key: cord- -ec qzurk authors: devaney, james; contreras, maya; laffey, john g title: clinical review: gene-based therapies for ali/ards: where are we now? date: - - journal: crit care doi: . /cc sha: doc_id: cord_uid: ec qzurk acute lung injury (ali) and acute respiratory distress syndrome (ards) confer substantial morbidity and mortality, and have no specific therapy. the accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ali/ards, suggest that the disease may be amenable to gene-based therapies. ongoing advances in our understanding of the pathophysiology of ali/ards have revealed multiple therapeutic targets for gene-based approaches. strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ali/ards have all demonstrated promise in preclinical models. despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ali/ards remains to be realized. multiple barriers to effective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection efficiency of nonviral delivery methods. deficits remain in our knowledge regarding the optimal molecular targets for gene-based approaches. encouragingly, recent progress in overcoming these barriers offers hope for the successful translation of gene-based approaches for ali/ards to the clinical setting. gene-based therapy involves the insertion of genes or smaller nucleic acid sequences into cells and tissues to replace the function of a defective gene, or to alter the production of a specifi c gene product, in order to treat a disease. gene therapy can be classifi ed into germline and somatic gene therapies. germline approaches modify the sperm or egg prior to fertilization and confer a stable heritable genetic modifi cation. somatic gene approaches use gene therapy to alter the function of mature cells. commonly used somatic gene therapy strategies include the overexpression of an existing gene and/or the insertion of smaller nucleic acid sequences into cells to alter the production of an existing gene. ali/ards may be suitable for gene-based therapies as it is an acute but relatively transient process [ ] , requiring short-lived gene expression, obviating the need for repeated therapies and reducing the risk of an adverse immunological response. th e distal lung epithelium is selectively accessible via the tracheal route of administration, allowing targeting of the pulmonary epithelium [ ] . th e pulmonary vasculature is also relatively accessible, as the entire cardiac output must transit this circulation. antibodies that bind antigens selectively expressed on the pulmonary endothelial surface can be complexed to gene vectors to facilitate selective targeting following intravenous administration [ ] . it is also possible to use gene-based strategies to target other cells central to the pathogenesis of ali/ards, such as leuko cytes and abstract acute lung injury (ali) and acute respiratory distress syndrome (ards) confer substantial morbidity and mortality, and have no specifi c therapy. the accessibility of the distal lung epithelium via the airway route, and the relatively transient nature of ali/ ards, suggest that the disease may be amenable to gene-based therapies. ongoing advances in our understanding of the pathophysiology of ali/ards have revealed multiple therapeutic targets for genebased approaches. strategies to enhance or restore lung epithelial and/or endothelial cell function, to strengthen lung defense mechanisms against injury, to speed clearance of infection and to enhance the repair process following ali/ards have all demonstrated promise in preclinical models. despite three decades of gene therapy research, however, the clinical potential for gene-based approaches to lung diseases including ali/ ards remains to be realized. multiple barriers to eff ective pulmonary gene therapy exist, including the pulmonary architecture, pulmonary defense mechanisms against inhaled particles, the immunogenicity of viral vectors and the poor transfection effi ciency of nonviral delivery methods. defi cits remain in our knowledge regarding the optimal molecular targets for genebased approaches. encouragingly, recent progress in overcoming these barriers off ers hope for the successful translation of gene-based approaches for ali/ards to the clinical setting. fi bro blasts [ ] . furthermore, gene-therapy-based approaches off er the potential to selectively target diff erent phases of the injury and repair process. th e potential to target specifi c aspects of the injury and repair processes such as epithelial-mesenchymal transition, fi brosis, fi brinolysis, coagulopathy and oxidative stress with these approaches is also clear. gene therapy requires the delivery of genes or smaller nucleic acid sequences into the cell nucleus using a carrier or vector. th e vector enables the gene to overcome barriers to entry into the cell, and to make its way to the nucleus to be transcribed and translated itself or to modulate transcription and/or translation of other genes. both viral and nonviral vector systems have been developed (table ) . viral vectors are the most eff ective and effi cient way of getting larger nucleic acid sequences, particularly genes, into cells (table ) . th e viral genome is modifi ed to remove the parts necessary for viral replication. th is segment is then replaced with the gene of interesttermed a transgene -coupled to a promoter that drives its expression. th e modifi ed genome is then encapsulated with viral proteins. following