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Abstract A major challenge to tuberculosis (TB) vaccine development is the lack of a validated immune correlate of protection. Mycobacterial growth inhibition assays (MGIAs) represent an unbiased measure of the ability to control mycobacterial growth in vitro. A successful MGIA could be applied to preclinical and clinical post-vaccination samples to aid in the selection of novel vaccine candidates at an early stage and provide a relevant measure of immunogenicity and protection. However, assay harmonisation is critical to ensure that comparable information can be extracted from different vaccine studies. As part of the FP7 European Research Infrastructures for Poverty Related Diseases (EURIPRED) consortium, we aimed to optimise the direct MGIA, assess repeatability and reproducibility, and harmonise the assay across different laboratories. We observed an improvement in repeatability with increased cell number and increased mycobacterial input. Furthermore, we determined that co-culturing in static 48-well plates compared with rotating 2 ml tubes resulted in a 23% increase in cell viability and a 500-fold increase in interferon-gamma (IFN-γ) production on average, as well as improved reproducibility between replicates, assay runs and sites. Applying the optimised conditions, we report repeatability to be <5% coefficient of variation (CV), intermediate precision to be <20% CV, and inter-site reproducibility to be <30% CV; levels within acceptable limits for a functional cell-based assay. Using relevant clinical samples, we demonstrated comparable results across two shared sample sets at three sites. Based on these findings, we have established a standardised operating procedure (SOP) for the use of the direct PBMC MGIA in TB vaccine development. Highlights • Optimised assay conditions for direct PBMC MGIA determined. • Standardised operating procedure for use in TB vaccine studies established. • Consistency across three sites using clinically-relevant samples demonstrated. [ABSTRACT FROM AUTHOR]

Abstract The Hawaiian deep-slope (75–400 m) Deep7 bottomfish fishery consists of seven (i.e., six snappers and one grouper) species. This study developed a sampling survey and modeling methodology for estimating biomass for this complex in the Main Hawaiian Islands. The island-wide fishery-independent sampling survey using two gears (commercial fishers with hook-and-line, 3D stereo camera) was conducted to generate estimates of relative abundance- and biomass-at-length for the complex. A length-based modeling approach was applied to the opakapaka fishery and survey size-structured abundance data, life history demography, and total fishery catches to estimate a feasible range of effective sampling area for the standard survey gear (i.e., cameras). These sampling area estimates were then used to expand survey estimates of relative biomass to population total biomass. The longer-term focus of this effort is to improve stock assessments of the Deep7 complex. The survey and modeling methods developed in this study provide the underpinnings of an integrated information and modeling system for assessment that enables multiple levels of comparison and validation with respect to data sources (fishery-dependent and fishery-independent) and modeling approaches (biomass-dynamic and cohort-structured). [ABSTRACT FROM AUTHOR]

Background Vaccination with Bacillus Calmette Guerin (BCG) protects infants against childhood tuberculosis however the immune mechanisms involved are not well understood. Further elucidation of the infant immune response to BCG will aid with the identification of immune correlates of protection against tuberculosis and with the design of new improved vaccines. The purpose of this study was to investigate BCG-induced CD4+ T-cell responses in blood samples from infants for cytokine secretion profiles thought to be important for protection against tuberculosis and compare these to PBMC-mediated in vitro mycobacterial growth inhibition. Methods Blood from BCG-vaccinated or unvaccinated infants was stimulated overnight with Mycobacterium tuberculosis ( M. tb ) purified protein derivative (PPD) or controls and intracellular cytokine staining and flow cytometry used to measure CD4+ T-cell responses. PBMC cryopreserved at the time of sample collection were thawed and incubated with live BCG for four days following which inhibition of BCG growth was determined. Results PPD-specific IFNγ+TNFα+IL-2+CD4+ T-cells represented the dominant T-cell response at 4 months and 1 year after infant BCG. These responses were undetectable in age-matched unvaccinated infants. IL-17+ CD4+ T-cells were significantly more frequent in vaccinated infants at 4 months but not at 1-year post-BCG. PBMC-mediated inhibition of mycobacterial growth was significantly enhanced at 4 months post-BCG as compared to unvaccinated controls. In an analysis of all samples with both datasets available, mycobacterial growth inhibition correlated significantly with the frequency of polyfunctional (IFNγ+TNFα+IL-2+) CD4+ T-cells. Conclusions These data suggest that BCG vaccination of infants induces specific polyfunctional T-helper-1 and T-helper-17 responses and the ability, in the PBMC compartment, to inhibit the growth of mycobacteria in vitro. We also demonstrate that polyfunctional T-helper-1 cells may play a role in growth inhibition as evidenced by a significant correlation between the two. [ABSTRACT FROM AUTHOR]

