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NR2F2 loss in the endothelial lining of blood vessels is associated with cardiovascular disease. Here, NR2F2-silenced human endothelial cells were inflammatory, proliferative, hypermigratory, and apoptosis-resistant with increased oxidant stress and endothelial-to-mesenchymal transition. DKK1 was induced in NR2F2-silenced endothelial cells, while co-silencing NR2F2 and DKK1 prevented NR2F2-loss-associated abnormalities in endothelial signaling and phenotype. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating vascular diseases associated with endothelial dysfunction.

Spleen tyrosine kinase (SYK) is a previously unidentified therapeutic target that inhibits neutrophil and macrophage activation in coronavirus disease 2019 (COVID-19). Fostamatinib, a SYK inhibitor, was studied in a phase 2 placebo-controlled randomized clinical trial and was associated with improvements in many secondary end points related to efficacy. Here, we used a multiomic approach to evaluate cellular and soluble immune mediator responses of patients enrolled in this trial. We demonstrated that SYK inhibition was associated with reduced neutrophil activation, increased circulation of mature neutrophils (CD10 + CD33 − ), and decreased circulation of low-density granulocytes and polymorphonuclear myeloid-derived suppressor cells (HLA-DR − CD33 + CD11b − ). SYK inhibition was also associated with normalization of transcriptional activity in circulating monocytes relative to healthy controls, an increase in frequency of circulating nonclassical and HLA-DR hi classical monocyte populations, and restoration of interferon responses. Together, these data suggest that SYK inhibition may mitigate proinflammatory myeloid cellular and soluble mediator responses thought to contribute to immunopathogenesis of severe COVID-19.

Background COVID-19 disease severity and outcomes have been linked to high antibody titers and a dysregulated neutrophil immune response. Here we query associations and connections between the endogenous SARS-CoV-2 antibody response and neutrophil activation in COVID-19. Methods Baseline serum or plasma samples from 57 patients hospitalized on oxygen with COVID-19 were used to perform; 1) quantitative measurements of SARS-CoV-2 specific antibodies using a luciferase-based immunoprecipitation system assay, 2) quantitative measurements of neutrophil specific biomarkers using Luminex technology, and 3) neutrophil extracellular traps (NETs) as measured by myeloperoxidase-DNA (MPO-DNA) complexes by ELISA. Absolute neutrophil count (ANC) and immature granulocyte count (IGC) were measured from complete blood counts (CBC). Antibody levels were compared by disease severity using Wilcoxon rank-sum test and correlations were generated between antibody levels and neutrophil activation markers using Spearman’s correlation (SC). Results In a cohort of hospitalized patients, severe/critical COVID-19 was associated with higher levels of nucleocapsid-IgA (p=0.011) as well as spike-IgG (p= 0.0007) compared to moderate disease, while spike-IgA and nucleocapsid-IgG showed similar associations, trending towards significance (Figure 1A). Levels of IgG-spike and IgG-nucleocapsid both had significant correlations with the ANC (SC 0.33, p = 0.029; SC 0.38 p = 0.012). All four antibody titers showed strong correlations with IGC, lactoferrin and lipocalin-2, evidence of emergency granulopoiesis. Further, S100A9, a component calprotectin correlated with spike-IgG and nucleocapsid-IgA levels (SC 0.29, p = 0.030, SC 0.29 p = 0.029). Lastly, we found circulating NETs correlated with spike IgA levels (SC 0.38 p = 0.006), and its correlations with IgG-spike and IgA-nucleocapsid additionally approached significance with NETs levels as well (Figure 1B). Antibody Levels Correlate with Disease Severity and Neutrophil Activation Markers Figure 1: A) Levels of anti-Spike and anti-Nucleocapsid IgA and IgG levels measured in the serum of 57 unvaccinated hospitalized COVID-19 patients. Moderate illness represents ordinal scale 5 requiring low flow oxygen, while severe/critical patients represent ordinal scale 6 and 7, requiring high flow oxygen, non-invasive or mechanical ventilation, respectively. P values are compared by a Wilcoxon ranked sum test. B) Heatmap showing Spearman correlations between levels of anti-Spike and anti-Nucleocapsid IgA and IgG and markers of neutrophil activation. P values for individual correlations are represented in parentheses. MPO (myeloperoxidase), ANC (absolute neutrophil count), S100A9 (S100 calcium binding protein A9). Conclusion Higher anti-spike and anti-nucleocapsid IgG and IgA levels associate with more severe COVID-19 illness. Further, endogenous SARS-CoV-2 specific antibody levels associate with markers of emergency granulopoiesis and neutrophil activation. Inhibiting antibody mediated neutrophil activation may improve outcomes in COVID-19. Disclosures All Authors: No reported disclosures.

