pacientes com RSCcPN (16/38), assim como os com RSCsPN (11/26) apresentaram amostras positivas para biofilmes bacterianos, mas não observou essa positividade no grupo controle (0/26). A análise para bactérias intracelulares demonstrou a presença em 31,5% de pacientes com RSCcPN (12/38), 19,2% em RSCsPN (5/26) e 0% nos controles (0/26). No estudo por FISH, 58% dos pacientes com RSCcPN (18/31) apresentaram positividade para S. aureus intracelular, seguido de 54% nos com RSCsPN (13/24) e em nenhum caso dos 24 analisados do grupo controle. Na avaliação por ELISA, apenas um paciente com RSCcPN foi positivo para a presença de superantígenos estafilocócicos. A avaliação da associação de biofilme bacteriano na superfície mucosa à MEV com bactéria intracelular à MET e com S. aureus intracelular por FISH nos dois diferentes grupos de RSC com e sem pólipo nasal, não mostrou diferença estatisticamente significativa. Conclusão: Foi observada uma maior prevalêcia de biofilmes e bactérias intracelulares em indivíduos com RSC com ou sem pólipo nasal, comparado a Resumo controles. Não houve diferença significativa dentre os grupos de RSC, com e sem pólipo nasal para a presença de biofilmes e bactérias intracelulares. Não houve associação entre a presença de biofilme e bactéria intracelular em pacientes com RSC. Os achados do presente estudo indicam que tanto biofilmes na superfície mucosa quanto microrganismos intracelulares podem estar envolvidos na fisiopatogenia da RSC.
Introduction: Although the pathophysiology of chronic rhinosinusitis (CRS) has not yet been fully elucidated, due to its heterogeneity and multifactorial etiology, there is a growing body of evidence that bacteria play a significant role in the genesis or perpetuation of chronic inflammation. One of the possible forms of acting are bacterial biofilms, which are commonly found in patients with CRS, and are associated with poor clinical outcomes in these patients. In addition to biofilms, there are some evidence pointing out that some bacterial species, especially Staphylococcus aureus (S. aureus), are able to invade into epithelial cells and remain viable intracellulary. Finally, it has been demonstrated that patients with CRS with nasal polyps (CRSwNP) have a high association with the presence of staphylococcal superantigens in the respiratory mucosa, responsible for the stimulation of marked local inflammatory responses. Although these different bacterial forms are well described in CRS, it is still unclear how they are associated in these individuals. Objectives: To evaluate the correlation between the presence of biofilms, intracellular bacteria expression and S. aureus superantigens in CRS patients (with and without nasal polyposis) compared to a control group. Casuistic and Methods: We evaluated the prevalence of bacterial biofilms, intracellular bacteria and the presence of bacterial superantigens in individuals with CRSwNP, without nasal polyp (CRSsNP) and controls, evaluating the association of prevalence distribution of these different groups (Fisher exact test, level of significance set at p<0.05). The biofilms were defined by morphological characteristics by scanning electron microscopy, intracellular bacteria were analyzed by transmission electron microscopy and fluorescence in situ hybridization (FISH) for S. aureus, and S. aureus A-E superantigens were quantified by ELISA. Ninety individuals were included, divided into 38 patients with CRSwNP, 26 patients with CRSsNP and 26 control patients. Results: 42% of patients with CRSwNP (16/38) as well as those with CRSsNP (11/26) presented positive samples for bacterial biofilms, while none of the control patients (0/26) had positive samples. The analysis for intracellular bacteria showed the presence in 31.5% of patients with CRSwNP (12/38), 19.2% in CRSsNP (5/26) and 0% in control patients (0/26). In the FISH study, 58% of patients with CRSwNP (18/31) presented intracellular S. aureus positivity, followed by 54% in patients with CRSsNP (13/24) and in none of the 24 analyzed in the control group. In the ELISA evaluation, only one patient with CRSwNP was positive for the presence of staphylococcal superantigens. The evaluation of the association of bacterial biofilm on the mucosal surface (SEM) with intracellular bacteria (MET) and with intracellular S. aureus by FISH in the two different groups of CRS (with and without nasal polyps) did not show a statistically significant difference. Conclusion: We found a higher prevalence of biofilms and intracellular bacteria in individuals with CRS, either with and without nasal polyps. There was no significant difference between the groups of CRS, with and without nasal polyp, for the presence of biofilms or intracellular bacteria. There was no significant diference on the association of biofilms and intracellular bacteria on pacientes with CRS. Our data indicate that both biofilms on the mucosal surface and intracellular microorganisms may be involved in the pathophysiology of CRS.

A fibrose cística (FC) é uma doença genética irreversível, mas os avanços no tratamento têm aumentado a expectativa de vida dos pacientes. O acometimento das vias aéreas superiores, principalmente por alteraçõesdos seios paranasais aos exames de imagem, é prevalente nestes pacientes, embora muitos apresentem poucos sintomas. Poucos trabalhos abordam as características e o manejo das doenças nasossinusais em pacientes adultos com FC. O acometimento nasossinusal, além de ter provável influência nas exacerbações pulmonares, pode afetar negativamente a qualidade de vida.Objetivos:(1) Identificar as características e o grau de acometimento das vias aéreas superiores;(2) Estabelecer associações com as manifestações clínicas e determinar preditores na pontuação do questionário SNOT-22.Métodos: A metodologia adotada para a presenteteseconsistiu na elaboração de três artigos: (1) Artigo original de revisão sistemática sobre achados tomográficos de seios paranasais em pacientes com FC; (2) Artigo de revisão narrativa sobre diagnóstico e tratamento da rinossinusite crônica (RSC) em pacientes com FC; e (3) Artigo original de estudo transversal e prospectivo sobre manifestações nasossinusais e avaliação da qualidade de vida pelo questionário SNOT-22 em pacientes adultos com FC. A revisão da literatura fundamentou-se na busca por artigos com as evidências mais recentes sobre o assunto nos bancos de dados Medline, Embase, Web of Science, Lilacs, Scielo e Cochrane. O estudo transversal consistiu na avaliação de pacientes adultos com FC clinicamente estáveis, sendo submetidos a avaliação clínica, exames de função pulmonar, endoscopia nasal e tomografia computadorizada de seios da face. Todos os pacientes responderam o questionário SNOT-22.Resultados: A literatura relata que os achados tomográficos mais comuns nos pacientes com FC são a opacificação dos seios paranasais, a presença de hipoplasia ou aplasia dos seios frontal e esfenoidal, o subdesenvolvimento pansinusal e a medialização da parede nasal lateral. Quando sintomática, a RSC com pólipos nasais pode afetar a qualidade de vida e desencadear as exacerbações pulmonares, já que os seios paranasais podem ser colonizados por bactérias patogênicas, principalmente a Pseudomonas aeruginosa. Esta bactéria tem papel crucial na morbidade e mortalidade após o transplante pulmonar em pacientes com FC. Embora o tratamento clínico das vias aéreas superiores seja indicado no manejo inicial, a indicação é muitas vezes extrapolada de estudos sobre RSC na população geral. No estudo original da tese, uma idade média maior, idade de diagnóstico mais tardio, sintomas de rinite crônica e critérios clínicos para rinossinusite foram mais observados em pacientes com pontuação maior no SNOT-22. Na análise de regressão múltipla, houve associação positiva da idade e presença de P. aeruginosa no escarro com a pontuação no SNOT-22.Em concordância com a literatura, o estudo também revelou uma alta prevalência de alterações tomográficas, sendo a aplasia/hipoplasia do seio esfenoidal o achado mais frequente.Conclusão:Apesar dasinúmeras alterações tomográficas, os pacientes relatam pouca intensidade dos sintomas nasossinusais. A idade e a presença da P. aeruginosa foram fatores associados a maior pontuação no SNOT-22. Mais estudos são necessários para compreender melhor o acometimento das vias aéreas superiores e melhorar o manejo da RSC na FC, a fim de preservar a função pulmonar, mas evitandoa indicação de procedimentos invasivos e a exposição radiológica desnecessária.
Cystic fibrosis (CF) is an irreversible genetic disease, but advances in treatment have increased the life expectancy of patients. Involvement of upper airways, especially by pathological changes in sinus imaging, is prevalent in these patients, although few exhibit symptoms. There are few studies about characteristics and management of sinonasal diseases in adult CF patients. Sinonasal involvement may initiate pulmonary exacerbations and negatively affect quality of life. Objectives: To identify characteristics and degree of involvement of upper airways, establishing associations with clinical manifestations and determine predictors in SNOT-22 questionnaire score. Methods: The methodology adopted for this thesis included the elaboration of three articles: (1) original systematic review article aboutparanasal sinuses CT findings in CF patients; (2) narrative review article about diagnosis and treatment of chronic rhinosinusitis (CRS) in CF patients; and (3) original article about crosssectional prospective study of sinonasal manifestations and assessment of quality of life by SNOT-22 questionnaire in adult CF patients. The literature review was based on search of articles with the latest evidence on the subject in databases Medline, Embase, Web of Science, Lilacs, Scielo and Cochrane. The cross-sectional study consisted in evaluation of adult CF patients clinically stable. They underwent clinical evaluation, pulmonary function tests, nasal endoscopy and paranasal sinuses CT scan. All patients answered SNOT-22 questionnaire. Results:Literature reports that the most common CT findings in CF patients areparanasal sinuses opacification, presence of sphenoid and frontal sinuses hypoplasia or aplasia, pansinusal underdevelopment and medial bulging oflateral nasal wall. When symptomatic, CRS with nasal polyps can affect quality of life and trigger pulmonary exacerbations. It is explained since paranasal sinuses may be colonized by pathogenic bacteria, especially Pseudomonas aeruginosa. This bacterium plays a crucial role in morbidity and mortality after lung transplantation in CF patients. Clinical treatment of upper airways is indicated as first management, but this indication is often extrapolated from studies on CRS in general population. In the original study, a high average age, age of later diagnosis, symptoms of chronic rhinitis and clinical criteria for rhinosinusitis were more frequently observed in patients with high SNOT-22 scores. In multiple regression analysis, there was a positive association between age and the presence of P. aeruginosa in sputum with the SNOT-22 score. According to literature, this study also revealed a high prevalence of tomographic alterations.Sphenoid sinus aplasia or hypoplasia was the most common finding. Conclusion: Despite CT findings, patients report little intensity of sinonasal symptoms. Age and presence of P. aeruginosa were associated with higher SNOT-22 scores. The most important is to preserve lung function, but avoinding unnecessary invasive procedures and radiation exposure. More studies are needed to better understand the involvement of upper airways and improve management of CRS in CF.

