O Complexo Burkholderia cepacia (CBc) é um grupo de 17 espécies intimamente relacionadas que estão associadas à deterioração pulmonar e aumento da mortalidade em pacientes com Fibrose Cística (FC). Essas espécies variam entre si em relação à prevalência, quadros clínicos e virulência. Pouco é conhecido em relação ao perfil de resistência aos antimicrobianos. Uma vez estabelecida a infecção, a abordagem terapêutica e as medidas de controle atualmente adotadas são baseadas no CBc, sem considerar cada espécie em particular. O objetivo deste estudo foi determinar a prevalência das espécies do CBc em pacientes atendidos em dois centros de referência no Rio de Janeiro, bem como estabelecer perfis de resistência a antimicrobianos e avaliar a diversidade molecular entre as espécies. Cem amostras do CBc isoladas de 38 pacientes com FC no período de janeiro de 2010 a fevereiro de 2012 foram identificadas por métodos fenotípicos e pelo sequenciamento do gene recA. As CIMs para amicacina, aztreonam, ceftazidima, trimetoprim/sulfametoxazol e tobramicina foram determinadas por microdiluição e a genotipagem das espécies foi realizada por PFGE com a enzima SpeI. B. vietnamiensis (44%) foi a espécie mais prevalente, seguida de B. cenocepacia IIIA (36%), B. multivorans (10%), B. cenocepacia IIIB (1%) e B. stabilis (1%). Cinco por cento das amostras não foram identificadas. B. vietnamiensis foi identificada em mais da metade dos pacientes (58,3%). Foram observadas diferenças no perfil de susceptibilidade entre as espécies do CBc. B. cenocepacia IIIA foi a espécie que apresentou as maiores taxas de resistência aos antimicrobianos, sobretudo para trimetoprim/ sulfametoxazol (80,5%), principal antimicrobiano utilizado no tratamento de infecções causadas pelo CBc. Amostras com perfis MDR ocorreram em todas as espécies, destacando-se o perfil A, resistente simultaneamente aos cinco antimicrobianos, observado em 58,8% das amostras de B.cenocepacia IIIA. A análise do polimorfismo genético mostrou que, apesar de B. vietnamiensis ter sido a espécie mais prevalente, a ocorrência de nove grupos clonais sugere que a aquisição dessas cepas tenha se dado a partir de uma fonte ambiental comum. Para B. cenocepacia IIIA, 52,9% das amostras foram atribuídas a um mesmo grupo clonal (BcA), compartilhado entre nove pacientes atendidos em um mesmo centro de referência. Oitenta por cento dessas amostras apresentaram ainda resistência a todos os antimicrobianos testados. Os dados mostram que, mesmo com o emprego de técnicas moleculares, é difícil a identificação do CBc em nível de espécie; que B. cenocepacia IIIA é caracterizada por índices de resistência superiores às outras espécies e que a transmissão cruzada entre os indivíduos aponta para a necessidade do estabelecimento de medidas de vigilância do CBc nos centros de referência. / The Burkholderia cepacia complex (BCC) is a group of 17 closely related species that are associated with pulmonary deterioration and increased mortality in patients with Cystic Fibrosis (CF). These species differ from each other in prevalence, clinical status and virulence. Little is known about the profile of antimicrobial resistance. Once the infection, the therapeutic approach and the control measures currently adopted are based on BcC, without considering each particular species. The aim of this study was to determine the prevalence of BcC species in patients from two reference centers in Rio de Janeiro, as well as establishing antimicrobial resistance profiles and assess the molecular diversity among them. One hundred samples of BcC isolates from 38 CF patients from January 2010 to February 2012 were identified by phenotypic methods and by sequencing the recA gene. The MIC for amikacin, aztreonam, ceftazidime, trimethoprim /sulfamethoxazole and tobramycin were determined by microdilution species and genotyping was carried out by PFGE with the enzyme SpeI. B. vietnamiensis (44%) was the most prevalent species, followed by B. cenocepacia IIIA (36%), B. multivorans (10%), B. cenocepacia IIIB (1%) and B. stabilis (1%). Five percent of the samples were not identified. B. vietnamiensis was identified in over half of patients (58.3%). There were differences in susceptibility profiles among BcC species. B. cenocepacia IIIA showed the highest rates of antimicrobial resistance, particularly to trimethoprim/ sulfamethoxazole (80.5%), primary antimicrobial used to treat infections caused by BcC. Samples with MDR profiles were observed for all species, highlighting the profile A, simultaneously resistant to five antibiotics, observed in 58.8% of B.cenocepacia IIIA samples. The analysis of genetic polymorphism showed that despite B. vietnamiensis was the most prevalent species, the occurrence of nine clonal groups suggests that these strains acquisition has taken place from a common environmental source. For B. cenocepacia IIIA, 52.9% of the samples were assigned to the same clonal group (BcA), shared among nine patients treated at a single referral center. Eighty percent of these samples also showed resistance to all antimicrobials tested. The data show that, even with the use of molecular techniques, the identification of BcC on species level is difficult; that B. cenocepacia IIIA is characterized by higher levels of resistance to other species and that the cross transmission between individuals points to the need for the establishment of BcC surveillance in reference centers.

