Cytidine 5'-Diphosphocholine Corrects Alveolar Type II Cell Mitochondrial Dysfunction in Influenza-infected Mice.

Development of acute respiratory distress syndrome (ARDS) in influenza A virus (IAV)-infected mice is associated with inhibition of ATII (alveolar type II) epithelial cell de novo phosphatidylcholine synthesis, and administration of the phosphatidylcholine precursor cytidine 5'-diphosphocholine (CDP-choline) attenuates IAV-induced acute respiratory distress syndrome in mice. We hypothesized inhibition of phosphatidylcholine synthesis would also impact the function of ATII cell mitochondria. To test this hypothesis, adult C57BL/6 mice of both sexes were inoculated intranasally with 10,000 pfu/mouse influenza A/WSN/33 (H1N1). Control mice were mock-infected with virus diluent. Mice were treated with saline vehicle or CDP-choline (100 µg/mouse i.p.) once daily from 1 to 5 days postinoculation (dpi). ATII cells were isolated by a standard lung digestion protocol at 6 dpi for analysis of mitochondrial function. IAV infection increased uptake of the glucose analog fludeoxyglucose F 18 by the lungs and caused a switch from oxidative phosphorylation to aerobic glycolysis as a primary means of ATII cell ATP synthesis by 6 dpi. Infection also induced ATII cell mitochondrial depolarization and shrinkage, upregulation of PGC-1α, decreased cardiolipin content, and reduced expression of mitofusin 1, OPA1, DRP1, complexes I and IV of the electron transport chain, and enzymes involved in cardiolipin synthesis. Daily CDP-choline treatment prevented the declines in oxidative phosphorylation, mitochondrial membrane potential, and cardiolipin synthesis resulting from IAV infection but did not fully reverse the glycolytic shift. CDP-choline also did not prevent the alterations in mitochondrial protein expression resulting from infection. Taken together, our data show ATII cell mitochondrial dysfunction after IAV infection results from impaired de novo phospholipid synthesis, but the glycolytic shift does not.

Postexposure Liponucleotide Prophylaxis and Treatment Attenuates Acute Respiratory Distress Syndrome in Influenza-infected Mice.

There is an urgent need for new drugs for patients with acute respiratory distress syndrome (ARDS), including those with coronavirus disease (COVID-19). ARDS in influenza-infected mice is associated with reduced concentrations of liponucleotides (essential precursors for de novo phospholipid synthesis) in alveolar type II (ATII) epithelial cells. Because surfactant phospholipid synthesis is a primary function of ATII cells, we hypothesized that disrupting this process could contribute significantly to the pathogenesis of influenza-induced ARDS. The goal of this study was to determine whether parenteral liponucleotide supplementation can attenuate ARDS. C57BL/6 mice inoculated intranasally with 10,000 plaque-forming units/mouse of H1N1 influenza A/WSN/33 virus were treated with CDP (cytidine 5'-diphospho)-choline (100 µg/mouse i.p.) ± CDP -diacylglycerol 16:0/16:0 (10 µg/mouse i.p.) once daily from 1 to 5 days after inoculation (to model postexposure influenza prophylaxis) or as a single dose on Day 5 (to model treatment of patients with ongoing influenza-induced ARDS). Daily postexposure prophylaxis with CDP-choline attenuated influenza-induced hypoxemia, pulmonary edema, alterations in lung mechanics, impairment of alveolar fluid clearance, and pulmonary inflammation without altering viral replication. These effects were not recapitulated by the daily administration of CTP (cytidine triphosphate) and/or choline. Daily coadministration of CDP-diacylglycerol significantly enhanced the beneficial effects of CDP-choline and also modified the ATII cell lipidome, reversing the infection-induced decrease in phosphatidylcholine and increasing concentrations of most other lipid classes in ATII cells. Single-dose treatment with both liponucleotides at 5 days after inoculation also attenuated hypoxemia, altered lung mechanics, and inflammation. Overall, our data show that liponucleotides act rapidly to reduce disease severity in mice with severe influenza-induced ARDS.

Depletion of microRNA-451 in response to allergen exposure accentuates asthmatic inflammation by regulating Sirtuin2.