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Rinosinusite crónica fúngica invasiva é uma infeção rara e potencialmente agressiva, caracterizada por obstrução nasal, dor facial e hipósmia, secundárias à infiltração da mucosa, submucosa, ossos ou vasos sanguíneos dos seios paranasais. Conidiobolus é uma etiologia muito rara de Rinosinusite Crónica Fúngica Invasiva, predominando em zonas de florestas tropicas, não sendo encontrado normalmente na Europa.Pretendemos apresentar o primeiro caso de Rinosinusite Crónica Fúngica Invasiva por Conidiobolus diagnosticada num doente português.Apresentamos um doente do sexo masculino, 65 anos de idade, com obstrução nasal progressiva, cefaleia frontal bilateral e hiposmia com 8 meses de evolução. Foi diagnosticada uma rinosinusite crónica fúngica invasiva com hipertrofia dos cornetos inferiores e mucosite nasal ulcerativa. A identificação fúngica foi possível através de biopsias realizadas cirurgicamente, observação macroscópica de colónias, observação microscópica de micelas e técnicas de biologia molecular. O doente foi tratado com Anfotericina B lipossómica, tendo sido seguido durante 3 anos sem intercorrências.A Rinosinusite Crónica Fúngica Invasiva por Conidiobolus já não se encontra restrita às florestas tropicais, devendo os clínicos europeus estar atentos a esta etiologia. O diagnóstico é apenas possível através de fortes suspeições conjugadas com o trabalho conjunto de cirurgiões e patologistas.Rinosinusite crónica fúngica invasiva é uma infeção rara e potencialmente agressiva, caracterizada por obstrução nasal, dor facial e hipósmia, secundárias à infiltração da mucosa, submucosa, ossos ou vasos sanguíneos dos seios paranasais. Conidiobolus é uma etiologia muito rara de Rinosinusite Crónica Fúngica Invasiva, predominando em zonas de florestas tropicas, não sendo encontrado normalmente na Europa.Pretendemos apresentar o primeiro caso de Rinosinusite Crónica Fúngica Invasiva por Conidiobolus diagnosticada num doente português.Apresentamos um doente do sexo masculino, 65 anos de idade, com obstrução nasal progressiva, cefaleia frontal bilateral e hiposmia com 8 meses de evolução. Foi diagnosticada uma rinosinusite crónica fúngica invasiva com hipertrofia dos cornetos inferiores e mucosite nasal ulcerativa. A identificação fúngica foi possível através de biopsias realizadas cirurgicamente, observação macroscópica de colónias, observação microscópica de micelas e técnicas de biologia molecular. O doente foi tratado com Anfotericina B lipossómica, tendo sido seguido durante 3 anos sem intercorrências.A Rinosinusite Crónica Fúngica Invasiva por Conidiobolus já não se encontra restrita às florestas tropicais, devendo os clínicos europeus estar atentos a esta etiologia. O diagnóstico é apenas possível através de fortes suspeições conjugadas com o trabalho conjunto de cirurgiões e patologistas.
Chronic invasive fungal rhinosinusitis is a rare and potentially aggressive infection, characterized by nasal obstruction due to the presence of fungal hyphae infiltrating the mucosa, submucosa, bone, or blood vessels of the paranasal sinuses, facial pain and hyposmia. Conidiobolus is a very rare cause of chronic invasive fungal rhinosinusitis. This fungus predominates in tropical forests and usually is not present in Europe. We aim to present the first case of chronic invasive fungal rhinosinusitis due to Conidiobolus diagnosed in a Portuguese patient. We present a Caucasian 65 years old male patient with progressive nasal obstruction, bilateral frontal headache and a hyposmia with 8 months of evolution. He was diagnosed a chronic invasive rhinosinusitis associated with hypertrophied inferior turbines and ulcerative nasal mucositis. The identification of Conidiobolus was performed in samples from surgical excision biopsies, by macroscopic observation of the colony, microscopic observation of the mycelium and molecular biology techniques. The patient was treated using liposomal B amphotericin and followed up for 3 years without intercurrences.Chronic invasive fungal rhinosinusitis by conidiobolus is not restricted to tropical forests anymore. European physicians must be aware of this possibility. Diagnosis is only possible by strong suspicious and conjugated efforts between surgeons and pathologists.Chronic invasive fungal rhinosinusitis is a rare and potentially aggressive infection, characterized by nasal obstruction due to the presence of fungal hyphae infiltrating the mucosa, submucosa, bone, or blood vessels of the paranasal sinuses, facial pain and hyposmia. Conidiobolus is a very rare cause of chronic invasive fungal rhinosinusitis. This fungus predominates in tropical forests and usually is not present in Europe. We aim to present the first case of chronic invasive fungal rhinosinusitis due to Conidiobolus diagnosed in a Portuguese patient. We present a Caucasian 65 years old male patient with progressive nasal obstruction, bilateral frontal headache and a hyposmia with 8 months of evolution. He was diagnosed a chronic invasive rhinosinusitis associated with hypertrophied inferior turbines and ulcerative nasal mucositis. The identification of Conidiobolus was performed in samples from surgical excision biopsies, by macroscopic observation of the colony, microscopic observation of the mycelium and molecular biology techniques. The patient was treated using liposomal B amphotericin and followed up for 3 years without intercurrences.Chronic invasive fungal rhinosinusitis by conidiobolus is not restricted to tropical forests anymore. European physicians must be aware of this possibility. Diagnosis is only possible by strong suspicious and conjugated efforts between surgeons and pathologists.

Chronic rhinosinusitis (CRS) is a heterogeneous disease characterised by inflammation of the nose and paranasal sinuses, which results in nasal obstruction, rhinorrhoea, post-nasal drip, facial pressure, and alterations in smell. The pathophysiology of CRS is complex and involved interactions between the host, microbial flora, and environment. Studies have shown that the mucosa of CRS sufferers demonstrates signs of defective barrier function, although little is known about the contributing factors to this process. Understanding epithelial barrier dysfunction in the gastrointestinal, skin, and pulmonary systems highlights the various contributing factors to this process, which may be paralleled in the paranasal sinuses. It appears that bacterial mediated mechanism, inflammatory surroundings, and the divalent metal zinc are important in these systems. Staphylococcus aureus has been implicated in the pathogenesis and persistence of CRS, poorer wound healing, and increased disease severity. Additionally, it is known that S. aureus secretes an unknown factor that perturbs the airway barrier in-vitro. Previous research has suggested that zinc concentrations may be lower in CRS, particularly in patients with nasal polyposis, however little is known about the consequences of localised zinc deficiency in CRS. This thesis examines the S. aureus secretome to elucidate the factor or factors involved in barrier disruption. Furthermore, the role of zinc in mucosal barrier integrity and CRS has been delineated.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2018

Chronic Rhinosinusitis (CRS) is a recalcitrant disease, characterized by headache, nasal discharge / blockage, which substantially impairs daily functioning and negatively affect quality of life. Endoscopic Sinus Surgery (ESS) is an important treatment option for CRS, but has variable success rates. Biofilms are well organised heterogeneous communities of microbes embedded in a mosaic of extracellular matrix, adherent to biotic / abiotic surfaces. As they are resistant to host defences and medical treatments, they have been touted as possible pathogenic factors in CRS, which may perpetuate the recurrent and recalcitrant character of the disease and negatively affect treatment outcomes. This thesis encompasses research undertaken to enhance our understanding about the effect that presence and types of biofilms have on the clinical profile and treatment outcomes of patients suffering with chronic rhinosinusitis. An in-vitro model of fungal biofilms and a potential tool to assay in-vivo mucosal biofilms on sinonasal tissues has also been described. Chapter 1 of the thesis comprehensively reviews the scientific literature pertaining to biofilms and CRS, and exhaustively evaluates the evidence present in relation to bacterial and fungal biofilms in CRS. Chapter 2 describes a study to investigate the effect of biofilms on outcomes following ESS in CRS patients using internationally accepted standardised symptom scores, quality of life measures and endoscopy scores to assess the disease. It showed that patients with biofilms presented with more severe disease before surgery, and after surgery had persistent symptoms, ongoing mucosal inflammation and infections necessitating extra post-operative visits and multiple antibiotic treatments. This study thus strengthened the evidence for the role that biofilms may play in recalcitrant CRS. Chapter 3 describes a further subgroup analysis of the above patients in whom the specific organisms forming the biofilms were identified and how patients with specific biofilm types progressed after surgery was studied. Patients with polymicrobial biofilms suffered more severe disease and had worse post-surgery mucosal outcomes requiring more post–operative visits. S.aureus biofilms played a dominant role in negatively affecting outcomes of ESS with persisting post-operative symptoms, ongoing mucosal inflammation and infections. Chapter 4 describes an in-vitro model characterizing A. fumigatus biofilm formation on primary human sinonasal epithelium cultures under different growth conditions. 3-dimensional biofilm structures with parallel-packed and cross-linked hyphae, channels/passages, extracellular matrix (ECM) encasing the hyphae, were formed. Biofilms formed under flow conditions displayed more robust and faster growth kinetics as compared to those under static conditions, with extensive ECM production. Chapter 5 investigates application of an analysis program ‘COMSTAT 2’ for assaying & quantitatively describing the 3-dimensional in-vivo biofilm structures observed via confocal scanning microscopy on sino-nasal mucosal samples. This can be used for temporal analysis of biofilm development, comparison of different types of biofilms formed under controlled conditions, analysis of influence of varying environmental factors on biofilms and the efficacy of different antibiofilm treatments. Chapter 6 summarises and discusses the salient features of the studies included in this thesis which has attempted to characterize fungal and bacterial biofilms and the impact they may have in CRS patients.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2011

碩士
高雄醫學大學
公共衛生學系公共衛生學碩士班
103
Background: Several studies have reported the association between asthma and chronic rhinosinusitis with/without nasal polyps. However, this relationship has not been investigated demographically . The aim of this study was to utilize the Taiwan National Health Insurance database to analyze the association between asthma and the risk of chronic rhinosinusitis patterns in order to provide information for clinical applications. Methods: Data were obtained from the National Health Insurance Research Database (NHIRD) of Taiwan from 1997 to 2009. The study was divided into cohort and matched case-control analysis of two parts, which were described as follows: Cohort study included cases with a new primary diagnosis of asthma (ICD-9: 493) between 2000 and 2008. These cases were compared in sex-, age-, residence-, and insurance premium-matched controls, and both groups were followed up until the end of 2009 for instances of chronic rhinosinusitis with/without nasal polyps (CRS w/s NP), defined as ICD-9 codes CRS (473, 473.0, 473.1, 473.2, 473.3, 473.8, and 473.9), with/without NP (471, 471.0, 471.1, 471.8, and 471.9). Both of CRSwNP and CRSsNP analysis were performed. Competing risk-adjusted Cox regression analyses were applied after adjusting for sex, age, residence, insurance premium, steroid use (topical or systemic), hyperlipidemia, diabetes, hypertension, coronary artery disease, Charlson comorbidity index and mortality. In matched case-control study, case group were diagnosed with chronic rhinosinusitis with/without nasal polyps (CRS w/s NP), defined as ICD-9 codes CRS (473, 473.0, 473.1, 473.2, 473.3, 473.8, and 473.9), with/without NP (471, 471.0, 471.1, 471.8, and 471.9) between 2000 and 2009. In the matched case-control study the control groups were matched according to sex, age, residence, insurance premium with the population rate of 1:4 from non-CRS population. Conditional logistic regression models analyses were used to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) between the asthma and CRS w/s NP risk, adjusted for other types of steroid drugs and comorbidities. Results: In cohort study, among 81,462 subjects, 58 developed CRSwNP and 799 developed CRSsNP with a mean (SD) follow-up period of 5.8 (2.4) years. Asthma was an independent predictor of CRSwNP in the fully adjusted model (HR =1.80; 95% CI = 1.02-3.17; P=0.041). Among the CRSsNP analysis, asthma was also an independent predictor of CRSsNP in the fully adjusted model (HR =2.62; 95% CI = 2.23-3.08; P<0.001). In matched case-control analysis, a total of 1204 subjects were identified as CRSwNP, including 107 with asthma before diagnosed as CRSwNP；a total of 4816 subjects were identified as Non-CRSwNP, including 155 with asthma before diagnosed as Non-CRSwNP between 2000-2009. A total of 11308 people were identified as CRSsNP, including 1617 with asthma before diagnosed as CRSsNP；a total of 45232 subjects were identified as Non-CRSsNP, including 1960 subjects with asthma before diagnosed as Non-CRSsNP between 2000-2009. Conditions logistic regression analysis for which steroid use, hyperlipidemia, diabetes, hypertension, coronary artery disease, and Charlson comorbidity index were adjusted, asthma was positively associated with CRSwNP (Odds-ratio= 2.49; 95% CI = 1.89–3.30; P < 0.001). Asthma was also positively associated with CRSsNP (Odds-ratio= 3.10; 95% CI = 2.87–3.34; P < 0.001). Conclusion: Results of this study show that asthma was associated with an increased risk of CRS w/s NP. The result is consistency with previous reports and confirms that there is an relationship between asthma and CRS w/s NP. It is evident that, in Taiwan, asthma has increased effect on the risk of developing CRSsNP than CRSwNP.

