An assessment of the alcohol content of medicines commonly used in paediatric liver and cystic fibrosis patients

Poster session - Paediatric cystic fibrosis and liver patients can be prescribed a number of different pharmaceutical preparations, often in liquid form - It is not uncommon for alcohol to be present in liquid preparations, often as a solvent - Although the quantity of alcohol present can be low, patients taking a number of alcohol-containing preparations may be at risk of toxicity - It has been found that only a few of the preparations used for both paediatric cystic fibrosis and liver patients contain alcohol - The quantity of alcohol present in these preparations is low and should not cause toxicity, even when the products are used in combination

Effects of recombinant human DNase and hypertonic saline on airway inflammation in children with cystic fibrosis

Recombinant human DNase (rhDNase) is an established treatment in cystic fibrosis (CF), but it may liberate cationic mediators bound to DNA in the airways. An alternative mucolytic therapy is hypertonic saline (HS); however, HS may potentiate neutrophilic inflammation. We compared the effect of rhDNase and HS on cationic proinflammatory mediators in CF sputum. In a randomized, crossover trial, 48 children with CF were allocated consecutively to 12 weeks of once-daily 2.5 mg rhDNase, alternate-day 2.5 mg rhDNase, and twice-daily 7% HS. Sputum levels of total interleukin-8 (IL-8), free IL-8, myeloperoxidase, eosinophil cationic protein, and neutrophil elastase (NE) activity were measured before and after each treatment. The change in mediator levels from baseline with daily rhDNase and HS was not significant; however, with alternate-day rhDNase, there was an increase in free IL-8. When changes in mediator levels with daily rhDNase were compared with alternate-day rhDNase and HS, no significant differences were detected. Only changes in NE activity were associated with changes in lung function. In summary, we were unable to show that rhDNase or HS promote airway inflammation in CF.

Understanding the link between transglutaminase and the induction of fibrosis in cystic fibrosis (CF)

The emerging role of the multifunctional enzyme, Transglutaminase 2 (TG2) in Cystic Fibrosis (CF) has been linked to its increased expression and intracellular transamidating activity. However, a full understanding of the molecular mechanisms involved still remains unclear despite numerous studies that have attempted to delineate this process. These mechanisms include the NFκB and TGFβ1 pathway amongst others. This study reveals for the first time that the development of fibrosis in CF is due to a TG2-driven epithelial to mesenchymal transition (EMT) via a mechanism involving the activation of the pro-fibrotic cytokine TGFβ1. Using a human ΔF508/W1282X CFTR CF mutant bronchial cell (IB3-1), its CFTR corrected “add-back” cell (C38) as well as a primary human bronchial epithelial cell (HBEC), elevated TG2 levels in the CFTR mutant IB3 cell were shown to activate latent TGFβ1 leading to increased levels found in the culture medium. This activation process was blocked by the presence of cell-permeable and impermeable TG2 inhibitors while inhibition of TGFβ1 receptors blocked TG2 expression. This demonstrates the direct link between TG2 and TGFβ1 in CF. The presence of active cell surface TG2 correlated with an increase in the expression of EMT markers, associated with the CF mutant cells, which could be blocked by the presence of TG2 inhibitors. This was mimicked using the “addback” C38 cell and the primary human bronchial epithelial cell, HBEC, where an increase in TG2 expression and activity in the presence of TGFβ1 concurred with a change in cell morphology and an elevation in EMT marker expression. Conversely, a knockdown of TG2 in the CF mutant IB3 cells illustrated that an inhibition of TG2 blocks the increase in EMT marker expression as well as causing an increase in TEER measurement. This together with an increase in the migration profile of the CF mutant IB3 cell against the “add-back” C38 cell suggests that TG2 drives a mesenchymal phenotype in CF. The involvement of TG2 activated TGFβ1 in CF was further demonstrated with an elevation/inhibition of p- SMAD 2 and 3 activation in the presence of TGFβ1/TG2 cell-permeable/impermeable inhibitors respectively. The use of a comparative airway cell model where bronchial epithelial cells were cultured at the air liquid interface (ALI) confirmed the observations in submerged culture depicting the robustness of the model and reiterated the importance of TG2 in CF. Using a CFTR corrector combined with TG2 inhibitors, this study showed that the correction and stabilisation of the ΔF508 CFTR mutation in the mutant cell forged an increase in matured CFTR copies trafficking to the apical surface by circumventing proteosomal degradation. Thus the results presented here suggests that TG2 expression is elevated in the CFTR mutant bronchial cell via a TGFβ1 driven positive feedback cycle whereby activation of latent TGFβ1 by TG2 leads in turn to an elevation in its own expression by TGFβ1. This vicious cycle then drives EMT in CF ultimately leading to lung remodelling and fibrosis. Importantly, TG2 inhibition blocks TGFβ1 activation leading to an inhibition of EMT and further blocks the emerging fibrosis, thus stabilizing and supporting the maturation, trafficking and conductance of CFTR channels at the apical surface.

