Understanding the effectiveness of different exercise training programme designs on V̇O2peak in COPD: a component network meta-analysis.

Pulmonary rehabilitation programmes including aerobic training improve cardiorespiratory fitness in patients with COPD, but the optimal programme design is unclear. We used random effects additive component network meta-analysis to investigate the relative effectiveness of different programme components on fitness measured by V̇O2peak in COPD. The included 59 studies involving 2191 participants demonstrated that V̇O2peak increased after aerobic training of at least moderate intensity with the greatest improvement seen following high intensity training. Lower limb aerobic training (SMD 0.56 95% CI 0.32;0.81, intervention arms=86) and the addition of non-invasive ventilation (SMD 0.55 95% CI 0.04;1.06, intervention arms=4) appeared to offer additional benefit but there was limited evidence for effectiveness of other exercise and non-exercise components.

Diagnostic accuracy of natriuretic peptide screening for left ventricular systolic dysfunction in the community: systematic review and meta-analysis.

AIMS: Heart failure (HF) is a global health burden and new strategies to achieve timely diagnosis and early intervention are urgently needed. Natriuretic peptide (NP) testing can be used to screen for left ventricular systolic dysfunction (LVSD), but evidence on test performance is mixed, and international HF guidelines differ in their recommendations. Our aim was to summarize the evidence on diagnostic accuracy of NP screening for LVSD in general and high-risk community populations and estimate optimal screening thresholds. METHODS: We searched relevant databases up to August 2020 for studies with a screened community population of over 100 adults reporting NP performance to diagnose LVSD. Study inclusion, quality assessment, and data extraction were conducted independently and in duplicate. Diagnostic test meta-analysis used hierarchical summary receiver operating characteristic curves to obtain estimates of pooled accuracy to detect LVSD, with optimal thresholds obtained to maximize the sum of sensitivity and specificity. RESULTS: Twenty-four studies were identified, involving 26 565 participants: eight studies in high-risk populations (at least one cardiovascular risk factor), 12 studies in general populations, and four in both high-risk and general populations combined. For detecting LVSD in screened high-risk populations with N-terminal prohormone brain natriuretic peptide (NT-proBNP), the pooled sensitivity was 0.87 [95% confidence interval (CI) 0.73-0.94] and specificity 0.84 (95% CI 0.55-0.96); for BNP, sensitivity was 0.75 (95% CI 0.65-0.83) and specificity 0.78 (95% CI 0.72-0.84). Heterogeneity between studies was high with variations in positivity threshold. Due to a paucity of high-risk studies that assessed NP performance at multiple thresholds, it was not possible to calculate optimal thresholds for LVSD screening in high-risk populations alone. To provide an indication of where the positivity threshold might lie, the pooled accuracy for LVSD screening in high-risk and general community populations were combined and gave an optimal cut-off of 311 pg/mL [sensitivity 0.74 (95% CI 0.53-0.88), specificity 0.85 (95% CI 0.68-0.93)] for NT-proBNP and 49 pg/mL [sensitivity 0.68 (95% CI 0.45-0.85), specificity 0.81 (0.67-0.90)] for BNP. CONCLUSIONS: Our findings suggest that in high-risk community populations NP screening may accurately detect LVSD, potentially providing an important opportunity for diagnosis and early intervention. Our study highlights an urgent need for further prospective studies, as well as an individual participant data meta-analysis, to more precisely evaluate diagnostic accuracy and identify optimal screening thresholds in specifically defined community-based populations to inform future guideline recommendations.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43.

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus' ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Change in V˙O2peak in Response to Aerobic Exercise Training and the Relationship With Exercise Prescription in People With COPD: A Systematic Review and Meta-analysis.

