text: DSpace Community: http://hdl.handle.net/2381/6 2015-03-30T16:15:37Z 2015-03-30T16:15:37Z Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells. Abbasian, Nima Burton, James O. Herbert, Karl E. Tregunna, Barbara-Emily Brown, Jeremy R. Ghaderi-Najafabadi, Maryam Brunskill, Nigel J. Goodall, Alison H. Bevington, Alan http://hdl.handle.net/2381/31860 2015-03-13T10:26:57Z 2015-03-11T17:03:29Z Title: Hyperphosphatemia, Phosphoprotein Phosphatases, and Microparticle Release in Vascular Endothelial Cells. Authors: Abbasian, Nima; Burton, James O.; Herbert, Karl E.; Tregunna, Barbara-Emily; Brown, Jeremy R.; Ghaderi-Najafabadi, Maryam; Brunskill, Nigel J.; Goodall, Alison H.; Bevington, Alan Abstract: Hyperphosphatemia in patients with advanced CKD is thought to be an important contributor to cardiovascular risk, in part because of endothelial cell (EC) dysfunction induced by inorganic phosphate (Pi). Such patients also have an elevated circulating concentration of procoagulant endothelial microparticles (MPs), leading to a prothrombotic state, which may contribute to acute occlusive events. We hypothesized that hyperphosphatemia leads to MP formation from ECs through an elevation of intracellular Pi concentration, which directly inhibits phosphoprotein phosphatases, triggering a global increase in phosphorylation and cytoskeletal changes. In cultured human ECs (EAhy926), incubation with elevated extracellular Pi (2.5 mM) led to a rise in intracellular Pi concentration within 90 minutes. This was mediated by PiT1/slc20a1 Pi transporters and led to global accumulation of tyrosine- and serine/threonine-phosphorylated proteins, a marked increase in cellular Tropomyosin-3, plasma membrane blebbing, and release of 0.1- to 1-μm-diameter MPs. The effect of Pi was independent of oxidative stress or apoptosis. Similarly, global inhibition of phosphoprotein phosphatases with orthovanadate or fluoride yielded a global protein phosphorylation response and rapid release of MPs. The Pi-induced MPs expressed VE-cadherin and superficial phosphatidylserine, and in a thrombin generation assay, they displayed significantly more procoagulant activity than particles derived from cells incubated in medium with a physiologic level of Pi (1 mM). These data show a mechanism of Pi-induced cellular stress and signaling, which may be widely applicable in mammalian cells, and in ECs, it provides a novel pathologic link between hyperphosphatemia, generation of MPs, and thrombotic risk. 2015-03-11T17:03:29Z Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin Leung, C. Dudkina, N. V. Lukoyanova, N. Hodel, A. W. Farabella, I. Pandurangan, A. P. Jahan, Nasrin Damaso, Mafalda Pires Osmanovic, D. Reboul, C. F. Dunstone, M. A. Andrew, Peter W. Lonnen, Rana Topf, M. Saibil, H. R. Hoogenboom, B. W. http://hdl.handle.net/2381/31848 2015-03-12T02:02:12Z 2015-03-11T09:52:03Z Title: Stepwise visualization of membrane pore formation by suilysin, a bacterial cholesterol-dependent cytolysin Authors: Leung, C.; Dudkina, N. V.; Lukoyanova, N.; Hodel, A. W.; Farabella, I.; Pandurangan, A. P.; Jahan, Nasrin; Damaso, Mafalda Pires; Osmanovic, D.; Reboul, C. F.; Dunstone, M. A.; Andrew, Peter W.; Lonnen, Rana; Topf, M.; Saibil, H. R.; Hoogenboom, B. W. Abstract: Membrane attack complex/perforin/cholesterol-dependent cytolysin (MACPF/CDC) proteins constitute a major superfamily of pore-forming proteins that act as bacterial virulence factors and effectors in immune defence. Upon binding to the membrane, they convert from the soluble monomeric form to oligomeric, membrane-inserted pores. Using real-time atomic force microscopy (AFM), electron microscopy (EM), and atomic structure fitting, we have mapped the structure and assembly pathways of a bacterial CDC in unprecedented detail and accuracy, focussing on suilysin from Streptococcus suis. We show that suilysin assembly is a noncooperative process that is terminated before the protein inserts into the membrane. The resulting ring-shaped pores and kinetically trapped arc-shaped assemblies are all seen to perforate the membrane, as also visible by the ejection of its lipids. Membrane insertion requires a concerted conformational change of the monomeric subunits, with a marked expansion in pore diameter due to large changes in subunit structure and packing. Description: Figure supplement 1. Symmetry of suilysin prepores and pores, determined by negative-stain EM. DOI: 10.7554/eLife.04247.004 Figure supplement 2. Resolution curves for EM maps. DOI: 10.7554/eLife.04247.005 Figure supplement 3. Electrostatic potential maps and interacting residues. DOI: 10.7554/eLife.04247.006 Figure supplement 4. Comparison between prepore and crystal structure conformations. DOI: 10.7554/eLife.04247.007 Accession codes for EM maps: prepore EMD-12698, pore EMD-12711. 2015-03-11T09:52:03Z Temperature dependent bacteriophages of a tropical bacterial pathogen Shan, Jinyu Korbsrisate, S. Withatanung, P. Lazar Adler, Natalie Clokie, Martha R. J. Galyov, Edouard E. http://hdl.handle.net/2381/31813 2015-03-07T02:02:30Z 2015-03-06T16:40:07Z Title: Temperature dependent bacteriophages of a tropical bacterial pathogen Authors: Shan, Jinyu; Korbsrisate, S.; Withatanung, P.; Lazar Adler, Natalie; Clokie, Martha R. J.; Galyov, Edouard E. Abstract: There is an increasing awareness of the multiple ways that bacteriophages (phages) influence bacterial evolution, population dynamics, physiology, and pathogenicity. By studying a novel group of phages infecting a soil borne pathogen, we revealed a paradigm shifting observation that the phages switch their lifestyle according to temperature. We sampled soil from an endemic area of the serious tropical pathogen Burkholderia pseudomallei, and established that podoviruses infecting the pathogen are frequently present in soil, and many of them are naturally occurring variants of a common virus type. Experiments on one phage in the related model B. thailandensis demonstrated that temperature defines the outcome of phage-bacteria interactions. At higher temperatures (37◦C), the phage predominantly goes through a lytic cycle, but at lower temperatures (25◦C), the phage remains temperate. This is the first report of a naturally occurring phage that follows a lytic or temperate lifestyle according to temperature.These observations fundamentally alter the accepted views on the abundance, population biology and virulence of B. pseudomallei. Furthermore, when taken together with previous studies, our findings suggest that the phenomenon of temperature dependency in phages is widespread. Such phages are likely to have a profound effect on bacterial biology, and on our ability to culture and correctly enumerate viable bacteria. 2015-03-06T16:40:07Z MLP a novel Mucin-like cancer biomarker Aldali, Jehad Abdulrazaq Z. http://hdl.handle.net/2381/31812 2015-03-07T02:02:44Z 2015-03-06T16:33:07Z Title: MLP a novel Mucin-like cancer biomarker Authors: Aldali, Jehad Abdulrazaq Z. Abstract: Malignant ovarian tumours pose a significant risk to women all over the world. Ovarian cancers are the fifth most common cancers among women in the UK. Mortality due to ovarian malignancies is unacceptably high and the disease is often called a "silent killer" as the early stages of ovarian malignancies present with a little to no symptoms. Ovarian cancer is therefore mostly diagnosed at a very late stage of tumour progression when treatment is hampered by metastastatic spread of the primary tumour. Ovarian tumours express and release a previously undescribed novel mucin-like protein (MLP), which is absent in normal ovarian tissue or benign ovarian cysts (Schwaeble, W and Walker, R unpublished results). This novel mucin-like protein is a glycoprotein composed of a protein backbone, which is highly glycosylated with O-linked oligosaccharides. The high degree of glycosylation accounts for most the molecular weight of the native glycoprotein. The aim of this project was to develop a reliable diagnostic assay for the early and non-invasive detection of ovarian malignancies using specific monoclonal antibodies for MLP detection. The early detection of ovarian malignancies poses a so far unmet clinical necessity. The presently used tumour-associated biomarkers (mainly based on the relatively non-specific tumour marker CA-125) unfortunately all fail to detect ovarian malignancies at an early stage. Recombinant MLP was produced in a bacterial expression system, purified, analysed by Western blotting and used to raise polyclonal antibodies for the development of an MLP- specific sandwich ELISA. Eight different hybridomas producing mouse monoclonal antibodies against recombinant human MLP were also grow up and supernatant tested. Three of the eight monoclonal antibodies were shown to recognise native, glycosylated MLP as well as the recombinant antigen. These three were characterised in detail by ELISA, immunohistochemical staining of tissue sections and Western blot analysis. Preliming experiments were performed using the three monoclonal antibodies and the goat anti-MLP antiserum to investigate the properties of the antibodies and their sutability for establishing a sandwish ELISA able to detect the presence of MLP in serum for the development of a test for the early diagnosis of ovarian cancer. 2015-03-06T16:33:07Z Elevated levels of procoagulant plasma microvesicles in dialysis patients. Burton, James O. Hamali, Hassan A. Singh, Ruchir Abbasian, Nima Parsons, Ruth Patel, Amit K. Goodall, Alison H. Brunskill, Nigel J. http://hdl.handle.net/2381/31771 2015-03-06T02:02:05Z 2015-03-05T12:02:22Z Title: Elevated levels of procoagulant plasma microvesicles in dialysis patients. Authors: Burton, James O.; Hamali, Hassan A.; Singh, Ruchir; Abbasian, Nima; Parsons, Ruth; Patel, Amit K.; Goodall, Alison H.; Brunskill, Nigel J. Abstract: Cardiovascular (CV) death remains the largest cause of mortality in dialysis patients, unexplained by traditional risk factors. Endothelial microvesicles (EMVs) are elevated in patients with traditional CV risk factors and acute coronary syndromes while platelet MVs (PMVs) are associated with atherosclerotic disease states. This study compared relative concentrations of circulating MVs from endothelial cells and platelets in two groups of dialysis patients and matched controls and investigated their relative thromboembolic risk. MVs were isolated from the blood of 20 haemodialysis (HD), 17 peritoneal dialysis (PD) patients and 20 matched controls. Relative concentrations of EMVs (CD144(+ ve)) and PMVs (CD42b(+ ve)) were measured by Western blotting and total MV concentrations were measured using nanoparticle-tracking analysis. The ability to support thrombin generation was measured by reconstituting the MVs in normal plasma, using the Continuous Automated Thrombogram assay triggered with 1µM tissue factor. The total concentration of MVs as well as the measured sub-types was higher in both patient groups compared to controls (p<0.05). MVs from HD and PD patients were able to generate more thrombin than the controls, with higher peak thrombin, and endogenous thrombin potential levels (p<0.02). However there were no differences in either the relative quantity or activity of MVs between the two patient groups (p>0.3). Dialysis patients have higher levels of circulating procoagulant MVs than healthy controls. This may represent a novel and potentially modifiable mediator or predictor of occlusive cardiovascular events in these patients. Description: PMCID: PMC3732282 2015-03-05T12:02:22Z Inflammatory factors and exercise in chronic kidney disease. Dungey, Maurice Hull, Katherine L. Smith, Alice C. Burton, James O. Bishop, Nicolette C. http://hdl.handle.net/2381/31770 2015-03-06T02:02:06Z 2015-03-05T11:57:00Z Title: Inflammatory factors and exercise in chronic kidney disease. Authors: Dungey, Maurice; Hull, Katherine L.; Smith, Alice C.; Burton, James O.; Bishop, Nicolette C. Abstract: Patients with chronic kidney disease frequently present with chronic elevations in markers of inflammation, a condition that appears to be exacerbated by disease progression and onset of haemodialysis. Systemic inflammation is interlinked with malnutrition and muscle protein wasting and is implicated in a number of morbidities including cardiovascular disease: the most common cause of mortality in this population. Research in the general population and other chronic disease cohorts suggests that an increase in habitual activity levels over a prolonged period may help redress basal increases in systemic inflammation. Furthermore, those populations with the highest baseline levels of systemic inflammation appear to have the greatest improvements from training. On the whole, the activity levels of the chronic kidney disease population reflect a sedentary lifestyle, indicating the potential for increasing physical activity and observing health benefits. This review explores the current literature investigating exercise and inflammatory factors in the chronic kidney disease population and then attempts to explain the contradictory findings and suggests where future