Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Dimethyl fumarate in patients admitted to hospital with COVID-19 (RECOVERY): a randomised, controlled, open-label, platform trial

Dimethyl fumarate (DMF) inhibits inflammasome-mediated inflammation and has been proposed as a treatment for patients hospitalised with COVID-19. This randomised, controlled, open-label platform trial (Randomised Evaluation of COVID-19 Therapy [RECOVERY]), is assessing multiple treatments in patients hospitalised for COVID-19 (NCT04381936, ISRCTN50189673). In this assessment of DMF performed at 27 UK hospitals, adults were randomly allocated (1:1) to either usual standard of care alone or usual standard of care plus DMF. The primary outcome was clinical status on day 5 measured on a seven-point ordinal scale. Secondary outcomes were time to sustained improvement in clinical status, time to discharge, day 5 peripheral blood oxygenation, day 5 C-reactive protein, and improvement in day 10 clinical status. Between 2 March 2021 and 18 November 2021, 713 patients were enroled in the DMF evaluation, of whom 356 were randomly allocated to receive usual care plus DMF, and 357 to usual care alone. 95% of patients received corticosteroids as part of routine care. There was no evidence of a beneficial effect of DMF on clinical status at day 5 (common odds ratio of unfavourable outcome 1.12; 95% CI 0.86-1.47; p = 0.40). There was no significant effect of DMF on any secondary outcome

Conditional gene expression in Chlamydia trachomatis using the tet system.

Chlamydia trachomatis is maintained through a complex bi-phasic developmental cycle that incorporates numerous processes that are poorly understood. This is reflective of the previous paucity of genetic tools available. The recent advent of a method for transforming Chlamydia has enabled the development of essential molecular tools to better study these medically important bacteria. Critical for the study of Chlamydia biology and pathogenesis, is a system for tightly controlled inducible gene expression. To accomplish this, a new shuttle vector was generated with gene expression controlled by the Tetracycline repressor and anhydryotetracycline. Evaluation of GFP expression by this system demonstrated tightly controlled gene regulation with rapid protein expression upon induction and restoration of transcription repression following inducer removal. Additionally, induction of expression could be detected relatively early during the developmental cycle and concomitant with conversion into the metabolically active form of Chlamydia. Uniform and strong GFP induction was observed during middle stages of the developmental cycle. Interestingly, variable induced GFP expression by individual organisms within shared inclusions during later stages of development suggesting metabolic diversity is affecting induction and/or expression. These observations support the strong potential of this molecular tool to enable numerous experimental analyses for a better understanding of the biology and pathogenesis of Chlamydia

Effect of anhydrotetracycline on <i>C. trachomatis</i>.

<p>L929 cells were infected with wild-type <i>C</i>. <i>trachomatis</i> L2 and then incubated in the presence of various concentrations of ATc throughout the infection (0 to 24 hpi). Cultures were fixed at 24 hpi and stained for MOMP (green). Representative images from three different concentrations are shown (A). Additionally, the same samples (and additional samples) were analyzed using Cell Profiler to calculate the average inclusion size (B). Over 700 inclusions were analyzed at each concentration. Separately, infected L929 cell cultures were treated with different concentrations of ATc and lysed at 24 hpi. Lysates were used to infect a fresh monolayer (in the absence of ATc) and infection levels were enumerated at 24 hpi using immunofluorescence microscopy. Percent inhibition of progeny was determined relative to a lysate from an untreated control (C).</p

Detection of GFP gene transcription by qPCR.

<p>L929 cell cultures infected with pASK-GFP-L2 transformed <i>C</i>. <i>trachomatis</i> were induced with 10 ng/mL ATc from 20-24 hpi. The culture medium was then replaced with ATc-free medium and samples were collected at the indicated time points for processing and analysis of GFP transcription by qPCR. Relative expression levels are in comparison to the uninduced sample. Error bars indicate standard deviation from two separate experiments.</p

Shuttle vector with GFP expression under Tet control.

<p>The <i>E</i>. <i>coli</i> cloning vector pASK-IBA33plus, with GFP inserted under Tet control, was ligated to the wild-type plasmid from <i>C</i>. <i>trachomatis</i> strain L2 using PCR-generated restriction endonuclease sites (see Materials and Methods for details). A variation of this shuttle vector was later constructed with a gene encoding mKate2 inserted at the indicated <i>Kas</i>I site.</p

Detection of constitutive mKate2 and inducible GFP.

<p>L929 cell cultures infected with pASK-GFP/mKate2-L2 transformed <i>C</i>. <i>trachomatis</i> were induced at 24 hpi and images were then taken at indicated time points. Representative images from an uninduced sample and a sample induced with 10 ng/mL ATc at 24 hpi are shown in (A). Representative images of a sample induced with 10 ng/mL ATc at 16 hpi (from a separate experiment) are shown in (B). White arrows highlight the relatively few <i>C</i>. <i>trachomatis</i> cells within the inclusion that did not express detectable GFP at 19 hpi and 20 hpi.</p

GFP expression after induction at 24 hpi.

<p>L929 cell cultures infected with pASK-GFP-L2 transformed <i>C</i>. <i>trachomatis</i> were induced with various concentrations of ATc at 24 hpi. Images were captured every 10 minutes for a total of 6 hours. Representative images from the culture induced with 10 ng/mL and an uninduced control are shown (A). The images were also analyzed using the Otsu segmentation algorithm to determine the mean fluorescence intensities in arbitrary units within the inclusions (B). Three microscope fields were averaged for each sample with approximately 40 infected cells per field. The mean fluorescence intensity of an uninduced sample was also calculated at each time point and the resulting values were subtracted from the induced samples at the corresponding time points.</p

Induction of GFP with lower ATc concentrations.

<p>L929 cells were infected with pASK-GFP/mKate2-L2 transformed <i>C</i>. <i>trachomatis</i>. At 24 hpi, samples were induced for 4 hours with the indicated concentrations of ATc. After induction, soluble GFP and mKate2 in each sample was quantified using a microplate fluorometer. To normalize samples, GFP expression was divided by mKate2 expression and the resulting ratio was graphed (A). Increases observed in GFP fluorescence of samples induced with 0.25 ng/mL or higher were statistically significant compared to the 0 ng/mL sample (P < 0.01; Student’s <i>t</i> test). Induction from 16 to 20 hpi was also tested and the resulting GFP and mKate2 expression quantified as described above (B). All ATc concentrations tested for this induction period resulted in statistically significant increases in GFP expression. GFP fluorescence from the experiments shown in panels A and B are also graphed with mKate2 normalization (C). Error bars indicate standard deviation of three separate samples.</p