Energy expenditure and the body cell mass in Cystic Fibrosis

Poor nutritional status in patients with cystic fibrosis (CF) is associated with severe lung disease, and possible causative factors include inadequate intake, malabsorption, and increased energy requirements. Body cell mass (which can be quantified by measurement of total body potassium) provides an ideal standard for measurements of energy expenditure. The aim of this study was to compare resting energy expenditure (REE) in patients with CF with both predicted values and age-matched healthy children and to    determine whether REE was related to either nutritional status or pulmonary function. REE was measured by indirect calorimetry and body cell mass by scanning with total body potassium in 30 patients with CF (12 male, mean AGE = 13.07 ± 0.55 y) and 18 healthy children (six male, mean AGE = 12.56 ± 1.25 y). Nutritional status was expressed as a percentage of predicted total body potassium. Lung function was measured in the CF group by spirometry and expressed as the percentage of predicted forced expiratory volume in 1 s. Mean REE was significantly increased in the patients with CF compared with healthy children (119.3 ± 3.1% predicted versus 103.6 ± 5% predicted, P < 0.001) and, using multiple regression techniques, REE for total body potassium was significantly increased in patients with CF (P = 0.0001). There was no relation between REE and nutritional status or pulmonary disease status in the CF group. In conclusion, REE is increased in children and adolescents with CF but is not directly related to nutritional status or pulmonary disease.

Cystic fibrosis in children of the eastern Arabian peninsula : a clinical, spatial and genetic study

Aim: The aim of this thesis is to describe the process by which the inherited disease, cystic fibrosis, (CF) was recognised as an important clinical entity in the United Arab Emirates (UAE) and the Sultanate of Oman (Oman). It examines the clinical presentation of the first patients and assesses their degree of severity. Further, it describes the first studies carried out to determine the underlying CF mutations associated with the disease in the UAE and Oman. An estimate is offered of the birth frequency of the condition. Overall, the cultural, geographical and historical aspect of the societies in which the disease occurs is stressed. Methods: An initial literature search was carried out using Medline of any literature pertaining to the Arab World and CF. this was read and classified into the relevance to Arabs in general, the Middle East and then specifically the Arab (Persian) Gulf societies. Thereafter, a clinic was established at Tawam Hospital, Al Ain, UAE, for children presenting With chronic respiratory disease that could serve as a national referral centre. It was run by the Author as a service of the Paediatric Department of the UAE University Medical School. I sent a letter to every Paediatrician working in the UAE informing them of our clinic and offering our services for the diagnosis and management of chronic respiratory disease in children. This was based on the author's experience as a respiratory paediatrician in Australia and New Zealand and as the Professor of Paediatrics in the UAE. No such service then existed in the UAE. Funding was sought to establish a research programme and develop a molecular genetics laboratory in the UAE Medical School. A series of successful research applications provided the grants to commence the investigations. Once a small number of children had been identified as having CF from those referred to the respiratory clinic, the initial project was to assess and report their clinical presentation. Following this an early start was made on the identification of the mutations responsible. Once these were established an attempt was made to estimate the frequency of the condition at birth. Additional clinical studies revolved around assessing the severity of the condition that was associated with the main mutations that were identified. A clinical comparison was made with those with the mutation AF508 and the other main mutation, despite the obvious limitation of small numbers then available. Radiological assessment was made to evaluate the progression of the disease. The final aspect of the study was to assess patients from Oman and compare their findings and mutations with the neighbouring UAE. Based on information gained hypotheses are proposed regarding the spread of the gene mutation by population drift. Thesis outline: A literature review is presented in the form of a critique on the disease and a resume of the relevant aspects of the genetics of CF. Additionally, facts about the two countries' geography and history are presented. Finally, knowledge about CF mutations and population origins from other areas is presented. The second main section deals with the clinical features of the disorder as it presents in the UAE. Molecular findings are then presented and details of the common mutation found in Bedouin Arabs. Hypotheses are then presented based on the information gathered. Results: CF is not a rare disease in the Arab children of the UAE and Oman. These findings refute previous reports of CF being a rare or non-existent disease in Arabs. The condition presents with a severe clinical picture, with early colonisation of the respiratory tract with staphylococcus, haemophilus and pseudomonas organisms, even with conventional CF management practices in place. The CF mutation S549R is prevalent in Arabs of Bedouin stock, while AF508 is found in those of Baluch origin. The former may be descendants of Arabs who left southern Arabia and travelled to the Trucial Coast at the time of the destruction of the great dam at Marib. The origins of this mutation may lie in the area that corresponds to the modern Republic of Yemen. The latter groups are descendants of those who came originally from Baluchistan. It is hypothesised also that the ancestral home of the AF508 mutation may be in the geographical area now known as Baluchistan, that spans three separate modern political territories. The evidence presented supports the concept that the S549R mutation may be associated with a severe, if not the severest, clinical pattern recognised. It equates with that seen with the homozygous AF508 genotype. The absence of an additional mutation in the promoter region accounts for the different clinical pattern seen in previously described patients. Conclusions: There needs to be a major awareness of the presence of CF as a severe clinical disease in the children of the Gulf States. The clinical presentation and findings support the concept of under recognition of the disease. Climatic conditions put the children at special risk of hyponatraemia and electrolyte imbalance. The absence of surviving adults with the disease suggests premature deaths have occurred, but the high fertility rates have maintained the gene pool for this recessive disorder.