research is required. Description: PMCID: PMC3666228 2015-03-05T11:57:00Z Temporal Assessment Of Airway Remodeling In Severe Asthma Using Quantitative Computed Tomography Gupta, Sumit Hartley, Ruth Singapuri, Amisha Hargadon, Beverly Monteiro, William Pavord, I. D. Sousa, A. R. Marshall, R. P. Subramanian, D. Parr, D. Entwisle, J. J. Siddiqui, Salman Raj, Vimal Brightling, Christopher E. http://hdl.handle.net/2381/31606 2015-02-05T02:01:33Z 2015-02-04T17:27:52Z Title: Temporal Assessment Of Airway Remodeling In Severe Asthma Using Quantitative Computed Tomography Authors: Gupta, Sumit; Hartley, Ruth; Singapuri, Amisha; Hargadon, Beverly; Monteiro, William; Pavord, I. D.; Sousa, A. R.; Marshall, R. P.; Subramanian, D.; Parr, D.; Entwisle, J. J.; Siddiqui, Salman; Raj, Vimal; Brightling, Christopher E. 2015-02-04T17:27:52Z CIM (R) monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles Adriaenssens, E. M. Lehman, S. M. Vandersteegen, K. Vandenheuvel, D. Philippe, Didier L. Cornelissen, A. Clokie, Martha R.J Garcia, A. J. De Proft, M. Maes, M. Lavigne, R. http://hdl.handle.net/2381/31376 2015-01-08T02:01:41Z 2015-01-07T12:33:29Z Title: CIM (R) monolithic anion-exchange chromatography as a useful alternative to CsCl gradient purification of bacteriophage particles Authors: Adriaenssens, E. M.; Lehman, S. M.; Vandersteegen, K.; Vandenheuvel, D.; Philippe, Didier L.; Cornelissen, A.; Clokie, Martha R.J; Garcia, A. J.; De Proft, M.; Maes, M.; Lavigne, R. Abstract: The use of anion-exchange chromatography was investigated as an alternative method to concentrate and purify bacterial viruses, and parameters for different bacteriophages were compared. Chromatography was performed with Convective Interactive Media® monoliths, with three different volumes and two matrix chemistries. Eleven morphologically distinct phages were tested, infecting five different bacterial species. For each of the phages tested, a protocol was optimized, including the choice of column chemistry, loading, buffer and elution conditions. The capacity and recovery of the phages on the columns varied considerably between phages. We conclude that anion-exchange chromatography with monoliths is a valid alternative to the more traditional CsCl purification, has upscaling advantages, but it requires more extensive optimization. 2015-01-07T12:33:29Z IL-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo Jackson, David J. Makrinioti, Heidi Rana, Batika M. J. Shamji, Betty W. H. Trujillo-Torralbo, Maria-Belen Footitt, Joseph del-Rosario, Jerico Telcian, Aurica G. Nikonova, Alexandra Zhu, Jie Aniscenko, Julia Gogsadze, Leila Bakhsoliani, Eteri Traub, Stephanie Dhariwal, Jaideep Porter, James Hunt, Duncan Hunt, Toby Hunt, Trevor Stanciu, Luminita A. Khaitov, Musa Bartlett, Nathan W. Edwards, Michael R. Kon, Onn Min Mallia, Patrick Papadopoulos, Nikolaos G. Akdis, Cezmi A. Westwick, John Edwards, Matthew J. Cousins, David J. Walton, Ross P. Johnston, Sebastian L. http://hdl.handle.net/2381/31365 2014-12-23T02:01:37Z 2014-12-22T15:51:51Z Title: IL-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo Authors: Jackson, David J.; Makrinioti, Heidi; Rana, Batika M. J.; Shamji, Betty W. H.; Trujillo-Torralbo, Maria-Belen; Footitt, Joseph; del-Rosario, Jerico; Telcian, Aurica G.; Nikonova, Alexandra; Zhu, Jie; Aniscenko, Julia; Gogsadze, Leila; Bakhsoliani, Eteri; Traub, Stephanie; Dhariwal, Jaideep; Porter, James; Hunt, Duncan; Hunt, Toby; Hunt, Trevor; Stanciu, Luminita A.; Khaitov, Musa; Bartlett, Nathan W.; Edwards, Michael R.; Kon, Onn Min; Mallia, Patrick; Papadopoulos, Nikolaos G.; Akdis, Cezmi A.; Westwick, John; Edwards, Matthew J.; Cousins, David J.; Walton, Ross P.; Johnston, Sebastian L. Abstract: Rationale: Rhinoviruses are the major cause of asthma exacerbations; however, its underlying mechanisms are poorly understood. We hypothesized that the epithelial cell–derived cytokine IL-33 plays a central role in exacerbation pathogenesis through augmentation of type 2 inflammation. Objectives: To assess whether rhinovirus induces a type 2 inflammatory response in asthma in vivo and to define a role for IL-33 in this pathway. Methods: We used a human experimental model of rhinovirus infection and novel airway sampling techniques to measure IL-4, IL-5, IL-13, and IL-33 levels in the asthmatic and healthy airways during a rhinovirus infection. Additionally, we cultured human T cells and type 2 innate lymphoid cells (ILC2s) with the supernatants of rhinovirus- infected bronchial epithelial cells (BECs) to assess type 2 cytokine production in the presence or absence of IL-33 receptor blockade. Measurements and Main Results: IL-4, IL-5, IL-13, and IL-33 are all induced by rhinovirus in the asthmatic airway in vivo and relate to exacerbation severity. Further, induction of IL-33 correlates with viral load and IL-5 and IL-13 levels. Rhinovirus infection of human primary BECs induced IL-33, and culture of human T cells and ILC2s with supernatants of rhinovirus-infected BECs strongly induced type 2 cytokines. This induction was entirely dependent on IL-33. Conclusions: IL-33 and type 2 cytokines are induced during a rhinovirus-induced asthma exacerbation in vivo. Virus-induced IL-33 and IL-33 – responsive T cells and ILC2s are key mechanistic links between viral infection and exacerbation of asthma. IL-33 inhibition is a novel therapeutic approach for asthma exacerbations. 2014-12-22T15:51:51Z Inflammatory markers in and the treatment of acute pre-school viral wheeze Oommen, Abraham http://hdl.handle.net/2381/30509 2014-12-16T02:27:38Z 2014-12-15T10:40:03Z Title: Inflammatory markers in and the treatment of acute pre-school viral wheeze Authors: Oommen, Abraham Abstract: Asthma in preschool children is characterised by recurrent viral cold triggered wheezy episodes (PVW) and in the majority may be phenotypically distinct from atopic asthma. In order to address this, I performed four interrelated studies on children (1-5 years) presenting to hospital with acute PVW, and 'normal' and 'atopic' controls. The study on eosinophil activation performed by analysing urinary (u) Eosinophil Protein X (EPX) showed that uEPX was increased in acute PVW compared to both controls, with no association between uEPX and IgE. uEPX fell on convalescence, but was not predictive of asthma symptoms two years later. The investigation on cysteinyl leukotrienes, using urinary (u) Leukotriene E4 (LTE4) showed for the first time, its relationship with wheeze and atopy. Elevated uLTE4 in acute PVW fell rapidly in recovery. uLTE4 correlated with IgE, and the fall in uLTE4 at recuperation was seen only in the high IgE subgroup. The neutrophil activation study was novel, and examined the expression of adhesion molecule L-selectin. In acute PVW this was reduced on systemic neutrophils and the soluble L-selectin in serum was increased compared to controls.;Children included in the clinical trial were allocated to a high- and low-primed stratum by measuring serum Eosinophil Cationic Protein and EPX, and randomised to receive parent-initiated prednisolone or placebo for the next PVW. 108 children were randomised to placebo and 109 to prednisolone. No difference in mean day- and night-time symptom score, salbutamol usage and need for hospitalisation was noted. Sub analysis within either eosinophil strata also was similar. 2014-12-15T10:40:03Z Expression and function of TR7/8 in monocytes and macrophages Boghdadi, Ghada http://hdl.handle.net/2381/30507 2014-12-16T02:27:36Z 2014-12-15T10:40:02Z Title: Expression and function of TR7/8 in monocytes and macrophages Authors: Boghdadi, Ghada Abstract: We have studies the response of Mo subsets to the TLR 7/8 ligand R848 (Resiquimod). Whole blood stimulation demonstrated a 2 fold higher level of TNF protein in the CD14+CD16+ Mo as measured by intracellular cytokine staining (p < 0.001). CD14+CD16+ Mo expressed 2 fold more of TLR7 protein and 1.3 fold less of TLR8 protein than CD14++ Mo. ssRNA that represents the natural ligand for human TLR8 induced lower TNF production in CD14+CD16+ Mo. Taken together, these data raise the possibility that the higher TLR7 protein level is essential in determining higher TNF production induced by R848 in CD14+CD16+ Mo. Little is known about the differentiation of CD14+CD16+ into macrophages (Mf). Therefore, we investigated the ability of Mo subsets cultured with M-CSF in vitro for 3 days to generate monocytes-derived macrophages (MDM). CD14+CD16+ MDM expressed higher levels of HLA-DR and TLR7 protein and lower levels of CD14 and TLR8 protein compared to CD14++ MDM while levels of CD68 and CD16 were similar. R848 induced TNF production in both MDM subsets. We analysed Mo subsets of patients with recurrent warts for TNF expression in response to R848. Compared with the normal donors, Mo subsets from patients with recurrent warts did not respond to R848 at 100ng/ml whereas at 1 mug/ml CD14+CD16+ Mo produced TNF but at a significantly lower level compared to controls (p < 0.005). These data indicate that the failure to respond to TLR7 ligand may contribute to the patients' susceptibility to recurrent warts. 2014-12-15T10:40:02Z Non-invasive assessment of pollutant particles in the lower airway of children and adults Kulkarni, Neeta Sushrut http://hdl.handle.net/2381/30508 2014-12-16T02:27:37Z 2014-12-15T10:40:02Z Title: Non-invasive assessment of pollutant particles in the lower airway of children and adults Authors: Kulkarni, Neeta Sushrut Abstract: Epidemiological studies in children suggest that inhalation of carbonaceous particulate matter (PM) adversely affects children's respiratory health. However, the relationship between markers of PM exposure and the quantity of particles entering the paediatric airway is unclear. The hypothesis was that, the carbon in alveolar macrophages (AM) reflects individual exposure of healthy children to PM. The aim was to quantify carbon in AM in extreme (biomass smoke) and with fossil fuel exposure. For fossil fuel exposure the aim was also to establish the relationship between median carbon loading per AM per child and: i) markers of exposures at the home address and ii) lung function iii) markers of airway inflammation. Healthy children were recruited from schools. Exercise parameters recorded. Environmental smoke exposure was excluded by salivary cotinine. AM were sampled by induced sputum. Median carbon area was determined by image analysis of digitized light microscopic images of AM. The median area of AM carbon was higher in Ethiopian women compared with UK adults, (p = 0.0002). Median carbon area in Ethiopian children was higher than UK children (p = 0.0002). In fossil-fuel exposure group, a weak, but significantly positive, association was found between median carbon area and modelled primary PM10 (p = 0.022), and a stronger inverse association between median carbon area and FEV1% predicted (p = 0.004), and FEF25-75% predicted (p = 0.004). There was no significant association between median carbon area and exercise variables and markers of airway inflammation. 2014-12-15T10:40:02Z Molecular mechanisms in down-regulation of IL-10 gene expression by IFN-gamma Schaefer, Annette http://hdl.handle.net/2381/30505 2014-12-16T02:27:33Z 2014-12-15T10:40:01Z Title: Molecular mechanisms in down-regulation of IL-10 gene expression by IFN-gamma Authors: Schaefer, Annette Abstract: Interleukin-10 (IL-10) is a suppressive cytokine that down-regulates immune response and inflammation. It was shown previously that IL-10 expression is up-regulated after stimulation with LPS and that IFNgamma is able to down-regulate LPS-induced IL-10 expression.;In the present study, the cis- and trans-elements involved in the IFNgamma-mediated down-regulation of LPS-induced IL-10 expression in the human B-cell line RPMI 8226.1 were analysed. The inhibition of IL-10 trans-activation activity by IFNgamma was time-dependent. A stronger effect was seen when cells were primed with IFNgamma for 2 hours previous to stimulation with LPS. The presence of the STAT-binding site in the IL-10 promoter was sufficient to mediate the down-regulation of LPS-induced IL-10 expression. Of note, stimulation with IFNgamma alone had a slight increasing effect on IL-10 promoter activity in some constructs tested.;In the nucleus, tyrosine-phosphorylated STAT1 and STAT3 were detected in response to IFNgamma and LPS, respectively. After stimulation with both IFNgamma and LPS, binding of the respective STAT1 and STAT3 binding factors to the STAT site of the IL-10 promoter was demonstrated by EMS and supershift assays.;Chromatin immunoprecipitation showed that binding of STAT3 to the IL-10 promoter in response to LPS does occur in intact cells and that this is inhibited by IFNgamma in vivo, possibly by the recruitment of STAT1.;In addition, IFNgamma inhibited the expression of LPS-induced IL-10 mRNA in cells of the RPMI 8226.1 cell line and in human blood mononuclear cells (PBMC). Finally, the expression of IL-10 protein in response to LPS was also down-regulated by IFNgamma.;In conclusion, the present study shows that IFNgamma down-regulates the LPS-induced expression of IL-10 at the promoter, mRNA and protein level by inhibiting binding of STAT3 to the IL-10 promoter. 