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Background Biomarkers that can risk-stratify children with influenza virus lower respiratory infection may identify patients for targeted intervention. Early elevation of alveolar-related proteins in the bloodstream in these patients could indicate more severe lung damage portending worse outcomes. Methods We used a mouse model of human influenza infection and evaluated relationships between lung pathophysiology and surfactant protein D (SP-D), SP-A, and Club cell protein 16 (CC16). We then measured SP-A, SP-D and CC16 levels in plasma samples from 94 children with influenza-associated acute respiratory failure (PICFLU cohort), excluding children with underlying conditions explaining disease severity. We tested for associations between levels of circulating proteins and disease severity including the diagnosis of acute respiratory distress syndrome (ARDS), mechanical ventilator, intensive care unit and hospital days, and hospital mortality. Results Circulating SP-D showed a greater increase than SP-A and CC16 in mice with increased alveolar-vascular permeability following influenza infection. In the PICFLU cohort, SP-D was associated with moderate-severe ARDS diagnosis (p=0.01) and with mechanical ventilator (r =0.45, p=0.002), ICU (r=0.44, p=0.002), and hospital days (r = 0.37, p=0.001) in influenza infected children without bacterial coinfection. Levels of SP-D were lower in children with secondary bacterial pneumonia (p=0.01) and not associated with outcomes. CC16 and SP-A levels did not differ with bacterial coinfection and were not consistently associated with severe outcomes. Conclusions SP-D has potential as an early circulating biomarker reflecting degree of lung damage caused directly by influenza virus infection in children. Secondary bacterial pneumonia alters SP-D biomarker performance. This article is protected by copyright. All rights reserved.

Background Chronic obstructive pulmonary disease (COPD) exacerbations are heterogenous and profoundly impact the disease trajectory. Bioactive lipid lysophosphatidic acid (LPA) has been implicated in airway inflammation but the significance of LPA in COPD exacerbation is not known. The aim of the study was to investigate the utility of serum LPA species (LPA16:0, 18:0, 18:1, 18:2, 20:4) as biomarkers of COPD exacerbation. Patients and methods LPA species were measured in the baseline placebo sera of a COPD randomized controlled trial. Tertile levels of each LPA were used to assign patients into biomarker high, medium, and low subgroups. Exacerbation rate and risk were compared among the LPA subgroups. Results The levels of LPA species were intercorrelated (rho 0.29–0.91). Patients with low and medium levels of LPA (LPA16:0, 20:4) had significantly higher exacerbation rate compared to the respective LPA-high patients [estimated rate per patient per year (95% CI)]: LPA16:0-low = 1.2 (0.8–1.9) (p = 0.019), LPA16:0-medium = 1.3 (0.8–2.0) (p = 0.013), LPA16:0-high = 0.5 (0.2–0.9); LPA20:4-low = 1.4 (0.9–2.1) (p = 0.0033), LPA20:4-medium = 1.2 (0.8–1.8) (p = 0.0089), LPA20:4-high = 0.4 (0.2–0.8). These patients also had earlier time to first exacerbation (hazard ratio (95% CI): LPA16:0-low = 2.6 (1.1–6.0) (p = 0.028), LPA16:0-medium = 2.7 (1.2–6.3) (p = 0.020); LPA20.4-low = 2.8 (1.2–6.6) (p = 0.017), LPA20:4-medium = 2.7 (1.2–6.4) (p = 0.021). Accordingly, these patients had a significant increased exacerbation risk compared to the respective LPA-high subgroups [odd ratio (95% CI)]: LPA16:0-low = 3.1 (1.1–8.8) (p = 0.030), LPA16:0-medium = 3.0 (1.1–8.3) (p = 0.031); LPA20:4-low = 3.8 (1.3–10.9) (p = 0.012), LPA20:4-medium = 3.3 (1.2–9.5) (p = 0.025). For the other LPA species (LPA18:0, 18:1, 18:2), the results were mixed; patients with low and medium levels of LPA18:0 and 18:2 had increased exacerbation rate, but only LPA18:0-low patients had significant increase in exacerbation risk and earlier time to first exacerbation compared to the LPA18:0-high subgroup. Conclusions The study provided evidence of association between systemic LPA levels and exacerbation in COPD. Patients with low and medium levels of specific LPA species (LPA16:0, 20:4) had increased exacerbation rate, risk, and earlier time to first exacerbation. These non-invasive biomarkers may aid in identifying high risk patients with dysregulated LPA pathway to inform risk management and drug development.

