INTRODUÇÃO: a doença nasossinusal em população pediátrica com fibrose cística (FC) é um tema ainda pouco explorado, sendo incerto o papel da tomografia de seios da face (TCSF) para avaliação e seguimento desses pacientes. OBJETIVOS: avaliar se existe correlação entre sintomas nasossinusais e genótipo, colonização crônica porPseudomonasaeruginosa, volume expiratório forçado no primeiro Segundo (VEF1), índice de massa corporal (IMC), Escore de Shwachman-Kulczycki, Escore tomográfico de Bhalla e achados tomográficos nasossinusais em crianças e em adolescentes com FC. DELINEAMENTO: estudo transversal. MATERIAIS E MÉTODOS:Foram avaliados 61 pacientes com idades entre dois e 16 anos que mantinham acompanhamento no Centro Pediátrico de Fibrose Císticado Hospital de Clínicas de Porto Alegre e tivessem feito TCSF. O estudo foi retrospectivo, mediante análise de prontuários, sendo obtidos dados do período de 2003 a 2013. Asimagens tomográficas de seios paranasais foram pontuadas mediante o escore de LundMackay e descritas. Naqueles pacientes com duas TCSFs, foram feitas avaliações nos dois momentos. Os valores de VEF1e de IMC foram convertidos para escore z. RESULTADOS: Os pacientes com colonização crônica por Pseudomonasaeruginosae aqueles com colapso pulmonar apresentaram maior escore de LundMackay (p= 0,04 e 0,01 respectivamente). Não houve diferença entre o escore de LundMackay entre sintomáticos e assintomáticos do ponto de vista nasossinusal (p= 0,45). Entre os pacientes que apresentavam TCSF em dois momentos, aqueles sem bronquiectasias na TCT (tomografia computadorizada de tórax), tiveram redução no escore de Lund- Mackay, ao contrário daqueles que as apresentavam (p=0,03). Além disso, a variação do escore de Bhalla correlacionou-se positivamente e o escore de Shwachman-Kulczycki, negativamente, com a variação do escore deLundMackay (r = 0,74, p =0,01 e r= -0,85 p<0,01). CONCLUSÕES: Demonstramos haver associação entre achados da TCSF, colonização crônica por Pseudomonasaeruginosa e escore clínico e achados tomográficos de tórax. São necessários estudos que avaliem o seguimento de maior número de pacientes e o benefício da abordagem terapêuticanasossinusal naqueles que apresentem deterioração pulmonar, apesar de controlados os demais fatores associados ao seu agravamento. / INTRODUCTION: Sinonasaldisease in pediatric population with cystic fibrosis (CF) is not a much explored theme, being uncertain the role of sinonasal computed tomography (NCT) in evaluation and follow up of such patients. OBJECTIVES: To evaluate the correlation betweensinonasal symptoms and genotype, Pseudomonas aeruginosachronic colonization, Forced Expiratory Volume at 1.0 second (FEV1), body mass index (BMI), Shwachman-Kulczycki score, Bhalla thoracic tomography score and sinonasal findings in children and adolescents with CF. DESIGN: cross-sectional study. MATERIALS AND METHODS: Sixty one patients between two and 16 years old, who were followed up at the Cystic Fibrosis Pediatric Center at Hospital de Clínicas de Porto Alegre and had undergone NCT were included in the study. Data were obtained retrospectively through chart review, from studies obtained from 2003 to 2013. Tomographic paranasal sinus images were described and graded according with Lund-Mackay score. Patients who had two NCT exams had their data evaluated at both moments. FEV1 and BMI values were converted to z scores. RESULTS: Pseudomonas aeruginosa chronically colonized patients and those with lung collapse on thoracic tomography (TCT) had higher Lund Mackay score (p= 0,04 and 0,01 respectively).There was no difference between Lund Mackay score in sinonasal symptomatic and asymptomaticpatients (p= 0,45). Among patients who had a NCT at two moments those without bronchiectasis in TCT had a reduction in Lund- Mackay score, as compared to those who presented them (p=0,03). Besides, Bhalla score changescorrelated positively and Shwachman-Kulczycki, negatively with Lund- Mackay score variation(r = 0,74, p =0,01 and r= -0,85 p<0,01). CONCLUSIONS: We demonstrated there is an association between NCT, Pseudomonas aeruginosa chronic colonizationand TCT findings and clinical Shwachman score.Studies evaluating follow-up of a greater number of patients and benefits ofsinonasaltherapeuticalapproachesin those who show pulmonary deterioration, despite of other possible worseningfactors,are necessary.