INTRODUÇÃO: apesar da identificação de mais de 1500 mutações para o gene CFTR (cystic fibrosis transmembrane conductance regulator), o teste do suor ainda é o teste diagnóstico para Fibrose Cística (FC). O teste quantitativo de iontoforese por pilocarpina (TQIP) é o padrão-ouro para coleta do suor e análise do cloro, mas está sujeito a erros se não for realizado por técnicos qualificados. Embora a técnica de coleta do suor pelo sistema macroduct® e análise pela condutividade seja simples e tenha boa correlação com os níveis de cloro em estudos prévios, a mesma ainda é considerada como um teste de triagem para FC. O melhor ponto de corte para confirmar ou afastar a FC pelo método da condutividade deve ser ainda estabelecido. OBJETIVOS: comparar os valores de cloro no suor obtidos pelo teste quantitativo da iontoforese pela pilocarpina (teste clássico) com os valores de condutividade do suor obtido pelo sistema de coleta por macroduct® (teste simplificado) em pacientes com e sem FC e em uma amostra aleatória de pacientes em investigação para FC. O custo e o tempo despendidos na execução de cada teste foram também analisados na fase inicial do estudo. MÉTODOS: o teste do suor, pelas duas técnicas, foi realizado simultaneamente em pacientes com e sem FC e posteriormente em pacientes em investigação diagnóstica da doença no período de fevereiro/2006 a outubro/2008. Os pontos de corte para a condutividade para excluir ou diagnosticar FC foram < 75 e ? 90 mmol/L, respectivamente, e para o teste clássico cloro ? 60 e > 60 mmol/L. Na fase inicial da pesquisa (casos com e sem FC) foram utilizadas tabelas de contingência para os cálculos de sensibilidade (S), especificidade (E), valor preditivo positivo (VPP) e negativo (VPN), além do teste exato de Fisher para avaliar a associação entre os testes e a presença ou ausência de FC. Na amostra aleatória de pacientes usou-se a curva ROC também para os cálculos de S, E, VPP e VPN e também para calcular a área sob a curva entre os testes, e, em ambas as fases da pesquisa, para avaliar sua acurácia. Os respectivos intervalos de confiança de 95% (IC95%) também foram analisados. Para avaliação da concordância entre os testes, na amostra aleatória de pacientes, utilizou-se o coeficiente de kappa e o teste de McNemar. Aplicou-se o teste de Wilcoxon para se comparar os tempos na execução de cada teste, sendo considerados significativos quando p<0,05. RESULTADOS: 52 pacientes com FC (29M/23F; 1,5 a 18,2 anos) realizaram o teste do suor pelas duas técnicas, apresentando valores medianos de cloro e condutividade no suor de 114 e 122 mmol/L, respectivamente. A condutividade foi ? 95 mmol/L em todos os pacientes, conferindo ao teste 100% de sensibilidade (IC95%: 93,1 a 100%). Cinquenta pacientes sem FC (24M/26F; 0,5 a 12,5 anos) apresentaram valores medianos de cloro e condutividade no suor de 15,5 e 30 mmol/L, respectivamente. Em todos os casos a condutividade foi < 70 mmol/L, conferindo ao teste 100% de especificidade (IC95%: 92,9 a 100%). Foram então realizados 918 testes nos pacientes em investigação para FC, mas, em 180, as amostras foram inadequadas. Dos 738 testes realizados pelas duas técnicas, em 714 pacientes se afastou a FC, encontrando-se mediana de cloro de 11 mmol/L (variação: 3 a 137 mmol/L) e de condutividade de 25 mmol/L (variação: 14 a 138 mmol/L). Foram confirmados 24 pacientes com FC, encontrando-se uma mediana de cloro de 87 mmol/L (variação: 54 a 132 mmol/L) e de condutividade de 103 mmol/L (variação: 50 a 126 mmol/L). Pela curva ROC, com valores de condutividade > 90 mmol/L, obteve-se S= 83,3%, E= 99,7%, VPP= 90,9% e VPN= 99,4% para o diagnóstico de FC. Com valores de condutividade < 75 mmol/L praticamente se pôde excluir o diagnóstico de FC (VPN=99,7%; IC95%:99,0-100%). Houve excelente concordância entre o teste clássico e o simplificado, tanto pelo valor de kappa (0,934; IC95% 0,86 a 1,009), quanto pelo teste de McNemar (p=1,0000). O tempo despendido na execução dos testes foi significativamente menor com o teste simplificado (p<0,0001) e o custo do método simplificado foi discretamente inferior. CONCLUSÕES: o teste da condutividade do suor, seja em pacientes com diagnóstico previamente conhecido (com ou sem FC) ou quando realizado aleatoriamente, mostrou resultados superponíveis ao teste clássico e foi capaz de diferenciar pacientes com e sem FC. O teste simplificado apresentou alta sensibilidade e especificidade e houve excelente