This thesis embodies research investigating the role that bacterial biofilms play in the pathogenesis of chronic rhinosinusitis (CRS). It focuses on their detection on the sinus mucosa of CRS patients and the implications of their presence. Finally, it addresses deficiencies in the innate immune system that may predispose to their development in this condition. Bacterial biofilms are structural assemblages of microbial cells that encase themselves in a protective self-produced matrix and irreversibly attach to a surface. Their extreme resistance to both the immune system as well as medical therapies has implicated them as playing a potential role in the pathogenesis of many chronic diseases. Although their role in many diseases is now well established, their objective presence and importance in CRS remains largely unknown. Chapter 1 of this thesis reviews the current literature pertaining to CRS and biofilms and critically evaluates the small body of research relating to this topic. Chapter 2 describes the development of a sheep model to study the role of bacterial biofilms in rhinosinusitis. It compares the use of traditional electron microscopy (EM) and more recent confocal scanning laser microscopy (CSLM) in the detection of biofilms on the surface of sinus mucosa. The results of this study inferred a causal relationship between biofilms and the macroscopic changes that accompany rhinosinusitis. Furthermore it illustrated the superiority that CSLM has over EM in the imaging of biofilms on sinus mucosa Chapter 3 and 4 outline the results of human studies utilizing the more objective CSLM to evaluate the prevalence of bacterial biofilms on the sinus mucosa of CRS patients and their effect on post-operative mucosal healing. The results of these studies demonstrated a biofilm prevalence of approximately 50% in the CRS population studied and suggested, that biofilm presence may predispose to adverse post-operative outcomes following sinus surgery. Chapter 5 and 6 describe experiments examining the level of the innate immune system’s anti-biofilm peptide lactoferrin, in patients with CRS. Lactoferrin was found to be downregulated at both an mRNA and protein level in the majority of CRS patients, with biofilm positive patients demonstrating the most significant reduction. In summary, this thesis provides further evidence that bacterial biofilms play a major role in the pathogenesis and disease persistence in a subset of CRS patients. Deficiencies in components of the innate immune system, such as lactoferrin, may play an important role in the predisposition of certain individuals to the initial development of bacterial biofilms.
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Thesis (Ph.D.) -- University of Adelaide, School of Medicine 2008

This thesis embodies research investigating the role that bacterial biofilms play in the pathogenesis of chronic rhinosinusitis (CRS). It focuses on their detection on the sinus mucosa of CRS patients and the implications of their presence. Finally, it addresses deficiencies in the innate immune system that may predispose to their development in this condition. Bacterial biofilms are structural assemblages of microbial cells that encase themselves in a protective self-produced matrix and irreversibly attach to a surface. Their extreme resistance to both the immune system as well as medical therapies has implicated them as playing a potential role in the pathogenesis of many chronic diseases. Although their role in many diseases is now well established, their objective presence and importance in CRS remains largely unknown. Chapter 1 of this thesis reviews the current literature pertaining to CRS and biofilms and critically evaluates the small body of research relating to this topic. Chapter 2 describes the development of a sheep model to study the role of bacterial biofilms in rhinosinusitis. It compares the use of traditional electron microscopy (EM) and more recent confocal scanning laser microscopy (CSLM) in the detection of biofilms on the surface of sinus mucosa. The results of this study inferred a causal relationship between biofilms and the macroscopic changes that accompany rhinosinusitis. Furthermore it illustrated the superiority that CSLM has over EM in the imaging of biofilms on sinus mucosa Chapter 3 and 4 outline the results of human studies utilizing the more objective CSLM to evaluate the prevalence of bacterial biofilms on the sinus mucosa of CRS patients and their effect on post-operative mucosal healing. The results of these studies demonstrated a biofilm prevalence of approximately 50% in the CRS population studied and suggested, that biofilm presence may predispose to adverse post-operative outcomes following sinus surgery. Chapter 5 and 6 describe experiments examining the level of the innate immune system’s anti-biofilm peptide lactoferrin, in patients with CRS. Lactoferrin was found to be downregulated at both an mRNA and protein level in the majority of CRS patients, with biofilm positive patients demonstrating the most significant reduction. In summary, this thesis provides further evidence that bacterial biofilms play a major role in the pathogenesis and disease persistence in a subset of CRS patients. Deficiencies in components of the innate immune system, such as lactoferrin, may play an important role in the predisposition of certain individuals to the initial development of bacterial biofilms.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine 2008

Chronic rhinosinusitis (CRS) is a heterogenous disease characterised by the symptomatic inflammation of the nose and paranasal sinuses for more than 12 weeks. These symptoms include nasal obstruction, nasal discharge, facial pain and pressure, resulting in a considerable impairment of a patients’ quality of life. CRS is subcategorised into two types based on the absence (CRSsNP) and presence of nasal polyps (CRSwNP) visualised within the middle meatus. Interestingly, although CRSsNP patients may lack easily identifiable polyps, the mucosa of these patients may show variable degrees of polypoid change. This raises the question as to whether the proposed classification system is an over simplification and that CRSsNP and CRSwNP in fact only represent two extremes of phenotype along a broader spectrum of immunologically different disease processes. Recently, research into CRS has identified a dysregulated immune response as a major contributor to the aetiopathology of disease, however few studies have utilised flow cytometry to phenotype the cells present. This thesis examines both the local and systemic populations of different adaptive and innate immune cells in the tissue and blood of CRSsNP and CRSwNP patients along different degrees of polypoid change within the same patient.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, Adelaide Medical School, 2017.

Includes author's previously published paper.
Bibliography: leaves 187-224.
[7], xvii, 229 leaves, [6] : ill. (some col.), maps (col.), plates (chiefly col.) ; 30 cm.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, School of Medicine, Discipline of Surgery, 2006

The research described in this PhD thesis follows an extensive literature review of the role of the medical and surgical management of CRS. Despite the utilization of surgery to alleviate the symptoms of CRS refractory to medical therapy, there are clear deficiencies in our understanding of what type of surgery to perform, and how extensive this surgery should be so as to maximize long-term symptom alleviation and control. Particular controversy exists regarding addressing the frontal sinus with a wide variety of philosophies employed, but with limited scientific rationale to support such approaches. Chapter two describes a prospective study to validate a quality of life tool, the Adelaide Disease Severity Score. This study showed a simple 5 question tool directly related to sinus symptoms and visual analogue quality of life score correlated very highly with other more complex rhinological quality of life tools – the SNOT 20/22. It further correlated with radiological disease burden (Lund Mackay CT score) and endoscopic disease (Lund Kennedy endoscopic score) burden. This study validated our use of this tool to measure quality of life and symptom improvement in patients undergoing surgery. Chapter three describes a detailed retrospective study of the outcomes of primary frontal sinus surgery. This is the largest study in the literature of primary frontal surgery and forms the basis to support an approach where the diseased frontal sinus should be addressed surgically to optimize long-term outcomes. It also identified that certain anatomical factors such as a narrow frontal ostium seemed to play a role in persistence of symptoms. This raised questions as to whether these outcomes were as successful for revision and extended frontal sinus surgery. Were there identifiable risk factors for success and failure? The fourth chapter describes the outcomes of primary and revision standard frontal sinus surgery and investigates which patient, anatomical and disease factors were poor prognostic factors for failure. It identified a select cohort of patients that would benefit not just from frontal sinus surgery, but extended frontal sinus surgery (EMLP) in the first instance. The final chapter investigates the outcomes of extended frontal sinus surgery (EMLP) and seeks to determine the risk factors for its success and failure. This study found that the EMLP had excellent outcomes in the majority of patients, but there was a significant minority of patients that had persistence of symptoms. The relevance of the host immune system response to sinonasal microorganisms, and anatomical risk factors was also explored and lays open the basis for further study.
Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2014