The pseudomonas aeruginosa secreted protein PA2934 decreases apical membrane expression of the cystic fibrosis transmembrane conductance regulator

We previously reported that Pseudomonas aeruginosa PA14 secretes a protein that can reduce the apical membrane expression of the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Here we report that we have used a proteomic approach to identify this secreted protein as PA2394, and we have named the gene cif, for CFTR inhibitory factor. We demonstrate that Cif is a secreted protein and is found associated with outer membrane-derived vesicles. Expression of Cif in Escherichia coli and purification of the C-terminal six-His-tagged Cif protein showed that Cif is necessary and sufficient to mediate the reduction in apical membrane expression of CFTR and a concomitant reduction in CFTR-mediated Cl− ion secretion. Cif demonstrates epoxide hydrolase activity in vitro and requires a highly conserved histidine residue identified in α/β hydrolase family enzymes to catalyze this reaction. Mutating this histidine residue also abolishes the ability of Cif to reduce apical membrane CFTR expression. Finally, we demonstrate that the cif gene is expressed in the cystic fibrosis (CF) lung and that nonmucoid isolates of P. aeruginosa show greater expression of the gene than do mucoid isolates. We propose a model in which the Cif-mediated decrease in apical membrane expression of CFTR by environmental isolates of P. aeruginosa facilitates the colonization of the CF lung by this microbe.

c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl− currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

Effect of bronchoalveolar lavage-directed therapy on pseudomonas aeruginosa infection and structural lung injury in children with cystic fibrosis

Context Early pulmonary infection in children with cystic fibrosis leads to increased morbidity and mortality. Despite wide use of oropharyngeal cultures to identify pulmonary infection, concerns remain over their diagnostic accuracy. While bronchoalveolar lavage (BAL) is an alternative diagnostic tool, evidence for its clinical benefit is lacking.

Objective To determine if BAL-directed therapy for pulmonary exacerbations during the first 5 years of life provides better outcomes than current standard practice relying on clinical features and oropharyngeal cultures.

Design, Setting, and Participants The Australasian Cystic Fibrosis Bronchoalveolar Lavage (ACFBAL) randomized controlled trial, recruiting infants diagnosed with cystic fibrosis through newborn screening programs in 8 Australasian cystic fibrosis centers. Recruitment occurred between June 1, 1999, and April 30, 2005, with the study ending on December 31, 2009.

Interventions BAL-directed (n=84) or standard (n=86) therapy until age 5 years. The BAL-directed therapy group underwent BAL before age 6 months when well, when hospitalized for pulmonary exacerbations, if Pseudomonas aeruginosa was detected in oropharyngeal specimens, and after P aeruginosa eradication therapy. Treatment was prescribed according to BAL or oropharyngeal culture results.