No description available.

A Fibrose Cística, também conhecida como Mucoviscidose, é uma doença crônica de origem autossômica recessiva e até o momento incurável. O presente estudo tem como objetivo compreender a experiência da Fibrose Cística no contexto familiar de crianças portadoras. Os participantes do estudo são crianças com Fibrose Cística em acompanhamento em um hospital-escola do interior do estado de São Paulo e suas famílias. Utilizou-se o método etnográfico, com coleta de dados realizada a partir da observação participante e entrevistas abertas, realizadas em visitas programadas no domicílio e locais significativos para a vida familiar. Como resultados obtivemos que a experiência da doença, como processo subjetivo, interpessoal e contínuo pode ser aproximada à história de vida das crianças, bem como daqueles que compartilham situações de vida e cuidado a esses pacientes. A busca por significados da doença, por suporte social e pela redução do estigma permearam esta experiência nas três dimensões temporais da vida dos participantes. O estudo revela dimensões significativas do viver com FC as quais influenciam a adesão à terapêutica e os processos de socialização. Concluindo, observou-se que a experiência da doença pode se apresentar como abordagem relevante na busca por um cuidado integral. / Cystic Fibrosis, also known as Mucoviscidosis, is a chronic disease of autosomal recessive origin and so far incurable. The present study aimed to understand the experience of Cystic Fibrosis in the familiar context of children carriers. The subjects of the study are children with Cystic Fibrosis under follow-up at a university hospital from an interior city in São Paulo state and their families. It was used the ethnographic method, the data collection was done through participant observation and open interviews, during scheduled visits at the family domicile and significant places for the family life. The results show that the disease experience, as a subjective, interpersonal and continuous process, can be approximated to the childs life history, as well as those who share the patients situations of life and care. The search for the disease meanings, social support and reduction of the stigma permeated this experience in the three temporal dimensions of the participants life. The study reveals significant dimensions of living with CF which influence the treatment adherence and the socialization processes. It was observed that the disease experience can be a relevant approach in the search for a comprehensive care.

A infecção crônica das vias aéreas por Pseudomonas aeruginosa (PA) é a principal causa de morbidade e mortalidade em pacientes com fibrose cística (FC), devido à contínua degradação do tecido pulmonar, que leva ao declínio da função pulmonar, gerada pela infecção e pelo processo inflamatório. O objetivo do presente estudo foi analisar características genéticas de PA que levam à sua adaptação às vias aéreas destes pacientes com infecção pulmonar crônica, atendidos no Centro de Referência em FC do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP e relacionar com resultados de tipagem molecular, resistência a antibióticos, cronicidade e dados clínicos dos pacientes em acompanhamento clínico no período de julho/2011 a abril/2014. As características genéticas dos isolados investigados englobam pesquisa de 18 genes de virulência e genes do sistema quorum sensing (genes lasR e rhlR), associação entre mutações e conversão para fenótipo mucoide (operon algTmucABD) e caracterização de linhagens hipermutantes (genes mutS e mutL). A identificação de P. aeruginosa foi realizada por PCR e MALDI-TOF, que mostraram alta concordância. Foram considerados os dados clínicos dos pacientes: índice de massa corpórea, escore de Shwachman, medidas de capacidade vital forçada e volume expiratório forçado no primeiro segundo. A porcentagem de pacientes com infecção pulmonar crônica por PA observada foi similar aos dados disponíveis na literatura, entretanto, a alta incidência em pacientes jovens foi preocupante. O perfil de macrorrestrição do DNA genômico por PFGE se mostrou útil para definição de colonização crônica/intermitente em associação com critérios clínicos e, juntamente com a detecção de mutações nos genes mucA e mucD confirmaram transmissão interpacientes. Foi observada alta ocorrência dos genes de fatores de virulência pesquisados para grande maioria dos isolados de pacientes crônicos. A