The most common cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation is δF508 and this causes cystic fibrosis (CF). Animal models that recapitulate the human disease phenotype are critical to understanding pathophysiologic mechanisms in CF and developing therapies. New CF models in the pig and ferret have been generated that develop lung, pancreatic, liver, and intestinal pathologies that reflect disease in CF patients. Species-specific biology in the processing of CFTR has demonstrated that pig and mouse δF508-CFTR proteins are more effectively processed to the apical membrane of airway epithelia than human δF508-CFTR. The processing behavior of ferret wild-type (WT) and δF508-CFTR proteins remain unknown and such information is important to predicting the utility of a δF508-CFTR ferret. To this end, we sought to compare processing, membrane stability, and function of human and ferret WT- and δF508-CFTR proteins in a heterologous expression system using HT1080, HEK293T, BHK21, and Cos7 cells, as well as human and ferret CF polarized airway epithelia. Analysis of the protein processing and stability by metabolic pulse-chase and surface On-Cell Western blots revealed that WT-fCFTR half-life and membrane stability were increased relative to WT-hCFTR. Furthermore, in BHK21, Cos7, and CuFi cells, human and ferret δF508-CFTR processing was negligible, while low levels of processing of δF508-fCFTR could be seen in HT1080 and HEK293T cells. Only the WT-fCFTR, but not δF508-fCFTR, produced functional cAMP-inducible chloride currents in both CF human and ferret airway epithelia. Further elucidation of the mechanism responsible for elevated fCFTR protein stability may lead to new therapeutic approaches to augment CFTR function. These findings also suggest that generation of a ferret CFTRδF508/δF508 animal model may be useful. Furthermore, in the CFTR and CFTR+/+ ferret model we have characterized abnormalities in the bioelectric properties of the trachea, stomach, intestine and gallbladder of newborn CF ferrets. Short circuit current (ISC) analysis of CF and WT tracheas revealed the following similarities and differences: 1) amiloride sensitive sodium currents were similar between genotypes, 2) responses to 4,4'-diisothiocyano-2,2'-stilbene disulphonic acid (DIDS) were ~4-fold greater in CF animals, suggesting elevated baseline chloride transport through non-CFTR channels, and 3) as expected, there was a lack of IBMX/forskolin-stimulated and GlyH-101-inhibited currents in CF animals due to the lack of CFTR. CFTR mRNA and protein was present throughout all levels of the WT ferret and IBMX/forskolin-inducible ISC was only observed in WT animals. Interestingly, IBMX/forskolin-inducible intestinal ISC in WT animals was not inhibited by the CFTR inhibitor GlyH-101 or bumetanide. The luminal pH of the CF ferret stomach was significantly decreased relative to the controls, while both genotypes maintained near neutral pH along the length of the intestine. The WT stomach and gallbladder exhibited significantly enhanced IBMX/forskolin ISC responses and inhibition by GlyH-101 relative to CF samples. These findings demonstrate that multiple organs affected by disease in the CF ferret have bioelectric abnormalities consistent with the lack of cAMP-mediated chloride transport.

Cystic fibrosis (CF) is a lethal genetic disease that affects 30,000 people in the United States alone. While the disease affects organs throughout the body, it is the lung disease that is the primary cause of morbidity and mortality for people with the disease. CF lung disease is characterized by thick and sticky mucus that obstructs the airways, acute and chronic bacterial infections, and chronic inflammation and remodeling. Thanks to the creation of the CF pig, it is now possible to study the manifestations of CF lung disease at birth. The CF pig develops spontaneous lung disease, similar to that found in humans with CF, making it the ideal model for our studies. One of the critical findings that revealed in studies of the CF pig is that airway surface liquid (ASL) bactericidal activity is impaired in CF at birth, and this activity is pH dependent. Because infants and children with CF tend to suffer greater morbidity from respiratory viruses than non-CF infants and children, we sought to determine if ASL has antiviral activity and if that activity is reduced in newborn CF pigs. We found that pre-incubating either tracheal or nasal ASL from wild-type pigs reduced the infectivity of various recombinant viruses expressing an eGFP or GFP reporter gene. Those viruses include Sendai virus (SeV-eGFP), respiratory syncytial virus (RSV-GFP), the PR8 strain of influenza virus A (PR8-eGFP), and adenovirus (Ad-eGFP), indicating ASL has broad-spectrum antiviral activity. Nasal secretions from newborn CF pigs had strikingly reduced antiviral activity against SeV-eGFP and Ad-eGFP compared to nasal secretions from WT littermates. Unlike what was observed for ASL antibacterial activity, nasal secretion antiviral activity was not affected by pH, nor was it affected by bicarbonate concentration, one of the molecules that drives pH in the airways. However, when we mixed CF and WT nasal secretions at different ratios, we found the antiviral activity to follow a linear trend, with antiviral activity increasing as the percentage of WT nasal secretions increased. This suggests that one or more components of nasal secretions are found less abundantly in CF nasal secretions compared to WT nasal secretions, leading to reduced antiviral activity in CF. The CF pig has facilitated a much greater understanding of the early stages of CF lung disease. This model will allow us to determine what antiviral components are lacking in the CF airways and why they are reduced in CF.

