The inhibition of adenovirus vector expression by MVA was also co

The inhibition of adenovirus vector expression by MVA was also confirmed through in vitro experiments. Furthermore, the suppression factor(s) included an undefined soluble protein, besides cytokines such as type I IFN. Two viral vectors were used in this study: One vector was an E1/3-deleted adenovirus vector expressing the secreted alkaline phosphatase SEAP gene (Ad-SEAP), HIVIIIB gp160 Env (Ad-HIV)

[4], the green fluorescent protein (Ad-GFP) or mCherry fluorescent protein (Ad-Cherry). Another vector was modified vaccinia virus Ankara expressing HIVBH2 gp160 Env and a report gene LacZ (MVA-HIV, a kind gift from Dr. Bernard Moss, National Institutes of Health, Rockville, MD) or the green fluorescent protein (MVA-GFP). The Ad vector was propagated in HEK293 cells and purified over Afatinib in vivo CsCl as described elsewhere [5]. The total concentration of virions in each preparation was calculated by using the following formula: 1 OD260=1012 viral particle (vp)/ml1 OD260=1012 viral particle (vp)/ml The MVA virus was propagated in the BHK21 cell line and purified by one round of ultracentrifuge over 36% sucrose. The MVA virus was titrated Selleck Gefitinib in the BHK21 cell line to determine the number of plaque forming units (pfu). Eight-week-old BALB/c mice (H-2Dd) were purchased from

Japan SLC Inc. (Shizuoka, Japan). The mice were immunized with an intramuscular injection of 1010 vp of Ad-HIV and Ad-GFP, 107 pfu of MVA-HIV, or 105–7 pfu of MVA-GFP. All experiments were performed in accordance with the guidelines of the University

Animal Care and Use Committee of the Animal Research Center, Yokohama City University Graduate School of Medicine. The assay was performed as described previously [25]. The H-2Dd/p18 tetramer (RGPGRAFVTI; synthesized by NIH Tetramer Core Facility, Atlanta, GA) labeled with phycoerythrin (PE) was used for the tetramer assay. Briefly, 100 μl of heparinized whole mouse blood was stained with 0.25 μg of fluorescein isothiocyanate (FITC-conjugated) anti-mouse CD8a antibody (clone 53-6.7; eBioscience, San Diego, CA), along with 0.05 μg of tetramer reagent at room temperature for 30 min. The cells were all then fixed with 100 μl of OptiLyse B-Lysing solution (Beckman Coulter, Marseille Cedex, France) at room temperature for 10 min. Erythrocytes were lysed by adding 1 ml of H2O and washed with phosphate-buffered saline (PBS). To detect antigen-specific memory T cells, the cells were co-stained with PE-p18 tetramer, FITC-anti CD8 antibody, 0.1 μg of phycoerythrin/cyanin7 (PE Cy7)-conjugated anti-mouse CD62L antibody (clone MEL-14; Biolegend, San Diego, CA), and 0.25 μg of Alexa Fluor 647-conjugated anti-mouse CD127 antibody (clone SB/199; AbD Serotec, Oxford, UK), similar to the tetramer assay described herein. The p18 tetramer+CD62L+CD127+CD8 T cells and p18 tetramer+CD62L−CD127+CD8 T cells were respectively defined as central memory (CM) CD8 T cells and effector memory (EM) CD8 T cells.

First, a vaccine would need to be rigorously shown to induce full

First, a vaccine would need to be rigorously shown to induce full protection, rather than inducing partial protection which could lead to unrecognized latent infection. Therefore, such a vaccine would

need to a) prevent chancre development associated with primary disease and the lesions associated with secondary disease to abolish transmission of T. pallidum and HIV and b) inhibit treponemal dissemination throughout the host to prevent corresponding disease progression and establishment of CS. Second, the vaccine candidate(s) would need to be effective in generating a Th1 response and opsonic antibodies due to the critical role that opsonophagocytosis plays in T. pallidum clearance during infection. And third, the vaccine candidate(s) must be selected to ensure the vaccine is broadly protective against many T. pallidum strains. These complex requirements are very unlikely to be met using a single treponemal protein, and thus it is probable Compound C research buy that an effective syphilis vaccine will constitute a multi-component formulation. After almost a century of research, significant insight has been provided