2014-12-15T10:40:01Z Development of new vaccine strategies against Streptocarpus pneumoniae Smith, Claire Mary http://hdl.handle.net/2381/30506 2014-12-16T02:27:35Z 2014-12-15T10:40:01Z Title: Development of new vaccine strategies against Streptocarpus pneumoniae Authors: Smith, Claire Mary Abstract: In this thesis I have shown that peptide mimotopes of both serotype 6B and 9V pneumococcal polysaccharide, following vaccination, can induce specific and anti-CPS antibodies and can protect mice against developing pneumococcal disease. This was achieved using previously described monoclonal antibodies (mAb) directed against the capsular polysaccharide of S. pneumoniae serotype 6B and 9V. These mAbs were shown to protect mice in passive immunisation experiments, and were then used to screen phage-displayed peptide libraries. In total four peptide mimics of serotype 9V pneumococcal polysaccharide and seven mimics of serotype 6B pneumococcal polysaccharide were identified. The peptides were conjugated to keyhole limpet haemocyanin (KLH) and were used to immunise mice. Two peptides, MP7 and MP13, were found significantly protect mice against a lethal challenge with S. pneumoniae and induce anti- capsule specific antibodies. This project showed that the poor immunogenicity of capsular polysaccharide could be improved using peptide mimics.;Secondly, this thesis confronts the problem of high vaccine production costs by developing a transgenic plant capable of manufacturing pneumococcal polysaccharide. The four genes involved in type 3 pneumococcal capsular polysaccharide synthesis are closely linked on the bacterial chromosome, arranged within a single locus (cassette). I cloned a copy of the serotype 3 capsule synthase gene, cps3S, into a plant expression vector, which was transformed into tobacco plants by Agrobacterium-mediated gene transfer. After cultivating a second generation of transgenic plants, we have shown the stable integration of the cps3S gene and production of type 3 pneumococcal polysaccharide. This discovery aims to confront the high vaccine production costs associated with pneumococcal vaccines. 2014-12-15T10:40:01Z Human respiratory cilia Chilvers, Mark Alexander http://hdl.handle.net/2381/30503 2014-12-16T02:27:31Z 2014-12-15T10:40:00Z Title: Human respiratory cilia Authors: Chilvers, Mark Alexander Abstract: Human respiratory cilia densely line the airways and beat continually removing mucus and debris from the respiratory tract. Ciliary damage may be primary, from genetic causes, or secondary due to a variety of toxins, bacteria, or viruses. This may result in a reduction in ciliary beat frequency and abnormalities of ciliary beat pattern. Digital high-speed imaging has been presented as a technique to measure both ciliary beat frequency and beat pattern. This has been evaluated against existing methods and found to be a gold standard. Using this method the ciliary beat pattern has been evaluated in detail for the first time. Cilia were found to beat with a forward power stroke and a recovery stroke within the same plane. Using digital high speed imaging normal reference ranges have been evaluated for both a paediatric and young adult population. Data has been collected for ciliary beat frequency, beat pattern and ultrastructural parameters. Having established normal reference ranges it has been possible to evaluate ciliary beat frequency, beat pattern and ultrastructure inpatients with primary ciliary dyskinesia. Different beat patterns were found to be associated with different ultrastructural defects. Digital high-speed imaging is a gold standard for evaluation of ciliary beat frequency and beat pattern. With the availability of normal reference ranges it can be used confidently as a diagnostic and research method.. 2014-12-15T10:40:00Z Regulation of metabolic acidosis-induced skeletal muscle wasting Pickering, Warren Paul http://hdl.handle.net/2381/30504 2014-12-16T02:27:32Z 2014-12-15T10:40:00Z Title: Regulation of metabolic acidosis-induced skeletal muscle wasting Authors: Pickering, Warren Paul Abstract: This work aims to investigate metabolic acidosis induced skeletal muscle protein wasting, and the inter-relationships between: metabolic acidosis, glucocorticoids (GC), and the ubiquitin proteasome system in this process.;Experiments were undertaken using three key themes and models: 1. The role of glucocorticoids and apoptosis in the process of protein degradation (PD) in L6G8C5 myoblasts in a cell culture system of metabolic acidosis utilizing dexamethasone and the glucocorticoid antagonist RU38486. Stimulation of PD in these cells by acid and GC does not appear to be an artefact of apoptosis or dedifferentiation, but differentiation state does determine whether PD responds spontaneously to acid or (as in vivo) only does so in the presence of GC. 2. The ability of RU38486 to pharmacologically antagonize the suggested permissive effect of glucocorticoid in an in vivo model of acidosis-induced muscle wasting. RU38486 did not prevent the acidosis-induced muscle wasting in this model despite demonstration of significant GC receptor blockade. 3. The role of the ATP-dependent ubiquitin proteasome system in the malnutrition of patients treated by peritoneal dialysis. When serum bicarbonate increased in these patients weight and body mass index increased significantly as did plasma BCAA. Muscle levels of ubiquitin mRNA decreased significantly; serum tumour necrosis factor-a also decreased. These results indicate that even a small correction of serum bicarbonate improves nutritional status, and provide evidence for down-regulation of BCAA degradation and muscle proteolysis via the ubiquitin proteasome system. Whether acidosis and inflammatory cytokines (such as TNF-alpha) interact to impair nutrition remains to be determined. 2014-12-15T10:40:00Z Genomic mosaicism in the opportunistic pathogen pseudomonas aeruginosa and its contribution to virulence Harrison, Ewan Michael http://hdl.handle.net/2381/30501 2014-12-16T02:27:28Z 2014-12-15T10:39:59Z Title: Genomic mosaicism in the opportunistic pathogen pseudomonas aeruginosa and its contribution to virulence Authors: Harrison, Ewan Michael Abstract: Pseudomonas aeruginosa is a ubiquitous Gram negative opportunistic pathogen which belongs to the gamma subclass of proteobacteria. P. aeruginosa genomes consist of a highly conserved core component making up ~90% of the total genome and a highly variable accessory genome that makes up the remaining ~10% of the organism's DNA blueprint.;The extent and nature of tRNA-integrated genomic islands in the available sequenced P. aeruginosa genomes and clinical isolates were defined by both in silico comparative genomics methods and in vitro approaches. These investigations demonstrated that certain tRNA sites seemed to be favoured for integration and that most strains exhibited divergent tRNA site island-occupation profiles.;The contribution of three P. aeruginosa genomic islands PAPI-1, PAPI-2 and the tRNAPro21 island in strain PA14 to fitness and virulence were evaluated. By creating 'en bloc' deletant mutants that lacked islands and testing the resulting phenotype of the mutants alongside the wild-type parent, three islands (PAPI-1, PAPI-2 and tRNAPro21 island) were demonstrated to be important to full virulence of strain PA14.;The dynamics of site-specific integration of the PAPI-1 island into both of its cognate target attB sites were examined. A quantitative real-time PCR approach was used to measure the copy number of all 'states' of PAPI-1: chromosomally-integrated forms present at each of the two tRNALys sites, extra-chromosomal circular entities, and empty versions of the two attB sites. The pattern of occupation was clearly dynamic. Prior occupation of the tRNA Lys10 site by an alternative genomic island in PAOl and PA14 appeared to hinder integration of PAPI-1 into this occupied tRNA Lys10 site through an as yet unknown mechanism. This hypothesis was supported by evidence that the deletion of the original occupying island allowed PAPI-1 to integrate at high frequency into this site. However, surprisingly this effect was found to be variable as several independently derived deletant mutants behaved distinctly. These studies also identified a potential link between the number of chromosomally-integrated copies of PAPI-1 and the stability of the island.;Several approaches, including the introduction of a third attB site within a 'neutral' genomic location combined with DNA signature copy number assays and pulsed field gel electrophoresis to identify large scale inversions, deletions or translocations involving rrn operons or tRNA genes were used to further investigate this mysterious 'dynamic-yet-stable genome'. The results demonstrated that rrn operon-mediated inversions are a constantly occurring in P. aeruginosa populations. Each strain does not have a single genomic orientation but rather a dominant orientation with other genomic variants also present within the population. 2014-12-15T10:39:59Z IgE-associated enhancement of allergen presentation and T cell activation in atopic dermatitis Cooper, Dawn. http://hdl.handle.net/2381/30502 2014-12-16T02:27:29Z 2014-12-15T10:39:59Z Title: IgE-associated enhancement of allergen presentation and T cell activation in atopic dermatitis Authors: Cooper, Dawn. Abstract: Previous research has suggested that IgE-facilitated allergen presentation may play a key role in the pathogenesis of atopic dermatitis. This is the process by which allergen-specific IgE bound to Fcepsilon receptors on antigen presenting cells is able to enhance the uptake and presentation of allergen and thus the activation of allergen-specific T cells. However, until now IgE-facilitated allergen presentation has only been demonstrated in systems biased to maximise this process. This present work sought to determine whether IgE-mediated enhancement of allergen presentation could be demonstrated in vitro using human peripheral blood mononuclear cells from atopic dermatitis donors cultured in the presence of 10% autologous serum containing allergen-specific IgE. PBMC from 11/20 atopic dermatitis patients responded in a dose-dependent manner to Der p with maximal stimulation index (mSI) values of 3.7-70.7, whereas normal donors did not respond (mSI < 2.7, n=8). Heating of autologous serum at 56°C for 60 minutes, conditions which irreversibly denature IgE, caused major reductions in peripheral blood mononuclear cell responses to Der p (p=0.002, paired t test, n=9). Incubation of peripheral blood mononuclear cells with blocking monoclonal antibodies against the high (FcepsilonRI) and low (CD23) affinity IgE receptors produced inconsistent inhibition of responses to in vitro (n=6). Flow cytometric measurements on peripheral blood leucocytes from AD patients further indicated that IgE-receptor blocking antibodies were unable to displace monocyte surface bound IgE, even after 24 hours in culture. However, peripheral blood mononuclear cell responses to Der p were significantly reduced in cultures containing 5% IgE depleted serum compared to unmodified serum (mean mSI 8.8 and 20.6 respectively, p<0.03, n=6). These experiments support the concept that T cell proliferation in individual atopic dermatitis patients may be influenced by an IgE-dependent mechanism, probably IgE-facilitated allergen presentation. 2014-12-15T10:39:59Z Membrane spanning 4A gene family expression and function in human mast cells Cruse, Glenn http://hdl.handle.net/2381/30500 2014-12-16T02:27:26Z 2014-12-15T10:39:58Z Title: Membrane spanning 4A gene family expression and function in human mast cells Authors: Cruse, Glenn Abstract: Mast cells are major effector cells of allergic and inflammatory disease. Thus understanding mechanisms of mast cell biology are of great interest to cell biology research. This study aimed to identify the expression and function of a newly identified gene family, the MS4A family, in human mast cells. This study identified 8 gene variants expressed in mast cells including 2 novel variants. All of the expressed genes were cloned and GFP and adenoviral constructs were generated. Quantitative RT-PCR demonstrated that all expressed gene variants were differentially regulated by mast cell stimulation with IgE, IgE/anti-lgE and stem cell factor (SCF) suggesting roles in mast cell biology. Transfections demonstrated that most proteins were trafficked to the cytoplasmic membrane, but some were trafficked to the nuclear membrane. This intracellular localisation of the MS4A family may be critical for their function. This was exemplified in this study by the function of a novel truncation (MS4A2trunc) of MS4A 2 (the beta chain of FceRI). MS4A2trunc was trafficked to the nuclear membrane, whereas MS4A 2 was trafficked to the cytoplasmic membrane. Overexpression of MS4A 2 using adenoviral transduction had no apparent effect on mast cell survival or proliferation. However, overexpression of MS4A2trunc profoundly inhibited mast cell proliferation and induced apoptosis via G2 phase cell cycle arrest. The removal of SCF from mast cell cultures upregulated the expression of MS4A2trunc. In addition, the slowly replicating primary human lung mast cells, and the mast cell line LAD-2 expressed MS4A2trunc. However, I was unable to demonstrate expression of MS4A2trunc in the rapidly replicating c-KIT gain-of-function mutated mast cell line HMC-1. Thus loss of expression of MS4A2trunc may be an important step in the development of mast cell neoplasia. This study has identified an entirely novel function for the MS4A 2 gene and has opened numerous avenues for future research on the MS4A family. 