The incidence of asthma has increased from 5.5% to near 8% of the population, which is a major health concern. The hallmarks of asthma include eosinophilic airway inflammation that is associated with chronic airway remodeling. Allergic airway inflammation is characterized by a complex interplay of resident and inflammatory cells. MicroRNAs (miRNAs) are small noncoding RNAs that function as posttranscriptional modulators of gene expression. However, the role of miRNAs, specifically miR-451, in the regulation of allergic airway inflammation is unexplored. Our previous findings showed that oxidant stress regulates miR-451 gene expression in macrophages during an inflammatory process. In this paper, we examined the role of miR-451 in regulating macrophage phenotype using an experimental poly-allergenic murine model of allergic airway inflammation. We found that miR-451 contributes to the allergic induction of CCL17 in the lung and plays a key role in proasthmatic macrophage activation. Remarkably, administration of a Sirtuin 2 (Sirt2) inhibitor diminished alternate macrophage activation and markedly abrogated triple-allergen [dust mite, ragweed, Aspergillus fumigatus (DRA)]-induced lung inflammation. These data demonstrate a role for miR-451 in modulating allergic inflammation by influencing allergen-mediated macrophages phenotype.

ATP catabolism by tissue nonspecific alkaline phosphatase contributes to development of ARDS in influenza-infected mice.

Influenza A viruses are highly contagious respiratory pathogens that are responsible for significant morbidity and mortality worldwide on an annual basis. We have shown previously that influenza infection of mice leads to increased ATP and adenosine accumulation in the airway lumen. Moreover, we demonstrated that A1-adenosine receptor activation contributes significantly to influenza-induced acute respiratory distress syndrome (ARDS). However, we found that development of ARDS in influenza-infected mice does not require catabolism of ATP to adenosine by ecto-5'-nucleotidase (CD73). Hence, we hypothesized that increased adenosine generation in response to infection is mediated by tissue nonspecific alkaline phosphatase (TNAP), which is a low-affinity, high-capacity enzyme that catabolizes nucleotides in a nonspecific manner. In the current study, we found that whole lung and BALF TNAP expression and alkaline phosphatase enzymatic activity increased as early as 2 days postinfection (dpi) of C57BL/6 mice with 10,000 pfu/mouse of influenza A/WSN/33 (H1N1). Treatment at 2 and 4 dpi with a highly specific quinolinyl-benzenesulfonamide TNAP inhibitor (TNAPi) significantly reduced whole lung alkaline phosphatase activity at 6 dpi but did not alter TNAP gene or protein expression. TNAPi treatment attenuated hypoxemia, lung dysfunction, histopathology, and pulmonary edema at 6 dpi without impacting viral replication or BALF adenosine. Treatment also improved epithelial barrier function and attenuated cellular and humoral immune responses to influenza infection. These data indicate that TNAP inhibition can attenuate influenza-induced ARDS by reducing inflammation and fluid accumulation within the lung. They also further emphasize the importance of adenosine generation for development of ARDS in influenza-infected mice.

MiR-155 deletion reduces ischemia-induced paralysis in an aortic aneurysm repair mouse model: Utility of immunohistochemistry and histopathology in understanding etiology of spinal cord paralysis.

Spinal cord paralysis is relatively common after surgical repair of thoraco-abdominal aortic aneurysm (TAAA) and its etiology is unknown. The present study was designed to examine the histopathology of the disease and investigate whether miR-155 ablation would reduce spinal cord ischemic damage and delayed hindlimb paralysis induced by aortic cross-clamping (ACC) in our mouse model. The loss of locomotor function in ACC-paralyzed mice correlated with the presence of extensive gray matter damage and central cord edema, with minimal white matter histopathology. qRTPCR and Western blotting showed that the spinal cords of wild-type ACC mice that escaped paralysis showed lower miR-155 expression and higher levels of transcripts encoding Mfsd2a, which is implicated in the maintenance of blood-brain barrier integrity. In situ based testing demonstrated that increased miR-155 detection in neurons was highly correlated with the gray matter damage and the loss of one of its targets, Mfsd2a, could serve as a good biomarker of the endothelial cell damage. In vitro, we demonstrated that miR-155 targeted Mfsd2a in endothelial cells and motoneurons and increased endothelial cell permeability. Finally, miR-155 ablation slowed the progression of central cord edema, and reduced the incidence of paralysis by 40%. In sum, the surgical pathology findings clearly indicated that the epicenter of the ischemic-induced paralysis was the gray matter and that endothelial cell damage correlated to Mfsd2a loss is a good biomarker of the disease. MiR-155 targeting therefore offers new therapeutic opportunity for edema caused by traumatic spinal cord injury and diagnostic pathologists, by using immunohistochemistry, can clarify if this mechanism also is important in other ischemic diseases of the CNS, including stroke.

