3,532 research outputs found

    Characterising Post-mortem Bacterial Translocation Under Clinical Conditions Using 16S rRNA Gene Sequencing in Two Animal Models

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    Sudden unexpected death in infancy (SUDI) is the sudden and unexpected death of an apparently healthy infant occurring within the first year of life where the cause is not immediately obvious. It is believed that a proportion of unexplained infant deaths are due to an infection that remains undiagnosed. The interpretation of post-mortem microbiology results is difficult due to the potential false-positives, a source of which is post-mortem bacterial translocation. Post-mortem bacterial translocation is the spread of viable bacteria from highly colonised sites to extra-intestinal tissues. We hypothesise that although post-mortem bacterial translocation occurs, when carcasses are kept under controlled routine clinical conditions it is not extensive and can be defined using 16S rRNA gene sequencing. With this knowledge, implementation of the 16S rRNA gene sequencing technique into routine clinical diagnostics would allow a more reliable retrospective diagnosis of ante-mortem infection. Therefore, the aim of this study was to establish the extent of post-mortem bacterial translocation in two animal models to establish a baseline sequencing signal for the post-mortem process. To do this we used 16S rRNA gene sequencing in two animal models over a 2 week period to investigate (1) the bacterial community succession in regions of high bacterial colonisation, and (2) the bacterial presence in visceral tissues routinely sampled during autopsy for microbiological investigation. We found no evidence for significant and consistent post-mortem bacterial translocation in the mouse model. Although bacteria were detected in tissues in the piglet model, we did not find significant and consistent evidence for post-mortem bacterial translocation from the gastrointestinal tract or nasal cavity. These data do not support the concept of significant post-mortem translocation as part of the normal post-mortem process

    Impaired function of endothelial progenitor cells in children with primary systemic vasculitis

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    INTRODUCTION: Previously, we demonstrated that children with active systemic vasculitis (SV) have higher circulating CD34 + CD133 + KDR+ endothelial progenitor cells (EPC); the function of these EPCs, and their relationship with disease activity in vasculitis remains largely unexplored. We hypothesized that although EPC numbers are higher, EPC function is impaired in active SV of the young. The aims of this study were therefore to: 1. investigate the relationship between disease activity and EPC function in children with SV; and 2. study the influence of systemic inflammation on EPC function by investigating the effects of hyperthermia and TNF-α on EPC function. METHODS: We performed a cross-sectional study of unselected children with SV with different levels of disease activity attending a single center (Great Ormond Street Hospital, London) between October 2008 and December 2014. EPCs were isolated from peripheral blood of children with SV, and healthy child controls. EPC function was assessed by their potential to form colonies (EPC-CFU), and ability to form clusters and incorporate into human umbilical vein endothelial cell (HUVEC) vascular structures in matrigel. The effects of hyperthermia and TNF-α on EPC function were also studied. RESULTS: Twenty children, median age 12-years (5-16.5; nine males) were studied. EPC-CFU and the number of EPC clusters formed on matrigel were significantly reduced in children with active vasculitis compared with healthy controls (p = 0.02 for EPC-CFU; p = 0.01 for EPC cluster formation). Those with active vasculitis had lower EPC-CFU and EPC cluster formation than those with inactive disease, although non-significantly so. In addition, EPC incorporation into matrigel HUVEC networks was lower in children with SV compared with healthy children, irrespective of disease activity. Ex-vivo pre-treatment of EPC with hyperthermia impaired EPC function; TNF-α down-regulated EPC expression of CD18/CD11b and resulted in decreased incorporation into HUVEC networks. CONCLUSIONS: Whilst our previous work showed that circulating CD34 + EPC numbers are well preserved, this study revealed that EPC function is significantly impaired in children with vasculitis. It is possible that the chronic inflammatory milieu associated with vasculitis may impair EPC function, and thus contribute to an unfavourable balance between endothelial injury and repair. The mechanism of this remains to be established, however

    Paediatric Behçet's disease: a UK tertiary centre experience

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    There are currently limited data regarding paediatric Behçet's disease (BD), particularly in the UK. We describe the clinical spectrum, treatment and outcome of BD, and explore the relative sensitivities of the criteria for the diagnosis of BD in a UK paediatric cohort. Single retrospective case note review of children with a clinical diagnosis of BD presenting between 1987 and 2012. Demographics, clinical features, treatment and outcomes were recorded. The sensitivities of the International Study Group (ISG) and International Criteria for BD (ICBD) criteria were explored. BD disease activity was calculated using the Behçet's Disease Activity Index (BDAI). Forty-six patients (22 male) were identified. Median age of onset was 4.87 (0.04-15.71) years; median time to diagnosis was 3.74 (0.25-13.48) years. Clinical features were recurrent oral ulceration (97.8 %), recurrent genital ulceration (73.9 %), gastrointestinal (58.7 %), musculoskeletal (47.83 %), cutaneous (23.9 %) involvement and uveitis (2 %). Recurrent genital ulceration was more common in female patients (P = 0.044). Thirty-seven patients (80.4 %) fulfilled the ICBD criteria; only 12 patients (26.1 %) fulfilled the ISG criteria. BDAI score at diagnosis was 7/20 (0-10/20) and significantly decreased to 5/20 (0-9/20) (P < 0.0001) at latest follow-up. The commonest systemic treatment was colchicine (76.1 %); anti-TNFα treatment was reserved for severe cases (15.5 %). Paediatric BD in the UK may present very early in life, sometimes with a family history, and with a low incidence of ocular involvement. Diagnostic delay is common. The majority of our patients required systemic therapy; anti-TNFα was reserved for severe cases and has largely superseded the use of thalidomide

