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Tuberculosis is a major global health problem and is one of the top 10 causes of death worldwide. There is a pressing need for new treatments that circumvent emerging antibiotic resistance. Mycobacterium tuberculosis parasitises macrophages, reprogramming them to establish a niche in which to proliferate, therefore macrophage manipulation is a potential host-directed therapy if druggable molecular targets could be identified. The pseudokinase Tribbles1 (Trib1) regulates multiple innate immune processes and inflammatory profiles making it a potential drug target in infections. Trib1 controls macrophage function, cytokine production, and macrophage polarisation. Despite wide-ranging effects on leukocyte biology, data exploring the roles of Tribbles in infection in vivo are limited. Here, we identify that human Tribbles1 is expressed in monocytes and is upregulated at the transcript level after stimulation with mycobacterial antigen. To investigate the mechanistic roles of Tribbles in the host response to mycobacteria in vivo, we used a zebrafish Mycobacterium marinum (Mm) infection tuberculosis model. Zebrafish Tribbles family members were characterised and shown to have substantial mRNA and protein sequence homology to their human orthologues. trib1 overexpression was host-protective against Mm infection, reducing burden by approximately 50%. Conversely, trib1 knockdown/knockout exhibited increased infection. Mechanistically, trib1 overexpression significantly increased the levels of proinflammatory factors il-1β and nitric oxide. The host-protective effect of trib1 was found to be dependent on the E3 ubiquitin kinase Cop1. These findings highlight the importance of Trib1 and Cop1 as immune regulators during infection in vivo and suggest that enhancing macrophage TRIB1 levels may provide a tractable therapeutic intervention to improve bacterial infection outcomes in tuberculosis.

Our hospital replaced the format for delivering portable antimicrobial prescribing guidance from a paper-based pocket guide to a smartphone application (app). We used this opportunity to assess the relationship between its use and the attitudes and behaviours of antimicrobial prescribers. We used 2 structured cross-sectional questionnaires issued just prior to and 3 months following the launch of the smartphone app. Ordinal Likert scale responses to both frequencies of use and agreement statements permitted quantitative assessment of the relationship between variables. The smartphone app was used more frequently than the pocket guide it replaced (p < 0.01), and its increased use was associated with sentiments that the app was useful, easy to navigate and its content relevant. Users who used the app more frequently were more likely to agree that the app encouraged them to challenge inappropriate prescribing by their colleagues (p = 0.001) and were more aware of the importance of antimicrobial stewardship (p = 0.005). Reduced use of the app was associated with agreement that senior physicians' preferences for antimicrobial prescribing would irrespectively overrule guideline recommendations (p = 0.0002). Smartphone apps are an effective and acceptable format to deliver guidance on antimicrobial prescribing. Our findings suggest that they may empower users to challenge incorrect prescribing, breaking well-established behaviours, and thus supporting vital stewardship efforts in an era of increased antimicrobial resistance. Future work will need to focus on the direct impact on drug prescriptions as well as identifying barriers to implementing smartphone apps in other clinical settings.

T cells contribute to immune protection and pathogenesis in tuberculosis, but measurements of polyclonal responses have failed to resolve correlates of outcome. We report the temporal evaluation of the human in vivo clonal repertoire of Mycobacterium tuberculosis ( Mtb )-reactive T cell responses, by T cell receptor (TCR) sequencing at the site of the tuberculin skin test, as a model for a standardised antigenic challenge. Initial non-selective recruitment of T cells is followed by enrichment of Mtb -reactive clones arising from oligoclonal T cell proliferation. We introduce a modular computational pipeline, Metaclonotypist, to sensitively cluster distinct TCRs with shared epitope specificity, which we apply here to establish a catalogue of public Mtb -reactive HLA-restricted T cell metaclones. Although most in vivo Mtb -reactive T cells are private, 10 metaclones were sufficient to identify Mtb -T cell reactivity across our study population (N≥128), indicating striking population level immunodominance of specific TCR-peptide interactions that may inform patient stratification and vaccine development. T cells contribute to protection and pathogenesis in tuberculosis. Here the authors sequence T cell receptor repertoires in human skin biopsies from the site of the tuberculin skin test and show enrichment of clonotypes reactive to Mycobacterium tuberculosis using a computational pipeline metaclonotypist to identify distinct TCRs predicted to share peptide-MHC reactivity across participants, as an approach to explore T cell correlates of tuberculosis disease-risk stratification and vaccine efficacy.