Lethal H1N1 influenza A virus infection alters the murine alveolar type II cell surfactant lipidome.

Alveolar type II (ATII) epithelial cells are the primary site of influenza virus replication in the distal lung. Development of acute respiratory distress syndrome in influenza-infected mice correlates with significant alterations in ATII cell function. However, the impact of infection on ATII cell surfactant lipid metabolism has not been explored. C57BL/6 mice were inoculated intranasally with influenza A/WSN/33 (H1N1) virus (10,000 plaque-forming units/mouse) or mock-infected with virus diluent. ATII cells were isolated by a standard lung digestion protocol at 2 and 6 days postinfection. Levels of 77 surfactant lipid-related compounds of known identity in each ATII cell sample were measured by ultra-high-performance liquid chromatography-mass spectrometry. In other mice, bronchoalveolar lavage fluid was collected to measure lipid and protein content using commercial assay kits. Relative to mock-infected animals, ATII cells from influenza-infected mice contained reduced levels of major surfactant phospholipids (phosphatidylcholine, phosphatidylglycerol, and phosphatidylethanolamine) but increased levels of minor phospholipids (phosphatidylserine, phosphatidylinositol, and sphingomyelin), cholesterol, and diacylglycerol. These changes were accompanied by reductions in cytidine 5'-diphosphocholine and 5'-diphosphoethanolamine (liponucleotide precursors for ATII cell phosphatidylcholine and phosphatidylethanolamine synthesis, respectively). ATII cell lamellar bodies were ultrastructurally abnormal after infection. Changes in ATII cell phospholipids were reflected in the composition of bronchoalveolar lavage fluid, which contained reduced amounts of phosphatidylcholine and phosphatidylglycerol but increased amounts of sphingomyelin, cholesterol, and protein. Influenza infection significantly alters ATII cell surfactant lipid metabolism, which may contribute to surfactant dysfunction and development of acute respiratory distress syndrome in influenza-infected mice.

Ecto-5'-nucleotidase CD73 modulates the innate immune response to influenza infection but is not required for development of influenza-induced acute lung injury.

Extracellular nucleotides and nucleosides are important signaling molecules in the lung. Nucleotide and nucleoside concentrations in alveolar lining fluid are controlled by a complex network of surface ectonucleotidases. Previously, we demonstrated that influenza A/WSN/33 (H1N1) virus resulted in increased levels of the nucleotide ATP and the nucleoside adenosine in bronchoalveolar lavage fluid (BALF) of wild-type (WT) C57BL/6 mice. Influenza-induced acute lung injury (ALI) was highly attenuated in A1-adenosine receptor-knockout mice. Because AMP hydrolysis by the ecto-5'-nucleotidase (CD73) plays a central role in and is rate-limiting for generation of adenosine in the normal lung, we hypothesized that ALI would be attenuated in C57BL/6-congenic CD73-knockout (CD73-KO) mice. Infection-induced hypoxemia, bradycardia, viral replication, and bronchoconstriction were moderately increased in CD73-KO mice relative to WT controls. However, postinfection weight loss, pulmonary edema, and parenchymal dysfunction were not altered. Treatment of WT mice with the CD73 inhibitor 5'-(α,ß-methylene) diphosphate (APCP) also had no effect on infection-induced pulmonary edema but modestly attenuated hypoxemia. BALF from CD73-KO and APCP-treated WT mice contained more IL-6 and CXCL-10/IFN-γ-induced protein 10, less CXCL-1/keratinocyte chemoattractant, and fewer neutrophils than BALF from untreated WT controls. BALF from APCP-treated WT mice also contained fewer alveolar macrophages and more transforming growth factor-ß than BALF from untreated WT mice. These results indicate that CD73 is not necessary for development of ALI following influenza A virus infection and suggest that tissue-nonspecific alkaline phosphatase may be responsible for increased adenosine generation in the infected lung. However, they do suggest that CD73 has a previously unrecognized immunomodulatory role in influenza.