into the correlates of protection in the rabbit model. However, successful vaccine development will depend upon extending our understanding MG-132 research buy of the correlates of protection in humans by fostering exchange of information and samples between the basic research laboratories and the clinics. Development of appropriate and effective adjuvants is essential and is likely to require the participation

of industry. Within the realm of research there needs to be the application of large-scale “omics” experimental approaches and data analyses to enhance our understanding of factors such as differential gene and protein expression among T. pallidum subspecies and T. pallidum subspecies pallidum strains. And, most importantly, there needs to be an enhanced effort to conclusively determine the identity of surface-exposed antigens. This includes the OMPs, but also requires that the field pursue non-protein antigens including membrane lipids and post-translational modifications such as glycosylation or methylation Mephenoxalone of exposed proteins. The field has been focussing on the “easier” protein antigens, perhaps at its peril. The accomplishment of these goals will require attracting a larger number of trained syphilis basic scientists to the field and a commitment of continual and enhanced training and research support that is commensurate with technical barriers and the high cost of performing T. pallidum research. The successful development of vaccines for a developing world market is challenging, as the average timeline for development of a new vaccine is 8-18.5 years at an estimated cost of $200–$900 million [97]. However, there is already a significant precedent for the support of pharmaceutical and biotechnology companies in the development of vaccines for diseases that disproportionately affect people in the developing world.

06 × 10−2/site/year (95% HPD 9 53 × 10−3 to 1 05 × 10−2) This is

06 × 10−2/site/year (95% HPD 9.53 × 10−3 to 1.05 × 10−2). This is Sorafenib nmr similar to the report (1.12 × 10−2/site/year) for VP1 sequences of A-Iran-05 viruses [13]; but higher than those reported by others [26], [27], [28], [29], [30], [31] and [32]. The high evolutionary rate of serotype A viruses in the ME is resulting in emergence of new variants in the region. An unbiased analysis of capsid sequences of the 51 A-Iran-05 viruses revealed 692 nt substitutions at 637 sites distributed

across the region (Fig. 1B). Out of these, 80.05% of nt substitutions were found to be synonymous (silent) and 19.95% were non-synonymous (non-silent). Forty seven sites were identified to have been substituted twice and four were substituted three times. At one site (VP2-134) the

first two bases of the codon were mutated encoding 5 different aa (P->T/S/L/H). This residue is located very close to residues VP2-132 and 133 that were reported as critical by mar-mutant studies for A10 virus [9]. In addition, the residue at this position has been reported to strongly influence the binding of antigenic site-2 mAbs in serotype O viruses [16]. Out of the four find protocol sites with three nt substitutions (encoding 2–4 aa residues), three were present in VP3 and one in VP1 (Table 1A). The analysis of the capsid aa residues of A-Iran-05 viruses revealed 140 substitutions at 101 sites across the capsid (Fig. 2A) with some sites having 2–5 alternate aa (Table 1B). Interestingly, sequences for VP1-204 encoded five different aa and exhibited nt changes at all the three positions within the codon as did VP1-196, with changes at all the three positions of the codon giving rise to four alternative aa. In addition, the non-synonymous nt substitutions were not equally distributed across the capsid coding regions: there were several local areas where the dN/dS ratio was higher than in other parts of the sequence alignment

(Fig. 2B). One region in VP3 (57–65), two in VP2 (75–76 and 130–134) and eight regions in VP1 (52–53, 83–84, 92–105, 131–132, 137–141, 145–152, 168–171 and 192–204) had dN/dS ratio of >1 indicative of sites under strong positive selection. Investigation of aa variability Histone demethylase across the capsid of the A-Iran-05 viruses revealed VP4 to be highly conserved and VP1 least conserved (Fig. 3A); similar to an earlier report [13]. The residues with a score greater than 0.75 (3 in VP2, 6 in VP3 and 12 in VP1) are shown in Fig. 3B-D indicating that over 50% of the residues with very high variability scores were present in VP1 (Fig. 3A). All these residues were found to be surface-exposed, except one residue in the N-terminus of VP1 (position 28) and one in N-terminus of VP3 (position 8) (Fig. 3C and D).