resistência aos antibióticos pesquisados dos isolados de P. aeruginosa foi baixa e está de acordo com a literatura nacional e internacional e com a antibioticoterapia adotada no hospital. Não foi observada resistência aos carbapenêmicos e às fluoroquinolonas devido à presença de genes de resistência plasmideais. As mutações no gene mucA foram o principal mecanismo de conversão para o fenótipo mucoide e o fenótipo revertente não-mucoide ocorreu principalmente por mutações no gene algT. Foram detectadas novas mutações nos genes mutS e mutL que também suportam a ideia que hipermutação em PA está associada com mutações do sistema mismatch de reparo do DNA. O sistema quorum sensing dos isolados estudados está parcialmente prejudicado devido às várias mutações no gene lasR, mas todos conservam o gene rhlR intacto, que sustenta alguma atividade quorum sensing envolvida na produção de fatores de virulência importantes. Pacientes com infecção pulmonar crônica por PA com isolamento de outros bacilos gram-negativos não-fermentadores apresentaram maior alteração da função pulmonar quando comparados com pacientes com infecção pulmonar crônica por PA com ou sem isolamento de Staphylococcus aureus. As alterações presentes no operon algTmucABD, quorum sensing e hipermutabilidade contribuem para a cronicidade dos pacientes com FC em relação à infecção por P. aeruginosa. / The chronic airway infection by P. aeruginosa (PA) is the leading cause of morbidity and mortality in cystic fibrosis (CF) patients, due to continuous degradation of the pulmonary tissue. This leads to decline in lung function, which is generated by the related infection and inflammation. The aim of this study was to analyze genetic characteristics associated with the adaptation of PA to the airways of patients with chronic pulmonary infection attended at the CF Reference Center from the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto - USP; and to correlate these findings with the results of molecular typing, antibiotic resistance, chronicity and clinical data of patients in clinical follow-up from July/2011 to April/2014. The genetic characteristics of isolates investigated includes the research of 18 virulence genes and the quorum sensing system genes (lasR and rhlR genes), association between mutations and conversion to the mucoid phenotype (algTmucABD operon), and characterization of hypermutable strains (mutS and mutL genes). Identification of PA was performed by PCR and MALDI-TOF, which showed a high correlation. The patients\' clinical data considered were: body mass index, Shwachman score, forced vital capacity measures and forced expiratory volume in one second. The percentage of patients with chronic PA infection observed was similar to the data available in the literature; however, a worrying high incidence in young patients was noticed. The macrorestriction profile of genomic DNA by PFGE proved to be useful to define chronic/intermittent colonization in association with clinical criteria and it confirmed interpatient transmission, in combination with the detection of mutations in the mucA and mucD genes. High occurrence of virulence genes was detected for the vast majority of isolates from chronic CF patients. Antibiotic resistance for PA isolates was low and is in accordance with national and international literature and antibiotic therapy adopted in the hospital. There was no resistance to carbapenems and fluoroquinolones by the presence of plasmid mediated resistance genes. Mutations in the mucA gene were the main mechanism to conversion to mucoidy, and the non-mucoid revertants occurred mainly by mutations in the algT gene. New mutations in mutS and mutL genes were detected, which support the idea that hypermutation in PA is associated with mutations in the DNA mismatch repair system. The quorum sensing system of the isolates is partially damaged due to several mutations in the lasR gene, but all isolates maintain an intact rhlR gene, which holds some quorum sensing activity with production of important virulence factors. Patients with chronic PA infection with isolation of other non-fermenting gram-negative rods had greater change in lung function compared with patients with chronic PA infection with or without isolation of Staphylococcus aureus. The changes presented in the algTmucABD operon, quorum sensing and hypermutability contribute to the chronicity of CF patients in relation to infection by P. aeruginosa.