Introduction Chronic rhinosinusitis with nasal polyps (CRSwNP) is a common disease, with high morbidity and chronicity, but its exact etiology is still unclear and remains a difficult to treat condition. Considering the emerging consensus that chronic rhinosinusitis (CRS) results from a dysfunctional host-environment interaction, this study pretends to clarify exogenous and endogenous factors, which can contribute to the occurrence and perpetuation of sinonasal mucosa inflammation observed in CRSwNP. The aims of this study are: 1) To evaluate endoscopic sinus surgery (ESS) efficacy in CRSwNP treatment and to establish prognostic factors for disease recurrence; 2) To compare the prevalence of nasal polyps (NP) in a group of workers with occupational dust exposure and in a control group; 3) To characterize systemic immunological alterations that occur in patients with CRSwNP compared to controls; 4) To clarify the role of food allergy in CRSwNP disease, comparing serum levels of food specific IgE and IgG antibodies in cases and controls. Material and Methods 1) Retrospective observational study in 85 patients with CRSwNP submitted to ESS and a minimum follow-up of 9 months. Patients’ demographics, occupational exposure, comorbidities, previous nasal surgeries, pre and postoperative symptoms and ENT examination findings, CT results, medical and surgical treatment information were collected from medical records. 2) Cross-sectional study with a random sample of textile (n=215) and retail store employees (n=101). Clinical data was gathered through a systematic interview, which included RhinoQOL-pv and CATTM questionnaires. A systematic endoscopic nasal examination was performed using a 0º rigid endoscope and Lund-Kennedy endoscopic score was determined for each participant. 3) Case-control study with 37 CRSwNP patients and 34 controls without CRS. Clinical data was gathered through a systematic interview. CT scan, skin prick test, spirometry, immunological parameters (leukocyte differential count, immunoglobulin classes and IgG subclasses) and 25-hydroxyvitamin D (25-HOD), alpha1-antitrypsin (A1AT) and C-reactive protein (CRP) dosage in serum specimens were obtained. 4) Case-control study with 33 patients with CRSwNP and 31 controls without CRS. Clinical data was gathered through a systematic interview (including the application of Food Frequency Questionnaire (QFA)). ELISA tests using OmegaDiagnostics® kit with 40 food allergens for detection of specific IgG antibodies were performed and food specific IgE antibodies were determined by immunoassay using ImmunoCAP™ against 11 food antigens. Statistical analysis was performed using SPSS v.23. Results 1) All rhinologic symptoms improved after ESS. The major and minor complications prevalences were 1.2% and 15.3%, respectively. Disease recurrence occurred in 31% of cases, but only 7% required surgical reintervention. Multivariate logistic regression analysis identified occupational dust exposure (p=0.001) and non-atopic asthma (p=0.012) as independent predictive variables in CRSwNP recurrence, unlike the other tested variables: age, sex, atopic asthma, allergic rhinitis, smoking habits, nasal polyps endoscopic grade, Lund-Mackay score and postoperative topical corticoid use. The adjusted logistic model had a ROC area under curve of 0.82 (p<0.001; CI95%: [0.73; 0.91]). 2) 316 participants were included in the study, i.e. 215 textile workers and 101 retail store workers. NP were found in 19 subjects (8.8%) among textile workers and none in the control group (p=0.001). The prevalence of NP increased by age strata (p=0.03) and by years of dust exposition (p=0.017). Polypoid degeneration of the middle turbinate was more prevalent in the exposed group (p=0.001) with Lund-Kennedy scoring also higher (p<0.001). RhinoQOL-pv and CATTM questionnaires had both significantly higher scores among textile employees. 3) A significantly higher eosinophil (p<0.001) and basophil relative count (p=0.022) and a lower relative neutrophil count (p=0.013) were found among CRSwNP group. Patients with CRSwNP had higher IgG1 (p=0.022), but lower IgG2 (p=0.014) and IgG3 (p=0.018) serum levels compared to controls. IgG4, total IgG, IgA, IgM and IgE serum levels did not differ between groups, as well as the prevalence of immunoglobulin classes or IgG subclasses deficiency; 25-HOD, A1AT and CRP dosage had also no significant difference. 4) The overall sum of food IgG antibodies was significantly lower in CRSwNP compared to control group (p=0.012), and this difference was also observed for different specific IgG antibodies (corn, soya, grain legumes, pear and apple, berries, citric fruit). In controls a positive correlation between IgG1 and the sum of food IgG antibodies was seen (p=0.049) but in CRSwNP group a negative correlation was found (p=0.048). Significant higher level of IgG1 was found among CRSwNP patients (p=0.041). Levels of serum specific IgE antibodies against the different studied food allergens, as well as the sum of food IgE antibodies, did not differ significantly between the groups. Conclusions Endoscopic sinus surgery proved to be an effective treatment in CRSwNP, but with a considerable disease recurrence. The first study of this investigation demonstrated that occupational dust exposure and non-atopic asthma are independent predictive factors of disease recurrence risk. The epidemiologic study performed, based on endoscopy, was pioneer in the evaluation of occupational exposure to dust impact on NP prevalence and the results pointed to an important association between them by demonstrating a significantly higher prevalence of the disease among textile workers. This investigation also showed a distinct systemic immunologic profile in CRSwNP patients compared to controls, and the variation observed in peripheral relative leukocyte count and the systemic IgG1 subclass shift are similar to what is known to happen in nasal polyp tissue. Concerning food allergy, it does not seem to have an important role in CRSwNP etiopathogenesis, whether if it is IgG or IgE-mediated. Moreover, the observed suppression of specific IgG antibodies against food allergens, its negative correlation with IgG1 and the raised IgG1 serum levels in CRSwNP, can be related to deviated IgG responses against other targets (e.g. airborne particles) and warrants future investigation.
Introdução A rinossinusite crónica (RSC) é uma doença inflamatória crónica do nariz e seios perinasais, que engloba dois fenótipos clínicos: a Rinossinusite Crónica sem Pólipos Nasais (RSCsPN) e a Rinossinusite Crónica com Pólipos Nasais (RSCcPN). Esta última destaca-se pela presença de formações polipoides, hiperplásicas, pedunculadas e edematosas nas cavidades nasais e seios perinasais, geralmente de forma bilateral. A RSCcPN é uma entidade clínica comum, com elevada morbilidade e cronicidade, já descrita no tempo do Antigo Egipto (2000 a.C.) e sobre a qual a investigação científica tem incidido de forma intensa nas últimas duas décadas. No entanto, apesar de toda a pesquisa realizada, a RSCcPN continua a ser um enigma na história da Medicina, permanecendo como uma doença idiopática de prevalência desconhecida, cuja fisiopatologia é em grande parte oculta. Estas incertezas refletem-se na eficácia limitada dos tratamentos disponíveis e explicarão, em parte, a elevada refratariedade da doença ao tratamento médico e cirúrgico. As principais limitações no estudo desta patologia têm sido: a sua sintomatologia inespecífica que dificulta o diagnóstico diferencial com outras patologias nasossinusais; os estudos epidemiológicos pouco fiáveis que estimam a prevalência da doença e avaliam os seus fatores de risco baseando-se em questionários sobre sintomas; a frequente ausência de diferenciação entre os tipos de RSC (RSCsPN e RSCcPN) em diferentes estudos e a inclusão de subtipos de doença (ex. RSCcPN no contexto de Fibrose Quística, Discinésias Ciliares primárias, Vasculites), que sendo casos raros e com mecanismos fisiopatológicos particulares, deverão ser alvo de estudos individualizados. Tendo em conta o consenso atual de que a RSC resultará de uma interação disfuncional entre hospedeiro-ambiente, pretende-se estudar fatores de risco exógenos e endógenos que possam estar na origem e perpetuação da inflamação da mucosa nasal que caracteriza a RSCcPN. Este trabalho tem como objetivos: 1) Avaliar a eficácia da cirurgia endoscópica nasossinusal (CENS) no tratamento da RSCcPN e estabelecer fatores prognósticos de recidiva da doença; 2) Comparar a prevalência da polipose nasal (PN) num grupo de trabalhadores com e sem exposição ocupacional a poeiras; 3) Caracterizar e comparar alterações imunológicas sistémicas dos doentes com RSCcPN versus grupo controlo; 4) Clarificar o papel da alergia alimentar na RSCcPN, comparando os níveis séricos de anticorpos IgE e IgG específicos contra antigénios alimentares em casos e controlos. Material e Métodos 1) Estudo observacional retrospetivo de 85 doentes submetidos a CENS e com um follow-up mínimo de 9 meses. Os dados demográficos, a exposição ocupacional, as comorbilidades, a história cirúrgica prévia, os sintomas pré e pós-operatórios, os dados do exame ORL, resultados da TC e a informação sobre o tratamento médico e cirúrgico foram obtidos através da revisão dos processos clínicos. A análise estatística foi efetuada com recurso ao SPSS v.23. A estatística descritiva foi utilizada na caracterização da amostra. Utilizou-se o teste de McNemar na comparação dos sintomas pré e pós-operatórios. Os doentes com e sem recidiva de RSCcPN foram divididos em dois grupos independentes e foram comparados para múltiplos fatores: na avaliação da associação entre a recidiva e variáveis categóricas utilizou-se o teste de Qui-Quadrado (ou o teste exato de Fisher quando não se verificavam as assunções necessárias à execução do teste anterior); no estudo de associação entre a recidiva e variáveis quantitativas utilizou-se o teste de Mann-Whitney. Realizou-se uma regressão logística multivariada para avaliar a existência de fatores preditivos independentes na recidiva da polipose nasal. O teste de razão de verossimilhança, o teste de Hosmer e Lemeshow, a área sob a curva ROC foram realizados/calculados para avaliação do modelo criado, e procedeu-se à determinação do coeficiente de Nagelkerke’s. O teste de Wald’s e o teste de score foram obtidos para cada variável independente, assim como o Odds Ratio e seu intervalo de confiança a 95%. 2) Estudo epidemiológico transversal numa amostra randomizada de trabalhadores têxteis (n=215) e de trabalhadores de venda a retalho (n=101). Realizou-se uma entrevista clínica sistematizada, que incluiu os questionários RhinoQOL-pv e CATTM, e uma avaliação endoscópica com ótica rígida 0º, com determinação do score endoscópico de Lund-Kennedy em cada participante. A análise estatística foi efetuada com recurso ao SPSS v.23. Utilizou-se a estatística descritiva na caracterização dos dois grupos de trabalhadores e procedeu-se à sua comparação. Na comparação de variáveis categóricas entre os grupos, utilizou-se o teste de Qui-Quadrado (ou teste exato de Fisher, quando adequado) e na comparação de variáveis quantitativas utilizou-se o teste de Mann-Whitney. O teste Binomial foi utilizado na comparação da prevalência de PN no grupo de trabalhadores têxteis com a prevalência de outros estudos publicados na literatura. 3) Estudo caso-controlo de 37 doentes com RSCcPN e 34 controlos sem RSC. Os dados clínicos foram obtidos por entrevista clínica e exame ORL. Realizaram-se TC do nariz e seios perinasais, teste cutâneo de Prick, espirometria; determinação dos parâmetros imunológicos no plasma (contagem diferencial de leucócitos, classes e subclasses de imunoglobulinas) e também dos níveis de 25-hidroxivitamina D (25-HOD), alfa-1-antitripsina (A1AT) e proteína C reativa (PCR). A análise estatística foi efetuada com recurso ao SPSS v.23. Utilizou-se a estatística descritiva na caracterização do grupo de casos e controlos. O teste de Mann-Whitney foi utilizado na comparação de variáveis contínuas entre os dois grupos e o teste de Qui-Quadrado ou o teste exato de Fisher na comparação de variáveis categóricas. Realizou-se uma subanálise com o teste de Kruskal-Wallis na comparação dos parâmetros analíticos entre 3 grupos (grupo controlo sem doenças respiratórias crónicas inferiores (DRCI), grupo RSCcPN sem DRCI e grupo RSCcPN com DRCI), seguido de comparações múltiplas interpares com o teste post-hoc de Dunn. 4) Estudo caso-controlo de 33 doentes com RSCcPN e 31 controlos sem RSC. Os dados clínicos foram obtidos por entrevista clínica, incluindo a aplicação do Questionário de Frequência Alimentar (QFA). Realizou-se o teste de ELISA com o kit OmegaDiagnostics® com 40 antigénios alimentares para determinação de anticorpos IgG específicos e procedeu-se ao teste de imunoensaio usando o ImmunoCAP™ na avaliação de anticorpos IgE específicos contra 11 antigénios alimentares. Procedeu-se à análise estatística dos dados obtidos, com recurso ao SPSS v.23. Utilizou-se a estatística descritiva na caracterização dos dois grupos. O teste de Mann-Whitney foi utilizado na comparação de variáveis quantitativas entre os dois grupos, enquanto o teste de Qui-Quadrado ou o teste exato de Fisher foi utilizado na comparação de variáveis categóricas. O teste não-paramétrico de Spearman foi utilizado na avaliação de correlação entre variáveis quantitativas. Resultados 1) Houve uma melhoria significativa de todos os sintomas rinológicos após a CENS. As prevalências de complicações major e minor foram de 1,2 e 15,3%, respetivamente. A proporção de doentes com RSCcPN com recidiva da patologia após CENS foi de 31%, com 7% a necessitar de reintervenção cirúrgica. 60% dos doentes com RSCcPN reportaram exposição ocupacional a poeiras, das quais 90,4% correspondiam a poeiras de baixo peso molecular (BPM) (<5KDa). Os doentes com exposição ocupacional a poeiras apresentaram uma recorrência da doença significativamente superior ao grupo não exposto (48% vs 3%, p=5,5x10-6). A análise de regressão logística multivariada identificou a exposição ocupacional a poeiras (p=0,001, OR=38,02, IC95%: [4,18; 345,69]) e a asma não-atópica (p=0,012, OR=8,65, IC95%: [1,62; 46,16]) como fatores preditivos independentes de recidiva da RSCcPN ao contrário das outras variáveis analisadas: idade, sexo, asma atópica, rinite alérgica, hábitos tabágicos, classificação endoscópica da polipose nasal, score de Lund-Mackay e uso pós-operatório de corticoide tópico. O modelo logístico apresentou área sob a curva ROC de 0,82 (p<0,001; IC95%: [0,73; 0,91]). O teste de razão de verossimilhança do modelo criado obteve um p=1,2x10-7, o teste de Hosmer e Lemeshow demonstrou um p=0,503 e o coeficiente de Nagelkerke’s foi de 0,44. 2) A PN foi diagnosticada em 19 participantes do grupo dos trabalhadores têxteis (8,8%) e em nenhum do grupo controlo (p=0,001). A prevalência da PN aumentou conforme o estrato etário (p=0,03) e dependendo do número de anos de exposição às poeiras (p=0,017). A degenerescência polipoide do corneto médio foi mais prevalente no grupo exposto (p=0,001), que também obteve um score de Lund-Kennedy mais elevado (p<0,001). No RhinoQOL-pv e no CATTM obtiveram-se scores significativamente mais elevados entre os trabalhadores têxteis. A prevalência de PN nos trabalhadores têxteis (8.8%) foi significativamente superior às prevalências reportadas em estudos endoscópicos prévios (2,7% e 5,5%; p<0,001 e p=0,029, respetivamente). 3) No grupo dos doentes com RSCcPN a prevalência de doenças respiratórias crónicas inferiores (DRCI) foi significativamente superior ao grupo controlo (p<0,001), ao contrário da patologia atópica que não diferiu. Nos doentes com RSCcPN obteve-se uma contagem relativa de eosinófilos (p<0,001) e de basófilos (p=0,022) no plasma significativamente mais elevada do que no grupo controlo, ao contrário dos neutrófilos cuja contagem foi significativamente menor (p=0,013). Os doentes com RSCcPN apresentaram níveis mais elevados de IgG1 (p=0,022), mas mais reduzidos de IgG2 (p=0,014) e IgG3 (p=0,018) comparativamente aos controlos. Essas diferenças observadas foram mais evidentes nos doentes com RSCcPN e DRCI concomitante. Os níveis de IgG4, IgG total, IgA, IgM e IgE não diferiram entre os grupos, assim como a prevalência das deficiências de classes e subclasses de imunoglobulinas; os níveis de 25-HOD, A1AT e PCR também não diferiram de forma significativa. 4) No grupo com RSCcPN verificou-se uma concentração total de anticorpos alimentares do tipo IgG significativamente menor do que a do grupo controlo (p=0,012); esta diferença foi também observada para diferentes anticorpos IgG específicos (milho, soja, leguminosas, maçã e pera, frutos vermelhos, citrinos). No grupo controlo verificou-se uma correlação positiva entre os níveis de IgG1 séricos e a soma da concentração dos anticorpos IgG alimentares (p=0,049). Pelo contrário, no grupo com RSCcPN observou-se uma correlação negativa entra essas variáveis (p=0,048). Os níveis de IgG1 encontravam-se significativamente elevados no grupo com RSCcPN (p=0,041). Os níveis séricos de IgE específicas contra os diferentes alergénios alimentares avaliados, bem como a concentração total de IgE específicas alimentares, não diferiram de forma estatisticamente significativa entre os grupos. Conclusões Apesar da cirurgia endoscópica ser um tratamento eficaz na RSCcPN, com benefícios óbvios na resolução de sintomas no pós-operatório, a recorrência da doença é considerável. O primeiro estudo deste trabalho demonstrou que a exposição ocupacional a poeiras e a asma não-atópica são fatores preditivos independentes de recidiva da doença. A identificação da exposição a partículas de BPM como principal exposição ocupacional reportada é também relevante. Estas partículas de BPM têm sido associadas ao risco de desenvolver asma ocupacional (não-atópica) e ao contrário das partículas de alto peso molecular (APM) que atuam por mecanismos IgE-mediados, não têm os seus mecanismos de ação bem estabelecidos. A distinção realizada entre asma atópica e não-atópica permitiu clarificar o impacto da asma nos resultados pós-operatórios, que era até então controverso. O estudo epidemiológico realizado, baseado em endoscopia, foi pioneiro na avaliação do impacto da exposição ocupacional a poeiras na prevalência da PN e aponta para uma importante associação entre ambas, ao demonstrar uma prevalência da doença significativamente elevada nos trabalhadores têxteis. Os resultados deste trabalho alertam para um relevante problema de Saúde Pública, reforçando a necessidade de medidas de proteção dos trabalhadores expostos a poeiras (ex. uso de máscara com filtros apropriados) e a necessidade de controlo no funcionamento dos sistemas de exaustão de partículas e filtros de ar que garantam a qualidade do ar. Depreende-se assim que os doentes com RSCcPN devem sempre que possível trabalhar em ambientes livres de poeiras, de forma a reduzir o risco de recidiva e melhorar o seu prognóstico. Outros estudos epidemiológicos serão necessários na avaliação de outro tipo de exposições ocupacionais, tentando se possível comparar o impacto de exposição a poeiras de BPM e APM na prevalência de RSCcPN. O estudo prospetivo, clínico-laboratorial, demonstrou ainda que os doentes com RSCcPN apresentam um perfil imune sistémico distinto dos controlos, com variações na contagem diferencial de leucócitos e um desvio IgG1 a nível das subclasses de IgG. Estas diferenças estão de acordo com o que tem sido reportado a nível local, nos pólipos nasais. De notar, que essas diferenças eram mais marcadas nos doentes com DRCI, o que reforça o conceito de “one airway, one disease”. Relativamente ao estudo sobre o impacto da alergia alimentar na RSCcPN, esta não parece ter um papel relevante na sua etiopatogenia, seja esta resposta imune IgG ou IgE mediada. Além do mais, observou-se uma supressão de anticorpos IgG específicos contra antigénios alimentares nos doentes com RSCcPN, uma correlação negativa da sua soma com os níveis séricos de IgG1 e valores de IgG1 significativamente elevados nestes doentes. Esta supressão poderá estar relacionada com um desvio da resposta imune IgG-mediada contra outros agentes (p.ex. partículas inalantes) na RSCcPN e deverá ser investigada no futuro.