Background: Cystic fibrosis (CF) is a genetically inherited disease which adversely affects the respiratory and gastrointestinal systems. Malnutrition is a major clinical problem in individuals with the disease. Nutritional interventions are warranted as improvements in nutritional status could improve the rates of morbidity and mortality associated with the disease. The review of the literature indicated the need to develop a behavioural-based nutrition prevention program in order for children to achieve CF dietary requirements and appropriate pancreatic enzyme replacement therapy.Methods: The intervention program, Go and Grow with CF, and nutrition and pancreatic enzyme knowledge and self-management questionnaires were developed for children with CF and their carers as part of this thesis. Social learning theory constructs which particularly assist children in achieving desirable behaviours were applied during the development of the Go and Grow with CF program. The program consisted of workshops and a home-based course.Fifty eight children with cystic fibrosis, aged 2 to 11 years, and their carers participated in a clinical trial that was designed to assess the effects of the Go and Grow with CF pilot program on knowledge, self-management, behaviour, dietary intake and body composition, using anthropometry. Process evaluation was conducted on the pilot program and on the clinic-wide implementation of the revised Go and Grow with CF program. The revised program included the Australian Pancreatic Enzyme Replacement Therapy Guidelines and the effects of fat-based dosing were assessed with a cohort of 29 children with CF-related pancreatic insufficiency aged 1 to 13 years.Results: Similar to the process evaluation of the pilot program, 100% of carers who completed the revised home-based course indicated that they would recommend Go and Grow with CF to other families ++ / with a child who has CF. The 'objective assessment of knowledge indicated a significant m improvement in' children's knowledge in the short-term. There were no statistically significant improvements in any of the other parameters assessed. The lack of significant improvements in self-management, behaviour, dietary intake and anthropometry may have been because the program had no effect, the parameters assessed or the instruments used (particularly the questionnaires) were not sufficiently sensitive, the sample size (which was determined by the CF population available) was too small or the duration of the intervention and follow-up was too short.Conclusion: Carers' unanimous recommendation of Go and Grow with CF, together with high levels of perceived learning, reported increase in confidence and improvement in children's knowledge in the short-term, indicate the benefits of the program.Although there was no statistically significant improvement in the anthropometric measurements after the intervention, 'the extensive data obtained during this study suggest that measurements of height and weight may underestimate the presence of poor nutritional status. It is likely that comprehensive assessments of body composition of children with CF would be useful in detecting mild degrees of malnutrition and in providing information about the effects of nutritional status on morbidity and mortality associated with the disease.Fat-based pancreatic enzyme replacement therapy dosing warrants further investigation given that parents had a strong preference for this method and that fat absorption remains abnormal in the majority of individuals who have pancreatic insufficiency. Evaluation of all pancreatic enzyme replacement therapy dosing methods are needed and this research suggests that dose should be assessed on a meal and snack basis, rather than just on daily intake, in order ++ / for levels of adherence to be examined.The apparent absence of a long-term effect of a single exposure to the program on knowledge suggests that regular, ongoing education and counselling is required by families to reinforce aspects related to the child's current stage of development and disease status.

"16th September 2002." Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). Bibliography: leaves xxix-li. This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway.

"16th September 2002." / Accompanying CD contains 2 MPEG clips with accompanying text, and a copy in PDF format of: Recovery of airway cystic fibrosis transmembrane conductance regulator function in mice with cystic fibrosis after single-dose lentivirus-mediated gene transfer / M. Limberis ... [et al.], published in Human gene therapy vol. 13 (2002). / Bibliography: leaves xxix-li. / xxvii, 213, li leaves : ill., plates (some col.) ; 30 cm. + 1 CD-ROM (4 3/4 in.) / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis focuses on modulating the physical barriers of the airway epithelium with mild detergents, so as to enhance gene transfer by a HIV-1 based lentivirus vector in vivo. The efficiency of the gene transfer was evaluated in the nasal airway of C57B1/6 mice using the Lac Z marker gene. This demonstration of lentivirus-mediated in vivo recovery of CFTR function in CF airway epithelium illustrated the potential of combining a pre-conditioning of the airway surface with a simple and brief HIV-1 based gene transfer vector exposure to produce therapeutic gene expression in the intact airway. / Thesis (Ph.D.)--University of Adelaide, Dept. of Paediatrics, 2003

