S-nitrosogluthatione is an endogenous substance, present at decreased levels in the lungs of CF patients and was recently found to induce mature CFTR in airway epithelial CF cell lines. We show that S-nitrosoglutathione in physiological concentrations increases the presence of ΔF508 CFTR in the cell membrane and induces cAMP dependent chloride transport in cystic fibrosis airway epithelial cells. The properties of S-nitrosoglutathione include other potential benefits for the CF patient and make this agent an interesting candidate for pharmacological treatment of CF that needs to be further evaluated. Genistein was found to increase the chloride efflux in both normal and ΔF508 cells without stimulation of cAMP elevating agents and without prior treatment with phenylbutyrate. Genistein, in concentrations close to those that can be detected in plasma after a high soy diet, could induce chloride efflux in cells with the ΔF508 CFTR mutation and its possible use in the treatment of CF should therefore be further investigated. Studies on nasal epithelial cells from CF patients showed cAMP dependent chloride efflux in some of the patients with severe genotypes. This may complicate in vitro evaluation of clinical treatment of these patients. The presence of cAMP dependent chloride transport did not necessarily lead to a milder phenotype. Other factors than CFTR may influence the clinical development of the disease. Cystic fibrosis (CF) is the most common monogenetic disease among Caucasians. A defective cAMP regulated chloride channel (cystic fibrosis transmembrane conductance regulator, CFTR) in epithelial cells leads to viscous mucus, bacterial infections, inflammation and tissue damage in the lungs that cause death in 95% of the cystic fibrosis patients. There is no cure for the disease although existing treatment has dramatically prolonged the life expectancy. The aim of this thesis was to study pharmacological agents for their ability to restore the cellular deficiency in CF airway epithelial cells. X-ray microanalysis, MQAE fluorescence and immunocytochemistry were used to evaluate the effects.

Cystic fibrosis (CF) is the most common lethal genetic disease in the white population. It is due to mutations in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), a protein that functions mainly as a cAMP-activated chloride channel. The disease impairs ion and water transport in epithelia-lined organs such as airways, digestive tract, reproductive epithelium and sweat glands. At present the only therapy is symptomatic and development of curative treatment depends on uncovering the links between the defective CFTR and the disease, as well as on improving end-point measurements. A method has been established for studying ion transport in an easily accessible cell type (nasal epithelial cells) from normal and cystic fibrosis patients by X-ray microanalysis. This method represents a rather simple and direct way of measuring simultaneously several chemical elements of biological interest. Studies of chloride transport by means of a fluorescent indicator (MQAE) in nasal epithelial cells from CF patients showed that the phenotype cannot exclusively be explained by the CFTR activity in patients with severe genotype. A common Portuguese CFTR mutation (A561E) causes protein mislocalization in the endoplasmic reticulum similar to the most common CF mutation (ΔF508) and thus it should be possible to treat it with the same pharmacological strategies. Chronic treatment of CF airway epithelial cells with nanomolar concentrations of colchicine increased the chloride efflux via chloride channels other than CFTR, strengthening the notion that colchicine could be beneficial to CF patients. Successful in vitro transfection of CF airway epithelial cells with cationic vectors was possible with short incubation times. Heparin added at the end of the transfection incubation time could help to maintain the viability of the cells, without interfering with the transfection efficiency. It seems possible that heparin could be an adjuvant for non-viral mediated gene therapy.