Adherence to treatment in children and adolescents with cystic fibrosis:a cross-sectional, multi-method study investigating the influence of beliefs about treatment and parental depressive symptoms

Background: Adherence to treatment is often reported to be low in children with cystic fibrosis. Adherence in cystic fibrosis is an important research area and more research is needed to better understand family barriers to adherence in order for clinicians to provide appropriate intervention. The aim of this study was to evaluate adherence to enzyme supplements, vitamins and chest physiotherapy in children with cystic fibrosis and to determine if any modifiable risk factors are associated with adherence. Methods: A sample of 100 children (≤18 years) with cystic fibrosis (44 male; median [range] 10.1 [0.2-18.6] years) and their parents were recruited to the study from the Northern Ireland Paediatric Cystic Fibrosis Centre. Adherence to enzyme supplements, vitamins and chest physiotherapy was assessed using a multi-method approach including; Medication Adherence Report Scale, pharmacy prescription refill data and general practitioner prescription issue data. Beliefs about treatments were assessed using refined versions of the Beliefs about Medicines Questionnaire-specific. Parental depressive symptoms were assessed using the Center for Epidemiologic Studies Depression Scale. Results: Using the multi-method approach 72% of children were classified as low-adherers to enzyme supplements, 59% low-adherers to vitamins and 49% low-adherers to chest physiotherapy. Variations in adherence were observed between measurement methods, treatments and respondents. Parental necessity beliefs and child age were significant independent predictors of child adherence to enzyme supplements and chest physiotherapy, but parental depressive symptoms were not found to be predictive of adherence. Conclusions: Child age and parental beliefs about treatments should be taken into account by clinicians when addressing adherence at routine clinic appointments. Low adherence is more likely to occur in older children, whereas, better adherence to cystic fibrosis therapies is more likely in children whose parents strongly believe the treatments are necessary. The necessity of treatments should be reinforced regularly to both parents and children.

Nicotine exposure reduces cell viability but induces anti-inflammatory effects in human cystic fibrosis bronchial epithelial cells

Background: Mouse models of cystic fibrosis (CF) fail to truly represent the respiratory pathology. We have consequently developed human airways cell culture models to address this. The impact of cigarette smoke within the CF population is well documented, with exposure being known to worsen lung function. As nicotine is often perceived to be a less harmful component of tobacco smoke, this research aimed to identify its effects upon viability and inflammatory responses of CF (IB3-1) and CF phenotype corrected (C38) bronchial epithelial cells. Methods: IB3-1 and C38 cell lines were exposed to increasing concentrations of nicotine (0.55-75μM) for 24 hours. Cell viability was assessed via Cell Titre Blue and the inflammatory response with IL-6 and IL-8 ELISA. Results: CF cells were more sensitive; nicotine significantly (P<0.05) reduced cell viability at all concentrations tested, but failed to have a marked effect on C38 viability. Whilst nicotine induced anti-inflammatory effects in CF cells with a significant reduction in IL-6 and IL-8 release, it had no effect on chemokine release by C38 cells. Conclusion: CF cells may be more vulnerable to inhaled toxicants than non-CF cells. As mice lack a number of human nicotinic receptor subunits and fail to mimic the characteristic pathology of CF, these data emphasise the importance of employing relevant human cell lines to study a human-specific disease.

A novel regulatory role for tissue transglutaminase in epithelial-mesenchymal transition in cystic fibrosis

Cystic fibrosis (CF) is a genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) for which there is no overall effective treatment. Recent work indicates tissue transglutaminase (TG2) plays a pivotal intracellular role in proteostasis in CF epithelia and that the pan TG inhibitor cysteamine improves CFTR stability. Here we show TG2 has another role in CF pathology linked with TGFβ1 activation and signalling, induction of epithelial-mesenchymal transition (EMT), CFTR stability and induction of matrix deposition. We show that increased TG2 expression in normal and CF bronchial epithelial cells increases TGFβ1 levels, promoting EMT progression, and impairs tight junctions as measured by Transepithelial Electric Resistance (TEER) which can be reversed by selective inhibition of TG2 with an observed increase in CFTR stability. Our data indicate that selective inhibition of TG2 provides a potential therapeutic avenue for reducing fibrosis and increasing CFTR stability in CF.

