Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro

BACKGROUND: Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. RESULTS: A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. CONCLUSIONS: This study highlights the value of RNA-seq in identifying novel immunomodulatory mechanisms that underlie host-mycobacterial pathogen interactions during infection, including possible complex post-transcriptional regulation of host gene expression involving antisense RNA

Predicting participation in group parenting education in an Australian sample: The role of attitudes, norms, and control factors

We examined the theory of planned behavior (TPB) in predicting intentions to participate in group parenting education. One hundred and seventy-six parents (138 mothers and 38 fathers) with a child under 12 years completed TPB items assessing attitude, subjective norms, perceived behavioral control (PBC), and two additional social influence variables (self-identity and group norm). Regression analyses supported the TPB predictors of participation intentions with self-identity and group norm also significantly predicting intentions. These findings offer preliminary support for the TPB, along with additional sources of social influence, as a useful predictive model of participation in parenting education

Protocol for the challenge non-typhoidal Salmonella (CHANTS) study: a first-in-human, in-patient, double-blind, randomised, safety and dose-escalation controlled human infection model in the UK

Introduction Invasive non-typhoidal Salmonella (iNTS) serovars are a major cause of community-acquired bloodstream infections in sub-Saharan Africa (SSA). In this setting, Salmonella enterica serovar Typhimurium accounts for two-thirds of infections and is associated with an estimated case fatality rate of 15%–20%. Several iNTS vaccine candidates are in early-stage assessment which—if found effective—would provide a valuable public health tool to reduce iNTS disease burden. The CHANTS study aims to develop a first-in-human Salmonella Typhimurium controlled human infection model, which can act as a platform for future vaccine evaluation, in addition to providing novel insights into iNTS disease pathogenesis. Methods and analysis This double-blind, safety and dose-escalation study will randomise 40–80 healthy UK participants aged 18–50 to receive oral challenge with one of two strains of S. Typhimurium belonging to the ST19 (strain 4/74) or ST313 (strain D23580) lineages. 4/74 is a global strain often associated with diarrhoeal illness predominantly in high-income settings, while D23580 is an archetypal strain representing invasive disease-causing isolates found in SSA. The primary objective is to determine the minimum infectious dose (colony-forming unit) required for 60%–75% of participants to develop clinical or microbiological features of systemic salmonellosis. Secondary endpoints are to describe and compare the clinical, microbiological and immunological responses following challenge. Dose escalation or de-escalation will be undertaken by continual-reassessment methodology and limited within prespecified safety thresholds. Exploratory objectives are to describe mechanisms of iNTS virulence, identify putative immune correlates of protection and describe host–pathogen interactions in response to infection. Ethics and dissemination Ethical approval has been obtained from the NHS Health Research Authority (London—Fulham Research Ethics Committee 21/PR/0051; IRAS Project ID 301659). The study findings will be disseminated in international peer-reviewed journals and presented at national/international stakeholder meetings. Study outcome summaries will be provided to both funders and participants. Trial registration number NCT0587015

Mycolactone Diffuses into the Peripheral Blood of Buruli Ulcer Patients - Implications for Diagnosis and Disease Monitoring.

BACKGROUND: Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU), is unique among human pathogens in its capacity to produce a polyketide-derived macrolide called mycolactone, making this molecule an attractive candidate target for diagnosis and disease monitoring. Whether mycolactone diffuses from ulcerated lesions in clinically accessible samples and is modulated by antibiotic therapy remained to be established. METHODOLOGY/PRINCIPAL FINDING: Peripheral blood and ulcer exudates were sampled from patients at various stages of antibiotic therapy in Ghana and Ivory Coast. Total lipids were extracted from serum, white cell pellets and ulcer exudates with organic solvents. The presence of mycolactone in these extracts was then analyzed by a recently published, field-friendly method using thin layer chromatography and fluorescence detection. This approach did not allow us to detect mycolactone accurately, because of a high background due to co-extracted human lipids. We thus used a previously established approach based on high performance liquid chromatography coupled to mass spectrometry. By this means, we could identify structurally intact mycolactone in ulcer exudates and serum of patients, and evaluate the impact of antibiotic treatment on the concentration of mycolactone. CONCLUSIONS/SIGNIFICANCE: Our study provides the proof of concept that assays based on mycolactone detection in serum and ulcer exudates can form the basis of BU diagnostic tests. However, the identification of mycolactone required a technology that is not compatible with field conditions and point-of-care assays for mycolactone detection remain to be worked out. Notably, we found mycolactone in ulcer exudates harvested at the end of antibiotic therapy, suggesting that the toxin is eliminated by BU patients at a slow rate. Our results also indicated that mycolactone titres in the serum may reflect a positive response to antibiotics, a possibility that it will be interesting to examine further through longitudinal studies