Cystic fibrosis (CF) is one of the most common fatal inherited diseases, most prevalent among Caucasians. CF is caused by a mutation in the gene coding for the cystic fibrosis transmembrane conductance regulator (CFTR), which helps to create sweat, digestive juices, and airway surface liquid (ASL). The airways are covered with a thin layer of fluid, the airway surface liquid, in which the cilia bathe. Defective or absent CFTR leads to a defective water and ion transport in the epithelial cells, which results in viscous mucus, defective ciliary clearance, bacterial inflammation and tissue damage. The volume and composition of ASL are important in the pathogenesis of cystic fibrosis and it is therefore relevant to determine its composition. However, there are a number of difficulties in determining the ionic composition of the ASL due to its small volume. Literature data vary from very hypotonic to markedly hypertonic. These controversial data inspired the development of a simple method for determining the elemental composition of the ASL in different animal species and humans. Two techniques were developed to determine the composition of ASL, from which indirect information on chloride transport in airway epithelium can be obtained. In the first method, tissue is removed from the animals under anesthesia, frozen and analyzed in the frozen-hydrated state. In the second method, the ASL is collected with small dextran (Sephadex) beads; the dried beads are then analyzed by X-ray microanalysis. The Sephadex-bead method appears more accurate compared to the frozen-hydrated samples. Both methods were applied to collect tracheal and/or nasal fluid in pigs, normal and transgenic cystic fibrosis mice, the fluid covering the apical surface of normal bronchial cells (16HBE14o-) and a cystic fibrosis human bronchial cell line (CFBE41o-), and finally nasal fluid in healthy and diseased subjects. The ionic composition of the ASL was isotonic both in pigs and healthy human subjects. CF patients had much higher levels of Na and Cl ions than healthy subjects. The ASL under control conditions was hypotonic in mice and cell cultures, whereas the concentrations of Na and Cl ions in the species with the ΔF508 mutation or absent CFTR were significantly higher than in the corresponding controls. It was also demonstrated that the ionic composition of the ASL can be influenced by pharmacological treatment. The study confirms earlier findings that CFTR also is involved in bicarbonate transport. Mist tent therapy has been tested in the study of a treatment for CF patients, in order to hydrate the viscous mucus. But the effect of mist tent therapy on ion concentrations in the ASL appeared to be short-lived, although no patients became chronically colonized with pseudomonads while on nocturnal mist tent therapy.

The opportunistic bacterial pathogen Pseudomonas aeruginosa uses type IV pili (T4P) for adherence to, and rapid colonization of, surfaces via twitching motility. T4P are formed from thousands of pilin (PilA) subunits. Two groups of P. aeruginosa pilins were described previously (I and II), distinguished by protein length and sequence. PilA_I was glycosylated with an O-antigen subunit through the action of PilO/TfpO, encoded downstream of pilA_I. To determine if additional pilin variants existed, analysis of the pilin locus of >300 P. aeruginosa strains from a variety of environments was conducted. Three additional pilin alleles were discovered, each of which was invariantly associated with a unique, previously unidentified, downstream gene(s): pilA_III+tfpY, pilAIV+tfpW+tfpX, pilA_V+tfpZ. This survey also revealed that strains with group I T4P were more commonly associated with respiratory infections than strains with other pilins, suggesting that glycosylated T4P may confer a colonization advantage in this environment. The newly identified group IV pilin, represented by strain Pa5196, migrated aberrantly through SDS-PA gels, suggesting it was also glycosylated, a hypothesis confirmed by periodic acid-Schiff staining and mass spectrometry (MS) analyses. Disruption of Pa5196 O-antigen biosynthesis did not prevent the production of glycosylated pilins, demonstrating that these pilins were modified in a novel manner, unlike group I pilins. Using MS, nuclear magnetic resonance spectroscopy and site-directed mutagenesis, the Pa5196 pilins were shown to be uniquely modified with homo-oligosaccharides of mycobacterial-like α-1,5-D-arabinofuranose at multiple locations. Residues Thr64 and Thr66, located on the αβ-loop region of the protein, appear to be the preferred, but not exclusive sites of modification, each being modified with up to four D-Araf sugars. This region of the pilin is partially surface-exposed in the pilus, therefore modification of these sites may influence the surface chemistry of the fibre. Residues Ser81, Ser82, Ser85 and Ser89, located in the β-strand region, were also modified, mainly with mono- and disaccharides. Bioinformatic analyses and mutagenesis of TfpW suggest that this novel protein is an arabinosyltransferase necessary for PilA_IV modification. This research has increased our understanding of the complexity of this virulence factor, and may aid in development of new therapeutics for P. aeruginosa and mycobacterial infections.

Aim: The aim with the current study was to examine the physical activity levels (PAL) in a group of children and adolescents with cystic fibrosis (CF) and compare PAL-values between boys and girls. Further, the aim was to look at the total energy expenditure (TEE) estimated with an activity diary and measured with the accelerometer and compare values between these two measurement methods. Method: The sample consisted of 29 children and adolescents diagnosed with CF where PAL and TEE was measured during a three-day registration with activity diary and the ActiCal© accelerometer. The data was analyzed with the Mann-Whitney U-test. Results: The results indicated that the girls had a slightly higher median PAL-value than the boys. In addition, the range between the lowest and highest PAL-value was larger among the boys. The lowest PAL-value was lower among the boys than among the girls and the highest value was similar to the girls’ highest PAL-value. Overall, the subjective estimation of the PAL-values were slightly higher than what was objectively measured by the accelerometer. There was a slight difference between TEE estimated with the activity diary compared to the TEE measured by the accelerometer. There was no significant difference in PAL-values between boys and girls. Conclusion: There was no significant difference in PAL-value between boys and girls and that there was a slight difference in TEE estimated with the activity diary and measured with the accelerometer. Both methods of data collection are reliable enough when used together to produce a valid estimation of CF- children’s activity levels and TEE. The fact that both PAL and TEE values were higher in the activity diary may be because the participants perceived a higher level of physical activity then what was objectively measured with the accelerometer.

