Interleukin-13 promotes susceptibility to chlamydial infection of the respiratory and genital tracts

Chlamydiae are intracellular bacteria that commonly cause infections of the respiratory and genital tracts, which are major clinical problems. Infections are also linked to the aetiology of diseases such as asthma, emphysema and heart disease. The clinical management of infection is problematic and antibiotic resistance is emerging. Increased understanding of immune processes that are involved in both clearance and immunopathology of chlamydial infection is critical for the development of improved treatment strategies. Here, we show that IL-13 was produced in the lungs of mice rapidly after Chlamydia muridarum (Cmu) infection and promoted susceptibility to infection. Wild-type (WT) mice had increased disease severity, bacterial load and associated inflammation compared to IL-13 deficient (−/−) mice as early as 3 days post infection (p.i.). Intratracheal instillation of IL-13 enhanced bacterial load in IL-13−/− mice. There were no differences in early IFN-g and IL-10 expression between WT and IL-13−/− mice and depletion of CD4+ T cells did not affect infection in IL-13−/− mice. Collectively, these data demonstrate a lack of CD4+ T cell involvement and a novel role for IL-13 in innate responses to infection. We also showed that IL-13 deficiency increased macrophage uptake of Cmu in vitro and in vivo. Moreover, the depletion of IL-13 during infection of lung epithelial cells in vitro decreased the percentage of infected cells and reduced bacterial growth. Our results suggest that enhanced IL-13 responses in the airways, such as that found in asthmatics, may promote susceptibility to chlamydial lung infection. Importantly the role of IL-13 in regulating infection was not limited to the lung as we showed that IL-13 also promoted susceptibility to Cmu genital tract infection. Collectively our findings demonstrate that innate IL-13 release promotes infection that results in enhanced inflammation and have broad implications for the treatment of chlamydial infections and IL-13-associated diseases.

TLR2, but Not TLR4, is required for effective host defence against Chlamydia respiratory tract infection in early life

Chlamydia pneumoniae commonly causes respiratory tract infections in children, and epidemiological investigations strongly link infection to the pathogenesis of asthma. The immune system in early life is immature and may not respond appropriately to pathogens. Toll-like receptor (TLR)2 and 4 are regarded as the primary pattern recognition receptors that sense bacteria, however their contribution to innate and adaptive immunity in early life remains poorly defined. We investigated the role of TLR2 and 4 in the induction of immune responses to Chlamydia muridarum respiratory infection, in neonatal wild-type (Wt) or TLR2-deficient (−/−), 4−/− or 2/4−/− BALB/c mice. Wt mice had moderate disease and infection. TLR2−/− mice had more severe disease and more intense and prolonged infection compared to other groups. TLR4−/− mice were asymptomatic. TLR2/4−/− mice had severe early disease and persistent infection, which resolved thereafter consistent with the absence of symptoms in TLR4−/− mice. Wt mice mounted robust innate and adaptive responses with an influx of natural killer (NK) cells, neutrophils, myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, and activated CD4+ and CD8+ T-cells into the lungs. Wt mice also had effective production of interferon (IFN)γ in the lymph nodes and lung, and proliferation of lymph node T-cells. TLR2−/− mice had more intense and persistent innate (particularly neutrophil) and adaptive cell responses and IL-17 expression in the lung, however IFNγ responses and T-cell proliferation were reduced. TLR2/4−/− mice had reduced innate and adaptive responses. Most importantly, neutrophil phagocytosis was impaired in the absence of TLR2. Thus, TLR2 expression, particularly on neutrophils, is required for effective control of Chlamydia respiratory infection in early life. Loss of control of infection leads to enhanced but ineffective TLR4-mediated inflammatory responses that prolong disease symptoms. This indicates that TLR2 agonists may be beneficial in the treatment of early life Chlamydia infections and associated diseases.

Correcting for bias when estimating the cost of hospital-acquired infection : an analysis of lower respiratory tract infections in non-surgical patients

Hospital acquired infections (HAI) are costly but many are avoidable. Evaluating prevention programmes requires data on their costs and benefits. Estimating the actual costs of HAI (a measure of the cost savings due to prevention) is difficult as HAI changes cost by extending patient length of stay, yet, length of stay is a major risk factor for HAI. This endogeneity bias can confound attempts to measure accurately the cost of HAI. We propose a two-stage instrumental variables estimation strategy that explicitly controls for the endogeneity between risk of HAI and length of stay. We find that a 10% reduction in ex ante risk of HAI results in an expected savings of £693 ($US 984).