    Shallow vs deep learning architectures for white matter lesion segmentation in the early stages of multiple sclerosis

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    In this work, we present a comparison of a shallow and a deep learning architecture for the automated segmentation of white matter lesions in MR images of multiple sclerosis patients. In particular, we train and test both methods on early stage disease patients, to verify their performance in challenging conditions, more similar to a clinical setting than what is typically provided in multiple sclerosis segmentation challenges. Furthermore, we evaluate a prototype naive combination of the two methods, which refines the final segmentation. All methods were trained on 32 patients, and the evaluation was performed on a pure test set of 73 cases. Results show low lesion-wise false positives (30%) for the deep learning architecture, whereas the shallow architecture yields the best Dice coefficient (63%) and volume difference (19%). Combining both shallow and deep architectures further improves the lesion-wise metrics (69% and 26% lesion-wise true and false positive rate, respectively).Comment: Accepted to the MICCAI 2018 Brain Lesion (BrainLes) worksho

    A critical role for ATF2 transcription factor in the regulation of E-selectin expression in response to non-endotoxin components of Neisseria meningitidis

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    Vascular injury is a serious complication of sepsis due to the gram-negative bacterium Neisseria meningitidis. One of the critical early steps in initiating this injury is via the interaction of leucocytes, particularly neutrophils, with adhesion molecules expressed on inflamed endothelium. We have previously demonstrated that both lipopolysaccharide (LPS) and non-LPS components of meningococci can induce very high levels of expression of the vascular endothelial cell adhesion molecule E-selectin, which is critical for early tethering and capture of neutrophils onto endothelium under flow. Using an LPS-deficient strain of meningococcus, we showed that very high levels of expression can be induced in primary endothelial cells, even in the context of weak activation of the major host signal transduction factor [nuclear factor-κB (NF-κB)]. In this study, we show that the particular propensity for N. meningitidis to induce high levels of expression is regulated at a transcriptional level, and demonstrate a significant role for phosphorylation of the ATF2 transcription factor, likely via mitogen-activated protein (MAP) kinases, on the activity of the E-selectin promoter. Furthermore, inhibition of E-selectin expression in response to the lpxA- strain by a p38 inhibitor indicates a significant role of a p38-dependent MAPK signalling pathway in ATF2 activation. Collectively, these data highlight the role that LPS and other bacterial components have in modulating endothelial function and their involvement in the pathogenesis of meningococcal sepsis. Better understanding of these multiple mechanisms induced by complex stimuli such as bacteria, and the specific inflammatory pathways they activate, may lead to improved, focused interventions in both meningococcal and potentially bacterial sepsis more generally

    N-acetylation and phosphorylation of Sec complex subunits in the ER membrane.

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    BACKGROUND: Covalent modifications of proteins provide a mechanism to control protein function. Here, we have investigated modifications of the heptameric Sec complex which is responsible for post-translational protein import into the endoplasmic reticulum (ER). It consists of the Sec61 complex (Sec61p, Sbh1p, Sss1p) which on its own mediates cotranslational protein import into the ER and the Sec63 complex (Sec63p, Sec62p, Sec71p, Sec72p). Little is known about the biogenesis and regulation of individual Sec complex subunits. RESULTS: We show that Sbh1p when it is part of the Sec61 complex is phosphorylated on T5 which is flanked by proline residues. The phosphorylation site is conserved in mammalian Sec61ß, but only partially in birds, and not in other vertebrates or unicellular eukaryotes, suggesting convergent evolution. Mutation of T5 to A did not affect the ability of mutant Sbh1p to complement the growth defect in a Δsbh1Δsbh2 strain, and did not result in a hypophosphorylated protein which shows that alternate sites can be used by the T5 kinase. A survey of yeast phosphoproteome data shows that Sbh1p can be phosphorylated on multiple sites which are organized in two patches, one at the N-terminus of its cytosolic domain, the other proximal to the transmembrane domain. Surprisingly, although N-acetylation has been shown to interfere with ER targeting, we found that both Sbh1p and Sec62p are cotranslationally N-acetylated by NatA, and N-acetyl-proteome data indicate that Sec61p is modified by the same enzyme. Mutation of the N-acetylation site, however, did not affect Sec62p function in posttranslational protein import into the ER. Disabling NatA resulted in growth retardation, but not in co- or posttranslational translocation defects or instability of Sec62p or Sbh1p. CONCLUSIONS: We conclude that N-acetylation of transmembrane and tail-anchored proteins does not interfere with their ER-targeting, and that Sbh1p phosphorylation on T5, which is not present in Sbh2p, plays a non-essential role specific to the Sec61 complex.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