A multi-faceted diagnostic approach to lung infections in patients with cystic fibrosis

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

Presumptive Identification of the Fungal Communities in Cystic Fibrosis Patients' Sputum Using Amplicon Length Heterogeneity Polymerase Chain Reaction

This thesis would not have been possible without the aid of my family, friends, laboratory members, and professors. First and foremost, I would like to thank Dr. Kalai Mathee for allowing me to enter her lab in August 2007 and enabling to embark on this journey. This experience has transformed me into more mature scientist, teaching me how to ask the right questions and the process needed to solve them. I would also like to acknowledge Dr. Lisa Schneper. She has helped me throughout the whole process, by graciously giving me input at every step of the way. I would like to express gratitude to Dr. Jennifer Richards for all her input in writing the thesis. She has been a great teacher and being in her class has been a pleasure. Moreover, I would like to thank all the committee members for their constructive criticism throughout the process. When I entered the lab in August, there was one person who literally was by my side, Melissa Doud. Without your input and guidance I would not have even been able to do these experiments. I would also like to thank you and Dr. Light for allowing me to meet some cystic fibrosis patients. It has allowed me to put a face on the disease, and help the patients' fight. For a period before I had entered the lab, Ms. Doud had an apprentice, who started the fungal aspect of the project, Caroline Veronese. Her initial work has enabled me to prefect the protocols and complete the ITS 1 region.One very unique aspect about Dr. Mathee's lab is the camaraderie. I would like to thank all the lab members for the good times in and out of the lab. These individuals have been able to make smile and laugh in parties and lab meetings. I would like to individually thank Balachandar Dananjeyan, Deepak Balasubramanian, and V arinderpal Singh Pannu for all the PCR help and Natalie Maricic for the laughs and being a great classmate. Last, but not least, I would like to acknowledge my family and friends for their support and keeping me sane: Cecilia, my mother, Mohammad, my father, Amir, my older brother, Billal, my younger brother, Ouday Akkari and Stephanie De Bedout, my best friends.

Investigating an Alternative Treatment to Cystic Fibrosis: Testing the Effect of Panax ginseng C.A. Meyer extracts on Pseudomonas aeruginosa

The predominant pathogen found in the lungs of cystic fibrosis (CF) patients is Pseudomonas aeruginosa. The success of the infection is partially due to virulence factor production, which is regulated by quorum sensing (QS) signaling. Currently, antibiotics are used to treat the infection, but resistant forms of P. aeruginosa have evolved, necessitating alternative treatments. Previous animal studies showed that treatment with extracts from the Chinese herb Panax ginseng C.A. Meyer reduced bacterial load resulting in a favorable immune response. It is hypothesized that ginsenosides, the major bioactive compounds in ginseng, is responsible for this effect. This study explores the role of ginseng extracts in attenuating P. aeruginosa virulence. A sequential extraction was performed using hexane, methylene chloride, methanol, and water. High performance liquid chromatography (HPLC) analysis showed the methanol and water ginseng extracts contained the known ginsenosides Rb1, Rb2, Rc, Rd, Re, and Rg1• All extracts were tested on biomonitor strains of Agrobacterium tumefaciens,Chromobacterium violaceum, and P. aeruginosa. Antibacterial and anti-QS activity were assessed using a disc diffusion assay. This was then followed by thin layer chromatography (TLC) bioautographic assay to further separate active compounds. The hexane and dichloromethane extracts, that lacked ginsenosides, displayed antibacterial activity against C. violaceum, whereas methanol and water extracts had anti-QS activity. The results of the bioassay with the pure ginsenoside standards showed that they lack antibacterial or anti-QS activity. Our results indicate that there are bioactive compounds, other than ginsenosides, that are the cause of antibacterial effects and anti-QS in the ginseng extracts.

