環氧合酶-2(COX-2)是在花生四烯酸(AA)轉化為前列腺素H₂(PGH₂)的過程中最重要的限速酶，PGH2再進一步被合成為各種前列腺素，包括前列腺素E₂(PGE₂), 因此，COX-2在前列腺素的合成中起著舉足輕重的作用。COX-2在受到例如感染和炎症等刺激的情況下被誘導，迅速大量地產生。越來越多的證據證明瞭COX-2在許多細胞反應和病理生理過程中起重要作用, 其中, 對COX-2在炎症中的作用研究最深入。 / 囊性纖維化病(CF)是一種由於編碼囊性纖維化跨膜轉導調節器(CFTR)基因的突變所引起的常染色體隱性遺傳疾病。CFTR是在上皮細胞中廣泛表達的環磷酸腺苷(cAMP)依賴的陰離子通道。愈來愈多的證據顯示, CF的呼吸道上皮處於過量炎症因子和前列腺素的微環境中, 最終導致了在CF肺部病變中觀察到的超炎症反應. 但其中的機制仍未闡明. 本研究觀察到, 相對於野生型人類支氣管上皮細胞系(16HBE14o-), CF的人類支氣管上皮細胞系(CFBE41o-)中NFκB的活化, COX-2的表達和PGE₂的產量增加. 此外, CFTR基因敲除小鼠顯示出升高的NFκB活性和COX-2表達水準, 提示CFTR基因的缺失介導了超炎症反應的信號. 我們還驗證了一條PKA和CREB參與介導的PGE₂產生的正回饋通路. 更重要的是, 在CFBE41o-細胞中過表達CFTR顯著地抑制了COX-2的表達. 用LPS或者PGE₂處理16HBE14o-細胞導致了野生型CFTR表達的顯著升高. 這些實驗結果提示了CFTR可能參與對COX-2/PGE₂的負調節. 因此, CFTR負調節PGE₂介導的炎症反應. 這個調節機制的缺陷可能導致在CF炎症反應的組織中觀察到的過量的NFκB活化和過量PGE₂產生. / 我們證實了睾丸中也存在這條CFTR負調節COX-2/PGE₂的通路. 由於隱睾處於比陰囊溫度高的腹腔中, 在隱睾中, 我們觀察到了高溫導致的CFTR下調,伴隨著COX-2的上調以及緊密連接蛋白(ZO-1, occludin)的下調. 這種CFTR和COX-2的負相關在小鼠睾丸高熱動物模型以及CFTR基因敲除小鼠模型中也被證實. 為了模擬隱睾的病理狀況, 我們提高原代睾丸支援細胞的培養溫度至37°C. 與在32°C培養條件下的對照細胞相比, 37C培養的支持細胞中CFTR表達顯著下調, 而COX-2表達顯著上調. 用CFTR的抑制劑CFTRinh-172處理支持細胞48小時後, COX-2的表達也上升了. 抑制或者敲除支持細胞中的CFTR都引起了ZO-1和occludin表達水準的下降, 從而損傷了支持細胞間的緊密連接. NFκB或者PGE₂的抑制劑都能逆轉ZO-1和occludin表達水準的下降. PGE₂同樣導致了支援細胞間緊密連接的損傷. 以上結果提示CFTR對緊密連接的調節作用是通過NFκB/COX-2/PGE₂通路實現的. 本研究闡明了在支持細胞中, CFTR通過負調節NFκB/COX-2/PGE₂通路調節緊密連接, 從而參與了隱睾導致的生精障礙的病理過程. / 總之, 本研究論證了CFTR/COX-2/PGE₂通路在CF呼吸道的超炎症反應以及隱睾導致的生精障礙兩個病理過程中的作用, 說明了CFTR在呼吸系統和男性生殖系統中維持細胞因子穩態的重要作用. CF肺中CFTR的缺失或者隱睾病中CFTR表達水準的下降可能導致了呼吸道中過剩炎症反應和生精障礙. / Cyclooxygenase-2 (COX-2) is a pivotal rate-limiting enzyme responsible for the production of prostaglandins by converting arachidonic acid (AA) to prostaglandin H₂ (PGH₂), which is further metabolized to various prostaglandins, including PGE₂. COX-2 is inducible and increases dramatically upon stimulation, such as infection and inflammation. Accumulating evidences have demonstrated the important role of COX-2 in many cellular responses and pathophysiological processes, especially inflammation. / Cystic Fibrosis (CF) is an autosomal recessive disorder caused by mutations of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), a cAMP-dependent anion channel expressed in many epithelia. Accumulating evidence suggests that CF airway epithelia are overwhelmed by excessive inflammatory cytokines and prostaglandins (PGs), which eventually lead to the over-inflammatory condition observed in CF lung disease. However, the exact underlying mechanism remains elusive. In this study, we observed increased COX-2 expression and over-production of prostaglandin E₂ (PGE₂) in human CF bronchial epithelial cell line (CFBE41o-) with elevated NFκB activity compared to a wild-type bronchial epithelial cell line (16HBE14o-). Moreover, we demonstrated that CFTR knockout mice had inherently higher levels of COX-2 and NFκB activity, supporting the notion that lack of CFTR results in hyper-inflammatory signaling. In addition, we identified a positive feedback loop for production of PGE₂ involving PKA and transcription factor, CREB. More importantly, overexpression of wild-type CFTR significantly suppressed COX-2 expression in CFBE41o- cells, and wild-type CFTR protein expression was significantly increased when 16HBE14o- cells were challenged with LPS as well as PGE₂, indicating possible involvement of CFTR in the negative regulation of COX-2/PGE₂. These results suggest that CFTR is a negative regulator of PGE₂-mediated inflammatory response, defect of which may result in excessive activation of NFκB, leading to over production of PGE2 as seen in inflammatory CF tissues. / This negative regulation of COX-2/PGE₂ pathway by CFTR was also identified in the testis in the present study. Downregulation of CFTR accompanied by upregulation of COX-2/PGE₂ and downregulation of tight junction proteins, including ZO-1 and occludin, were observed in a cryptorchidism mouse model with elevated testis in the abdomen, at which the temperature is several degrees higher than that in the scrotum. The inverse correlation of CFTR and COX-2 was further confirmed in a mouse testis hyperthermia model and in CF mice. Culturing primary Sertoli cells at a temperature of 37°C, which mimics the pathological condition of cryptorchidism, led to a significant decrease in CFTR and increase in COX-2 expression compared to the physiological condition of 32°C. Increase of COX-2 expression was also detected 48 hours after administrating CFTRinh-172 to the cells. Inhibition or knockdown of CFTR led to decreased ZO-1 and occludin expression and impaired tight junction in Sertoli cells, which could be mimicked by PGE₂, but reversed by NFκB and COX-2 inhibitors, suggesting that regulation of tight junction