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1.  A national survey of the diagnosis and management of suspected ventilator-associated pneumonia 
BMJ Open Respiratory Research  2014;1(1):e000066.
Background
Ventilator-associated pneumonia (VAP) affects up to 20% of patients admitted to intensive care units (ICU). It is associated with increased morbidity, mortality and healthcare costs. Despite published guidelines, variability in diagnosis and management exists, the extent of which remains unclear. We sought to characterise consultant opinions surrounding diagnostic and management practice for VAP in the UK.
Methods
An online survey was sent to all consultant members of the UK Intensive Care Society (n=∼1500). Data were collected regarding respondents’ individual practice in the investigation and management of suspected VAP including use of diagnostic criteria, microbiological sampling, chest X-ray (CXR), bronchoscopy and antibiotic treatments.
Results
339 (23%) responses were received from a broadly representative spectrum of ICU consultants. All respondents indicated that microbiological confirmation should be sought, the majority (57.8%) stating they would take an endotracheal aspirate prior to starting empirical antibiotics. Microbiology reporting services were described as qualitative only by 29.7%. Only 17% of respondents had access to routine reporting of CXRs by a radiologist. Little consensus exists regarding technique for bronchoalveolar lavage (BAL) with the reported volume of saline used ranging from 5 to 500 mL. 24.5% of consultants felt inadequately trained in bronchoscopy.
Conclusions
There is wide variability in the approach to diagnosis and management of VAP among UK consultants. Such variability challenges the reliability of the diagnosis of VAP and its reported incidence as a performance indicator in healthcare systems. The data presented suggest increased radiological and microbiological support, and standardisation of BAL technique, might improve this situation.
doi:10.1136/bmjresp-2014-000066
PMCID: PMC4275666  PMID: 25553248
Respiratory Infection; Pneumonia; Assisted Ventilation
2.  Impact of Xpert MTB/RIF for TB Diagnosis in a Primary Care Clinic with High TB and HIV Prevalence in South Africa: A Pragmatic Randomised Trial 
PLoS Medicine  2014;11(11):e1001760.
Helen Cox and colleagues investigate the impact Xpert MTB/RIF for diagnosing patients with presumptive tuberculosis in a large primary care clinic in Khayelitsha, Cape Town.
Please see later in the article for the Editors' Summary
Background
Xpert MTB/RIF is approved for use in tuberculosis (TB) and rifampicin-resistance diagnosis. However, data are limited on the impact of Xpert under routine conditions in settings with high TB burden.
Methods and Findings
A pragmatic prospective cluster-randomised trial of Xpert for all individuals with presumptive (symptomatic) TB compared to the routine diagnostic algorithm of sputum microscopy and limited use of culture was conducted in a large TB/HIV primary care clinic. The primary outcome was the proportion of bacteriologically confirmed TB cases not initiating TB treatment by 3 mo after presentation. Secondary outcomes included time to TB treatment and mortality. Unblinded randomisation occurred on a weekly basis. Xpert and smear microscopy were performed on site. Analysis was both by intention to treat (ITT) and per protocol.
Between 7 September 2010 and 28 October 2011, 1,985 participants were assigned to the Xpert (n = 982) and routine (n = 1,003) diagnostic algorithms (ITT analysis); 882 received Xpert and 1,063 routine (per protocol analysis). 13% (32/257) of individuals with bacteriologically confirmed TB (smear, culture, or Xpert) did not initiate treatment by 3 mo after presentation in the Xpert arm, compared to 25% (41/167) in the routine arm (ITT analysis, risk ratio 0.51, 95% CI 0.33–0.77, p = 0.0052).