2014-12-15T10:39:58Z Lipid bodies in mycobacteria Sherratt, Anna Louise http://hdl.handle.net/2381/30499 2014-12-16T02:27:25Z 2014-12-15T10:39:57Z Title: Lipid bodies in mycobacteria Authors: Sherratt, Anna Louise Abstract: A survey of clinical samples revealed that LBs are a universal feature of tubercle bacilli in sputum. A number of conditions including hypoxia, Nitric Oxide (NO) exposure, pH, heat and cold shock were shown to promote LB formation in M. tuberculosis in vitro. The formation of LBs in NO exposed M. tuberculosis was shown to correlate with the level of antibiotic tolerance displayed by the population. Antibiotic tolerance was thought to be a result of transitory growth arrest; however attempts to assess the growth status of LB positive M. tuberculosis cells were unsuccessful. The morphology of LBs in mycobacteria varied according to the growth condition of the cell and may be due to a change in lipid composition. The mechanism by which LBs are formed in mycobacteria remains unknown; however, there was some evidence to suggest that it follows a scheme similar to that which has been previously demonstrated in Rhodococcus opacus..;It was concluded that LB formation in mycobacteria may depend on a number of environmental factors, including conditions that promote growth arrest. The formation of LBs in M. tuberculosis may anticipate antibiotic tolerance. The presence of LBs in sputum tubercle bacilli may be used to assess treatment response in patients with tuberculosis; however, it remains to be shown that LB positive M. tuberculosis cells in vitro represent the physiological LB positive sputum bacilli. 2014-12-15T10:39:57Z Human toll - like receptor 9 (hTLR9) expression and function in haematological and non-haematological malignancies Assaf, Areej Mashhour Tawfiq http://hdl.handle.net/2381/30498 2014-12-16T02:27:22Z 2014-12-15T10:39:54Z Title: Human toll - like receptor 9 (hTLR9) expression and function in haematological and non-haematological malignancies Authors: Assaf, Areej Mashhour Tawfiq Abstract: This thesis addresses the expression and function of human TLR9 in haematological and non-haematological tumour cells. I have shown that most B linage tumour cell lines expressed TLR9, with the exception of myeloma cell lines U266, Karpas 707H and the EBV-lymphoblastoid HMy2 cell line, whereas all non-haematological cell lines tested were negative or very weakly positive. TLR9 positive B-cells/B-cell lines, responded to CpG-ODN activation by activating intracellular signalling pathways, cytokine release, surface marker upregulation and cellular proliferation, whereas TLR9 negative cells did not.;Data on the ability of pre-treatment with low doses of CpG-ODN to induce a refractory state (tolerance) on Burkitt's lymphoma BJAB cells indicates that low doses of CpG-ODN tolerised NF-kappaB activation and, to some extent, cellular proliferation and surface marker upregulation. Moreover, reactivating NF-kappaB on BJAB cells after being tolerised with low dose of CpG-ODN is time dependent with the tolerising effect lasting up to two weeks after a single dose of CpG-ODN.;Finally, inhibiting the signalling pathways (ERK, p38MAPK, PI3K/AKTand NF-kappaB) with selective inhibitors U0126, SB203580, LY294002 and Curcumin respectively, has shown different effects on basal and CpG-ODN activated surface marker expression, cellular proliferation and cytokine release. Furthermore, different signalling pathways mediate different functional effects of CpG-ODN/TLR9 activation, and suggest that ERK and p38 MAPK pathways are involved in CpG-ODN mediated NF-kappaB activation. This work should shed a new light on the mechanisms of action of CpG activation on TLR5 signalling in immune and non-immune cells and on its immunotherapeutic use in the treatment of cancer. 2014-12-15T10:39:54Z Characterising the mobile genome of Shigella Lonnen, James David http://hdl.handle.net/2381/30495 2014-12-16T02:27:18Z 2014-12-15T10:39:53Z Title: Characterising the mobile genome of Shigella Authors: Lonnen, James David Abstract: Nineteen Shigella strains were probed for the presence of GIs using a high throughput PCR screen (tRIP) across 16 tRNA gene integration hotspots. Putative GIs were then investigated using a chromosome walking technique (SGSP-PCR). Representative PCR amplicons were sequenced to get a snapshot of the islands contents. Islands of particular interest were characterised further using allelic exchange and marker rescue to capture clones that harbour larger portions of the GI and subsequent sequencing and analysis.;Using SGSP-PCR, 81% of the putative GI occupied tRNA loci were characterised, and sequencing analysis found they all contain island DNA, indicating that tRIP followed by SGSP-PCR is a robust strategy for GI discovery in unsequenced strains; also this method should be applicable to a broad range of microorganisms.;At lest 54% of the islands identified harbour phage-like integrase genes, strongly supporting the notion that many of these elements arose following acquisition of horizontally acquired integrative GIs. The frequent presence of integrase genes also highlights the potential role of bacteriophage in the original and/or ongoing dissemination of DNA in Shigella..;Only one novel GI was discovered; it has classic prophage-like features and contains completely novel DNA, indicating that while Shigella has a plastic genome, it is a highly specialised human pathogen that has undergone considerable pathoadaptive genome reduction.;The major development from this study is evidence that a number of key Shigella virulence determinants are independently mobile and not only localised to a single family of islands; this significantly increases their potential to spread by HGT across Shigella and could contribute to the rapid emergence of new endemic strains. 2014-12-15T10:39:53Z Enhancement of cellular adaption to physical training in chronic obstructive pulmonary disease : a randomised placebo controlled trial of creatine supplementation Deacon, Sarah Jane http://hdl.handle.net/2381/30497 2014-12-16T02:27:21Z 2014-12-15T10:39:53Z Title: Enhancement of cellular adaption to physical training in chronic obstructive pulmonary disease : a randomised placebo controlled trial of creatine supplementation Authors: Deacon, Sarah Jane Abstract: Dietary creatine supplementation (CrS) has been shown to augment high-intensity exercise training in athletes and healthy elderly, thereby increasing muscle mass. Uptake is increased by exercise and is likely to be most beneficial when combined with training. Previous small studies looking at the effects of CrS during pulmonary rehabilitation in subjects with COPD have shown conflicting results.;Hypothesis: CrS in association with aerobic exercise and resistance training will usefully augment the benefits of pulmonary rehabilitation.;Methods: This hypothesis was tested by a randomised double blind, placebo-controlled, parallel group trial of CrS during PR in patients with COPD. One hundred subjects with COPD (mean (SD) age 68.2 (8.2) years, FEV1 44.0 (19.6) percent predicted) were randomised to receive creatine (22g/day loading for 5-days, maintenance 3.76g/d throughout PR) or placebo (lactose) supplements during 7-weeks of PR encompassing aerobic and resistance exercises. Baseline, post-loading and post-rehabilitation measurements included pulmonary function, body composition, peripheral muscle strength and functional performance (shuttle walking tests and cycle ergometry).;A protocol was developed for testing isokinetic strength in subjects with COPD, using a Cybex II dynamometer. 2014-12-15T10:39:53Z Moleulcar characterisation of lipoprotein processing in streptococcus uberis Denham, Emma Louise http://hdl.handle.net/2381/30496 2014-12-16T02:27:19Z 2014-12-15T10:39:53Z Title: Moleulcar characterisation of lipoprotein processing in streptococcus uberis Authors: Denham, Emma Louise Abstract: Streptococcus uberis is a common cause of bovine mastitis and the lipoprotein MtuA has been shown to be essential for growth in milk and virulence. The enzymes responsible for processing lipoproteins in other Gram positive bacteria are lipoprotein diacylglyceryl transferase (Lgt) which acts to anchor lipoproteins to the membrane and lipoprotein signal peptidase (Lsp) which cleaves the signal peptide. S. uberis mutants containing lesions in lgt and lsp uncovered several novel phenotypes. A number of additional proteins were shown to be present in extracellular fractions prepared from lgt- and lgt-/lsp- mutants when compared to the equivalent fraction prepared from wild type bacteria. Atypical processing of MtuA and other lipoproteins within the signal peptide was shown to occur, indicating the presence of an activity capable of shaving such proteins from the membrane in the absence of Lgt and Lsp activity. MtuA was shown to be released into the extracellular space by Wetern blot; the size closely resembled that of wild type protein in both the lgt- and lgt-/lsp- mutants. A metallopeptidase that can be inhibited by phosphoramidon and metal ion chelating agents may be responsible for the activity that results in these proteins being alternatively processed. MtuA in the lsp- mutant had a molecular weight that corresponded to full length MtuA and remained localised in the membrane as seen in the wild type. During late log phase a second form of MtuA with a lower molecular weight was detected. A mutant containing insertions in both lsp and the gene encoding the S. uberis homologue to the Enterococcus faecalis was studied. Enhanced expression of pheromone (eep) suggested that this metallopeptidase was also able to cleave the signal peptide of MtuA. 2014-12-15T10:39:53Z The role of hypoxia and its interaction with the PI3K/Akt pathway in the pathogenesis of malignant pleural mesothelioma Stewart, Duncan John http://hdl.handle.net/2381/30493 2014-12-16T02:27:15Z 2014-12-15T10:39:52Z Title: The role of hypoxia and its interaction with the PI3K/Akt pathway in the pathogenesis of malignant pleural mesothelioma Authors: Stewart, Duncan John Abstract: Carbonic Anhydrase (CA) IX, a surrogate marker of hypoxia, is upregulated downstream of the stabilisation of hypoxia-inducible factor (HIF)-1alpha and is over-expressed in solid tumours. Protein Kinase B, or Akt, known to have important intracellular roles including resistance to apoptosis, is activated in human malignancies and upregulated in hypoxic conditions.;This body of work examined the expression of CA IX and phosphorylated Akt (pAkt) in tumour samples from patients with MPM, correlating expression with established prognostic factors. The role of pAkt and HIF-1alpha in the survival of an MPM cell line exposed to hypoxic conditions was also examined. Tumour samples from 200 patients with MPM were stained using pAkt and CA IX specific antibodies. The effect of hypoxia on apoptosis was evaluated in 4 mesothelioma cell lines and 1 benign mesothelial cell line, in the presence or absence of the phosphatidylinositol-3-kinase inhibitor, LY294002.;There was a positive association between the level of CA IX and pAkt staining, implying that intra-tumoural hypoxia may be a stimulus for Akt phosphorylation. On multivariate analysis increased expression of nuclear pAkt was found to be associated with a poor survival. In-vitro cell culture work showed that, although pAkt is expressed in normoxic conditions in the cell lines studied, in the JU77 cell line the rates of apoptosis were significantly increased in hypoxic conditions when the phosphorylation of Akt was blocked by LY294002.;This work provides evidence for the anti-apoptotic role of pAkt in hypoxic conditions in solid human malignancies. Phospho-Akt may represent a novel therapeutic target in MPM. 2014-12-15T10:39:52Z Development of monocyte and macrophage subpopulations Aldraihim, Asaad http://hdl.handle.net/2381/30494 2014-12-16T02:27:16Z 2014-12-15T10:39:52Z Title: Development of monocyte and macrophage subpopulations Authors: Aldraihim, Asaad Abstract: The mechanism by which monocytes develop into different macrophages in vivo is not well understood. This study examines the in vitro development of different types of monocytes-derived macrophages (MDM) from the two main blood monocyte subsets (CD14++ Mo and CD14+16+Mo).;Culture of CD14++Mo and CD14+16 +Mo with macrophage colony-stimulating factor (M-CSF) for 7 days generated two phenotypically and functionally different types of macrophage. The hallmark of CD14+16+MDM is the expression of CD209 (DC-SIGN), while CD14++MDM remains CD209 negative. In addition, CD14 +16+MDM show higher expression of CD14 and human leukocyte antigen-DR (HLA-DR), reflecting the ability to bind lipopolysaccharide (LPS) and for antigen presentation respectively. Moreover, alternative activation of macrophages with interleukin-13 (IL-13) led to the generation of macrophages with an almost similar phenotype.;Functional analysis of cells cultured with M-CSF revealed that a higher percentage of CD14+16+MDM were able to interact with microspheres. Looking at different mycobacteria, I found that both macrophages generated showed low ability to interact with M. chelonae and M. smegmatis. On the other hand, interaction with M. bovis Bacille-Calmette-Guerin (BCG) was strong and the number of bacteria interacting with CD14+16+MDM was higher than in CD14++MDM.;In conclusion, I succeeded in generating from the two blood monocytes subsets two different types of macrophage with distinct phenotypes. Moreover, they showed remarkably different functions with respect to their ability to interact with foreign materials. This evidence is the first to show that the type of monocytes can determine the type of macrophage generated. 