by CFTR is mediated by NFκB /COX-2/PGE₂ pathway. This study illustrates that CFTR may be involved in regulating testicular tight junctions through its negative regulation of NFκB/COX-2/PGE₂ pathway in Sertoli cells, defect of which may result in spermatogenesis defect in cryptorchidism. / Taken together, the present study has demonstrated the role of CFTR/ NFκB /COX-2/PGE₂ pathway in two pathological processes, exaggerated inflammation in CF airway and defective spermatogenesis in cryptorchidism, indicating that CFTR is critical for maintaining cytokine homeostasis in respiratory system and male reproductive system. Defect of CFTR in CF lung and downregulation of CFTR in cryptorchidism may contribute to the excessive lung inflammation and impaired spermatogenesis respectively. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Jing. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 109-121). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENT --- p.vi / LIST OF PUBLICATIONS --- p.vii / ABBREVIATIONS --- p.xii / LIST OF FIGURES AND TABLES --- p.xvi / Chapter 1 --- Chpter 1: Overview --- p.1 / Chapter 1.1 --- CFTR and Cystic Fibrosis --- p.1 / Chapter 1.1.1 --- Cystic Fibrosis --- p.1 / Chapter 1.1.2 --- Structure of CFTR --- p.2 / Chapter 1.1.3 --- Mutations of CFTR --- p.2 / Chapter 1.1.4 --- Channel and signal transduction function of CFTR --- p.3 / Chapter 1.1.5 --- Interaction of CFTR with other proteins --- p.4 / Chapter 1.1.6 --- Regulation of CFTR --- p.5 / Chapter 1.2 --- COX-2 and PGE₂ --- p.6 / Chapter 1.2.1 --- Biosynthesis of PGE₂ --- p.6 / Chapter 1.2.2 --- Pathophysiologic roles of COX-2 and PGE₂ --- p.7 / Chapter 1.2.3 --- Role of COX-2/PGE₂ in inflammation --- p.7 / Chapter 1.2.4 --- Regulation of COX-2 --- p.8 / Chapter 1.2.4.1 --- Regulation of COX-2 by NF-κB --- p.9 / Chapter 1.2.4.2 --- Regulation of COX-2 by CREB --- p.10 / Chapter 1.3 --- Link between CFTR and NF-κB --- p.11 / Chapter 1.4 --- General hypothesis and aims of study --- p.12 / Chapter 2 --- Chapter 2: CFTR negatively regulates COX-2/PGE₂ positive loop in feedback loop in inflammation --- p.13 / Chapter 2.1 --- Introduction --- p.13 / Chapter 2.1.1 --- Airway inflammation in Cystic Fibrosis --- p.13 / Chapter 2.1.2 --- Current theories on the causes of pulmonary inflammation in CF --- p.13 / Chapter 2.1.2.1 --- Theory one --- p.14 / Chapter 2.1.2.2 --- Theory two --- p.16 / Chapter 2.1.3 --- Role of airway epithelia in CF airway inflammation --- p.16 / Chapter 2.1.4 --- Link between CFTR and NF-κB in pulmonary inflammation --- p.17 / Chapter 2.1.5 --- Link between CFTR and COX-2/PGE₂ in pulmonary inflammation --- p.18 / Chapter 2.1.6 --- Hypothesis and aims of study --- p.18 / Chapter 2.2 --- Materials and methods --- p.20 / Chapter 2.2.1 --- Cell culture materials --- p.20 / Chapter 2.2.2 --- Animals --- p.20 / Chapter 2.2.3 --- Chemicals, drugs and assay kits --- p.20 / Chapter 2.2.4 --- Antibodies --- p.22 / Chapter 2.2.5 --- Cell culture. --- p.22 / Chapter 2.2.6 --- Animal models and procedures --- p.23 / Chapter 2.2.7 --- Manipulation of RNA and QRT-PCR --- p.23 / Chapter 2.2.8 --- Manipulation of protein and Western blot --- p.25 / Chapter 2.2.9 --- Histological and morphological --- p.27 / Chapter 2.2.9.1 --- Tissue section. --- p.28 / Chapter 2.2.9.2 --- Hematoxylin and eosin staining --- p.28 / Chapter 2.2.9.3 --- Immunohistochemistry --- p.28 / Chapter 2.2.10 --- PGE₂ EIA --- p.29 / Chapter 2.2.11 --- Statistical analysis --- p.30 / Chapter 2.3 --- Results --- p.30 / Chapter 2.3.1 --- Increased expression of NF-κB and COX-2 in the lung of CF mice --- p.31 / Chapter 2.3.2 --- Defect of CFTR leads to increased COX-2 expression in CF cell line --- p.31 / Chapter 2.3.3 --- Increased expression of COX-2 in CF cells is attributed to NF-κB activation --- p.33 / Chapter 2.3.4 --- A positive feedback loop from PGE₂ to COX-2 is mediated by PGE₂/cAMP/PKA/p-CREB pathway --- p.34 / Chapter 2.3.5 --- PGE₂ increase the expression of CFTR protein in 16HBE14o- but not in CFBE41o- cells --- p.35 / Chapter 2.4 --- Discussion --- p.47 / Chapter 2.5 --- Conclusion --- p.51 / Chapter 3 --- Chapter 3: Role of CFTR/COX-2/PGE₂ Pathway in the Regulation of Junctional Complex Proteins in Sertoli Cells and its Implication in Spermatogenesis Defect in Cryptorchidism --- p.53 / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.1.1 --- Spermatogenesis.p53 / Chapter 3.1.1.1 --- Structure of the seminiferous tubules --- p.53 / Chapter 3.1.1.2 --- Role of Sertoli cells in spermatogenesis --- p.55 / Chapter 3.1.1.3 --- Role of junctional complexes in spermatogenesis --- p.55 / Chapter 3.1.2 --- Junctional complexes in the testis --- p.59 / Chapter 3.1.2.1 --- Tight Junction --- p.59 / Chapter 3.1.2.2 --- Anchoring Junction. --- p.60 / Chapter 3.1.2.3 --- Cross talk between TJs and AJs --- p.60 / Chapter 3.1.3 --- Cryptorchidism --- p.61 / Chapter 3.1.3.1 --- Causes and consequences of Cryptorchidism --- p.61 / Chapter 3.1.3.2 --- Elevated temperature caused by cryptorchidism greatly contributes to defective spermatogenesis --- p.62 / Chapter 3.1.3.3 --- Changes of Sertoli cells in cryptorchidim contributing to defective spermatogenesis. --- p.62 / Chapter 3.1.3.4 --- Disruption of junctional complexes in heat shock and cryptorchidism. --- p.65 / Chapter 3.1.4 --- CFTR and spermatogenesis --- p.66 / Chapter 3.1.4.1 --- Expression of CFTR in Sertoli cells in testis --- p.66 / Chapter 3.1.4.2 --- Temperature sensitive processing of CFTR protein --- p.66 / Chapter 3.1.4.3 --- CFTR and junctional complex --- p.67 / Chapter 3.1.4.4 --- CFTR and male reproduction --- p.68 / Chapter 3.1.4.5 --- Role of CFTR in spermatogenesis --- p.68 / Chapter 3.1.5 --- Prostaglandins and male fertility --- p.69 / Chapter 3.1.5.1 --- Expression of COX-2 in testis. --- p.69 / Chapter 3.1.5.2 --- Role of prostaglandins in spermatogenesis --- p.70 / Chapter 3.1.5.3 --- Regulation of junctional complexes by PGE₂ --- p.70 / Chapter 3.1.5.4 --- Prostaglandins in cryptorchidism --- p.72 / Chapter 3.1.6 --- Hypothesis and aims of study --- p.73 / Chapter 3.2 --- Materials and Methods --- p.74 / Chapter 3.2.1 --- Cell culture materials --- p.74 / Chapter 3.2.2 --- Drugs and Reagents --- p.74 / Chapter 3.2.3 --- Antibodies --- p.74 / Chapter 3.2.4 --- Animals --- p.75 / Chapter 3.2.4.1 --- Mice artificial cryptorchidism model --- p.75 / Chapter 3.2.4.2 --- Mice testes hyperthermia model --- p.75 / Chapter 3.2.5 --- Sertoli cell primary culture --- p.76 / Chapter 3.2.6 --- siRNA against CFTR and transfection --- p.76 / Chapter 3.2.7 --- Examination of assembly and destruction of assembly of inter-Sertoli TJs --- p.77 / Chapter 3.2.8 --- Manipulation of RNA and Real-Time Quantitative RT-PCR (QRT-PCR) --- p.77 / Chapter 3.2.9 --- Manipulation of protein and western blot --- p.77 / Chapter 3.2.10 --- Histological and morphological studies --- p.78 / Chapter 3.2.10.1 --- Immunofluorescence of ZO-1 Staining in Sertoli cells --- p.78 / Chapter 3.2.10.2 --- Immunofluorescent staining of ZO-1, Occludin and β-Catenin in testes --- p.78 / Chapter 3.2.11 --- PGE₂ EIA --- p.79 / Chapter 3.2.12 --- Statistical Analysis --- p.79 / Chapter 3.3 --- Results --- p.79 / Chapter 3.3.1 --- Downregulation of CFTR is associated with upregulation of COX-2 in mice cryptorchidism model, mice testes hyperthermia model, and CF mice testes --- p.79 / Chapter 3.3.2 --- Negative regulation of COX-2 by CFTR is mediated by NF-κB --- p.81 / Chapter 3.3.3 --- Decreased tight junction proteins expression and increased anchoring junction proteins expression in cryptorchid testes. --- p.81 / Chapter 3.3.4 --- Elevation of culture temperature results in downregulation of CFTR and upregulation of COX-2 in primary cultured rat sertoli cells --- p.82 / Chapter 3.3.5 --- Defect of functional CFTR leads to increased COX-2 expression. --- p.83 / Chapter 3.3.6 --- CFTR regulates TJ protein expression and TJ formation through NF-κB/COX-2/PGE₂. --- p.83 / Chapter 3.4 --- Discussion --- p.100 / Chapter 3.5 --- Conclusion --- p.104 / Chapter 4 --- Chapter 4: General Discussion --- p.105 / Chapter 4.1 --- The immunosuppressive function of PGE₂ in CF lung disease and cryptorchidism-induced infertility. --- p.105 / Chapter 4.2 --- Importance of CFTR/ NF-κB /COX-2/PGE₂ pathway in inflammation-based diseases. --- p.106 / Chapter 4.3 --- Possible implications of CFTR/NF-κB /COX-2/PGE₂ pathway in cancer --- p.107 / Chapter 4.4 --- Concluding remarks --- p.108

Master of Science / Biochemistry and Molecular Biophysics / John M. Tomich / NC-1007 (CK₄-M2GlyR) (PARVGLGITTVLTMTTQSSGSRAKKKK) is a synthetic peptide modeled after the second transmembrane segment of the spinal cord glycine receptor’s α-subunit, and has demonstrates the capacity to oligomerize to form transmembrane channels with Cl[superscript]- permselectivity. While studies into the effects of truncation on both CK[subcript]4 (C-terminal tetra-lysl adducted) and NK[subscript]4 (N-terminal tetra-lysl adducted) led to more control over solution aggregation in the NK[subscript]4 variant, the work presented explore whether C-terminal sequential substitutions with a tryptophan residue could similarly stabilize the aqueous structure in monomeric form or further define the pore