delivery to the target site, the virus binds to the host cell, enters the cytoplasm and releases its payload into the nucleus (figure ). th e size of trans gene that can be used depends on the capsid size. a number of diff erent viral vectors have been used in preclinical lung injury studies to date. adenoviruses have double-stranded dna genomes, have demonstrated promise in preclinical models [ , ] and are well tolerated at low to intermediate doses in humans [ , ] . advantages include their ease of production, the high effi ciency at which they can infect the pulmonary epithelium [ , ] and that they can deliver relatively large transgenes. a disadvantage of adenoviruses is their immunogenicity, particularly in repeated doses [ ] . newer adenoviral vectors, in which much of the immuno genicity has been removed, hold promise [ ] . while adenovirus-mediated gene transfer in the absence of epithelial damage is relatively ineffi cient [ ] , this may be less of a problem in ali/ards that is characterized by widespread epithelial damage. adeno-associated viruses (aavs) are single-stranded dna parvoviruses that are replication defi cient [ ] . a substantial proportion of the human population has been exposed to aavs but the clinical eff ects are unknown. aav vectors have a good safety profi le, and are less immunogenic compared with other viruses, although anti bodies do develop against aav capsid proteins that can compromise repeat administration. aav vectors can insert genes at a specifi c site on chromosome . th e packaging capacity of the virus is limited to . kb, restricting the size of the transgene that can be used. aavs are less effi cient in transducing cells than adenoviral vectors. successful aav vector gene transfer has been demon strated in multiple lung cell types including lung progenitor cells, in both normal and naphthaleneinduced ali lungs [ ] . aav serotypes have specifi c tissue tropisms, due to diff erent capsid proteins that bind to specifi c cell membrane receptors. aav- [ ] and avv- [ ] exhibit enhanced tropism for the pulmonary epi thelium [ , ] . aavs can transduce nondividing cells and result in long-lived transgene expression. aav vectors have been used in clinical trials in cystic fi brosis patients, underlining their safety profi le [ , ] . th ese rna viruses can transfect nondividing cells such as mature airway epithelial cells [ ] . th e virus stably but randomly integrates into the genome and expression is likely to last for the lifetime of the cell (~ days). th e transgene can be transmitted post mitosis, and there is also a risk of tumorigenesis if the transgene integrates near an oncogene. th e development of leukemias in children following gene therapy for severe combined immunodefi ciency highlights this risk [ , ] . while lentiviral vectors may be useful to correct a gene defi ciency associated with increased risk of ali, the long-lived gene expression of lentiviral delivered genes may be more suitable for chronic diseases than for ali/ards. nonviral delivery systems, while generally less effi cient than viral vectors in transfecting the lung epithelium, are increasingly used to deliver smaller dna/rna molecules (table ). strategies include the use of dna-lipid and dna-polymer complexes and naked dna/rna oligonucleotides, such as sirna [ ] , decoy oligo nucleo tides [ ] and plasmid dna [ ] . nonviral delivery systems are less immunogenic than viral vector-based approaches, and can be generated in large amounts at relatively low cost. plasmid vectors are composed of closed circles of doublestranded dna. as naked and plasmid dna contain no proteins for attachment to cellular receptors, there is no specifi c targeting to diff erent cell types and thus it is essential that the dna is placed in close contact with the desired cell type. th ese limitations make this approach less relevant clinically. th e therapeutic dna is held within a sphere of lipids, termed a lipoplex, or within a sphere of polymers, such as polyethyleneimine, termed a polyplex. lipoplexes and polyplexes act to protect the dna, facilitate binding to the target cell membrane and also trigger endocytosis of the complex into the cell, thereby enhancing gene expression. th ese systems can be modifi ed to include a targeting peptide for a specifi c cell type, such as airway epithelial cells [ ] . th ese complexes effi ciently and safely transfect airway epithelial cells [ ] , and they have demonstrated promise in human studies [ ] . sirnas are dsrna molecules of to nucleotides that can regulate the expression of specifi c genes. specifi c sirnas reduce infl ammation-associated lung injury in table . viral vector-delivered gene therapy relatively easily produced immunogenic [ ] adenoviral transfer of genes for a surfactant (dsdna genome) effi ciently transfect lung enzyme [ ] , angiopoietin- [ ] , hsp- [ ] , epithelium [ , ] apolipoprotein a- [ ] , and na + ,k + -atpase pump can deliver larger genes [ ] genes attenuate experimental ali well tolerated in lower doses [ , ] adenoviral delivery of il- gene attenuates zymosan ali at low doses, but is harmful at high doses [ ] adeno-associated virus good safety profi le; less limited transgene size aav vector gene transfer demonstrated in multiple vectors (ssdna genome) immunogenic diffi cult to produce in large lung cell types including progenitor cells in both inherently replication defi cient quantities normal lungs and following