Bromodomains are conserved structural modules responsible for recognizing acetylated-lysine residues on histone tails and other transcription-associated proteins, such as transcription factors and co-factors. Owing to their important functions in the regulation of ordered gene transcription in chromatin, bromodomains of the BET family proteins have recently been shown as druggable targets for a wide array of human diseases, including cancer and inflammation. Here we review the structural and functional features of the bromodomains and their small-molecule inhibitors. Additional new insights provided herein highlight the landscape of the ligand binding sites in the bromodomains that will hopefully facilitate further development of new inhibitors with optimal affinity and selectivity. [ABSTRACT FROM AUTHOR]

Background Thymic stromal lymphopoietin (TSLP) and IL-33 are considered important initiators of type 2 immunity. In asthmatic patients allergic inflammatory responses are associated with increased lung homing of bone marrow–derived CD34 + hematopoietic progenitor cells (HPCs), which include eosinophil lineage–committed progenitor cells. In this study we investigated the role of TSLP and IL-33 in the recruitment of progenitor cells to the airways in asthmatic subjects. Objectives We sought (i) to examine the effect of allergen inhalation challenge on expression levels of receptors for TSLP (thymic stromal lymphopoietin receptor [TSLPR] and CD127) and IL-33 (ST2) and (ii) investigate the functional effects of these cytokines on HPCs. Methods Consenting patients with mild atopic asthma (n = 19) with an FEV 1 of 70% or greater and methacholine PC 20 of 16 mg/mL or less were recruited. Blood- and sputum-extracted progenitors were phenotyped by flow cytometry before and 24 hours after allergen challenge. Functional responses, including cytokine production and migration to TSLP and IL-33, were assessed in vitro . Results Significant increases in mature eosinophil, HPC, and eosinophil lineage–committed progenitor cell counts in sputum were observed 24 hours after allergen and were associated with a significant allergen-induced increase in HPCs expressing TSLPR, CD127, and ST2. Pre-exposure to TSLP and IL-33 primed the migration of HPCs to a potent progenitor cell chemoattractant, stromal cell–derived factor 1α (CXCL12). Incubation with TSLP and IL-33 stimulated significant production of IL-5 and IL-13, but not IL-4, by HPCs. This priming effect was inhibited by blocking antibodies to TSLPR and ST2, respectively, and IL-13 receptor α1 in both scenarios. Conclusions In allergic asthmatic responses increased lung homing of HPCs may be orchestrated by TSLP and IL-33 through an IL-13–dependent axis. [ABSTRACT FROM AUTHOR]

