Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST–XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at −80°C for at least 60 days. M30-Apoptosense™ plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at −80°C, while at 37°C it had a half-life of 80–100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited

TNF-α is involved in activating DNA fragmentation in skeletal muscle

Intraperitoneal administration of 100 μg kg−1 (body weight) of tumour necrosis factor-α to rats for 8 consecutive days resulted in a significant decrease in protein content, which was concomitant with a reduction in DNA content. Interestingly, the protein/DNA ratio was unchanged in the skeletal muscle of the tumour necrosis factor-α-treated animals as compared with the non-treated controls. Analysis of muscle DNA fragmentation clearly showed enhanced laddering in the skeletal muscle of tumour necrosis factor-α-treated animals, suggesting an apoptotic phenomenon. In a different set of experiments, mice bearing a cachexia-inducing tumour (the Lewis lung carcinoma) showed an increase in muscle DNA fragmentation (9.8-fold) as compared with their non-tumour-bearing control counterparts as previously described. When gene-deficient mice for tumour necrosis factor-α receptor protein I were inoculated with Lewis lung carcinoma, they were also affected by DNA fragmentation; however the increase was only 2.1-fold. These results suggest that tumour necrosis factor-α partly mediates DNA fragmentation during experimental cancer-associated cachexia

The disruption of proteostasis in neurodegenerative diseases

Cells count on surveillance systems to monitor and protect the cellular proteome which, besides being highly heterogeneous, is constantly being challenged by intrinsic and environmental factors. In this context, the proteostasis network (PN) is essential to achieve a stable and functional proteome. Disruption of the PN is associated with aging and can lead to and/or potentiate the occurrence of many neurodegenerative diseases (ND). This not only emphasizes the importance of the PN in health span and aging but also how its modulation can be a potential target for intervention and treatment of human diseases.info:eu-repo/semantics/publishedVersio

Risk of adverse outcomes in patients with underlying respiratory conditions admitted to hospital with COVID-19:a national, multicentre prospective cohort study using the ISARIC WHO Clinical Characterisation Protocol UK

Background Studies of patients admitted to hospital with COVID-19 have found varying mortality outcomes associated with underlying respiratory conditions and inhaled corticosteroid use. Using data from a national, multicentre, prospective cohort, we aimed to characterise people with COVID-19 admitted to hospital with underlying respiratory disease, assess the level of care received, measure in-hospital mortality, and examine the effect of inhaled corticosteroid use. Methods We analysed data from the International Severe Acute Respiratory and emerging Infection Consortium (ISARIC) WHO Clinical Characterisation Protocol UK (CCP-UK) study. All patients admitted to hospital with COVID-19 across England, Scotland, and Wales between Jan 17 and Aug 3, 2020, were eligible for inclusion in this analysis. Patients with asthma, chronic pulmonary disease, or both, were identified and stratified by age (<16 years, 16–49 years, and ≥50 years). In-hospital mortality was measured by use of multilevel Cox proportional hazards, adjusting for demographics, comorbidities, and medications (inhaled corticosteroids, short-acting β-agonists [SABAs], and long-acting β-agonists [LABAs]). Patients with asthma who were taking an inhaled corticosteroid plus LABA plus another maintenance asthma medication were considered to have severe asthma. Findings 75 463 patients from 258 participating health-care facilities were included in this analysis: 860 patients younger than 16 years (74 [8·6%] with asthma), 8950 patients aged 16–49 years (1867 [20·9%] with asthma), and 65 653 patients aged 50 years and older (5918 [9·0%] with asthma, 10 266 [15·6%] with chronic pulmonary disease, and 2071 [3·2%] with both asthma and chronic pulmonary disease). Patients with asthma were significantly more likely than those without asthma to receive critical care (patients aged 16–49 years: adjusted odds ratio [OR] 1·20 [95% CI 1·05–1·37]; p=0·0080; patients aged ≥50 years: adjusted OR 1·17 [1·08–1·27]; p<0·0001), and patients aged 50 years and older with chronic pulmonary disease (with or without asthma) were significantly less likely than those without a respiratory condition to receive critical care (adjusted OR 0·66 [0·60–0·72] for those without asthma and 0·74 [0·62–0·87] for those with asthma; p<0·0001 for both). In patients aged 16–49 years, only those with severe asthma had a significant increase in mortality compared to those with no asthma (adjusted hazard ratio [HR] 1·17 [95% CI 0·73–1·86] for those on no asthma therapy, 0·99 [0·61–1·58] for those on SABAs only, 0·94 [0·62–1·43] for those on inhaled corticosteroids only, 1·02 [0·67–1·54] for those on inhaled corticosteroids plus LABAs, and 1·96 [1·25–3·08] for those with severe asthma). Among patients aged 50 years and older, those with chronic pulmonary disease had a significantly increased mortality risk, regardless of inhaled corticosteroid use, compared to patients without an underlying respiratory condition (adjusted HR 1·16 [95% CI 1·12–1·22] for those not on inhaled corticosteroids, and 1·10 [1·04–1·16] for those on inhaled corticosteroids; p<0·0001). Patients aged 50 years and older with severe asthma also had an increased mortality risk compared to those not on asthma therapy (adjusted HR 1·24 [95% CI 1·04–1·49]). In patients aged 50 years and older, inhaled corticosteroid use within 2 weeks of hospital admission was associated with decreased mortality in those with asthma, compared to those without an underlying respiratory condition (adjusted HR 0·86 [95% CI 0·80−0·92]). Interpretation Underlying respiratory conditions are common in patients admitted to hospital with COVID-19. Regardless of the severity of symptoms at admission and comorbidities, patients with asthma were more likely, and those with chronic pulmonary disease less likely, to receive critical care than patients without an underlying respiratory condition. In patients aged 16 years and older, severe asthma was associated with increased mortality compared to non-severe asthma. In patients aged 50 years and older, inhaled corticosteroid use in those with asthma was associated with lower mortality than in patients without an underlying respiratory condition; patients with chronic pulmonary disease had significantly increased mortality compared to those with no underlying respiratory condition, regardless of inhaled corticosteroid use. Our results suggest that the use of inhaled corticosteroids, within 2 weeks of admission, improves survival for patients aged 50 years and older with asthma, but not for those with chronic pulmonary disease

