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Cell surface receptors such as adhesion molecules and connexins are involved in interactions between cells and their surroundings. They play important roles in the normal function of healthy tissues and in the responses of cells to injury. In many cases aberrant repair mechanisms are thought to result in disease and this thesis will assess the expression of cell adhesion molecules (principally the pi integrins) and the connexin43 gap junction protein in normal and diseased kidney The expression of pi integrins on normal human mesangial cells was localised using the alkaline phosphatase anti-alkaline phosphatase immunochemical technique (APAAP) and Western blotting. Expression of mRNA coding for integrins was also assessed using reverse-transcription polymerase chain reaction (RT-PCR). Human mesangial cells in culture expressed the a2, a3, av, pi av and P3 integrin chains. Messenger RNA was detected for these integrin subunits plus the al, a4, a5 and a6 chains. Normal human kidney sections were stained using APAAP and monoclonal antibodies towards a wide range of integrin chains. Within the glomerulus, mesangial cells express al, a2 and pi, epithelial cells a3, av and pi and endothelial cells al, a5 and pi. Tubules express a2, a3, a6, av and pi and the interstitium al and pi. In renal biopsies from patients with IgA disease the main alterations in integrin expression were upregulation of a2, a3, av and pi on damaged tubules, with increased pi expression and de novo a5 and av staining within areas of interstitial damage. These changes were replicated in a wide range of other renal pathologies and correlate with the degree of tubulointerstitial histological damage. Connexin43 (Cx43) is distributed extensively on normal human kidney, particularly on glomerular epithelial cells and intra- and extra-glomerular endothelium. Human mesangial cells in vitro express Cx43 protein and its coding mRNA. There is, however, no expression of Cx43 by the mesangium in vivo. In biopsies from patients with inflammatory renal disease there is strong expression of Cx43 on infiltrating inflammatory cells, in areas of interstitial damage and on damaged tubules. The pattern of Cx43 expression in inflammatory renal disease was very similar to that of intercellular cell adhesion molecule-1 and vascular cell adhesion molecule-1. The work in this thesis has demonstrated the large repertoire of cell surface receptors expressed on normal kidney. The principal alterations in diseased kidney are found within the tubulointerstitum. The potential relevance of these changes in the pathogenesis of renal disease are discussed and possible future avenues of research are suggested.

Fibroblasts are abundant mesenchymal cells present in all tissues in a quiescent state, which contribute to wound healing when activated. Cytokine transforming growth factor-β1 (TGF-β1) stimulates fibroblast-myofibroblast differentiation, which induces extracellular matrix secretion, tissue contraction and promotes cancer cell migration. Hence, chronic activity of stromal myofibroblasts correlates with a poor prognosis for cancer and organ fibrosis patients. Therefore, modulating myofibroblast activity may reduce the severity of these diseases. Previous research suggests blockade of transmembrane integrin receptors expressed by fibroblasts prevents TGF-β1- induced differentiation, indicating integrins are attractive therapeutic targets. However, fibroblasts derived from different organs exhibit heterogeneity, although their integrin expression and integrin-regulated differentiation has not been directly compared. The aim of my research was 1) to understand and compare how integrins regulate TGF-β1-induced activation of fibroblasts derived from normal skin, lung and breast tissue; 2) to examine the global gene expression of TGF-β1-treated lung fibroblasts; 3) to identify novel therapeutic targets that modulate TGF-β1-induced activation of lung fibroblasts using a drug library. qPCR showed skin, lung and breast fibroblasts differentially expressed TGF-β1- induced activation markers, including ACTA2, FN1, TIMP3, CTGF and SERPINE1, in addition to integrin genes for α1, α4, α11 and β3. Small-molecule inhibitors of αv integrins only reduced the invasion of TGF-β1-exposed skin fibroblasts, but not lung or breast fibroblasts. siRNA against α11, β3 and β5 decreased TGF-β1-induced collagen contraction and activation marker expression in skin and lung fibroblasts, while α1 siRNA prevented collagen contraction by breast fibroblasts only. RNA sequencing of TGF-β1-treated lung fibroblasts revealed pro-inflammatory and profibrotic pathways were significantly enriched, while screening TGF-β1-treated lung fibroblasts with a FDA-approved drug library identified 46 hits that significantly reduced α-smooth muscle actin and fibronectin expression. Overall, genes are differentially expressed in TGF-β1-treated skin, lung and breast fibroblasts, while different integrins in each fibroblast appear to regulate invasion, TGF-β1-induced collagen contraction and gene expression. RNA sequencing revealed TGF-β1 promotes the expression of a pro-tumour signature in lung fibroblasts and several novel therapeutic targets that modulate the activation of lung fibroblasts have been identified. Understanding these integrin-dependent and independent mechanisms will facilitate the generation of myofibroblast-targeted treatments for cancer and organ fibrosis.

