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1.  Development of a Gas Chromatography-Mass Spectrometry Method for the Quantification of Glucaric Acid Derivatives in Beverage Substrates 
A gas chromatography-mass spectrometry (GC-MS) method using the standard addition methodology was developed for the determination of glucuronolactone (GL) and glucuronic acid (DGuA) in four beverages categorized as detoxification, recovery, or energy drinks. The method features a precolumn derivatization step with a combination of BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide) and TMCS (trimethylchlorosilane) to silylate the analytes. The sample pretreatment required no extraction, filtration, or reduction step prior to the injection. The quantification of the analytes was performed using a five-point standard addition protocol. The proposed method presented excellent intraday precision (%RSD < 10) and linearity for GL calibration curves (correlation coefficients > 0.995) and acceptable linearity for DGuA calibration curves (correlation coefficients > 0.97). The estimated limits of detection (LOD) and quantification (LOQ) for GL ranged from 0.006 ppm to 0.14 ppm, and 0.02 ppm to 0.47 ppm, respectively. The estimated LOD and LOQ for DGuA determination ranged, respectively, from 0.06 ppm to 1.1 ppm and 0.2 ppm to 3.8 ppm. The results demonstrated that the method should be regarded as a reliable alternative to the simultaneous determination of GL and DGuA.
doi:10.1155/2014/402938
PMCID: PMC4082866  PMID: 25024704
2.  Cathelicidin Host Defence Peptide Augments Clearance of Pulmonary Pseudomonas aeruginosa Infection by Its Influence on Neutrophil Function In Vivo 
PLoS ONE  2014;9(6):e99029.
Cathelicidins are multifunctional cationic host-defence peptides (CHDP; also known as antimicrobial peptides) and an important component of innate host defence against infection. In addition to microbicidal potential, these peptides have properties with the capacity to modulate inflammation and immunity. However, the extent to which such properties play a significant role during infection in vivo has remained unclear. A murine model of acute P. aeruginosa lung infection was utilised, demonstrating cathelicidin-mediated enhancement of bacterial clearance in vivo. The delivery of exogenous synthetic human cathelicidin LL-37 was found to enhance a protective pro-inflammatory response to infection, effectively promoting bacterial clearance from the lung in the absence of direct microbicidal activity, with an enhanced early neutrophil response that required both infection and peptide exposure and was independent of native cathelicidin production. Furthermore, although cathelicidin-deficient mice had an intact early cellular inflammatory response, later phase neutrophil response to infection was absent in these animals, with significantly impaired clearance of P. aeruginosa. These findings demonstrate the importance of the modulatory properties of cathelicidins in pulmonary infection in vivo and highlight a key role for cathelicidins in the induction of protective pulmonary neutrophil responses, specific to the infectious milieu. In additional to their physiological roles, CHDP have been proposed as future antimicrobial therapeutics. Elucidating and utilising the modulatory properties of cathelicidins has the potential to inform the development of synthetic peptide analogues and novel therapeutic approaches based on enhancing innate host defence against infection with or without direct microbicidal targeting of pathogens.
doi:10.1371/journal.pone.0099029
PMCID: PMC4041793  PMID: 24887410
3.  Functional characterisation of human pulmonary monocyte-like cells in lipopolysaccharide-mediated acute lung inflammation 
Background
We have previously reported the presence of novel subpopulations of pulmonary monocyte-like cells (PMLC) in the human lung; resident PMLC (rPMLC, HLA-DR+CD14++CD16+cells) and inducible PMLC (iPMLC, HLA-DR+CD14++CD16- cells). iPMLC are significantly increased in bronchoalveolar lavage (BAL) fluid following inhalation of lipopolysaccharide (LPS). We have carried out the first functional evaluation of PMLC subpopulations in the inflamed lung, following the isolation of these cells, and other lineages, from BAL fluid using novel and complex protocols.
Methods
iPMLC, rPMLC, alveolar macrophages (AM), neutrophils, and regulatory T cells were quantified in BAL fluid of healthy subjects at 9 hours post-LPS inhalation (n = 15). Cell surface antigen expression by iPMLC, rPMLC and AM and the ability of each lineage to proliferate and to undergo phagocytosis were investigated using flow cytometry. Basal cytokine production by iPMLC compared to AM following their isolation from BAL fluid and the responsiveness of both cell types following in vitro treatment with the synthetic corticosteroid dexamethasone were assessed.
Results
rPMLC have a significantly increased expression of mature macrophage markers and of the proliferation antigen Ki67, compared to iPMLC. Our cytokine data revealed a pro-inflammatory, corticosteroid-resistant phenotype of iPMLC in this model.