registry in such a way as to promote an increase ion permeability. Tryptophan was substituted for amino acids in the 18[superscript]th, 19[superscript]th, 20[superscript]th, and 21[superscript]st positions of the peptide sequence (SSGS, respectively), and changes in aggregation profiles, secondary structure, and channel ion permeability were observed. Synthesized peptides show circular dichroism spectral profiles indicating that the studied tryptophan substitutions did not result in a reduction of the characteristic helicity of the peptide; however, the tryptophan substitution also did little to decrease solution aggregation as demonstrated by comparative studies by reverse-phase high- performance liquid chromatography. All peptides demonstrated channel activity, directly measured by recordings of transepithelial short-circuit current. with profiles that suggest trends in electrostatic interactions and membrane registry relative to substitution position. One peptide in particular, NC-1007 S21W displayed atypical activity, which could not be effectively described by the standard Hill-based model but may be indicative of an ill-defined registry due to the substituted peptide’s proximity to another strongly pore-defining residue. Further studies in the effects of sequence modification to channel-forming peptides will elucidate how sequences may be altered to optimize synthetic peptide solubility, resistance to in-solution aggregation, and ability to form selective and permeable ion channels. The understanding gained from this study will improve our ability to develop peptides that could serve as a therapeutic treatments for a number of endogenous channelopathies.

Clinical research in cystic fibrosis (CF) requires study endpoints that are sensitive to airways disease, repeatable and non-invasive. Despite significant advances in the treatment of CF, lung function assessments continue to rely on the forced expiratory volume in 1 second (FEV1). Although simple to perform, it lacks sensitivity, is difficult for younger subjects, and changes over time. An alternative method of assessing lung physiology is to derive measures of ventilation heterogeneity from inert gas washout tests. In early lung disease, measures of gas mixing appear to be more sensitive than spirometry. In addition, since only tidal breathing is required, they are more physiological and are more straightforward for younger subjects. Widespread use has been impaired by the lack of a robust and cost effective gas analyser technology. The work presented in this thesis concerns the adaptation, validation and then use of a novel gas analyser (Innocor) in a clinical system for the performance of multiple breath washouts. Lung clearance index (LCI), a simple measure of ventilation heterogeneity, has been calculated from washouts in 52 adults with CF and 50 healthy controls. LCI was more sensitive to disease than FEV1 in CF, being elevated in 11 of the 12 CF patients with normal spirometry. In healthy subjects, LCI has been shown to be repeatable and reproducible, with a narrow range of normal that is stable over a wide age range. In a separate study of 19 patients, LCI has also been shown to improve with treatment of an exacerbation in CF. Correlation with changes in other biochemical (serum CRP, peripheral blood white cell count, sputum IL-8, sputum neutrophil) clinical (symptom score) or structural (computed tomography) markers was poor. Short term change in LCI has also been demonstrated in CF patients in response to chest physiotherapy, although there was considerable heterogeneity of response in terms of both LCI and volume of lung ventilated by tidal breathing (as measured by washout functional residual capacity). In addition to LCI, multiple breath phase III slope analysis has been performed on washouts of CF patients and healthy controls, and this has been compared to other measures of lung physiology. Proposed measures of convective and diffusive gas mixing have been shown to be unreliable in CF. These studies have also been the first to demonstrate multi-centre use of washout tests as endpoints. The technology described here offers the possibility of a simple and reliable system for performing multiple breath washouts, though at present it is not available commercially. The studies have added to the understanding of the utility and reliability of washout tests, as well as some of their limitations. It is hoped that in future LCI will be an important clinical endpoint in therapeutic intervention studies in CF, and that it will also offer new ways to follow changes in lung physiology in other diseases.