2014-12-15T10:39:52Z Characterisation of Mannan Binding Lectin associated serine proteases MASP-2, MASP-3, and of the MASP-2 related plasma protein MAp19 in the chicken : evidence for the absence of MASP-1 in birds Khan, Saeed-ul-Hassan. http://hdl.handle.net/2381/30492 2014-12-16T02:27:13Z 2014-12-15T10:39:51Z Title: Characterisation of Mannan Binding Lectin associated serine proteases MASP-2, MASP-3, and of the MASP-2 related plasma protein MAp19 in the chicken : evidence for the absence of MASP-1 in birds Authors: Khan, Saeed-ul-Hassan. Abstract: Very recently a novel pathway of complement activation, termed the lectin pathway, has been described. In this pathway, three serum components act as sugar recognition molecules, Mannan Binding Lectin (MBL) and two members of the ficolin family (H-ficolin and L-Ficolin). In human and rodents, these three molecules associate with serine proteases, the so-called MBL associated serine proteases (MASP-1, MASP-2 and MASP-3) and a MASP-2 related non-protease MAp19. Activation of complement via the lectin pathway occurs when MBL or ficolins interact with carbohydrate structures present on yeast, bacteria and viruses. This interaction is translated into the activation of the complement cascade by the conversion of MASP-2 proenzyme (it is not yet clear if and how MASP-1 participates in this process) into its enzymatically active form. The functions of MASP-3 and MAp19 are unknown. The present PhD project was aimed at the characterisation of chicken MASPs and MAp19. Complete MASP-3 specific cDNA and partial MASP-2 and MAp19 specific cDNAs were isolated from chicken liver by RT-PCR, cDNA library screening and RACE technique. Northern blot analyses on RNA prepared from livers from seven birds provided evidence for the absence of MASP-l, while MASP-3 is expressed. In situ hybridisation experiments on chicken embryos revealed that while MASP-2 mRNA is expressed only in the liver, MASP-3 mRNA is also synthesised in extra-hepatic locations e.g. organs of nervous system, urinary system and gastrointestinal tract. Finally C4 cleavage assays were done to measure the lectin pathway activity in sera of birds in response to microbial surfaces and microbial carbohydrates. To conclude, this project is the first description of the primary structure and site of synthesis of MASP-2, MAp19 and MASP-3 mRNA in sauropsides. 2014-12-15T10:39:51Z New insight into the molecular mechanisms of corticosteroid resistance in asthma Chachi, Latifa http://hdl.handle.net/2381/29242 2014-11-05T02:01:35Z 2014-11-04T16:25:22Z Title: New insight into the molecular mechanisms of corticosteroid resistance in asthma Authors: Chachi, Latifa Abstract: Although corticosteroids are very efficient in managing asthma and other inflammatory diseases, a small percentage of patients affected by "severe asthma" fail to respond even to high doses of oral glucocorticoids. It is therefore important to try to understand the potential mechanisms behind this insensitivity to corticosteroid therapy in order to be able to effectively control asthma in this patient subset. We have decided to focus on one particular channel called K[subscript Ca]3.1, a calcium-activated potassium channel. First, emerging evidence in the literature to date has strongly supported a significant role for K[subscript Ca]3.1 channel in the pathophysiology of asthma. K[subscript Ca]3.1 channel is expressed by several inflammatory and structural airway cells including mast cells and human bronchial smooth muscle (HBSM). Therefore these channels might serve as new targets for the treatment of lung diseases. Here we established a cellular model of corticosteroid insensitivity consisting of primary HBSM cells exposed to two cytokines TNF-α and IFN-γ. Under these conditions, HBSMC exhibit a marked production of different pro-asthmatic chemokines like CCL5, CX3CL1, CCL11 and CXCL10 that are completely resistant to corticosteroid treatment. In this model, we found that although K[subscript Ca]3.1 channel expression did not change between healthy control, asthmatic and COPD subjects, K[subscript Ca]3.1 channel blockers (ICA-17043 and TRAM-34) were able to inhibit the production of corticosteroid-resistant chemokines either directly via the suppression of gene expression or indirectly via the restoration of the anti-inflammatory action of fluticasone. We also found that K[subscript Ca]3.1 channel blockers restored cell sensitivity to corticosteroid in cytokine-treated HBSMC by re-establishing the transactivation function of fluticasone via the prevention of dephosphorylation of Glucocorticoid Receptors (GRα) at Ser[superscript 211] and induction of anti-inflammatory genes such as Glucocorticoid-induced leucine zipper (GILZ). The likely mechanism of this restoration of corticosteroid sensitivity by K[subscript Ca]3.1 channel blockers is via the inhibition of protein phosphatase 5 (PP5) expression found to be up-regulated in steroid resistant conditions. 2014-11-04T16:25:22Z Early pulmonary rehabilitation for exacerbations of chronic obstructive pulmonary disease Greening, Neil James http://hdl.handle.net/2381/29155 2014-10-10T01:01:39Z 2014-10-09T11:48:05Z Title: Early pulmonary rehabilitation for exacerbations of chronic obstructive pulmonary disease Authors: Greening, Neil James Abstract: Exacerbations are key events in the natural history of chronic obstructive pulmonary disease (COPD), with limited recovery of physical performance, and the highest cause of readmission in the UK. This thesis explores the impact of exacerbations in COPD and chronic respiratory disease. In the first study I have investigated the effects of an early rehabilitation intervention on healthcare utilisation, strength and exercise capacity by conducting a large randomised control trial. Using a sub-group of this cohort I have then explored factors that predict hospital readmission. Finally I have conducted a study of single leg neuromuscular electrical stimulation (NMES) in stable COPD, alongside a resistance training group. No difference was seen following early rehabilitation in hospitalisation, healthcare utilisation or physical performance. A number of unexpected findings were noted, including an increase in 12 month mortality in the intervention group and large functional recovery in the usual care group. Using multivariate analysis three risk factors for hospital readmission were identified, including quadriceps cross sectional area, using ultrasound. In the stable state NMES was seen to significantly increase muscle mass from baseline, comparable to changes seen using resistance training. In summary early rehabilitation in chronic respiratory disease does not impact on future hospitalisation. Identification of those with rehabilitation potential is required as the hospitalised population represent a frail group, with advanced disease. 2014-10-09T11:48:05Z IL-9 is a key component of memory TH cell peanut-specific responses from children with peanut allergy Brough, Helen A. Cousins, David J. Munteanu, Alina Wong, Yuen Fei Sudra, Asha Makinson, Kerry Stephens, Alick C. Arno, Matthew Ciortuz, Liviu Lack, Gideon Turcanu, Victor http://hdl.handle.net/2381/29140 2014-10-08T01:01:31Z 2014-10-07T13:48:00Z Title: IL-9 is a key component of memory TH cell peanut-specific responses from children with peanut allergy Authors: Brough, Helen A.; Cousins, David J.; Munteanu, Alina; Wong, Yuen Fei; Sudra, Asha; Makinson, Kerry; Stephens, Alick C.; Arno, Matthew; Ciortuz, Liviu; Lack, Gideon; Turcanu, Victor Abstract: Background : Differentiation between patients with peanut allergy (PA) and those with peanut sensitization (PS) who tolerate peanut but have peanut-specific IgE, positive skin prick test responses, or both represents a significant diagnostic difficulty. Previously, gene expression microarrays were successfully used to identify biomarkers and explore immune responses during PA immunotherapy. Objective : We aimed to characterize peanut-specific responses from patients with PA, subjects with PS, and atopic children without peanut allergy (NA children). Methods : A preliminary exploratory microarray investigation of gene expression in peanut-activated memory TH subsets from 3 children with PA and 3 NA children identified potential PA diagnostic biomarkers. Microarray findings were confirmed by using real-time quantitative PCR in 30 subjects (12 children with PA, 12 children with PS, and 6 NA children). Flow cytometry was used to identify the TH subsets involved. Results : Among 12,257 differentially expressed genes, IL9 showed the greatest difference between children with PA and NA children (45.59-fold change, P < .001), followed by IL5 and then IL13. Notably, IL9 allowed the most accurate classification of children with PA and NA children by using a machine-learning approach with recursive feature elimination and the random forest algorithm. Skin- and gut-homing TH cells from donors with PA expressed similar TH2- and TH9-associated genes. Real-time quantitative PCR confirmed that IL9 was the highest differentially expressed gene between children with PA and NA children (23.3-fold change, P < .01) and children with PS (18.5-fold change, P < .05). Intracellular cytokine staining showed that IL-9 and the TH2-specific cytokine IL-5 are produced by distinct TH populations. Conclusion : In this study IL9 best differentiated between children with PA and children with PS (and atopic NA children). Mutually exclusive production of IL-9 and the TH2-specific cytokine IL-5 suggests that the IL-9–producing cells belong to the recently described TH9 subset. 2014-10-07T13:48:00Z Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33 Pfeffer, P. E. Chen, Y. H. Woszczek, G. Matthews, N. C. Chevretton, E. Gupta, A. Saglani, S. Bush, A. Corrigan, C. Cousins, David J. Hawrylowicz, C. M. http://hdl.handle.net/2381/29130 2014-10-04T01:01:30Z 2014-10-03T13:32:48Z Title: Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33 Authors: Pfeffer, P. E.; Chen, Y. H.; Woszczek, G.; Matthews, N. C.; Chevretton, E.; Gupta, A.; Saglani, S.; Bush, A.; Corrigan, C.; Cousins, David J.; Hawrylowicz, C. M. Abstract: Vitamin D enhances production of sST2, the soluble decoy receptor for pro - inflammatory IL - 33 . This likely contributes to the association between vitamin D sufficiency and improved asthma control. Description: The file associated with this record is embargoed until 12 months after the date of publication. The final published version may be available through the links above. 