There are a number of studies reporting rotavirus strain distribu

There are a number of studies reporting rotavirus strain distribution in animals or humans in India but they do not provide any geographic or temporal comparisons of distribution among animals and humans [14], [18], [23] and [24]. This is also similar to the lack of such reports worldwide with only a few studies that have compared the strains isolated from animals Pexidartinib concentration and humans simultaneously in the same region [25] and [26]. In this study, we aimed to provide data on the disease burden and strain prevalence of rotavirus in animals and humans in our region and investigate interspecies transmission

by comparison of circulating genotypes using hemi-nested PCR typing for common human G- and P-types. In addition, a G10 rotavirus strain isolated for the first time with combination of P[15] in India was characterized by partial genome sequence analysis.

Stool samples were collected from children aged less than five years, admitted to the hospital between January 2003 and May 2006 for diarrhea, defined as the passage of three or more watery stools in a 24-h period [27]. The severity of diarrhea was assessed using the Vesikari scoring system [28]. Information was collected on duration of diarrhea, maximum number of stools passed per day, duration and peak frequency of vomiting, degree of fever, presence and severity of dehydration and treatment. An episode was considered Docetaxel cost mild for scores 0–5, moderate for a score of 6–10, severe for a score of 11–15 and very severe for scores 16–20. Diarrheal samples from animals were collected from a veterinary clinic and several dairy farms near Vellore between February 2007 and May 2008. At the dairy farms, diarrheal samples from cows alone were collected, while from the veterinary clinic, samples from cows, buffaloes, bullocks and goats were collected. Animal stool samples were subjected to proteinase K (2 μg/ml in 20 mM Tris, pH 7.5, 10 mM EDTA, and 0.1% SDS) treatment for 1 h followed by CC41 extraction [29]. From the stool samples of hospitalized

children, RNA was extracted using Trizol™ reagent [30]. cDNA was synthesized from see more the extracted viral RNA through reverse transcription in the presence of random hexamers. Amplification of the VP6 gene was performed using primers described previously [31]. G and P typing were performed using VP7 and VP4 specific multiplex hemi-nested RT-PCRs for common human genotypes, as described previously [32], [33] and [34]. Forward and reverse primers for the amplification of each segment other than VP7, VP6, VP4 and NSP4 to characterize G10P[15] strain were obtained from a published protocol [35]. PCR cycling conditions were determined based on the melting temperatures (Tm) of the primer pairs used for each PCR. When strains failed to genotype or genotypes needed to be confirmed, the first round PCR products generated through the use of consensus primers were sequenced and the genotype determined by sequence and phylogenetic analysis.

Alternatively, it is

speculated that our findings may be

Alternatively, it is

speculated that our findings may be explained by some form of immunological tolerance following 2 or 3 PCV-7 doses. Our findings indicate that PCV-7/PPV-23 compared to the PCV-7 primary series without a booster should offer superior protection from pneumococcal disease lasting at least 5 months following the 12 month PPV-23. A recent study of asthmatic children aged 2–5 years underwent sequential immunization of PCV-7 followed by PPV-23 either 2 or 10 months post PCV-7 [37]. Antibody concentrations for PCV-7 and 2 non-PCV-7 serotypes (5 and 7F) were higher following the PPV-23 booster than after PCV-7 alone [37]. Despite superior antibody concentrations being demonstrated for PCV-7/PPV-23 compared with this website PCV-7/PCV-7, we would not advise PCV-7/PPV-23 for 3 reasons. Firstly, superior vaccine efficacy using PCV-7/PPV-23 against clinical disease has not been demonstrated. A study of vaccine

efficacy against acute otitis media found that a PCV-7/PPV-23 NVP-BKM120 clinical trial compared to a PCV-7/PCV-7 schedule had similar results despite higher antibodies generated post PCV-7/PPV-23 [12]. This may be due to inferior quality of antibodies being produced following PPV-23. However previous studies have found that the quality of antibody, measured by avidity or opsonophagocytic activity, can differ in those that have received PPV-23 or PCV-7 as a booster, however results have been conflicting and therefore inconclusive [8], [10], [38], [39] and [40]. Finnish studies have shown the concentration

