Background. Segregation, a procedure where people with Cystic Fibrosis (CF) are restricted or discouraged from social mixing, is used increasingly to prevent crossinfection in CF Centres. However, research is sparse about the impact of segregation, particularly the potential negative psychosocial impact, on young people with CF. Objectives. To gain an in-depth understanding about the experience of hospitalisation under segregation and its impact from the perspective of the young person with CF. Method. In-depth interviews were conducted with 14 young people with CF aged 7 to 17 years at the end of their two-week admission for IV antibiotics. Interpretative Phenomenological Analysis (JP A) was used to analyse transcribed interviews. Results. The young people could be categorised into two groups in terms of their experiential history of hospitalisation. Segregation was 'protective, and better than expected' for relatively 'inexperienced' participants; whereas it was deemed to be 'restrictive, and led to a difficult stay' by 'experienced' participants. Three common themes appeared to characterise the views of all participants regarding the issue of cross-infection: 'wanting to reduce the risk', 'the importance of contact with similar others' and 'difficult choices'. A range of coping strategies were discernible from the data with the main connecting theme regarding coping as 'being resilient'. Discussion. The themes emanating from the data are discussed in terms of Lazarus and Folkman's Transactional Model of Stress and Coping and the concept of peer support. The results suggest that there may be four ways in which segregation impacts negatively upon young people with CF in terms of: (1) anxiety about bacterial infection, (2) loss of CF peers, (3) learning to cope with limited social contact during hospitalisation, and (4) fragmentation of the CF community. The findings are discussed in relation to existing research and in terms of their clinical implications.

No description available.

Excessive neutrophilic inflammation of the airways in response to infection is characteristic for patients with CF. There is also an important but not fully understood role for platelets. Previous studies established increased copper levels in the circulation and in the sputum in CF. Inhaled tobramycin was suggested to have an anti-inflammatory effect beyond eradicating Pseudomonas aeruginosa. This study tested the hypothesis that tobramycin has anti-inflammatory and anti-oxidant efficacy due to its ability to bind copper into a copper-tobramycin complex. A copper-tobramycin complex was synthesised and the UV-VIS spectrum analysed. Neutrophil migration through a TNF-α-stimulated human lung microvascular endothelial cell layer towards thrombin-activated platelets was measured. The role of CFTRinh-172 on neutrophil transendothelial migration was assessed. Endothelial tobramycin uptake and CFTR expression were assessed using immunocytochemistry. Endothelial oxidative stress was measured using a fluorescent indicator. Neutrophils were stimulated to measure reactive oxygen species (ROS) production and neutrophil elastase (NE) activity, spectrophotometrically. Platelet and endothelium-derived NAP-2 and IL-8, respectively, contributed to neutrophil transendothelial migration. Copper-tobramycin was shown to be more effective than tobramycin in limiting migration of neutrophils. Both, tobramycin and copper-tobramycin accumulated in endothelial cells via a heparan sulphate-dependent mechanism, decreased intracellular ROS and increased endothelial surface CFTR expression. CFTRinh-172 failed to create an inflammatory profile in endothelium. Copper-tobramycin decreased extracellular superoxide released by activated neutrophils, and displaced NE from sites of encryption, making it more susceptible to inhibition by α1-antitrypsin. The antibiotic tobramycin was demonstrated to be a multi-potent drug with additional anti-inflammatory and anti-oxidant properties. These effects, desirable in CF treatment, are due to copper binding.