2014-10-03T13:32:48Z Distinct conformations of the chemokine receptor CCR4 with implications for its targeting in allergy Viney, Jonathan M. Phillips, Rhian M. Meiser, Andrea Patel, Pallavi Pease, James E. Andrew, David P. Barton, Nicholas P. Hall, David A. Lennartz-Walker, Melissa Cousins, David J. http://hdl.handle.net/2381/29116 2014-09-26T01:01:30Z 2014-09-25T11:23:19Z Title: Distinct conformations of the chemokine receptor CCR4 with implications for its targeting in allergy Authors: Viney, Jonathan M.; Phillips, Rhian M.; Meiser, Andrea; Patel, Pallavi; Pease, James E.; Andrew, David P.; Barton, Nicholas P.; Hall, David A.; Lennartz-Walker, Melissa; Cousins, David J. Abstract: CC chemokine receptor 4 (CCR4) is expressed by Th2 and regulatory T cells and directs their migration along gradients of the chemokines CCL17 and CCL22. Both chemokines and receptor are upregulated in allergic disease, making CCR4 a therapeutic target for the treatment of allergy. We set out to assess the mechanisms underlying a previous report that CCL22 is a dominant ligand of CCR4, which may have implications for its therapeutic targeting. Human T cells expressing endogenous CCR4 and transfectants engineered to express CCR4 were assessed for receptor function, using assays of calcium release, chemotaxis, receptor endocytosis, and ligand binding. Despite the two ligands having equal potency in calcium flux and chemotaxis assays, CCL22 showed dominance in both receptor endocytosis assays and heterologous competitive binding assays. Using two different CCR4-specific Abs, we showed that CCR4 exists in at least two distinct conformations, which are differentially activated by ligand. A major population is activated by both CCL17 and CCL22, whereas a minor population is activated only by CCL22. Mutation of a single C-terminal residue K310 within a putative CCR4 antagonist binding site ablated activation of CCR4 by CCL17, but not by CCL22, despite having no effect on the binding of either ligand. We conclude that CCL17 and CCL22 are conformationally selective ligands of CCR4 and interact with the receptor by substantially different mechanisms. This finding suggests that the selective blockade of CCR4 in allergy may be feasible when one CCR4 ligand dominates, allowing the inhibition of Th2 signaling via one ligand while sparing regulatory T cell recruitment via another. The Journal of Immunology, 2014, 192: 3419-3427. © Copyright 2014 by The American Association of Immunologists, Inc. All rights reserved. Description: Full text of this item is not currently available on the LRA. The final published version may be available through the links above. 2014-09-25T11:23:19Z Sphingosine-1-phosphate induces pro-remodelling response in airway smooth muscle cells. Fuerst, Elisabeth Foster, Holly R. Ward, Jeremy P. T. Corrigan, Christopher J. Cousins, David J. Woszczek, Grzegorz http://hdl.handle.net/2381/29026 2014-08-09T01:01:24Z 2014-08-08T08:51:52Z Title: Sphingosine-1-phosphate induces pro-remodelling response in airway smooth muscle cells. Authors: Fuerst, Elisabeth; Foster, Holly R.; Ward, Jeremy P. T.; Corrigan, Christopher J.; Cousins, David J.; Woszczek, Grzegorz Abstract: Background : Increased proliferation of airway smooth muscle (ASM) cells leading to hyperplasia and increased ASM mass is one of the most characteristic features of airway remodelling in asthma. A bioactive lipid, sphingosine-1-phosphate (S1P), has been suggested to affect airway remodelling by stimulation of human ASM cell proliferation. Objective : To investigate the effect of S1P on signalling and regulation of gene expression in ASM cells from healthy and asthmatic individuals. Methods : ASM cells grown from bronchial biopsies of healthy and asthmatic individuals were exposed to S1P. Gene expression was analysed using microarray, real-time PCR and western blotting. Receptor signalling and function was determined by mRNA knockdown and intracellular calcium mobilisation experiments. Results : S1P potently regulated the expression of more than 80 genes in human ASM cells, including several genes known to be involved in the regulation of cell proliferation and airway remodelling (HBEGF, TGFB3, TXNIP, PLAUR, SERPINE1, RGS4). S1P acting through S1P[subscript 2] and S1P[subscript 3] receptors activated intracellular calcium mobilisation and extracellular signal-regulated and Rho-associated kinases to regulate gene expression. S1P-induced responses were not inhibited by corticosteroids and did not differ significantly between ASM cells from healthy and asthmatic individuals. Conclusion : S1P induces a steroid-resistant, pro-remodelling pathway in ASM cells. Targeting S1P or its receptors could be a novel treatment strategy for inhibiting airway remodelling in asthma. Description: The file associated with this record is embargoed until 12 months after the date of first publication. The final published version may be available through the links above. 2014-08-08T08:51:52Z Fibronectin is a TH1-specific molecule in human subjects Sandig, Hilary McDonald, Joanne Gilmour, Jane Arno, Matthew Lee, Tak H. Cousins, David J. http://hdl.handle.net/2381/29025 2014-09-26T01:01:22Z 2014-08-08T08:29:10Z Title: Fibronectin is a TH1-specific molecule in human subjects Authors: Sandig, Hilary; McDonald, Joanne; Gilmour, Jane; Arno, Matthew; Lee, Tak H.; Cousins, David J. Abstract: Background : T[Subscript H]1 cell–mediated immunity is essential for host defense against a variety of intracellular pathogens, such as mycobacteria, salmonella, and Leishmania species. A major T[subscript H]1-mediated effector mechanism involves the IFN-γ–induced killing of the pathogen by infected macrophages. Objectives : The range of known T[subscript H]1-specific effector molecules is limited, especially in human subjects. We sought to identify novel effector molecules that might be involved in T[subscript H]1-mediated pathogen clearance. Methods : We performed microarray-based analysis of human T[subscript H]1 and T[subscript H]2 cells to identify T[subscript H]1-specific molecules. These analyses identified the extracellular matrix molecule fibronectin as a highly expressed T[subscript H]1-specific molecule. We examined the expression of fibronectin in a variety of human cell types by using real-time RT-PCR, ELISA, and Western blotting. We also studied the role of fibronectin in modulating monocyte phenotype using in vitro culture. Results : We show that human T[subscript H]1 cells constitutively express and secrete fibronectin after in vitro differentiation from naive precursors. Furthermore, we demonstrate that ex vivo human T[subscript H]1 cells selectively express fibronectin when compared with T[subscript H]2 cells. The predominant isoform of fibronectin expressed by T[subscript H]1 cells contains additional domains of the protein responsible for α4β1 integrin binding and activation of Toll-like receptor 4. We show that treatment of monocytes with T[subscript H]1 cell–derived fibronectin induces expression of the proinflammatory cytokine IL-6 while inhibiting IL-10 expression. Conclusions : Because fibronectin also plays a major role in the attachment and opsonization of numerous intracellular pathogens, we propose that it might be a critical molecule produced by T[subscript H]1 cells involved in pathogen eradication. 2014-08-08T08:29:10Z Human Th2 cells selectively express the orexigenic peptide, pro-melanin-concentrating hormone Sandig, Hilary McDonald, Joanne Gilmour, Jane Arno, Matthew Lee, Tak H. Cousins, David J. http://hdl.handle.net/2381/29024 2014-08-08T08:56:38Z 2014-08-07T15:54:42Z Title: Human Th2 cells selectively express the orexigenic peptide, pro-melanin-concentrating hormone Authors: Sandig, Hilary; McDonald, Joanne; Gilmour, Jane; Arno, Matthew; Lee, Tak H.; Cousins, David J. Abstract: Th1 and Th2 cells represent the two main functional subsets of CD4+ T helper cell, and are defined by their cytokine expression. Human Th1 cells express IFNγ, whilst Th2 cells express IL-4, IL-5, and IL-13. Th1 and Th2 cells have distinct immunological functions, and can drive different immunopathologies. Here, we show that in vitro-differentiated human Th2 cells highly selectively express the gene for pro-melanin-concentrating hormone (PMCH), using real-time RT-PCR, enzyme immunoassay, and Western blot analysis. PMCH encodes the prohormone, promelanin-concentrating hormone (PMCH), which is proteolytically processed to produce several peptides, including the orexigenic hormone melanin-concentrating hormone (MCH). PMCH expression by Th2 cells was activation responsive and increased throughout the 28-day differentiation in parallel with the expression of the Th2 cytokine genes. MCH immunoreactivity was detected in the differentiated Th2 but not Th1 cell culture supernatants after activation, and contained the entire PMCH protein, in addition to several smaller peptides. Human Th1 and Th2 cells were isolated by their expression of IFNγ and CRTH2, respectively, and the ex vivo Th2 cells expressed PMCH upon activation, in contrast to the Th1 cells. Because Th2 cells are central to the pathogenesis of allergic diseases including asthma, expression of PMCH by activated Th2 cells in vivo may directly link allergic inflammation to energy homeostasis and may contribute to the association between asthma and obesity. Description: Full text of this item is not currently available on the LRA. The final published version may be available through the links above. 2014-08-07T15:54:42Z Clostridium difficile phages : Still difficult? Hargreaves, Katherine R. Clokie, Martha R. J. http://hdl.handle.net/2381/28990 2014-07-18T13:53:50Z 2014-07-18T13:53:21Z Title: Clostridium difficile phages : Still difficult? Authors: Hargreaves, Katherine R.; Clokie, Martha R. J. Abstract: Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however, developing suitable phages is challenging. In this review we summarize the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics. Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage–host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution. No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using "whole-phages" are such that they remain a major research focus. Whilst we don't envisage working with C. difficile phages will be problem-free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen. 2014-07-18T13:53:21Z Lower limb muscle dysfunction in COPD : The effects of resistance training and nutritional supplementation Menon, Manoj Kumar http://hdl.handle.net/2381/28982 2014-07-17T01:01:23Z 2014-07-16T08:58:23Z Title: Lower limb muscle dysfunction in COPD : The effects of resistance training and nutritional supplementation Authors: Menon, Manoj Kumar Abstract: Introduction: Weakness and wasting of the lower limb muscles is a frequent extra-pulmonary manifestation of Chronic Obstructive Pulmonary Disease (COPD). Although the underlying lung pathology is largely irreversible, the impact of the disease on the lower limbs is potentially treatable. Resistance training (RT) is an effective intervention for improving lower limb function in COPD, while nutritional supplementation as an adjunct to RT can provide additional benefits in older adults. The functional impact of this therapeutic combination and the accompanying muscle cellular and molecular changes has not been previously explored in COPD. Methods: This thesis explored the functional, molecular, and cellular responses of the lower limb muscles in COPD patients in response to high-intensity quadriceps isokinetic RT. The primary hypothesis was that RT-induced increases in lower limb muscle mass and strength in COPD patients would be mediated through the expression of genes and proteins associated with muscle mass regulation in humans (chapter 6). Secondary aims were to determine whether protein-carbohydrate supplementation at the time of training would augment the gains in functional performance in COPD (chapter 5), to study the muscle inflammatory and satellite cell profile in response to RT (chapter 7), and to measure the training-induced changes in lower limb muscle mass using portable ultrasound (chapter 8). Results: All participants demonstrated significant improvements in lower limb muscle mass and strength following RT, but protein-carbohydrate supplementation did not provide additional benefits. Absolute work done during RT was lower throughout in COPD when compared to healthy controls (HCs). Except for myogenic signalling, the increased expression of anabolic, catabolic, and transcription factor proteins was blunted in COPD patients, and nutritional supplementation did not alter this response. Inflammatory cells in the lower limb muscles increased in response to acute RT in COPD, but returned to baseline with chronic training. Portable ultrasound was shown to be a reproducible and sensitive technique for measuring changes in lower limb muscle mass following RT in COPD. Conclusions: This thesis demonstrates that the potential for lower limb muscle rehabilitation in response to RT is preserved in COPD, while post-exercise protein-carbohydrate supplementation did not augment the functional or molecular responses. Except for myogenic proteins, the molecular responses to training were uncoupled from the functional gains, and more closely related to the absolute workloads performed during training. 