Distribution of respiratory droplets in enclosed environments under different air distribution methods

Dispersion characteristics of respiratory droplets in indoor environments are of special interest in controlling transmission of airborne diseases. This study adopts an Eulerian method to investigate the spatial concentration distribution and temporal evolution of exhaled and sneezed/coughed droplets within the range of 1.0~10.0μm in an office room with three air distribution methods, i.e. mixing ventilation (MV), displacement ventilation (DV), and under-floor air distribution (UFAD). The diffusion, gravitational settling, and deposition mechanism of particulate matters are well accounted in the one-way coupling Eulerian approach. The simulation results find that exhaled droplets with diameters up to 10.0μm from normal respiration process are uniformly distributed in MV, while they are trapped in the breathing height by thermal stratifications in DV and UFAD, resulting in a high droplet concentration and a high exposure risk to other occupants. Sneezed/coughed droplets are diluted much slower in DV/UFAD than in MV. Low air speed in the breathing zone in DV/UFAD can lead to prolonged residence of droplets in the breathing zone.

Biochemical characterization of Aprataxin, the protein deficient in Ataxia with Oculomotor Apraxia type 1

Neurodegenerative disorders are heterogenous in nature and include a range of ataxias with oculomotor apraxia, which are characterised by a wide variety of neurological and ophthalmological features. This family includes recessive and dominant disorders. A subfamily of autosomal recessive cerebellar ataxias are characterised by defects in the cellular response to DNA damage. These include the well characterised disorders Ataxia-Telangiectasia (A-T) and Ataxia-Telangiectasia Like Disorder (A-TLD) as well as the recently identified diseases Spinocerebellar ataxia with axonal neuropathy Type 1 (SCAN1), Ataxia with Oculomotor Apraxia Type 2 (AOA2), as well as the subject of this thesis, Ataxia with Oculomotor Apraxia Type 1 (AOA1). AOA1 is caused by mutations in the APTX gene, which is located at chromosomal locus 9p13. This gene codes for the 342 amino acid protein Aprataxin. Mutations in APTX cause destabilization of Aprataxin, thus AOA1 is a result of Aprataxin deficiency. Aprataxin has three functional domains, an N-terminal Forkhead Associated (FHA) phosphoprotein interaction domain, a central Histidine Triad (HIT) nucleotide hydrolase domain and a C-terminal C2H2 zinc finger. Aprataxins FHA domain has homology to FHA domain of the DNA repair protein 5’ polynucleotide kinase 3’ phosphatase (PNKP). PNKP interacts with a range of DNA repair proteins via its FHA domain and plays a critical role in processing damaged DNA termini. The presence of this domain with a nucleotide hydrolase domain and a DNA binding motif implicated that Aprataxin may be involved in DNA repair and that AOA1 may be caused by a DNA repair deficit. This was substantiated by the interaction of Aprataxin with proteins involved in the repair of both single and double strand DNA breaks (XRay Cross-Complementing 1, XRCC4 and Poly-ADP Ribose Polymerase-1) and the hypersensitivity of AOA1 patient cell lines to single and double strand break inducing agents. At the commencement of this study little was known about the in vitro and in vivo properties of Aprataxin. Initially this study focused on generation of recombinant Aprataxin proteins to facilitate examination of the in vitro properties of Aprataxin. Using recombinant Aprataxin proteins I found that Aprataxin binds to double stranded DNA. Consistent with a role for Aprataxin as a DNA repair enzyme, this binding is not sequence specific. I also report that the HIT domain of Aprataxin hydrolyses adenosine derivatives and interestingly found that this activity is competitively inhibited by DNA. This provided initial evidence that DNA binds to the HIT domain of Aprataxin. The interaction of DNA with the nucleotide hydrolase domain of Aprataxin provided initial evidence that Aprataxin may be a DNA-processing factor. Following these studies, Aprataxin was found to hydrolyse 5’adenylated DNA, which can be generated by unscheduled ligation at DNA breaks with non-standard termini. I found that cell extracts from AOA1 patients do not have DNA-adenylate hydrolase activity indicating that Aprataxin is the only DNA-adenylate hydrolase in mammalian cells. I further characterised this activity by examining the contribution of the zinc finger and FHA domains to DNA-adenylate hydrolysis by the HIT domain. I found that deletion of the zinc finger ablated the activity of the HIT domain against adenylated DNA, indicating that the zinc finger may be required for the formation of a stable enzyme-substrate complex. Deletion of the FHA domain stimulated DNA-adenylate hydrolysis, which indicated that the activity of the HIT domain may be regulated by the FHA domain. Given that the FHA domain is involved in protein-protein interactions I propose that the activity of Aprataxins HIT domain may be regulated by proteins which interact with its FHA domain. We examined this possibility by measuring the DNA-adenylate hydrolase activity of extracts from cells deficient for the Aprataxin-interacting DNA repair proteins XRCC1 and PARP-1. XRCC1 deficiency did not affect Aprataxin activity but I found that Aprataxin is destabilized in the absence of PARP-1, resulting in a deficiency of DNA-adenylate hydrolase activity in PARP-1 knockout cells. This implies a critical role for PARP-1 in the stabilization of Aprataxin. Conversely I found that PARP-1 is destabilized in the absence of Aprataxin. PARP-1 is a central player in a number of DNA repair mechanisms and this implies that not only do AOA1 cells lack Aprataxin, they may also have defects in PARP-1 dependant cellular functions. Based on this I identified a defect in a PARP-1 dependant DNA repair mechanism in AOA1 cells. Additionally, I identified elevated levels of oxidized DNA in AOA1 cells, which is indicative of a defect in Base Excision Repair (BER). I attribute this to the reduced level of the BER protein Apurinic Endonuclease 1 (APE1) I identified in Aprataxin deficient cells. This study has identified and characterised multiple DNA repair defects in AOA1 cells, indicating that Aprataxin deficiency has far-reaching cellular consequences. Consistent with the literature, I show that Aprataxin is a nuclear protein with nucleoplasmic and nucleolar distribution. Previous studies have shown that Aprataxin interacts with the nucleolar rRNA processing factor nucleolin and that AOA1 cells appear to have a mild defect in rRNA synthesis. Given the nucleolar localization of Aprataxin I examined the protein-protein interactions of Aprataxin and found that Aprataxin interacts with a number of rRNA transcription and processing factors. Based on this and the nucleolar localization of Aprataxin I proposed that Aprataxin may have an alternative role in the nucleolus. I therefore examined the transcriptional activity of Aprataxin deficient cells using nucleotide analogue incorporation. I found that AOA1 cells do not display a defect in basal levels of RNA synthesis, however they display defective transcriptional responses to DNA damage. In summary, this thesis demonstrates that Aprataxin is a DNA repair enzyme responsible for the repair of adenylated DNA termini and that it is required for stabilization of at least two other DNA repair proteins. Thus not only do AOA1 cells have no Aprataxin protein or activity, they have additional deficiencies in PolyADP Ribose Polymerase-1 and Apurinic Endonuclease 1 dependant DNA repair mechanisms. I additionally demonstrate DNA-damage inducible transcriptional defects in AOA1 cells, indicating that Aprataxin deficiency confers a broad range of cellular defects and highlighting the complexity of the cellular response to DNA damage and the multiple defects which result from Aprataxin deficiency. My detailed characterization of the cellular consequences of Aprataxin deficiency provides an important contribution to our understanding of interlinking DNA repair processes.

