Bioconjugation of Green Fluorescent Protein via an Unexpectedly Stable Cyclic Sulfonium Intermediate

Bioconjugation of superfolder GFP involving the formation of an unusually stable, and unprecedented, cyclic sulfonium species is described. This sulfonium can undergo smooth reaction with a range of nucleophiles to give sulfur-, selenium- and azide-modified GFP derivatives in high conversions

In vitro and in vivo effects of salbutamol on neutrophil function in acute lung injury

Background: Intravenous salbutamol (albuterol) reduces lung water in patients with the acute respiratory distress syndrome (ARDS). Experimental data show that it also reduces pulmonary neutrophil accumulation or activation and inflammation in ARDS. Aim: To investigate the effects of salbutamol on neutrophil function. Methods: The in vitro effects of salbutamol on neutrophil function were determined. Blood and bronchoalveolar lavage (BAL) fluid were collected from 35 patients with acute lung injury (ALI)/ARDS, 14 patients at risk from ARDS and 7 ventilated controls at baseline and after 4 days’ treatment with placebo or salbutamol (ALI/ARDS group). Alveolar–capillary permeability was measured in vivo by thermodilution (PiCCO). Neutrophil activation, adhesion molecule expression and inflammatory cytokines were measured. Results: In vitro, physiological concentrations of salbutamol had no effect on neutrophil chemotaxis, viability or apoptosis. Patients with ALI/ARDS showed increased neutrophil activation and adhesion molecule expression compared with at risk-patients and ventilated controls. There were associations between alveolar– capillary permeability and BAL myeloperoxidase (r = 0.4, p = 0.038) and BAL interleukin 8 (r = 0.38, p = 0.033). In patients with ALI/ARDS, salbutamol increased numbers of circulating neutrophils but had no effect on alveolar neutrophils. Conclusion: At the onset of ALI/ARDS, there is increased neutrophil recruitment and activation. Physiological concentrations of salbutamol did not alter neutrophil chemotaxis, viability or apoptosis in vitro. In vivo, salbutamol increased circulating neutrophils, but had no effect on alveolar neutrophils or on neutrophil activation. These data suggest that the beneficial effects of salbutamol in reducing lung water are unrelated to modulation of neutrophil-dependent inflammatory pathways

Type XVIII collagen degradation products in acute lung injury

Introduction: In acute lung injury, repair of the damaged alveolar-capillary barrier is an essential part of recovery. Endostatin is a 20 to 28 kDa proteolytic fragment of the basement membrane collagen XVIII, which has been shown to inhibit angiogenesis via action on endothelial cells. We hypothesised that endostatin may have a role in inhibiting lung repair in patients with lung injury. The aims of the study were to determine if endostatin is elevated in the plasma/bronchoalveolar lavage fluid of patients with acute lung injury and ascertain whether the levels reflect the severity of injury and alveolar inflammation, and to assess if endostatin changes occur early after the injurious lung stimuli of one lung ventilation and lipopolysaccharide (LPS) challenge. Methods: Endostatin was measured by ELISA and western blotting. Results: Endostatin is elevated within the plasma and bronchoalveolar lavage fluid of patients with acute lung injury. Lavage endostatin reflected the degree of alveolar neutrophilia and the extent of the loss of protein selectivity of the alveolar-capillary barrier. Plasma levels of endostatin correlated with the severity of physiological derangement. Western blotting confirmed elevated type XVIII collagen precursor levels in the plasma and lavage and multiple endostatin-like fragments in the lavage of patients. One lung ventilation and LPS challenge rapidly induce increases in lung endostatin levels. Conclusions: Endostatin may adversely affect both alveolar barrier endothelial and epithelial cells, so its presence within both the circulation and the lung may have a pathophysiological role in acute lung injury that warrants further evaluation

Vitamin D deficiency contributes directly to the acute respiratory distress syndrome (ARDS)

Rationale: Vitamin D deficiency has been implicated as a pathogenic factor in sepsis and intensive therapy unit mortality but has not been assessed as a risk factor for acute respiratory distress syndrome (ARDS). Causality of these associations has never been demonstrated. Objectives: To determine if ARDS is associated with vitamin D deficiency in a clinical setting and to determine if vitamin D deficiency in experimental models of ARDS influences its severity. Methods: Human, murine and in vitro primary alveolar epithelial cell work were included in this study. Findings: Vitamin D deficiency (plasma 25(OH)D levels 600 genes. In a clinical setting, pharmacological repletion of vitamin D prior to oesophagectomy reduced the observed changes of in vivo measurements of alveolar capillary damage seen in deficient patients. Conclusions: Vitamin D deficiency is common in people who develop ARDS. This deficiency of vitamin D appears to contribute to the development of the condition, and approaches to correct vitamin D deficiency in patients at risk of ARDS should be developed

Interferon gamma release assays for Diagnostic Evaluation of Active tuberculosis (IDEA): test accuracy study and economic evaluation