Chronic Rhinosinusitis (CRS) is characterised by a plethora of symptoms that patients suffer as a result of chronic inflammation of the sinus mucosa. These are associated with chronic relapsing infections caused by mucosal biofilms that are predominantly due to Staphylococcus aureus. CRS is a prevalent disease affecting around 10% of the general population in Western societies with significant impact on the socio-economical life. However, the pathophysiology of CRS and exact role of biofilms and S. aureus infections are poorly understood. Therefore, understanding the mechanism by which these micro-organisms and biofilms affect the mucosa and relate to chronic inflammation and relapsing infections is subject of intense ongoing research worldwide. The first part of this thesis studied the pathogenicity of S. aureus clinical isolates isolated from patients who were suffering from CRS and that were grown in planktonic form and biofilm form. We collected the exoproteins secreted from those isolates and studied their effect on in-vitro models of the nasal epithelial barrier. As a result of this study we found S. aureus biofilm exoproteins disrupt the mucosal barrier structure in a time- and dose- dependent manner and are toxic to the barrier. This damage to the mucosal barrier is thought to play an important role in the etiopathogenesis of CRS. In the second part of the thesis we explored the ability of S. aureus biofilms to induce a host immune response and we studied the relationship between S. aureus biofilm properties and host immune responses. S. aureus clinical isolates harvested from CRSwNP patients exhibited higher S. aureus biofilm exoprotein production and higher metabolic activity than S. aureus biofilms from CRSsNP patients. There was a significant strong positive correlation between the number of lymphocytes or eosinophils and the concentration of biofilm exoproteins secreted by corresponding clinical isolates and their metabolic activity. In contrast, exoprotein concentrations from planktonic cells did not correlate with inflammatory cell infiltration. The gradual increase in inflammation and Th2 polarisation in relation to S. aureus biofilm properties seen in that study imply that biofilm exoproteins might trigger inflammation in those patients. The third part of this thesis involved studying the resistance pattern of S. aureus in planktonic and biofilm form, and to know if they were any different in their sensitivity to commonly used antibiotics used in routine clinical practice. By doing so it was our aim to show that persistence and severity of symptoms in recalcitrant (rCRS) disease is due to biofilms that resist treatment to conventional antibiotics, many times higher than their planktonic counterparts. We were able to show that S. aureus biofilm have unique properties of excessive exoprotein production which was directly proportionate to their biomass and metabolic activity. The minimum dosage for biofilm eradication (MBEC) was much higher than routinely used minimum inhibitory concentration (MIC) of planktonic forms of the organisms. MBEC values furthermore correlated with inflammatory cell counts, giving us valuable insights into the mechanisms by which the organism evade treatment and induce human suffering. This study helps us to design future experiments in-vivo and study human response conditions thereby paving ways as a model for future therapies that could target the microorganism’s exoprotein production.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2020

Chronic rhinosinusitis (CRS) is chronic inflammation of the sinonasal mucosa. It is a disease of significant impact on public health, one that affects about 10-15% of the population. Functional Endoscopic Sinus Surgery (FESS) is the “gold standard” surgical treatment for CRS; its original philosophy or concepts are based upon the sinonasal mucociliary clearance studies by Messerklinger and Stammberger, which emphasize the role of the osteo-meatal complex (OMC). However, although the success rate of FESS is about 90%, there is a subgroup of patients who exhibit no improvement, and thus require repeated surgeries. This subgroup of patients suffers from refractory chronic rhinosinusitis (rCRS), which is the main focus of this thesis. In this thesis the current understanding of the pathogenesis and causes of surgical failure in CRS are reviewed. This thesis presents the hypothesis that our understanding of the pathogenesis of CRS has advanced since the original concepts of FESS were put forward, and that patients who develop rCRS have other pathogenic features that cannot be addressed by these concepts. We revisit middle turbinate lateralization (MTL) as a surgery-related factor of rCRS in Chapter 6, and we pose the question: Is MTL a complication associated with worse surgical outcomes, or just a harmless sequela, of the surgical destabilization of the middle turbinate during sinus surgery? Our findings show that MTL plays a role in surgical failure and requiring revision surgery, but suggest that the clinical significance of MTL may be related to frontal sinus obstruction and not necessarily to the OMC. We then present two novel hypotheses: the inflammatory load hypothesis in Chapter 7, and the irreversible disease hypothesis in Chapter 8. In Chapter 9, we investigate nasal polyp recurrence in CRS with Nasal Polyposis (CRSwNP) as an important cause of rCRS. We study the patterns of polyp recurrence and the clinical factors associated with more aggressive recurrence. The findings show that firstly, comorbid factors such as asthma and aspirin sensitivity contribute to the disease load and rCRS; and secondly, that more aggressive surgical removal of that disease load and maximal opening of the sinuses through a frontal drillout procedure improve the surgical outcome and disease control for these rCRS patients. We then proceed to investigate the relevance of our two novel hypotheses to refractory CRSwNP through a histopathological study in Chapter 10. We also describe the evolution of the inflammatory load in patients with rCRS from first to second surgery, a topic rarely addressed in the literature. We found that a higher inflammatory load is present in patients who fail surgery and go on to develop refractory CRS, when compared to patients who respond to surgery, with a particular significance to the eosinophilic load. In summary, our findings suggest that the inflammatory load is associated with long-term surgical outcomes. The recommendation based upon findings in this thesis is that surgery offered for CRS should be viewed as a tool for addressing and controlling disease load, and not just for the conservative clearance of disease of the OMC.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2015.

Chapter 1 of this thesis reviews the current literature on Chronic Rhinosinusitis (CRS) including its aetiology and the role of bacterial biofilms in CRS. It also presents an updated review on all current and emerging topical anti-biofilm options in the management of CRS. Chapter 2 describes the optimisation of an in vivo model using Chitogel (CG) as a drug delivery vehicle for anti-biofilm agents to treat Staphylococcus aureus biofilms. Budesonide and Mupirocin were incorporated as they have some efficacy in the management of recalcitrant CRS in the form of topical irrigations. This study showed that Chitogel- Budesonide-Mupirocin gel significantly reduces S. aureus biofilms in vivo and is the first study to suggest an alternative mode of topical drug delivery to the sinuses. Chapter 3 furthers the study of topical anti-biofilm gel by incorporating novel antimicrobial agents Deferiprone and Gallium Protoporphyrin (DG) into Chitogel. DG has a synergistic anti-biofilm effect against S. aureus and Methicillin Resistant S. aureus (MRSA) biofilms by targeting the iron metabolism pathway that is crucial for bacterial growth and survival. This study has shown that Chitogel- Deferiprone- Gallium Protoporphyrin is safe and effective in eradicating S. aureus biofilms in vivo. Chapter 4 explores the potential wound healing properties of Deferiprone in vitro. In this study Deferiprone was shown to delay primary nasal fibroblast migration without affecting epithelial migration, decrease collagen and reactive oxygen species (ROS) production and reduce interleukin 6 (IL-6) production. This anti-inflammatory potential and ability to limit scar tissue formation has wide prospective applications in the field of sinus surgery. Chapter 5 explores the long-term safety of S. aureus bacteriophage (NOV012) in vivo as a novel treatment option in CRS. Bacteriophage (Phage) therapy was first proposed as an antibacterial treatment in the 1910s and has been recently revived in the face of a global antibiotic resistance crisis. In this study we have shown that S. aureus phage are safe as a topical sinus irrigation in vivo for up to 3 weeks. Chapter 6 furthers the study of Pseudomonas aeruginosa phage (CT-PA) in vivo. Topical sinus irrigation of CT-PA was safe and effective in reducing P. aeruginosa biofilms in vivo over 3 weeks. Chapter 7 explores the translation of our in vivo studies into the first phase-1 clinical trial investigating the safety and preliminary efficacy of phage in S. aureus CRS. It was safe and well tolerated up to 3×109 PFU twice daily for 14 days with no dose-limiting side effects. Preliminary efficacy indicated favourable outcomes across all cohorts with 2 out of 9 patients achieving eradication of infection. Chapter 8 presents a pilot study investigating the evidence behind alternative treatments like colloidal silver (CS) in recalcitrant CRS. This is the first clinical study looking at the safety and efficacy of CS as a topical sinus irrigation in the management of CRS despite it being widely available for purchase over-the-counter. Twice daily CS sinonasal rinses for 10 days is safe but not superior to culture-directed oral antibiotics. We believe that a larger study and further optimisation of treatment duration could further the potential of this therapy. Chapter 9 investigates the role of manuka honey (MH) sinus irrigations with augmented methylglyoxal (MGO) in recalcitrant CRS. Twice daily MH rinses for 14 days have not been shown to be superior to culture-directed oral antibiotics and saline rinses in bacterial eradication but have shown significant improvement in endoscopic scores comparable to control. In the face of a global antibiotic resistance crisis, now more than ever, clinicians are practising more judicious use of systemic oral antibiotics. With an eye to the future, we hope that this thesis on novel topical anti-biofilm therapies would spur further research and someday offer clinicians viable alternatives to systemic oral antibiotics in the management of recalcitrant CRS.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2020

Objectives: To study the expression of cathelicidin antimicrobial peptides (CAMP) and surfactant protein D (SP-D) in patients with chronic rhinosinusitis (CRS) and eosinophilic mucus chronic rhinosinusitis (EMCRS) and by a nasal explant in vitro model cultured with fungal allergens. Methods: Nasal biopsies from 59 CRS and EMCRS patients, stratified into Allergic fungal sinusitis (AFS), Nonallergic fungal eosinophilic sinusitis (NAFES), and Nonallergic nonfungal eosinophilic sinusitis (NANFES) were studied by quantitative real-time (q)RTPCR, Western blot, immunostaining and ELISA. Nasal tissue from CRS and EMCRS patients were cultured with increasing concentrations of fungal allergens in a nasal explant in vitro model for 24 hours and CAMP and SP-D mRNA and protein levels in response to the fungi were determined by qRT-PCR and ELISA. Results: The expression of CAMP mRNA was significantly increased in EMCRS patients compared to CRS patients (p=0.0004). By immunohistochemistry, expression of CAMP was localised to nasal epithelial, submucosal glands and inflammatory subepithelial cells. Western blotting demonstrated the presence of CAMP in the study patients. Culturing nasal explants with fungal allergens demonstrated significant upregulation of CAMP mRNA expression in CRS, but not EMCRS patients, by Aspergillus (mean 4-fold increase) and Alternaria (mean 6-fold increase) extracts with a significant dose-response effect (p<0.001). CAMP protein levels in the nasal tissue from CRS patients increased in response to Alternaria (p<0.05). In contrast, with EMCRS patients the expression of CAMP peptide in nasal tissue increased with Aspergillus (p<0.001) but decreased with Alternaria. Staining for SP-D was detected in the submucosal glands from the nasal biopsies in all patient groups except for AFS. By ELISA, SP-D was undetectable in AFS and decreased in NAFES, NANFES, and CRS compared to controls. CRS patients cultured with Aspergillus and Alternaria allergens in the in vitro nasal explant model induced significant upregulation of SP-D mRNA (p<0.0001). In contrast, NANFES nasal tissue explants cultured with Aspergillus allergens induced downregulation of SP-D and only a modest upregulation of SP-D mRNA to Alternaria allergens. Conclusion: This study demonstrates expression of cathelicidin antimicrobial peptides and surfactant proteins in nasal mucosa supporting its potential role in innate defences against inhaled pathogens. There is significant upregulation of CAMP mRNA in the EMCRS group implying an increased inflammatory state. In vitro, CAMP is significantly upregulated at the mRNA and protein level in CRS tissue explants to Aspergillus and Alternaria allergens. However, EMCRS tissue cultured with Alternaria in vitro does not demonstrate increased CAMP at the mRNA or protein level. The expression of SP-D in nasal tissue is reported for the first time. SP-D expression in the CRS, but not the EMCRS group, is upregulated in vitro by Aspergillus and Alternaria. The EMCRS group compared to CRS group demonstrate abnormal CAMP and SP-D expression to common fungal allergens. These important findings in understanding the pathogenesis of chronic rhinosinusitis are discussed in this thesis and may provide potential novel therapies for chronic rhinosinusitis in the future.
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Thesis(PhD)-- School of Medicine, 2007