concordância entre os testes. O tempo de execução foi mais rápido e o custo inferior ao teste clássico. / INTRODUCTION: despite the identification of over 1500 CFTR (cystic fibrosis transmembrane conductance regulator) gene mutations, the sweat test is still the diagnostic test for cystic fibrosis (CF). The quantitative pilocarpine iontophoresis test (QPIT) is the gold-standard method for collection of sweat and chloride analyses, but is subjected to errors if not performed by qualified technicians. Although the technique using the macroduct system for sweat collection and the conductivity analysis is simpler and has good correlation with chloride levels in previous studies, it is still considered a screening test for CF. The best cut-off point of sweat conductivity to confirm or rule out CF must yet be established. OBJECTIVES: to compare the sweat chloride values obtained by the quantitative pilocarpine iontophoresis test (classic test) with sweat conductivity analysis obtained by the macroduct (simplified test) in patients with a confirmed CF diagnosis, in patients without CF and in a random sample of patients being investigated for CF. The cost and time spent to perform each test were also analysed in the initial phase of the study. METHODS: both techniques of sweat test were simultaneously performed initially in patients with CF, afterwards in patients in whom CF had been ruled out and finally in patients referred for a sweat test between February 2006 and October 2008. The cut-off values for sweat conductivity to exclude or diagnose CF were = 90 mmol/L and for the QPIT were sweat chloride ? 60 e > 60 mmol/L, respectively. Contingency tables were used in the initial phase of the study (cases with or without CF) for calculation of sensitivity (Se), specificity (Sp), positive (PPV) and negative predictive value (NPV) and Fisher\'s exact test was used to assess the association between the tests and the presence or absence of CF. ROC curve was used in the random sample of patients also for calculation of Se, Sp, PPV and NPV and also to calculate the area under the curve between both tests in both phases of the study to assess their accuracy. The respective 95% confidence intervals (95%CI) were also analysed. Kappa coefficient and McNemar tests were used for evaluation of agreement between the tests in the random sample of patients. Wilcoxon test was used to compare the time spent to perform each test, with the significant difference set at p < 0.05. RESULTS: in 52 CF patients (29M/23F, age range 1.5 to 18.2y) the median value of sweat Cl and conductivity were 114 and 122 mmol/L, respectively. All patients had sweat conductivity values above 95 mmol/L (100% sensitivity; 95%CI: 93.1 to 100%). In 50 patients without CF (24M/26F, age range 6m to 12.5y) the median value of sweat Cl and conductivity were 15.5 and 30 mmol/L, respectively. All patients had conductivity values bellow 70 mmol/L (100% specificity; 95%CI: 92.9 to 100%). Nine hundred and eighteen tests were then performed in patients being investigated for CF but 180 had inadequate samples. Of the 738 tests performed with both techniques in 714 CF was ruled out, with median values of sweat Cl of 11 mmol/L (range: 3 to 137 mmol/L) and of conductivity of 25 mmol/L (range: 14 to 138 mmol/L). Twenty four patients had a diagnosis of CF presenting a median sweat Cl of 87 mmol/L (range: 54 to 132 mmol/L) and a median conductivity value of 103 mmol/L (range: 50 to 126 mmol/L). The ROC curve showed that with a conductivity value > 90 mmol/L sensitivity of 83.3%, specificity of 99.7%, PPV of 90.9% and NPV of 99.4% was obtained to diagnose CF. The best conductivity cut-off value to exclude CF was < 75 mmol/L (NPV=99.7%; IC95%:99.0-100%). Good agreement were observed between the tests (kappa: 0.934; IC95% 0.86 a 1.009; McNemar test: p=1.0000). The time spent to perform the tests was significantly lower with the simplified test (p<0.0001) and the cost was slightly lower with the conductivity test. CONCLUSIONS: sweat conductivity performed in patients with a known CF or non-CF diagnosis or randomly applied in subjects referred for a sweat test showed similar results as the classic test and could differentiate patients with or without CF. Conductivity test had a high sensitivity and specificity and good agreement was observed between the techniques. The time spent to perform the tests was lower with the simplified test, as well as the cost.