In the phase III SYNAPSE study, mepolizumab reduced nasal polyp (NP) size and nasal obstruction in chronic rhinosinusitis with NP. We sought to assess the efficacy of mepolizumab in patients from SYNAPSE grouped by comorbid asthma, aspirin-exacerbated respiratory disease (AERD), and baseline blood eosinophil count (BEC). SYNAPSE, a randomized, double-blind, 52-week study (NCT03085797), included patients with severe bilateral chronic rhinosinusitis with NP eligible for surgery despite intranasal corticosteroid treatment. Patients received 4-weekly subcutaneous mepolizumab 100 mg or placebo plus standard of care for 52 weeks. Coprimary end points were change in total endoscopic NP score (week 52) and nasal obstruction visual analog scale score (weeks 49-52). Subgroup analyses by comorbid asthma and AERD status, and post hoc by BEC, were exploratory. Analyses included 407 patients (289 with asthma; 108 with AERD; 371 and 278 with BEC counts ≥150 or ≥300 cells/μL, respectively). The proportion of patients with greater than or equal to 1-point improvement from baseline in NP score was higher with mepolizumab versus placebo across comorbid diseases (asthma: 52.9% vs 29.5%; AERD: 51.1% vs 20.6%) and baseline BEC subgroups (<150 cells/μL: 55.0% vs 31.3%; ≥150 cells/μL: 49.5% vs 28.1%; <300 cells/μL: 50.7% vs 29.0%; ≥300 cells/μL: 50.4% vs 28.1%). A similar trend was observed in patients without comorbid asthma or AERD. More patients had more than 3-point improvement in nasal obstruction VAS score with mepolizumab versus placebo across comorbid subgroups. Mepolizumab reduced polyp size and nasal obstruction in chronic rhinosinusitis with NP regardless of the presence of comorbid asthma or AERD. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: Natural regulatory T (Treg) cells are implicated in the regulation of the inflammatory response in patients with allergic asthma. Objectives: We sought to determine changes in Treg cell numbers in the airways and peripheral blood of isolated early responder (IER) versus dual responder (DR) subjects with mild allergic asthma before and after allergen challenge. Methods: Induced sputum was collected from 22 subjects with allergic asthma (10 IERs and 12 DRs) and peripheral blood collected from 8 DRs with allergic asthma at 0, 7, and 24 hours after allergen challenge. Treg cells were identified by using fluorescently labeled antibodies to CD4 and forkhead box protein 3 and enumerated by using flow cytometry. Results: There was a significant increase in the percentage of sputum CD4+ cells 24 hours after allergen challenge in both IERs and DRs. The percentage of sputum Treg cells significantly decreased 24 hours after challenge in DRs but not IERs. This change was significantly correlated with the magnitude of the late asthmatic response. There was also a significant increase in the absolute number of sputum CD4+ cells and Treg cells at 24 hours in DRs only. The ratio of the number of Treg cells to CD4+ cells at 24 hours was significantly smaller in DRs compared with that in IERs. None of the above changes were observed in peripheral blood. Conclusions: DRs exhibit a diminished percentage of airway Treg cells after allergen challenge that is not observed in IERs and a significantly lower ratio of Treg cells to CD4+ cells, which might contribute to the development of the late asthmatic response. [Copyright &y& Elsevier]

Abstract: The efficacy of no-take marine reserves (NTMRs) to enhance and sustain regional coral reef fisheries was assessed in Dry Tortugas, Florida, through 9 annual fishery-independent research surveys spanning 2 years before and 10 years after NTMR implementation. A probabilistic sampling design produced precise estimates of population metrics of more than 250 exploited and non-target reef fishes. During the survey period more than 8100 research dives utilizing SCUBA Nitrox were optimally allocated using stratified random sampling. The survey domain covered 326km2, comprised of eight reef habitats in four management areas that offered different levels of resource protection: the Tortugas North Ecological Reserve (a NTMR), Dry Tortugas National Park (recreational angling only), Dry Tortugas National Park Research Natural Area (a NTMR), and southern Tortugas Bank (open to all types of fishing). Surveys detected significant changes in population occupancy, density, and abundance within management zones for a suite of exploited and non-target species. Increases in size, adult abundance, and occupancy rates were detected for many principal exploited species in protected areas, which harbored a disproportionately greater number of adult spawning fishes. In contrast, density and occupancy rates for aquaria and non-target reef fishes fluctuated above and below baseline levels in each management zone. Observed decreases in density of exploited species below baseline levels only occurred at the Tortugas Bank area open to all fishing. Our findings indicate that these NTMRs, in conjunction with traditional fishery management control strategies, are helping to build sustainable fisheries while protecting the fundamental ecological dynamics of the Florida Keys coral-reef ecosystem. [Copyright &y& Elsevier]