A Fibrose Cística (FC) é uma doença genética, até o momento incurável, que junto às demais condições crônicas, exigem cuidados que transcendem a esfera biológica da doença e do cuidado e necessita de estratégias inovadoras para seu manejo. Esse estudo teve por objetivo compreender o itinerário terapêutico de crianças com Fibrose Cística, a partir da perspectiva deles próprios. Para tanto, nos apoiamos no quadro teórico e metodológico conformado pela antropologia médica, etnografia e a hermenêutica interpretativa. Participaram do estudo, 7 crianças entre 8 a 10 anos, em tratamento em um hospital escola do interior do estado de São Paulo e seus familiares. Os dados foram coletados por quatro meses com cada criança partindo-se do referencial da etnografia, mediante observação participante, entrevistas e confecções de materiais que permitiram a participação infantil. Os resultados foram compreendidos em três atos interpretativos que conformam o círculo hermenêutico: As várias facetas da Fibrose Cística; Caminhos e cuidados: avaliações sobre o tratamento e Escolhas, caminhos e cuidados: o itinerário terapêutico de crianças com Fibrose Cística. A partir dos resultados, interpretamos que os modelos explicativos existentes na FC atualmente estão circunscritos em círculos concêntricos dentro dos setores e entre os diferentes atores envolvidos gerando avaliações fragmentadas e diferentes possibilidades de tratamento. As interpretações nos levaram a refletir a respeito da hegemonia do paradigma da saúde pautado no positivismo e suas implicações para o cuidado integral. As possibilidades de mudança aparecem ao considerarmos a etnografia e a hermenêutica enquanto referenciais para a prática bem como a centralidade e a potencialidade da criança como agente da mudança. / Cystic Fibrosis (CF) is a genetic disease which, along with other chronic conditions, calls for care that transcends the biological aspects of the disease and requires innovative strategies for its management. The aim of this study was to understand the therapeutic itinerary of children with cystic fibrosis through their point of view. To achieve this, we were supported by theoretical and methodological framework composed by medical anthropology, ethnography and interpretative hermeneutic. The participants were 7 children between the ages of 8 and 10 who were in treatment for CF at a University Hospital in the state of São Paulo and their relatives. Data were obtained during 4 months with each child using the ethnography method through participant observation, interviews and handmade materials that allowed children to participate. The results were understood in 3 interpretative acts that shaped the hermeneutic circle: Plenty faces of Cystic Fibrosis; Paths and care:treatment evaluations and Choices, paths and care: the therapeutic itinerary of children with Cystic Fibrosis. After analyzing the results, we interpreted that the explanatory models currently available in Cystic Fibrosis are limited in circles in the sectors and between the different actors involved what causes fragmented evaluations and different possibilities of treatment. Those interpretations lead us to reflect the hegemony of the health paradigm that is based on positivism and its implication for integral care. The possibilities of change appear when using the ethnography and the hermeneutic theories in the clinical practice and when considering the centrality and the potentiality of the children as transition agents.