Pseudomonas aeruginosa is a common cause of chronic respiratory infection in cystic fibrosis (CF). Infection is established within the lung epithelial mucus layer, through adhesion to mucins. Terminal residues on mucin oligosaccharide chains are highly sulphated and sialylated, which increases their resistance to degradation by bacterial enzymes. However, a number of microbes display mucin sulphatase activity, including P. aeruginosa. Using ion chromatography, the levels of sulphation on different respiratory mucins and the availability of inorganic sulphate in CF sputum were quantified, and the ability of clinical P. aeruginosa isolates to desulphate mucin was tested by providing mucin as a sole sulphur source for growth. All tested P. aeruginosa strains isolated from the CF lung were able to use human respiratory mucin as source of sulphur for growth, whereas other non-clinical Pseudomonas species were not. However, measured levels of inorganic sulphate in CF sputum suggest that bacteria resident in the lung have sufficient inorganic sulphate for growth and are unlikely to require access to mucin-sulphur as a sulphur source during chronic infection. This was confirmed when expression of sulphate-repressed P. aeruginosa genes, atsK and msuE, were found by quantitative PCR to be repressed in CF sputum. These results indicate that sulphate-starvation is unlikely to occur in pathogens residing in CF sputum and, therefore, mucin desulphation may have an alternative purpose in the association between P. aeruginosa and CF airways.Transcriptomic studies showed enhanced expression of 5 main islands on the P. aeruginosa genome in the presence of mucin as a sulphur source, when compared to sulphate. These islands include general sulphur-starvation response gene clusters, encoding desulphurizing enzymes AtsA, SsuD and MsuD, plus the locus PA2083-PA2094. This locus has not been characterised but encodes a putative sulphonatase, an extracellular-function (ECF) type sigma factor, with associated TonB-dependent transducer, and Major Facilitator Superfamily transporters. Transcriptional studies of this locus in response to various sulphur sources revealed that the locus comprises two transcriptional units under sulphate-limited conditions, and putative σ70-type promoters were identified using 5’-RACE and sequence alignment. Transcriptional regulation of the locus is contributed to by the encoded ECF-type σ factor and anti-σ factor, as a mutant carrying only a disrupted copy of these genes displayed a lack of transcriptional downregulation of the locus in the presence of sulphate. The influence of mucin on transcription levels of the locus was also investigated by RT-qPCR, showing that for maximum transcriptional levels both sulphate-limitation and the presence of mucin are required. However, despite repression of P. aeruginosa sulphate-regulated genes in CF sputum, the level of expression of the locus PA2083-PA2094 in CF sputum was comparable to that of P. aeruginosa culture grown in sulphate-limited conditions. The influence of the lung environment may, therefore, have a greater impact on expression levels of the locus than seen in in vitro studies with mucin. To further investigate the role of the locus, mutants were constructed and screened for changes in their ability to utilise a range of sulphur sources, including mucin, for growth. However, none of the mutants showed significant change in their growth patterns in response to any of the other sulphur sources tested, suggesting that the locus may be involved in desulphurization of a compound not tested in this study or may be functionally replaced by other organosulphur utilisation pathways in its absence. With the aim of identifying genes involved in mucin desulphurization, a P. aeruginosa transposon library was generated, combining the high-throughput nature of a random library with the variable expression reporter capabilities afforded by a promoterless GFP insert. The GFP reporter transposon produced varying fluorescence levels over time during growth of individual mutants, based on the activity of the promoter upstream of the transposon insertion site. A preliminary method was devised using fluorescence-activated cell-sorting to isolate mutants displaying altered GFP expression levels in response to sulphate availability and to mucin. Overall, this work explores the prevalence and importance of mucin desulphurization by P. aeruginosa, with relation to cystic fibrosis lungs, and provides some insight into the transcriptional patterns of the P. aeruginosa locus PA2083 to PA2094.

For the first time, an in vitro cystic fibrosis (CF) artificial sputum model (ASM) was found to support the growth and survival of a clinical epidemic strain of meticillin-resistant Staphylococcus aureus (EMRSAl6-252). Specific components, which included mucin. DNA and others, were removed from ASM and the physiological impact of this was fully explored using viable counts and light microscopy. As CF patients are known to develop cystic fibrosis-related diabetes or CFRD, glucose was added to ASM (GASM), to explore the physiological impact of glucose on the growth and survival MRSA252. Total RNA was extracted from the corresponding log phases of MRSA252 grown in brain heart infusion (BHI) as a laboratory control, as well as ASM and GASM. RNA was extracted in order to conduct microarray analysis. MRSA252 DNA was used as a control. RNA (from the samples) was labelled with Cy5 and control DNA was labelled with Cy3. Once labelled and amplified, the Cy5/Cy3 mixture was then purified and hybridised onto an array containing seven sequenced S. aureus : genomes (N315, Mu50, MW2, MRSA252, MSSA476, COL and NCTC8325).

No description available.