Abstract: Principles of statistical sampling design were used to guide refinement of a 30-year multispecies fishery-independent diver visual survey of population abundance and size structure of more than 250 exploited and non-target fishes in the Florida coral reef ecosystem. Reef habitat features and no-take marine reserves (NTMRs) were used to partition the 885km2 sampling domain into sub-areas (or strata) to control the variation of fish density. For the period 1999–2008, survey precision of population density and abundance (CV, coefficient of variation, ratio of standard error to mean) ranged from 7% to 20% for the majority of 13 primary exploited species in the Florida Keys and Dry Tortugas regions. Population sustainability metrics like species average length in the exploited life stage were comparable between our fishery-independent survey and fishery-dependent catch-sampling. The survey design also performed well for non-target fishes, yielding CVs between 6% and 15% for population density for the majority of 36 species. Sampling efficiency was improved over time via an iterative learning process by which past survey data was used to refine the stratification and allocation schemes of future surveys. We show how survey data are used to support multispecies stock assessments, evaluate the effectiveness of NTMRs, and assess ecosystem condition for the reef fish community. [Copyright &y& Elsevier]

Abstract: The reliable stereochemical assignment of flexible molecules, such as acyclic polypropionates is an enormously challenging task. This is illustrated by the NMR chemical shifts for a complete set of sixteen diastereomeric stereopentads whose experimental data is reported here for the first time. Although the experimental spectra are very similar to each other, analysis of the similarity between the shifts of different diastereoisomers reveals that some diastereoisomers are much more distinctive than others. In addition, the NMR shifts of the sixteen compounds have also been calculated using DFT GIAO calculations, and the use of our recently developed CP3 parameter for structure assignment is illustrated for these molecules. Even in cases where the experimental spectra are very similar, our CP3 parameter makes possible the correct assignment of pairs of diastereoisomers with high confidence. [ABSTRACT FROM AUTHOR]

Abstract: IFNγ plays an important part in immunity to tuberculosis (TB), but although it is necessary, it is not on its own sufficient for protection against TB. To identify other cytokines that play a role in the protection against TB induced by BCG vaccination, immune responses were compared between vaccinated and unvaccinated infants from the UK where BCG is known to provide protection. Twenty-one cytokines and chemokines were tested in supernatants from diluted whole blood cultures that had been stimulated for 6 days with Mycobacterium tuberculosis PPD. For 15 out of 21 of the cytokines tested responses were much higher in BCG vaccinated infants than in unvaccinated infants. These included: pro-inflammatory cytokines; IFNγ (median 1705pg/ml vs. 1.6pg/ml in vaccinated and unvaccinated infants, respectively), TNFα (median 226pg/ml vs. 18pg/ml), as well as IL-2, IL-1α and IL-6; TH2 cytokines: IL-4, IL-5 and IL-13 (median 104pg/ml vs. 1.6pg/ml); the regulatory cytokine IL-10 (median response 96pg/ml vs. 8pg/ml); the TH17 cytokine IL-17, chemokines (IP-10, MIP-1α and IL-8) and growth factors (GM-CSF and G-CSF). The greatest increase in cytokine production in BCG vaccinees compared to unvaccinated infants was seen with IFNγ. While responses for many cytokines were correlated with the IFNγ response, others including IL-17 and IL-10 were not. The pattern of cytokine induction following BCG vaccination is complex and measurement of one of two cytokines does not reveal the whole picture of vaccine-induced protection. [Copyright &y& Elsevier]