of antibodies required for 50% killing was higher [38] and that the avidity of such antibodies was Digestive enzyme lower after PCV-7/PPV-23 compared with PCV-7/PCV-7 [8], [39] and [41]. In contrast, another study in Finland using the 11-valent pneumococcal conjugate vaccine showed that opsonophagocytic activity was better in the group that received a PPV-23 booster at 12–15 months than those that had the conjugate booster [40]. A study in Israeli children who received 1 dose of the 7-valent pneumococcal polysaccharide-meningococcal outer membrane protein complex conjugate vaccine followed by either a conjugate or PPV-23 booster, achieved similar opsonic antibody titers in each group for the 1 serotype tested (6B) [8]. Data from the assessment of functional antibody responses in our study documenting the avidity to 23 serotypes and opsonophagocytic activity to 8 serotypes will be forthcoming. Secondly, conjugate vaccines are the only vaccines that provide mucosal immunity. As nasopharyngeal (NP) carriage is an antecedent event in IPD, the reduction or prevention of NP carriage reduces the transmission of pneumococci and prevents IPD in the vaccinated individual and provides herd immunity [42], [43] and [44]. In contrast, pneumococcal polysaccharide vaccines have shown no effect on pneumococcal carriage [20], [21], [22], [23] and [24].

025–0 0025% of total CD4 T cells [57] The background responses o

025–0.0025% of total CD4 T cells [57]. The background responses of most assays in naïve mice (<0.05% CD4 T cells) may obscure such populations [57]. Indeed, recent studies have had to employ enrichment of tetramer+ cells [58], to allow detection of rare TCM cells in BCG vaccinates [19] and [22]. Other in vitro expansion approaches, such as cultured ELISPOT [59] may also help to resolve this population. Therefore, we cannot

rule out the existence of undetected BCG-specific TCM. The GSK1120212 ic50 existence of potential TCM cells has been demonstrated in adoptive-transfer experiments, where cells with a potential TCM phenotype (CD62Lhi/CD45RBhi, but unknown for CCR7) conferred modest protection [12] and [60]. In the absence of a robust TCM response, other potential mechanisms of protection in BCG abbreviated mice may include alternate T cell subsets secreting cytokines not examined in this study (e.g. TH17 [13]), or undetected CD8 T cells, B cells or ‘innate’ cell activation and imprinting [61]. Current models for assessing TB vaccines

compare performance against the BCG ‘gold standard’, which likely include persistent bacilli and thus active TEM responses. This may account for the inability to improve upon BCG often reported [62]. A model where protection is assessed against only long-term memory, such as the abbreviation this website method used here, or other strategies to remove constant priming; may allow an enhanced ‘window of protection’ and subsequent identification of vaccines with potential for improved performance. This report has implications for the interpretation of immunity in pre-clinical models, with predominant responses dependent on antigen persistence. Therefore, studies which include such persistent BCG, not only demand a vaccine candidate to outperform the ‘gold standard’ in the face of constantly

primed TEffector and TEM responses; but also confound interpretation of the immunological analyses, with the dominant responses induced by live BCG undoubtedly obscuring the immune responses responsible for long-term memory-mediated protection. This underscores the importance of understanding the mechanisms of T cell memory. Conceived and TCL designed the experiments: PJH DAK. Performed the experiments: DAK CGP. Analyzed the data: DAK PJH. Wrote the paper: DAK PJH. This work was funded by the Department for Environment, Food and Rural Affairs, United Kingdom under grant number SE3266 ( We are especially grateful for the excellent services provided by the AHVLA Animal Services Unit. We would like to thank Dr Belinda Dagg at the National Institute for Biological Standards and Control (NIBSC, UK) for advice regarding antibiotic preparation and administration.

In this study, we evaluated the immune responses induced by synth

In this study, we evaluated the immune responses induced by synthetic vaccine particles (SVP) carrying covalently bound or entrapped TLR agonist co-delivered with encapsulated antigen (either in the same or in separate nanoparticle preparations). We hypothesized that such an approach may provide a two-pronged benefit by enabling a focused delivery of antigen and adjuvant and hence enhancing immunogenicity while preventing systemic exposure of the TLR agonist, which can result in excessive systemic cytokine release. Indeed, encapsulation of TLR agonist changed the dynamics of cytokine induction in vitro and in vivo.