Burkholderia cepacia complex (Bcc) species are opportunistic pathogens in patients with cystic fibrosis (CF), which are able to cause lethal infections. The Bcc are inherently resistant to most classes of antibiotics, which makes successful treatment problematic. Lipid A (also known as endotoxin), the hydrophobic anchor of lipopolysaccaride (LPS), is the bio-active component of LPS. One of several unique characteristics of the lipid A of the Bcc, is the permanent attachment of 4-amino-4-deoxy-L-arabinose (L-Ara4N) to the lipid A molecule. Also, the genes involved in L-Ara4N biosynthesis are necessary for viability in B. cenocepacia. Here we present research on lipid A biosynthesis, modi cation, and transport in the Bcc and highlight promising antimicrobial targets. The synthetic antibiotic CHIR-090 is an inhibitor of LpxC, an enzyme involved in the lipid A biosynthetic pathway. I investigated the activity of CHIR-090 against the Bcc and found that sensitivity to this antibiotic was both species- and strain-specific. CHIR-090 displayed MICs between 0.1 and 12.5 μg/ml against a panel of B. multivorans, the most prevalent Burkholderia species in CF. The species- and strain-specific sensitivity towards CHIR-090 was further explored and a strong correlation was found between the presence of a unique open reading frame, named LpxC2, in resistant species. To address the problem of multiple drug-resistance of the Bcc, we investigated the activity of the pyridoxal 50-phosphate (PLP)-dependent enzyme inhibitor cycloserine (CS) against the Bcc. CS is used as a second line of defense against M. tuberculosis. The activity of the D-enantiomer of CS (DCS) against the Bcc was tested and displayed MICs between 2 and 128 μg/ml and acted bactericidal towards the Bcc. Additionally, DCS inhibition of recombinant ArnB from B. cenocepacia J2315, a PLP-dependent enzyme necessary for viability in the Bcc, was studied. ArnB was inhibited reversibly by DCS. ArnB was further explored as a promising drug-target in the Bcc, but only CS has been identified as an inhibitor so far. In this thesis it was attempted to find the reason why is L-Ara4N modification of lipid A necessary for viability in B. cenocepacia. Therefore, two proteins were characterised, which are involved in lipid A transport: LptA, the periplasmic lipid A binding protein, and LptB, the cytoplasmic ATP-ase. LptA was found to be able to bind both modified and unmodified lipid A in vitro and therefore is not L-Ara4N specific. Furthermore, LptA could bind deep-rough-, rough-, and smooth- LPS, similar to that described for Escherichia coli LptA. The kinetic parameters of LptB were determined in vitro (kcat = 5.71 min-1 and KM = 0.88 mM), and were comparable to E. coli LptB. The ATP-ase activity of LptB was not influenced by the presence of any forms of LPS (modified or non-modified). Therefore, we concluded that both B. cenocepacia J2315 LptA and LptB are not L-Ara4N specific.

La mucoviscidose est une maladie causée par des mutations du gène cftr entraînant des défauts importants de la protéine CFTR. La mutation la plus fréquente (F508del) est caractérisée par un repliement incorrect conduisant à la rétention de la protéine dans le RE.L’accumulation de CFTR-F508del dans le RE, l’inflammation et les infections vont déclencher un stress du RE dans les cellules épithéliales ainsi que l’UPR. Cette dernière est une réponse adaptative déclenchée par le stress du RE et permet de rétablir l’homéostasie de ce compartiment. L’UPR est constituée de trois voies majeures dont l’une d’entre elles est activée dans les cellules exprimant un CFTR-F508del. Il s’agit de la voie ATF6 qui est de plus responsable de la répression transcriptionnelle du CFTR, ce qui en fait une cible thérapeutique potentielle. Nous avons montré que son inhibition conduit à l’amélioration de la fonction duCFTR-F508del et à l’augmentation de sa présence à la membrane des cellules.Nous nous sommes également intéressés au Mg2+ et au TRPM7, le régulateur principal de la [Mg2+]i dans les cellules. Nous avons émis l’hypothèse que TRPM7 était en partie responsable de l’activation d’ATF6 dans les cellules exprimant un CFTR-F508del. Le but de cette seconde partie du projet était donc tout d’abord d’étudier la relation existante entre le Mg2+, TRPM7 et le CFTR. Nous avons montré qu’il existait des différences de [Mg2+]i selon le type de mutation du CFTR exprimé par les cellules. Ces différences sont en partie dues à un défaut d’activation de TRPM7, lui-même probablement lié à un défaut du CFTR. En augmentant l’activité de TRPM7 par du Naltriben, nous avons pu montrer un effet potentialisant sur leCFTR-G551D. / Cystic fibrosis is caused by mutations in the cftr gene resulting in several defaults on the CFTR protein. The most frequent mutation is F508del which is characterized by an incorrect folding causing its retention within the ER. CFTR-F508del protein accumulation in the ER, inflammation and infections will trigger the ER stress in epithelial cells, as well as UPR. UPR constitutes an adaptive response of the ER in order to restore ER’s homeostasis. UPR consists in three major pathways. Among them, one is activated in cells expressing CFTR-F508del protein. The ATF6 pathway of UPR is responsible of the transcriptional repression of CFTR, which makes of it a potential therapeutic target. We showed that the inhibition of ATF6 leads to the improvement of CFTR-508del function, as well as its increased presence in the cellular membrane. We were also interested in Mg2+ and TRPM7, the main regulator of [Mg2+]i. We suspected that TRPM7 is, at least in part, responsible for the activation of ATF6 in cells expressing the mutant CFTR-F508del. Thus, the second part of my work was focused on the study of the relationship between Mg2+, TRPM7 and CFTR. We showed the existence of [Mg2+]I differences according to CFTR mutant expressed in cells. These differences are the result of an altered TRPM7 activation, probably in link with the mutated CFTR’s malfunction. We proved that increasing TRPM7 activity by Naltriben treatment potentiates CFTR-G551D.