Lysophospholipids are bioactive signaling molecules derived from cell membrane glycerophospholipids or sphingolipids and are highly regulated under normal physiological conditions. Lysophosphatidic acids (LPAs) are a class of lysophospholipids that act on G-protein-coupled receptors to exert a variety of cellular functions. Dysregulation of phospholipase activity and consequently LPA synthesis in serum have been linked to inflammation, such as seen in chronic obstructive pulmonary disease (COPD). The accurate measurement of phospholipids is critical for evaluating their dysregulation in disease. In this study, we optimized experimental parameters for the sensitive measurement of LPAs. We validated the method based on matrix, linearity, accuracy, precision, and stability. An investigation into sample extraction processes emphasized that the common practice of including low concentration of hydrochloric acid in the extraction buffer causes an overestimation of lipid recovery. The liquid chromatography gradient was optimized to separate various lysophospholipid classes. After optimization, detection limits of LPA were sufficiently sensitive for subsequent analysis, ranging from 2 to 8 nM. The validated workflow was applied to a cohort of healthy donor and COPD patient sera. Eight LPA species were identified, and five unique species of LPA were quantified. Most LPA species increased significantly in COPD patients compared to healthy donors. The correlation between LPAs and other demographic parameters was further investigated in a sample set of over 200 baseline patient sera from a COPD clinical trial. For the first time, LPAs other than the two most abundant and readily detectable moieties are quantified in COPD patients using validated methods, opening the door to downstream biomarker evaluation in respiratory disease.

The mechanisms responsible for driving endogenous airway hyperresponsiveness (AHR) in the form of exercise-induced bronchoconstriction (EIB) are not fully understood. We examined alterations in airway phospholipid hydrolysis, surfactant degradation, and lipid mediator release in relation to AHR severity and changes induced by exercise challenge. Paired induced sputum ( n = 18) and bronchoalveolar lavage (BAL) fluid ( n = 11) were obtained before and after exercise challenge in asthmatic subjects. Samples were analyzed for phospholipid structure, surfactant function, and levels of eicosanoids and secreted phospholipase A2group 10 (sPLA2-X). A primary epithelial cell culture model was used to model effects of osmotic stress on sPLA2-X. Exercise challenge resulted in increased surfactant degradation, phospholipase activity, and eicosanoid production in sputum samples of all patients. Subjects with EIB had higher levels of surfactant degradation and phospholipase activity in BAL fluid. Higher basal sputum levels of cysteinyl leukotrienes (CysLTs) and prostaglandin D2(PGD2) were associated with direct AHR, and both the postexercise and absolute change in CysLTs and PGD2levels were associated with EIB severity. Surfactant function either was abnormal at baseline or became abnormal after exercise challenge. Baseline levels of sPLA2-X in sputum and the absolute change in amount of sPLA2-X with exercise were positively correlated with EIB severity. Osmotic stress ex vivo resulted in movement of water and release of sPLA2-X to the apical surface. In summary, exercise challenge promotes changes in phospholipid structure and eicosanoid release in asthma, providing two mechanisms that promote bronchoconstriction, particularly in individuals with EIB who have higher basal levels of phospholipid turnover.