Background Interferon gamma release assays (IGRAs) are blood tests recommended for the diagnosis of tuberculosis (TB) infection. There is currently uncertainty about the role and clinical utility of IGRAs in the diagnostic workup of suspected active TB in routine NHS clinical practice. Objectives To compare the diagnostic accuracy and cost-effectiveness of T-SPOT.TB® (Oxford Immunotec, Abingdon, UK) and QuantiFERON® TB GOLD In-Tube (Cellestis, Carnegie, VIC, Australia) for diagnosis of suspected active TB and to estimate the diagnostic accuracy of second-generation IGRAs. Design Prospective within-patient comparative diagnostic accuracy study. Setting Secondary care. Participants Adults (aged ≥ 16 years) presenting as inpatients or outpatients at 12 NHS hospital trusts in London, Slough, Oxford, Leicester and Birmingham with suspected active TB. Interventions The index tests [T-SPOT.TB and QuantiFERON GOLD In-Tube (QFT-GIT)] and new enzyme-linked immunospot assays utilising novel Mycobacterium tuberculosis antigens (Rv3615c, Rv2654, Rv3879c and Rv3873) were verified against a composite reference standard applied by a panel of clinical experts blinded to IGRA results. Main outcome measures Sensitivity, specificity, predictive values and likelihood ratios were calculated to determine diagnostic accuracy. A decision tree model was developed to calculate the incremental costs and incremental health utilities [quality-adjusted life-years (QALYs)] of changing from current practice to using an IGRA as an initial rule-out test. Results A total of 363 patients had active TB (culture-confirmed and highly probable TB cases), 439 had no active TB and 43 had an indeterminate final diagnosis. Comparing T-SPOT.TB and QFT-GIT, the sensitivities [95% confidence interval (CI)] were 82.3% (95% CI 77.7% to 85.9%) and 67.3% (95% CI 62.1% to 72.2%), respectively, whereas specificities were 82.6% (95% CI 78.6% to 86.1%) and 80.4% (95% CI 76.1% to 84.1%), respectively. T-SPOT.TB was more sensitive than QFT-GIT (relative sensitivity 1.22, 95% CI 1.14 to 1.31; p < 0.001), but the specificities were similar (relative specificity 1.02, 95% CI 0.97 to 1.08; p = 0.3). For both IGRAs the sensitivity was lower and the specificity was higher for human immunodeficiency virus (HIV)-positive than for HIV-negative patients. The most promising novel antigen was Rv3615c. The added value of Rv3615c to T-SPOT.TB was a 9% (95% CI 5% to 12%) relative increase in sensitivity at the expense of specificity, which had a relative decrease of 7% (95% CI 4% to 10%). The use of current IGRA tests for ruling out active TB is unlikely to be considered cost-effective if a QALY was valued at £20,000 or £30,000. For T-SPOT.TB, the probability of being cost-effective for a willingness to pay of £20,000/QALY was 26% and 21%, when patients with indeterminate test results were excluded or included, respectively. In comparison, the QFT-GIT probabilities were 8% and 6%. Although the use of IGRAs is cost saving, the health detriment is large owing to delay in diagnosing active TB, leading to prolonged illness. There was substantial between-patient variation in the tests used in the diagnostic pathway. Limitations The recruitment target for the HIV co-infected population was not achieved. Conclusions Although T-SPOT.TB was more sensitive than QFT-GIT for the diagnosis of active TB, the tests are insufficiently sensitive for ruling out active TB in routine clinical practice in the UK. Novel assays offer some promise. Future work The novel assays require evaluation in distinct clinical settings and in immunosuppressed patient groups. Funding This project was funded by the National Institute for Health Research (NIHR) Health Technology Assessment programme and the NIHR Health Protection Research Unit in Respiratory Infections, Imperial College London, London, UK

The international WAO/EAACI guideline for the management of hereditary angioedema – the 2017 revision and update

Abstract Hereditary Angioedema (HAE) is a rare and disabling disease. Early diagnosis and appropriate therapy are essential. This update and revision of the global guideline for HAE provides up-to-date consensus recommendations for the management of HAE. In the development of this update and revision of the guideline, an international expert panel reviewed the existing evidence and developed 20 recommendations that were discussed, finalized and consented during the guideline consensus conference in June 2016 in Vienna. The final version of this update and revision of the guideline incorporates the contributions of a board of expert reviewers and the endorsing societies. The goal of this guideline update and revision is to provide clinicians and their patients with guidance that will assist them in making rational decisions in the management of HAE with deficient C1-inhibitor (type 1) and HAE with dysfunctional C1-inhibitor (type 2). The key clinical questions covered by these recommendations are: 1) How should HAE-1/2 be defined and classified?, 2) How should HAE-1/2 be diagnosed?, 3) Should HAE-1/2 patients receive prophylactic and/or on-demand treatment and what treatment options should be used?, 4) Should HAE-1/2 management be different for special HAE-1/2 patient groups such as pregnant/lactating women or children?, and 5) Should HAE-1/2 management incorporate self-administration of therapies and patient support measures? This article is co-published with permission in Allergy and the World Allergy Organization Journal

Why Are Outcomes Different for Registry Patients Enrolled Prospectively and Retrospectively? Insights from the Global Anticoagulant Registry in the FIELD-Atrial Fibrillation (GARFIELD-AF).