2014-07-16T08:58:23Z The role of complement properdin in murine infection with Listeria monocytogenes Mohamed M Mohamed, Fatima http://hdl.handle.net/2381/28975 2014-07-11T01:01:29Z 2014-07-10T14:57:15Z Title: The role of complement properdin in murine infection with Listeria monocytogenes Authors: Mohamed M Mohamed, Fatima Abstract: Properdin or complement factor P is a conserved serum glycoprotein of the immune defence. It plays a role in strengthening the activation of complement, a system of proteins essential in the first line defence against infection. Properdin is the only positive regulator and plays a major role in regulating the alternative pathway of the complement system, an effector system of the innate immune response, by binding and stabilising two specific converting enzyme complexes, which are normally labile (C3bBb and C3bBbC3b). Mouse models have shown that complement, in particular complement receptor 3 (CR3) and complement 5 (C5), contributes to survival of infection with Listeria monocytogenes. The purpose of the project was to characterise the contribution of properdin in the response to L. monocytogenes (EGD-e), a Gram-positive, intracellular pathogen, which can cause severe infectious disease in human and animals, by using in vitro and in vivo methods. In vitro assays for the first time point to the significant role of properdin in infection with L. monocytogenes: using dendritic cells and macrophages derived from the bone marrows of properdin-deficient (KO) and wild type mice (WT), cells from WT mice showed greater intracellular load of viable L. monocytogenes at an early time point. Cells from KO mice produced less IFN-γ and nitric oxide compared to cells from WT mice and showed less surface expression of CD40. In addition properdin is found to react as a hypoxia sensitive gene; its expression in WT macrophages was also significantly decreased after infection compared to uninfected cells. In vivo experiments demonstrated for the first time that properdin is necessary in the survival of acute murine listeriosis: properdin-deficient mice were more susceptible to intravenous infection with L. monocytogenes compared to wild type mice, and had a greater systemic IFN-gamma and splenic IL-17A response and greater disease severity with impaired M1 type activation. In conclusion, these findings show that properdin is essential in survival of murine listeriosis and in sustaining a cellular response to the intracellular pathogen L. monocytogenes. 2014-07-10T14:57:15Z The investigation of intrinsic abnormalities in the airway smooth muscle cells in asthma Sutcliffe, Amanda Jane http://hdl.handle.net/2381/28935 2014-06-20T01:01:33Z 2014-06-19T13:17:17Z Title: The investigation of intrinsic abnormalities in the airway smooth muscle cells in asthma Authors: Sutcliffe, Amanda Jane Abstract: Rationale: Airway smooth muscle dysfunction is a cardinal feature of asthma that is believed to contribute to the symptoms and recurrent exacerbations. However, the mechanisms driving this dysfunction are not fully understood. Hypothesis: Intrinsic abnormalities of the ASM from asthma exist and contribute to the functional differences in vitro which correlate with clinical characteristics Methods: Primary airway smooth muscle cells from healthy control subjects and asthmatics were used. Baseline protein and gene expression studies were assessed by mass spectrometry and microarrays. Functional differences were assessed, such as survival, proliferation, migration, wound healing and contraction. Oxidative stress DNA damage was assessed by immunohistology and comet assay, reactive oxygen species (ROS) quantification, and the role of NADPH oxidase (NOX4) and superoxide dismutase (SOD2) were assessed by quantitative gene expression and pharmacological inhibitors, mimetic and gene silencing. Results: In ex vivo airway smooth muscle cultures, asthmatic smooth muscle dysfunction persisted, indicating exaggerated contractility and pro-inflammatory mediator release compared to ASM from healthy controls. The oxidative burden and damage in asthmatic ASM was increased with increased expression of NOX4 which negatively correlated to the airflow obstruction. Agonist induced hyper-contractile response was abrogated by using pharmacological inhibition or by silencing the mRNA. Summary: Intrinsic abnormalities of the airway smooth muscle exist in asthma. Asthmatic ASM cells possess intrinsic abnormalities in gene and protein expression, synthetic mediator production and exaggerated agonist induced contraction. Increased expression of NOX4 in asthmatic ASM promotes the susceptibility of a hyper-contractile response, implicating NOX4 as a potential therapeutic target for the treatment of asthma. 2014-06-19T13:17:17Z Studies on the phenotypes of Mycobacterium tuberculosis in sputum Binjomah, Abdulwahab Zaid A. http://hdl.handle.net/2381/28914 2014-06-17T01:01:25Z 2014-06-16T10:04:32Z Title: Studies on the phenotypes of Mycobacterium tuberculosis in sputum Authors: Binjomah, Abdulwahab Zaid A. Abstract: Transcriptional, cytological and culture-based analyses of Mycobacterium tuberculosis (Mtb) in sputum have revealed multiple traits indicating the presence of a persister-like or dormant mycobacterial population. Between patients, variable proportions of bacilli in sputum appear to be slow or non-growing, contain lipid bodies (LBs) and depend on exogenous Resuscitation promoting factors for growth. More recently by using Auramine O/Nile-red staining the presence of non-acid-fast (NAF) Mtb-like bacilli containing abundant LBs has been noted. Based on these findings, Mtb in sputum may present in multiple populations and express distinctive transmission adapted phenotypes. Identifying these phenotypes and replicating them in in vitro settings may lead to important new understanding of Mtb in vivo. To study the suspected NAF Mtb cells in sputum, immunofluorescence (IF), peptide nucleic acid (PNA) probe and fluorescence Kinyoun methods were developed and studied. The IF and PNA methods detected only minor components of sputum and variable proportions of in vitro grown cells. Various conditions such as freeze thawing, growth phase and biofilm cultures were shown to alter Auramine NAF proportions. In contrast the fluorescence Kinyoun method labelled the majority of Mtb cells in the preparations studied and provides a promising method for future studies when combined with a suitable LB stain. The capacity of biofilm cultures to replicate the Mtb bacillary populations in sputum was studied. Three phases of biofilm cultures (pellicle, planktonic and attached layers) were studied for gene expression, cytological, growth, antibiotic tolerance and [superscript 3]H-uracil labelling properties comparable to the Mtb phenotypes seen in sputum. The three layers replicated to differing degrees the sputum phenotypes including LB and NAF content, and modest Rpf-dependancy. Attached and planktonic cells gave well correlated transcriptional patterns. Overall, it appears plausible that biofilm grown cells in patient's lungs could contribute to the populations seen in sputum. 2014-06-16T10:04:32Z Characterising IgA1 solution structures in IgA nephropathy Vennard, Owen Learoy http://hdl.handle.net/2381/28828 2014-05-22T01:01:21Z 2014-05-21T10:59:21Z Title: Characterising IgA1 solution structures in IgA nephropathy Authors: Vennard, Owen Learoy Abstract: IgA nephropathy (IgAN) remains the commonest pattern of primary glomerulonephritis seen worldwide and results in the development of end-stage renal failure in over 30% of affected individuals. It is characterised by IgA1 containing immune complex formation and deposition within the mesangium; the reasons for which are unknown. IgAN is associated with aberrant serum O-glycoforms, characterised by over representation of IgA1 molecules displaying undergalactosylation. While a number of studies have postulated a pathogenic role for the IgA1 hinge region O-glycans in IgAN, none have investigated the impact of altered glycosylation on its three-dimensional shape. A combination of small-angle X-ray scattering (SAXS), and analytical ultracentrifugation (AUC) experiments were used to determine whether the monomeric IgA1 (mIgA1) conformational shape was altered in IgAN. Serum IgA1 from a healthy subject and three patients with IgAN with differing IgA1galactosylation were isolated using jacalin affinity chromatography. Monomeric IgA1 species were isolated by FPLC. Relative hinge region O-galactosylation was determined using a standardised helix aspersa agglutinin (HAA) lectin assay. The Guinier X-ray radius of gyration (R[subscript G]) for the healthy control, and the IgAN mIgA1 samples with both elevated and reduced galactosylation were 6.10–6.30 nm. This agreed well with that previously reported for mIgA1 and indicated that under the experimental conditions mIgA1 did not adopt a significantly altered conformation. The AUC sedimentation coefficients for all samples were 6.2 S, in agreement with this. The distance distribution curve P(r) gave an overall length (L) of 21 nm for the healthy mIgA1 control and 22–25 nm for IgAN-associated mIgA1. Purified IgAN-associated IgA1 monomers with the reduced O-galactosylation thus displayed an increased molecular length L, indicating a more extended arrangement in comparison to the healthy mIgA1 control. Elevated O-galactosylation however did not alter the length of mIgA1. The formation of dimer species was observed by AUC for all samples. Although the initial data analyses do not suggest major conformation change, these results provide a promising insight into the potential for altered O-galactosylation in IgA1 to promote self-aggregation, and unravelling of its hinge region which may expose neoantgenic epitopes for autoantibodies and alter IgA-receptor and IgA-extracellular matrix interactions. 2014-05-21T10:59:21Z 6th Annual Postgraduate Student Conference, 1st & 2nd April 2014, Program & Abstracts http://hdl.handle.net/2381/28820 2014-05-21T01:01:06Z 2014-05-20T13:40:01Z Title: 6th Annual Postgraduate Student Conference, 1st & 2nd April 2014, Program & Abstracts Description: This is the programme (including abstracts) of the 6th Annual Postgraduate Student Conference, 1st & 2nd April 2014. 2014-05-20T13:40:01Z Glycosyltransferase expression in IgA[superscript +] B cells in IgA nephropathy Boyd, Joanna Kathryn Fiona http://hdl.handle.net/2381/28816 2014-05-17T01:01:33Z 2014-05-16T15:14:47Z Title: Glycosyltransferase expression in IgA[superscript +] B cells in IgA nephropathy Authors: Boyd, Joanna Kathryn Fiona Abstract: IgA nephropathy is a common kidney disease classically presenting in the second to third decade with episodic haematuria following mucosal infection. Up to a third of patients will develop progressive renal failure requiring renal replacement therapy. IgA nephropathy is diagnosed on renal biopsy by finding deposits containing the immunoglobulin IgA in the renal mesangium. Deposited IgA has particular characteristics: it is typically polymeric, of the IgA1 subtype and the IgA1 hinge region tends to be poorly galactosylated when compared to circulating IgA1 from healthy subjects. IgA is synthesised by IgA-committed B cells usually in response to mucosal antigen encounter. Differential glycosylation of the IgA1 hinge region appears to be important in the normal human immune response and is controlled by specific glycosyl- and sialyl-transferases. Abnormalities in glycosyl- or sialyltransferase expression are therefore predicted to play a role in the generation of poorly galactosylated IgA1 which deposits in the renal mesangium of patients with IgA nephropathy. My aim was to define glycosyltransferase expression in circulating IgA-secreting B cells from patients and compare expression patterns with healthy subjects. We isolated IgA[superscript +] plasmablasts and memory B cells from peripheral blood samples by flow cytometry and interrogated them by quantitative PCR for mRNA expression of the glycosyltransferase C1GalT1 and its molecular chaperone Cosmc, both known to be important in IgA1 hinge region galactosylation. We found that IgA+ plasmablasts but not IgA[superscript +] memory B cells from patients tended to express higher levels of both C1GalT1 and Cosmc when compared to healthy subjects. This confirms previous suggestions that an enzyme abnormality is not global and we show that it may be localised to the IgA[superscript +] plasmablast population. Decreased IgA1 hinge region galactosylation would be predicted to be associated with lower levels of C1GalT1 expression and we speculate that our results demonstrate a compensatory response to poor galactosylation. 2014-05-16T15:14:47Z New approach for the treatment of pneumococcal diseases Pires Damaso, Mafalda http://hdl.handle.net/2381/28799 2014-05-08T01:01:25Z 2014-05-07T12:20:55Z Title: New approach for the treatment of pneumococcal diseases Authors: Pires Damaso, Mafalda Abstract: Pneumolysin (PLY) is a proven virulence factor of Streptococcus pneumoniae, deeply involved in the development of pneumococcal invasive diseases. The antibacterial and adjunctive therapies currently used in the management of pneumococcal infection do not address the pneumolysin-induced cytotoxicity. Therefore, even if patients survive the infection, they may suffer from long-term disabilities as a consequence of the exposure to the toxin. This research seeks to address this problem by developing new approaches to the treatment of pneumococcal diseases, based on peptides or small molecules that target pneumolysin and inhibit its activity. The peptides and small molecules were evaluated for their capacity to inhibit the haemolytic activity of pneumolysin in vitro. In total, four peptides and six small molecules were selected for in vivo tests. In vivo experiments were performed using a pneumonia model, where each animal was administered PBS or peptide / small molecule in PBS intranasally, every 6 hours after intranasal infection. One peptide (P1) and three small molecules (SM6, SM10 and SM14) improved significantly the outcome of the treatment group over the control group, when looking at the CFU in the blood, disease sign score and survival comparison. Moreover, two small molecules (SM10 and SM14) tested ex vivo, showed the capacity to abolish pneumolysin cytotoxic effect on the ciliary function of rat ependymal cells. The pneumolysin inhibitors identified in this work were pointed as potential candidates to further testing in drug discovery. 