Conclusions
These data emphasise the presence of functionally distinct subpopulations of the monocyte/macrophage lineage in the human lung in experimental acute lung inflammation.
doi:10.1186/1476-9255-11-9
PMCID: PMC4032498  PMID: 24684897
Monocytes; Macrophages; Acute lung inflammation; Lipopolysaccharide; Multiparameter flow cytometry; Corticosteroid
4.  GeneXpert MTB/RIF Version G4 for Identification of Rifampin-Resistant Tuberculosis in a Programmatic Setting 
Journal of Clinical Microbiology  2014;52(2):635-637.
A recent Cochrane review estimated GeneXpert MTB/RIF specificity for rifampin resistance as 98% (95% confidence interval [CI], 97 to 99), based on results from earlier test versions. The measured positive predictive value of the new generation test from programmatic implementation in Cape Town, South Africa, was 99.5% (95% CI, 98.5 to 100), confirming excellent specificity.
doi:10.1128/JCM.02517-13
PMCID: PMC3911341  PMID: 24478501
6.  Monocytes Control Second-Phase Neutrophil Emigration in Established Lipopolysaccharide-induced Murine Lung Injury 
Rationale: Acute lung injury (ALI) is an important cause of morbidity and mortality, with no currently effective pharmacological therapies. Neutrophils have been specifically implicated in the pathogenesis of ALI, and there has been significant research into the mechanisms of early neutrophil recruitment, but those controlling the later phases of neutrophil emigration that characterize disease are poorly understood.
Objectives: To determine the influence of peripheral blood monocytes (PBMs) in established ALI.
Methods: In a murine model of LPS-induced ALI, three separate models of conditional monocyte ablation were used: systemic liposomal clodronate (sLC), inducible depletion using CD11b diphtheria toxin receptor (CD11b DTR) transgenic mice, and antibody-dependent ablation of CCR2hi monocytes.
Measurements and Main Results: PBMs play a critical role in regulating neutrophil emigration in established murine LPS-induced lung injury. Gr1hi and Gr1lo PBM subpopulations contribute to this process. PBM depletion is associated with a significant reduction in measures of lung injury. The specificity of PBM depletion was demonstrated by replenishment studies in which the effects were reversed by systemic PBM infusion but not by systemic or local pulmonary infusion of mature macrophages or lymphocytes.
Conclusions: These results suggest that PBMs, or the mechanisms by which they influence pulmonary neutrophil emigration, could represent therapeutic targets in established ALI.
doi:10.1164/rccm.201112-2132OC
PMCID: PMC3480527  PMID: 22822022
acute lung injury; LPS; monocytes; neutrophils
7.  Serial Magnetic Resonance Imaging in Hypoplastic Left Heart Syndrome Gives Valuable Insight Into Ventricular and Vascular Adaptation 
Objectives
This study sought to investigate changes in magnetic resonance imaging (MRI) ventricular volumes and vascular dimensions before hemi-Fontan (HF) and before total cavopulmonary connection (TCPC) in children with hypoplastic left heart syndrome (HLHS).
Background
The systemic right ventricle (RV) in HLHS is subject to significant changes in volume loading throughout the surgical stages of palliation, particularly after the HF.
Methods
Fifty-eight patients had paired pre-HF and pre-TCPC MRI for assessment of changes of RV volumes, neoaortic flow, and vascular dimensions.
Results
Comparison of pre-HF and pre-TCPC MRI results showed a decrease of indexed RV end-diastolic volume and end-systolic volume (98 ml/m2 to 87 ml/m2 and 50 ml/m2 to 36 ml/m2, respectively) with stroke volume remaining constant (49 ml/m2 vs. 51 ml/m2), leading to an increased RV ejection fraction (51% vs. 59%). These findings persisted after excluding the 3 patients who underwent tricuspid valve repair as part of their HF procedure. Indexed RV end-diastolic volume plotted against neoaortic stroke volume demonstrated a Frank-Starling–like curve that shifted upward after HF. The indexed distal left and right cross-sectional pulmonary artery areas were reduced after HF.