Four studies investigating the assessment of glycaemic control in cystic fibrosis are presented within this thesis. The first was a validation study of continual glucose monitoring (CGM) in cystic fibrosis (CF). 50 stable adults with CF underwent home CGM for 3 days, during which time they attended the CF centre for OGTT. Gold standard fasting (0 hour) plasma glucose and 2 hour plasma glucose values during OGTT were compared with concurrent CGM sensor glucose values using a 'limits of agreement' analysis. CGM was found to be valid in adults with CF, with its accuracy being consistent with that published in non-CF populations. The next investigation compared OGTT with CGM with several objectives: to determine whether OGTT is a relevant and adequate measure of glycaemia in CF, find out whether CGM could offer a superior alternative to OGTT and explore whether OGTT and CGM results are associated with prior change in lung function and weight in adults with CF. Data from the first study was used to show that the OGTT can only identify abnormal glycaemic control in CF at a late stage, and that CGM is a more relevant reflection of everyday glycaemia in CF. No correlation was found between prior change in lung function and nutritional status in CF and glycaemia measured by OGTT or CGM. The subsequent study investigated whether CGM could identify early abnormal glycaemic control in CF. This involved ten non-CF healthy controls undergoing the same study protocol as the 50 stable adults with CF, to determine 'normal' glycaemic control parameters. Of 25 CF patients with normal glucose tolerance by OGTT, 19 (76%) had significantly higher mean and/or variability of CGM levels than healthy controls. This lead to changes in their management, including 2 subjects being commenced on insulin therapy. The final investigation was a questionnaire study, asking the 50 CF patients to provide information on their experience of undergoing CGM. 58% of patients responded, with replies indicating that they found CGM broadly acceptable, interfering little in their lives and that their experiences were generally positive. This insight into patients' experiences of CGM can be used to guide future clinical and research roles for this tool. These studies have provided novel data regarding the assessment of glycaemic in CF. Information captured by CGM has greater relevance to CF patients' daily lives than OGTT. CGM can identify early problems with glycaemic control leading to changes in management that may not be detected by conventional measures. CGM offers potential in further clinical application and research to improve the lives and outcomes for adults with CF.

