World TB Day 2018: The Challenge of Drug Resistant Tuberculosis.

On 24th March, the world commemorates the day in 1882 when Dr Robert Koch announced his discovery of Mycobacterium tuberculosis (MTB). Over 130 years later, tuberculosis (TB) continues to affect individuals, communities, and entire health systems and economies. Koch unsuccessfully tried to 'cure' TB, and despite major advances in other areas of medicine, control of TB remains elusive- in 2016 TB was the leading infectious cause of death. The STOP TB partnership and World Health Organization (WHO) have announced their theme for World TB Day 2018 "Wanted: Leaders for a TB-Free World. You can make history. End TB." This theme recognizes that TB is much larger than any one person, institute or discipline of research, and provides an opportunity for us to reflect on the major challenges and consider how we, as a scientific community, can work together and take the lead to address the global crisis of drug-resistant TB (DR-TB)

Analysis tools to quantify dissemination of pathology in zebrafish larvae

We describe new open source software called QuantiFish for rapid quantitation of fluorescent foci in zebrafish larvae, to support infection research in this animal model. QuantiFish extends the conventional measurements of bacterial load and number of bacterial foci to include measures for dissemination of infection. These are represented by the proportions of bacteria between foci and their spatial distribution. We showcase these measures by comparison of intravenous and hindbrain routes of Mycobacterium marinum infection, which are indistinguishable by measurement of bacterial load and not consistently differentiated by the number of bacterial foci. The intravenous route showed dose dependent dissemination of infection, reflected by increased spatial dispersion of bacteria and lower proportions of bacteria distributed across many foci. In contrast, hindbrain infection resulted in localised disease, limited to a smaller area and higher proportions of bacteria distributed across fewer foci. The application of QuantiFish may extend beyond models of infection, to study other pathologies such as metastatic cancer

Brands in international and multi‐platform expansion strategies: economic and management issues

Powerful media branding has historically facilitated successful international expansion on the part of magazine and other content forms including film and TV formats. Multi-platform expansion is now increasingly central to the strategies of media companies and, as this chapter argues, effective use of branding in order to engage audiences effectively and to secure a prominent presence across digital platforms forms a core part of this. Drawing on original research into the experience of UK media companies, this chapter highlights some of the key economic, management and socio-cultural issues raised by the ever-increasing role of brands and branding in the strategies of international and multi-platform expansion that are increasingly common- place across media

Shigella sonnei infection of zebrafish reveals that O-antigen mediates neutrophil tolerance and dysentery incidence.

Funder: Lister Institute of Preventive Medicine; funder-id: http://dx.doi.org/10.13039/501100001255Shigella flexneri is historically regarded as the primary agent of bacillary dysentery, yet the closely-related Shigella sonnei is replacing S. flexneri, especially in developing countries. The underlying reasons for this dramatic shift are mostly unknown. Using a zebrafish (Danio rerio) model of Shigella infection, we discover that S. sonnei is more virulent than S. flexneri in vivo. Whole animal dual-RNAseq and testing of bacterial mutants suggest that S. sonnei virulence depends on its O-antigen oligosaccharide (which is unique among Shigella species). We show in vivo using zebrafish and ex vivo using human neutrophils that S. sonnei O-antigen can mediate neutrophil tolerance. Consistent with this, we demonstrate that O-antigen enables S. sonnei to resist phagolysosome acidification and promotes neutrophil cell death. Chemical inhibition or promotion of phagolysosome maturation respectively decreases and increases neutrophil control of S. sonnei and zebrafish survival. Strikingly, larvae primed with a sublethal dose of S. sonnei are protected against a secondary lethal dose of S. sonnei in an O-antigen-dependent manner, indicating that exposure to O-antigen can train the innate immune system against S. sonnei. Collectively, these findings reveal O-antigen as an important therapeutic target against bacillary dysentery, and may explain the rapidly increasing S. sonnei burden in developing countries

Relative Contributions of Extracellular and Internalized Bacteria to Early Macrophage Proinflammatory Responses to Streptococcus pneumoniae.

