The ALSFRS-R Summit: a global call to action on the use of the ALSFRS-R in ALS clinical trials

The Amyotrophic Lateral Sclerosis Functional Rating Scale (ALSFRS) was developed more than 25 years ago as an instrument to monitor functional change over time in patients with ALS. It has since been revised and extended to meet the needs of high data quality in ALS trials (ALSFRS-R), however a full re-validation of the scale was not completed. Despite this, the scale has remained a primary outcome measure in clinical trials. We convened a group of clinical trialists to discuss and explore opportunities to improve the scale and propose alternative measures. In this meeting report, we present a call to action on the use of the ALSFRS-Revised scale in clinical trials, focusing on the need for (1) harmonization of the ALSFRS-R administration globally, (2) alignment on a set of recommendations for clinical trial design and statistical analysis plans (SAPs), and (3) use of additional outcome measures

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Boosting Bioluminescence Neuroimaging: An Optimized Protocol for Brain Studies

<div><p>Bioluminescence imaging is widely used for optical cell tracking approaches. However, reliable and quantitative bioluminescence of transplanted cells in the brain is highly challenging. In this study we established a new bioluminescence imaging protocol dedicated for neuroimaging, which increases sensitivity especially for noninvasive tracking of brain cell grafts. Different D-Luciferin concentrations (15, 150, 300 and 750 mg/kg), injection routes (iv, ip, sc), types of anesthesia (Isoflurane, Ketamine/Xylazine, Pentobarbital) and timing of injection were compared using DCX-Luc transgenic mice for brain specific bioluminescence. Luciferase kinetics was quantitatively evaluated for maximal photon emission, total photon emission and time-to-peak. Photon emission followed a D-Luciferin dose-dependent relation without saturation, but with delay in time-to-peak increasing for increasing concentrations. The comparison of intravenous, subcutaneous and intraperitoneal substrate injection reflects expected pharmacokinetics with fastest and highest photon emission for intravenous administration. Ketamine/Xylazine and Pentobarbital anesthesia showed no significant beneficial effect on maximal photon emission. However, a strong difference in outcome was observed by injecting the substrate pre Isoflurane anesthesia. This protocol optimization for brain specific bioluminescence imaging comprises injection of 300 mg/kg D-Luciferin pre Isoflurane anesthesia as an efficient and stable method with a signal gain of approx. 200% (compared to 150 mg/kg post Isoflurane). Gain in sensitivity by the novel imaging protocol was quantitatively assessed by signal-to-noise calculations of luciferase-expressing neural stem cells grafted into mouse brains (transplantation of 3,000–300,000 cells). The optimized imaging protocol lowered the detection limit from 6,000 to 3,000 cells by a gain in signal-to-noise ratio.</p> </div

Substrate concentration modulates luciferase activity.

<p>a, b) The PE_max and AUC values increase exponentially with the D-Luciferin concentration. c) The time for maximal luciferase activity is dependent on the substrate concentration. d) The slope of initial photon emission kinetics is concentration dependent. (* statistically significant difference with p≤0.05 to standard protocol 150 mg/kg post-Iso in post hoc comparison with Sidak correction).</p

Photon emission is substantially increased by the modified BLI protocol.

<p>a) Representative images (equally scaled) for each cell number grafted into nude mice acquired with the standard protocol (upper row) and with the advanced protocol (lower row) reveal the objective sensitivity benefit, which is also represented in the quantitative SNR values. b) Correlation between photon emission and cell number revealed a linear relationship under in vivo conditions, with a steeper slope for the novel protocol, indicating the increased sensitivity.</p

Characterization of Luc2-expressing NSCs.

<p>a) Efficient NSC transduction and selection process (by FACS and antibiotics) was confirmed by the homogeneous expression of the fluorescent reporter copGFP, which directly reflects Luc2 expression because of the T2A linker element (microscopic images 20× magnification, 50 µm scale bar). b) Reporter gene expression had no impact on cell viability, as confirmed with the PrestoBlue assay (data of 5 independent measurements presented as relative fluorescent units, RFU). c, d) Quantitative analysis of NSC<i>^Luc2+</i> dilution series (1 min acquisition at 37°C with 30 µg/ml D-Luciferin) revealed a linear correlation between photon emission and cell number, as well as SNR in vitro.</p

The photon flux maximum and timing are dependent on the route of substrate administration.

<p>a, b) The PE_max and AUC increase corresponding to the physiologically expected biodistribution for sc, ip and iv substrate administration, reflected by the characteristic time activity curves. c) Maximal photon flux is reached at minimal time for iv injections followed by ip and sc (* statistically significant difference with p≤0.05 to standard protocol 150 mg/kg post-Iso in post hoc comparison with Sidak correction).</p

Photon flux is modulated by the type of anesthesia.

<p>a) PE_max and AUC were decreased under Ketamine/Xylazine conditions but not different for Pentobarbital compared to Isoflurane. b, c) Representative time activity curves showing the anesthesia dependent signal behavior leading to delayed time-to-peak for Ketamine/Xylazine, but no clear difference between Isoflurane and Pentobarbital anesthesia. (* statistically significant difference with p≤0.05 to standard protocol 150 mg/kg post-Iso in post hoc comparison with Sidak correction).</p

Luciferase inhibition by Isoflurane is avoided by substrate administration before anesthesia onset.

<p>a, b) Difference in PE_max and AUC under pre/post Isoflurane conditions becomes more pronounced with increasing substrate concentration. c) The order of application between Isoflurane anesthesia and substrate had no impact on the time-to-peak for 15, 150 and 300 mg/kg D-Luciferin. (* statistically significant difference with p≤0.05 to standard protocol 150 mg/kg post-Iso in post hoc comparison with Sidak correction;+statistically significant difference with p≤0.05 between pre and post condition in post hoc comparison with Sidak correction).</p