BACKGROUND: Despite the wide-ranging benefits of pulmonary rehabilitation, conflicting results remain regarding whether people with COPD can improve their peak oxygen uptake (V˙O2peak) with aerobic training. RESEARCH QUESTION: The goal of this study was to investigate the effect of aerobic training and exercise prescription on V˙O2peak in COPD. STUDY DESIGN AND METHODS: A systematic review was performed by using MEDLINE, Embase, Cumulative Index to Nursing and Allied Health Literature, and Cochrane databases for all studies measuring V˙O2peak prior to and following supervised lower-limb aerobic training in COPD. A random effects meta-analysis limited to randomized controlled trials comparing aerobic training vs usual care was conducted. Other study designs were included in a secondary meta-analysis and meta-regression to investigate the influence of program and patient factors on outcome. RESULTS: A total of 112 studies were included (participants, N = 3,484): 21 controlled trials (n = 489), of which 13 were randomized (n = 288) and 91 were uncontrolled (n = 2,995) studies. Meta-analysis found a moderate positive change in V˙O2peak (standardized mean difference, 0.52; 95% CI, 0.34-0.69) with the intervention. The change in V˙O2peak was positively associated with target duration of exercise session (P = .01) and, when studies > 1 year duration were excluded, greater total volume of exercise training (P = .01). Similarly, the change in V˙O2peak was greater for programs > 12 weeks compared with those 6 to 12 weeks when adjusted for age and sex. However, reported prescribed exercise intensity (P = .77), training modality (P > .35), and mode (P = .29) did not affect V˙O2peak. Cohorts with more severe airflow obstruction exhibited smaller improvements in V˙O2peak (P < .001). INTERPRETATION: Overall, people with COPD achieved moderate improvements in V˙O2peak through supervised aerobic training. There is sufficient evidence to show that programs with greater total exercise volume, including duration of exercise session and program duration, are more effective. Reduced effects in severe disease suggest alternative aerobic training methods may be needed in this population. CLINICAL TRIAL REGISTRATION: PROSPERO; No.: CRD42018099300; URL: https://www.crd.york.ac.uk/prospero/.

Construction of a pneumolysin deficient mutant in streptococcus pneumoniae serotype 1 strain 519/43 and phenotypic characterisation.

Streptococcus pneumoniae capsular serotype 1 continues to pose a huge infectious disease burden in low- and middle-income countries, particularly in West Africa. However, studies on this important serotype have been hampered by the inability to genetically modify these strains. In this study we have genetically modified a serotype 1 strain (519/43), the first time that this has been achieved for this serotype, providing the methodology for a deeper understanding of its biology and pathogenicity. As proof of principle we constructed a defined pneumolysin mutant and showed that it lost its ability to lyse red blood cells. We also showed that when mice were infected intranasally with the mutant 519/43Δply there was no significant difference between the load of bacteria in lungs and blood when compared to the wild type 519/43. When mice were infected intraperitoneally there were significantly fewer bacteria recovered from blood for the mutant 519/43Δply strain, although all mice still displayed signs of disease. Our study demonstrates S. pneumoniae serotype 1 strains can be genetically manipulated using our methodology and demonstrate that the ability to cause pneumonia in mice is independent of active pneumolysin for the 519/43 serotype 1 strain.

Dietary zinc and the control of Streptococcus pneumoniae infection.

Human zinc deficiency increases susceptibility to bacterial infection. Although zinc supplementation therapies can reduce the impact of disease, the molecular basis for protection remains unclear. Streptococcus pneumoniae is a major cause of bacterial pneumonia, which is prevalent in regions of zinc deficiency. We report that dietary zinc levels dictate the outcome of S. pneumoniae infection in a murine model. Dietary zinc restriction impacts murine tissue zinc levels with distribution post-infection altered, and S. pneumoniae virulence and infection enhanced. Although the activation and infiltration of murine phagocytic cells was not affected by zinc restriction, their efficacy of bacterial control was compromised. S. pneumoniae was shown to be highly sensitive to zinc intoxication, with this process impaired in zinc restricted mice and isolated phagocytic cells. Collectively, these data show how dietary zinc deficiency increases sensitivity to S. pneumoniae infection while revealing a role for zinc as a component of host antimicrobial defences.

The First Histidine Triad Motif of PhtD Is Critical for Zinc Homeostasis in Streptococcus pneumoniae.

Streptococcus pneumoniae is the world's foremost human pathogen. Acquisition of the first row transition metal ion zinc is essential for pneumococcal colonization and disease. Zinc is acquired via the ATP-binding cassette transporter AdcCB and two zinc-binding proteins, AdcA and AdcAII. We have previously shown that AdcAII is reliant upon the polyhistidine triad (Pht) proteins to aid in zinc recruitment. Pht proteins generally contain five histidine (His) triad motifs that are believed to facilitate zinc binding and therefore play a significant role in pneumococcal metal ion homeostasis. However, the importance and potential redundancy of these motifs have not been addressed. We examined the effects of mutating each of the five His triad motifs of PhtD. The combination of in vitro growth assays, active zinc uptake, and PhtD expression studies show that the His triad closest to the protein's amino terminus is the most important for zinc acquisition. Intriguingly, in vivo competitive infection studies investigating the amino- and carboxyl-terminal His triad mutants indicate that the motifs have similar importance in colonization. Collectively, our new insights into the contributions of the individual His triad motifs of PhtD, and by extension the other Pht proteins, highlight the crucial role of the first His triad site in zinc acquisition. This study also suggests that the Pht proteins likely play a role beyond zinc acquisition in pneumococcal virulence.