As infecções pneumocócicas são uma importante causa de morbi-mortalidade entre as crianças. Até 2000, era disponível apenas a vacina pneumocócica polissacarídica 23valente, de uso a partir dos 2 anos de idade. Essa vacina era recomendada para crianças com fibrose cística (FC) e para as asmáticas em uso de corticóide oral, dentre outras recomendações. A partir de 2000, licenciou-se a vacina pneumocócica conjugada 7valente, com grande impacto contra infecções causadas pelos sorotipos vacinais. Nos países onde as crianças não são universalmente vacinadas com essa vacina, as recomendações permanecem as mesmas. Atualmente, os adultos asmáticos estão incluídos nas recomendações para vacinação pneumocócica nos EUA. Há poucos estudos sobre o risco de doença pneumocócica em crianças asmáticas per si e naquelas com fibrose cística e sobre a resposta à vacina pneumocócica conjugada. Salienta-se que ainda não há um critério estabelecido para avaliar a resposta sorológica a essa vacina. Recentemente, foi sugerido o critério de 0,35mcg/ml por Elisa para se correlacionar com proteção para doença invasiva pneumocócica. Objetivou-se determinar a concentração dos anticorpos contra os sorotipos vacinais contidos na vacina pneumocócica conjugada 7valente em crianças com asma moderada em uso de corticóide inalatório e em crianças com fibrose cística; avaliando-as pelos critérios de 0,35mcg/ml, 1,3mcg/ml e aumento de 4 vezes o título pós em relação ao pré-vacinal, para cada sorotipo e para a vacina, considerando-se a positividade para 5 sorotipos. Foram avaliadas 18 crianças em cada grupo. A mediana da idade foi de 82,5m nas asmáticas e 69,5m naquelas com FC. Foi colhida amostra para sorologia pré-vacinação e outra após 2 doses da vacina conjugada. As concentrações de anticorpos para os sorotipos vacinais foram quantificadas pelo Elisa. Para 0,35mcg/ml de corte, a grande maioria nos dois grupos já era positiva à inclusão para os sorotipos vacinais e à vacina. Considerando-se o valor de 1,3mcg/ml, entre os que eram negativos, as crianças asmáticas responderam entre 66,7% (9V) e 100% (14), e as com FC, entre 50% (19F) e 100% (6B e 14); e, em relação à resposta vacinal para esse nível, as asmáticas apresentaram 81,8% de resposta, enquanto as com FC, 91,7%. Avaliando-se pelo aumento de 4 vezes o título pós em relação ao pré-vacinal, a melhor resposta aos sorotipos, nos asmáticos, foi de 33,3% (4, 6B, 14 e 18C), e a nos com FC, 61,1% para o 6B; em termos de resposta vacinal, obteve-se 16,7% e 44,4%, para as asmáticas e aquelas com FC, respectivamente. Não houve interferência da vacinação prévia com a vacina pneumocócica polissacarídica. As medianas dos títulos pós em relação aos pré-vacinais, para os sorotipos, nos dois grupos, apresentaram um aumento significante. Apesar de boa parte das crianças apresentarem uma positividade elevada à inclusão, aquelas que eram negativas tenderam a apresentar uma boa resposta à vacina. / Pneumococcal infections are an important morbi-mortality cause among children. Until 2000, it was only available the 23-valent polysaccharide pneumococcal vaccine for children over two years old. This vaccine was recommended for cystic fibrosis (CF) children and to asthmatics children in use of oral corticosteroids, among other recommendations. From 2000, it was licensed the 7-valent conjugated pneumococcal vaccine, with a great impact against the infections caused by the vaccine serotypes. In the countries that dont make a universally use of this vaccine for children, the recommendations remain the same. At the present time, asthmatic adults are included for the pneumococcal vaccine recommendations in the United States. There are few studies about pneumococcal disease risk with cystic fibrosis children and asthmatics, per si, and about the conjugated pneumococcal vaccine response. It points out that there are no a definitive criteria or evaluation established for the serology response for this vaccine. It was suggested, recently, that the level of 0,35mcg/ml, measured by ELISA, is adequate to correlate with the invasive pneumococcal disease protection. The goal of this study was to determine the antibodies concentration of the seven vaccine serotypes from 7-valent