In people living with HIV (PLHIV), we sought to test the hypothesis that long term anti-retroviral therapy restores the normal T cell repertoire, and investigate the functional relationship of residual repertoire abnormalities to persistent immune system dysregulation. We conducted a case-control study in PLHIV and HIV-negative volunteers, of circulating T cell receptor repertoires and whole blood transcriptomes by RNA sequencing, complemented by metadata from routinely collected health care records. T cell receptor sequencing revealed persistent abnormalities in the clonal T cell repertoire of PLHIV, characterized by reduced repertoire diversity and oligoclonal T cell expansion correlated with elevated CD8 T cell counts. We found no evidence that these expansions were driven by cytomegalovirus or another common antigen. Increased frequency of long CDR3 sequences and reduced frequency of public sequences among the expanded clones implicated abnormal thymic selection as a contributing factor. These abnormalities in the repertoire correlated with systems level evidence of persistent T cell activation in genome-wide blood transcriptomes. The diversity of T cell receptor repertoires in PLHIV on long term anti-retroviral therapy remains significantly depleted, and skewed by idiosyncratic clones, partly attributable to altered thymic output and associated with T cell mediated chronic immune activation. Further investigation of thymic function and the antigenic drivers of T cell clonal selection in PLHIV are critical to efforts to fully re-establish normal immune function.

The incidence of multi-drug resistant ESBL-associated urinary tract infections (UTIs) is increasing globally. Patients with abnormal renal tract anatomy and other co-morbidities are at increased risk of complicated UTI and ESBL-associated infections. The duration and safety of OPAT for this cohort of patients is unknown. This study aims to provide an evidence base to support decision-making regarding duration of antibiotic treatment for complicated UTIs. We retrospectively reviewed all patients receiving ertapenem with or without adjunctive fosfomycin for complicated UTIs in the OPAT service of our tertiary infectious diseases hospital. All data had been collected prospectively as part of routine clinical care. Our primary outcomes were microbiological and clinical cure of UTI. We identified 33 treatment episodes of ertapenem use for UTIs. 76% episodes related to pyelonephritis or urosepsis diagnoses. Renal tract abnormalities or prior urological surgery were present in 45% of patients. The median duration of appropriate parenteral antibiotic therapy in our study was 6 days. Clinical cure was achieved with short-course parenteral treatment alone in 81% of patients and this increased to 96% when adjunctive fosfomycin was used. There was a single treatment failure resulting in hospital admission. Short duration ertapenem via OPAT with or without adjunctive fosfomycin is safe and effective for the treatment of complicated UTIs. Further studies are required to inform optimal treatment strategies and publication of guidelines in this field.

Increased risk of tuberculosis (TB) associated with HIV-1 infection is primarily attributed to deficient T helper (Th)1 immune responses, but most people with active TB have robust Th1 responses, indicating that these are not sufficient to protect against disease. Recent findings suggest that favourable outcomes following Mycobacterium tuberculosis infection arise from finely balanced inflammatory and regulatory pathways, achieving pathogen control without immunopathology. We hypothesised that HIV-1 and antiretroviral therapy (ART) exert widespread changes to cell mediated immunity, which may compromise the optimal host protective response to TB and provide novel insights into the correlates of immune protection and pathogenesis. We sought to define these effects in patients with active TB by transcriptional profiling of tuberculin skin tests (TST) to make comprehensive molecular level assessments of in vivo human immune responses at the site of a standardised mycobacterial challenge. We showed that the TST transcriptome accurately reflects the molecular pathology at the site of human pulmonary TB, and used this approach to investigate immune dysregulation in HIV-1/TB co-infected patients with distinct clinical phenotypes associated with TST reactivity or anergy and unmasking TB immune reconstitution inflammatory syndrome (IRIS) after initiation of ART. HIV-1 infected patients with positive TSTs exhibited preserved Th1 responses but deficient immunoregulatory IL10-inducible responses. Those with clinically negative TSTs revealed profound anergy of innate as well as adaptive immune responses, except for preservation of type 1 interferon activity, implicated in impaired anti-mycobacterial immunity. Patients with unmasking TB IRIS showed recovery of Th1 immunity to normal levels, but exaggerated Th2-associated responses specifically. These mechanisms of immune dysregulation were localised to the tissue microenvironment and not evident in peripheral blood. TST molecular profiling categorised different mechanisms of immunological dysfunction in HIV-1 infection beyond the effects on CD4 T cells, each associated with increased risk of TB disease and amenable to host-directed therapies.