Plasma gelsolin (pGSN) is the secreted isoform of an intracellular actin remodeling protein found in high concentrations in human plasma. Clinical studies demonstrate reduced pGSN concentrations in several disease states, including severe trauma, burns, and sepsis. Markedly decreased pGSN concentrations in these conditions precede and predict adverse clinical outcomes. In this study, we measured pGSN in patients with chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent infections and dysregulated inflammation. pGSN was quantified using a sandwich ELISA in plasma from healthy volunteers, clinically stable CGD patients, and X-linked CGD carriers and in sera from 12 CGD patients undergoing bone marrow transplantation. pGSN was also quantified in healthy volunteers challenged with intravenous endotoxin. pGSN concentrations were lower in CGD patients without active infection or systemic inflammation compared with healthy control subjects. In CGD patients undergoing bone marrow transplantation, pGSN concentrations increased significantly following successful transplant. X-linked carriers of CGD had normal pGSN. Despite reduction of pGSN in CGD patients, we did not detect significant changes in pGSN over 24 h following challenge of healthy volunteers with intravenous endotoxin (4 ng/kg) that elicited a febrile response. We describe, for the first time, significantly lower pGSN in clinically stable patients with CGD compared with age- and sex-matched healthy volunteers. Low pGSN levels in CGD patients significantly increased following bone marrow transplantation. X-linked carriers of CGD had normal pGSN. In healthy volunteers challenged with intravenous endotoxin, pGSN is not an acute phase reactant.

PURPOSE: To develop an isotropic high-resolution stack-of-spirals UTE sequence for pulmonary imaging at 0.55 Tesla by leveraging a combination of robust respiratory-binning, trajectory correction, and concomitant-field corrections. METHODS: A stack-of-spirals golden-angle UTE sequence was used to continuously acquire data for 15.5 minutes. The data was binned to a stable respiratory phase based on superoinferior readout self-navigator signals. Corrections for trajectory errors and concomitant field artifacts, along with image reconstruction with conjugate gradient SENSE, were performed inline within the Gadgetron framework. Finally, data were retrospectively reconstructed to simulate scan times of 5, 8.5, and 12 minutes. Image quality was assessed using signal-to-noise, image sharpness, and qualitative reader scores. The technique was evaluated in healthy volunteers, patients with coronavirus disease 2019 infection, and patients with lung nodules. RESULTS: The technique provided diagnostic quality images with parenchymal lung SNR of 3.18 ± 0.0.60, 4.57 ± 0.87, 5.45 ± 1.02, and 5.89 ± 1.28 for scan times of 5, 8.5, 12, and 15.5 minutes, respectively. The respiratory binning technique resulted in significantly sharper images (p < 0.001) as measured with relative maximum derivative at the diaphragm. Concomitant field corrections visibly improved sharpness of anatomical structures away from iso-center. The image quality was maintained with a slight loss in SNR for simulated scan times down to 8.5 minutes. Inline image reconstruction and artifact correction were achieved in

Observational studies suggest obesity is associated with sepsis survival, but these studies are small, fail to adjust for key confounders, measure body mass index at inconsistent time points, and/or use administrative data to define sepsis. To estimate the relationship between body mass index and sepsis mortality using detailed clinical data for case detection and risk adjustment.Retrospective cohort analysis of a large clinical data repository.One-hundred thirty-nine hospitals in the United States.Adult inpatients with sepsis meeting Sepsis-3 criteria.Body mass index in six categories: underweight (body mass index18.5 kg/m), normal weight (body mass index = 18.5-24.9 kg/m), overweight (body mass index = 25.0-29.9 kg/m), obese class I (body mass index = 30.0-34.9 kg/m), obese class II (body mass index = 35.0-39.9 kg/m), and obese class III (body mass index ≥ 40 kg/m).Multivariate logistic regression with generalized estimating equations to estimate the effect of body mass index category on short-term mortality (in-hospital death or discharge to hospice) adjusting for patient, infection, and hospital-level factors. Sensitivity analyses were conducted in subgroups of age, gender, Elixhauser comorbidity index, Sequential Organ Failure Assessment quartiles, bacteremic sepsis, and ICU admission.From 2009 to 2015, we identified 55,038 adults with sepsis and assessable body mass index measurements: 6% underweight, 33% normal weight, 28% overweight, and 33% obese. Crude mortality was inversely proportional to body mass index category: underweight (31%), normal weight (24%), overweight (19%), obese class I (16%), obese class II (16%), and obese class III (14%). Compared with normal weight, the adjusted odds ratio (95% CI) of mortality was 1.62 (1.50-1.74) for underweight, 0.73 (0.70-0.77) for overweight, 0.61 (0.57-0.66) for obese class I, 0.61 (0.55-0.67) for obese class II, and 0.65 (0.59-0.71) for obese class III. Results were consistent in sensitivity analyses.In adults with clinically defined sepsis, we demonstrate lower short-term mortality in patients with higher body mass indices compared with those with normal body mass indices (both unadjusted and adjusted analyses) and higher short-term mortality in those with low body mass indices. Understanding how obesity improves survival in sepsis would inform prognostic and therapeutic strategies.