Asthma is a common chronic inflammatory condition which affects over 300 million people worldwide. Thickening of the subepithelial layer is a key feature of asthmatic airways and the extent of thickening has been correlated with severity of asthma and increased exacerbations. Recent epidemiological studies have shown a link between fungal sensitisation primarily with Aspergillus fumigatus (A. fumigatus) and exacerbations of asthma leading to increased morbidity and mortality. The airway epithelium acts as an initial defence barrier to inhaled allergens such as A. fumigatus and emerging evidence suggests that as well as orchestrating an allergic immune response, it initiates aspects of airway wall remodelling including subepithelial thickening. However, induction of a profibrogenic response by the airway epithelium following exposure to inhaled fungi associated with severe asthma has not been well documented. The epithelial expression and production of the profibrotic growth factors, TGF-β1, TGF-β2, IL-6, endothelin-1 and periostin, selected as implicated in the aetiology of asthma and their profibrotic activity, were investigated in response to both A. fumigatus spores and culture filtrate in vitro. Furthermore, in vivo chronic inhalation models using either live spores or culture filtrate from two different strains of A. fumigatus (AF293 and CEA10) were used to determine the ability of the fungi to induce murine airway wall remodelling. In vitro, spores from both strains were able to induce the expression and production of IL-6 and endothelin-1 from human bronchial epithelial cells but none of the other profibrotic growth factors. In vivo, despite spores from both strains inducing expression and production of IL-6 and endothelin-1, only CEA10 spores caused significant subepithelial collagen deposition however, both strains induced α-SMA, a myofibroblast and smooth muscle marker around the airways. As a secreted factor was suspected of driving airway wall remodelling, subsequent studies used culture filtrate produced by the two strains, AF293, a low and CEA10, a high protease producer in basal medium. Only AF293 culture filtrate induced IL-6 and endothelin-1 from human bronchial epithelial cells in vitro. However, in vivo, culture filtrate from both strains was able to induce IL-6 and endothelin-1 expression, with AF293 causing a more profound subepithelial collagen deposition and significantly increased α-SMA abundance. It was hypothesised that epithelial-derived endothelin-1 drives airway wall remodelling and hence Endothelin receptor A was inhibited in the in vivo culture filtrate inhalation model. A significant reduction in subepithelial collagen deposition and α-SMA localisation around the airways was demonstrated in mice receiving an Endothelin receptor A antagonist compared with culture filtrate alone. This thesis indicates that A. fumigatus exposure can drive features of airway wall remodelling such as subepithelial fibrosis possibly through the epithelial production of profibrotic growth factor, endothelin-1.

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

Background: Lung clearance index (LCI), obtained by multiple breath washout testing (MBW), is a sensitive measure of lung disease in infants. It has been identified as a particularly suitable endpoint for clinical trials in cystic fibrosis (CF), but has potential applications in many other conditions. However, MBW in infants presents a number of technical challenges. Conventional MBW is based on simultaneous measurement of flow and gas. These two signals are then aligned and combined to derive expired gas volumes and measures of ventilation inhomogeneity: this process becomes increasingly vulnerable to errors in gas signal alignment at rapid respiratory rates. At present, no existing system for infant MBW meets all the criteria set out in international guidelines, and there is no simple method of assessing lung function outside research laboratories in this population. This thesis describes an alternative method of performing MBW in infants. In this method, expired gas is collected and analysed to derive functional residual capacity (FRC) and LCI. There is no need to simultaneously measure flow, and therefore no need for the complicated step of integrating flow and gas signals. Dead space is also significantly reduced by removing the flowmeter. Methods: In the first phase of testing, an existing lung model was modified to generate realistic infant breathing parameters with high accuracy. The prototype system was modified to improve accuracy and subsequently tested at FRC of 100-250mls with respiratory rates of 20-60min-1. In the second phase, testing proceeded to an in vivo pilot study of the novel method in children with cystic fibrosis and healthy controls. Practical applicability of the system was determined by the number of successful duplicate tests, and within-subject repeatability. Comparison was made with LCI measurements obtained using a respiratory mass spectrometer, currently considered the gold standard for infant LCI. Results: In a total of 103 tests performed in the lung model, overall mean error (standard deviation) of FRC measurement was -1.0(3.3)%, with 90% of tests falling within +/-5%. 13 patients were excluded from the clinical study due to being unsedated or inadequately sedated and therefore failing to tolerate the test. A total of 25 patients (7 children with CF, 18 healthy control children) were deemed to be adequately sedated at the start of the test, of these 20 patients (7 with CF) successfully underwent duplicate testing (80% success rate). Mean FRC for healthy controls was 19.5ml/kg, and mean LCI 6.45. For children with CF, mean FRC was 21.8ml/kg and mean LCI 6.98. Mean within-subject coefficient of variation for FRC was 7.18% and for LCI 5.94%. Of 4 infants assessed with both the novel method and the respiratory mass spectrometer, there was good correlation in FRC measurement (mean difference -8.1%). Comparison of LCI with the mass spectrometer was affected by technical difficulties with the test; in those patients who underwent technically adequate tests with both methods, mean difference in LCI between the two methods was 1.65%. Discussion: FRC measurement using the novel method has superior accuracy in vitro than previously described systems. Data from the pilot study suggest that this is a feasible and reproducible method of performing LCI in infants and young children, as long as they are adequately sedated. Results in both children with CF and controls fall within the expected range, and well within accuracy limits set by international guidelines. However, the system and testing protocol could be further improved to reduce the number of technically inadequate tests having to be excluded. This could provide a more accessible alternative to previously described systems for infant MBW.