Potential Application of Phage Therapy Against Pseudomonas aeruginosa Biofilm Infection in Cystic Fibrosis Patients

Majority of the microbial activity in humans is in the form of biofilms i.e. an Exopolysaccharide-enclosed bacterial mass. Unlike planktonic cells and the cells on the surface of the biofilm, the biofilm-embedded cells are more resistant to the effects of the antibiotics and the host cellular defense mechanisms. A combination of biofilm growth and inherent resistance prevents effective antibiotics treatment of Pseudomonas aeruginosa infections including those in patients with cystic fibrosis. This has lead to an increasing interest in alternative modalities of treatment. Thus, phages that multiply in situ, only in the presence of susceptible hosts can be used as natural, self-limiting, and deeply penetrating antibacterial agents. The objective of this study is to identify effective phages against a collection of P. aeruginosa isolates (PCOR strains) including the prototype PAOl and the isogenic constitutively alginate-producing PD0300 strains.These PCOR strains were tested against six phages (P105, P134, P140, P168, P175B and P182). Analysis shows 69 % of the PCOR isolates are sensitive and the rest are resistant to all six phages. These phages were then tested for their ability to inhibit biofilm formation using a modified biofilm assay. The analysis demonstrated that the sensitive strains showed increased resistance but none of the sensitive strains from the initial screening were resistant. Using the minimum biofilm eradication concentration (MBEC) assay for biofilm formation, the biofilm eradication ability of the phages was tested. The data showed that a higher volume of phage was required to eradicate preformed biofilms than the volume required to prevent colonization of planktonic cells. This data supports the idea of phage therapy more as a prophylactic treatment.

Molecular Mechanisms Involved in Regulating the Mucoid-to-Nonmucoid Conversion of Pseudomonas aeruginosa Associated with Cystic Fibrosis Patients

Pseudomonas aeruginosa is an opportunistic pathogen that has received attention because of its close association with cystic fibrosis (CF). Chronic pulmonary infection with the mucoid P. aeruginosa is the leading cause of mortality in CF patients. This bacterium has the ability to sense and adapt to the harsh environment in the CF lung by converting from a nonmucoid to a mucoid state. The mucoid phenotype is caused by overproduction of a polysaccharide called alginate. Alginate production is regulated by the algT/U operon containing five genes, algT/U-mucA-mucB-mucC-mucD. Alginate overproduction in CF isolates has been partially attributed to a loss-of-function mutation in mucA that results in the overexpression of algT. This mucoid phenotype is unstable, reverting to the nonmucoid form when the isolates are cultured outside of the CF lung. This study was undertaken to determine the mechanisms involved in the conversion from the mucoid to the nonmucoid form. Thirty-six spontaneous nonmucoid variants of a known mucoid isolate with a mucA mutation were analyzed. Ten of these isolates were complemented in trans by plasmids containing the algT operon and the algT gene. Chromosomal DNA was extracted and the mucA and algT genes were amplified by the polymerase chain reaction. Sequence analysis of the genes showed that these mutants retained the original mucA mutation but acquired secondary mutations in the algT gene.

Role of AmpR and alginate production in Pseudomonas aeruginosa biofilm antibiotic resistance associated with cystic fibrosis

Pseudomonas aeruginosa is an ubiquitous Gram-negative opportunistic pathogen that is commonly found in nosocomial infections, immunocompromised patients and burn victims. In addition, P. aeruginosa colonizes the lungs of cystic fibrosis patients, leading to chronic infection, which inevitably leads to their demise. In this research, I analyzed the factors contributing to P. aeruginosa antibiotic resistance, such as the biofilm mode of growth, alginate production, and 13-lactamase synthesis. Using the biofilm eradication assay (MBEC™ assay), I exposed P. aeruginosa to B-lactams (piperacillin, ceftazidime, and cefotaxime ), aminoglycosides ( amikacin, tobramycin and gentamicin), and a fluoroquinolone ( ciprofloxacin) at various concentrations. I analyzed the effects of biofilm on P. aeruginosa antibiotic resistance, and confirmed that the parent strain PAO 1 biofilms cells were > 100 times more resistant than planktonic (freefloating) cells. The constitutively alginate-producing strain PDO300 exhibited an altered resistance pattern as compared to the parent strain P AO 1. Finally, the role of AmpR, the regulator of ampC-encoded 13-lactamase expression was analyzed by determining the resistance of the strain carrying a mutation in the ampR gene and compared to the parent strain PAOl. It was confirmed that the loss of ampR contributes to increased antibiotic resistance.