Main Outcome Measures Primary outcomes at age 5 years were prevalence of P aeruginosa on BAL cultures and total cystic fibrosis computed tomography (CF-CT) score (as a percentage of the maximum score) on high-resolution chest CT scan.

Results Of 267 infants diagnosed with cystic fibrosis following newborn screening, 170 were enrolled and randomized, and 157 completed the study. At age 5 years, 8 of 79 children (10%) in the BAL-directed therapy group and 9 of 76 (12%) in the standard therapy group had P aeruginosa in final BAL cultures (risk difference, −1.7% [95% confidence interval, −11.6% to 8.1%]; P=.73). Mean total CF-CT scores for the BAL-directed therapy and standard therapy groups were 3.0% and 2.8%, respectively (mean difference, 0.19% [95% confidence interval, −0.94% to 1.33%]; P=.74).

Conclusion Among infants diagnosed with cystic fibrosis, BAL-directed therapy did not result in a lower prevalence of P aeruginosa infection or lower total CF-CT score when compared with standard therapy at age 5 years.

Trial Registration anzctr.org.au Identifier: ACTRN12605000665639

Prospective evaluation of respiratory exacerbations in children with cystic fibrosis from newborn screening to 5 years of age

Background Newborn screening allows novel treatments for cystic fibrosis (CF) to be trialled in early childhood before irreversible lung injury occurs. As respiratory exacerbations are a potential trial outcome variable, we determined their rate, duration and clinical features in preschool children with CF; and whether they were associated with growth, lung structure and function at age 5 years. Methods: Respiratory exacerbations were recorded prospectively in Australasian CF Bronchoalveolar Lavage trial subjects from enrolment after newborn screening to age 5 years, when all participants underwent clinical assessment, chest CT scans and spirometry. Results 168 children (88 boys) experienced 2080 exacerbations, at an average rate of 3.66 exacerbations per person-year; 80.1% were community managed and 19.9% required hospital admission. There was an average increase in exacerbation rate of 9% (95% CI 4% to 14%; p<0.001) per year of age. Exacerbation rate differed by site (p<0.001) and was 26% lower (95% CI 12% to 38%) in children receiving 12 months of prophylactic antibiotics. The rate of exacerbations in the first 2 years was associated with reduced forced expiratory volume in 1 s z scores. Ever having a hospitalmanaged exacerbation was associated with bronchiectasis (OR 2.67, 95% CI 1.13 to 6.31) in chest CT scans, and lower weight z scores at 5 years of age (coefficient -0.39, 95% CI -0.74 to -0.05). Conclusions Respiratory exacerbations in young children are markers for progressive CF lung disease and are potential trial outcome measures for novel treatments in this age group.

Costs of bronchoalveolar lavage-directed therapy in the first 5 years of life for children with cystic fibrosis.

To determine whether bronchoalveolar lavage (BAL)-directed therapy for infants and young children with cystic fibrosis (CF), rather than standard therapy, was justified on the grounds of a decrease in average costs and whether the use of BAL reduced treatment costs associated with hospital admissions.

Pseudomonas aeruginosa genotypes acquired by children with cystic fibrosis by age 5-years

BACKGROUND: We describe Pseudomonas aeruginosa acquisitions in children with cystic fibrosis (CF) aged ≤5-years, eradication treatment efficacy, and genotypic relationships between upper and lower airway isolates and strains from non-CF sources. METHODS: Of 168 CF children aged ≤5-years in a bronchoalveolar lavage (BAL)-directed therapy trial, 155 had detailed microbiological results. Overall, 201/271 (74%) P. aeruginosa isolates from BAL and oropharyngeal cultures were available for genotyping, including those collected before and after eradication therapy. RESULTS: Eighty-two (53%) subjects acquired P. aeruginosa, of which most were unique strains. Initial eradication success rate was 90%, but 36 (44%) reacquired P. aeruginosa, with genotypic substitutions more common in BAL (12/14) than oropharyngeal (3/11) cultures. Moreover, oropharyngeal cultures did not predict BAL genotypes reliably. CONCLUSIONS: CF children acquire environmental P. aeruginosa strains frequently. However, discordance between BAL and oropharyngeal strains raises questions over upper airway reservoirs and how to best determine eradication in non-expectorating children.