Both intracellular immune sensing and extracellular innate immune sensing have been implicated in initiating macrophage proinflammatory cytokine responses to Streptococcus pneumoniae The S. pneumoniae capsule, a major virulence determinant, prevents phagocytosis, and we hypothesized that this would reduce activation of host innate inflammatory responses by preventing activation of intracellular proinflammatory signaling pathways. We investigated this hypothesis in human monocyte-derived macrophages stimulated with encapsulated or isogenic unencapsulated mutant S. pneumoniae Unexpectedly, despite strongly inhibiting bacterial internalization, the capsule resulted in enhanced inflammatory cytokine production by macrophages infected with S. pneumoniae Experiments using purified capsule material and a Streptococcus mitis mutant expressing an S. pneumoniae serotype 4 capsule indicated these differences required whole bacteria and were not due to proinflammatory effects of the capsule itself. Transcriptional profiling demonstrated relatively few differences in macrophage gene expression profiles between infections with encapsulated S. pneumoniae and those with unencapsulated S. pneumoniae, largely limited to reduced expression of proinflammatory genes in response to unencapsulated bacteria, predicted to be due to reduced activation of the NF-κB family of transcription factors. Blocking S. pneumoniae internalization using cytochalasin D had minimal effects on the inflammatory response to S. pneumoniae Experiments using murine macrophages indicated that the affected genes were dependent on Toll-like receptor 2 (TLR2) activation, although not through direct stimulation of TLR2 by capsule polysaccharide. Our data demonstrate that the early macrophage proinflammatory response to S. pneumoniae is mainly dependent on extracellular bacteria and reveal an unexpected proinflammatory effect of encapsulated S. pneumoniae that could contribute to disease pathogenesis.IMPORTANCE Multiple extra- and intracellular innate immune receptors have been identified that recognize Streptococcus pneumoniae, but the relative contributions of intra- versus extracellular bacteria to the inflammatory response were unknown. We have shown that intracellular S. pneumoniae contributes surprisingly little to the inflammatory responses, with production of important proinflammatory cytokines largely dependent on extracellular bacteria. Furthermore, although we expected the S. pneumoniae polysaccharide capsule to block activation of the host immune system by reducing bacterial internalization and therefore activation of intracellular innate immune receptors, there was an increased inflammatory response to encapsulated compared to unencapsulated bacteria, which is likely to contribute to disease pathogenesis.This work was supported by grants from the Medical Research Council, UK: MR/K00168X/1 (to J.P.), G0700569 (to T.P.), G0600410 (to E.C.), and G0801211 (to G.T.) and Wellcome Trust grant WT076442 (to S.C.). C.H. received support from the Astor Foundation and GlaxoSmithKline through the University College London MBChB program. This work was undertaken at UCLH/UCL, which received a proportion of funding from the Department of Health’s NIHR Biomedical Research Centre’s funding scheme

In vivo molecular dissection of the effects of HIV-1 in active tuberculosis

Author Summary HIV-1 infected people have substantially increased risk of tuberculosis (TB) leading to a large burden of disease worldwide. We aimed to investigate how HIV-1 causes this effect by altering human immune responses. We measured the products of all immune genes at injection sites of sterilized TB under the skin, in order to look for differences between TB patients with and without HIV-1. We found that the predominant effect of early HIV-1 infection was to diminish a component of immune responses that contributes to prevention of harmful inflammation. In more advanced HIV-1, we found almost complete absence of any immune response to TB except for immune activity which is normally part of our defence against viruses, but may also weaken immune protection against TB. In some patients, TB becomes apparent after starting treatment for HIV-1. In these patients we found that most immune responses had recovered to normal levels, but that one type of response sometimes associated with asthma and allergies was exaggerated. Our findings provide new insights into how HIV-1 can affect immune responses and changes to the immune system that are associated with risk of TB, which will inform the development of new strategies to improve protective immunity