abstract: Introduction: Cystic fibrosis (CF) is the most common life-shortening autosomal recessive genetic disease affecting Caucasians. The disease is characterized by a dysfunctional cystic fibrosis transmembrane regulator (CFTR) protein and aberrant mucus accumulation that subsequently alters the physicochemical environment in numerous organ systems. These mucosal perturbations have been associated with inflammation and microbial dysbiosis, most notably in the lungs and gastrointestinal (GI) tract. Genistein, a soy isoflavone and dietary polyphenol, has been shown to modulate CFTR function in cell cultures and murine models, as well exert sex-dependent improvement of survival rates in a CF mouse model. However, it is unknown whether dietary genistein affects gut microbiome diversity and community structure in cystic fibrosis. This study sought to examine associations between dietary genistein treatment and gut microbiome diversity and community structure in a murine model of CF. Methods: Twenty-four male and female mice homozygous for the DF508 CFTR gene mutation were maintained on one of three diet regimens for a 45-day period (n=11, standard chow; n=7, Colyte-treated water and standard chow; n=6, 600 mg dietary genistein per kg body weight). One fecal pellet was collected per mouse post-treatment, and microbial genomic DNA was extracted from the fecal samples, quantified, amplified, and sequenced on the Illumina MiSeq platform. QIIME 2 was used to conduct alpha- and beta-diversity analyses on all samples. Results: Measures of alpha-diversity were significantly decreased in the dietary genistein group as compared to either standard chow or Colyte groups. Measures of beta-diversity showed that community structure differed significantly between dietary treatment groups; these differences were further illustrated by distinct clustering of taxa as shown by principal coordinates analysis plots. Conclusion: This 3-arm parallel experimental study showed that dietary genistein treatment was associated with decreased microbial diversity and differences in microbial community structure in DF508 mice. / Dissertation/Thesis / Masters Thesis Nutrition 2019

Se determinó el alelo delta F508 del gen cftr en familias de pacientes diagnosticados con fibrosis quística (FQ) en Lima. El grupo que participa pertenece a la Asociación Nacional Contra la Fibrosis Quística. Se estudiaron 21 personas pertenecientes a seis familias. En cada caso, los familiares adultos y los padres de los menores de edad firmaron un consentimiento informado, previo a su inclusión en el estudio. Después, se recolectaron muestras de sangre venosa para extraer ADN genómico de los leucocitos y se amplificaron regiones específicas del gen cftr por la reacción en cadena de la polimerasa con cebadores específicos diseñados para detectar la mutación delta F508 en el exón 10. Los productos amplificados fueron cortados con la enzima MboI. La frecuencia del alelo delta F508 fue de 11.09 %, distribuidos en un homocigoto delta F508 y tres heterocigotos delta F508/X. El resto de familiares no presentó la mutación delta F508. La frecuencia del alelo delta F508 en esta muestra es menor a la reportada en países europeos pero en América Latina se ha observado una gran heterogeneidad debido a los diferentes patrones de mestizaje. Además se realizaron el perfil lipídico y determinación de glucosa. / Tesis