Introduction COPD exacerbations are heterogeneous and can be triggered by bacterial, viral, or noninfectious insults. Exacerbations are also heterogeneous in neutrophilic or eosinophilic inflammatory responses. A noninvasive peripheral biomarker of COPD exacerbations characterised by bacterial/neutrophilic inflammation is lacking. Granulocyte-colony stimulating factor (G-CSF) is a key cytokine elevated during bacterial infection and mediates survival, proliferation, differentiation and function of neutrophils. Objective We hypothesised that high peripheral G-CSF would be indicative of COPD exacerbations with a neutrophilic and bacterial phenotype associated with microbial dysbiosis. Methods Serum G-CSF was measured during hospitalised exacerbation (day 0 or D0) and after 30 days of recovery (Day30 or D30) in 37 subjects. In a second cohort, serum and sputum cytokines were measured in 59 COPD patients during stable disease, at exacerbation, and at 2-weeks and 6-weeks following exacerbation. Results Serum G-CSF was increased during exacerbation in a subset of patients. These exacerbations were enriched for bacterial but not viral or type-2 biologies. The median serum G-CSF level was 1.6-fold higher in bacterial exacerbation compared to nonbacterial exacerbation (22 pg·mL−1 versus 13 pg·mL−1, p=0.0007). Serum G-CSF classified bacterial exacerbations with an area under the curve (AUC) for the receiver operating characteristic (ROC) curve equal to 0.76. Exacerbations with a two-fold or greater increase in serum G-CSF were characterised by neutrophilic inflammation, with increased sputum and blood neutrophils, and high sputum interleukin (IL)-1β, IL-6 and serum amyloid A1 (SAA1) levels. These exacerbations were preceded by dysbiosis, with decreased microbiome diversity and enrichment of respiratory pathogens such as Haemophilus and Moraxella. Furthermore, serum G-CSF at exacerbation classified neutrophilic-dysbiotic exacerbations (AUC for the ROC curve equal to 0.75). Conclusions High serum G-CSF enriches for COPD exacerbations characterised by neutrophilic inflammation with underlying bacterial dysbiosis., Noninvasive biomarkers to characterise #AECOPD subtypes are limited. High serum G-CSF enriches for COPD exacerbations associated with bacterial infection and neutrophilic inflammation preceded by lung microbial dysbiosis. https://bit.ly/3rck3M6

Introduction: Autotaxin-Lysophosphatidic acid (ATX-LPA) pathway has been associated with inflammatory lung conditions including COPD as it has pleiotropic roles in cell proliferation, lymphocyte homing and fibrosis. ATX functions primarily as lysophospholipase D to generate bioactive lipid LPA detected in blood and inflamed sites. Aims: We assessed if systemic LPA levels were associated with exacerbation frequency and severity in COPD patients. Methods: Mass spectrometry assays for LPA species (LPAs) were developed in-house using a custom lipid extraction method, and demonstrated good reproducibility and recovery. LPAs were measured in baseline serum samples from the placebo arm (n=136) of a COPD randomized controlled trial (NCT02546700). The relationship between LPA levels, baseline demographics, and other biomarkers were assessed using Mann-Whitney or Spearman method. Tertile levels of each LPA species were used to assign patients into biomarker high, mid, and low subgroups, and association with on-study exacerbation assessed by a Quasi-Poisson model. Results: Levels of LPA species were correlated with each other (Spearman rho 0.35 – 0.92), and were influenced by age, gender, region, and exacerbation history. After adjusting for these confounders, patients with the lowest tertile of LPAs had two times higher exacerbation rate in the 6-month follow-up, compared to patients with the highest tertile of LPAs (p Conclusions: These findings provide preliminary support that systemic LPAs are detectable in COPD and associate with exacerbation rate.