conjugated pneumococcal vaccine among children with moderate asthma in use of inhaled corticosteroids and with cystic fibrosis. It was considered the dosage 0,35mcg/ml and 1,3mcg/ml levels and the four-fold increase between pre- and post-immunization concentrations levels, to each serotype and to the vaccine (positivity for five serotypes or more) for positivity. Eighteen children were included in each study group. The age median was 82,5 months for the asthmatics and 69,5 months for the CF children. A blood sample was taken for pre-immunization serology and a second one after the second vaccine dose was given. The antibodies concentrations for the vaccine serotypes were measured by ELISA. Considering the 0,35mcg/ml levels, the majority of children, in both groups, was positive for vaccine serotypes and for the vaccine as well in the beginning. At the 1,3mcg/ml level, among the children with negative serology, asthmatic children responded between 66,7% (9V) and 100% (14), and those with CF, between 50% (19F) and 100% (6B e 14). Related to the vaccine response for this level, the asthmatics had a 81,8% response, while the CF childrens response was 91,7%. Evaluating for the four-fold increase between pre- and post-immunization concentrations, the best response observed for the vaccine serotypes was 33,3% (4, 6B, 14 e 18C) for the asthmatics. In the CF group the best result was 61,1% (6B). In terms of the vaccine response, it was observed that 16,7% and 44,4% were the results for both the asthmatics and CF group, respectively. The polysaccharide vaccine didnt interfere in the results. The medians of the pre- and post-immunization antibodies concentrations for the vaccine serotypes, in both groups, were significantly increased. Despite those children that were already positive for the criteria evaluated, at the first moment of the study, for those children that were negative, the majority had a positive serology towards the vaccination response.

A Fibrose Cística (FC) é uma condição crônica genética que pode acometer diversos órgãos. A complexidade dos aspectos presentes na vida de pessoas com FC desafia pesquisadores e profissionais de saúde a cuidar e avaliar sua saúde de maneira ampla. Pesquisas sobre Qualidade de Vida Relacionada à Saúde (QVRS) se propõem a investigar e avaliar o impacto de enfermidades e seu tratamento na vida das pessoas. O projeto DISABKIDS, desenvolvido simultaneamente em sete países europeus, disponibiliza instrumentos, para mensuração da QVRS de crianças e adolescentes com condições crônicas, dentre estes, um instrumento específico para a FC. Esse estudo metodológico, quantitativo, teve como objetivo realizar a etapa piloto referente a adaptação e validação cultural para o Brasil do DISABKIDS - Cystic Fibrosis Module®. Após a tradução e retrotradução os dados foram coletados junto a crianças e adolescentes de 8 a 18 anos e seus pais ou cuidadores. A coleta de dados foi realizada de janeiro a outubro de 2009, compondo uma amostra composta de 128 participantes em 4 estados do Brasil. Os resultados encontrados foram muito satisfatórios, demonstrando que o instrumento foi bem aceito e compreendido pelos participantes e, dentro os dez itens, apenas um foi adaptado culturalmente. Em relação às propriedades psicométricas iniciais o instrumento apresentou consistência interna aceitável, com valores sempre acima de 0,70 e não maiores que 0,85 para crianças e adolescentes e seus pais ou cuidadores em ambas dimensões, validade convergente muito satisfatória, para as duas dimensões, impacto e tratamento, em ambas as versões self e proxy, com valores de ajuste sempre acima de 75% e concordância substancial entre as respostas self e proxy tanto para a dimensão impacto como para a dimensão tratamento, com valor igual a 0,65.Os resultados encontrados apontam que o DISABKIDS-CFM® poderá se constituir um instrumento válido e confiável, podendo ser inserido na rotina de seguimento de pessoas com FC. / Cystic Fibrosis (CF) is a genetic chronic condition that can affect several organs. The complexity of the issues involved on the lives of people with CF challenges researchers and health professionals to care and evaluate their health broadly. Researches about