Deposition of combustion aerosols in the human respiratory tract : comparison of theoretical predictions with experimental data

Total deposition of petrol, diesel and environmental tobacco smoke (ETS) aerosols in the human respiratory tract for nasal breathing conditions was computed for 14 nonsmoking volunteers, considering the specific anatomical and respiratory parameters of each volunteer and the specific size distribution for each inhalation experiment. Theoretical predictions were 34.6% for petrol, 24.0% for diesel, and 18.5% for ETS particles. Compared to the experimental results, predicted deposition values were consistently smaller than the measured data (41.4% for petrol, 29.6% for diesel, and 36.2% for ETS particles). The apparent discrepancy between experimental data on total deposition and modeling results may be reconciled by considering the non-spherical shape of the test aerosols by diameter-dependent dynamic shape factors to account for differences between mobility-equivalent and volume-equivalent or thermodynamic diameters. While the application of dynamic shape factors is able to explain the observed differences for petrol and diesel particles, additional mechanisms may be required for ETS particle deposition, such as the size reduction upon inspiration by evaporation of volatile compounds and/or condensation-induced restructuring, and, possibly, electrical charge effects.

Long-term exposure to gaseous air pollutants and cardio-respiratory mortality in Brisbane, Australia

Air pollution is ranked by the World Health Organisation as one of the top ten contributors to the global burden of disease and injury. Exposure to gaseous air pollutants, even at a low level, has been associated with cardiorespiratory diseases (Vedal, Brauer et al. 2003). Most recent epidemiological studies of air pollution have used time-series analyses to explore the relationship between daily mortality or morbidity and daily ambient air pollution concentrations based on the same day or previous days (Hajat, Armstrong et al. 2007). However, most of the previous studies have examined the association between air pollution and health outcomes using air pollution data from a single monitoring site or average values from a few monitoring sites to represent the whole population of the study area. In fact, for a metropolitan city, ambient air pollution levels may differ significantly among the different areas. There is increasing concern that the relationships between air pollution and mortality may vary with geographical area (Chen, Mengersen et al. 2007). Additionally, some studies have indicated that socio-economic status can act as a confounder when investigating the relation between geographical location and health (Scoggins, Kjellstrom et al. 2004). This study examined the spatial variation in the relationship between long-term exposure to gaseous air pollutants (including nitrogen dioxide (NO2), ozone (O3) and sulphur dioxide (SO2)), and cardiorespiratory mortality in Brisbane, Australia, during the period 1996 - 2004.