naphthalene-induced aav- and aav- lung epithelial ali [ ] tropism [ , ] transduce nondividing cells long-lived gene expression used in clinical trials for cf [ , ] lentivirus vectors transduce nondividing cells [ ] oncogenesis risk due to lentiviral transfer of shrna to silence cd gene (rna genome) integrate stably but randomly integration into genome expression suppresses silica-induced lung fi brosis into the genome [ , ] in the rat [ ] nonviral gene-based strategies plasmid transfer (closed easily produced at low cost no specifi c cell targeting electroporation-mediated gene transfer of the dsdna circles) very ineffi cient na + ,k + -atpase rescues endotoxin-induced lung injury [ ] nonviral dna complexes complexes protect dna less effi cient than viral vectors cationic lipid-mediated transfer of the na + ,k + -(lipoplexes or polyplexes) complexes facilitate cellular atpase gene ameliorated high-permeability targeting [ ] pulmonary edema [ ] lipoplex-delivered il- gene decreased clp-induced ali [ ] systemic cationic polyethylenimine polyplexes incorporating indoleamine- , -dioxygenase decreased ischemia-reperfusion ali [ ] dna and rna easily produced at low cost no specifi c cell targeting specifi c sirnas reduce infl ammation-associated oligonucleotides (sirna, smaller molecules that can lung injury in humans [ ] and in animal models shrna, decoy easily enter cells [ , ] oligonucleotides) target regulation of specifi c genes shrna-based approaches have reduced lung injury in animal models [ , ] cell-delivered gene therapy humans [ ] and in animal models [ , ] . shrna is a single strand of rna that, when introduced into the cell, is reverse transcribed and integrated into the genome, becoming heritable. during subsequent transcription, the sequence generates an oligonucleotide with a tight hairpin turn that is processed into sirna. shrnas have reduced lung injury in animal models [ , ] . decoy oligonucleotides are double-stranded dna molecules of to nucleo tides, which bind to specifi c transcription factors to reduce expression of targeted genes, and have been successfully used in animal models [ , ] . an alternative approach is to use systemically delivered cells to deliver genes to the lung. th is approach has been used to enhance the therapeutic potential of stem cellssuch as mesenchymal stem/stromal cells, which demon strate promise in preclinical ali/ards models [ ] . fibroblasts have also been used to successfully deliver genes to the lung to attenuate ali [ ] . preliminary data from a clinical trial in pulmonary hypertension show that endothelial progenitor cells overexpressing endothelial nitric oxide synthase (nos ) decrease pulmonary vascular resistance [ ] , highlighting the potential of cell-delivered gene therapy for ali/ards. nebulization of genetic material into the lung is eff ective [ ] , safe and well tolerated [ , , ] . th e integrity of aav vectors [ , ] and adenoviral virus vectors [ ] are maintained post nebulization, as are cationic lipid vectors [ ] and dna and rna oligonucleotides [ ] . a number of gene therapy clinical trials have utilized nebulization to deliver the transgene to the lung [ , ] , but without clear clinical benefi t to date [ , ] . intravascular delivery approaches target the lung endothelium. th ese approaches have been successfully used in preclinical studies of cell-based gene therapies [ , ] , and also with vectors that incorporate components such as antibodies to target antigens on the lung endothelium [ ] . successful gene-based therapies require the delivery of high quantities of the gene or oligonucleotide to the pulmonary epithelial or endothelial surface, require effi cient entry into the cytoplasm of these large and insoluble nucleic acids, which then have to move from the cytoplasm into the nucleus, and activate transcription of its product. multiple barriers exist that hinder this process, not least the natural defense mechanisms of the lung, and additional diffi culties that exist in transducing the acutely injured lung (table ). limitations regarding delivery technologies and defi ciencies in our knowledge regarding the optimal molecular targets also reduce the effi cacy of these approaches. th e lung has evolved eff ective barriers to prevent the uptake of any inhaled foreign particles [ ] . while advantageous in minimizing the potential for uptake of external genetic material (for example, viral dna), these barriers make it more diffi cult to use gene-based therapies in the lung. barriers to entry of foreign genetic material into the lung include airway mucus and the epithelial lining fl uid, which traps and clears inhaled material. th e glycocalyceal barrier hinders contact with the cell membrane, while the tight intercellular epithelial junctions and limited luminal endocytosis further restrict entry of foreign material into the epithelial cells. transducing the acutely injured lung may be diffi cult, due to the presence of pulmonary edema, consolidated or collapsed alveoli, and additional extracellular barriers such as mucus. gene-based therapies targeted at the pulmonary epithelium may be less eff ective where there is extensive denudation of the pulmonary epithelium, as may occur in primary ards. encouragingly, there is some evidence to suggest that ali may not substantially impair viral gene transfer to the alveolar epithelium [ ] . th e key limitation of nonviral vector approaches has been their lack of effi