Substrates and inhibitors of human multidrug resistance associated proteins and the implications in drug development

Human contains 49 ATP-binding cassette (ABC) transporter genes and the multidrug resistance associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP4/ABCC4, MRP5/ABCC5, MRP6/ABCC6, MRP7/ABCC10, MRP8/ABCC11 and MRP9/ABCC12) belong to the ABCC family which contains 13 members. ABCC7 is cystic fibrosis transmembrane conductance regulator; ABCC8 and ABCC9 are the sulfonylurea receptors which constitute the ATP-sensing subunits of a complex potassium channel. MRP10/ABCC13 is clearly a pseudo-gene which encodes a truncated protein that is highly expressed in fetal human liver with the highest similarity to MRP2/ABCC2 but without transporting activity. These transporters are localized to the apical and/or basolateral membrane of the hepatocytes, enterocytes, renal proximal tubule cells and endothelial cells of the blood-brain barrier. MRP/ABCC members transport a structurally diverse array of important endogenous substances and xenobiotics and their metabolites (in particular conjugates) with different substrate specificity and transport kinetics. The human MRP/ABCC transporters except MRP9/ABCC12 are all able to transport organic anions, such as drugs conjugated to glutathione, sulphate or glucuronate. In addition, selected MRP/ABCC members may transport a variety of endogenous compounds, such as leukotriene C(4) (LTC(4) by MRP1/ABCC1), bilirubin glucuronides (MRP2/ABCC2, and MRP3/ABCC3), prostaglandins E1 and E2 (MRP4/ABCC4), cGMP (MRP4/ABCC4, MRP5/ABCC5, and MRP8/ABCC11), and several glucuronosyl-, or sulfatidyl steroids. In vitro, the MRP/ABCC transporters can collectively confer resistance to natural product anticancer drugs and their conjugated metabolites, platinum compounds, folate antimetabolites, nucleoside and nucleotide analogs, arsenical and antimonial oxyanions, peptide-based agents, and in concert with alterations in phase II conjugating or biosynthetic enzymes, classical alkylating agents, alkylating agents. Several MRP/ABCC members (MRPs 1-3) are associated with tumor resistance which is often caused by an increased efflux and decreased intracellular accumulation of natural product anticancer drugs and other anticancer agents. Drug targeting of these transporters to overcome MRP/ABCC-mediated multidrug resistance may play a role in cancer chemotherapy. Most MRP/ABCC transporters are subject to inhibition by a variety of compounds. Based on currently available preclinical and limited clinical data, it can be expected that modulation of MRP members may represent a useful approach in the management of anticancer and antimicrobial drug resistance and possibly of inflammatory diseases and other diseases. A better understanding of their substrates and inhibitors has important implications in development of drugs for treatment of cancer and inflammation.

All around Patrick

With the help from the RCH Education Institute which supports students with health conditions, Patrick Mackenzie, who has cystic fibrosis, is able to attend primary school.

A Pseudomonas aeruginosa toxin that hijacks the host ubiquitin proteolytic system

Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic pathogen chronically infecting the lungs of patients with chronic obstructive pulmonary disease (COPD), pneumonia, cystic fibrosis (CF), and bronchiectasis. Cif (PA2934), a bacterial toxin secreted in outer membrane vesicles (OMV) by P. aeruginosa, reduces CFTR-mediated chloride secretion by human airway epithelial cells, a key driving force for mucociliary clearance. The aim of this study was to investigate the mechanism whereby Cif reduces CFTR-mediated chloride secretion. Cif redirected endocytosed CFTR from recycling endosomes to lysosomes by stabilizing an inhibitory effect of G3BP1 on the deubiquitinating enzyme (DUB), USP10, thereby reducing USP10-mediated deubiquitination of CFTR and increasing the degradation of CFTR in lysosomes. This is the first example of a bacterial toxin that regulates the activity of a host DUB. These data suggest that the ability of P. aeruginosa to chronically infect the lungs of patients with COPD, pneumonia, CF, and bronchiectasis is due in part to the secretion of OMV containing Cif, which inhibits CFTR-mediated chloride secretion and thereby reduces the mucociliary clearance of pathogens.