Molecular epidemiology of respiratory viruses in a paediatric cohort from Indonesia

Acute lower respiratory tract infections (ALRTIs) are a common cause of morbidity and mortality among children under 5 years of age and are found worldwide, with pneumonia as the most severe manifestation. Although the incidence of severe disease varies both between individuals and countries, there is still no clear understanding of what causes this variation. Studies of community-acquired pneumonia (CAP) have traditionally not focused on viral causes of disease due to a paucity of diagnostic tools. However, with the emergence of molecular techniques, it is now known that viruses outnumber bacteria as the etiological agents of childhood CAP, especially in children under 2 years of age. The main objective of this study was to investigate viruses contributing to disease severity in cases of childhood ALRTI, using a two year cohort study following 2014 infants and children enrolled in Bandung, Indonesia. A total of 352 nasopharyngeal washes collected from 256 paediatric ALRTI patients were used for analysis. A subset of samples was screened using a novel microarray pathogen detection method that identified respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and human rhinovirus (HRV) in the samples. Real-time RT-PCR was used both for confirming and quantifying viruses found in the nasopharyngeal samples. Viral copy numbers were determined and normalised to the numbers of human cells collected with the use of 18S rRNA. Molecular epidemiology was performed for RSV A and hMPV using sequences to the glycoprotein gene and nucleoprotein gene respectively, to determine genotypes circulating in this Indonesian paediatric cohort. This study found that HRV (119/352; 33.8%) was the most common virus detected as the cause of respiratory tract infections in this cohort, followed by the viral pathogens RSV A (73/352; 20.7%), hMPV (30/352; 8.5%) and RSV B (12/352; 3.4%). Co-infections of more than two viruses were detected in 31 episodes (defined as an infection which occurred more than two weeks apart), accounting for 8.8% of the 352 samples tested or 15.4% of the 201 episodes with at least one virus detected. RSV A genotypes circulating in this population were predominantly GA2, GA5 and GA7, while hMPV genotypes circulating were mainly A2a (27/30; 90.0%), B2 (2/30; 6.7%) and A1 (1/30; 3.3%). This study found no evidence of disease severity associated either with a specific virus or viral strain, or with viral load. However, this study did find a significant association with co-infection of RSV A and HRV with severe disease (P = 0.006), suggesting that this may be a novel cause of severe disease.

Theoretical analysis of the motion and evaporation of exhaled respiratory droplets of mixed composition

The dynamics of droplets exhaled from the respiratory system during coughing or talking is addressed. A mathematical model is presented accounting for the motion of a droplet in conjunction with its evaporation. Droplet evaporation and motion are accounted for under two scenarios: 1) A well mixed droplet and 2) A droplet with inner composition variation. A multiple shells model was implemented to account for internal mass and heat transfer and for concentration and temperature gradients inside the droplet. The trajectories of the droplets are computed for a range of conditions and the spatial distribution and residence times of such droplets are evaluated.

Association between indoor air pollution measurements and respiratory health in women and children in Lao PDR

This article presents the results of a study on the association between measured air pollutants and the respiratory health of resident women and children in Lao PDR, one of the least developed countries in Southeast Asia. The study, commissioned by the World Health Organisation, included PM10, CO and NO2 measurements made inside 181 dwellings in nine districts within two provinces in Lao PDR over a 5- month period (12/05–04/06), and respiratory health information (via questionnaires and peak expiratory flow rate (PEFR) measurements) for all residents in the same dwellings. Adjusted odds ratios were calculated separately for each health outcome using binary logistic regression. There was a strong and consistent positive association between NO2 and CO for almost all questionnaire-based health outcomes for both women and children. Women in dwellings with higher measured NO2 had more than triple of the odds of almost all of the health outcomes, and higher concentrations of NO2 and CO were significantly associated with lower PEFR. This study supports a growing literature confirming the role of indoor air pollution in the burden of respiratory disease in developing countries. The results will directly support changes in health and housing policy in Lao PDR.