Infection of mice with influenza A/WSN/33 (H1N1) virus alters alveolar type II cell phenotype.

Influenza viruses cause acute respiratory disease of great importance to public health. Alveolar type II (ATII) respiratory epithelial cells are central to normal lung function and are a site of influenza A virus replication in the distal lung. However, the consequences of infection for ATII cell function are poorly understood. To determine the impact of influenza infection on ATII cells we used C57BL/6-congenic SP-C(GFP) mice that express green fluorescent protein (GFP) under the control of the surfactant protein-C (SP-C) promoter, which is only active in ATII cells. Most cells isolated from the lungs of uninfected SP-C(GFP) mice were GFP(+) but did not express the alveolar type I (ATI) antigen podoplanin (PODO). ATII cells were also EpCAM(+) and α2,3-linked sialosaccharide(+). Infection with influenza A/WSN/33 virus caused severe hypoxemia and pulmonary edema. This was accompanied by loss of whole lung GFP fluorescence, reduced ATII cell yields, increased ATII cell apoptosis, reduced SP-C gene and protein expression in ATII cell lysates, and increased PODO gene and protein levels. Flow cytometry indicated that infection decreased GFP(+)/PODO(-) cells and increased GFP(-)/PODO(+) and GFP(-)/PODO(-) cells. Very few GFP(+)/PODO(+) cells were detectable. Finally, infection resulted in a significant decline in EpCAM expression by PODO(+) cells, but had limited effects on α2,3-linked sialosaccharides. Our findings indicate that influenza infection results in a progressive differentiation of ATII cells into ATI-like cells, possibly via an SP-C(-)/PODO(-) intermediate, to replace dying or dead ATI cells. However, impaired SP-C synthesis is likely to contribute significantly to reduced lung compliance in infected mice.

TGF-ß-induced IL-6 prevents development of acute lung injury in influenza A virus-infected F508del CFTR-heterozygous mice.

As the eighth leading cause of annual mortality in the USA, influenza A viruses are a major public health concern. In 20% of patients, severe influenza progresses to acute lung injury (ALI). However, pathophysiological mechanisms underlying ALI development are poorly defined. We reported that, unlike wild-type (WT) C57BL/6 controls, influenza A virus-infected mice that are heterozygous for the F508del mutation in the cystic fibrosis transmembrane conductance regulator (HETs) did not develop ALI. This effect was associated with higher IL-6 and alveolar macrophages (AMs) at 6 days postinfection (d.p.i.) in HET bronchoalveolar lavage fluid (BALF). In the present study, we found that HET AMs were an important source of IL-6 at 6 d.p.i. Infection also induced TGF-ß production by HET but not WT mice at 2 d.p.i. TGF-ß neutralization at 2 d.p.i. (TGF-N) significantly reduced BALF IL-6 in HETs at 6 d.p.i. Neither TGF-N nor IL-6 neutralization at 4 d.p.i. (IL-6-N) altered postinfection weight loss or viral replication in either mouse strain. However, both treatments increased influenza A virus-induced hypoxemia, pulmonary edema, and lung dysfunction in HETs to WT levels at 6 d.p.i. TGF-N and IL-6-N did not affect BALF AM and neutrophil numbers but attenuated the CXCL-1/keratinocyte chemokine response in both strains and reduced IFN-γ production in WT mice. Finally, bone marrow transfer experiments showed that HET stromal and myeloid cells are both required for protection from ALI in HETs. These findings indicate that TGF-ß-dependent production of IL-6 by AMs later in infection prevents ALI development in influenza A virus-infected HET mice.

Activation of A1-adenosine receptors promotes leukocyte recruitment to the lung and attenuates acute lung injury in mice infected with influenza A/WSN/33 (H1N1) virus.