Systemic cytokine production observed with selleck chemicals llc free resiquimod (R848) was suppressed by its encapsulation within nanoparticles. At the same time, SVP-encapsulated

TLR agonists, but not free TLR agonists, promoted sustained cytokine induction in the local draining lymph node as well as a robust infiltration by APCs and, later, by antigen-responsive cells. SVP-encapsulated TLR7/8 and TLR9 ligands augmented humoral and cellular immune responses to both soluble and nanoparticle-delivered protein compared to that observed with free adjuvants. Furthermore, this augmentation did not require co-encapsulation of antigen and TLR agonist in the same SVP. OTX015 clinical trial Collectively, these data indicate that SVPs may enable the use of potent TLR agonists as novel adjuvants by targeting their activity to the draining lymph node and minimizing systemic exposure, thereby reducing adjuvant-related side effects. Six- to eight-week-old female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA, USA) or Taconic (Germantown, NY, USA). All animal protocols were reviewed and approved by IACUC in accordance with federal, state and city of Cambridge (MA, USA) regulations

and guidelines. Fresh murine splenocytes were cultivated in RPMI with 10% FBS and were assayed Non-specific serine/threonine protein kinase in 96-well plates at 20,000–50,000 cells/well. Cell lines J774 (murine macrophages), EL4 (H-2b murine thymoma), and E.G7-OVA (EL4 cells transfected with full the length gene encoding chicken OVA) were purchased from the ATCC (American Type Culture Collection, Rockville, MD, USA) and grown per manufacturer’s recommendations. R848 was purchased from Enzo Life Sciences (Farmingdale, NY, USA) or Princeton Global Synthesis (Bristol, PA, USA). Phosphorothioate (PS) or phosphodiester (PO) forms of CpG-1826 (5′-TCCATGACGTTCCTGACGTT-3′) were purchased either from Enzo Life Sciences or from Oligo Factory (Holliston, MA, USA). OVA was purchased from Worthington Biochemical Corporation (Lakewood, NJ, USA). Recombinant prostatic acid phosphatase (PAP) was expressed in Escherichia coli and purified by Virogen (Watertown, MA, USA). Aluminum hydroxide gel (alum) was purchased from Sigma–Aldrich (St. Louis, MO, USA).

Data were collected in 2006 The primary outcome of interest was

Data were collected in 2006. The primary outcome of interest was the number of falls in the six months after the initial mobility assessment. The definition of a fall used was ‘a person unintentionally coming to rest on the ground’ (Jensen et al 2002, Vu et al 2006). Participant medical notes and incident reports were audited find more at two-monthly intervals by the research physiotherapist for entries relating to falls. The putative predictors assessed were the individual items and total score of the Physical Mobility Scale (Nitz et al 2006).

The Physical Mobility Scale includes nine mobility tasks ranging from bed mobility to ambulation, which are scored on a six-point scale from full dependence (0) to highest independence (5). Item scores are summed to give a total score (0–45) representing overall mobility, with lower scores indicating greater mobility impairment. Physical Mobility Scale assessments were carried out by physiotherapists who were independent of the staff employed by the residential aged care facilities. Physical Mobility Scale assessments were completed at three time check details points: baseline, and at two and four months after the baseline assessment. Thus, multiple Physical Mobility Scale assessments and fall data were included for each resident. The association between Physical

Mobility Scale total score and item scores, and risk of falling was assessed using Prentice, Williams, and Peterson conditional risk set survival models for recurrent events (Prentice et al 1981). An advantage of these models over traditional survival models is that they can be applied to data that include multiple observations for each participant, eg, multiple risk factor assessments and multiple outcome events. The recurrent event models used in this analysis were based on data that included up to three Physical Mobility Scale score observations for each resident corresponding to the baseline, two, and four month assessments and additional observations for each fall event that occurred. Total scores were coded into a priori specified

score categories to allow non-linear associations to be explored. Five score categories were selected to ensure an adequate number of observations these in each category. Too few observations in categories can lead to predictive models that are unstable and may provide imprecise and inaccurate associations. Each Physical Mobility Scale total score category was entered in a univariable model to establish the risk, reported as a hazard ratio, of sustaining a fall for each Physical Mobility Scale total score category. The ability of the Physical Mobility Scale items and total score categories to discriminate fallers from non-fallers was also explored through Prentice, Williams, and Peterson conditional risk set survival models for recurrent events (Prentice et al 1981).