Overlapping functionality of the Pht proteins in zinc homeostasis of Streptococcus pneumoniae.

Streptococcus pneumoniae is a globally significant pathogen that causes a range of diseases, including pneumonia, sepsis, meningitis, and otitis media. Its ability to cause disease depends upon the acquisition of nutrients from its environment, including transition metal ions such as zinc. The pneumococcus employs a number of surface proteins to achieve this, among which are four highly similar polyhistidine triad (Pht) proteins. It has previously been established that these proteins collectively aid in the delivery of zinc to the ABC transporter substrate-binding protein AdcAII. Here we have investigated the contribution of each individual Pht protein to pneumococcal zinc homeostasis by analyzing mutant strains expressing only one of the four pht genes. Under conditions of low zinc availability, each of these mutants showed superior growth and zinc accumulation profiles relative to a mutant strain lacking all four genes, indicating that any of the four Pht proteins are able to facilitate delivery of zinc to AdcAII. However, optimal growth and zinc accumulation in vitro and pneumococcal survival and proliferation in vivo required production of all four Pht proteins, indicating that, despite their overlapping functionality, the proteins are not dispensable without incurring a fitness cost. We also show that surface-attached forms of the Pht proteins are required for zinc recruitment and that they do not contribute to defense against extracellular zinc stress.

Development of primary invasive pneumococcal disease caused by serotype 1 pneumococci is driven by early increased type I interferon response in the lung.

The pneumococcus is the world's foremost respiratory pathogen, but the mechanisms allowing this pathogen to proceed from initial asymptomatic colonization to invasive disease are poorly understood. We have examined the early stages of invasive pneumococcal disease (IPD) by comparing host transcriptional responses to an invasive strain and a noninvasive strain of serotype 1 Streptococcus pneumoniae in the mouse lung. While the two strains were present in equal numbers in the lung 6 h after intranasal challenge, only the invasive strain (strain 1861) had invaded the pleural cavity at that time point; this correlated with subsequent development of bacteremia in mice challenged with strain 1861 but not the noninvasive strain (strain 1). Progression beyond the lung was associated with stronger induction of the type I interferon (IFN-I) response in the lung at 6 h. Suppression of the IFN-I response through administration of neutralizing antibody to IFNAR1 (the receptor for type I interferons) led to significantly reduced invasion of the pleural cavity by strain 1861 at 6 h postchallenge. Our data suggest that strong induction of the IFN-I response is a key factor in early progression of invasive serotype 1 strain 1861 beyond the lung during development of IPD.

AdcA and AdcAII employ distinct zinc acquisition mechanisms and contribute additively to zinc homeostasis in Streptococcus pneumoniae.

Streptococcus pneumoniae is a globally significant human pathogen responsible for nearly 1 million deaths annually. Central to the ability of S. pneumoniae to colonize and mediate disease in humans is the acquisition of zinc from the host environment. Zinc uptake in S. pneumoniae occurs via the ATP-binding cassette transporter AdcCB, and, unusually, two zinc-binding proteins, AdcA and AdcAII. Studies have suggested that these two proteins are functionally redundant, although AdcA has remained uncharacterized by biochemical methods. Here we show that AdcA is a zinc-specific substrate-binding protein (SBP). By contrast with other zinc-binding SBPs, AdcA has two zinc-binding domains: a canonical amino-terminal cluster A-I zinc-binding domain and a carboxy-terminal zinc-binding domain, which has homology to the zinc-chaperone ZinT from Gram-negative organisms. Intriguingly, this latter feature is absent from AdcAII and suggests that the two zinc-binding SBPs of S. pneumoniae employ different modalities in zinc recruitment. We further show that AdcAII is reliant upon the polyhistidine triad proteins for zinc in vitro and in vivo. Collectively, our studies suggest that, despite the overlapping roles of the two SBPs in zinc acquisition, they may have unique mechanisms in zinc homeostasis and act in a complementary manner during host colonization.

Vaccination against Streptococcus pneumoniae using truncated derivatives of polyhistidine triad protein D.