Cystic fibrosis is an inherited pathological condition which can be treated but not cured and is ultimately life threatening. Those affected by cystic fibrosis require daily treatment to minimize the symptoms of the illness and retard the progression of pulmonary deterioration. An important component of the prophylactic therapy regime is chest physiotherapy which enhances the clearance of lung secretions. However, chest physiotherapy is time consuming and not always enjoyable, particularly for infants and toddlers. This study utilized an independent and repeated measures design to evaluate the effect of recorded music as an adjunct to daily routine chest physiotherapy on children's enjoyment, caregivers' enjoyment and caregivers' perception of time taken to complete the routine. Participants were caregivers of one or more children with cystic fibrosis who were aged between 4½ months and 24 months at the commencement of the clinical trial and required chest physiotherapy on a daily basis. / The children's cystic fibrosis care was managed by the Department of Thoracic Medicine at the Royal Children's Hospital in Victoria. Participants were randomly allocated into treatment and control groups and were involved in the study for 12 weeks. Participants in the treatment group were given the treatment tape: a specifically compiled music tape consisting of instrumental music and children's songs, newly composed for use as an adjunct to chest physiotherapy. Participants in the control group received no tape for the first 6 weeks, then received their choice from two commercially available, children's audiocassettes) both of which were familiar to the participants. Enjoyment and perception of time were assessed at the commencement of the trial and then twice more at 6-week intervals. After 6 weeks of using the treatment tape, children's and caregivers' enjoyment of chest physiotherapy increased significantly compared to no music. There was no change in perception of time taken to complete the chest physiotherapy after using the treatment tape. Use of the familiar music tape was not associated with significant increases in enjoyment for children or caregivers. Familiar music did not alter the perception of time taken to complete the routine. The results suggest that recorded music is an effective adjunct to daily chest physiotherapy which enhances caregivers' and children's enjoyment, particularly when that music is specifically compiled for use with the physiotherapy.

Genetic studies of lung disease in Cystic Fibrosis are faced with the challenge of identifying a severity measure that accounts for chronic disease progression and mortality attrition. Further, combining analyses across studies requires common phenotypes that are robust to study design and patient ascertainment. This thesis uses data from the North American Cystic Fibrosis Modifier Consortium (Canadian Consortium for CF Genetic Studies (CGS), Johns Hopkins University Twins and Siblings Study (TSS), and University of North Carolina/Case Western Reserve University Gene Modifier Study (GMS)), to calculate two novel phenotypes using age-specific CF percentile values of FEV1 (Forced Expiratory Volume in 1 second), with adjustment for CF age-specific mortality. The normalized residual, mortality adjusted (NoRMA) was designed for population based samples, while KNoRMA, using Kulich percentiles, is robust to sample ascertainment; both account for the effects of age-related disease progression and mortality attrition. NoRMA was computed for 2122 patients representing the Canadian CF population. KNoRMA was computed for these 2122 patients and also 1137 extreme phenotype patients in the GMS study and 1323 patients from multiple CF sib families in the TSS study. Phenotype was distributed in all three samples in a manner consistent with ascertainment differences, reflecting the lung disease severity of each individual in the underlying population. The new phenotype was highly correlated with the previously recommended mixed model phenotype1; 2, but computationally much easier and suited to studies with limited follow up time. As an example of its use, KNoRMA was used to test the association between locus variants in a previously published candidate gene, Transforming Growth Factor β1(TGFβ1), and lung function in CF, in an attempt to provide insight into discrepant results in the literature. A disease progression and mortality adjusted phenotype reduces the need for stratification or additional covariates, increasing statistical power and avoiding possible interpolation distortions.

Misfolding of membrane proteins as a result of mutations that disrupt their functions in substrate transport across the membrane or signal transduction is the cause of many significant human diseases. Yet, we still have a limited understanding of the direct consequences of these mutations on folding and function - a necessary step toward the rational design of corrective therapeutics. This thesis addresses the gap in understanding the residue-specific implications for folding through a series of experiments that utilize the cystic fibrosis transmembrane conductance regulator (CFTR) as a model in various contexts. We first examined the thermodynamic implications of mutations in the soluble nucleotide binding domain 1 (NBD1) of CFTR. We found that mutations can have a significant effect on thermodynamic stability that is masked in non-physiological conditions. Our studies were then focussed on a membrane-embedded hairpin CFTR fragment comprised of transmembrane segments 3 (TM3) and 4 (TM4) to evaluate the direct effects of mutations on folding in a systematic manner. It was found that the translocon-mediated membrane insertion of helices closely parallels a basic hydrophobic-aqueous partitioning event. This study was then extended to determine residue-specific effects on helix-helix association. We found that this process is not solely dependent on hydropathy, but there is a context dependence of these results with regard to residue position within the helix. Overall, these findings constitute a key step in relating mutation-derived effects on membrane protein folding to the underlying basis of human disease such as cystic fibrosis.

No description available.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains v, 111 p. : ill. Includes abstract. Includes bibliographical references (p. 67-76).