Rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART): Study protocol for a randomized controlled trial

Background: Acute respiratory distress syndrome (ARDS) is associated with high in-hospital mortality. Alveolar recruitment followed by ventilation at optimal titrated PEEP may reduce ventilator-induced lung injury and improve oxygenation in patients with ARDS, but the effects on mortality and other clinical outcomes remain unknown. This article reports the rationale, study design, and analysis plan of the Alveolar Recruitment for ARDS Trial (ART). Methods/Design: ART is a pragmatic, multicenter, randomized (concealed), controlled trial, which aims to determine if maximum stepwise alveolar recruitment associated with PEEP titration is able to increase 28-day survival in patients with ARDS compared to conventional treatment (ARDSNet strategy). We will enroll adult patients with ARDS of less than 72 h duration. The intervention group will receive an alveolar recruitment maneuver, with stepwise increases of PEEP achieving 45 cmH(2)O and peak pressure of 60 cmH2O, followed by ventilation with optimal PEEP titrated according to the static compliance of the respiratory system. In the control group, mechanical ventilation will follow a conventional protocol (ARDSNet). In both groups, we will use controlled volume mode with low tidal volumes (4 to 6 mL/kg of predicted body weight) and targeting plateau pressure <= 30 cmH2O. The primary outcome is 28-day survival, and the secondary outcomes are: length of ICU stay; length of hospital stay; pneumothorax requiring chest tube during first 7 days; barotrauma during first 7 days; mechanical ventilation-free days from days 1 to 28; ICU, in-hospital, and 6-month survival. ART is an event-guided trial planned to last until 520 events (deaths within 28 days) are observed. These events allow detection of a hazard ratio of 0.75, with 90% power and two-tailed type I error of 5%. All analysis will follow the intention-to-treat principle. Discussion: If the ART strategy with maximum recruitment and PEEP titration improves 28-day survival, this will represent a notable advance to the care of ARDS patients. Conversely, if the ART strategy is similar or inferior to the current evidence-based strategy (ARDSNet), this should also change current practice as many institutions routinely employ recruitment maneuvers and set PEEP levels according to some titration method.Hospital do Coracao (HCor) as part of the Program 'Hospitais de Excelencia a Servico do SUS (PROADI-SUS)'Brazilian Ministry of Healt

<i>Schistosoma mansoni Sm</i>KI-1 serine protease inhibitor binds to elastase and impairs neutrophil function and inflammation

<div><p>Protease inhibitors have important function during homeostasis, inflammation and tissue injury. In this study, we described the role of <i>Schistosoma mansoni Sm</i>KI-1 serine protease inhibitor in parasite development and as a molecule capable of regulating different models of inflammatory diseases. First, we determine that recombinant (r) <i>Sm</i>KI-1 and its Kunitz domain but not the C-terminal region possess inhibitory activity against trypsin and neutrophil elastase (NE). To better understand the molecular basis of NE inhibition by S<i>m</i>KI-1, molecular docking studies were also conducted. Docking results suggest a complete blockage of NE active site by <i>Sm</i>KI-1 Kunitz domain. Additionally, r<i>Sm</i>KI-1 markedly inhibited the capacity of NE to kill schistosomes. In order to further investigate the role of <i>Sm</i>KI-1 in the parasite, we designed specific siRNA to knockdown <i>Sm</i>KI-1 in <i>S</i>. <i>mansoni</i>. <i>SmKI-1</i> gene suppression in larval stage of <i>S</i>. <i>mansoni</i> robustly impact in parasite development <i>in vitro</i> and <i>in vivo</i>. To determine the ability of <i>Sm</i>KI-1 to interfere with neutrophil migration and function, we tested <i>Sm</i>KI-1 anti-inflammatory potential in different murine models of inflammatory diseases. Treatment with <i>Sm</i>KI-1 rescued acetaminophen (APAP)-mediated liver damage, with a significant reduction in both neutrophil recruitment and elastase activity. In the model of gout arthritis, this protein reduced neutrophil accumulation, IL-1β secretion, hypernociception, and overall pathological score. Finally, we demonstrated the ability of <i>Sm</i>KI-1 to inhibit early events that trigger neutrophil recruitment in pleural cavities of mice in response to carrageenan. In conclusion, <i>Sm</i>KI-1 is a key protein in <i>S</i>. <i>mansoni</i> survival and it has the ability to inhibit neutrophil function as a promising therapeutic molecule against inflammatory diseases.</p></div

<i>Sm</i>KI-1 treatment decreased inflammation after MSU-induced gout.