Cystic fibrosis (CF) lung disease is characterised by early airways infection and inflammation, chronic suppuration, frequent infective exacerbations and an increased influx of acute, and chronic inflammatory cells. The inflammatory process involves activation of many cell types including neutrophils, macrophages and epithelial cells, and leads ultimately to the development of progressive respiratory failure and death. Accurate assessment of the inflammatory process is a crucial part of disease monitoring and should allow appropriate evaluation of therapeutic interventions so as to maximize control of the respiratory sequelae of the disorder. Lung function markers such as FEV1 are insensitive and indirect. Direct but invasive methods such as fibreoptic bronchoscopy and biopsy are limited in application, repeatability and safety. Non-invasive methods of assessment are, therefore, attractive. Exhaled Breath Gases, Exhaled Breath Condensate and Induced Sputum provide potential for such measures. These techniques are safe, simple, repeatable and could assess all airways and can be used in children as young as 6 years. We hypothesised that biomarkers of inflammation in Cystic Fibrosis Lung Disease are measurable in samples collected noninvasively, and can be developed into clinically useful assays. These assays would have the ability to reflect the level of inflammation in the CF lungs as well as holding the potential to act as surrogate markers of CFTR function. Methods Non-invasive markers of inflammation in Cystic Fibrosis lung disease Methods. Exhaled breath gases, exhaled breath condensate, bronchoalveolar lavage fluid and induced sputum were investigated using a number of analysis techniques to identify the markers which best discriminated CF from non CF subjects. Analysis techniques used were electrochemical cells, chemiluminescene, ELISA, EIA, ion selective probes and mass spectrometry. Results Markers found to discriminate CF from non CF subjects were EBC pH and ammonium, and 38 proteomic markers were found in induced sputum. 21 proteomic markers were found in bronchoalveolar lavage fluid. One biomarker has been identified with confidence, Calgranulin A. Discussion A large component of the work of this thesis was focussed on exhaled breath condensate. Two markers, pH and Ammonium were different between the CF and control groups. The measurement of EBC pH and ammonium as markers of inflammation should be used in future gene therapy trials as they are cheap, quick and simple to perform Using clean techniques free from contamination, no proteins are repeatedly detectable in EBC using highly sensitive SELDI techniques. This technique reflects the highest sensitivity of any available proteomics instrument and therefore until new technologies become available, it would be incorrect to assay any proteins in EBC. The induced sputum proteomics study identified 38 independent markers of CF lung inflammation Therefore, sampling by collection of induced sputum should be used in gene therapy trials. The endpoints should be assessed by a combination of SELDI as an endpoint and by ELISA where this is available. The marker Calgranulin is likely to report on neutrophil recruitment to the lung. It is anticipated that this will be a sensitive marker of inflammation in the lung and it also has the potential to report on successful of gene transfer as it is raised in heterozygote carriers as well as homozygotes with CF. Therefore, the non-invasive technique induced sputum coupled to proteomic analysis would have the ability to reflect the level of inflammation in CF subjects and may also report on CFTR function.

No description available.

Class of 2012 Abstract / Specific Aims: We sought to determine the influence of genetic variation of ADRB2 on the airway response to albuterol in patients with CF when compared to matched healthy controls at baseline and at 60 minutes following the administration of albuterol (2.5mg diluted in 3ml normal saline). Methods: Baseline pulmonary function (forced vital capacity, FVC, forced expiratory flow in 1-second, FEV1, mid-maximal expiratory flow, MMF, and forced expiratory flow at 50% of the FVC) was assessed in 17 patients with CF and 31 healthy subjects. Main Results: As expected, the healthy group had higher baseline pulmonary function when compared to the CF group (FVC=97±3 vs. 83±5; FEV1=95±3 vs. 72±6; MMF=90±4 vs. 54±8, % predicted for healthy and CF, respectively, mean±SE, p<0.05 for all. We compared Arg16Arg to Arg16Gly/Gly16Gly subjects. There was no effect of genotype on the response to albuterol in healthy subjects. However, in the CF group, we found that the Arg16Arg group (n=6) had an attenuated response to β-agonist when compared to the Gly-containing group (n=11) (FVC=0±0.9 vs. 6±3: FEV1=3±1 vs. 7±4: MMF=12±3 vs. 12±5 % change, for Arg16Arg and Gly-containing groups, respectively, p<0.05 for FVC, p=0.06 for FEV1). Conclusions: These results demonstrate a differential response to β-agonists according to genetic variation of the ADRB2 at amino acid 16. Due to the differences in FVC and FEV1 but not in MMF, these data suggest that the genetic difference in airway function is primarily in bronchodilation of the larger airways.