Conclusions
In HLHS, serial MRI shows the adaptation of the systemic RV after HF with volume reduction in the context of a preserved stroke volume and an increased ejection fraction. The staged palliation in HLHS may be a risk factor particularly for reduced left pulmonary artery growth in itself as no factors investigated in this study were found to significantly impact on this.
doi:10.1016/j.jacc.2012.11.016
PMCID: PMC3573231  PMID: 23273398
hemi-Fontan operation; hypoplastic left heart syndrome; magnetic resonance imaging; Norwood procedure; remodeling of systemic right ventricle; 3D, 3-dimensional; EDV, end-diastolic volume; EF, ejection fraction; ESV, end-systolic volume; HF, hemi-Fontan; HLHS, hypoplastic left heart syndrome; HR, heart rate; iEDV, indexed end-diastolic volume; iESV, indexed end-systolic volume; iSV, indexed stroke volume; LPA, left pulmonary artery; LV, left ventricle; MRI, magnetic resonance imaging; RPA, right pulmonary artery; RV, right ventricle; SENSE, sensitivity encoding; SSFP, steady-state free precession; TCPC, total cavopulmonary connection; TR, tricuspid regurgitation
8.  Direct Identification of Nonreducing GlcNAc Residues on N-Glycans of Glycoproteins Using a Novel Chemoenzymatic Method 
Bioconjugate chemistry  2007;18(3):806-814.
The mutant β1,4-galactosyltransferase (β4Gal-T1), β4Gal-T1-Y289L, in contrast to wild-type β4Gal-T1, can transfer GalNAc from the sugar donor UDP-GalNAc to the acceptor, GlcNAc, with efficiency as good as that of galactose from UDP-Gal. Furthermore, the mutant can also transfer a modified sugar, C2 keto galactose, from its UDP derivative to O-GlcNAc modification on proteins that provided a functional handle for developing a highly sensitive chemoenzymatic method for detecting O-GlcNAc post-translational modification on proteins. We report herein that the modified sugar, C2 keto galactose, can be transferred to free GlcNAc residues on N-linked glycoproteins, such as ovalbumin or asialo-agalacto IgG1. The transfer is strictly dependent on the presence of both the mutant enzyme and the ketone derivative of the galactose. Moreover, the PNGase F treatment of the glycoproteins, which cleaves the N-linked oligosaccharide chain, shows that the modified sugar has been transferred to the N-glycan chains of the glycoproteins and not to the protein portion. The application of the mutant galactosyltransferase, β4Gal-T1-Y289L, to produce glycoconjugates carrying sugar moieties with reactive groups, is demonstrated. We envision a broad potential for this technology such as the possibilities to link cargo molecules to glycoproteins, such as monoclonal antibodies, via glycan chains, thereby assisting in the glycotargeting of drugs to the site of action or used as biological probes.
doi:10.1021/bc060341n
PMCID: PMC3534963  PMID: 17370997
9.  Subjective Evaluation of Right Ventricular Systolic Function in Hypoplastic Left Heart Syndrome: How Accurate Is It? 
Background
The geometry and heterogeneity of the right ventricle in hypoplastic left heart syndrome makes objective echocardiographic assessment of systolic function challenging. Consequently, subjective echocardiographic assessment of right ventricular (RV) function is still routinely undertaken. The aims of this study were to compare this with magnetic resonance imaging (MRI), investigate the impact of experience and training on the accuracy of subjective assessment, and critically analyze the role of echocardiography to detect impaired systolic function.
Methods
A retrospective analysis of prospectively acquired data was performed. Children with hypoplastic left heart syndrome underwent routine preoperative cardiac MRI and echocardiography under the same general anesthetic. Echocardiograms were reviewed, and members of the congenital heart disease team with differing echocardiography experience subjectively graded RV systolic function (good, moderate, or poor). This was compared with MRI-derived ejection fraction.
Results
Twenty-eight patients at different palliative stages were included. Twenty-eight observers were divided into five experience categories (congenital heart disease junior trainees to attending cardiologists). Median agreement was 47.6% (range, 31.4%–58.2%), with the lowest agreement among junior trainees and the highest among attending cardiologists. When used as a screening test for poor RV systolic function, the median sensitivity of echocardiography was 0.89 (range, 0.86–0.96), and median specificity was 0.45 (range, 0.26–0.55). The highest sensitivity was observed among junior trainees but with the lowest specificity. The highest specificity was observed among attending cardiologists (0.55).
Conclusions
Agreement between echocardiographic and MRI RV ejection fraction improves with experience but remains suboptimal. When used as a screening test for poor RV function, echocardiography is sensitive, but specificity is heavily influenced by operator experience.
doi:10.1016/j.echo.2012.09.020
PMCID: PMC3548410  PMID: 23098782
Magnetic resonance imaging; Echocardiography; Experience; Hypoplastic left heart syndrome; CI, Confidence interval; EF, Ejection fraction; HLHS, Hypoplastic left heart syndrome; MRI, Magnetic resonance imaging; RV, Right ventricular
10.  Regulation of Transforming Growth Factor-β1–driven Lung Fibrosis by Galectin-3 
Rationale: Idiopathic pulmonary fibrosis (IPF) is a chronic dysregulated response to alveolar epithelial injury with differentiation of epithelial cells and fibroblasts into matrix-secreting myofibroblasts resulting in lung scaring. The prognosis is poor and there are no effective therapies or reliable biomarkers. Galectin-3 is a β-galactoside binding lectin that is highly expressed in fibrotic tissue of diverse etiologies.