The yield of bacteriologically confirmed TB cases among patients with presumptive TB was 17% (167/1,003) with routine diagnosis and 26% (257/982) with Xpert diagnosis (ITT analysis, risk ratio 1.57, 95% CI 1.32–1.87, p<0.001). This difference in diagnosis rates resulted in a higher rate of treatment initiation in the Xpert arm: 23% (229/1,003) and 28% (277/982) in the routine and Xpert arms, respectively (ITT analysis, risk ratio 1.24, 95% CI 1.06–1.44, p = 0.013). Time to treatment initiation was improved overall (ITT analysis, hazard ratio 0.76, 95% CI 0.63–0.92, p = 0.005) and among HIV-infected participants (ITT analysis, hazard ratio 0.67, 95% CI 0.53–0.85, p = 0.001). There was no difference in 6-mo mortality with Xpert versus routine diagnosis. Study limitations included incorrect intervention allocation for a high proportion of participants and that the study was conducted in a single clinic.
Conclusions
These data suggest that in this routine primary care setting, use of Xpert to diagnose TB increased the number of individuals with bacteriologically confirmed TB who were treated by 3 mo and reduced time to treatment initiation, particularly among HIV-infected participants.
Trial registration
Pan African Clinical Trials Registry PACTR201010000255244
Please see later in the article for the Editors' Summary
Editors' Summary
Background
In 2012, about 8.6 million people developed active tuberculosis (TB)—a contagious mycobacterial disease that usually affects the lungs—and at least 1.3 million people died from the disease. Most of these deaths were in low- and middle-income countries, and a fifth were in HIV-positive individuals, who are particularly susceptible to TB. Mycobacterium tuberculosis, the bacterium that causes TB, is spread in airborne droplets when people with active disease cough or sneeze. The characteristic symptoms of TB include a cough, weight loss, and night sweats. Diagnostic tests for TB include microscopic examination of sputum (mucus coughed up from the lungs), growth (culture) of M. tuberculosis from sputum, and molecular tests (for example, the automated Xpert MTB/RIF test) that rapidly and accurately detect M. tuberculosis in sputum and determine its antibiotic resistance. TB can be cured by taking several antibiotics daily for at least six months, although the emergence of multidrug-resistant TB is making the disease harder to treat.
Why Was This Study Done?
To improve TB control, active disease needs to be diagnosed and treated quickly. However, sputum microscopy, the mainstay of TB diagnosis in many high-burden settings, fails to identify up to half of infected people, and mycobacterial culture (the “gold standard” of TB diagnosis) is slow and often unavailable in resource-limited settings. In late 2010, the World Health Organization recommended the routine use of the Xpert MTB/RIF test (Xpert) for TB diagnosis, and several low- and middle-income countries are now scaling up access to Xpert in their national TB control programs. But although Xpert performs well in ideal conditions, little is known about the impact of its implementation in routine (real-life) settings. In this pragmatic cluster-randomized trial, the researchers assess the health impacts of Xpert in a large TB/HIV primary health care clinic in South Africa, an upper-middle-income country that began to scale up access to Xpert for individuals showing symptoms of TB (individuals with presumptive TB) in 2011. A pragmatic trial asks whether an intervention works under real-life conditions; a cluster-randomized trial randomly assigns groups of people to receive alternative interventions and compares outcomes in the differently treated “clusters.”
What Did the Researchers Do and Find?
The researchers assigned everyone with presumptive TB attending a TB/HIV primary health care clinic in Cape Town to receive either Xpert for TB diagnosis or routine sputum microscopy and limited culture. Specifically, Xpert was requested on the routine laboratory request forms for individuals attending the clinic during randomly designated Xpert weeks but not during randomly designated routine testing weeks. During the 51-week trial, 982 individuals were assigned to the Xpert arm, and 1,003 were assigned to the routine testing arm, but because clinic staff sometimes failed to request Xpert during Xpert weeks, only 882 participants in the Xpert arm received the intervention. In an “intention to treat” analysis (an analysis that considers the outcomes of all the participants in a trial whether or not they received their assigned intervention), 13% of bacteriologically confirmed TB cases in the Xpert arm did not initiate TB treatment by three months after enrollment (the trial's primary outcome) compared to 25% in the routine testing arm. The proportion of participants with microbiologically confirmed TB and the proportion initiating TB treatment were higher in the Xpert arm than in the routine testing arm. Finally, the time to treatment initiation was lower in the Xpert arm than in the routine testing arm, particularly among HIV-infected participants.