Pseudomonas aeruginosa (PA) infections in Cystic Fibrosis (CF) patients are not easily cleared due to the conversion from a nonmucoid to a mucoid phenotype. Alginate is an acetylated exopolysaccharide produced by mucoid PA that is responsible for increased resistance to antibiotics, host phagocytic killing, and propagating biofilm formation. Understanding the interaction between PA and host cells is critical to understanding chronic infection and inflammation in CF. In order to investigate this, we used A549 pulmonary epithelial cells and murine alveolar macrophages (MH-S) to examine host response to nonmucoid versus mucoid PA infection. Adhesion assays in A549 pulmonary epithelial cells revealed that mucoid PA mutants adhere poorly compared to their nonmucoid counterparts. Similarly, phagocytosis assays using MH-S infected with PA revealed that mucoid PA are increasingly resistant to phagocytosis. The alginate acetylation mutant FRD1175 is more susceptible to phagocytic killing than alginate+ FRD1. Adherence and phagocytosis of mucoid FRD1 was increased by increasing the multiplicity of infection (MOI) from 50:1 to 500:1. Furthermore, confocal microscopy revealed that mucoid PA are inherently less inflammatory than nonmucoid strains in both A549 and MH-S. Increasing the MOI of mucoid FRD1 from 50:1 to 500:1 significantly increased caspase-1 activation in MH-S but not in A549, revealing that intensity of inflammatory signaling by epithelial cells is likely independent of increased adherence. FRD1175 infection in both A549 and MH-S revealed that alginate acetylation plays a significant role in reducing inflammasome activation. Western analysis revealed that PA does not actively induce TGF-β secretion by A549 epithelial cells. Similarly, NF-κB expression was reduced in both A549 and MH-S when infected with mucoid FRD strains, but not PA from the PAO background, suggesting FRD strains have accumulated additional mutations facilitating escape of inflammation. MH-S treated with cytochalasin D to block phagocytosis were still able to activate NF-κB signaling, suggesting NF-κB activation is adherence but not phagocytosis dependent. These data increase our understanding of the various mechanisms in which mucoid PA is able to evade host immune defenses and provides insight into potential therapies to treat PA infections.

Cystic Fibrosis (CF) is the most common hereditary genetic disorder among Caucasians. Pseudomonas aeruginosa is a major cause of morbidity in cystic fibrosis patients. Chronic infection with P. aeruginosa eventually occurs and is associated with a switch to biofilm formation of the bacteria. The symptoms and pathology of acute and chronic P. aeruginosa infections differ greatly. The first line of defense within the lung is the physical barrier of the lung epithelia. The examination of established biofilm interactions with lung epithelia is difficult. Here, I use the Calgary Biofilm Device co-culture system to conduct the concurrent analysis of established biofilms and planktonic bacteria with A549 lung cells. Comparison of P. aeruginosa biofilm and planktonic bacteria’s effects on A549 lung cells showed that planktonic bacteria caused more A549 cell rounding and death, while biofilm stimulated more IL-8 release by epithelial cells. Biofilm was shown to secrete significantly more Pseudomonal Elastase than planktonic, causing A549 morphological changes and loss of tight junctions. The antimicrobial peptide LL-37 was shown to differentially affect biofilm and planktonic bacteria. LL-37 caused a decrease in twitching of planktonic bacteria and exposure to LL-37 for 48 hours resulted in a decrease in elastase secretion likely due to down-regulated type 2 secretion. When established biofilms were compared with newly adherent biofilms, young biofilms were shown to have characteristics similar to both planktonic bacteria and mature biofilms. From this data we can follow the pattern of bacterial virulence as P. aeruginosa transitions from the planktonic mode of growth to the eventual mature biofilm that is associated with chronic infection. In conclusion, this study provides the foundation for a co-culture system that can be used to study the host-pathogen interactions of mammalian epithelia with established P. aeruginosa biofilms. The future adaptations of this model will better represent the in vivo characteristics of chronic lung infection to delineate ongoing virulence mechanisms of the bacteria causing host cell stimulation and damage. / May 2016

Achromobacter sp. e Burkholderia sp. são considerados patógenos problemáticos em pacientes com fibrose cística (FC), principalmente por apresentarem linhagens que podem ser transmissíveis e multidroga resistentes. Este trabalho teve como objetivo analisar isolados de Achromobacter e do complexo Burkholderia cepacia (CBc) de pacientes com FC atendidos no Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto (HCFMRP-USP) e no Hospital das Clínicas da Faculdade de Ciências Médicas de Campinas (HCFCM-UNICAMP): identificar gênero/espécies; avaliar a sensibilidade a antimicrobianos; investigar relações genéticas entre os isolados por Pulsed-field Gel Electrophoresis (PFGE); elucidar a taxonomia e epidemiologia molecular dos isolados por Multilocus Sequence Typing (MLST) e correlacionar os resultados com dados clínicos. Entre julho/2011 a setembro/2014, nos dois hospitais, as espécies mais prevalentes de Achromobacter e CBc