    Perinatal risk factors for neonatal encephalopathy: an unmatched case-control study

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    OBJECTIVE: Neonatal encephalopathy (NE) is the third leading cause of child mortality. Preclinical studies suggest infection and inflammation can sensitise or precondition the newborn brain to injury. This study examined perinatal risks factor for NE in Uganda. DESIGN: Unmatched case-control study. SETTING: Mulago National Referral Hospital, Kampala, Uganda. METHODS: 210 term infants with NE and 409 unaffected term infants as controls were recruited over 13 months. Data were collected on preconception, antepartum and intrapartum exposures. Blood culture, species-specific bacterial real-time PCR, C reactive protein and placental histology for chorioamnionitis and funisitis identified maternal and early newborn infection and inflammation. Multivariable logistic regression examined associations with NE. RESULTS: Neonatal bacteraemia (adjusted OR (aOR) 8.67 (95% CI 1.51 to 49.74), n=315) and histological funisitis (aOR 11.80 (95% CI 2.19 to 63.45), n=162) but not chorioamnionitis (aOR 3.20 (95% CI 0.66 to 15.52), n=162) were independent risk factors for NE. Among encephalopathic infants, neonatal case fatality was not significantly higher when exposed to early neonatal bacteraemia (OR 1.65 (95% CI 0.62 to 4.39), n=208). Intrapartum antibiotic use did not improve neonatal survival (p=0.826). After regression analysis, other identified perinatal risk factors (n=619) included hypertension in pregnancy (aOR 3.77), male infant (aOR 2.51), non-cephalic presentation (aOR 5.74), lack of fetal monitoring (aOR 2.75), augmentation (aOR 2.23), obstructed labour (aOR 3.8) and an acute intrapartum event (aOR 8.74). CONCLUSIONS: Perinatal infection and inflammation are independent risk factors for NE in this low-resource setting, supporting a role in the aetiological pathway of term brain injury. Intrapartum antibiotic administration did not mitigate against adverse outcomes. The importance of intrapartum risk factors in this sub-Saharan African setting is highlighted

    Next-generation cell line selection methodology leveraging data lakes, natural language generation and advanced data analytics

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    Cell line development is an essential stage in biopharmaceutical development that often lies on the critical path. Failure to fully characterise the lead clone during initial screening can lead to lengthy project delays during scale-up, which can potentially compromise commercial manufacturing success. In this study, we propose a novel cell line development methodology, referenced as CLD4, which involves four steps enabling autonomous data-driven selection of the lead clone. The first step involves the digitalisation of the process and storage of all available information within a structured data lake. The second step calculates a new metric referenced as the cell line manufacturability index (MICL) quantifying the performance of each clone by considering the selection criteria relevant to productivity, growth and product quality. The third step implements machine learning (ML) to identify any potential risks associated with process operation and relevant critical quality attributes (CQAs). The final step of CLD4 takes into account the available metadata and summaries all relevant statistics generated in steps 1–3 in an automated report utilising a natural language generation (NLG) algorithm. The CLD4 methodology was implemented to select the lead clone of a recombinant Chinese hamster ovary (CHO) cell line producing high levels of an antibody-peptide fusion with a known product quality issue related to end-point trisulfide bond (TSB) concentration. CLD4 identified sub-optimal process conditions leading to increased levels of trisulfide bond that would not be identified through conventional cell line development methodologies. CLD4 embodies the core principles of Industry 4.0 and demonstrates the benefits of increased digitalisation, data lake integration, predictive analytics and autonomous report generation to enable more informed decision making

    Specific Immunity to Cytomegalovirus in Pediatric Cardiac Transplantation

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    BACKGROUND: Cytomegalovirus (CMV) infection is implicated in endothelial dysfunction and graft damage after pediatric heart transplantation. CMV specific immune responses are thought to be necessary for CMV viral control but there is little data in pediatric heart transplantation. METHODS: We studied 28 consecutive pediatric heart transplant recipients for 1-year posttransplant. CMV-specific T cells expressing IFN-γ, TNF-α and IL-2 in response to ex-vivo stimulation with CMV lysates or peptides were measured. Circulating cytokines were measured in plasma. Generalised Additive Models were applied to the data to model changes of cell population dynamics over time. RESULTS: CMV-specific T cell mediated responses were impaired in the first 8 weeks posttransplant. During this period, 25% of patients had CMV viremia, of which those with viral loads ≥10,000 CMV DNA copies/mL were given ganciclovir. In this group, the frequency of CD4+ and CD8+ T cells producing IFN-γ and the CD8+CD57+GB+ T cell population increased at 12-24 weeks and remained elevated for the duration of the study. CONCLUSIONS: We have shown that CMV viremia is associated with CMV specific immune responses and increased CD8+CD57+GB+ cells at 1-year posttransplant, however early responses were not predictive of impending CMV viremia. It remains to be seen if the early CMV immune response detected is associated with endothelial and allograft damage, in light of previous studies demonstrating increased vasculopathy in pediatric patients with CMV viremia
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