What Do These Findings Mean?
These findings show that, in this primary health care setting, the provision of Xpert for TB diagnosis in individuals with presumptive TB provided benefits over testing that relied primarily on sputum microscopy. Notably, these benefits were seen even though a substantial proportion of individuals assigned to the Xpert intervention did not actually receive an Xpert test. The pragmatic nature of this trial, which aimed to minimize clinic disruption, and other aspects of the trial design may limit the accuracy and generalizability of these findings. Moreover, further studies are needed to discover whether the use of Xpert in real-life settings reduces the burden of TB illness and death over the long term. Nevertheless, these findings suggest that the implementation of Xpert has the potential to improve the outcomes of TB control programs and may also improve outcomes for individuals.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001760.
The World Health Organization provides information (in several languages) on all aspects of tuberculosis, including general information on tuberculosis diagnostics and specific information on the roll-out of the Xpert MTB/RIF test; further information about the World Health Organization's endorsement of Xpert MTB/RIF is included in a Strategic and Technical Advisory Group for Tuberculosis report; the “Global Tuberculosis Report 2013” provides information about tuberculosis around the world, including in South Africa
The Stop TB Partnership is working towards tuberculosis elimination and provides patient stories about tuberculosis (in English and Spanish); the Tuberculosis Vaccine Initiative (a not-for-profit organization) also provides personal stories about tuberculosis
The US Centers for Disease Control and Prevention has information about tuberculosis and its diagnosis (in English and Spanish)
The US National Institute of Allergy and Infectious Diseases also has detailed information on all aspects of tuberculosis
The South African National Tuberculosis Management Guidelines 2014 are available
The Foundation for Innovative New Diagnostics, a not-for-profit organization that helps to develop and introduce new diagnostic tests for tuberculosis, malaria, and neglected tropical diseases, has detailed information about the Xpert MTB/RIF test
More information about TB, HIV, and drug-resistant TB treatment in Khayelitsha, Cape Town, South Africa are provided by Médecins sans Frontières, South Africa
doi:10.1371/journal.pmed.1001760
PMCID: PMC4244039  PMID: 25423041
3.  Serial characterisation of monocyte and neutrophil function after lung resection 
BMJ Open Respiratory Research  2014;1(1):e000045.
Objectives
The primary aim of this prospective study was to perform a comprehensive serial characterisation of monocyte and neutrophil function, circulating monocyte subsets, and bronchoalveolar lavage (BAL) fluid after lung resection. A secondary aim was to perform a pilot, hypothesis-generating evaluation of whether innate immune parameters were associated with postoperative pneumonia.
Methods
Forty patients undergoing lung resection were studied in detail. Blood monocytes and neutrophils were isolated preoperatively and at 6, 24 and 48 h postoperatively. BAL was performed preoperatively and immediately postoperatively. Monocyte subsets, monocyte responsiveness to lipopolysaccharide (LPS) and neutrophil phagocytic capacity were quantified at all time points. Differential cell count, protein and cytokine concentrations were measured in BAL. Pneumonia evaluation at 72 h was assessed using predefined criteria.
Results
After surgery, circulating subsets of classical and intermediate monocytes increased significantly. LPS-induced release of proinflammatory cytokines from monocytes increased significantly and by 48 h a more proinflammatory profile was found. Neutrophil phagocytosis demonstrated a small but significant fall. Factors associated with postoperative pneumonia were: increased release of specific proinflammatory and anti-inflammatory cytokines from monocytes; preoperative neutrophilia; and preoperative BAL cell count.