Objectives: To study the expression of cathelicidin antimicrobial peptides (CAMP) and surfactant protein D (SP-D) in patients with chronic rhinosinusitis (CRS) and eosinophilic mucus chronic rhinosinusitis (EMCRS) and by a nasal explant in vitro model cultured with fungal allergens. Methods: Nasal biopsies from 59 CRS and EMCRS patients, stratified into Allergic fungal sinusitis (AFS), Nonallergic fungal eosinophilic sinusitis (NAFES), and Nonallergic nonfungal eosinophilic sinusitis (NANFES) were studied by quantitative real-time (q)RTPCR, Western blot, immunostaining and ELISA. Nasal tissue from CRS and EMCRS patients were cultured with increasing concentrations of fungal allergens in a nasal explant in vitro model for 24 hours and CAMP and SP-D mRNA and protein levels in response to the fungi were determined by qRT-PCR and ELISA. Results: The expression of CAMP mRNA was significantly increased in EMCRS patients compared to CRS patients (p=0.0004). By immunohistochemistry, expression of CAMP was localised to nasal epithelial, submucosal glands and inflammatory subepithelial cells. Western blotting demonstrated the presence of CAMP in the study patients. Culturing nasal explants with fungal allergens demonstrated significant upregulation of CAMP mRNA expression in CRS, but not EMCRS patients, by Aspergillus (mean 4-fold increase) and Alternaria (mean 6-fold increase) extracts with a significant dose-response effect (p<0.001). CAMP protein levels in the nasal tissue from CRS patients increased in response to Alternaria (p<0.05). In contrast, with EMCRS patients the expression of CAMP peptide in nasal tissue increased with Aspergillus (p<0.001) but decreased with Alternaria. Staining for SP-D was detected in the submucosal glands from the nasal biopsies in all patient groups except for AFS. By ELISA, SP-D was undetectable in AFS and decreased in NAFES, NANFES, and CRS compared to controls. CRS patients cultured with Aspergillus and Alternaria allergens in the in vitro nasal explant model induced significant upregulation of SP-D mRNA (p<0.0001). In contrast, NANFES nasal tissue explants cultured with Aspergillus allergens induced downregulation of SP-D and only a modest upregulation of SP-D mRNA to Alternaria allergens. Conclusion: This study demonstrates expression of cathelicidin antimicrobial peptides and surfactant proteins in nasal mucosa supporting its potential role in innate defences against inhaled pathogens. There is significant upregulation of CAMP mRNA in the EMCRS group implying an increased inflammatory state. In vitro, CAMP is significantly upregulated at the mRNA and protein level in CRS tissue explants to Aspergillus and Alternaria allergens. However, EMCRS tissue cultured with Alternaria in vitro does not demonstrate increased CAMP at the mRNA or protein level. The expression of SP-D in nasal tissue is reported for the first time. SP-D expression in the CRS, but not the EMCRS group, is upregulated in vitro by Aspergillus and Alternaria. The EMCRS group compared to CRS group demonstrate abnormal CAMP and SP-D expression to common fungal allergens. These important findings in understanding the pathogenesis of chronic rhinosinusitis are discussed in this thesis and may provide potential novel therapies for chronic rhinosinusitis in the future.
Thesis(PhD)-- School of Medicine, 2007

Trabalho Final do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introdução: O uso de questionários de qualidade de vida relacionados com a saúde tem vindo a aumentar na prática clínica para avaliar o impacto da intervenção médica e medir o resultado da intervenção de cuidados de saúde na qualidade de vida de um paciente. O questionário SNOT-22 foi considerado a ferramenta mais adequada para avaliar a rinossinusite crónica. Este estudo tem como objetivo determinar o impacto funcional do tratamento cirúrgico das doenças naso-sinusais na qualidade de vida dos pacientes, medido através um questionário de qualidade de vida, o SNOT-22.Métodos: Estudo observacional prospectivo com 52 pacientes com rinossinusite crónica submetidos a cirurgia que responderam ao questionário SNOT-22-pt. Os dados foram recolhidos no período pré-operatório e 3 meses após a cirurgia e foram analisados para determinar a consistência interna e a capacidade de resposta do questionário SNOT-22.Resultados: Este estudo constatou que houve uma diminuição estatisticamente significativa (P <0.0001, t = 9.643) nos scores de SNOT-pt 22 referidos pelos pacientes (média 21) em comparação com antes da cirurgia (média 51), mostrando capacidade de resposta clínica. O SNOT-22-pt mostrou um alto nível de consistência interna (alfa de Cronbach de 0.895). O tamanho total do efeito foi de 1.91, que é considerado grande. A diferença minimamente importante foi 28, o que significa que uma mudança de menos de 28 pontos não é percebida pelo paciente como uma melhoria real. O score pós-operatório normal é inferior a 25 pontos.Conclusão: Verificamos que a versão Portuguesa do questionário SNOT-22 é um instrumento válido e de fácil utilização para avaliar pacientes com RSC, pois demonstrou alta consistência interna, capacidade de resposta e facilidade de interpretação clínica. O SNOT-22-pt deve ser usado como uma ferramenta para facilitar a prática clínica de rotina na avaliação do impacto da RSC na qualidade de vida de um paciente e também para medir a eficácia das intervenções cirúrgicas.
Introduction: Health-related quality of life questionnaires use has been increasing in clinical practice to assess the impact of medical intervention and measure the outcome of health care intervention in a patient quality of life. The SNOT-22 questionnaire has been considered the most appropriate tool to evaluate chronic rhinosinusitis. This study aims to determine the functional impact of surgical treatment of nasosinusal diseases on patients' quality of life, measured by a quality of life questionnaire, the SNOT-22.Methods: Prospective observational study with 52 patients with chronic rhinosinusitis submitted to surgery who answered the SNOT-22-pt questionnaire. Data were collected preoperatively and 3 months after surgery and analysed to determine the internal consistency and responsiveness of the SNOT-22 questionnaire.Results: This study found that there was a statistically significant (P < 0,0001, t = 9,643) decrease in patient reported SNOT-22-pt scores (mean 21) compared to before surgery (mean 51), showing clinical responsiveness. SNOT-22-pt showed a high level of internal consistency (Cronbach’s alpha of 0.895). The overall effect size was 1.91, which is considered large. The minimally important difference was 28, which means that a change of less than 28 points is not perceived by the patient as a real improvement. Normal post operative scores cut-off are below 25 points.Conclusion: We found that the Portuguese version of the SNOT-22 questionnaire is a valid and easy to use instrument to evaluate patients with CRS, as it demonstrated a high internal consistency, responsiveness and easy clinical interpretability. The SNOT-22-pt should be used as a tool to facilitate routine clinical practice to assess the impact of CRS on a patient’s quality of life and also to measure the effectiveness of surgical interventions.

Chronic rhinosinusitis (CRS) is a debilitating condition characterised by critical inflammation of the mucosa of the nose and paranasal sinuses. Effecting up to 14% of the world’s population CRS severely impacts a patient’s quality of life. The aetiology of CRS is complex and relatively undefined encompassing a multitude of contributing factors. Bacterial infection is one factor thought to play a role in the pathogenesis of CRS. More specifically biofilm forms of the bacterial species Staphylococcus aureus have been shown to negatively influence post-operative progression. Current practice treatment strategies often fail to remove biofilms from the mucosa of the nose. It is therefore of import to develop novel anti-biofilm therapeutics. Our understanding of the epidemiology of S. aureus infections and biofilms in CRS is also limited. Increasing our epidemiological knowledge would help in the development of effective treatment strategies against recurrent infections. Investigation into the epidemiology of S. aureus infections was undertaken by collecting S. aureus isolates from mucous and biofilm structures of CRS patients. The clonal type of each isolate was then compared to the other isolates using pulse field gel-electrophoresis. Results of this study indicated that the majority of patients experiencing recurrent infections maintained the same clonal type. Furthermore the study suggested that long-term antibiotic therapy in some patients can lead to the development of bacterial antibiotic resistance. Therefore development of a novel antibacterial therapy outside of antibiotics is required. A potential anti-biofilm therapy both eliminating and preventative in nature is the application of bacteriophage. Bacteriophage (phage) are viruses that specifically target, infect and destroy bacterial cells. Initially in vitro study was undertaken to assess the anti-biofilm activity of a phage cocktail specific for S. aureus (CT-SA) using a minimal biofilm eradication assay plate. S. aureus isolates from CRS patients were grown to mature biofilm form and treated with CT-SA for 48hrs. Following treatment biofilm biomass was determined by staining bacteria with a Live/Dead BacLight stain, imaging the biofilm using confocal scanning laser microscopy and determining biofilm biomass using software COMSTAT2. Results showed CT-SA significantly reduced S. aureus biofilms of susceptible strains. Results also indicated that a cocktail of phage was superior to use of a single phage as it reduced the frequency of bacterial resistant to the phage treatment. Following on from in vitro work, the safety and efficacy of CT-SA was assessed in vivo using a sheep model of frontal sinusitis associated with S. aureus infections. CT-SA was also combined with ethylenediaminetetraaceticacid (EDTA) to observe if these therapies would synergise. Results indicated both CT-SA and EDTA were safe for short term topical application to the sinus regions. Furthermore both CT-SA and EDTA individually significantly reduced S. aureus biofilm levels in the frontal sinus, but were not seen to synergise. Work conducted in this thesis has helped lead towards development of a novel anti-S. aureus biofilm agent. Future translation of CT-SA to a clinical trial setting may not only reduce or remove S. aureus biofilm from CRS patient noses but also improve their symptomatology and quality of life.
Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Medicine, 2015.

“Surgery is a profession defined by its authority to cure by means of bodily invasion. The brutality and risks of opening a living person's body have long been apparent, the benefits only slowly and haltingly worked out”, says Atul Rawande on reviewing 200 yrs. of Surgery as a specialty in NEJM. My research focuses on working out these benefits, specifically looking at reduction of scar tissue formation in ENT, Abdominal & Spine surgery. Scar tissue formation is an outcome of healing process that can be excessive due to inflammation or infection and thereby has the ability to curtail the benefits or warrant revision surgery. Multiple strategies have been tested and employed thus far and none have given favourable results without causing additional harm or economic burden in health care costs. I propose to use a hydrogel synthesized by combining Chitosan and Dextran aldehyde -Chitin is an exoskeleton extracted polymer and Dextran Aldehyde a sugar, with added noveldrugs Deferiprone and Gallium Protoporphyrin providing additional anti scaring and antibiotic properties which could potentially augment the healing properties of the gel. I have conducted 3 types of studies. There are 2 animal studies and a Phase 1 Human clinical trial. The animal studies are an abdominal surgery rat model and a spine surgery sheep model. These studies show the safety and efficacy of this chitogel-drug combination at various dosages and illustrate the healing benefits of gel-drug combination.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2021