Background Coronavirus disease 2019 (COVID-19) requiring hospitalization is characterized by robust antibody production, dysregulated immune response, and immunothrombosis. Fostamatinib is a novel spleen tyrosine kinase inhibitor that we hypothesize will ameliorate Fc activation and attenuate harmful effects of the anti-COVID-19 immune response. Methods We conducted a double-blind, randomized, placebo-controlled trial in hospitalized adults requiring oxygen with COVID-19 where patients receiving standard of care were randomized to receive fostamatinib or placebo. The primary outcome was serious adverse events by day 29. Results A total of 59 patients underwent randomization (30 to fostamatinib and 29 to placebo). Serious adverse events occurred in 10.5% of patients in the fostamatinib group compared with 22% in placebo (P = .2). Three deaths occurred by day 29, all receiving placebo. The mean change in ordinal score at day 15 was greater in the fostamatinib group (-3.6 ± 0.3 vs -2.6 ± 0.4, P = .035) and the median length in the intensive care unit was 3 days in the fostamatinib group vs 7 days in placebo (P = .07). Differences in clinical improvement were most evident in patients with severe or critical disease (median days on oxygen, 10 vs 28, P = .027). There were trends toward more rapid reductions in C-reactive protein, D-dimer, fibrinogen, and ferritin levels in the fostamatinib group. Conclusion For COVID-19 requiring hospitalization, the addition of fostamatinib to standard of care was safe and patients were observed to have improved clinical outcomes compared with placebo. These results warrant further validation in larger confirmatory trials. Clinical Trials Registration Clinicaltrials.gov, NCT04579393.

Messenger RNA (mRNA) vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are highly effective at inducing protective immunity. However, weak antibody responses are seen in some individuals, and cellular correlates of immunity remain poorly defined, especially for B cells. Here we used unbiased approaches to longitudinally dissect primary antibody, plasmablast, and memory B cell (MBC) responses to the two-dose mRNA-1273 vaccine in SARS-CoV-2-naive adults. Coordinated immunoglobulin A (IgA) and IgG antibody responses were preceded by bursts of spike-specific plasmablasts after both doses but earlier and more intensely after dose 2. While antibody and B cell cellular responses were generally robust, they also varied within the cohort and decreased over time after a dose-2 peak. Both antigen-nonspecific postvaccination plasmablast frequency after dose 1 and their spike-specific counterparts early after dose 2 correlated with subsequent antibody levels. This correlation between early plasmablasts and antibodies remained for titers measured at 6 months after vaccination. Several distinct antigen-specific MBC populations emerged postvaccination with varying kinetics, including two MBC populations that correlated with 2- and 6-month antibody titers. Both were IgG-expressing MBCs: one less mature, appearing as a correlate after the first dose, while the other MBC correlate showed a more mature and resting phenotype, emerging as a correlate later after dose 2. This latter MBC was also a major contributor to the sustained spike-specific MBC response observed at month 6. Thus, these plasmablasts and MBCs that emerged after both the first and second doses with distinct kinetics are potential determinants of the magnitude and durability of antibodies in response to mRNA-based vaccination.

Neutrophil extracellular traps (NETs) contribute to immunothrombosis and have been associated with mortality in coronavirus disease 2019 (COVID-19). We stimulated donor neutrophils with plasma from patients with COVID-19 and demonstrated that R406 can abrogate the release of NETs. These data provide evidence for how fostamatinib may mitigate neutrophil-associated mechanisms contributing to COVID-19 immunopathogenesis.

Bacterial products including endotoxin have been administered to humans over the past century as therapeutic, diagnostic, and experimental agents. The acute phase response is the early immediate host response to infection or tissue injury and results in fever, leukocytosis, changes in vascular permeability, and altered metabolic responses in various organs. Humoral defense mechanisms are rapidly activated after endotoxin administration. The contact system composed of Factors XII, XI, prekallikrein, and high molecular weight kininogen amplifies several host inflammatory responses including the initiation of coagulation via the intrinsic pathway, activation of plasminogen and neutrophils, as well as the generation of bradykinin, a potent vasodilator. Cytokines serve as important links to amplify the inflammatory response initiated by endotoxin. Hepatic synthetic function is rapidly upregulated in response to endotoxin. Endotoxin has been implicated in the pathogenesis of gram-negative bacterial pneumonia as well as the development of acute and chronic lung inflammation due to occupational dust exposure.

Ancestral genetic exchange between members of many important bacterial pathogen groups has resulted in phylogenetic relationships better described as networks than as bifurcating trees. In certain cases, these reticulated phylogenies have resulted in phenotypic and molecular overlap that challenges the construction of practical approaches for species identification in the clinical microbiology laboratory. Burkholderia cepacia complex (Bcc), a betaproteobacteria species group responsible for significant morbidity in persons with cystic fibrosis and chronic granulomatous disease, represents one such group where network-structured phylogeny has hampered the development of diagnostic methods for species-level discrimination. Here, we present a phylogeny-informed proteomics approach to facilitate diagnostic classification of pathogen groups with reticulated phylogenies, using Bcc as an example. Starting with a set of more than 800 Bcc and Burkholderia gladioli whole-genome assemblies, we constructed phylogenies with explicit representation of inferred interspecies recombination. Sixteen highly discriminatory peptides were chosen to distinguish B. cepacia, Burkholderia cenocepacia, Burkholderia multivorans, and B. gladioli and multiplexed into a single, rapid liquid chromatography-tandem mass spectrometry multiple reaction monitoring (LC-MS/MS MRM) assay. Testing of a blinded set of isolates containing these four Burkholderia species demonstrated 50/50 correct automatic negative calls (100% accuracy with a 95% confidence interval [CI] of 92.9 to 100%), and 70/70 correct automatic species-level positive identifications (100% accuracy with 95% CI 94.9 to 100%) after accounting for a single initial incorrect identification due to a preanalytic error, correctly identified on retesting. The approach to analysis described here is applicable to other pathogen groups for which development of diagnostic classification methods is complicated by interspecies recombination.