Objectives: To examine the role of galectin-3 in pulmonary fibrosis.
Methods: We used genetic deletion and pharmacologic inhibition in well-characterized murine models of lung fibrosis. Further mechanistic studies were performed in vitro and on samples from patients with IPF.
Measurements and Main Results: Transforming growth factor (TGF)-β and bleomycin-induced lung fibrosis was dramatically reduced in mice deficient in galectin-3, manifest by reduced TGF-β1–induced EMT and myofibroblast activation and collagen production. Galectin-3 reduced phosphorylation and nuclear translocation of β-catenin but had no effect on Smad2/3 phosphorylation. A novel inhibitor of galectin-3, TD139, blocked TGF-β–induced β-catenin activation in vitro and in vivo and attenuated the late-stage progression of lung fibrosis after bleomycin. There was increased expression of galectin-3 in the bronchoalveolar lavage fluid and serum from patients with stable IPF compared with nonspecific interstitial pneumonitis and controls, which rose sharply during an acute exacerbation suggesting that galectin-3 may be a marker of active fibrosis in IPF and that strategies that block galectin-3 may be effective in treating acute fibrotic exacerbations of IPF.
Conclusions: This study identifies galectin-3 as an important regulator of lung fibrosis and provides a proof of principle for galectin-3 inhibition as a potential novel therapeutic strategy for IPF.
doi:10.1164/rccm.201106-0965OC
PMCID: PMC3410728  PMID: 22095546
fibrosis; epithelial cells; fibroblasts
11.  The use of Z-scores in paediatric cardiology 
Annals of Pediatric Cardiology  2012;5(2):179-184.
Z-scores are a means of expressing the deviation of a given measurement from the size or age specific population mean. By taking account of growth or age, Z-scores are an excellent means of charting serial measurements in paediatric cardiological practice. They can be applied to echocardiographic measurements, blood pressure and patient growth, and thus may assist in clinical decision-making.
doi:10.4103/0974-2069.99622
PMCID: PMC3487208  PMID: 23129909
Blood pressure; cardio Z; echocardiography; paediatric cardiology; Z-score
12.  Quantitative Analysis of Phospholipids Using Nanostructured Laser Desorption Ionization Targets§ 
Lipids  2011;46(5):469-477.
Since its introduction as an ionization technique in mass spectrometry, matrix assisted laser desorption ionization (MALDI) has been applied to a wide range of applications. Quantitative small molecule analysis by MALDI, however, is limited due to the presence of intense signals from the matrix coupled with non-homogeneous surfaces. The surface used in nanostructured laser desorption ionization (NALDI) eliminates the need for a matrix and the resulting interferences, and allows for quantitative analysis of small molecules. This study was designed to analyze and quantitate phospholipid components of liposomes.
Here we have developed an assay to quantitate the DPPC and DC8,9PC in liposomes by NALDI following various treatments. To test our method we chose to analyze a liposome system composed of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DC8,9PC (1,2-bis (tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine), as DC8,9PC is known to undergo cross-linking upon treatment with UV (254 nm) and this reaction converts the monomer into a polymer.
First, calibration curves for pure lipids (DPPC and DC8,9PC) were created using DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) as an internal standard. The calibration curve for both DPPC and DC8,9PC showed an R2 of 0.992, obtained using the intensity ratio of analyte and internal standard. Next, DPPC:DC8,9PC liposomes were treated with UV radiation (254 nm). Following this treatment, lipids were extracted from the liposomes and analyzed. The analysis of the lipids before and after UV exposure confirmed a decrease in the signal of DC8,9PC of about 90%. In contrast, there was no reduction in DPPC signal.
doi:10.1007/s11745-010-3493-1
PMCID: PMC3238685  PMID: 21327726
NALDI; MALDI; mass spectrometry; lipids; quantitation; drug delivery
13.  Cutaneous signs are important in the diagnosis of the rare neoplasia syndrome Carney complex 
European journal of pediatrics  2009;168(11):1401-1404.