foram A. xylosoxidans e B. vietnamiensis, respectivamente. Os antibióticos mais efetivos contra isolados de Achromobacter sp. de pacientes do HCFMRP-USP foram imipenem e meropenem e do HCFCM-UNICAMP foram meropenem e ceftazidima. Os antibióticos mais efetivos contra CBc de pacientes do HCFMRP-USP foram sulfametoxazol-trimetoprim e meropenem e do HCFCM-UNICAMP foram ceftazidima e meropenem. Houve suspeita de contaminação cruzada entre alguns pacientes que apresentaram isolados com o mesmo perfil de PFGE. No HCFMRP-USP, isolados de B. vietnamiensis de pacientes diferentes tiveram o mesmo perfil de PFGE e apenas 2 pacientes tinham infecção crônica. No HCFCM-UNICAMP, isolados de B. cenocepacia IIIB de 4 pacientes apresentaram o mesmo pulsotipo, porém nenhum dos pacientes tinha infecção crônica. Isolados de B. vietnamiensis e B. multivorans de pacientes diferentes no HCFCM-UNICAMP também apresentaram o mesmo pulsotipo, e apenas um paciente colonizado por B. multivorans tinha infecção crônica. No HCFCM-UNICAMP, isolados de Achromobacter apresentaram perfis únicos de PFGE, enquanto que no HCFMRP-USP houve suspeita de contaminação cruzada somente entre pacientes colonizados por A. xylosoxidans, sendo que 3 destes pacientes estavam com infecção crônica. Nos dois hospitais, 17 STs foram identificados em isolados do CBc, 14 deles pela primeira vez e 3 STs (ST17, ST369 e ST911) apresentaram distribuição intercontinental. Em isolados de pacientes dos dois hospitais foram identificados alguns STs em comum (STs 1056, 1057, 369 e 911), o que pode sugerir ancestral comum. No total, 6 STs diferentes foram identificados em isolados de A. xylosoxidans de pacientes do HCFMRP-USP, dos quais 3 STs apareceram pela primeira vez e os outros 3 STs apresentaram distribuição intercontinental. Nenhuma das espécies apresentou linhagens epidêmicas descritas. Os pacientes colonizados cronicamente por A. xylosoxidans apresentaram valores de escore de Shwachman, índice de massa corporal (IMC) e função pulmonar menos preservados e exacerbações ligeiramente mais frequentes do que pacientes colonizados por bactérias do CBc. Este estudo possibilitou a correta identificação dos patógenos proporcionando a adoção de medidas de controle mais efetivas e tratamentos mais adequados, além de atualização do banco de dados epidemiológicos, o que facilita a análise colaborativa multicêntrica e auxilia no controle de infecção global destes patógenos. / Achromobacter sp. and Burkholderia sp. are troublesome pathogens in cystic fibrosis (CF) patients, mainly because they may have transmissible and multidrug resistant strains. The aim of this study was to analyze the Achromobacter and Burkholderia cepacia complex (Bcc) isolates from CF patients treated at the Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto (HCFMRP-USP) and Hospital das Clínicas da Faculdade de Ciências Médicas de Campinas (HCFCM-UNICAMP); to identify genus/species; to evaluate antimicrobial susceptibility; to investigate clonal relatedness among isolates by Pulsed-field Gel Electrophoresis (PFGE); to elucidate taxonomy and molecular epidemiology of the isolates by Multilocus Sequence Typing (MLST), and to relate the results to clinical data. Between July/2011 and September/2014, in both hospitals, the most prevalent species of Achromobacter and Bcc were A. xylosoxidans and B. vietnamiensis, respectively. The most effective antibiotics against Achromobacter sp. isolates of patients from HCFMRP-USP were imipenem and meropenem, and from HCFCM-UNICAMP were meropenem and ceftazidime. The most effective antibiotics against Bcc isolates of patients from HCFMRP-USP were sulfamethoxazole-trimethoprim and meropenem, and from HCFCM-UNICAMP were ceftazidime and meropenem. Cross-contamination was suspected among some patients who presented isolates with the same PFGE profile. In HCFMRP-USP, isolates of B. vietnamiensis from different patients showed the same PFGE profile, and only 2 patients had chronic infection. In HCFCM-UNICAMP, isolates of B. cenocepacia IIIB of 4 patients showed the same pulsetype, but none of the patients had chronic infection. Isolates of B. vietnamiensis and B. multivorans from different patients from HCFCM-UNICAMP also showed the same pulsetype, and only one patient colonized by B. multivorans had chronic infection. In HCFCM-UNICAMP, Achromobacter isolates showed unique profiles of PFGE, whereas in HCFMRP-USP cross-contamination was only suspected among patients colonized by A. xylosoxidans, and 3 of these patients had chronic infection. In both hospitals, 17 STs were identified in Bcc isolates, 14 of them for the first time and 3 STs (ST17, ST369 and ST911) presented intercontinental distribution. In both hospitals, some common STs (STs 1056, 1057, 369 and 911) were identified, which may suggest a common ancestor. In total, 6 different STs were identified in A. xylosoxidans isolates of patients from HCFMRP-USP, of which 3 STs were identified for the first time, and the other 3 STs presented intercontinental distribution. None of the species presented described epidemic strains. Patients chronically colonized by A. xylosoxidans showed less preserved Shwachman score, body mass index (BMI) and lung function, and slightly more frequent exacerbations than patients colonized by Bcc bacteria. This study provided the correct identification of the pathogens, allowing the adoption of more effective control measures and adequate treatments, besides updating the epidemiological database, which facilitates the multicentric collaborative analysis and assists in the control of global infection of these pathogens

