Platelet-Activating Factor Receptor Plays a Role in Lung Injury and Death Caused by Influenza A in Mice

Influenza A virus causes annual epidemics which affect millions of people worldwide. A recent Influenza pandemic brought new awareness over the health impact of the disease. It is thought that a severe inflammatory response against the virus contributes to disease severity and death. Therefore, modulating the effects of inflammatory mediators may represent a new therapy against Influenza infection. Platelet activating factor (PAF) receptor (PAFR) deficient mice were used to evaluate the role of the gene in a model of experimental infection with Influenza A/WSN/33 H1N1 or a reassortant Influenza A H3N1 subtype. The following parameters were evaluated: lethality, cell recruitment to the airways, lung pathology, viral titers and cytokine levels in lungs. The PAFR antagonist PCA4248 was also used after the onset of flu symptoms. Absence or antagonism of PAFR caused significant protection against flu-associated lethality and lung injury. Protection was correlated with decreased neutrophil recruitment, lung edema, vascular permeability and injury. There was no increase of viral load and greater recruitment of NK1.1+ cells. Antibody responses were similar in WT and PAFR-deficient mice and animals were protected from re-infection. Influenza infection induces the enzyme that synthesizes PAF, lyso-PAF acetyltransferase, an effect linked to activation of TLR7/8. Therefore, it is suggested that PAFR is a disease-associated gene and plays an important role in driving neutrophil influx and lung damage after infection of mice with two subtypes of Influenza A. Further studies should investigate whether targeting PAFR may be useful to reduce lung pathology associated with Influenza A virus infection in humans

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

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 <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

Usefulness of ECG criteria to rule out left ventricular hypertrophy in older individuals with true left bundle branch block: an observational study

Abstract Background Advanced age is associated with both left bundle branch block (LBBB) and hypertension and the usefulness of ECG criteria to detect left ventricular hypertrophy (LVH) in patients with LBBB is still unclear. The diagnostic performance and clinical applicability of ECG-based LVH criteria in patients with LBBB defined by stricter ECG criteria is unknown. The aim of this study was to compare diagnostic accuracy and clinical utility of ECG criteria in patients with advanced age and strict LBBB criteria. Methods Retrospective single-center study conducted from Jan/2017 to Mar/2018. Patients undergoing both ECG and echocardiogram examinations were included. Ten criteria for ECG-based LVH were compared using LVH defined by the echocardiogram as the gold standard. Sensitivity, specificity, predictive values, likelihood ratios, AUC, and the Brier score were used to compare diagnostic performance and a decision curve analysis was performed. Results From 4621 screened patients, 68 were included, median age was 78.4 years, (IQR 73.3–83.4), 73.5% with hypertension. All ECG criteria failed to provide accurate discrimination of LVH with AUC range between 0.54 and 0.67, and no ECG criteria had a balanced tradeoff between sensitivity and specificity. No ECG criteria consistently improved the net benefit compared to the strategy of performing routine echocardiogram in all patients in the decision curve analysis within the 10–60% probability threshold range. Conclusion ECG-based criteria for LVH in patients with advanced age and true LBBB lack diagnostic accuracy or clinical usefulness and should not be routinely assessed

NLRP3 inflammasome-mediated neutrophil recruitment and hypernociception depend on leukotriene B4 in a murine model of gout

Objective Deposition of monosodium urate monohydrate (MSU) crystals in the joints promotes an intense inflammatory response and joint dysfunction. This study evaluated the role of the NLRP3 inflammasome and 5-lipoxygenase (5-LOX)derived leukotriene B4 (LTB4) in driving tissue inflammation and hypernociception in a murine model of gout. Methods. Gout was induced by injecting MSU crystals into the joints of mice. Wild-type mice and mice deficient in NLRP3, ASC, caspase 1, interleukin-1 beta (IL-1 beta), IL-1 receptor type I (IL-1RI), IL-18R, myeloid differentiation factor 88 (MyD88), or 5-LOX were used. Evaluations were performed to assess neutrophil influx, LTB4 activity, cytokine (IL-1 beta, CXCL1) production (by enzyme-linked immunosorbent assay), synovial microvasculature cell adhesion (by intravital microscopy), and hypernociception. Cleaved caspase 1 and production of reactive oxygen species (ROS) were analyzed in macrophages by Western blotting and fluorometric assay, respectively. Results. Injection of MSU crystals into the knee joints of mice induced neutrophil influx and neutrophildependent hypernociception. MSU crystal-induced neutrophil influx was CXCR2-dependent and relied on the induction of CXCL1 in an NLRP3/ASC/caspase 1/IL-1 beta/MyD88-dependent manner. LTB4 was produced rapidly after injection of MSU crystals, and this was necessary for caspase 1-dependent IL-1 beta production and consequent release of CXCR2-acting chemokines in vivo. In vitro, macrophages produced LTB4 after MSU crystal injection, and LTB4 was relevant in the MSU crystalinduced maturation of IL-1 beta. Mechanistically, LTB4 drove MSU crystal-induced production of ROS and ROS-dependent activation of the NLRP3 inflammasome. Conclusion. These results reveal the role of the NLRP3 inflammasome in mediating MSU crystalinduced inflammation and dysfunction of the joints, and highlight a previously unrecognized role of LTB4 in driving NLRP3 inflammasome activation in response to MSU crystals, both in vitro and in vivo.Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil)Fundacao de Amparo a Pesquisas do Estado de Minas Gerais (FAPEMIG, Brazil)Fundacao de Amparo a Pesquisas do Estado de Minas Gerais (Fapemig, Brazil