Health related Quality of Life (HRQoL) are proposed to investigate and evaluate the impact of illness and its treatment on people\'s lives. The DISABKIDS project, developed simultaneously in seven European countries, provides instruments to measure the HRQoL of children and adolescents with chronic conditions, among them, a specific instrument to CF. This quantitative methodological study, aimed to make the pilot phase of cultural adaptation and validation for Brazil DISABKIDS-Cystic Fibrosis Module®. After translation and back translations the data were collected from children and adolescents aged 8 to 18 and their parents or caregivers. Data collection was conducted from January to October 2009, and the sample was 128 participants in 4 Brazil\'s states. The results were very satisfactory, showing that the instrument was well accepted and understood by participants and within the ten items, only one was culturally adapted. The initial psychometric properties of the instrument showed acceptable internal consistency, with values always above 0.70 and no greater than 0.85 for children and adolescents and their parents or caregivers in both dimensions, very satisfactory convergent validity for the two dimensions , impact and treatment, in both versions, self and proxy, with scale fit values always above 75% and substantial agreement between self and proxy answer to both dimensions impact and treatment, with a value of 0.65. The results show that the DISABKIDS-CFM ® could constitute a valid and reliable instrument and can be inserted in the routine follow-up of people with CF.

L'utilisation de virus de bactéries ou bactériophages pourrait être un complément efficace à l’antibiothérapie. Mon travail a porté sur la caractérisation de bactériophages dirigés contre l’espèce Pseudomonas aeruginosa, pathogène opportuniste responsable d'infections des voies respiratoires des patients atteints de mucoviscidose.J'ai tout d'abord déterminé la sensibilité des souches mucoviscidosiques au Pyophage (un cocktail de phages thérapeutiques Géorgien) et identifié six phages lytiques de quatre genres différents. Environ 15% des souches sont résistantes au Pyophage. Ensuite, en utilisant les souches cliniques multi-résistantes aux phages comme bactérie d’enrichissement, 32 phages ont été obtenus à partir des eaux usées de France et Côte d’Ivoire. Tous les phages analysés sont caudés et distribués au sein de dix genres parmi lesquels six exclusivement lytiques. J'ai identifié des souches bactériennes qui demeurent insensibles à tous les phages. J'ai montré que le système CRISPRs-Cas n'est pas associé à la résistance des souches aux phages lytiques. / The use of viruses of bacteria commonly called bacteriophages could constitute an efficient complement to antibiotics. During my PhD, I have characterized phages infecting the opportunistic pathogen Pseudomonas. aeruginosa, responsible for lung infections in cystic fribrosis patients. Firstly, I investigated the efficiency of Pyophage (a cocktail of phages therapeutic Georgian) on clinical P. aeruginosa strains and recovered six lytic phages from four different genus. The Pyophage appears to be unactive on approximately 15% of clinical strains. Secondly, and using multi-phages resistant strains as enrichment bacteria, 32 phages were isolated from waste water of France and Côte d’Ivoire. All phages are tailed and distributed within ten different genus including six exclusively lytic. I identified bacterial strains which remain insensitive to all phages. I also demonstrated that the CRISPRs-cas system plays no role in the resistance of strains to lytic phages.

The purpose of this review was to identify the history of and advances in cystic fibrosis (CF). New treatment plans, medication developments, and a historical perspective of airway clearance therapy (ACT) will be presented. The importance of treatment compliance and time management in the care of cystic fibrosis patients will also be discussed. Furthermore, the development of cystic fibrosis clinics and the pivotal role they play in the treatment of the disease will be addressed. Lastly, a brief discussion concerning the need for and process of lung transplantation will be reported.