Background: Retrospective and prospective observational studies are designed to reflect real-world evidence on clinical practice, but can yield conflicting results. The GARFIELD-AF Registry includes both methods of enrolment and allows analysis of differences in patient characteristics and outcomes that may result. Methods and Results: Patients with atrial fibrillation (AF) and ≥1 risk factor for stroke at diagnosis of AF were recruited either retrospectively (n = 5069) or prospectively (n = 5501) from 19 countries and then followed prospectively. The retrospectively enrolled cohort comprised patients with established AF (for a least 6, and up to 24 months before enrolment), who were identified retrospectively (and baseline and partial follow-up data were collected from the emedical records) and then followed prospectively between 0-18 months (such that the total time of follow-up was 24 months; data collection Dec-2009 and Oct-2010). In the prospectively enrolled cohort, patients with newly diagnosed AF (≤6 weeks after diagnosis) were recruited between Mar-2010 and Oct-2011 and were followed for 24 months after enrolment. Differences between the cohorts were observed in clinical characteristics, including type of AF, stroke prevention strategies, and event rates. More patients in the retrospectively identified cohort received vitamin K antagonists (62.1% vs. 53.2%) and fewer received non-vitamin K oral anticoagulants (1.8% vs . 4.2%). All-cause mortality rates per 100 person-years during the prospective follow-up (starting the first study visit up to 1 year) were significantly lower in the retrospective than prospectively identified cohort (3.04 [95% CI 2.51 to 3.67] vs . 4.05 [95% CI 3.53 to 4.63]; p = 0.016). Conclusions: Interpretations of data from registries that aim to evaluate the characteristics and outcomes of patients with AF must take account of differences in registry design and the impact of recall bias and survivorship bias that is incurred with retrospective enrolment. Clinical Trial Registration: - URL: http://www.clinicaltrials.gov . Unique identifier for GARFIELD-AF (NCT01090362)

Improved risk stratification of patients with atrial fibrillation: an integrated GARFIELD-AF tool for the prediction of mortality, stroke and bleed in patients with and without anticoagulation.

OBJECTIVES: To provide an accurate, web-based tool for stratifying patients with atrial fibrillation to facilitate decisions on the potential benefits/risks of anticoagulation, based on mortality, stroke and bleeding risks. DESIGN: The new tool was developed, using stepwise regression, for all and then applied to lower risk patients. C-statistics were compared with CHA2DS2-VASc using 30-fold cross-validation to control for overfitting. External validation was undertaken in an independent dataset, Outcome Registry for Better Informed Treatment of Atrial Fibrillation (ORBIT-AF). PARTICIPANTS: Data from 39 898 patients enrolled in the prospective GARFIELD-AF registry provided the basis for deriving and validating an integrated risk tool to predict stroke risk, mortality and bleeding risk. RESULTS: The discriminatory value of the GARFIELD-AF risk model was superior to CHA2DS2-VASc for patients with or without anticoagulation. C-statistics (95% CI) for all-cause mortality, ischaemic stroke/systemic embolism and haemorrhagic stroke/major bleeding (treated patients) were: 0.77 (0.76 to 0.78), 0.69 (0.67 to 0.71) and 0.66 (0.62 to 0.69), respectively, for the GARFIELD-AF risk models, and 0.66 (0.64-0.67), 0.64 (0.61-0.66) and 0.64 (0.61-0.68), respectively, for CHA2DS2-VASc (or HAS-BLED for bleeding). In very low to low risk patients (CHA2DS2-VASc 0 or 1 (men) and 1 or 2 (women)), the CHA2DS2-VASc and HAS-BLED (for bleeding) scores offered weak discriminatory value for mortality, stroke/systemic embolism and major bleeding. C-statistics for the GARFIELD-AF risk tool were 0.69 (0.64 to 0.75), 0.65 (0.56 to 0.73) and 0.60 (0.47 to 0.73) for each end point, respectively, versus 0.50 (0.45 to 0.55), 0.59 (0.50 to 0.67) and 0.55 (0.53 to 0.56) for CHA2DS2-VASc (or HAS-BLED for bleeding). Upon validation in the ORBIT-AF population, C-statistics showed that the GARFIELD-AF risk tool was effective for predicting 1-year all-cause mortality using the full and simplified model for all-cause mortality: C-statistics 0.75 (0.73 to 0.77) and 0.75 (0.73 to 0.77), respectively, and for predicting for any stroke or systemic embolism over 1 year, C-statistics 0.68 (0.62 to 0.74). CONCLUSIONS: Performance of the GARFIELD-AF risk tool was superior to CHA2DS2-VASc in predicting stroke and mortality and superior to HAS-BLED for bleeding, overall and in lower risk patients. The GARFIELD-AF tool has the potential for incorporation in routine electronic systems, and for the first time, permits simultaneous evaluation of ischaemic stroke, mortality and bleeding risks. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier for GARFIELD-AF (NCT01090362) and for ORBIT-AF (NCT01165710)