<p>Mice were treated with r<i>Sm</i>KI-1 (10 mg/kg) or PBS vehicle i.v. 15 min prior MSU injection. Then, animals were challenged with intra-articular knee injection of MSU (100μg/cavity). Mice were grouped as MSU control, PBS control, MSU+<i>Sm</i>KI-1-treatment and PBS+<i>Sm</i>KI-1-treatment. Tissue inflammation was evaluated by <b>(a)</b> relative numbers of neutrophil in periarticular tissue determined by MPO assay, <b>(b)</b> total cells and <b>(c)</b> neutrophil recruitment in the synovial cavity, <b>(d)</b> IL-1β production measured by ELISA in the periarticular knee tissue and <b>(e)</b> joint dysfunction as noted by the increase nociceptive response of mice to mechanical stimulation using an electronic paw pressure meter test 15 hrs after MSU or PBS (control vehicle) injection. <b>(f)</b> Representative photographs of hematoxylin and eosin-stained sections of knee joints of mice after 15 hrs of injection with vehicle or MSU crystals (100μg/joint). Leukocyte infiltration and hyperplasia of the synovial membrane are indicated by black arrows. (<b>g</b>) Neutrophil recruitment in the synovial cavity of mice infected with <i>S</i>. <i>mansoni</i>. Mice were grouped as PBS control, PBS infected, MSU control and MSU infected. ND = not detected. Results are the mean ± SEM of n = 6 per group. Asterisks indicate statistically significant differences of r<i>Sm</i>KI-1 compared to MSU-vehicle group *p< 0.05 or ** p< 0.005. An asterisk also indicates statistically significant differences of <i>S</i>. <i>mansoni</i> infection versus control mice that received MSU, p<0.05.</p

<i>Sm</i>KI-1 reduces hepatic APAP-induced injury.

<p><b>(a)</b> MPO and <b>(b)</b> Elastase activities were measured in r<i>Sm</i>KI-1 treated mice during liver APAP-induced hepatotoxicity. Treated-mice received <i>Sm</i>KI-1 (10 mg/kg) or PBS vehicle i.v. 15 min prior APAP administration (600 mg/kg). <b>(c)</b> Number of neutrophils per field of view (FOV) in the liver of r<i>Sm</i>KI-1 treated animals. <b>(d)</b> Liver confocal intravital microscopy showing neutrophil (anti-GR1 PE in red) migration into necrotic sites (Sytox green staining) following 24 hours of APAP challenge. Scale bar = 100 μm. <b>(e)</b> Left panels represent histology of hematoxylin and eosin-stained liver sections, scale bar = 300μm. In right panels, liver damage is highlighted. <b>(f)</b> serum ALT levels confirmed severe liver damage in APAP group and reduction of liver necrosis in mice treated with APAP+<i>Sm</i>KI-1. Results are the mean ± SEM of n = 6 per group. An asterisk indicate statistically significant differences of r<i>Sm</i>KI-1 compared to APAP group (p< 0.05) or ** p< 0.005.</p

<i>Sm</i>KI-1 Kunitz type domain sequence and structure.

<p>(<b>a</b>) Multiple Sequence Alignment between the SmKI-1 protein from <i>Schistosoma mansoni</i>, and its homologous proteins performed using Clustal Omega, refined using BoxShade and then manually. The residues that are similar are shaded in gray, identical in dark-black and in yellow absolutely conserved cysteine residues. (<b>b</b>) Disordered Probability Prediction showing the structured Kunitz domain and unstructured C-terminal region. Analysis performed using COILS algorithms available at the Expasy website. (<b>c</b>) Schematic representation and linear view of the domains of the full-length SmKI-1 protein showing the Kunitz domain with the three disulfide bounds arrangements. (<b>d</b>) 3D protein structure of the <i>Sm</i>KI-1 Kunitz domain modeled using MODELLER v9.17.</p

Recombinant <i>Sm</i>KI-1 and its Kunitz domain inhibit serine proteases and protect <i>S</i>. <i>mansoni</i> against neutrophil elastase.