Combination of 16S rRNA variable regions provides a detailed analysis of bacterial community dynamics in the lungs of cystic fibrosis patients

Chronic bronchopulmonary bacterial infections remain the most common cause of morbidity and mortality among patients with cystic fibrosis (CF). Recent community sequencing work has now shown that the bacterial community in the CF lung is polymicrobial. Identifying bacteria in the CF lung through sequencing can be costly and is not practical for many laboratories. Molecular techniques such as terminal restriction fragment length polymorphism or amplicon length heterogeneity-polymerase chain reaction (LH-PCR) can provide many laboratories with the ability to study CF bacterial communities without costly sequencing. The aim of this study was to determine if the use of LH-PCR with multiple hypervariable regions of the 16S rRNA gene could be used to identify organisms found in sputum DNA. This work also determined if LH-PCR could be used to observe the dynamics of lung infections over a period of time. Nineteen samples were analysed with the V1 and the V1_V2 region of the 16S rRNA gene. Based on the amplicon size present in the V1_V2 region, Pseudomonas aeruginosa was confirmed to be in all 19 samples obtained from the patients. The V1 region provided a higher power of discrimination between bacterial profiles of patients. Both regions were able to identify trends in the bacterial population over a period of time. LH profiles showed that the CF lung community is dynamic and that changes in the community may in part be driven by the patient's antibiotic treatment. LH-PCR is a tool that is well suited for studying bacterial communities and their dynamics.

A Multi-Faceted Diagnostic Approach to Lung Infections in Patients with Cystic Fibrosis

One in 3,000 people in the US are born with cystic fibrosis (CF), a genetic disorder affecting the reproductive system, pancreas, and lungs. Lung disease caused by chronic bacterial and fungal infections is the leading cause of morbidity and mortality in CF. Identities of the microbes are traditionally determined by culturing followed by phenotypic and biochemical assays. It was first thought that the bacterial infections were caused by a select handful of bacteria such as S. aureus, H. influenzae, B. cenocepacia, and P. aeruginosa. With the advent of PCR and molecular techniques, the polymicrobial nature of the CF lung became evident. The CF lung contains numerous bacteria and the communities are diverse and unique to each patient. The total complexity of the bacterial infections is still being determined. In addition, only a few members of the fungal communities have been identified. Much of the fungal community composition is still a mystery. This dissertation addresses this gap in knowledge. A snap shot of CF sputa bacterial community was obtained using the length heterogeneity-PCR community profiling technique. The profiles show that south Florida CF patients have a unique, diverse, and dynamic bacterial community which changes over time. The identities of the bacteria and fungi present were determined using the state-of-the-art 454 sequencing. Sequencing results show that the CF lung microbiome contains commonly cultured pathogenic bacteria, organisms considered a part of the healthy core biome, and novel organisms. Understanding the dynamic changes of these identified microbes will ultimately lead to better therapeutical interventions. Early detection is key in reducing the lung damage caused by chronic infections. Thus, there is a need for accurate and sensitive diagnostic tests. This issue was addressed by designing a bacterial diagnostic tool targeted towards CF pathogens using SPR. By identifying the organisms associated with the CF lung and understanding their community interactions, patients can receive better treatment and live longer.

Prevalence and control of Clostridium difficile in patients with cystic fibrosis

The aim of this thesis was to investigate the high prevalence of Clostridium difficile in patients with cystic fibrosis (CF), and to control its dissemination. To determine the carriage rate of C. difficile in CF patients, 60 patients were tested for C. difficile and its toxin. In total, 50% of patients were found to be asymptomatic carriers of C. difficile despite toxin being detected in 31.66% of patients. Ribotyping of the C. difficile isolates revealed 16 distinct ribotypes, including the hyper virulent RT078. All isolates were sensitive to both Vancomycin and Metronidazole. The effect of CF and its treatment on the gut microbiota of CF patients was assessed by 16s sequencing of the gut microbiota of 68 CF patients. When compared to a healthy control group, CF patient gut microbiota was found to be less diverse and had an increased Firmicutes to Bacteriodetes ratio. Interestingly, CF patients who were carriers of C. difficile had a less diverse gut microbiota than C. difficile negative CF patients. Multilocus sequence typing was found to be comparable to PCR-ribotyping for typing C. difficile isolates from high risk patient groups. The sequence type ST 26 is potentially associated with CF patients as all seven isolates were found in this group and this sequence type has been previously reported in CF patients in a geographically distinct study. The bacteriophage ФCD6356 was assessed as a targeted antimicrobial against C. difficile in an ex-vivo model of the human distal colon. Despite reducing viable C. difficile by 1.75 logs over 24 hours, this bacteriophage was not suitable due to its lysogenic nature. Following treatment, all surviving C. difficile were immune to reinfection due to prophage integration. However, the ФCD6356 encoded endolysin was capable of reducing viable C. difficile by 2.9 over 2 hours in vitro after being cloned and expressed in Escherichia coli.