The opportunistic bacterial pathogen Pseudomonas aeruginosa uses type IV pili (T4P) for adherence to, and rapid colonization of, surfaces via twitching motility. T4P are formed from thousands of pilin (PilA) subunits. Two groups of P. aeruginosa pilins were described previously (I and II), distinguished by protein length and sequence. PilA_I was glycosylated with an O-antigen subunit through the action of PilO/TfpO, encoded downstream of pilA_I. To determine if additional pilin variants existed, analysis of the pilin locus of >300 P. aeruginosa strains from a variety of environments was conducted. Three additional pilin alleles were discovered, each of which was invariantly associated with a unique, previously unidentified, downstream gene(s): pilA_III+tfpY, pilAIV+tfpW+tfpX, pilA_V+tfpZ. This survey also revealed that strains with group I T4P were more commonly associated with respiratory infections than strains with other pilins, suggesting that glycosylated T4P may confer a colonization advantage in this environment. The newly identified group IV pilin, represented by strain Pa5196, migrated aberrantly through SDS-PA gels, suggesting it was also glycosylated, a hypothesis confirmed by periodic acid-Schiff staining and mass spectrometry (MS) analyses. Disruption of Pa5196 O-antigen biosynthesis did not prevent the production of glycosylated pilins, demonstrating that these pilins were modified in a novel manner, unlike group I pilins. Using MS, nuclear magnetic resonance spectroscopy and site-directed mutagenesis, the Pa5196 pilins were shown to be uniquely modified with homo-oligosaccharides of mycobacterial-like α-1,5-D-arabinofuranose at multiple locations. Residues Thr64 and Thr66, located on the αβ-loop region of the protein, appear to be the preferred, but not exclusive sites of modification, each being modified with up to four D-Araf sugars. This region of the pilin is partially surface-exposed in the pilus, therefore modification of these sites may influence the surface chemistry of the fibre. Residues Ser81, Ser82, Ser85 and Ser89, located in the β-strand region, were also modified, mainly with mono- and disaccharides. Bioinformatic analyses and mutagenesis of TfpW suggest that this novel protein is an arabinosyltransferase necessary for PilA_IV modification. This research has increased our understanding of the complexity of this virulence factor, and may aid in development of new therapeutics for P. aeruginosa and mycobacterial infections.

Objective: The aim of this study was to compare equations used to calculate BMR to values of BMR obtained from measurements in order to determine the most suitable equation to be used on children and adolescents with CF. Design: The participants where children and adolescents with CF. Data was collected from the division of Clinical Nutrition at Uppsala Akademiska Sjukhus. Age, weight, height, BMI, BMR, FFM and FM was recorded from 27 measurements. BMR from the participants collected from the indirect respiratory calorimetry was compared to the results obtained from five equations used to obtain a calculated value of BMR. Results: Tverskayas equation was best correlated with the measured BMR for the whole group. The equation underestimated BMR for the participants with a measured BMR over 1400 kcal/24h and overestimated BMR for participants with a measured BMR under 1400 kcal/24h. Similar results could be seen in the subgroups boys, girls and adolescents. Maffeis equation can be applied on the subgroup children. However, larger studies are needed to guarantee the results. Conclusion: It is desirable that an equation for calculating BMR would be developed that is specifically developed for children and adolescents with a low to normal BMI that takes into consideration a heightened metabolism and is therefore suitable for children and adolescents with CF. / Syfte: Att utifrån uppmätt BMR jämföra olika ekvationer för att hitta den mest lämpliga ekvationen för beräknandet av ett förutsätt värde av basalmetabolism hos en grupp barn och ungdomar med cystisk fibros. Metod: Deltagarna var barn och ungdomar med CF. Data till arbetet hämtades från enheten för klinisk nutrition vid Uppsala Akademiska sjukhus. Ålder, vikt, längd, BMI, BMR, FFM och FM samlades in från totalt 27 mättillfällen. Deltagarnas BMR, som uppmätts genom indirekta respiratorisk kalorimetri, jämfördes med fem ekvationer som räknar ut BMR utifrån olika antropometriska mått. Resultat: Tverskayas ekvation överensstämde bäst med uppmätt BMR i gruppen som helhet. Ekvationen underskattade dock BMR hos deltagare med ett BMR-behov över 1400 kcal/dygn och överskattade BMR hos deltagare med ett BMR-behov lägre än 1400 kcal/dygn. Liknande resultat sågs i undergrupperna pojkar, flickor och ungdomar. Maffeis ekvation skulle möjligen kunna tillämpas på gruppen barn, dock behöver större studier utföras för att kunna dra säkra slutsatser. Slutsats: Det vore önskvärt att det utvecklas en ekvation för BMR, särskilt utvecklad för barn och ungdomar med lågt till normalt BMI, som tar hänsyn till förhöjt BMR och som därmed lämpar sig särskilt för barn och ungdomar med CF.