Tracheobronchial submucosal glands (SMGs) develop from clusters of epithelial progenitor cells basally orientated within the surface airway epithelium called primordial glandular placodes (PGPs). Signal transduction events that coordinate the transitional process from PGPs into fully developed SMGs consisting of intricately branched networks of tubular secretary structures are still poorly understood. Wnt/β-catenin dependent induction of lymphoid enhancing factor-1 (Lef-1) expression in PGP progenitor/stem cells is required for SMG formation and maturation in the airway. In an effort to better understand the regulatory mechanisms that control Lef-1 during airway SMG development, I have studied its transcriptional regulation. I discovered that Sox2 expression is predominantly confined to the surface airway epithelium (SAE) and is repressed as Lef-1 is induced within PGPs. Deletion of Sox2 in polarized primary airway epithelia significantly enhances Lef-1 mRNA expression. Consequently, my hypothesis is that Sox2 functions as a negative regulator of Lef-1 expression in the SAE. I demonstrated that Sox2 modulates the expression of Lef-1 both independent and dependent on Wnt/β-catenin signaling. I discovered that a Sox2-binding site located in the Wnt Responsive Element (WRE) region of the 2.5Kb Lef-1 promoter is required for Sox2-mediated inhibition of β-catenin-dependent Lef-1 promoter transcription. It is important to understand the biology of SMG development because SMGs are the major mucus-producing structures in the proximal airway and are important in regulating the innate immunity of the lung in response to various neural signals. SMG ducts have also been proposed as a potential protective niche for slowly cycling progenitor cells (SCPCs). Hence, aberrant SMG function is thought to aggravate the pathoprogression of lung disease. Cystic fibrosis (CF) is a disease caused by a defect in the gene that encodes a chloride ion channel called cystic fibrosis transmembrane conductance regulator (CFTR). The absence of CFTR in serous cells within SMG ducts contributes to defective airway secretion, which alters the microenvironment within SMGs. I hypothesized that the glandular SCPC niche may be dysfunctional in CF. I reported that the neural peptide, calcitonin gene-related peptide (CGRP) activates CFTR-dependent SMG secretions and that this signaling pathway is hyperactivated in CF human, pig, ferret, and mouse SMGs. CFTR-deficient mice failed to maintain glandular SCPCs following airway injury, suggesting that the glandular SCPC niche may be dysfunctional in CF. CGRP levels increase following airway injury and function as an injury-inducible mitogen that stimulates progenitor cell proliferation. However, components of the receptor for CGRP (RAMP1 and CLR) were expressed in a very small subset of SCPCs, suggesting that CGRP indirectly stimulates SCPC proliferation through paracrine mechanisms. This discovery may have important implications for injury/repair mechanisms in the CF airway.

Cystic fibrosis (CF) is the most common autosomal recessive genetic disease in Caucasian populations. CF affects multiple organ systems including pancreas, liver, intestines, sweat glands, and male reproductive organs, however the leading cause of morbidity and mortality in CF patients is chronic lung disease. CF is caused by a mutant cystic fibrosis transmembrane conductance regulator (CFTR) gene which leads to chloride (Cl-) and bicarbonate (HCO3-) anion dysregulation at the airway surface. Without adequate anion exchange, thick, viscous mucus accumulates at the airway surface allowing bacterial colonization to occur. Complementing CFTR in the appropriate airway cells restores the anion channel activity in CFTR-deficient cells. The ultimate goal for CF gene therapy is to design an integrating vector that would lead to persistent and efficient expression of CFTR in the airways. Performing gene therapy experiments is dependent upon a relevant animal model. The CF pig is a large animal model similar in size, anatomy, and physiology to humans. Importantly, the CF pig recapitulates human lung disease. From the CF pig, we have learned much about CF lung disease and have developed relevant assays to measure anion channel correction. We have learned that loss of CFTR leads to a decreased airway surface ASL pH, bacterial killing ability, and increased mucus viscosity. Standardized assays have been developed to evaluate the change in current by Ussing chambers, ASL pH, bacterial killing in vivo and ASL pH and viscosity on primary airway cultures in vitro. Ultimately, these metrics allow us to make conclusions about the efficiency of CFTR restoration. Viral vectors are promising candidates for CF gene therapy. Viral vectors such as adenovirus (Ad), adeno-associated virus (AAV), and pseudotyped lentiviral vectors such as feline immunodeficiency virus (FIV) or human immunodeficiency virus (HIV) can efficiently transduce airway cells and express CFTR. Ad and AAV have both been tested in CF clinical trials, but CFTR expression was transient, if detected at all. Understanding vector biology and overcoming barriers in the lung have allowed us to improve vector delivery to the airways. However, the next major hurdle was achieving persistent expression. Ad and AAV are both transiently expressing vectors, and vector readministration is implausible due to the presence of neutralizing antibodies that develop against the vector. Creating a hybrid nonviral/viral vector in which the integrating nonviral piggyBac transposon system is delivered by an Ad or AAV vector has allowed us to achieve persistent expression in mice. In a third integrating vector system, lentiviral vectors have historically been challenging to work with due to low titer levels. However, improvement in vector purification methods have allowed us to validate a lentiviral vector as a viable gene therapy option. In total, we have validated three integrating vector systems by restoring CFTR to CF pigs to correct the phenotypic defect.