Although memory is pivotal to consciousness and without it no perceptual judgment or thinking is possible, Nyāya epistemology does not accept memory as a knowledge source (pramāņa). Prof Matilal elucidates and defends Udayana’s justification for calling into question the knowledgehood or even truth of any recollection. Deepening Matilal’s argument, this paper first shows why, if a remembering reproduces exactly the original experience from which it borrows its truth-claim, then there is a mismatch between the time of experience and the time of recall and the remembering ends up being false. To correct that error, if we change the tense in the content of recollection, the added past-ness goes beyond the original experience and violates the purely reproductive nature of memory. The paper ends by responding to this Nyāya position using arguments from Dvaita Vedānta and Jaina epistemology where remembering can be veridical and memory is accepted as an important knowledge source. The additional element of past-ness (a sense of “back-then”) cannot be derived from sense perception. It has to be a spontaneous contribution of the inner sense.

It is not a semantic accident that four key notions of social ethics are also key con- cepts of theater. These are the concepts of character, playing a part /role, performance, and acting. Of course, one could object that there is a touch of pun in this claim: A character in a drama is not quite the same as good or bad character in a virtue ethics; acting in theater is mere play-acting, whereas acting in social and personal life is serious business. But the distinction between play and serious business does not mean that the former is any less important than the latter. In Homo Ludens, his mag- isterial study of the play-element in culture, Johan Huizinga remarks: "Play is a thing by itself. The play-concept as such is of a higher order than is seriousness. For, seri- ousness seeks to exclude play, whereas play can very well include seriousness." 1 "The wise and learned," said Fu Yi (first century c.e.) in the preface to his famous tract On Dance, "should kno w how to balance playfulness and seriousness." 2 The Shakespearean fool often gives us the most serious insights not only into the plot and characters of the drama but into our own lives. "Chunyu Kun's sardonic laughter steered King Wei of Qi to a new course of action. Jester Meng's song of irony earned the firewood carrier his just reward. Jester Zhan's jocular reenactment of the atten- dant's discomfort (in front of the king) helped cut those men's long shift by half. These performances have real worthy impact too!" 3 To find out who is really the murderer of his father, Hamlet needed to watch the reactions of the prime suspects to a dramatic "enactment" of a similar fictional murder, because how we react to theater often 'betrays' what sort of persons we are in real life. A conscientious person of firm wisdom (sthita-prajna), whether in the Bhaga- vadgītā or in the Analects, performs actions not out of personal need, desire, or interest but in order to set an example to the community around him or her. This is not showing off: An element of demonstration or showing others and becoming conscious of being watched by others is central thus to both dramatic and socially exemplary action. In classical Chinese, Indian, and Greek cultures, theater (including music and dance) has been used from time immemorial as a spontaneous method of public aesthetic education. And it is increasingly clear, for contemporary television- and entertainment-addicted globalized youth as well as ancient secular humanity that, as Wittgenstein remarked, "Ethics and Aesthetics are one." 4 A film and a life can have a "good run" in the same ethico-aesthetic sense in w hich a team can have good "innings" in cric ket. Instead of focusing, therefore, on the distinction between pretending to do something and seriously doing it, between performing on stage and performing in real life, which would be invoked to distinguish play-acting from

SummaryThe NLRP3 inflammasome assembles in response to danger signals, triggering self-cleavage of procaspase-1 and production of the proinflammatory cytokine IL-1β. Although virus infection activates the NLRP3 inflammasome, the underlying events remain incompletely understood. We report that virus activation of the NLRP3 inflammasome involves the 2′,5′-oligoadenylate (2-5A) synthetase(OAS)/RNase L system, a component of the interferon-induced antiviral response that senses double-stranded RNA and activates endoribonuclease RNase L to cleave viral and cellular RNAs. The absence of RNase L reduces IL-1β production in influenza A virus-infected mice. RNA cleavage products generated by RNase L enhance IL-1β production but require the presence of 2′,3′-cyclic phosphorylated termini characteristic of RNase L activity. Additionally, these cleavage products stimulate NLRP3 complex formation with the DExD/H-box helicase, DHX33, and mitochondrial adaptor protein, MAVS, which are each required for effective NLRP3 inflammasome activation. Thus, RNA cleavage events catalyzed by RNase L are required for optimal inflammasome activation during viral infections.