We describe a 15-year-old boy who presented with a stroke. Brain MRI imaging showed thalamic and multiple cerebral infarcts. An echocardiogram revealed multiple atrial masses, which were resected. Histological examination confirmed multiple atrial myxomas. Further clinical examination of the patient revealed subtle buccal and peri-oral lentigenes. The diagnosis of Carney complex was made clinically. The patient was subsequently diagnosed with testicular seminomas and a cutaneous angiomyxoma. Genetic investigation revealed a pathological mutation in the PRKAR1A gene. We review the reported manifestations and presentations of Carney complex, along with current diagnostic guidelines. We emphasise the importance of recognising the cutaneous manifestations of this rare autosomal dominantly inherited neoplasia syndrome.
doi:10.1007/s00431-009-0935-y
PMCID: PMC3138206  PMID: 19219454
14.  The Human Cathelicidin LL-37 Preferentially Promotes Apoptosis of Infected Airway Epithelium 
Cationic host defense peptides are key, evolutionarily conserved components of the innate immune system. The human cathelicidin LL-37 is an important cationic host defense peptide up-regulated in infection and inflammation, specifically in the human lung, and was shown to enhance the pulmonary clearance of the opportunistic pathogen Pseudomonas aeruginosa in vivo by as yet undefined mechanisms. In addition to its direct microbicidal potential, LL-37 can modulate inflammation and immune mechanisms in host defense against infection, including the capacity to modulate cell death pathways. We demonstrate that at physiologically relevant concentrations of LL-37, this peptide preferentially promoted the apoptosis of infected airway epithelium, via enhanced LL-37–induced mitochondrial membrane depolarization and release of cytochrome c, with activation of caspase-9 and caspase-3 and induction of apoptosis, which only occurred in the presence of both peptide and bacteria, but not with either stimulus alone. This synergistic induction of apoptosis in infected cells was caspase-dependent, contrasting with the caspase-independent cell death induced by supraphysiologic levels of peptide alone. We demonstrate that the synergistic induction of apoptosis by LL-37 and Pseudomonas aeruginosa required specific bacteria–epithelial cell interactions with whole, live bacteria, and bacterial invasion of the epithelial cell. We propose that the LL-37–mediated apoptosis of infected, compromised airway epithelial cells may represent a novel inflammomodulatory role for this peptide in innate host defense, promoting the clearance of respiratory pathogens.
doi:10.1165/rcmb.2009-0250OC
PMCID: PMC2993089  PMID: 20097832
cationic host defense peptide; antimicrobial peptide; innate immunity; Pseudomonas; apoptosis
15.  The Human Cathelicidin LL-37 Preferentially Promotes Apoptosis of Infected Airway Epithelium 
Cationic host defence peptides are key, evolutionarily conserved components of the innate immune system. The human cathelicidin LL-37 is an important cationic host defence peptide upregulated in infection and inflammation, including in the human lung, and has been shown to enhance the pulmonary clearance of the opportunistic pathogen Pseudomonas aeruginosa in vivo by as yet undefined mechanisms. In addition to direct microbicidal potential, LL-37 can modulate inflammation and immune mechanisms in host defence against infection, including the capacity to modulate cell death pathways. We demonstrate that at physiologically relevant concentrations of LL-37, this peptide preferentially promoted the apoptosis of infected airway epithelium, via enhanced LL-37-induced mitochondrial membrane depolarisation and release of cytochrome c, with activation of caspases -9 and -3 and induction of apoptosis, which only occurred in the presence of both peptide and bacteria, but not with either stimulus alone. This synergistic induction of apoptosis in infected cells was caspase-dependent, contrasting with the caspase-independent cell death induced by supra-physiological levels of peptide alone. We demonstrate that the synergistic induction of apoptosis by LL-37 and P. aeruginosa required specific bacteria-epithelial cell interaction with whole, live bacteria, and bacterial invasion of the epithelial cell. We propose that LL-37-mediated apoptosis of infected, compromised airway epithelial cells might represent a novel inflammomodulatory role for this peptide in innate host defence, promoting clearance of respiratory pathogens.
doi:10.1165/rcmb.2009-0250OC
PMCID: PMC2993089  PMID: 20097832
LL-37; cathelicidin; Cationic host defence peptide; antimicrobial peptide; airway epithelium; innate immunity; Pseudomonas; lung; apoptosis; cell death; Bax; Caspase; cystic fibrosis
17.  Epidemic Levels of Drug Resistant Tuberculosis (MDR and XDR-TB) in a High HIV Prevalence Setting in Khayelitsha, South Africa 
PLoS ONE  2010;5(11):e13901.
Background
Although multidrug-resistant tuberculosis (MDR-TB) is emerging as a significant threat to tuberculosis control in high HIV prevalence countries such as South Africa, limited data is available on the burden of drug resistant tuberculosis and any association with HIV in such settings. We conducted a community-based representative survey to assess the MDR-TB burden in Khayelitsha, an urban township in South Africa with high HIV and TB prevalence.