Conclusions
We conclude that postoperative lung inflammation is associated with specific changes in the cellular innate immune response, a better understanding of which may improve patient selection and prediction of complications in the future.
doi:10.1136/bmjresp-2014-000045
PMCID: PMC4212786  PMID: 25478189
Innate Immunity; Thoracic Surgery
4.  Differential response to bacteria, and TOLLIP expression, in the human respiratory tract 
BMJ Open Respiratory Research  2014;1(1):e000046.
Objectives
The observation that pathogenic bacteria are commonly tolerated in the human nose, yet drive florid inflammation in the lung, is poorly understood, partly due to limited availability of primary human cells from each location. We compared responses to bacterial virulence factors in primary human nasal and alveolar cells, and characterised the distribution of Toll-interacting protein (TOLLIP; an inhibitor of Toll-like receptor (TLR) signalling) in the human respiratory tract.
Methods
Primary cells were isolated from nasal brushings and lung tissue taken from patients undergoing pulmonary resection. Cells were exposed to lipopolysaccharide, lipoteichoic acid, peptidoglycan, CpG-C DNA or tumour necrosis factor (TNF). Cytokines were measured in cell supernatants. TOLLIP was characterised using quantitative real-time PCR and immunofluorescence.
Results
In primary alveolar, but not primary nasal, cells peptidoglycan significantly increased secretion of interleukin (IL)-1β, IL-6, IL-8, IL-10 and TNF. TLR2 expression was significantly higher in alveolar cells and correlated with IL-8 production. TOLLIP expression was significantly greater in nasal cells.
Conclusion
In conclusion, primary human alveolar epithelial cells are significantly more responsive to peptidoglycan than primary nasal epithelial cells. This may partly be explained by differential TLR2 expression. TOLLIP is expressed widely in the human respiratory tract, and may contribute to the regulation of inflammatory responses.
doi:10.1136/bmjresp-2014-000046
PMCID: PMC4212710  PMID: 25478190
Innate Immunity
5.  Nonvisual Complex Spike Signals in the Rabbit Cerebellar Flocculus 
The Journal of Neuroscience  2014;34(9):3218-3230.
In addition to the well-known signals of retinal image slip, floccular complex spikes (CSs) also convey nonvisual signals. We recorded eye movement and CS activity from Purkinje cells in awake rabbits sinusoidally oscillated in the dark on a vestibular turntable. The stimulus frequency ranged from 0.2 to 1.2 Hz, and the velocity amplitude ranged from 6.3 to 50°/s. The average CS modulation was evaluated at each combination of stimulus frequency and amplitude. More than 75% of the Purkinje cells carried nonvisual CS signals. The amplitude of this modulation remained relatively constant over the entire stimulus range. The phase response of the CS modulation in the dark was opposite to that during the vestibulo-ocular reflex (VOR) in the light. With increased frequency, the phase response systematically shifted from being aligned with contraversive head velocity toward peak contralateral head position. At fixed frequency, the phase response was dependent on peak head velocity, indicating a system nonlinearity. The nonvisual CS modulation apparently reflects a competition between eye movement and vestibular signals, resulting in an eye movement error signal inferred from nonvisual sources. The combination of this error signal with the retinal slip signal in the inferior olive results in a net error signal reporting the discrepancy between the actual visually measured eye movement error and the inferred eye movement error derived from measures of the internal state. The presence of two error signals requires that the role of CSs in models of the floccular control of VOR adaption be expanded beyond retinal slip.
doi:10.1523/JNEUROSCI.3080-13.2014
PMCID: PMC3935084  PMID: 24573280
accessory optic system; climbing fiber; complex spike; inferior olive; prepositus hypoglossi; Purkinje cell
6.  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
7.  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
8.  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
9.  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
11.  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
12.  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
13.  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
14.  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
15.  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
16.  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
17.  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
18.  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
19.  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
20.  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
22.  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
23.  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
24.  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
25.  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

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