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.

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.

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.

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.

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.

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

Introduction Bacterial biofilms have been implicated in the pathogenesis of Chronic Rhinosinusitis (CRS). This thesis consists of a number of separate studies. The results of each study were designed to help provide an evolution of knowledge that could be applied to our subsequent investigations on the topic of bacterial biofilms and chronic rhinosinusitis. In vitro studies were utilized to document the capacity of CRS bacteria to form biofilms as well as to investigate the efficacy of various antimicrobials at high concentrations. Additionally, an in vivo sheep model was developed to examine different biofilm detection techniques. Finally, a study of CRS patients was conducted to investigate the incidence of biofilm related sinus disease. Methods Our in vitro studies used 96 well crystal violet microtiter plate assays to determine the biofilm growth characteristics of S.aureus isolated from patients with CRS. Established biofilms were then subjected various antimicrobial agents, and the degree of biofilm reduction calculated to examine their potential for sinus biofilm treatment. A sheep sinusitis model involved performing endoscopic sinus surgery, occlusion of frontal sinus ostia and the introduction of bacteria. Mucosal specimens were subsequently examined for the presence of bacterial biofilms using transmission electron microscopy (TEM), scanning electron microscopy (SEM) and confocal scanning laser microscopy (CSLM). CSLM was also used in a prospective study to document the presence bacterial biofilms on the mucosa of patients with CRS compared to controls. Results The findings of in vitro experiments revealed that not all isolates were capable of forming biofilms. Of the antibiotics tested, only Mupirocin was capable of reducing biofilm mass by 90% in all isolates. The animal model showed considerable variation in biofilm detection rates. The CSLM biofilm detection rate was 100% in obstructed sinuses with bacteria introduced, whereas TEM detected only 66%. Both these objective measures failed to identify biofilms in control groups. SEM found biofilms in all experimental groups including controls. CSLM analysis of CRS patients found Bacterial biofilms in 44% and no biofilms in controls. Conclusion The demonstration of biofilms in the sheep model for sinusitis and biofilms on the mucosal specimens of patients with CRS, and the ability of bacteria in CRS to form biofilms in vitro, further supports the hypothesis that biofilms play a role in the pathogenesis of CRS. CSLM is the modality of choice in documenting the presence of bacterial biofilms on sinus mucosal surfaces due to the inherent flaws of sampling error and subjectivity of TEM and SEM. Finally, CRS is a multi-factorial disease, topical Mupirocin via nasal irrigation may be a therapeutic option in patients with likely S.aureus biofilms. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1367183 / Thesis (M.S.) - University of Adelaide, School of Medicine, 2009

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.