Wang Xiaofei. / "June 2002." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (p. 152-167). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

Both SM and ST reduced ALT elevation in rats in the prevention study. In the treatment study, ALT of all rats was resolved. Only ST reduced the fibrosis in both prevention and treatment studies. Maximum reduction of fibrosis compared to control was 44.12% in the prevention group and 56.83% in the treatment group. Activated HSC was decreased and apoptosis increased in rats with improved fibrosis. / Conclusion. ST prevented formation of liver fibrosis and promoted resolution of established fibrosis in the rat model. These effects were mediated through induction of HSC apoptosis in the liver. (Abstract shortened by UMI.) / Hepatic fibrosis results from the wound healing response to prolonged liver insult such as chronic hepatitis. It represents an imbalance of fibrogenesis and fibrolysis, causing formation of scars. Activation and proliferation of hepatic stellate cells (HSC) is a key to fibrogenesis while apoptosis of HSC is associated with resolution of fibrosis. / Intense efforts are currently underway to evaluate potential anti-fibrotic agents in herbal medicine. The study hypothesized that herbs may resolve hepatic fibrosis through induction of apoptosis of HSC. In this study, the anti-fibrotic potentials of fourteen commonly used herbs were examined. The anti-fibrotic effect and the underlying mechanism of two herbs were further investigated in an animal model. / Method. Fourteen herbs including Angelica sinensis(AS), Astragalus membranaceus(AM), Cordyceps sinensis(CS), Curcuma wenyujin(CW), Carthamus tinctorius(CT), Curcuma kwangsinensis(CK), Bupleurum chinensis(BC), Ligusticum chuanxiong(LC), Paeconia lactiflora(PL), Prunus persiea(PP), Poria cocos(PC), Salvia miltorrhiza(SM), Schisandra chinensis(SC) and Stephania tetrandra(ST) were selected for screening based on documented safety and effectiveness, and availability in commercial extracts. These two herbs were also authenticated by chemical profiling using HPLC. / Result. For in vitro bioassay, five herbs, namely Angelica sinensis (AS), Carthamus tinctorius (CT), Ligusticum chuanxiong(LC), Salvia miltiorrhiza(SM) and Stephania tetrandra(ST) demonstrated both anti-proliferative and pro-apoptotic activities in T6. SM and ST showed highest potencies with 51.63% and 44.52% of T6 cells showing apoptotsis respectively. Fas and Bax expression was up-regulated and BclxL expression decreased in HSC after incubation with SM and ST. Fas ligand and Bcl2 expression remained unchanged. / Treatment of chronic liver disease with herbal medicine has been documented in ancient China. Nowadays, practitioners of traditional Chinese medicine (TCM) also use herbs to treat chronic liver disease and it is conceivable that such herbs redress the imbalance between fibrogenesis and fibrolysis. / Chor Sin Yee. / "July 2005." / Adviser: Joseph J. Y. Sung. / Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0172. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 196-217). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

In summary, the present findings have demonstrated the important roles of CFTR in prostatitis and cancer development, which may provide new insight into the understanding of the prostate in health and disease. The present findings may also have potential application in diagnosis and prognosis of cancer. / In the first part of the study, the possible role and a bacterial killing mechanism involving CFTR-mediated bicarbonate secretion in prostatitis were investigated in a rat prostate model. CFTR was found to be expressed in the epithelium of rat ventral prostate. Experiments using cultured rat primary prostate epithelial cells demonstrated that CFTR was involved in mediating bicarbonate extrusion across the prostate epithelium. The expression of CFTR and carbonic anhydrase II (CAII), a key enzyme involved in cellular HCO 3- production, along with several pro-inflammatory cytokines including IL-6, IL-1beta, TNF-alpha, was significantly up-regulated in the primary culture of rat prostate epithelial cells upon E.coli-LPS challenge. Inhibition of CFTR function in vitro or in vivo resulted in reduced bacterial killing by prostate epithelial cells or the prostate. High HCO3- content (>50mM), rather than alkaline pH, was found to be responsible for bacterial killing. The direct action of HCO 3- on bacterial killing was confirmed by its ability to suppress bacterial initiation factors in E coli. The relevance of the CFTR-mediated HCO3- secretion in human was demonstrated by the upregulated expression of CFTR and CAII in human prostatitis tissues. The present results have demonstrated that CFTR plays a previously undefined role in prostatitis and could be up-regulated during the inflammation in prostate as a host defense mechanism to increase bicarbonate secretion for bacterial killing. / In the second part of the study, the possible role of CFTR in prostate cancer development and the underlying mechanisms were investigated. Our results showed that the expression of CFTR and CAII in prostate was remarkably decreased in aged rat prostate. We observed that testosterone could up-regulate the expression of CFTR and