UNLABELLED: We have shown that bronchoalveolar epithelial A1-adenosine receptors (A1-AdoR) are activated in influenza A virus-infected mice. Alveolar macrophages and neutrophils also express A1-AdoRs, and we hypothesized that activation of A1-AdoRs on these cells will promote macrophage and neutrophil chemotaxis and activation and thereby play a role in the pathogenesis of influenza virus-induced acute lung injury. Wild-type (WT) C57BL/6 mice, congenic A1-AdoR knockout (A1-KO) mice, and mice that had undergone reciprocal bone marrow transfer were inoculated intranasally with 10,000 PFU/mouse influenza A/WSN/33 (H1N1) virus. Alternatively, WT mice underwent daily treatment with the A1-AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) from 1 day prior to inoculation. Infection increased bronchoalveolar lining fluid (BALF) adenosine comparably in WT and A1-KO mice. Infection of WT mice resulted in reduced carotid arterial O2 saturation (hypoxemia), lung pathology, pulmonary edema, reduced lung compliance, increased basal airway resistance, and hyperresponsiveness to methacholine. These effects were absent or significantly attenuated in A1-KO mice. Levels of BALF leukocytes, gamma interferon (IFN-γ), and interleukin 10 (IL-10) were significantly reduced in infected A1-KO mice, but levels of KC, IP-10, and MCP-1 were increased. Reciprocal bone marrow transfer resulted in WT-like lung injury severity, but BALF leukocyte levels increased only in WT and A1-KO mice with WT bone barrow. Hypoxemia, pulmonary edema, and levels of BALF alveolar macrophages, neutrophils, IFN-γ, and IL-10 were reduced in DPCPX-treated WT mice. Levels of viral replication did not differ between mouse strains or treatment groups. These findings indicate that adenosine activation of leukocyte A1-AdoRs plays a significant role in their recruitment to the infected lung and contributes to influenza pathogenesis. A1-AdoR inhibitor therapy may therefore be beneficial in patients with influenza virus-induced lung injury. IMPORTANCE: Because antiviral drugs are of limited efficacy in patients hospitalized for influenza virus-induced respiratory failure, there is an urgent need for new therapeutics that can limit the progression of lung injury and reduce influenza death rates. We show that influenza A virus infection results in increased production of the nucleoside adenosine in the mouse lung and that activation of A1-subtype adenosine receptors by adenosine contributes significantly to both recruitment of innate immune cells to the lung and development of acute lung injury following influenza virus infection. We also show that treatment with an A1-adenosine receptor antagonist reduces the severity of lung injury in influenza virus-infected mice. Our findings indicate that adenosine plays an important and previously unrecognized role in the innate immune response to influenza virus infection and suggest that drugs which can inhibit either generation of adenosine or activation of A1-adenosine receptors may be beneficial in treating influenza patients hospitalized for respiratory failure.

Mouse Models of Acute Respiratory Distress Syndrome: A Review of Analytical Approaches, Pathologic Features, and Common Measurements.

Acute respiratory distress syndrome (ARDS) is a severe pulmonary reaction requiring hospitalization, which is incited by many causes, including bacterial and viral pneumonia as well as near drowning, aspiration of gastric contents, pancreatitis, intravenous drug use, and abdominal trauma. In humans, ARDS is very well defined by a list of clinical parameters. However, until recently no consensus was available regarding the criteria of ARDS that should be evident in an experimental animal model. This lack was rectified by a 2011 workshop report by the American Thoracic Society, which defined the main features proposed to delineate the presence of ARDS in laboratory animals. These should include histological changes in parenchymal tissue, altered integrity of the alveolar capillary barrier, inflammation, and abnormal pulmonary function. Murine ARDS models typically are defined by such features as pulmonary edema and leukocyte infiltration in cytological preparations of bronchoalveolar lavage fluid and/or lung sections. Common pathophysiological indicators of ARDS in mice include impaired pulmonary gas exchange and histological evidence of inflammatory infiltrates into the lung. Thus, morphological endpoints remain a vital component of data sets assembled from animal ARDS models.

Depletion of the ubiquitin-binding adaptor molecule SQSTM1/p62 from macrophages harboring cftr ΔF508 mutation improves the delivery of Burkholderia cenocepacia to the autophagic machinery.