碩士
中國醫藥大學
醫務管理學研究所碩士班
94
Background： Chronic rhinosinusitis (CRS) is a prevalent disease among the general population and is recognized to have a great impact on a patient’s quality of life. No medical care procedure has been proved as treatment standard yet. Antibiotics, intranasal steroid, and endoscopic sinus surgery (ESS) are used to treat CRS, but none of these treatments have satisfactory long-term outcome. Recently fungal infection has been suggested to play an important role in the causation of CRS. In addition, traditional outcome measures of CRS often focus on the physiologic parameters and not enough attention on patient’s quality of life. Recently, health-related quality of life was advocated as one of the primary and important outcome variables. However there is still not any disease-specific questionnaire available for CRS in Taiwan. Objectives： We tested the hypothesis that intranasal antifungal treatment improves the quality of life and the objective nasal endoscopic scores and fungal burden of CRS in a prospective, double-blind, randomized, and placebo-controlled clinical trial. Another focus of this study is to evaluate the Chinese-version of RSOM-31 and the quality-of-life data from CRS patients in Taiwan. Methods： From September 2005 to March 2006, 40 CRS patients at Taichung Veterans General Hospital were recruited with 37 patients completed the trial. Patients applied 500 mL amphotericin B saline solution (0.04mg/mL) or placebo to each nostril twice a day by using pulsatile nasal irrigation. The nasal endoscopy, fungus culture rate and RSOM-31 were evaluated pre- and post-treatment. Results： The overall Cronbach’s alpha coefficient of Chinese-version RSOM-31 was 0.92, suggesting a high degree of internal consistency. The average Symptom-Impact score was 6.4, which was a little higher compared to Dr. Piccirillo’s score 5.8. The domains most affected were sleep and emotional. The amphotericin B group had a better quality-of-life improvement compared to the placebo group after treatment, especially in general symptoms and practical problems subscales. However the endoscopic disease-severity score and fungus culture rate were not significantly different between groups after treatment. Conclusions： The Chinese-version of RSOM-31 was proved to be a convenient and valid tool for evaluating the CRS health status and quality of life. Patients with amphotericin B nasal irrigation in the described dosing and time schedule appear to be both safe and effective and have better life quality compared to placebo. Keywords: Chronic rhinosinusitis, Quality of life, RSOM-31, Nasal irrigation, Amphotericin B

碩士
國立中興大學
高階經理人碩士在職專班
99
Chronic rhinosinusitis is one of the common infectious diseases. The prevalence rate and consumption of the related medical resource are increasing nearly everyday. The purpose of this study is to examine the health care utilization and related factors of chronic sinusitis patients. This source of the data came from claims data of beneficiaries of National Health Insurance from 2004 to 2008. ICD-9-CM with initial four codes as 471 or 473 were selected from the panel database. All statistical analyses were performed by using SAS 9.1 software. Multiple liner regression analysis was used to explore health services utilization and its related factors. The results indicated that within 5 years, a total of 98,412 visits in ambulatory care and emergency, belonging to 28,080 persons, and 2,338 persons in hospitalization were identified in this study. Male, age over 60 years, medical center, other division of visits, and Northern region branch had higher medical expenses in single visit outpatient and ambulatory care and emergency analysis of statistics. All kinds of ambulatory and emergency care expenses per person were significantly higher medical costs if male, age over 60 years, sinusitis without polyposis, sinusitis with co-morbidities (asthma, allergic rhinitis, otitis media, nasal septum deviation). The statistic analysis of hospitalization showed that: male, age over 60 years, and received functional endoscopic sinus surgery have higher medical expenses in hospitalization. According to the results of this study, we suggest that the health organizations could consider other supporting methods when chronic rhinosinusitis patients would be implemented with the case payment systems. In future studies, if questionnaire survery, patient clinical data, and other factors can be used simultaneously, it will make the research more perfect.

博士
中山醫學大學
醫學研究所
96
Objectives: This study aims to investigate the change of olfactory function in patients with chronic rhinosinusitis after functional endoscopic sinus surgery and explore the potential prognostic factors. Methods: Patients with chronic rhinosinusitis who had previously undergone functional endoscopic sinus surgery were enrolled in the study. On the day before FESS, olfactory function was evaluated by a symptom score, a phenyl ethyl alcohol odor detection threshold test (STT), the University of Pennsylvannia Smell Identification Test (UPSIT) and a short-term odor memory/discrimination test, and re-evaluated by the same methods 6 months after FESS. The potential prognostic factors (nasal obstruction, cross-sectional area of nasal cavity, rhinosinusitis severity, preoperative olfactory loss, nasal polyps, allergic rhinitis, concurrent septoplasty and turbinal surgery, postoperative steroid treatment) for improvement in olfaction after FESS were also evaluated in these patients. Results: A total of 70 patients with chronic rhinosinusitis were enrolled in the study. Fifty-two patients noticed their olfactory function was impaired before surgery, but the olfactory threshold was above –6 in 66 patients, and 62 patients’ UPSIT scores were below 30. After surgery, the olfactory function was improved in 27 patients using patients’ reports, in 30 patients by STT and in 36 patients by UPSIT. A good agreement existed between STT and UPSIT results and patients’ reports. Among potential prognostic factors, nasal obstruction, cross-sectional area of nasal cavity, rhinosinusitis severity, preoperative olfactory loss, nasal polyps and allergic rhinitis were not significantly reliable to predict improvement in olfaction after surgery, but patients with severe rhinosinusitis and severe preoperative olfactory loss and those with allergic rhinitis or without nasal polyps tended to show olfactory improvement. Concurrent septoplasty and turbinal surgery did not increase the rate of olfactory improvement after FESS, but postoperative use of nasal steroids did increase the rate of olfactory improvement, although the difference was not significant. Conclusion: Although many methods have been used to evaluate olfactory function, and the domains of olfactory function evaluated were not the same among these methods, our study showed that UPSIT should be the test of choice for evaluating olfactory function in patients with chronic rhinosinusitis. For these patients, disease severity, coexistence of nasal polyps and allergic rhinitis, and postoperative use of nasal steroids were found to be more reliable prognostic factors for improvement in olfaction after FESS, but these factors were not significantly reliable.

Chronic rhinosinusitis (CRS) is a complex and heterogenous disease characterised by nasal obstruction, facial pressure, rhinorrhoea, postnasal drip and reduction in smell. The pathophysiology of CRS is multifaceted with an interplay between host, environmental and microbial factors. Numerous hypothesise have been proposed to elucidate this complicated disease process, however, the jury is still out. Recently, the dysfunction in the mucosal barrier with associated pathogen invasion is described as a potential basis for the development of CRS. Studies have identified the overactivation of inflammatory cells with superfluous secretion of proinflammatory proteases and cytokines associated with the chronic inflammatory environment observed in CRS. Therefore, it is vital to understand the interactions between the host immunity with the mucosal barrier. Nasal mucus overproduction is a hallmark symptom of CRS and thus it is paramount to investigate its interaction with the mucosal barrier function in both healthy and CRS patients. Classically, nasal mucus from CRS patients are more viscous and contain increased levels of inflammatory cytokines. Previous research has found Pseudomonas aeruginosa elastase, an exoprotein, present in CRS nasal mucus associated with increased barrier dysfunction and worse symptom-scores. Furthermore, neutrophil infiltration is observed in CRS mucosa secondary to the persistent inflammation and bacterial activation. The collateral damage to host tissues secondary to the release of neutrophil-associated proteases against infections must be elucidated. Advances in mass spectrometry techniques allow for detailed analysis of differentially expressed proteins in nasal mucus. A systemic review of the nasal proteome will be conducted to determine the current progress and deficiencies within the literature. Based on these findings, the direct comparison of healthy and CRS mucus proteomes will be performed. By analysing the different proteins and cellular processes present in nasal mucus may assist in understanding the downstream manifestations of CRS. This thesis examines the interaction of nasal mucus with the nasal mucosal barrier function. Secondly, to ascertain the effects of neutrophil serine proteases on the mucosa barrier function. Lastly, to investigate and understand the differing mucus composition between healthy and CRS patients. Ultimately, through understanding the pathophysiology and various endotypes of CRS, we aim to direct future research to develop treatment regimens for numerous patients.
Thesis (Ph.D.) -- University of Adelaide, Adelaide Medical School, 2020

博士
輔仁大學
化學系
105
Chronic rhinosinusitis (CRS) is a chronic inflammatory disease of the sinonasal mucosa either accompanied by polyp formation (CRSwNP) or without polyps (CRSsNP). CRSsNP accounts for the majority of CRS cases and is characterized by fibrosis and neutrophilic inflammation. However, the pathophysiology of CRS, especially CRSsNP, remains unclear. In the fist part of the study, CRSsNP specimens were analyzed by immunohistochemistry and noted the submucosa, perivascular areas, and the mucous glands were highly expressed with fibroblasts. Therefore, the effects of bradykinin (BK), an autacoid known to participate in inflammation, were investigated in human CRSsNP nasal mucosa-derived fibroblasts (NMDFs). BK increased CXCL1 and -8 secretion and mRNA expression with EC50 ranging from 0.15~0.35 μM. Moreover, BK enhanced cell proliferation and upregulated the proinflammatory molecules expression, including cell adhesion molecules (CAMs) and cyclooxygenase (COX)-1 and -2 expressions. These effects functionally caused an increase in monocyte adhesion to fibroblast monolayer. The BK-induced CXCL1 and -8, cell proliferation, COX, and CAMs expressions were mainly through the B2 receptor, as demonstrated by the pharmacological intervention and siRNA knockdown assay. Accordingly, the B2R was preferentially expressed in the NMDFs than B1R. In parallel, the B2R was highly expressed in the CRSsNP than control specimens, while the B1R and kininogen (KNG)/BK expression was slightly increased in the CRSsNP mucosa. Collectively, we demonstrate here that fibroblasts, KNG/BK, and BKRs are overexpressed in CRSsNP mucosa and BK upregulates chemokine expression, proliferation, and proinflammatory molecules expression in NMDFs via B2R activation, which lead to a functional increase in monocyte-fibroblast interaction. This reveals a critical role of fibroblast, KNG/BK, and BKRs in CRSsNP development. Thromboxane A2 (TXA2), an arachidonic acid metabolite, participates in platelet aggregation and tissue inflammation. In the second part of the study, the CXCL1/8 chemokine and TXA2-TP receptor expression in the CRSsNP mucosa was investigated. The immunohistochemistry results indicated that CXCL1 and CXCL8 were highly expressed in the submucosal stroma of the CRSsNP mucosa compared with the corresponding expression in the controls; however, the TP receptors were expressed in both mucosa. Therefore, U46619 and IBOP, a TXA2 analog and TP agonist, were used to explore the role of TP activation in CXCL1/8 expression; both of these induced CXCL1/8 mRNA and protein expression in CRSsNP mucosa-derived fibroblasts. U46619 phosphorylated p38 MAPK, PI-3K-JNK, cyclic AMP (cAMP)/PKA, PKC, and cAMP response element (CREB). We observed that activation of cAMP/PKA, PKC, and CREB was the major cxcl1/8 gene transcription pathway and that the p38 MAPK pathway regulated CXCL1/8 secretion. Additional pharmacological and siRNA knockdown analyses revealed that activation of cAMP/PKA and PKCμ/PKD pathways were required for CREB phosphorylation and PKA/C crosstalk with the PI-3K-JNK pathway. The second part of our study provides the first evidence for abundant TP receptor and CXCL1/8 expression in CRSsNP mucosa and for TXA2 stimulation inducing CXCL1/8 expression in nasal fibroblasts primarily through PKA, PKCμ/PKD, and CREB-related pathways.

碩士
臺北醫學大學
醫學研究所
93
Chronic rhinosinusitis and nasal polyps mainly develop in the ethmoid sinus, maxillary sinus and middle turbinate area, often in relation to inflammatory and allergic conditions. Their exact etiology and pathogenesis are still under debate. Histologically, inflammatory cell infiltrations of nasal mucosa predominated by eosinophils are typical for such disease, and may be due to the chemotactic activity of chemokines specific for eosinophils. This findings suggest that nasal polyp is an inflammatory growth that is controlled by the local environment and the immunologic defense mechanism of the host. The CC-chemokine Eotaxin and RANTES (regulated on activation normal T cell expressed and secreted) have been postulated to be involved in the recruitment of eosinophils toward inflamed tissues. To explore their possible roles in chronic rhinosinusitis, we examined the concentraion of Eotaxin and RANTES protein in the serum of patients and correlated these results to the severity of disease graded by sinus CT imaging. Serum samples were obtained from 20 of patients undergoing endoscopic sinus surgery, and blood samples of 20 normal control subjects were also drawn. Enzyme-linked immunosorbent assay(ELISA) for Eotaxin and RANTES protein of serum was performed, and the relationship between these results and the Lund-MacKay rhinosinusitis scoring system and the percentage of peripheral eosinophil were investigated. In comparison to normal control group, we found significant elevation of Eotaxin and RANTES protein in the serum of experimental group(p<0.05). The levels of serum Eotaxin and RANTES in patients were correlated with the severity of the disease; with Eotaxin showed a more statistical significance. Besides, RANTES and Eotaxin levels were correlated with the percentage of peripheral eosinophil(p<0.05). These data suggest that, in patients of chronic rhinosinusitis with nasal polyp, disease severity correlates mainly with Eotaxin, and that RANTES may play a less important role in the mobilization of eosinophils from the blood into inflamed tissues. Further larger experiment was anticipated to validate this correlation.