Plasma gelsolin (pGSN) is the secreted isoform of an intracellular actin remodeling protein found in high concentrations in human plasma. Clinical studies demonstrate reduced pGSN concentrations in several disease states, including severe trauma, burns, and sepsis. Markedly decreased pGSN concentrations in these conditions precede and predict adverse clinical outcomes. In this study, we measured pGSN in patients with chronic granulomatous disease (CGD), a primary immunodeficiency characterized by recurrent infections and dysregulated inflammation. pGSN was quantified using a sandwich ELISA in plasma from healthy volunteers, clinically stable CGD patients, and X-linked CGD carriers and in sera from 12 CGD patients undergoing bone marrow transplantation. pGSN was also quantified in healthy volunteers challenged with intravenous endotoxin. pGSN concentrations were lower in CGD patients without active infection or systemic inflammation compared with healthy control subjects. In CGD patients undergoing bone marrow transplantation, pGSN concentrations increased significantly following successful transplant. X-linked carriers of CGD had normal pGSN. Despite reduction of pGSN in CGD patients, we did not detect significant changes in pGSN over 24 h following challenge of healthy volunteers with intravenous endotoxin (4 ng/kg) that elicited a febrile response. We describe, for the first time, significantly lower pGSN in clinically stable patients with CGD compared with age- and sex-matched healthy volunteers. Low pGSN levels in CGD patients significantly increased following bone marrow transplantation. X-linked carriers of CGD had normal pGSN. In healthy volunteers challenged with intravenous endotoxin, pGSN is not an acute phase reactant.

Guidelines-recommend thrombolytic therapy for pulmonary embolism in patients with severe hemodynamic compromise and low risk of bleeding. Thrombolytics in submassive pulmonary embolism have an unfavorable risk/benefit ratio and remain controversial. Based on our experience with extensive, lower extremity thrombi, nine patients with symptomatic, submassive pulmonary embolisms (five medical, four surgical) were treated with low-dose alteplase (

BACKGROUND Rapid identification of respiratory pathogens may facilitate targeted antimicrobial therapy. Direct identification of bacteria in bronchoalveolar lavage (BAL) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is confounded by interfering substances. We describe a method to identify unique peptide markers of 5 gram-negative bacteria by liquid chromatography–tandem mass spectrometry (LC-MS/MS) for direct pathogen identification in BAL. METHODS In silico translation and digestion were performed on 14–25 whole genomes representing strains of Acinetobacter baumannii, Moraxella catarrhalis, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Klebsiella pneumoniae. Peptides constituting theoretical core peptidomes in each were identified. Rapid tryptic digestion was performed; peptides were analyzed by LC-MS/MS and compared with the theoretical core peptidomes. High-confidence core peptides (false discovery rate RESULTS Five unique peptides with the highest abundance for each pathogen distinguished these pathogens with varied detection sensitivities. Peptide markers for A. baumannii and P. aeruginosa, when spiked simultaneously into inflamed BAL, were detected with as few as 3.6 (0.2) × 103 and 2.2 (0.6) × 103 colony-forming units, respectively, by targeted LC-MS/MS. CONCLUSIONS This proof-of-concept study shows the feasibility of identifying unique peptides in BAL for 5 gram-negative bacterial pathogens, and it may provide a novel approach for rapid direct identification of bacterial pathogens in BAL.

MicroRNA (miRNA) control gene transcription by binding to and repressing the translation of messenger RNA (mRNA). Their role in the acute respiratory distress syndrome (ARDS) is undefined.Blood leukocytes from 51 patients enrolled in a prior randomized trial of corticosteroids for ARDS were analyzed. After screening eight patients with microarrays for altered miRNA expression, 25 miRNAs were selected for further analysis using RT-PCR in all 51 patients.On day 0, the 51 patients had APACHE III score of 60.4 ± 17.7 and PaO2/FiO2 of 117 ± 49. 21 miRNA were expressed at increased levels in blood leukocytes at the onset of ARDS compared with healthy controls. These miRNA remained elevated at day 3 and increased further by day 7 (log2 fold change from 0.66 to 5.7 fold, P 0.05 compared to day 0). In a subgroup analysis (37 patients treated with corticosteroids and 14 treated with placebo), the interaction of miRNA expression over time and steroid administration was not significant suggesting that systemic corticosteroids had no effect on the miRNA detected in our study. In contrast, corticosteroids but not placebo decreased IL-6 and C-reactive protein at day 3 (P 0.001) demonstrating an early systemic anti-inflammatory response whereas both treatment arms had decreased values by day 7 (P 0.001).Expression of miRNA is increased in blood leukocytes of patients with ARDS at day 0 and day 3 and rises further by day 7, when systemic inflammation is subsiding. These effects appear independent of the administration of steroids, suggesting different inflammatory modifying roles for each in the resolving phases of ARDS.