Coral reef ecosystems are declining due to multiple interacting stressors. A bioassessment framework focused on stressor-response associations was developed to help organize and communicate complex ecological information to support coral reef conservation. This study applied the Biological Condition Gradient (BCG), initially developed for freshwater ecosystems, to fish assemblages of U.S. Caribbean coral reef ecosystems. The reef fish BCG describes how biological conditions changed incrementally along a gradient of increasing anthropogenic stress. Coupled with physical and chemical water quality data, the BGC forms a scientifically defensible basis to prioritize, protect and restore water bodies containing coral reefs. Through an iterative process, scientists from across the U.S. Caribbean used fishery-independent survey data and expert knowledge to develop quantitative decision rules to describe six levels of coral reef ecosystem condition. The resultant reef fish BCG provides an effective tool for identifying healthy and degraded coral reef ecosystems and has potential for global application. • An integrated ecologically-broad systems approach enhances coral reef ecosystem protection. • Biological Condition Gradients track ecosystem status as a function of anthropogenic stress. • The reef fish BGC model was effective in identification of impaired coral reef ecosystems • The reef fish BGC model has potential global application when calibrated with local data. [ABSTRACT FROM AUTHOR]

Understanding factors associated with varying efficacy of Bacillus Calmette-Guérin (BCG) would aid the development of improved vaccines against tuberculosis (TB). In addition, investigation of individual-level factors affecting mycobacterial-specific immune responses could provide insight into confounders of vaccine efficacy in clinical trials. Mycobacterial growth inhibition assays (MGIA) have been developed to assess vaccine immunogenicity ex vivo and provide a measure of immune function against live mycobacteria. In this study, we assessed the impact of immune cell phenotype, cytomegalovirus (CMV)-specific response and sex on ex vivo growth inhibition following historical BCG vaccination in a cohort of healthy individuals (n = 100). A higher frequency of cytokine-producing NK cells in peripheral blood was associated with enhanced ex vivo mycobacterial growth inhibition following historical BCG vaccination. A CMV-specific response was associated with T-cell activation, a risk factor for TB disease and we also observed an association between T-cell activation and ex vivo mycobacterial growth. Interestingly, BCG-vaccinated females in our cohort controlled mycobacterial growth better than males. In summary, our present study has shown that individual-level factors influence capacity to control mycobacterial growth following BCG vaccination and the MGIA could be used as a tool to assess how vaccine candidates may perform in different populations. [ABSTRACT FROM AUTHOR]

Background In patients with severe eosinophilic asthma, local maturation rather than systemic recruitment of mature cells might contribute to persistent airway eosinophilia. Group 2 innate lymphoid cells (ILC2s) are a major source of type 2 cytokines (IL-5 and IL-13) and can facilitate eosinophilic inflammatory responses in mouse models of asthma in the absence of CD4 + lymphocytes. This study investigated the potential role of ILC2s in driving chronic airway eosinophilia in patients with severe asthma, despite regular high-dose oral corticosteroid therapy. Methods In a cross-sectional study we enumerated blood and sputum ILC2s (lin − CD45 + 127 + ST2 + ) and levels of intracellular IL-5 and IL-13 in patients with severe asthma (n = 25), patients with steroid-naive mild atopic asthma (n = 19), and nonatopic control subjects (n = 5). Results were compared with numbers of CD4 + lymphocytes, eosinophil lineage–committed progenitors (eosinophilopoietic progenitor cells [EoPs]), and mature eosinophils. Results Significantly greater numbers of total and type 2 cytokine–producing ILC2s were detected in blood and sputum of patients with severe asthma compared to mild asthmatics. In contrast, intracellular cytokine expression by CD4 cells and EoPs within the airways did not differ between the asthmatic groups. In patients with severe asthma, although sputum CD4 + cells were more abundant than ILC2s and EoPs, proportionally, ILC2s were the predominant source of type 2 cytokines. In addition, there were significantly greater numbers of sputum IL-5 + IL-13 + ILC2s in patients with severe asthma whose airway eosinophilia was greater than 3%, despite normal blood eosinophil numbers (<300/μL). Conclusions Our findings suggest that ILC2s can promote the persistence of airway eosinophilia in patients with severe asthma through uncontrolled localized production of the type 2 cytokines IL-5 and IL-13, despite high-dose oral corticosteroid therapy. [ABSTRACT FROM AUTHOR]