INTRODUÇÃO: O tratamento da fibrose cística (FC) envolve o uso de medicamentos fornecidos através de nebulizadores e seu funcionamento adequado é essencial. OBJETIVOS: Avaliar o desempenho de nebulizadores a jato utilizados por pacientes com FC e comparar dois manômetros para avaliação dos compressores. MÉTODOS: Estudo descritivo, transversal, de avaliação dos nebulizadores usados pelos pacientes com FC da Associação Mineira de Assistência a Mucoviscidose. Os pacientes trouxeram compressores (Proneb Ultra®) e nebulizadores (Pari LC plus®) para avaliação. O desempenho do compressor foi avaliado por medidas de pressão operacional através dos manômetros PARI PG101® (PARI GmbH, Starnberg, Alemanha) e FSA analógico (Famabras, Itaquaquecetuba, Brasil).As variáveis de eficiência do nebulizador foram: O débito de volume nebulizado (DVN), taxa de oferta da medicação (TOR) e volume residual (VR) foram calculados por diferenças de peso de cada nebulizador após 10 minutos de nebulização de solução salina (2,5ml). O diâmetro médio de massa da partícula (DMM) foi calculado através da equação proposta por Standaert et al. A análise estatística incluiu o pacote R (v.2.15) e MINITAB, com alfa=0,05. O coeficiente Kappa foi calculado para avaliar concordância de valores entre os equipamentos, e curva ROC construída para calcular o valor aferido no manômetro FSA com melhor sensibilidade/especificidade, utilizando o manômetro PARI PG101® como referência. A associação entre valores de pressão, DVN, TOR e VR foi calculada pela correlação de Spearman. RESULTADOS: Avaliados 146 sistemas com tempo mediano de uso de 32(12-60) meses±36 meses. Cinquenta e sete (39%) não funcionaram adequadamente, com valores pressóricos inferiores à metade da referência. Os sistemas com funcionamento inadequado comprometeram as variáveis de eficiência dos nebulizadores A concordância entre os diferentes métodos de avaliação de acordo com a classificação; com funcionamento adequado e inadequado através do coeficiente Kappa foi 0,81(IC95%- 0,65-0,97), p<0,001. Na avaliação da sensibilidade e especificidade foi observado o ponto de corte de 23,5 PSI no manômetro FSA mostrou sensibilidade=99% e especificidade=79% (p<0,001). Houve associação significativa entre DVN, VR e pressões aferidas. CONCLUSÕES: Uma proporção significativa dos sistemas de nebulização não funcionou adequadamente. As variáveis de eficiência da nebulização estavam comprometidas indicando que a pressão gerada no compressor é um aspecto crítico na eficiência do tratamento. O método alternativo da avaliação dos compressores se apresentou adequado para ser utilizado nos compressores utilizados no tratamento da FC. / INTRODUCTION: Cystic fibrosis (CF) treatment of involves the use of medications supplied through nebulizers and their proper functioning is essential. OBJECTIVES: To evaluate the performance of jet nebulizers used by CF patients and to compare two pressure gauges Compressors evaluation. METHODS: This was a descriptive, cross - sectional study of the nebulizers used by patients with CF of the Mucoviscidosis Care Association of Minas Gerais. The patients brought compressors (Proneb Ultra®) and nebulizers (Pari LC plus®) for evaluation. The performance of the compressor was evaluated by operating pressure measurements using PARI PG101® manometers (PARI GmbH, Starnberg, Germany) and analog FSA (Famabras, Itaquaquecetuba, Brazil). The variables of efficiency of nebulization under study were: nebulizer delivery volume (NDV), drug output rate (DOR), and residual volume (RV), which were calculated by weighing each nebulizer before nebulization and 10 minutes after nebulization using a saline solution (2.5 mL). The mass median diameter (MMD) was calculated using the equation proposed by Standaert et al. Statistical analysis included the package R (v.2.15) and MINITAB, with alpha = 0.05. The Kappa coefficient was calculated to evaluate agreement of values between the equipment\'s, and ROC curve constructed to calculate the value measured in the FSA manometer with better sensitivity / specificity, using the PARI PG101® manometer as reference. The association between pressure values, NDV, DOR and RV was calculated by the Spearman correlation. RESULTS: We evaluated 146 systems with a median time of use of 32 (12-60) months ± 36 months. Fifty-seven (39%) did not function properly, with pressure values lower than half the reference. The systems with inadequate functioning compromised the efficiency variables of the nebulizers. The agreement between the different evaluation methods according to the classification; with adequate and inadequate functioning through the Kappa coefficient was 0.81 (95% CI -0.65-0.97), p <0.001. In the evaluation of sensitivity and specificity, the cut-off point of 23.5 PSI on the FSA manometer showed sensitivity = 99% and specificity = 79% (p <0.001). There was a significant association between NDV, DOR, RV and measured pressures. CONCLUSIONS: A significant number of the nebulizer systems were ineffective. The variables of nebulization efficiency were compromised, which indicated that the pressure generated by the compressor was a critical aspect for treatment efficiency. The alternative method of compressors evaluation was suitable for use in CF treatment routine.