Carbapenemase producing organisms (CPOs) represent an urgent public health threat, and the need for new rapid methods to detect these organisms has been widely recognized. CPOs carrying the Klebsiella pneumoniae carbapenemase (bla KPC ) gene have caused outbreaks globally with substantial attributable mortality. Here we describe the validation of a rapid MS method for the direct detection of unique tryptic peptides of the KPC protein in clinical bacterial isolates with an isolate-to-result time of less than 90 minutes. Using a genoproteomic discovery approach that combines theoretical peptidome analysis and liquid chromatography-tandem MS (LC-MS/MS), we selected three high abundance peptide markers of the KPC protein that can be robustly detected following rapid tryptic digestion. Protein BLAST analysis confirmed that the chosen peptide markers were unique to KPC. A blinded validation set containing 20 KPC-positive and 80 KPC-negative clinical isolates, performed in triplicate (300 runs) demonstrated 100% sensitivity and 100% specificity (60/60 positive identifications, 240/240 negative identifications) using defined rules for positive calls. The most robust tryptic peptide marker in the validation was LTLGSALAAPQR. The peptide discovery and detection methods validated here are general and should be broadly applicable to allow the direct and rapid detection of other resistance determinants.

There is significant interest in the development of mass spectrometry (MS) methods for antimicrobial resistance protein detection, given the ability of these methods to confirm protein expression. In this work, we studied the performance of a liquid chromatography, tandem MS multiple-reaction monitoring (LC-MS/MS MRM) method for the direct detection of the New Delhi metallo-β-lactamase (NDM) carbapenemase in clinical isolates. Using a genoproteomic approach, we selected three unique peptides (SLGNLGDADTEHYAASAR, AFGAAFPK, and ASMIVMSHSAPDSR) specific to NDM that were efficiently ionized and spectrally well-defined. These three peptides were used to build an assay with turnaround time of 90 min. In a blind set, the assay detected 21/24 bla(NDM)-containing isolates and 76/76 isolates with negative results, corresponding to a sensitivity value of 87.5% (95% confidence interval [CI], 67.6% to 97.3%) and a specificity value of 100% (95% CI, 95.3% to 100%). One of the missed identifications was determined by protein fractionation to be due to low (∼0.1 fm/μg) NDM protein expression (below the assay limit of detection). Parallel disk diffusion susceptibility testing demonstrated this isolate to be meropenem susceptible, consistent with low NDM expression. Total proteomic analysis of the other two missed identifications did not detect NDM peptides but detected other proteins expressed from the bla(NDM)-containing plasmids, confirming that the plasmids were not lost. The measurement of relative NDM concentrations over the entire isolate test set demonstrated variability spanning 4 orders of magnitude, further confirming the remarkable range that may be seen in levels of NDM expression. This report highlights the sensitivity of LC-MS/MS to variations in NDM protein expression, with implications for how this technology may be used.

Plasmodium infection depletes arginine, the substrate for nitric oxide synthesis, and impairs endothelium-dependent vasodilation. Increased conversion of arginine to ornithine by parasites or host arginase is a proposed mechanism of arginine depletion. We used high-performance liquid chromatography to measure plasma arginine, ornithine, and citrulline levels in Malawian children with cerebral malaria and in mice infected with Plasmodium berghei ANKA with or without the arginase gene. Heavy isotope-labeled tracers measured by quadrupole time-of-flight liquid chromatography-mass spectrometry were used to quantify the in vivo rate of appearance and interconversion of plasma arginine, ornithine, and citrulline in infected mice. Children with cerebral malaria and P. berghei-infected mice demonstrated depletion of plasma arginine, ornithine, and citrulline. Knock out of Plasmodium arginase did not alter arginine depletion in infected mice. Metabolic tracer analysis demonstrated that plasma arginase flux was unchanged by P. berghei infection. Instead, infected mice exhibited decreased rates of plasma arginine, ornithine, and citrulline appearance and decreased conversion of plasma citrulline to arginine. Notably, plasma arginine use by nitric oxide synthase was decreased in infected mice. Simultaneous arginine and ornithine depletion in malaria parasite-infected children cannot be fully explained by plasma arginase activity. Our mouse model studies suggest that plasma arginine depletion is driven primarily by a decreased rate of appearance.