Cystic fibrosis is the most common inherited lethal disease in Caucasians. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), of which the cftr ΔF508 mutation is the most common. ΔF508 macrophages are intrinsically defective in autophagy because of the sequestration of essential autophagy molecules within unprocessed CFTR aggregates. Defective autophagy allows Burkholderia cenocepacia (B. cepacia) to survive and replicate in ΔF508 macrophages. Infection by B. cepacia poses a great risk to cystic fibrosis patients because it causes accelerated lung inflammation and, in some cases, a lethal necrotizing pneumonia. Autophagy is a cell survival mechanism whereby an autophagosome engulfs non-functional organelles and delivers them to the lysosome for degradation. The ubiquitin binding adaptor protein SQSTM1/p62 is required for the delivery of several ubiquitinated cargos to the autophagosome. In WT macrophages, p62 depletion and overexpression lead to increased and decreased bacterial intracellular survival, respectively. In contrast, depletion of p62 in ΔF508 macrophages results in decreased bacterial survival, whereas overexpression of p62 leads to increased B. cepacia intracellular growth. Interestingly, the depletion of p62 from ΔF508 macrophages results in the release of the autophagy molecule beclin1 (BECN1) from the mutant CFTR aggregates and allows its redistribution and recruitment to the B. cepacia vacuole, mediating the acquisition of the autophagy marker LC3 and bacterial clearance via autophagy. These data demonstrate that p62 differentially dictates the fate of B. cepacia infection in WT and ΔF508 macrophages.

Heterozygosity for the F508del mutation in the cystic fibrosis transmembrane conductance regulator anion channel attenuates influenza severity.

BACKGROUND: Seasonal and pandemic influenza are significant public health concerns. Influenza stimulates respiratory epithelial Cl(-) secretion via the cystic fibrosis transmembrane conductance regulator (CFTR). The purpose of this study was to determine the contribution of this effect to influenza pathogenesis in mice with reduced CFTR activity. METHODS: C57BL/6-congenic mice heterozygous for the F508del CFTR mutation (HET) and wild-type (WT) controls were infected intranasally with 10 000 focus-forming units of influenza A/WSN/33 (H1N1) per mouse. Body weight, arterial O2 saturation, and heart rate were monitored daily. Pulmonary edema and lung function parameters were derived from ratios of wet weight to dry weight and the forced-oscillation technique, respectively. Levels of cytokines and chemokines in bronchoalveolar lavage fluid were measured by enzyme-linked immunosorbent assay. RESULTS: Relative to WT mice, influenza virus-infected HET mice showed significantly delayed mortality, which was accompanied by attenuated hypoxemia, cardiopulmonary dysfunction, and pulmonary edema. However, viral replication and weight loss did not differ. The protective HET phenotype was correlated with exaggerated alveolar macrophage and interleukin 6 responses to infection and was abrogated by alveolar macrophage depletion, using clodronate liposomes. CONCLUSIONS: Reduced CFTR expression modulates the innate immune response to influenza and alters disease pathogenesis. CFTR-mediated Cl(-) secretion is therefore an important host determinant of disease, and CFTR inhibition may be of therapeutic benefit in influenza.

SCARLET (Supplemental Citicoline Administration to Reduce Lung injury Efficacy Trial): study protocol for a single-site, double-blinded, placebo-controlled, and randomized Phase 1/2 trial of i.v. citicoline (CDP-choline) in hospitalized SARS CoV-2-infected patients with hypoxemic acute respiratory failure.