Characterization and applications of a Crimean-Congo hemorrhagic fever virus nucleoprotein-specific Affimer: Inhibitory effects in viral replication and development of colorimetric diagnostic tests.

peer reviewedCrimean-Congo hemorrhagic fever orthonairovirus (CCHFV) is one of the most widespread medically important arboviruses, causing human infections that result in mortality rates of up to 60%. We describe the selection of a high-affinity small protein (Affimer-NP) that binds specifically to the nucleoprotein (NP) of CCHFV. We demonstrate the interference of Affimer-NP in the RNA-binding function of CCHFV NP using fluorescence anisotropy, and its inhibitory effects on CCHFV gene expression in mammalian cells using a mini-genome system. Solution of the crystallographic structure of the complex formed by these two molecules at 2.84 Å resolution revealed the structural basis for this interference, with the Affimer-NP binding site positioned at the critical NP oligomerization interface. Finally, we validate the in vitro application of Affimer-NP for the development of enzyme-linked immunosorbent and lateral flow assays, presenting the first published point-of-care format test able to detect recombinant CCHFV NP in spiked human and animal sera

Rapid synchronous type 1 IFN and virus-specific T cell responses characterize first wave non-severe SARS-CoV-2 infections

Effective control of SARS-CoV-2 infection on primary exposure may reveal correlates of protective immunity to future variants, but we lack insights into immune responses before or at the time virus is first detected. We use blood transcriptomics, multiparameter flow cytometry, and T cell receptor (TCR) sequencing spanning the time of incident non-severe infection in unvaccinated virus-naive individuals to identify rapid type 1 interferon (IFN) responses common to other acute respiratory viruses and cell proliferation responses that discriminate SARS-CoV-2 from other viruses. These peak by the time the virus is first detected and sometimes precede virus detection. Cell proliferation is most evident in CD8 T cells and associated with specific expansion of SARS-CoV-2-reactive TCRs, in contrast to virus-specific antibodies, which lag by 1–2 weeks. Our data support a protective role for early type 1 IFN and CD8 T cell responses, with implications for development of universal T cell vaccines

Adherent Human Alveolar Macrophages Exhibit a Transient Pro-Inflammatory Profile That Confounds Responses to Innate Immune Stimulation

Alveolar macrophages (AM) are thought to have a key role in the immunopathogenesis of respiratory diseases. We sought to test the hypothesis that human AM exhibit an anti-inflammatory bias by making genome-wide comparisons with monocyte derived macrophages (MDM). Adherent AM obtained by bronchoalveolar lavage of patients under investigation for haemoptysis, but found to have no respiratory pathology, were compared to MDM from healthy volunteers by whole genome transcriptional profiling before and after innate immune stimulation. We found that freshly isolated AM exhibited a marked pro-inflammatory transcriptional signature. High levels of basal pro-inflammatory gene expression gave the impression of attenuated responses to lipopolysaccharide (LPS) and the RNA analogue, poly IC, but in rested cells pro-inflammatory gene expression declined and transcriptional responsiveness to these stimuli was restored. In comparison to MDM, both freshly isolated and rested AM showed upregulation of MHC class II molecules. In most experimental paradigms ex vivo adherent AM are used immediately after isolation. Therefore, the confounding effects of their pro-inflammatory profile at baseline need careful consideration. Moreover, despite the prevailing view that AM have an anti-inflammatory bias, our data clearly show that they can adopt a striking pro-inflammatory phenotype, and may have greater capacity for presentation of exogenous antigens than MDM

Effects of antiplatelet therapy on stroke risk by brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases: subgroup analyses of the RESTART randomised, open-label trial

Background Findings from the RESTART trial suggest that starting antiplatelet therapy might reduce the risk of recurrent symptomatic intracerebral haemorrhage compared with avoiding antiplatelet therapy. Brain imaging features of intracerebral haemorrhage and cerebral small vessel diseases (such as cerebral microbleeds) are associated with greater risks of recurrent intracerebral haemorrhage. We did subgroup analyses of the RESTART trial to explore whether these brain imaging features modify the effects of antiplatelet therapy