Background Mortality after smoke inhalation–associated acute lung injury (SI-ALI) remains substantial. Age and burn surface area are risk factors of mortality, whereas the impact of patient- and center-level variables and treatments on survival are unknown. Methods We performed a retrospective cohort study of burn and non-burn centers at 68 US academic medical centers between 2011 and 2014. Adult inpatients with SI-ALI were identified using an algorithm based on a billing code for respiratory conditions from smoke inhalation who were mechanically ventilated by hospital day 4, with either a length-of-stay ≥ 5 days or death within 4 days of hospitalization. Predictors of in-hospital mortality were identified using logistic regression. The primary outcome was the odds ratio for in-hospital mortality. Results A total of 769 patients (52.9 ± 18.1 years) with SI-ALI were analyzed. In-hospital mortality was 26% in the SI-ALI cohort and 50% in patients with ≥ 20% surface burns. In addition to age > 60 years (OR 5.1, 95% CI 2.53-10.26) and ≥ 20% burns (OR 8.7, 95% CI 4.55-16.75), additional risk factors of in-hospital mortality included initial vasopressor use (OR 5.0, 95% CI 3.16-7.91), higher diagnostic-related group–based risk-of-mortality assignment and lower hospital bed capacity (OR 2.3, 95% CI 1.23-4.15). Initial empiric antibiotics (OR 0.93, 95% CI 0.58-1.49) did not impact survival. These new risk factors improved mortality prediction by 9.9% (P Conclusions In addition to older age and major surface burns, mortality in SI-ALI is predicted by initial vasopressor use, higher diagnostic-related group–based risk-of-mortality assignment, and care at centers with

BACKGROUND Acinetobacter baumannii is a common nosocomial pathogen and strain-typing methods play an important role in hospital outbreak investigations and epidemiologic surveillance. We describe a method for identifying strain-specific peptide markers based on LC-MS/MS profiling of digested peptides. This method classified a test set of A. baumannii isolates collected from a hospital outbreak with discriminatory performance exceeding that of MALDI-TOF mass spectrometry. METHODS Following the construction of a species “pan-peptidome” by in silico translation and digestion of whole genome sequences, a hypothetical set of genome-specific peptides for an isolate was constructed from the disjoint set of the pan-peptidome and the isolate's calculated peptidome. The genome-specific peptidome guided selection of highly expressed genome-specific peptides from LC-MS/MS experimental profiles as potential peptide markers. The species specificity of each experimentally identified genome-specific peptide was confirmed through a Unipept lowest common ancestor analysis. RESULTS Fifteen A. baumannii isolates were analyzed to derive a set of genome- and species-specific peptides that could be used as peptide markers. Identified peptides were cross-checked with protein BLAST against a set of 22 A. baumannii whole genome sequences. A subset of these peptide markers was confirmed to be present in the actual peptide profiles generated by multiple reaction monitoring and targeted LC-MS/MS. The experimentally identified peptides separated these isolates into 6 strains that agreed with multilocus sequence typing analysis performed on the same isolates. CONCLUSIONS This approach may be generalizable to other bacterial species, and the peptides may be useful for rapid MS strain tracking of isolates with broad application to infectious disease diagnosis.

From September 2014 to April 2015, 6 persons who had occupational exposures to Zaire ebolavirus in West Africa received investigational agent rVSV-ZEBOV or TKM-100802 for postexposure prophylaxis and were monitored in the United States. All patients experienced self-limited symptoms after postexposure prophylaxis; none developed Ebola virus disease.

Racial disparities in sepsis outcomes have been previously reported. However, recently, there have been inconsistencies in identifying which socioeconomic variables, such as race, account for these disparities. The objective of this study was to perform a systematic review in order to examine the impact of race on sepsis-attributable mortality. Systematic searches for English-language articles identified through MEDLINE, EBSCOhost, PubMed, ERIC, and Cochrane Library databases from 1960 to 1 February 2017. Included studies examined sepsis outcomes in the context of sepsis incidence and/or mortality. Two investigators independently extracted data and assessed study quality. The meta-analysis was performed in accordance with the Cochrane Collaboration guidelines. Twenty-one studies adhered to the predefined selection criteria and were included in the review. Of the 21 studies, we pooled data from 6 studies comparing African American/Black race as a risk factor for sepsis-related mortality disparities (reference group being Caucasian/White). From the meta-analysis on these six studies, African American/Black race was found to have no statistical significant relationship with sepsis-related mortality (odds ratio 1.20, 95% CI, 0.81 to 1.77). Similar results were found for other races (Native Americans, Asians) and ethnicities (Hispanic/Latinos). On the basis of available evidence from a limited number of observation retrospective studies, race alone cannot fully explain sepsis-related disparities, especially sepsis-attributable mortality.

Phenotypic detection of the OXA-48-type class D β-lactamases in Enterobacteriaceae is challenging. We describe a rapid (less than 90 min) assay for the identification of OXA-48 family carbapenemases in subcultured bacterial isolates based on a genoproteomic approach. Following in silico trypsin digestion to ascertain theoretical core peptides common to the OXA-48 family, liquid chromatography-tandem mass spectrometry (LC-MS/MS) data-dependent acquisition was used to identify candidate peptide markers. Two peptides were selected based on performance characteristics: ANQAFLPASTFK, a core peptide common to all 12 OXA-48 family β-lactamase members, and YSVVPVYQEFAR, a highly specific peptide common to 11 of 12 OXA-48 family proteins providing the basis for an LC-MS/MS multiple reaction monitoring assay. An accuracy assessment was performed that included 98 isolates, 26 of which were OXA-48 positive. Two additional specificity assessments were performed including a mixture of isolates positive for OXA-48, KPC, NDM, VIM, and IMP carbapenemases. A combination of expert rules and expert judgment was applied by blinded operators to identify positive isolates. All isolates containing an OXA-48 family carbapenemase across all three test sets were correctly identified with no false positives, demonstrating 100% sensitivity (95% confidence interval [CI], 91.2% to 100%) and 100% specificity (95% CI, 96.2% to 100%) for the assay. These findings provide a framework for an LC-MS/MS-based method for the direct detection of OXA-48 family carbapenemases from cultured isolates that may have utility in predicting carbapenem resistance and tracking hospital outbreaks of OXA-48-carrying organisms.