Η λειτουργία των αναπνευστικών μυών σε ασθενείς με Κυστική Ίνωση (ΚΙ) μπορεί να εκτιμηθεί με τη μέτρηση της μέγιστης εισπνευστικής πίεσης (Pimax), της μέγιστης εκπνευστικής πίεσης (Pemax), του δείκτη πίεσης-χρόνου των αναπνευστικών μυών (PTImus), του ρυθμού μυϊκής χαλάρωσης (MRR) και του μέγιστου ρυθμού αύξησης πίεσης (MRPD). Ο σκοπός αυτής της μελέτης ήταν να μελετήσει τους δείκτες εκτίμησης της λειτουργίας των αναπνευστικών μυών σε ασθενείς με ΚΙ και την πιθανή επίδραση σπιρομετρικών και διατροφικών διαταραχών στη λειτουργία των αναπνευστικών μυών στους ασθενείς αυτούς. Επίσης να μελετήσει την πιθανή επίδραση της αεροβικής άσκησης στη λειτουργία των αναπνευστικών μυών σε ασθενείς με ΚΙ. Μελετήθηκε η λειτουργία των αναπνευστικών μυών με μέτρηση των PTImus, Pimax, Pemax, MRR και MRPD σε 140 ασθενείς με ΚΙ και σε ομάδα ελέγχου 140 υγιών μαρτύρων αντίστοιχης ηλικίας και φύλου. Η εκτίμηση της αναπνευστικής λειτουργίας περιλάμβανε μέτρηση του βιαίως εκπνεόμενου όγκου αέρα σε 1 δευτερόλεπτο (FEV1), της βιαίως εκπνεόμενης χωρητικότητας (FVC) και της βιαίως εκπνεόμενης ροής μεταξύ του 25% και του 75% της ζωτικής χωρητικότητας (MEF25-75). Η διατροφική εκτίμηση περιλάμβανε μέτρηση της περιφέρειας μυών μέσου βραχίονα (MAMC), της δερματικής πτυχής τρικεφάλου (TST), της μυϊκής επιφάνειας του βραχίονα (UAMA) και του δείκτη μάζας σώματος (BMI). Οι Pimax και Pemax βρέθηκαν σημαντικά ελαττωμένες σε ασθενείς με ΚΙ συγκρινόμενες με την ομάδα ελέγχου (p<0.01 και p<0.001 αντίστοιχα). Ο δείκτης PTImus βρέθηκε σημαντικά αυξημένος σε ασθενείς με ΚΙ σε σχέση με τα υγιή άτομα της ομάδας ελέγχου (p<0.05). Ο δείκτης PTImus βρέθηκε σημαντικά αυξημένος σε ασθενείς με ΚΙ και επηρεασμένους σπιρομετρικούς δείκτες. Επιπλέον, σε ασθενείς με ΚΙ ο δείκτης PTImus βρέθηκε να παρουσιάζει σημαντική αρνητική συσχέτιση με το δείκτη UAMA (p<0.05). Οι ασθενείς με ΚΙ και χαμηλές τιμές BMI δεν εμφάνισαν σημαντικά αυξημένες τιμές PTImus. Ο δείκτης MRR κατά την εκτέλεση δοκιμασίας Pemax (p<0.05) και ο δείκτης MRPD κατά την εκτέλεση δοκιμασίας Pemax (p<0.005) ήταν σημαντικά επηρεασμένοι σε ασθενείς με ΚΙ σε σχέση με την ομάδα ελέγχου. Οι ασκούμενοι ασθενείς με ΚΙ εμφάνισαν υψηλότερες τιμές μέγιστων αναπνευστικών πιέσεων και χαμηλότερες τιμές του δείκτη PTImus σε σχέση με μη ασκούμενους ασθενείς με ΚΙ. Συμπερασματικά, αυτή η μελέτη κατέδειξε ότι οι ασθενείς με ΚΙ εμφανίζουν υψηλότερες τιμές PTImus σε σχέση με το γενικό πληθυσμό. Ασθενείς με επηρεασμένους σπιρομετρικούς δείκτες όπως οι FEV1,FVC and MEF25-75, και διατροφικούς δείκτες όπως ο UAMA εμφανίζουν υψηλότερες τιμές PTImus σε σχέση με ασθενείς με φυσιολογικούς ή λιγότερο επηρεασμένους διατροφικούς και σπιρομετρικούς δείκτες. Η αεροβική άσκηση πιθανόν να επιδρά ευεργετικά στη διατήρηση της ισχύος των αναπνευστικών μυών σε ασθενείς με ΚΙ. / Respiratory muscle function in patients with Cystic Fibrosis (CF) can be assessed by measurement of maximal inspiratory pressure (Pimax), maximal expiratory pressure (Pemax), pressure time index of the respiratory muscles (PTImus), muscle relaxation rate (ΜRR) and maximum rate of pressure development (MRPD). This study aimed to examine respiratory muscle function indices in CF and the possible effect of pulmonary function and nutrition abnormalities to respiratory muscle performance, in patients with CF, as well as to investigate the possible effect of aerobic exercise in respiratory muscle function in CF. Respiratory muscle function by measurement of PTImus, Pimax, Pemax, MRR and MRPD was assessed in 140 CF patients and a control group of 140 healthy subjects matched as possible for age and gender. Pulmonary function evaluation consisted of forced expiratory volume in 1 sec (FEV1), forced vital capacity (FVC) and maximal expiratory flow between 25 and 75% of VC (MEF25-75). Nutritional assessment consisted of mid arm muscle circumference (MAMC), triceps skinfold thickness (TST), upper arm muscle area (UAMA) and body mass index (BMI). Pimax and Pemax were significantly lower in CF patients compared to the control group (p<0.01 and p<0.001 respectively). PTImus in CF patients compared to healthy controls was significantly increased (p<0.05). PTImus was significantly higher in CF patients with impaired pulmonary function. Furthermore, in CF patients, PTImus was significantly negatively related to UAMA (p<0.05). Patients with low BMI values did not have significantly higher PTImus values. MRR during Pemax (p<0.05) and MRPD during Pemax (p< 0.005) were significantly altered in CF compared to the control group. Exercising CF patients maintained higher maximal respiratory pressures and lower PTImus values compared to non-exercising patients. In conclusion this study demonstrated that CF patients exhibit higher PTImus values compared to the healthy population. Patients with affected pulmonary function parameters, such as FEV1, FVC and MEF25-75, and nutrition parameters such as UAMA exhibit higher PTImus values compared to CF patients with normal or less affected pulmonary function and nutrition indices. Exercise might exert a beneficial effect on respiratory muscle strength in patients with CF.