Alterations to sphingolipid metabolism are associated with increased pulmonary inflammation, but the impact of inflammatory mediators, such as neutrophil elastase (NE), on airway sphingolipid homeostasis remains unknown. NE is a protease associated CF lung disease progression, and can be found in up to micromolar concentrations in patient airways. While sphingolipids have been investigated in the context of CF, the focus has been on loss of cystic fibrosis transmembrane conductance regulator (CFTR) function. Here, we present a novel observation: oropharyngeal aspiration of NE increases airway ceramides in mice. Using a previously characterized mouse model of NE-induced inflammation, we demonstrate that NE increases de novo ceramide production, which is likely mediated via increased SPTLC2 levels. Inhibition of de novo sphingolipid synthesis using myriocin, an SPT inhibitor, decreases airway ceramide as well as the release of pro-inflammatory signaling molecules induced by NE. Furthermore, in a retrospective study of the sphingolipid content of CF sputum—the largest of its type in this patient cohort to date, we investigated the association between NE and sphingolipids. There were linear correlations between the concentration of active NE and ceramide, sphingomyelin, and monohexosylceramide moieties as well as sphingosine-1-phosphate. The presence of Methicillin-resistant Staphylococcus aureus (MRSA) positive culture and female gender both strengthened the association of NE and sphingolipids, but higher FEV1 % predicted weakened the association, and Pseudomonas aeruginosa had no effect on the association between NE and sphingolipids. These data suggest that NE may increase sphingolipids in CF airways as it did in our in vivo model, and that this association is stronger in patients that have worse lung function, are female, and whose lungs are colonized with MRSA. Modulating sphingolipid homeostasis could provide novel pharmacological approaches for alleviating pulmonary inflammation.

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR). CF affects multiple organ systems, but the major cause of morbidity and mortality is due to disease in the lungs. In theory, using gene therapy to deliver a correct copy of CFTR to the cells of the airway epithelium could result in a lifelong cure. Adeno-associated virus (AAV) is a single stranded DNA virus that is a promising candidate vector for gene therapy of multiple diseases, and numerous clinical trials are currently underway. Despite recent clinical successes, several challenges still impede wider application of AAV gene therapy to numerous diseases, including CF, as AAV-mediated gene transfer to the airways remains below the level needed for therapeutic efficacy for CF. We hypothesized that the low transduction efficiency of AAV in the airways could be overcome by using directed evolution of AAV in organotypic human and pig airway models, and in vivo in the lungs of pigs to select novel AAV capsid variants with improved infectious properties. We discovered a highly infectious, novel AAV that was a chimera of AAV2 and AAV5 with one point mutation (A581T) which we called AAV2.5T. We found that AAV2.5T mediated gene transfer significantly better than its parental serotypes, and corrected the chloride transport defect in CF human airway epithelial cultures. We determined that AAV2.5T developed increased binding to the apical surface of human airway epithelial cells, and that it has evolved to utilize specific 2,3N-linked sialic acid residues on the cell surface that mediate rapid internalization and subsequent infection. Thus, sialic acid serves as not just an attachment factor but is also required for AAV2.5T internalization, possibly representing an important rate-limiting step for other viruses that use sialic acids. Additionally, we utilized directed evolution in vivo in the lungs of pigs to select a novel AAV capsid that is identical to AAV2 except for five point mutations, which we called AAV2H22. We found that AAV2H22 mediated gene transfer to pig airway epithelial cultures significantly better than AAV2, and that it had evolved altered receptor binding. We also found that directed evolution in vitro in human and pig airway epithelial cultures results in the selection of distinct viruses for the two species, and that maintaining different selection stringencies results in the recovery of different AAV variants. Finally, we utilized Hoechst 33342, a DNA binding compound which was previously found to increase AAV transduction in cell lines, to increase AAV-mediated gene expression in primary human airway epithelia. We determined that the mechanism of this effect was due to activation of the CMV promoter. The findings from this research have significant implications for our understanding of AAV biology and for pulmonary gene therapy.