<p>Recombinant <i>Sm</i>KI-1, its Kunitz or C-terminal domains (100 nM) were tested as inhibitor of serino proteases: (<b>a</b>) Bovine Trypsin activity (100nM), (<b>b</b>) Human Neutrophil Elastase activity (300nM) and (<b>c</b>) Neutrophil-secreted elastase activity. In all experiments, bovine serum albumin (BSA, 300nM) was used as a negative control. Enzyme inhibition was detected over two-hour incubation with r<i>Sm</i>KI-1 or its Kunitz domain. Bars indicate each enzyme activity mean ± standard deviation. (<b>d</b>) Protective effect of rSmKI-1 (0.15 mg/mL) in cultured schistosomula treated with purified elastase (0.05 mg/mL). Bars represent live parasites ± standard deviation. Data are representative of at least three independent experiments. For (<b>a</b>) and (<b>b</b>), an asterisk indicate statistically significant differences of r<i>Sm</i>KI-1 or Kunitz domain compared to control group p< 0.05. For (<b>c</b>) and (<b>d</b>), ** asterisks indicate statistically significant differences of r<i>Sm</i>KI-1, compared to control group or elastase group p< 0.005. <b>(e)</b> Binding mode of <i>Sm</i>KI-1 Kunitz domain (purple) to neutrophil elastase (gray) predicted by docking with CLUSPRO 2.0. Residues from elastase catalytic triad (His⁷⁰, Asp¹¹⁷ and Ser²⁰²) are highlighted in orange sticks. (<b>f</b>) Detailed analysis of the docking predicted interface reveals residues involved in hydrogen bonds, a salt bridge and a π-stacking interaction (all interactions shown as green dashes). <i>Sm</i>KI-1 residues are represented and labeled in purple, NE residues in gray.</p

<i>Sm</i>KI-1 treatment reduces neutrophil migration into pleural cavity in response to carrageenan injection.

<p>After carrageenan injection (2mg/mL) into pleural cavity, animals received an intravenous dose of <i>Sm</i>KI-1 (10 mg/kg) or PBS (vehicle). Four hours later, we recovered cells by washing pleural cavity with PBS. Counting of (<b>a</b>) total cells and (<b>b</b>) neutrophils were performed by cytospin preparations. Specific cell populations in pleural fluid were also evaluated by flow cytometry, being the percentage of (<b>c</b>) neutrophils (Ly6G⁺CD11b⁺), (<b>d</b>) macrophages (F4/80⁺CD11b⁺), and (<b>e</b>) T lymphocytes (CD3⁺ cells) calculated from the total cell numbers. Results are expressed as the number of cells per cavity or percentage of cell subpopulations (mean ± SD) for each treated group (5–6 mice each). An Asterisk indicates statistically significant differences of carrageenan+<i>Sm</i>KI-1 compared to carrageenan vehicle group (p< 0.05).</p

ABC₂-SPH risk score for in-hospital mortality in COVID-19 patients

Objectives: The majority of available scores to assess mortality risk of coronavirus disease 2019 (COVID-19) patients in the emergency department have high risk of bias. Therefore, this cohort aimed to develop and validate a score at hospital admission for predicting in-hospital mortality in COVID-19 patients and to compare this score with other existing ones. Methods: Consecutive patients (≥ 18 years) with confirmed COVID-19 admitted to the participating hospitals were included. Logistic regression analysis was performed to develop a prediction model for in-hospital mortality, based on the 3978 patients admitted between March–July, 2020. The model was validated in the 1054 patients admitted during August–September, as well as in an external cohort of 474 Spanish patients. Results: Median (25–75th percentile) age of the model-derivation cohort was 60 (48–72) years, and in-hospital mortality was 20.3%. The validation cohorts had similar age distribution and in-hospital mortality. Seven significant variables were included in the risk score: age, blood urea nitrogen, number of comorbidities, C-reactive protein, SpO2/FiO2 ratio, platelet count, and heart rate. The model had high discriminatory value (AUROC 0.844, 95% CI 0.829–0.859), which was confirmed in the Brazilian (0.859 [95% CI 0.833–0.885]) and Spanish (0.894 [95% CI 0.870–0.919]) validation cohorts, and displayed better discrimination ability than other existing scores. It is implemented in a freely available online risk calculator (https://abc2sph.com/). Conclusions: An easy-to-use rapid scoring system based on characteristics of COVID-19 patients commonly available at hospital presentation was designed and validated for early stratification of in-hospital mortality risk of patients with COVID-19.</p