Methodology/Principal Findings
A cross-sectional survey was conducted among adult clinic attendees suspected for pulmonary tuberculosis in two large primary care clinics, together constituting 50% of the tuberculosis burden in Khayelitsha. Drug susceptibility testing (DST) for isoniazid and rifampicin was conducted using a line probe assay on positive sputum cultures, and with culture-based DST for first and second-line drugs. Between May and November 2008, culture positive pulmonary tuberculosis was diagnosed in 271 new and 264 previously treated tuberculosis suspects (sample enriched with previously treated cases). Among those with known HIV status, 55% and 71% were HIV infected respectively. MDR-TB was diagnosed in 3.3% and 7.7% of new and previously treated cases. These figures equate to an estimated case notification rate for MDR-TB of 51/100,000/year, with new cases constituting 55% of the estimated MDR-TB burden. HIV infection was not significantly associated with rifampicin resistance in multivariate analyses.
Conclusions/Significance
There is an extremely high burden of MDR-TB in this setting, most likely representing ongoing transmission. These data highlight the need to diagnose drug resistance among all TB cases, and for innovative models of case detection and treatment for MDR-TB, in order to interrupt transmission and control this emerging epidemic.
doi:10.1371/journal.pone.0013901
PMCID: PMC2981525  PMID: 21085569
18.  Association of FcγRIIa R131H polymorphism with idiopathic pulmonary fibrosis severity and progression 
Background
A significant genetic component has been described for idiopathic pulmonary fibrosis (IPF). The R131H (rs1801274) polymorphism of the IgG receptor FcγRIIa determines receptor affinity for IgG subclasses and is associated with several chronic inflammatory diseases. We investigated whether this polymorphism is associated with IPF susceptibility or progression.
Methods
In a case-control study, we compared the distribution of FcγRIIa R131H genotypes in 142 patients with IPF and in 218 controls using allele-specific PCR amplification.
Results
No differences in the frequency of FcγRIIa genotypes were evident between IPF patients and control subjects. However, significantly impaired pulmonary function at diagnosis was observed in HH compared to RR homozygotes, with evidence of more severe restriction (reduced forced vital capacity (FVC)) and lower diffusing capacity for carbon monoxide (DLCO). Similarly, increased frequency of the H131 allele was observed in patients with severe disease (DLCO < 40% predicted) (0.53 vs. 0.38; p = 0.03). Furthermore, the H131 allele was associated with progressive pulmonary fibrosis as determined by > 10% drop in FVC and/or > 15% fall in DLCO at 12 months after baseline (0.48 vs. 0.33; p = 0.023).
Conclusions
These findings support an association between the FcγRIIa R131H polymorphism and IPF severity and progression, supporting the involvement of immunological mechanisms in IPF pathogenesis.
doi:10.1186/1471-2466-10-51
PMCID: PMC2958991  PMID: 20929558
19.  C5a Mediates Peripheral Blood Neutrophil Dysfunction in Critically Ill Patients 
Rationale
Critically ill patients are highly susceptible to hospital-acquired infection. Neutrophil function in critical illness remains poorly understood.
Objectives
To characterize and define mechanisms of peripheral blood neutrophil (PBN) dysfunction in critically ill patients. To determine whether the inflamed lung contributes additional phagocytic impairment.
Methods
Prospective collection of blood and bronchoalveolar lavage fluid from patients with suspected ventilator-associated pneumonia and from age- and sex-matched volunteers; laboratory analysis of neutrophil functions.
Measurements and Main Results
Seventy-two patients and 21 volunteers were included. Phagocytic capacity of PBNs was 36% lower in patients than in volunteers (P < 0.0001). From several biologically plausible candidates only activated complement was significantly associated with impaired PBN phagocytosis (P < 0.0001). Phagocytosis was negatively correlated with serum C3a and positively correlated with expression of C5a receptor type 1 (CD88) on PBNs. C5a recapitulated impaired PBN phagocytosis and significantly down-regulated CD88 expression in vitro. C5a-mediated phagocytic impairment was prevented by blocking either CD88 or phosphoinositide 3-kinase, and completely reversed by granulocyte-macrophage colony-stimulating factor. C5a also impaired killing of Pseudomonas aeruginosa by, and migration of, PBNs, indicating that effects were not restricted to phagocytosis. Bronchoalveolar lavage fluid leukocytes from patients also demonstrated significantly impaired function, and lavage supernatant reduced phagocytosis in healthy neutrophils by 43% (P = 0.0001). However, lavage fluid did not affect CD88 expression and lavage-mediated impairment of phagocytosis was not blocked by anti-CD88 antibody.