Importance of patient bed pathways and length of stay differences in predicting COVID-19 hospital bed occupancy in England.

Background: Predicting bed occupancy for hospitalised patients with COVID-19 requires understanding of length of stay (LoS) in particular bed types. LoS can vary depending on the patient’s “bed pathway” - the sequence of transfers of individual patients between bed types during a hospital stay. In this study, we characterise these pathways, and their impact on predicted hospital bed occupancy. Methods: We obtained data from University College Hospital (UCH) and the ISARIC4C COVID-19 Clinical Information Network (CO-CIN) on hospitalised patients with COVID-19 who required care in general ward or critical care (CC) beds to determine possible bed pathways and LoS. We developed a discrete-time model to examine the implications of using either bed pathways or only average LoS by bed type to forecast bed occupancy. We compared model-predicted bed occupancy to publicly available bed occupancy data on COVID-19 in England between March and August 2020. Results: In both the UCH and CO-CIN datasets, 82% of hospitalised patients with COVID-19 only received care in general ward beds. We identified four other bed pathways, present in both datasets: “Ward, CC, Ward”, “Ward, CC”, “CC” and “CC, Ward”. Mean LoS varied by bed type, pathway, and dataset, between 1.78 and 13.53 days. For UCH, we found that using bed pathways improved the accuracy of bed occupancy predictions, while only using an average LoS for each bed type underestimated true bed occupancy. However, using the CO-CIN LoS dataset we were not able to replicate past data on bed occupancy in England, suggesting regional LoS heterogeneities. Conclusions: We identified five bed pathways, with substantial variation in LoS by bed type, pathway, and geography. This might be caused by local differences in patient characteristics, clinical care strategies, or resource availability, and suggests that national LoS averages may not be appropriate for local forecasts of bed occupancy for COVID-19. Trial registration: The ISARIC WHO CCP-UK study ISRCTN66726260 was retrospectively registered on 21/04/2020 and designated an Urgent Public Health Research Study by NIHR.</p

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals &lt;1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

CD4(+)CD25(+) regulatory T cells in health and disease

10.1111/j.1440-1681.2006.04401.xClinical and Experimental Pharmacology and Physiology335-6519-52