碩士
國立陽明大學
生理學研究所
104
Abstract Chronic rhinosinusitis (CRS) and allergic rhinitis (AR) are two of the most common upper airway diseases worldwide. Generally, CRS was regarded as a syndrome with multiple etiologies. Therefore, CRS may be a common endpoint for the interactions of different factors, including microbial factors, environmental factors, and host factors. Allergic rhinitis is defined by Allergic Rhinitis and its Impact on Asthma (ARIA) Guidelines as “a symptomatic disorder of the nose induced after allergen exposure by an IgE-mediated inflammation”. Recently, it has been noted that after exposure to allergens from snares, systemic humoral recirculation of allergic cells would nonspecifically recruit back to the diseased sinuses. In the past studies, climate factors, such as relative humidity, average daily temperature, and northeasterly winds were found to be related to worsening asthma, an allergic lower airway disease. However, the association between climate factors with CRS or AR has not been clarified. We conduct a retrospective analysis by using the National Health Insurance database during 2003 to 2012. The meteorological data will be obtained from the Central Weather Bureau of Taiwan, including relative humidity, average temperature. Five geographic zones and the related outpatient rates (Taipei city, Taichung city, Kaohsiung city, Hualien county, and I-Lan county) were analyzed. From 2003 to 2012, 476,833 patients were included in the study, with a male: female ratio of 1.02: 1. Twenty seven percent of patients were under eighteen years old. The outpatient rate of AR is increasing by time, but in CRS, it shows a decreasing pattern. The outpatient rate of AR presents with seasonal changes but CRS dose not. In AR, significant interactions between any single city/county and time, and generally, when temperature or relative humidity elevates, the rate of outpatient drops. In CRS, the rate of outpatient drops when temperature or relative humidity elevates, and variant interactions between any single city/county with temperature or relative humidity were found. The rates of outpatient of AR and asthma shows strong correlation (r=0.947, p<0.0001), and rates of outpatient of CRS and asthma also shows strong correlation by time (r=0.906, p<0.0001). In summary, the rates of outpatient of CRS and AR were influenced by two of the climate factors, relative humidity and temperature. In Taiwan, though the etiologies of CRS were multiple, it shows strong relation between CRS and atopy. The prevention of these two nasal diseases should be point on the prevention of allergen exposures.

Tese de doutoramento em Ciências Farmacêuticas, no ramo de Farmacologia e Farmacoterapia, apresentada à Faculdade de Farmácia da Universidade de Coimbra
Chronic rhinosinusitis is a persistent inflammation of the nasal cavity and paranasal sinuses mucosa with high prevalence and significant impact in patients´ quality of life. Although the exact etiology and pathophysiology of this multifactorial and difficult to treat disease are still unclear, there is growing evidence about the involvement of bacterial biofilms and their association with the refractory and chronic nature of the disease. Bacteria in biofilms show an increased resistance to antibiotics that can be as high as 1000-fold that of their corresponding planktonic form, which explains the lack of effectiveness of systemic administration of antibiotics in this clinical condition. High concentrations required to eradicate bacteria are difficult to attain without significant risks of systemic toxicity using the conventional routes of administration. Intranasal administration emerges as an alternative option to deliver drugs directly to the target site (sinonasal mucosa) achieving high local concentrations with minimal systemic exposure and consequent systemic adverse effects. The aim of the present thesis was to evaluate the potential of intranasal administration of fluoroquinolones by comparing their pharmacokinetic behavior after intranasal and intravenous delivery in Wistar rat biological matrices adequate to probe the risks and benefits of this topical strategy. The progression of experimental work led to the selection of ciprofloxacin and levofloxacin which are two of the most commonly used and well-known fluoroquinolones. To support the in vivo pharmacokinetic studies and thus achieve the above objectives, a high performance liquid chromatography method coupled with fluorescence detection was developed and validated to quantify ciprofloxacin and levofloxacin in rat nasal mucosa, plasma and olfactory bulb, probing drug efficacy as well as systemic and central nervous system safety, respectively. For intranasal administration a thermoreversible in situ gel was used to deliver ciprofloxacin and levofloxacin to Wistar rats at a dose of 0.24 mg/kg, a much lower dose than that administered by intravenous route, namely 10 mg/kg. After intranasal administration, markedly higher concentrations were attained in the anterior nasal mucosa compared to those found in the posterior region. This heterogeneous deposition pattern of formulation in nasal cavity contrasts with the intravenous homogeneous distribution and makes the application site more advantageous for this topical approach. Dose-normalized concentrations and exposure pharmacokinetic parameters obtained for nasal mucosa were two or one order of magnitude higher – in anterior and posterior nasal regions, respectively – by intranasal administration than by intravenous route. A similar comparison for plasma and olfactory bulb lead to the conclusion of a lower systemic exposure after topical intranasal administration and of a possible contribution of drug direct nose-to-brain transport that should be carefully taken into account. The results confirm the significant advantage of topical intranasal administration to deliver ciprofloxacin and levofloxacin to the biophase in comparison with the intravenous administration. Given the typically lower doses used for intranasal route, both systemic and central nervous system safe profiles were also demonstrated by the minimal or negligible values attained with the intranasal dose of 0.24mg/kg. Therefore, intranasal administration of topical-acting fluoroquinolones may represent a promising and safe alternative approach to be implemented in the management of chronic rhinosinusitis.
A rinossinusite crónica é uma inflamação persistente da mucosa da cavidade nasal e dos seios perinasais com elevada prevalência e um impacto significativo na qualidade de vida dos doentes. Embora a etiopatologia desta doença mulifatorial e difícil de tratar permaneça ainda por elucidar, existe evidência crescente do envolvimento de biofilmes bacterianos, sendo este o aspeto mais apontado para explicar a natureza crónica e refratária da doença. Com efeito as bactérias em biofilmes revelam uma resistência aos antibióticos que pode ser até 1000 vezes superior à da correspondente forma planctónica, o que explica a ineficácia terapêutica da administração sistémica de antibióticos. Utilizando vias convencionais de administração, as elevadas concentrações necessárias para erradicação das bactérias dificilmente se atingem sem riscos significativos de toxicidade sistémica. A administração tópica intranasal surge assim como uma estratégia alternativa para a entrega direta de fármacos na biofase (mucosa naso-sinusal) por forma a obter elevadas concentrações locais e exposição sistémica mínima, sem os efeitos adversos/tóxicos associados. O objetivo central desta dissertação foi a avaliação do potencial da administração intranasal de fluoroquinolonas, através da análise do risco/benefício envolvidos neste tipo de administração, utilizando matrizes biológicas estreitamente relacionadas com a segurança e a eficácia dos fármacos e comparando o respectivo comportamento farmacocinético após administração intranasal e intravenosa dos mesmos. A progressão do trabalho experimental conduziu à seleção da ciprofloxacina e levofloxacina, duas das fluoroquinolonas mais conhecidas e frequentemente utilizadas na clínica. Sendo a bioanálise um passo essencial de suporte aos estudos in vivo farmacocinéticos, procedeuse ao desenvolvimento e validação de uma técnica de cromatografia líquida de alta eficiência com deteção por fluorescência, para quantificar a ciprofloxacina e levofloxacina na mucosa nasal, plasma e bolbo olfativo de ratos Wistar. Um gel com propriedades termorreversíveis, capaz de gelificar na cavidade nasal, foi utilizado para a entrega tópica intranasal de uma dose de 0,24 mg/kg de ciprofloxacina e de levofloxacina, muito inferior à de 10 mg/kg administrada por via intravenosa. Após administração intranasal as concentrações obtidas na mucosa nasal da região anterior foram acentuadamente mais elevadas do que as encontradas na região posterior, esta distribuição heterogénea da formulação na cavidade nasal contrasta com a distribuição homogénea observada por administração intravenosa e põe em evidência a situação mais vantajosa para o local mais próximo da aplicação tópica. As concentrações e os parâmetros de exposição farmacocinéticos normalizados à dose obtidos na mucosa nasal por administração intranasal foram superiores aos da administração intravenosa - duas ordens de grandeza na região anterior e uma ordem de grandeza na região posterior. Uma comparação idêntica (portanto independente da dose) entre ambas as vias para o plasma e bolbo olfactivo permitiu inferir que a exposição sistémica foi inferior por administração intranasal e que a estreita relação do nariz com o sistema nervoso central não deve ser ignorada face os resultados obtidos no bolbo olfactivo. Confirma-se portanto a vantagem significativa da administração intranasal para entrega da ciprofloxacina e levofloxacina na biofase, face à administração intravenosa. Os valores negligenciáveis ou mínimos atingidos no plasma e bolbo olfactivo após administração de uma dose de 0,24 mg/kg demonstram um perfil seguro tanto a nível sistémico como a nível do sistema nervoso central. Em conclusão, a administração intranasal de fluoroquinolonas demonstrou ser uma abordagem promissora e segura, podendo representar um contributo importante no tratamento tópico da rinossinusite crónica.

碩士
臺灣大學
臨床醫學研究所
98
Background : Chronic rhinosinusitis (CRS) is defined as inflammation and infection involving the nasal mucosa and the adjacent sinus cavities. Clinically, the patients with CRS may suffer from symptoms of mucopurulent discharge, nasal obstruction, facial pain, and hyposmia. Antibiotics, saline irrigations, steroids, and anti-leukotrienes may be given for symptoms relief. However, episodes of recurrence or acute exacerbation occurred to these patients, and caused some irreversible changes in airway structure and function. The pathophysiology of frequent acute exacerbation in these patients is still unclear, making treatments particularly challenging. CpG-ODN refers to sequences that include an unmethylated cytosine and guanosine ,which are relatively common in the genomes of most bacteria and DNA viruses. CpG-ODN had been implicated as a major structural component of bacterial biofilm, and contributed to the persistence of chronic infection. Clinically, recalcitrant CRS is associated with biofilm formation, and biofilm may cause the unfavorable outcomes of surgery for CRS. However, the molecular significance of CpG-ODN in CRS is still unclear. We aim to investigate the immune-modulation effects of CpG-ODN ,and elucidate the role of biofilm in CRS. Hypothesis : 1.The existence of CpG-ODN may maintain a low profile of cytokines production in the sinus mucosa, and play a role in the persistence of inflammation in chronic rhinosinusitis. 2.During CRS with acute exacerbation, CpG–ODN synergistically up-regulate the immuno-reactions of pro-inflammatory mediators, augments intranasal cytokines release, and contribute to the complications and severity of CRS with acute exacerbation. Materials and Methods: Twenty patients who are diagnosed as CRS and underwent surgery were included prospectively. None of them receives any treatment at least one month before operation. The one who has atopy, asthma, or allergic rhinitis was previously excluded. The nasal epithelial cells harvested from nasal polyps were collected for ALI (air-liquid interface) culture. At the end of culture (Day 20 after confluence), CpG-ODN , IL-1β , and TNF-α were added in single regimen or in combination to stimulate the nasal epithelial cells. After 24 hours-treatment, the culture medium of each well was collected and immediately stored at -80℃for later IL-6,IL-8 and MCP-1 measurement using ELISA. Statistical analysis : The paired samples test was used for evaluation of differences between experiments and controls. ANOVA was used when there is a multiple sample comparison. P values less than 0.05 was considered significant. Results : 1.A model mimicking the in vivo microenvironment of stable CRS was created. 2.Intrinsic IL-1β and TNF-α levels were undetectable in this model, and IL-6 , IL-8 ,MCP-1 levels were detectable during 21-days culture period, with a trend toward homogeneity . 3.Extrinsic IL-1β and TNF-α were used to stimulate the cultured nasal epithelial cells, to initiate a condition resembles CRS with acute exacerbation. IL-1βinduced IL-6,IL-8 and MCP-1 expressions in a dose-dependent fashion, while TNF-α only induced these cytokines release insignificantly. 4. CpG–ODN induced MCP-1 expression with a dose-dependent fashion, but not in IL-6 and IL-8. 5. CpG-ODN synergistically potentiate IL-6,IL-8, and MCP-1 release in the presence of IL-1 β and TNF-α. Conclusion : CpG-ODN is associated with the chronicity of CRS. Elimination of such substances in the management of CRS is mandatory. CpG-ODN augments intranasal cytokines release in patients with acute exacerbated sinusitis. Strategies such as local debridement or short-term immuno-suppressants are needed to stop the vicious circle.