BACKGROUND: Resistance to all first-line antibiotics necessitates the use of less effective or more toxic “reserve” agents. Gram-negative bloodstream infections (GNBSIs) harboring such difficult-to-treat resistance (DTR) may have higher mortality than phenotypes that allow for ≥1 active first-line antibiotic. METHODS: The Premier Database was analyzed for inpatients with select GNBSIs. DTR was defined as intermediate/resistant in vitro to all ß-lactam categories, including carbapenems and fluoroquinolones. Prevalence and aminoglycoside resistance of DTR episodes were compared with carbapenem-resistant, extended-spectrum cephalosporin-resistant, and fluoroquinolone-resistant episodes using CDC definitions. Predictors of DTR were identified. The adjusted relative risk (aRR) of mortality was examined for DTR, CDC-defined phenotypes susceptible to ≥1 first-line agent, and graded loss of active categories. RESULTS: Between 2009–2013, 471 (1%) of 45011 GNBSI episodes at 92 (53.2%) of 173 hospitals exhibited DTR, ranging from 0.04% for Escherichia coli to 18.4% for Acinetobacter baumannii. Among patients with DTR, 79% received parenteral aminoglycosides, tigecycline, or colistin/polymyxin-B; resistance to all aminoglycosides occurred in 33%. Predictors of DTR included urban healthcare and higher baseline illness. Crude mortality for GNBSIs with DTR was 43%; aRR was higher for DTR than for carbapenem-resistant (1.2; 95% confidence interval, 1.0–1.4; P = .02), extended-spectrum cephalosporin-resistant (1.2; 1.1–1.4; P = .001), or fluoroquinolone-resistant (1.2; 1.0–1.4; P = .008) infections. The mortality aRR increased 20% per graded loss of active first-line categories, from 3–5 to 1–2 to 0. CONCLUSION: Nonsusceptibility to first-line antibiotics is associated with decreased survival in GNBSIs. DTR is a simple bedside prognostic measure of treatment-limiting coresistance.

Background Colistin (polymyxin E) and polymixin B are important bactericidal antibiotics used in the treatment of serious infections caused by multi-drug resistant Gram-negative organisms. Transferrable plasmid-mediated colistin resistance, conferred by the product of the mcr-1 gene, has emerged as a global healthcare threat. Consequently, the rapid detection of the MCR-1 protein in clinical bacterial isolates has become increasingly important. We used a genoproteomic approach to identify unique peptides of the MCR-1 protein that could be detected rapidly by liquid chromatography tandem mass spectrometry (LC–MS/MS). Methods MCR-1 tryptic peptides that were efficiently ionized and readily detectable were characterized in a set of mcr-1-containing isolates with triple quadrupole LC–MS. Three optimal peptides were selected for the development of a rapid multiple reaction monitoring LC–MS/MS assay for the MCR-1 protein. To investigate the feasibility of rapid detection of the MCR-1 protein in bacterial isolates using this assay, a blinded 99-sample test set was built that included three additional mcr-1-containing clinical isolates tested in triplicate (9 samples) and 90 negative control isolates. Results All of the mcr-1-containing isolates in the test set were accurately identified with no false positive detections by three independent, blinded operators, yielding an overall performance of 100% sensitivity and specificity for multiple operators. Among the three peptides tested in this study, the best performing was DTFPQLAK. The isolate-to-result time for the assay as implemented is less than 90 min. Conclusions This work demonstrates the feasibility of rapid detection of the MCR-1 protein in bacterial isolates by LC–MS/MS. Electronic supplementary material The online version of this article (10.1186/s12014-019-9228-2) contains supplementary material, which is available to authorized users.

Recent studies demonstrate that HDL's ability to promote cholesterol efflux from macrophages associates strongly with cardioprotection in humans independently of HDL-cholesterol (HDL-C) and apoA-I, HDL's major protein. However, the mechanisms that impair cholesterol efflux capacity during vascular disease are unclear. Inflammation, a well-established risk factor for cardiovascular disease, has been shown to impair HDL's cholesterol efflux capacity. We therefore tested the hypothesis that HDL's impaired efflux capacity is mediated by specific changes of its protein cargo. Humans with acute inflammation induced by low-level endotoxin had unchanged HDL-C levels, but their HDL-C efflux capacity was significantly impaired. Proteomic analyses demonstrated that HDL's cholesterol efflux capacity correlated inversely with HDL content of serum amyloid A (SAA)1 and SAA2. In mice, acute inflammation caused a marked impairment of HDL-C efflux capacity that correlated with a large increase in HDL SAA. In striking contrast, the efflux capacity of mouse inflammatory HDL was preserved with genetic ablation of SAA1 and SAA2. Our observations indicate that the inflammatory impairment of HDL-C efflux capacity is due in part to SAA-mediated remodeling of HDL's protein cargo.