CAII in vitro and in vivo , indicating that the declined male hormones during aging may be responsible for the observed age-dependent expression of CFTR. In the present study, we found that inhibition of CFTR enhanced cell proliferation/anti-apoptosis in the prostate primary epithelial cells. CFTR was detected in all examined prostate cell lines, but with relatively higher expression levels in immortalized cell lines (PZ-HPV-7, PNT1A, PNT2C2) than in cancer cell lines (PC-3, DU-145, LNCaP). Immunohistological studies showed that the expression of CFTR was dramatically reduced in prostate cancer specimens as compared to that in normal prostate tissues. Furthermore, our gain and loss of function studies showed that knockdown of CFTR profoundly enhanced cell proliferation, cell adhesion, invasion and migration, while inhibited apoptosis in prostate cancer cell lines, overexpression of CFTR dramatically suppressed tumorigenic phenotype of cancer cells. Soft agar anchorage-independent growth assay showed that knockdown of CFTR in prostate cancer cells increased the number of colonies formed in soft agar. More importantly, we demonstrated that CFTR knockdown promoted the tumor growth in vivo and forced overexpression of CFTR in prostate cancer cells and ultrasound-mediated gene transfer of CFTR inhibited xenograft tumor growth in vivo. Mechanistically, multiple mechanisms were identified to contribute to the CFTR- mediated tumor suppressive effects. Firstly, CFTR chloride channel function was implicated in the regulation of apoptosis in prostate cancer cells. Secondly, CFTR up-regulated the transcription level of miR-34a and miR-193b, both of which have been indicated as tumor suppressors in multiple cancers. Thirdly, 11 cancer-related genes were found to be up- or down-regulated by CFTR using PCR-array. These data demonstrated that CFTR may play an important role in prostate cancer development by acting as a tumor suppressor. / The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel conducting both Cl- and HCO3 -. It is expressed in epithelial cells of a wide variety of tissues. CFTR is also known to be expressed in human prostate; however, the physiological role of CFTR in the prostate and related diseases remains largely unknown. This thesis explored the biological roles of CFTR in prostatitis and cancer development. / Xie, Chen. / Adviser: Chan LiShaw Chang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 175-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Mutations in the eukaryotic integral membrane protein Cystic Fibrosis Transmembrane conductance Regulator (CFTR) cause the hereditary disease cystic fibrosis (CF). CFTR functions as an ion channel at the surface of epithelial cells and regulates the movement of chloride ions and water across the plasma membrane. CFTR is difficult to express and purify in heterologous systems due to its propensity to form insoluble aggregates and its susceptibility to degradation. Obtaining good yields of highly purified CFTR has proven problematic and contributes to our limited understanding of the structure and function of the protein. The most prevalent disease causing mutation, F508del, results in misfolded CFTR which is particularly unstable and is quickly targeted for degradation by the host system and is prevented from being trafficked to the plasma membrane. There are limited treatment options for patients with the F508del mutation and it is therefore of significant interest within CF research. New methods and assays are required to identify potential compounds which could correct the F508del mutation. This thesis investigates the use of Saccharomyces cerevisiae to express and purify codon optimised recombinant CFTR. The use of a green fluorescent protein (GFP) tag enabled quick and simple detection of CFTR in whole cells and after extraction from the plasma membrane. By optimising the culture conditions for CFTR expression and detergent solubilisation conditions, relatively high yields of full-length protein were obtained. When used as a chemical chaperone at the time of inducing CFTR expression, glycerol increased yields of full-length protein. Degradation of CFTR could be limited by inducing expression at an optimal cell density and by harvesting cells within a specific time window. CFTR was extracted by solubilisation in the mild detergent dodecyl-β-D-maltopyranoside (DDM) in the presence of up to 1 M NaCl with up to ~87% efficiency in some cases. Using a gene optimisation strategy in which additional purification tags and a yeast Kozak-like sequence were added, the human CFTR (hCFTR) protein was expressed and purified. Fluorescence microscopy revealed CFTR localisation at the periphery of yeast cells. Immunoaffinity chromatography facilitated by the GFP tag at the C terminus of CFTR produced protein of up to 95% purity. An assessment of the thermal stability of this highly purified CFTR using a fluorescent probe binding assay revealed a denaturation midpoint (Tm) of ~43 degC. The ability of this assay to determine the stability of CFTR is encouraging and there is the potential to further develop it in a high-throughput manner to identify compounds which stabilise the F508del protein and which may hold the key to developing new treatments for CF.