The present study focuses on adaptive metabolic steps adopted by neutrophils during inflammation, particularly during their recruitment into the cystic fibrosis (CF) airways. In CF, we previously described that airway neutrophils are alive and undergo reprogramming, featuring notably the activation of the anabolic mTOR pathway. The present work is based on specific properties of soluble ligands derived from the receptor-binding domains (RBD) of retroviral glycoprotein envelopes, which can be used for the detection of metabolite transporters at the cell surface. First, we validated the use of this new set of markers for the identification and characterization of the metabolic phenotype of CF leukocytes obtained from distinct compartments (blood and sputum). Second, by studying the metabolite transporter expression on blood neutrophils from CF or rheumatoid arthritis patients and control subjects, we distinguished metabolic phenotypes characteristic of specific inflammatory states. Then, we compared metabolite transporter expression between CF blood and airway neutrophils and showed that neutrophils undergo significant metabolic adaptation upon recruitment into the lungs. Finally, we demonstrated that CF airway neutrophils display significant transcriptional modulation and that despite their metabolic reprogramming, they remain functionally competent, thus adding an additional angle of approach to neutrophil studies with regard to inflammation, notably during CF airway disease.

Aim: This study aimedto illuminateawareness of hygiene regimens andinfection control guidelines for cystic fibrosis (CF) care at Scandinavian cystic fibrosis centers (SCFCs)and Norwegian cystic fibrosis satellite teams (NCFSTs).Method: We modified a knowledge, attitudes and practices (KAP) surveyused earlier for CF and infection control care teams in the United States. Our modified questionnaire sought responses regarding sputum cultures, hand hygiene, education and infection control measures in hospital/at home. We distributed individual questionnaires to 8SCFCs and 10 NCFSTs. Awareness exceeding 75% per item was considered sufficient for clinical practice. Results: Respondents represented different disciplines atSCFCs with 61 (37%) and 30 (58%) at NCFSTs core team professionals (mainly doctors, nurses and physiotherapists).SCFC respondents demonstrated higher KAP (range 49-100%) than NCFST (range 23-100%). Both groups showed a high awareness of outcome expectancy for selected guideline components (80-100%), but only moderate awareness regarding familiarity. Additionally, both SCFC and NCFST respondents demonstrated greater awareness of items connected closely to practical care and medical treatment(73% and 80% versus 93% and 74% respectively). As opposed, SCFC and NCFST respondentsshowed lower awareness of guideline content(49% and 23% respectively).Conclusion: Becauseinfection control is a cornerstone in CF care, reducing the risk of cross infection among CF patients in health-and non-health caresettings requireshealth care professionals to maintain increased awareness of CF guidelines. This study revealed generally good knowledge of infection control measures. Importantly, bestcarerequires continious collaboration and educationfor health care professionals and patients regardingthe written guidelines for CF infection control. / Hensikt:Studiens hensikt var å belyse etterlevelse av regimer for hygiene og infeksjonskontroll innen cystisk fibrose (CF) omsorg ved de seks skandinaviske CF sentrene (SCFC) og ti norske CF satelitt team (NCFST). Metode:Individuelle spørreskjema ble distribuert til seks SCFC og ti NCFST. Spørreskjemaet var tilpasset fra en tidligere Knowledge, Attitude and Practice (KAP) spørreundersøkelse for helsepersonell innen CF og smittevern i USA med spørsmål angående ekspektoratprøver, håndhygiene, opplæring og tiltak for infeksjonskontroll i sykehus og hjemme. Etterlevelse &gt;75% for alle komponenter var vurdert som tilfredsstillende i klinisk praksis.Resultat: Det var 61 (37 %) respondenter fra SCFC, og 30 (58 %) fra NSCFT, hovedsakelig leger, sykepleiere og fysioterapeuter. SCFChar flere ulike profesjoner medulike erfaringer fra klinisk virksomhet, og respondentene viser et høyt KAP nivå på de fleste komponenter av undersøkelsen (100-49%). NCFST representerer kjerneteamprofesjoner, og respondentene viser moderat til høyt nivå av KAP på de fleste komponenter ved anbefalinger og undersøkelsen (100-23 %). Et nøkkelfunn var det høye nivå av forventet resultat hos begge studiegrupper for alle utvalgte komponenter av undersøkelsen (100-80%), i kontrast til et mer moderat nivå av kunnskap om anbefalingene. Tiltak med nær tilknytning til klinisk praksis og medisinsk behandling viste høyere grad av etterlevelse hos begge grupper (73 % og 80 %, 93 % og 74 %), mens kunnskap om innhold i skriftlige anbefalinger var lavere (49 % og 23 %). Konklusjon: Infeksjonskontroll er en hjørnestein i CF behandling, og betydningen av helsepersonells etterlevelse av anbefalinger for CF infeksjonskontroll er essensielt for å redusere risiko for kryssmitte mellom CF pasienter i og utenforsykehus. Studien har avdekket generelt god kunnskap om tiltak for infeksjonskontroll, men også et behov for kontinuerlig samarbeid og opplæringfor helsepersonell og pasienteri skriftlige anbefalinger for CF infeksjonskontroll. / <p>ISBN 978-91-86739-62-1</p>

Bakgrund. Föräldrar upplever oro över att cystisk fibros ska få deras barn att uppleva sig annorlunda. På grund av en långvarig relation upplever sjuksköterskor det känslomässigt krävande att vårda en patient med cystisk fibros i livets slutskede. Problem. Få kunskap om den som är drabbad av CF upplever att sjukdomen är ett hinder för att leva. Syfte. Beskriva upplevelser av att leva med cystisk fibros. Metod. En kvalitativ ansats har använts i form av en litteraturstudie med beskrivande syntes av publicerade artiklar. Resultat. Det framkommer att människor med cystisk fibros har varierande upplevelser av begränsningar och autonomi. De upplever sig som normala utifrån sin livsstil samtidigt som de känner sig annorlunda. Ungdomar upplever svårigheter i vardagen och genomgår en process för att acceptera sjukdomen som en del av livet. Att uppleva ansvar över sin vård och sitt liv skapar upplevelse av självständighet vilket underlättar för den drabbade att uppleva sig som normal. Slutsatser. Det är svårt att acceptera ett liv med CF, den drabbade upplever sig som annorlunda samtidigt som de kämpar för att uppleva sig som normala. Ytterligare forskning behövs om vårdrelationen mellan sjuksköterska och patienten som är drabbad av CF. / Background. Parents experience concern because cystic fibrosis will make their children experience themselves different. Because of a lengthy nurse-patient relationship the nurses‟ experience it is emotionally demanding to care for a patient with cystic fibrosis in the final phase of life. Problem. To get knowledge if the one affected by cystic fibrosis experience that the disease is an obstacle in life. Purpose. To describe experiences of living with cystic fibrosis. Method. A qualitative approach was used in the form of a literature study with a descriptive synthesis of published articles. Results. It appears that people with cystic fibrosis experience varied perceptions of limitations and autonomy. They experience themselves as normal as seen from their lifestyle at the same time as they felt different. Young people experience difficulties in their everyday life and go through a process of accepting the disease as a part of life. To experience responsibility over your care and life creates an experience of independence which makes it easier for the ones‟ affected to experience themselves as normal. Conclusions. It is difficult to accept a life with cystic fibrosis, the affected ones experience themselves as different at the same time as they struggle to experience themselves as normal. Further research is needed about the nurse-patient relationship between the nurse and the patient that is affected by cystic fibrosis.