Conclusions
Critically ill patients have significant dysfunction of PBNs, which is mediated predominantly by activated complement. Further, profound complement-independent neutrophil dysfunction occurs in the inflamed lung.
doi:10.1164/rccm.200812-1928OC
PMCID: PMC2948533  PMID: 19324972
complement; natural immunity; intensive care; phagocytosis
20.  MRS-guided HDR brachytherapy boost to the dominant intraprostatic lesion in high risk localised prostate cancer 
BMC Cancer  2010;10:472.
Background
It is known that the vast majority of prostate cancers are multifocal. However radical radiotherapy historically treats the whole gland rather than individual cancer foci.
Magnetic resonance spectroscopy (MRS) can be used to non-invasively locate individual cancerous tumours in prostate. Thus an intentionally non-uniform dose distribution treating the dominant intraprostatic lesion to different dose levels than the remaining prostate can be delivered ensuring the maximum achievable tumour control probability.
The aim of this study is to evaluate, using radiobiological means, the feasibility of a MRS-guided high dose rate (HDR) brachytherapy boost to the dominant lesion.
Methods
Computed tomography and MR/MRS were performed for treatment planning of a high risk localised prostate cancer. Both were done without endorectal coil, which distorts shape of prostate during the exams.
Three treatment plans were compared:
- external beam radiation therapy (EBRT) only
- combination of EBRT and HDR brachytherapy
- combination of EBRT and HDR brachytherapy with a synchronous integrated boost to the dominant lesion
The criteria of plan comparison were: the minimum, maximum and average doses to the targets and organs at risk; dose volume histograms; biologically effective doses for organs at risk and tumour control probability for the target volumes consisting of the dominant lesion as detected by MR/MRS and the remaining prostate volume.
Results
Inclusion of MRS information on the location of dominant lesion allows a safe increase of the dose to the dominant lesion while dose to the remaining target can be even substantially decreased keeping the same, high tumour control probability. At the same time an improved urethra sparing was achieved comparing to the treatment plan using a combination of EBRT and uniform HDR brachytherapy.
Conclusions
MRS-guided HDR brachytherapy boost to dominant lesion has the potential to spare the normal tissue, especially urethra, while keeping the tumour control probability high.
doi:10.1186/1471-2407-10-472
PMCID: PMC2941503  PMID: 20809986
21.  Diagnostic importance of pulmonary interleukin-1β and interleukin-8 in ventilator-associated pneumonia 
Thorax  2009;65(3):201-207.
Background
Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP.
Methods
A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age- and sex-matched volunteers. Growth of pathogens at >104 colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from “non-VAP”. Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves.
Results
Seventy-two patients had recoverable lavage—24% had VAP. BALF interleukin-1β (IL-1β), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1α were significantly higher in the VAP group (all p<0.005). Using a cut-off of 10 pg/ml, BALF IL-1β generated negative likelihood ratios for VAP of 0.09. In patients with BALF IL-1β <10 pg/ml the post-test probability of VAP was 2.8%. Using a cut-off value for IL-8 of 2 ng/ml, the positive likelihood ratio was 5.03. There was no difference in cytokine levels between patients with sterile BALF and those with growth of <104 cfu/ml.
Conclusions
BALF IL-1β and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations.
doi:10.1136/thx.2009.122291
PMCID: PMC2866736  PMID: 19825784
22.  Prenatal diagnosis of pulmonary atresia: impact on clinical presentation and early outcome 
Aim
The impact of prenatal diagnosis on morbidity and mortality for certain types of congenital heart disease (obstructive left heart lesions and transposition of the great arteries) is well established. No data are available for lesions with duct dependent pulmonary flow. We aimed to assess the impact of prenatal diagnosis of pulmonary atresia on clinical presentation and neonatal outcome.
Method
Fifty‐eight newborns with pulmonary atresia presenting to our centre were identified retrospectively between 1997 and 2004 (prenatal diagnosis n = 37, postnatal n = 21). Anatomical sub‐types included intact ventricular septum (PAIVS, n = 33) and ventricular septal defect (PAVSD, n = 25); those with more complex anatomy were excluded.
Results
After adjusting for anatomical sub‐type, postnatally diagnosed infants were significantly more hypoxic at presentation (mean oxygen saturation 65% vs 84%). However, they presented early (median age 1 day) and prostaglandin E was initiated promptly (median 3 hours) with rapid improvement of oxygen saturations (interaction p<0.001). This resulted in no appreciable differences in terms of pH, base deficit, blood pressure or heart rate between the groups by the time of the first catheter/surgical intervention. Postnatal infants did not differ in terms of length of intensive care unit (p = 0.18) or hospital stay (p = 0.86), incidence of complications (p = 0.72), or mortality (p = 0.77). Multivariable analysis revealed a positive association between occurrence of complications and both degree of cyanosis at presentation (rather than postnatal diagnosis per se) and anatomy (PAIVS).