Objective: Prior reports suggested that infection with Helicobacter pylori was associated with respiratory diseases; pathogenetic mechanisms however, were not defined. We tested the hypothesis that VacA, an exotoxin of H. pylori, a gastric pathogen, was aspirated into the lung and could stimulate secretion of inflammatory cytokines by lung epithelial cells. Methods: The presence of VacA was determined by immunohistochemistry in surgical lung biopsy tissue samples from 72 patients with interstitial pneumonia. The effects of VacA on A549 human alveolar epithelial adenocarcinoma cells and normal human bronchial epithelial cells were determined. After incubation with VacA, the secretions of cytokines were measured by Multiplex LuminexR Assays. Results: VacA was detected with anti-VacA antibodies in bronchial epithelial cells and alveolar epithelial cells from 10 of 72 patients with interstitial pneumonia. VacA was more prevalent in lungs of patients with collagen vascular disease-associated interstitial pneumonia than in those of patients with idiopathic pulmonary fibrosis, nonspecific interstitial pneumonia and cryptogenic organizing pneumonia. Incubation of A549 cells and normal human bronchial epithelial cells with VacA for 24 h was cytotoxic, and resulted in vacuolation. VacA induced interleukin-8 production by A549 cells and normal human bronchial epithelial cells and interleukin-6 production by A549 cells. Based on multiplex screening, interleukin-8 and interleukin-6 were the primary secretory products induced by VacA. Conclusions: H. pylori VacA is present in human lung and can induce interleukin-8 and interleukin-6 production by human lung cells. VacA could have a role in the pathogenesis of respiratory diseases by its cytotoxic effects and by inducing the secretion of interleukin-8 and interleukin-6 by targeted airway epithelial cells., Biochemical and Biophysical Research Communications, 460(3), pp.721-726; 2015

The mortality rate of alveolar hemorrhage (AH) after allogeneic hematopoietic stem cell transplantation is greater than 60% with supportive care and high-dose steroid therapy. We performed a retrospective cohort analysis to assess the benefits and risks of recombinant human factor VIIa (rFVIIa) as a therapeutic adjunct for AH. Between 2005 and 2012, 57 episodes of AH occurred in 37 patients. Fourteen episodes (in 14 patients) were treated with steroids alone, and 43 episodes (in 23 patients) were treated with steroids and rFVIIa. The median steroid dose was 1.9 mg/kg/d (interquartile range [IQR], 0.8 to 3.5 mg/kg/d; methylprednisolone equivalents) and did not differ statistically between the 2 groups. The median rFVIIa dose was 41 μg/kg (IQR, 39 to 62 μg/kg), and a median of 3 doses (IQR, 2 to 17) was administered per episode. Concurrent infection was diagnosed in 65% of the episodes. Patients had moderately severe hypoxia (median PaO2/FiO2, 193 [IQR, 141 to 262]); 72% required mechanical ventilation, and 42% survived to extubation. The addition of rFVIIa did not alter time to resolution of AH (P = .50), duration of mechanical ventilation (P = .89), duration of oxygen supplementation (P = .55), or hospital mortality (P = .27). Four possible thrombotic events (9% of 43 episodes) occurred with rFVIIa. rFVIIa in combination with corticosteroids did not confer clear clinical advantages compared with corticosteroids alone. In patients with AH following hematopoietic stem cell transplantation, clinical factors (ie, worsening infection, multiple organ failure, or recrudescence of primary disease) may be more important than the benefit of enhanced hemostasis from rFVIIa.

Objective The incidence of thoracic injuries resulting from cardiopulmonary resuscitation (CPR) is not well characterized. We describe a case in which a CPR-associated atrial rupture was identified with ultrasound and successfully managed in the intensive care unit with a bedside thoracotomy and atrial repair. We then describe a systematic review with pooled data analysis of CPR-associated cardiovascular, pulmonary, pleural, and thoracic wall injuries. Data sources PubMed, Scopus, EMBASE, and Web of Science were searched to identify relevant published studies. Unpublished studies were identified by searching the Australian and New Zealand Clinical Trials Registry, World Health Organization International Clinical Trials Registry Platform, Cochrane Library, ClinicalTrials.gov, Current Controlled Trials, and Google. Study selection Inclusion criteria for the pooled analysis were any clinical or autopsy study in which (a) patients underwent cardiopulmonary resuscitation, (b) chest compressions were administered either manually or with the assistance of active compression–decompression devices, and (c) autopsy or dedicated imaging assessments were conducted to identify complications. Exclusion criteria for the pooled analysis were pre-clinical studies, case reports and abstracts. Data extraction Nine-hundred twenty-eight potentially relevant references were identified. Twenty-seven references met inclusion criteria. Data synthesis A systematic review of the literature is provided with pooled data analysis. Conclusions The incidence of reported CPR-associated cardiovascular and thoracic wall injuries varies widely. CPR with active compression–decompression devices has a higher reported incidence of cardiopulmonary injuries. Bedside ultrasound may be a useful adjunct to assess and risk-stratify patients to identify serious or life-threatening CPR-associated injuries.