Benveniste et al , Paris, France Therapy

of polymyositis

Benveniste et al., Paris, France Therapy

of polymyositis and dermatomyositis I. Marie, Rouen, France As reminded by D. Hilton-Jones in this issue’s review [1], the classification of myositides is currently changing. Since 1975, when Peter and Bohan [2] defined the diagnostic criteria for polymyositis (PM) and dermatomyositis (DM), the development of new pathological tools [3] and [4] permitted to refine the diagnosis criteria, but also, together with fundamental research in immunology [5] and neurosciences [4] to approach the various physiopathological events leading to the different acquired inflammatory and/or autoimmune myopathies. Beside the now “classical and well recognized” PM and DM, new insights have been this website done for the recognition of inclusion body myositis (IBM) [4] that must be distinguished from PM, but also, for the recognition of immune-mediated necrotizing myopathies (IMNM) [5] that clearly differ from inherited myopathies or dystrophies [6]. Among IMNM, some are related to the presence of particular specific auto-antibodies (anti-SRP), others are associated with neoplasia and the remaining are also recognized [7] for their property to be treatable by immunosuppressants. The recent discovery of a new auto-antibody specifically SNS-032 solubility dmso associated to IMNM (neither paraneoplastic,

nor anti-SRP positive) [8] highlights the potential toxic trigger role of statins in the genesis of IMNM/myositis, since the presence of this antibody was frequently associated with statin exposure [8]. A few weeks later, the same team also discovered

and published else the target of this antibody, which is the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) [9], the key enzyme in the cholesterol biosynthetic pathway specifically inhibited by statins. They also showed that statins up-regulate the expression of HMGCR on regenerative muscle fibers [9] (HMGCR being the major target of autoantibodies in statin-associated IMNM). Undoubtedly, commercial kits for the routine dosage of this auto-antibody will soon be available, facilitating the diagnosis of this condition. We will then see if all the myopathies due to the statins are due to the presence of this antibody. In the same vein, during the past few years, the burden of the dosages of the different myositis-specific (or associated) auto-antibodies has increased, an important step forward, since it may facilitate, at a modest cost, the diagnosis of these diseases. Within a very short time, we have now a routine access to the dosage of different antisynthetase antibodies anti-J0-1 (histidyl-tRNA synthetase), PL-7 (threonyl-tRNA synthetase), PL-12 (alanine-tRNA synthetase), OJ (isoleucil-tRNA synthetase), EJ (glycyl-tRNA synthetase), but also of anti-SRP, Mi-2, Ku, PM-Scl, RNP antibodies.

Since then, more large scale trials have been completed The inco

Since then, more large scale trials have been completed. The inconclusive result of the Cochrane review could be partially the result of comparing

treadmill walking with other mechanised walking (such as an electromechanical gait trainer) which may be expected to result in even more practice than treadmill walking. A systematic review examining electromechanical gait trainers only (Mehrholz et al 2010) found an increase in the likelihood of walking. We therefore planned a systematic review focusing broadly on any mechanically assisted walking, and comparing it with overground walking so that therapists and health administrators would have evidence to help guide decision making in terms of investing in mechanical walking equipment. In particular, we were interested in whether any benefits of mechanically assisted walking were still apparent in the long term or whether the effect was short lived. Clinicians still seem reluctant Regorafenib purchase to implement Selleckchem EPZ-6438 treadmill training for stroke patients due to a fear that an abnormal walking pattern will be practised (Hesse 2008) resulting in abnormal overground walking (Davies 1999). We were therefore interested in examining any aspects of walking commonly measured, such as speed and capacity, which would shed some light on whether this fear is reasonable. The specific research questions for this review were: 1. In subacute, non-ambulatory

patients after stroke, does mechanically assisted walking with body weight support result in more independent walking than overground walking in the short term? In order to make recommendations based on the highest level of evidence, this review included only randomised or quasi-randomised trials in which Tryptophan synthase patients undergoing inpatient stroke rehabilitation to enable them to walk were randomised to receive either mechanically assisted walking with body weight support or assisted overground walking. Searches were conducted of the following databases: MEDLINE (1966 to August Week

4 2009), CINAHL (1982 to August Week 4 2009), EMBASE (1980 to August Week 4 2009) and PEDro (to August Week 4 2009), without language restrictions for relevant articles. Search terms included words relating to stroke, exercise therapy, and locomotion (see Appendix 1 on the eAddenda for the full search strategy). In addition, we contacted authors about trials that we knew were in progress from trial registration. Title and abstracts were displayed and screened by one reviewer to identify relevant studies. Full paper copies of relevant studies were retrieved and their reference lists were screened. The methods of retrieved papers were extracted so that reviewers were blinded to authors, journals and outcomes and examined against predetermined inclusion criteria (Box 1) by two independent reviewers. Conflict of opinion was resolved by consensus after discussion with a third reviewer.