Conclusion
Postnatal diagnosis of pulmonary atresia is associated with greater cyanosis at presentation. However this does not translate into greater neonatal morbidity or mortality provided that early recognition and prompt initiation of prostaglandin E therapy occur.
doi:10.1136/adc.2006.093880
PMCID: PMC2675326  PMID: 16840499
23.  Diagnostic importance of pulmonary interleukin-1β and interleukin-8 in ventilator-associated pneumonia 
Thorax  2010;65(3):201-207.
Background
Ventilator-associated pneumonia (VAP) is the most commonly fatal nosocomial infection. Clinical diagnosis of VAP remains notoriously inaccurate. The hypothesis was tested that significantly augmented inflammatory markers distinguish VAP from conditions closely mimicking VAP.
Methods
A prospective, observational cohort study was carried out in two university hospital intensive care units recruiting 73 patients with clinically suspected VAP, and a semi-urban primary care practice recruiting a reference group of 21 age- and sex-matched volunteers.
Growth of pathogens at >104 colony-forming units (cfu)/ml of bronchoalveolar lavage fluid (BALF) distinguished VAP from “non-VAP”. Inflammatory mediators were quantified in BALF and serum. Mediators showing significant differences between patients with and without VAP were analysed for diagnostic utility by receiver operator characteristic (ROC) curves.
Results
Seventy-two patients had recoverable lavage—24% had VAP. BALF interleukin-1β (IL-1β), IL-8, granulocyte colony-stimulating factor and macrophage inflammatory protein-1α were significantly higher in the VAP group (all p<0.005). Using a cut-off of 10 pg/ml, BALF IL-1β generated negative likelihood ratios for VAP of 0.09. In patients with BALF IL-1β <10 pg/ml the post-test probability of VAP was 2.8%. Using a cut-off value for IL-8 of 2 ng/ml, the positive likelihood ratio was 5.03. There was no difference in cytokine levels between patients with sterile BALF and those with growth of <104 cfu/ml.
Conclusions
BALF IL-1β and IL-8 are amongst the strongest markers yet identified for accurately demarcating VAP within the larger population of patients with suspected VAP. These findings have potential implications for reduction in unnecessary antibiotic use but require further validation in larger populations.
doi:10.1136/thx.2009.122291
PMCID: PMC2866736  PMID: 19825784
Assisted ventilation; cytokine biology; bacterial infection; bronchoscopy; pneumonia
24.  Perceptions and Reactions with Regard to Pneumonic Plague 
Emerging Infectious Diseases  2010;16(1):120-122.
We assessed perceptions and likely reactions of 1,005 UK adults to a hypothetical terrorist attack involving pneumonic plague. Likely compliance with official recommendations ranged from good (98% would take antimicrobial drugs) to poor (76% would visit a treatment center). Perceptions about plague were associated with these intentions.
doi:10.3201/eid1601.081604
PMCID: PMC2874346  PMID: 20031056
Bioterrorism and preparedness; communication; health behavior; patient compliance; plague; public health; bacteria; respiratory infections; dispatch
25.  Are Londoners Prepared for an Emergency? A Longitudinal Study Following the London Bombings 
The UK government sees increasing individual preparedness as a priority, but the level of preparedness of people in the UK for a large-scale emergency is not known. The London bombings of July 7, 2005, affected many Londoners and may have altered their sense of vulnerability to a future terrorist attack. We used a longitudinal study design to assess individual preparedness within the same sample of Londoners at 2 points in time: immediately after the bombings (T1) and 7 to 8 months later (T2). A demographically representative sample of 1,010 Londoners participated in a phone interview at T1. Subsequently, at T2, 574 of the same people participated in a follow-up phone interview. At T1 51% of Londoners had made 4 or more relevant emergency plans; 48% had gathered 4 or more relevant supplies in case of emergency. There was evidence of increased preparedness at T2, by which time 90% had made 4 or more emergency plans. Ethnicity, low social status, and having felt a sense of threat during the bombings predicted increased preparedness between T1 and T2. Women in general, and women of low social status in particular, perceived themselves to be unprepared in the event of a future terrorist attack. In summary, Londoners show moderate levels of emergency preparedness, which increased following the London bombings. Although we cannot know whether this association is causal, the prospective nature of the study increases the likelihood that it is. However, preparedness is still patchy, and there are important demographic associations with levels of preparedness and perception of vulnerability. These findings have implications for future development of individual and community emergency preparedness policy.
doi:10.1089/bsp.2008.0043
PMCID: PMC2963593  PMID: 19117430

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