Microbial ligand costimulation drives neutrophilic steroid-refractory asthma

Funding: The authors thank the Wellcome Trust (102705) and the Universities of Aberdeen and Cape Town for funding. This research was also supported, in part, by National Institutes of Health GM53522 and GM083016 to DLW. KF and BNL are funded by the Fonds Wetenschappelijk Onderzoek, BNL is the recipient of an European Research Commission consolidator grant and participates in the European Union FP7 programs EUBIOPRED and MedALL. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Isometric force production parameters during normal and experimental low back pain conditions

BACKGROUND: The control of force and its between-trial variability are often taken as critical determinants of motor performance. Subjects performed isometric trunk flexion and extension forces without and with experiment pain to examine if pain yields changes in the control of trunk forces. The objective of this study is to determine if experimental low back pain modifies trunk isometric force production. METHODS: Ten control subjects participated in this study. They were required to exert 50 and 75% of their isometric maximal trunk flexion and extension torque. In a learning phase preceding the non painful and painful trials, visual and verbal feedbacks were provided. Then, subjects were asked to perform 10 trials without any feedback. Time to peak torque, time to peak torque variability, peak torque variability as well as constant and absolute error in peak torque were calculated. Time to peak and peak dF/dt were computed to determine if the first peak of dF/dt could predict the peak torque achieved. RESULTS: Absolute and constant errors were higher in the presence of a painful electrical stimulation. Furthermore, peak torque variability for the higher level of force was increased with in the presence of experimental pain. The linear regressions between peak dF/dt, time to peak dF/dt and peak torque were similar for both conditions. Experimental low back pain yielded increased absolute and constant errors as well as a greater peak torque variability for the higher levels of force. The control strategy, however, remained the same between the non painful and painful condition. Cutaneous pain affects some isometric force production parameters but modifications of motor control strategies are not implemented spontaneously. CONCLUSIONS: It is hypothesized that adaptation of motor strategies to low back pain is implemented gradually over time. This would enable LBP patients to perform their daily tasks with presumably less pain and more accuracy

Does the aldosterone: renin ratio predict the efficacy of spironolactone over bendroflumethiazide in hypertension? A clinical trial protocol for RENALDO (RENin-ALDOsterone) study

<p>Background: High blood pressure is an important determinant of cardiovascular disease risk. Treated hypertensives do not attain a risk level equivalent to normotensives. This may be a consequence of suboptimal blood pressure control to which indiscriminate use of antihypertensive drugs may contribute. Indeed the recent ALLHAT[1]study suggests that thiazides should be given first to virtually all hypertensives. Whether this is correct or whether different antihypertensive therapies should be targeted towards different patients is a major unresolved issue, which we address in this study.</p> <p>The measurement of the ratio of aldosterone: renin is used to identify hypertensive subjects who may respond well to treatment with the aldosterone antagonist spironolactone. It is not known if subjects with a high ratio have aldosteronism or aldosterone-sensitive hypertension is debated but it is important to know whether spironolactone is superior to other diuretics such as bendroflumethiazide in this setting.</p> <p>Methods/design: The study is a double-blind, randomised, crossover, controlled trial that will randomise 120 hypertensive subjects to 12 weeks treatment with spironolactone 50 mg once daily and 12 weeks treatment with bendroflumethiazide 2.5 mg once daily. The 2 treatment periods are separated by a 2-week washout period. Randomisation is stratified by aldosterone: renin ratio to include equal numbers of subjects with high and low aldosterone: renin ratios.</p> <p>Primary Objective – To test the hypothesis that the aldosterone: renin ratio predicts the antihypertensive response to spironolactone, specifically that the effect of spironolactone 50 mg is greater than that of bendroflumethiazide 2.5 mg in hypertensive subjects with high aldosterone: renin ratios.</p> <p>Secondary Objectives – To determine whether bendroflumethiazide induces adverse metabolic abnormalities, especially in subjects with high aldosterone: renin ratios and if baseline renin measurement predicts the antihypertensive response to spironolactone and/or bendrofluazide.</p> <p>Discussion: The numerous deleterious effects of hypertension dictate the need for a systematic approach for its treatment. In spite of various therapies, resistant hypertension is widely prevalent. Among various factors, primary aldosteronism is an important cause of resistant hypertension and is now more commonly recognised. More significantly, hypertensives with primary aldosteronism are also exposed to various other deleterious effects of excess aldosterone. Hence treating hypertension with specific aldosterone antagonists may be a better approach in this group of patients. It may lead on to better blood pressures with fewer medications.</p&gt