2014-05-07T12:20:55Z The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells Ashmole, Ian Duffy, S. Mark Leyland, Mark L. Bradding, Peter http://hdl.handle.net/2381/28712 2014-03-27T02:01:45Z 2014-03-26T13:51:39Z Title: The contribution of Orai(CRACM)1 and Orai(CRACM)2 channels in store-operated Ca2+ entry and mediator release in human lung mast cells Authors: Ashmole, Ian; Duffy, S. Mark; Leyland, Mark L.; Bradding, Peter Abstract: Background: The influx of extracellular Ca[superscript 2+] into mast cells is critical for the FcεR1-dependent release of preformed granule-derived mediators and newly synthesised autacoids and cytokines. The Orai(CRACM) ion channel family provide the major pathway through which this Ca[superscript 2+] influx occurs. However the individual role of each of the three members of the Orai channel family in Ca[superscript 2+] influx and mediator release has not been defined in human mast cells. Objective: To assess whether there might be value in targeting individual Orai family members for the inhibition of FcεRI-dependent human lung mast cells (HLMC) mediator release. Methods: We used an adenoviral delivery system to transduce HLMCs with shRNAs targeted against Orai1 and Orai2 or with cDNAs directing the expression of dominant-negative mutations of the three known Orai channels. Results: shRNA-mediated knockdown of Orai1 resulted in a significant reduction of approximately 50% in Ca[superscript 2+] influx and in the release of β-hexosaminidase (a marker of degranulation) and newly synthesized LTC[subscript 4] in activated HLMCs. In contrast shRNA knockdown of Orai2 resulted in only marginal reductions of Ca[superscript 2+] influx, degranulation and LTC4 release. Transduced dominant-negative mutants of Orai1, -2 and -3 markedly reduced Orai currents and completely inhibited HLMC degranulation suggesting that Orai channels form heteromultimers in HLMCs, and that Orai channels comprise the dominant Ca[superscript 2+] influx pathway following FceRI-dependent HLMC activation. Inhibition of Orai currents did not alter HLMC survival. In addition we observed a significant down-regulation of the level of CRACM3 mRNA transcripts together with a small increase in the level of CRACM1 and CRACM2 transcripts following a period of sustained HLMC activation. Conclusion and Clinical Relevance: Orai1 plays an important role in Ca[superscript 2+] influx and mediator release from HLMCs. Strategies which target Orai1 will effectively inhibit FcεRI-dependent HLMC activation, but spare off-target inhibition of Orai2 in other cells and body systems. 2014-03-26T13:51:39Z Genetic factors regulating lung vasculature and immune cell functions associate with resistance to pneumococcal infection Jonczyk, Magda S. Simon, Michelle Kumar, Saumya Fernandes, Vitor E. Sylvius, Nicolas Mallon, Ann-Marie Denny, Paul Andrew, Peter W. http://hdl.handle.net/2381/28710 2014-03-27T02:01:46Z 2014-03-26T11:56:12Z Title: Genetic factors regulating lung vasculature and immune cell functions associate with resistance to pneumococcal infection Authors: Jonczyk, Magda S.; Simon, Michelle; Kumar, Saumya; Fernandes, Vitor E.; Sylvius, Nicolas; Mallon, Ann-Marie; Denny, Paul; Andrew, Peter W. Abstract: Streptococcus pneumoniae is an important human pathogen responsible for high mortality and morbidity worldwide. The susceptibility to pneumococcal infections is controlled by as yet unknown genetic factors. To elucidate these factors could help to develop new medical treatments and tools to identify those most at risk. In recent years genome wide association studies (GWAS) in mice and humans have proved successful in identification of causal genes involved in many complex diseases for example diabetes, systemic lupus or cholesterol metabolism. In this study a GWAS approach was used to map genetic loci associated with susceptibility to pneumococcal infection in 26 inbred mouse strains. As a result four candidate QTLs were identified on chromosomes 7, 13, 18 and 19. Interestingly, the QTL on chromosome 7 was located within S. pneumoniae resistance QTL (Spir1) identified previously in a linkage study of BALB/cOlaHsd and CBA/CaOlaHsd F2 intercrosses. We showed that only a limited number of genes encoded within the QTLs carried phenotype-associated polymorphisms (22 genes out of several hundred located within the QTLs). These candidate genes are known to regulate TGFb signalling, smooth muscle and immune cells functions. Interestingly, our pulmonary histopathology and gene expression data demonstrated, lung vasculature plays an important role in resistance to pneumococcal infection. Therefore we concluded that the cumulative effect of these candidate genes on vasculature and immune cells functions as contributory factors in the observed differences in susceptibility to pneumococcal infection. We also propose that TGFbmediated regulation of fibroblast differentiation plays an important role in development of invasive pneumococcal disease. 2014-03-26T11:56:12Z CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells Moiseeva, Elena P. Straatman, Kees R. Leyland, Mark L. Bradding, Peter http://hdl.handle.net/2381/28693 2014-03-21T02:01:46Z 2014-03-20T13:02:03Z Title: CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells Authors: Moiseeva, Elena P.; Straatman, Kees R.; Leyland, Mark L.; Bradding, Peter Abstract: CADM1 is a major receptor for the adhesion of mast cells (MCs) to fibroblasts, human airway smooth muscle cells (HASMCs) and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM). Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion. 2014-03-20T13:02:03Z What does the talking? Quorum sensing signalling genes discovered in a bacteriophage genome Hargreaves, Katherine R. Kropinski, Andrew M. Clokie, Martha R. J. http://hdl.handle.net/2381/28646 2014-03-12T02:01:44Z 2014-03-11T13:18:37Z Title: What does the talking? Quorum sensing signalling genes discovered in a bacteriophage genome Authors: Hargreaves, Katherine R.; Kropinski, Andrew M.; Clokie, Martha R. J. Abstract: The transfer of novel genetic material into the genomes of bacterial viruses (phages) has been widely documented in several host-phage systems. Bacterial genes are incorporated into the phage genome and, if retained, subsequently evolve within them. The expression of these phage genes can subvert or bolster bacterial processes, including altering bacterial pathogenicity. The phage phiCDHM1 infects Clostridium difficile, a pathogenic bacterium that causes nosocomial infections and is associated with antibiotic treatment. Genome sequencing and annotation of phiCDHM1 shows that despite being closely related to other C. difficile myoviruses, it has several genes that have not been previously reported in any phage genomes. Notably, these include three homologs of bacterial genes from the accessory gene regulator (agr) quorum sensing (QS) system. These are; a pre-peptide (AgrD) of an autoinducing peptide (AIP), an enzyme which processes the prepeptide (AgrB) and a histidine kinase (AgrC) that detects the AIP to activate a response regulator. Phylogenetic analysis of the phage and C. difficile agr genes revealed that there are three types of agr loci in this species. We propose that the phage genes belonging to a third type, agr3, and have been horizontally transferred from the host. AgrB and AgrC are transcribed during the infection of two different strains. In addition, the phage agrC appears not to be confined to the phiCDHM1 genome as it was detected in genetically distinct C. difficile strains. The discovery of QS gene homologs in a phage genome presents a novel way in which phages could influence their bacterial hosts, or neighbouring bacterial populations. This is the first time that these QS genes have been reported in a phage genome and their distribution both in C. difficile and phage genomes suggests that the agr3 locus undergoes horizontal gene transfer within this species. 2014-03-11T13:18:37Z Factors affecting the frequency of lipid body positive tubercle bacilli in human sputum Tarekegn, Baye Gelaw http://hdl.handle.net/2381/28630 2014-03-06T02:01:48Z 2014-03-05T16:43:07Z Title: Factors affecting the frequency of lipid body positive tubercle bacilli in human sputum Authors: Tarekegn, Baye Gelaw Abstract: Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis (TB). Mtb cells contain intracellular lipid bodies (LBs) in sputum and the levels vary between patients. Previous reports showed the essential role of nitric oxide (NO) in killing Mtb in vivo and that Mtb forms LBs in vitro upon NO treatment. These treated populations are tolerant to isoniazid and rifampicin, but the reasons for varing LB levels in vivo are not understood. The objective of this study was to determine the association between fractional exhaled nitric oxide (FeNO) concentrations with the proportion of LB[superscript +ve] tubercle bacilli in sputum. The majority of TB patients (65%) showed ≤ 20.6 ppb FeNO concentration. FeNO concentration was weak but significantly associated with the proportion of LB[superscript +ve] tubercle bacilli in sputum. FeNO concentration was also significantly associated with the proportion of LB[superscript +ve] tubercle bacilli among HIV[superscript +ve] P[superscript -] and HIV[superscript –ve] P[superscript +]/TB patients. High esosinophil count was significantly associated with FeNO concentration in both HIV[superscript –ve] and HIV[superscript +ve] /TB patients infected with intestinal parasite. The CAS and EAL Mtb spoligotypes were the dominant Mtb spoligotypes in Gondar. FeNO concentration was significantly associated with the proportion of LB[superscript +ve] tubercle bacilli among TB patients infected with the CAS Mtb spoligotype (p<0.01; r²=0.323) but not among patients infected with the EAL. The anti-microbial susceptibility pattern showed an 11.9% either mono or multi-drug resistance. The average proportion of LB[superscript +ve] tubercle bacilli among drug resistant TB patients was relatively higher than the corresponding drug sensitive TB patients. The proportion of LB[superscript +ve] tubercle bacilli was also higher among MDR-TB patients. The association between FeNO concentrations with the proportion of LB[superscript +ve] tubercle bacilli raises questions regarding L-arginine supplementation during TB treatment. The association of FeNO concentration with the proportion of LB[superscript +ve] tubercle bacilli among specific Mtb spoligotypes may reflect difference in NO tolerance. 2014-03-05T16:43:07Z Death is associated with complement C3 depletion in cerebrospinal fluid of patients with pneumococcal meningitis Goonetilleke, U. R. Scarborough, M Ward, S. A. Hussain, S Kadioglu, A Gordon, S. B. http://hdl.handle.net/2381/28599 2014-02-22T02:02:07Z 2014-02-21T12:12:39Z Title: Death is associated with complement C3 depletion in cerebrospinal fluid of patients with pneumococcal meningitis Authors: Goonetilleke, U. R.; Scarborough, M; Ward, S. A.; Hussain, S; Kadioglu, A; Gordon, S. B. Abstract: Pneumococcal meningitis can lead to death or serious neurological sequelae as a result of the host inflammatory response. We investigated the association between host response protein expression and outcome in patients with pneumococcal meningitis. Cerebrospinal fluid (CSF) was obtained from 80 patients with pneumococcal meningitis (40 nonsurvivors and 40 survivors) and 10 normal controls. Candidate proteins were analyzed for an association with survival. Complement C3 levels were 5-fold lower in nonsurvivors than in survivors (P < 0.05). This C3 reduction was not associated with lower levels in serum, indicating a compartmentalized CSF response. Transferrin levels were significantly higher in CSF (but not serum) from nonsurvivors than in CSF from survivors, suggestive of blood-brain barrier damage. Classical apoptosis proteins caspase 3 and apoptosis-inducing factor were not present in CSF. Expression of creatine kinase BB in clinically infected CSF suggested neuronal necrosis, but there was no clear association between level of expression and clinical outcome. Increased blood-brain barrier permeability and complement C3 depletion may have a role in determining outcome from bacterial meningitis. Therapeutic use of citicoline or caspase inhibitors is unlikely to have beneficial effects in patients with meningitis. IMPORTANCE We previously identified proteins associated with clinical outcome in patients diagnosed with pneumococcal meningitis in a pilot proteomics study of cerebrospinal fluid (CSF). In this article, we have quantitatively assayed specific proteins identified from this previous proteomics analysis along with proteins associated with cell death by using Western blotting. 2014-02-21T12:12:39Z