Background Idiopathic pulmonary fibrosis (IPF) is one of a number of interstitial lung diseases (ILDs) that result in extensive and chronic pulmonary fibrosis. In IPF pathology, immunological dysfunction has been identified as a contributing factor to the ongoing fibrotic process, implicating cells and mechanisms of both the innate and humoral immune response. Due to the complex and diverse range of cells and mediators involved in IPF, the pathology is still poorly understood. Evidence of complement activation through the classical pathway in IPF lungs implies a role for IgG in the pathology. The active IgG in IPF may be autoreactive in nature, as IgG that target antigens of alveolar epithelial cells have been. Two autoantibodies in IPF, anti-periplakin IgG and anti-Hsp72 IgG, have been associated with poorer prognoses in IPF patients. The association of anti-Hsp72 IgG with IPF patient outcomes has not been validated and little work has been done to study the underlying mechanisms of autoantibodies in IPF pathogenesis. Hypothesis Anti-Hsp72 IgG is associated with poorer outcomes in IPF, and may induce alveolar macrophages to exhibit a pro-fibrotic phenotype. Aims The aims were to:  Optimise an ELISA for anti-Hsp72 IgG detection and determine any association of anti-Hsp72 IgG with IPF patient outcomes  Determine the location of anti-Hsp72 IgG producing cells and detect if Hsp72-IgG complexes are present in IPF patients’ lungs  Explore a potential underlying pro-fibrotic mechanism through which anti-Hps72 IgG modulates macrophage function. Results The presence of anti-Hsp72 IgG was determined in ILD patient and healthy control bronchoalveolar lavage fluid (BALf) and serum. A novel anti-Hsp72 IgG ELISA was developed and optimised and then compared against a commercial anti-Hsp72 IgGAM ELISA which became available during the PhD. Progression in IPF was defined by a decrease of ≥10% vital capacity (VC) over twelve months. Serum anti-Hsp72 IgG(AM) did not associate with changes in VC over 12 months. In contrast, BALf anti-Hsp72 IgG(AM) concentrations were elevated in IPF non-progressors. Patients with high BALf anti-Hsp72 IgGAM, had improved survival compared patient with low anti-Hsp72 IgGAM (adjusted HR 0.39, 95% CI 0.16-0.92; p=0.032) In contrast there was no association between anti-Hsp72 IgG and survival. Detection of anti-Hsp72 IgG subtypes in the serum and BALf of IPF patients revealed no significant difference in anti-Hsp72 IgG subtype detection levels between progressors and non-progressors. BALf anti-Hsp72 IgG1 levels were associated with a significantly lower rate of decline in VC over twelve months than patients with no detectable anti-Hsp72 IgG1. The presence of Hsp72-IgG complexes was confirmed by detection in purified IgG from IPF patient BALf. Immuno-histological detection of C4d deposition in the lungs of IPF patients coincided in areas of Hsp72 expression in alveolar epithelium. Summary These findings do not validate serum and-Hsp72 IgG as a biomarker for IPF. They support a role for anti-Hsp72 IgG in IPF, but associate with decreased rates of lung function decline and increased patient survival. Data also suggests that the decreased rate of decline may be related to specific anti-Hsp72 IgG subtype expression. The immune-histological data further suggests that anti-Hsp72 IgG may be targeting Hsp72 expressed by lung epithelium. Therefore these findings support a role for immunological dysfunction in IPF, but further work is required to determine the underlying mechanism.