Numerous gene signatures, or modules have been described to evaluate the immune cell composition in transcriptomes of multicellular tissue samples. However, significant diversity in module gene content for specific cell types is associated with heterogeneity in their performance. In order to rank modules that best reflect their purported association, we have generated the modular discrimination index (MDI) score that assesses expression of each module in the target cell type relative to other cells. We demonstrate that MDI scores predict modules that best reflect independently validated differences in cellular composition, and correlate with the covariance between cell numbers and module expression in human blood and tissue samples. Our analyses demonstrate that MDI scores provide an ordinal summary statistic that reliably ranks the accuracy of gene expression modules for deconvolution of cell type abundance in transcriptional data.

Making hypothesis-led interrogations of whole-genome transcriptomic datasets is a challenge. Transcriptional modules define specific biological processes, but multiple modules have now been published with overlapping annotations. In this study we aimed to generate and validate the modular discrimination index (MDI) score, ranking the accuracy and resolution of any cell type specific module to assess cellular enrichment in tissues from transcriptional datasets. We made use of publicly available transcriptomic datasets of purified immune cell types to compare the expression of modules annotated to reflect the expression of different immune cells. MDI scores were derived from the relative expression of each module in its annotated cell type, relative to all other cells. Independent datasets from human tissue and blood samples were then used to validate the MDI score in relation to the relative enrichment of different modules' gene expression. We found that 26 T-cell modules varied both in their composition and in their gene expression in a tuberculin skin test, a prototypical T cell driven cell inflammatory process. To address this heterogeneity, we assessed the accuracy of cell-type modules, by calculating MDI scores for all published T cell, B cell, natural killer cell and neutrophil modules. MDI scores strongly predicted each module's ability to show histologically proven cellular enrichment in a variety of independent tissue samples: T cells in psoriatic skin, B cells in lung fibrosis, and neutrophils in erythema nodosum leprosum skin lesions. In addition, MDI scores were also validated in relation to changes in cell frequency in blood: modules with the greatest MDI score were most predictive of each module's association with cell numbers for both high frequency cells (neutrophils r2=0·545, p=0·01) and low frequency cells (B cells r2=0·865, p=0·01) in blood. Finally, we showed that modules with the highest MDI score elicited the T cell enrichment seen in the tuberculin skin test, whereas this was most often not detected by low MDI scoring modules. This study shows that MDI scoring provides a robust and reproducible assessment of a transcriptional module's accuracy and resolution, and should be determined before using modular deconvolution of tissue samples. The MDI scoring framework can be extended and applied across a range of disciplines for which annotated modules could be used to interrogate transcriptional datasets. Wellcome Trust.

The impact of anti-tumor necrosis factor (TNF) therapies on inducible TNF-dependent activity in humans has never been evaluated . We aimed to test the hypothesis that patients responding to anti-TNF treatments exhibit attenuated TNF-dependent immune responses at the site of an immune challenge. We developed and validated four context-specific TNF-inducible transcriptional signatures to quantify TNF bioactivity in transcriptomic data. In anti-TNF treated rheumatoid arthritis (RA) patients, we measured the expression of these biosignatures in blood, and in skin biopsies from the site of tuberculin skin tests (TSTs) as a human experimental model of multivariate cell-mediated immune responses. In blood, anti-TNF therapies attenuated TNF bioactivity following stimulation. However, at the site of the TST, TNF-inducible gene expression and genome-wide transcriptional changes associated with cell-mediated immune responses were comparable to that of RA patients receiving methotrexate only. These data demonstrate that anti-TNF agents in RA patients do not inhibit inducible TNF activity at the site of an acute inflammatory challenge , as modeled by the TST. We hypothesize instead that their therapeutic effects are limited to regulating TNF activity in chronic inflammation or by alternative non-canonical pathways.

Herpes Simplex Virus‐1 is a common infectious agent, but the precise detail of entry and infection of cells has only now begun to be clarified. Four viral surface glycoproteins (gB, gD, gH and gL) are required. This review summarises the known structure and function of each of these essential viral envelope glycoproteins, and explores what is known about their close cooperation with each other in mediating cellular membrane fusion. It is suggested that, following gD binding to one of its entry receptors, membrane fusion is mediated by gB and the heterodimer gH/gL. Significantly, these four entry glycoproteins also play a key role in the interaction between HSV and the host immune system. The glycoproteins serve an important role as targets of adaptive immunity. However, recent studies have demonstrated that the same proteins also play a key role in initiating the early innate immune response to HSV. Understanding the complex functions of these HSV proteins may be essential for successful development of vaccines for HSV. Copyright © 2007 John Wiley & Sons, Ltd.