Polyhistidine triad protein D (PhtD) has been described as a promising vaccine candidate for use against Streptococcus pneumoniae, but there has been a lack of examination of its structure and of which region(s) of the protein are targeted by protective immune responses. In this study, we purified recombinant truncated derivatives of PhtD and examined their secondary structural composition, as well as their capacity to bind antibodies from polyclonal murine serum generated against the full length protein. This allowed the identification of a particularly immunogenic fragment of PhtD, which was also purified and characterised. The truncated derivatives were tested as vaccine antigens in mouse models of pneumococcal sepsis and colonisation, using alum and E. coli heat labile toxin B subunit respectively as adjuvants. These experiments revealed that whilst the immunogenic region identified may be a promising candidate to protect against sepsis, the full length PhtD was ineffective at conferring significant protective immunity. These results are significant for the potential for PhtD to be used in novel vaccines, which are currently being tested in clinical trials.

Surface association of Pht proteins of Streptococcus pneumoniae.

Streptococcus pneumoniae is a major human pathogen responsible for massive global morbidity and mortality. The pneumococcus attaches a variety of proteins to its cell surface, many of which contribute to virulence; one such family are the polyhistidine triad (Pht) proteins PhtA, PhtB, PhtD, and PhtE. In this study, we have examined the mechanism of Pht surface attachment using PhtD as a model. Analysis of deletion and point mutants identified a three-amino-acid region of PhtD (Q27-H28-R29) that is critical for the process. The analogous region in PhtE was also necessary for its attachment to the cell surface. Furthermore, we show that a large proportion of the total amount of each Pht protein is released into bacterial culture supernatants. Other surface proteins were also released, albeit to lesser extents, and this was not due to pneumococcal autolysis. The extent of release of surface proteins was strain dependent and was not affected by the capsule. Lastly, we compared the fitness of wild-type and ΔphtABDE pneumococci in vivo in a mouse coinfection model. Release of Pht proteins by the wild type did not complement the mutant strain, consistent with surface-attached rather than soluble forms of the Pht proteins playing the major role in virulence. The significant degree of release of Pht proteins from intact bacteria may have implications for the use of these proteins in novel vaccines.

Polyhistidine triad proteins of pathogenic streptococci.

The polyhistidine triad (Pht) proteins are an intriguing family of proteins found on the surface of members of the genus Streptococcus. Their defining feature is the presence of multiple copies of the eponymous His triad motif HxxHxH. This review focuses on the Pht proteins of Streptococcus pneumoniae, which contribute to virulence and are leading candidates for inclusion in protein-based pneumococcal vaccines. They appear to have multiple functions, including metal ion homeostasis, evasion of complement deposition and adherence of bacteria to host cells. Across the streptococci, there are many Pht homologs, which can be grouped according to structural features. Critically, there is considerable potential to use members of the Pht protein family as components of vaccines targeted at other streptococci.

Identification of genes that contribute to the pathogenesis of invasive pneumococcal disease by in vivo transcriptomic analysis.

Streptococcus pneumoniae (the pneumococcus) continues to be responsible for a high level of global morbidity and mortality resulting from pneumonia, bacteremia, meningitis, and otitis media. Here we have used a novel technique involving niche-specific, genome-wide in vivo transcriptomic analyses to identify genes upregulated in distinct niches during pathogenesis after intranasal infection of mice with serotype 4 or 6A pneumococci. The analyses yielded 28 common, significantly upregulated genes in the lungs relative to those in the nasopharynx and 25 significantly upregulated genes in the blood relative to those in the lungs in both strains, some of which were previously unrecognized. The role of five upregulated genes from either the lungs or the blood in pneumococcal pathogenesis and virulence was then evaluated by targeted mutagenesis. One of the mutants (ΔmalX) was significantly attenuated for virulence in the lungs, two (ΔaliA and ΔilvH) were significantly attenuated for virulence in the blood relative to the wild type, and two others (ΔcbiO and ΔpiuA) were completely avirulent in a mouse intranasal challenge model. We also show that the products of aliA, malX, and piuA are promising candidates for incorporation into multicomponent protein-based pneumococcal vaccines currently under development. Importantly, we suggest that this new approach is a viable complement to existing strategies for the discovery of genes critical to the distinct stages of invasive pneumococcal disease and potentially has broad application for novel protein antigen discovery in other pathogens such as S. pyogenes, Haemophilus influenzae type b, and Neisseria meningitidis.