The ΔF508-CFTR mutation results in increased biofilm formation by Pseodomonas aeruginosa by increasing iron availability

Enhanced antibiotic resistance of Pseudomonas aeruginosa in the cystic fibrosis (CF) lung is thought to be due to the formation of biofilms. However, there is no information on the antibiotic resistance of P. aeruginosa biofilms grown on human airway epithelial cells or on the effects of airway cells on biofilm formation by P. aeruginosa. Thus we developed a coculture model and report that airway cells increase the resistance of P. aeruginosa to tobramycin (Tb) by >25-fold compared with P. aeruginosa grown on abiotic surfaces. Therefore, the concentration of Tb required to kill P. aeruginosa biofilms on airway cells is 10-fold higher than the concentration achievable in the lungs of CF patients. In addition, CF airway cells expressing ΔF508-CFTR significantly enhanced P. aeruginosa biofilm formation, and ΔF508 rescue with wild-type CFTR reduced biofilm formation. Iron (Fe) content of the airway in CF is elevated, and Fe is known to enhance P. aeruginosa growth. Thus we investigated whether enhanced biofilm formation on ΔF508-CFTR cells was due to increased Fe release by airway cells. We found that airway cells expressing ΔF508-CFTR released more Fe than cells rescued with WT-CFTR. Moreover, Fe chelation reduced biofilm formation on airway cells, whereas Fe supplementation enhanced biofilm formation on airway cells expressing WT-CFTR. These data demonstrate that human airway epithelial cells promote the formation of P. aeruginosa biofilms with a dramatically increased antibiotic resistance. The ΔF508-CFTR mutation enhances biofilm formation, in part, by increasing Fe release into the apical medium.

Chemotoxicity of doxorubicin and surface expression of P-glycoprotein (MDR1) is regulated by the Pseudomonas aeruginosa toxin Cif

P-glycoprotein (Pgp), a member of the adenosine triphosphate-binding cassette (ABC) transporter superfamily, is a major drug efflux pump expressed in normal tissues, and is overexpressed in many human cancers. Overexpression of Pgp results in reduced intracellular drug concentration and cytotoxicity of chemotherapeutic drugs and is thought to contribute to multidrug resistance of cancer cells. The involvement of Pgp in clinical drug resistance has led to a search for molecules that block Pgp transporter activity to improve the efficacy and pharmacokinetics of therapeutic agents. We have recently identified and characterized a secreted toxin from Pseudomonas aeruginosa, designated cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif). Cif reduces the apical membrane abundance of CFTR, also an ABC transporter, and inhibits the CFTR-mediated chloride ion secretion by human airway and kidney epithelial cells. We report presently that Cif also inhibits the apical membrane abundance of Pgp in kidney, airway, and intestinal epithelial cells but has no effect on plasma membrane abundance of multidrug resistance protein 1 or 2. Cif increased the drug sensitivity to doxorubicin in kidney cells expressing Pgp by 10-fold and increased the cellular accumulation of daunorubicin by 2-fold. Thus our studies show that Cif increases the sensitivity of Pgp-overexpressing cells to doxorubicin, consistent with the hypothesis that Cif affects Pgp functional expression. These results suggest that Cif may be useful to develop a new class of specific inhibitors of Pgp aimed at increasing the sensitivity of tumors to chemotherapeutic drugs, and at improving the bioavailability of Pgp transport substrates.

An appraisal of the histopathological assessment of liver fibrosis.

One of the most important aspects of the histopathological assessment of liver biopsies in the setting of chronic liver disease is determination of the degree of fibrosis and architectural change. Most of the work in this regard has been concerned with chronic viral hepatitis. This article attempts to assess critically our current and historical biopsy practice, from subjective fibrosis scoring systems to biopsy sample size; and the appropriate use of the data that scoring systems
generate in the research and clinical setting. An understanding of the limitations of each of the components of the fibrosis assessment process can help to devise appropriate protocols to ensure that the information obtained is optimised, and its degree of reliability appreciated. It is only from this starting point that recently promulgated antifibrotic medications and ‘‘non-invasive’’ liver fibrosis assessment techniques can be evaluated properly.