CaCO₃ treatment of AIA mice has no anti-inflammatory and anti-nociceptive effects.

<p>AIA mice were treated as described in Material and Methods. The numbers of neutrophils in the synovial cavity (A), and the relative units of neutrophils in periarticular tissue, as determined by myeloperoxidase assay (B) were assessed 24 hours after injection of 10 μg mBSA or sterile saline (control) in knee joint of immunized mice. Hypernociception is presented as the change (Δ) in withdrawal threshold (in grams) (C). Representative H&E images of control (D), AIA + Vehicle (E), AIA + <i>L</i>. <i>muelleri</i> (F), AIA + CaCO₃ (G) mice. AIA+ vehicle and AIA + CaCO₃ groups (E and G, respectively) presented histopathological evidence of joint inflammation (inflammatory infiltrate [arrows], synovia hyperplasia [arrowheads], alteration of tissue architecture) compared with the other groups (D, F). Scale bars: 100 μm. Other parameters were evaluated as follows: (H), quantification of AIA arthritis index (described in Materials and Methods); and (I), quantification of proteoglycan loss, expressed in %. Results are presented as the mean and SEM results from 5 mice per group. * <i>P</i> <0.05 versus control mice; # for <i>P</i> < 0.05 versus vehicle-treated arthritic mice.</p

<i>In vivo</i> flow cytometry analyses from popliteal lymph node cells and <i>ex vivo</i> splenocyte antigen stimulation after <i>L</i>. <i>muelleri</i> treatment.

<p>AIA mice were treated as described before in Material and Methods. Twenty-four hours after joint challenge, the popliteal lymph node was collected and cells were isolated for quantifying the total cells numbers (Neubauer chamber) and assaying activated CD4 and DCs populations by cellular staining with labeled antibodies and FACS analysis. Results are expressed as numbers of total (A) or activated CD4⁺CD25⁺ (B), CD11c⁺CD86⁺ (C) cells in each population. In D-F, splenocytes were stimulated <i>ex vivo</i> with RPMI medium, 2 μg.mL⁻¹ of Con-A or 100 μg.mL⁻¹ of <i>L</i>. <i>muelleri</i> and culture supernatants were harvested 48 hours later for IFN-γ, IL-17 and IL-10 measurement by ELISA. Results are expressed as pg.mL⁻¹ of culture supernatant. Bars show the mean and SEM results from 5 mice per group. * <i>P</i> <0.05 versus control mice; # for <i>P</i> < 0.05 versus vehicle-treated arthritic mice.</p

Comparative analysis between different days of <i>L</i>.<i>muelleri</i> treatment on the reduction of inflammatory response in AIA.

<p>The treatments with L. muelleri (100mg/kg) were performed twice a day during 5 or 10 consecutive days. The results are presented as the mean and SEM from 7 mice per group.</p><p>* <i>P</i> <0.05 <i>versus</i> control mice;</p><p># for <i>P</i> < 0.05 <i>versus</i> vehicle-treated arthritic mice.</p><p>Comparative analysis between different days of <i>L</i>.<i>muelleri</i> treatment on the reduction of inflammatory response in AIA.</p

Effects of <i>L</i>.<i>muelleri</i> on AIA in mice.

<p>AIA mice were treated with vehicle (CMC 0.5% in filtered water) or with different doses (1, 10 or 100 mg.kg⁻¹) of <i>L</i>. <i>muelleri</i>, orally, twice a day, during 10 days before antigen challenge in knee joint. The number of neutrophils in the synovial cavity (A), and the relative units of neutrophils in periarticular tissue, as determined by myeloperoxidase assay (B) were assessed 24 hours after 10 μg of mBSA or sterile saline (control) in knee joint of immunized mice. The concentrations of the chemokines CXCL1 (C) and CXCL2 (D) in the periarticular tissue were evaluated by ELISA. In E, hypernociception is presented as the change (Δ) in withdrawal threshold (in grams), calculated by subtracting the zero-time mean measurements from the time-interval mean measurements. The results are presented as the mean and SEM from 7 mice per group. * <i>P</i> <0.05 <i>versus</i> control mice; # for <i>P</i> < 0.05 <i>versus</i> vehicle-treated arthritic mice.</p