BACKGROUND: The SARS CoV-2 pandemic has resulted in more than 1.1 million deaths in the USA alone. Therapeutic options for critically ill patients with COVID-19 are limited. Prior studies showed that post-infection treatment of influenza A virus-infected mice with the liponucleotide CDP-choline, which is an essential precursor for de novo phosphatidylcholine synthesis, improved gas exchange and reduced pulmonary inflammation without altering viral replication. In unpublished studies, we found that treatment of SARS CoV-2-infected K18-hACE2-transgenic mice with CDP-choline prevented development of hypoxemia. We hypothesize that administration of citicoline (the pharmaceutical form of CDP-choline) will be safe in hospitalized SARS CoV-2-infected patients with hypoxemic acute respiratory failure (HARF) and that we will obtain preliminary evidence of clinical benefit to support a larger Phase 3 trial using one or more citicoline doses. METHODS: We will conduct a single-site, double-blinded, placebo-controlled, and randomized Phase 1/2 dose-ranging and safety study of Somazina® citicoline solution for injection in consented adults of any sex, gender, age, or ethnicity hospitalized for SARS CoV-2-associated HARF. The trial is named "SCARLET" (Supplemental Citicoline Administration to Reduce Lung injury Efficacy Trial). We hypothesize that SCARLET will show that i.v. citicoline is safe at one or more of three doses (0.5, 2.5, or 5 mg/kg, every 12 h for 5 days) in hospitalized SARS CoV-2-infected patients with HARF (20 per dose) and provide preliminary evidence that i.v. citicoline improves pulmonary outcomes in this population. The primary efficacy outcome will be the SpO2:FiO2 ratio on study day 3. Exploratory outcomes include Sequential Organ Failure Assessment (SOFA) scores, dead space ventilation index, and lung compliance. Citicoline effects on a panel of COVID-relevant lung and blood biomarkers will also be determined. DISCUSSION: Citicoline has many characteristics that would be advantageous to any candidate COVID-19 therapeutic, including safety, low-cost, favorable chemical characteristics, and potentially pathogen-agnostic efficacy. Successful demonstration that citicoline is beneficial in severely ill patients with SARS CoV-2-induced HARF could transform management of severely ill COVID patients. TRIAL REGISTRATION: The trial was registered at www. CLINICALTRIALS: gov on 5/31/2023 (NCT05881135). TRIAL STATUS: Currently enrolling.

Contrast transthoracic echocardiography and the placement of a bicaval dual-lumen catheter in a Swine model of venovenous extracorporeal membrane oxygenation.

Placement of a bicaval dual-lumen (BCDL) catheter demands sophisticated visualization in patients to assure proper positioning in order to administer single-site venovenous extracorporeal membrane oxygenation (VV ECMO). Large animal models are needed and thus appropriate procedures to assure anatomic and functional cannula placement would assist in experimental design and procedures. This report describes the use of agitated blood and saline transthoracic contrast echocardiography to confirm appropriate placement and function of the BCDL catheter in a swine model of VV ECMO. Five consecutive common crossbred piglets had confirmation using this technique with assurances of cannulation while not significantly altering experimental time and procedures. Researchers studying VV ECMO in large animal models may want to consider this method of confirmation of BCDL catheter placement.

Postinfection A77-1726 treatment improves cardiopulmonary function in H1N1 influenza-infected mice.

Acute respiratory disease is associated with significant morbidity and mortality in influenza. Because antiviral drugs are only effective early in infection, new agents are needed to treat nonvaccinated patients presenting with late-stage disease, particularly those who develop acute respiratory distress syndrome. We found previously that the de novo pyrimidine synthesis inhibitor A77-1726 reversed the influenza-induced impairment of alveolar fluid clearance. Patients with acute respiratory distress syndrome and intact alveolar fluid clearance demonstrate lower mortality than those with compromised fluid clearance. We therefore investigated the effects of treatment with nebulized A77-1726 (67.5 mg/kg) on indices of cardiopulmonary function relevant to the diagnosis of acute respiratory distress syndrome. BALB/cAnNCr mice (8-12 wk old) were inoculated intranasally with 10,000 plaque-forming units/mouse influenza A/WSN/33 (H1N1). Pulse oximetry was performed daily. Alveolar fluid clearance, lung water, and lung mechanics were measured at 2 and 6 days after inoculation in live, ventilated mice by BSA instillation, magnetic resonance imaging, and forced-oscillation techniques, respectively. A77-1726 treatment at 1 day after inoculation delayed mortality. Treatment on Days 1 or 5 reduced viral replication on Day 6, and improved alveolar fluid clearance, peripheral oxygenation, and cardiac function. Nebulized A77-1726 also reversed influenza-induced increases in lung water content and volume, improved pulmonary mechanics, reduced bronchoalveolar lavage fluid ATP and neutrophil content, and increased IL-6 concentrations. The ability of A77-1726 to improve cardiopulmonary function in influenza-infected mice and to reduce the severity of ongoing acute respiratory distress syndrome late in infection suggests that pyrimidine synthesis inhibitors are promising therapeutic candidates for the management of severe influenza.