ciency in mediating gene transfer and transgene expression in the airway epithelium. viral vectors are immunogenic, due to the protein coat of the viral vector, and the immune response is related to both vector dose and number of administrations. th e potential to limit administration to a single dose in ali/ards may reduce this risk. however, the development of an infl amma tory response resulting in death following administration of a fi rst-generation adenoviral vector highlights the risks involved [ ] . additional limitations of viral vectors include transgene size, which is limited by the size of the capsid that encloses the viral genes. th e therapeutic potential of gene therapy for ali/ards is underlined by a growing body of literature demon strating effi cacy in relevant preclinical models. in considering the clinical implications of these studies, it is important to acknowledge that animal models of ards do not fully replicate the complex pathophysiological changes seen in the clinical setting. th is is highlighted by the fact that many pharmacologic strategies demonstrating considerable promise in preclinical studies were later proven ineff ective in clinical trials. nevertheless, these studies provide insights into the clinical potential of these strategies. adenovirus-mediated transfer of a gene that enhances surfactant production improves lung function and confers resistance to pseudomonas aeruginosa infection ( figure ) [ ] . adenovirus-delivered superoxide dismutase and catalase genes protected against hyperoxic-induced, but not ischemia-reperfusion-induced, lung injury [ ] . more recent studies have demonstrated the therapeutic potential of overexpression of a number of genes, including angio poietin- [ ] , hsp- [ ] , apolipo protein a- [ ] , defensin β [ ] and the na + ,k + -atpase pump [ ] . in contrast, overexpression of il- β can directly cause ali [ ] , while overexpression of suppressor of cytokine signal ing- worsens immune-complex-induced ali [ ] . intriguingly, intra tracheal administration of adenoviral vector incor porating il- , prior to zymosan-induced lung injury, improved survival at a lower dose but was ineff ective and even harmful at higher doses [ ] . an early murine study demonstrated that cationic lipidmediated transfer of the na + ,k + -atpase gene ameliorated high-permeability pulmonary edema [ ] . electroporationassisted gene transfer of plasmids encoding for na + ,k + -atpase reverses endotoxin-induced lung injury [ ] . th e lipoplex-delivered il- gene decreased lung and systemic organ injury induced by cecal ligation and puncture in mice [ ] . systemically administered cationic polyethyleni mine polyplexes incorporating indoleamine- , -dioxyge nase transduced pulmonary endo thelial cells and decreased lung ischemia-reper fusion injury [ ] . nf-κb decoy oligonucleotides, incorporated into viral vectors, attenuate systemic sepsis-induced lung injury when administered intravenously (figure ) [ ] . in animal models, both intratracheal [ , ] and intra venously [ , ] administered sirna successfully silence their target genes. shrna-based approaches have been used to suppress silica-induced lung fi brosis [ ] and to ameliorate lung ischemia-reperfusion-induced lung injury [ ] . more recently, aerosolization of sirna that targets respiratory syncytial virus viral replication was safe and potentially eff ective in patients post lung transplant with respiratory syncytial virus infection [ ] , clearly illustrating the therapeutic potential of these approaches for ali/ards. mei and colleagues enhanced the effi cacy of mesen chymal stem/stromal cells in endotoxin-induced ali by transducing them to overexpress angiopoeitin- (figure ) [ ] . mesenchymal stem/stromal cells overexpressing il- decreased alveolar infi ltration of cd and cd t cells following lung ischemia-reperfusion injury [ ] . bone marrow stem cells expressing keratinocyte growth factor attenuate bleomycin-induced lung injury [ ] . non stem cells can also be used to deliver genes to the injured lung [ ] . fibroblasts overexpressing angiopoeitin- attenuate endotoxin-induced lung injury [ ] , while fi broblasts overexpressing vascular endothelial growth factor and endothelial nitric oxide synthase can attenuate or even reverse endotoxin-induced ali [ ] . advances in the identifi cation of therapeutic targets, improvements in viral and nonviral vector technologies, and regulation of gene-based therapies by temporal and spatial targeting off er the potential to translate the therapeutic promise of gene-based therapies for ali/ ards to the clinical setting (table ) . viral vectors remain the focus of intensive research to optimize their effi ciency, to minimize their immuno genicity and to enhance their tissue specifi city [ , , , ] . strategies to develop less immunogenic vectors have focused on modifying the naturally occurring proteins in the viral coat [ ] . much research has been devoted to searching and characterizing both naturally occurring [ ] and engineered capsid variants from mammalian species [ ] . capsid protein modification has also been used to enhance tissue specifi city [ ] . envelope protein pseudotyping involves encapsulating the modifi ed genome from one virus, such as simian immuno defi ci ency virus, with envelope proteins from another virus, such as vesicular stomatitic virus. th is encapsu lation can enhance the therapeutic potential of viral vectors, by combining the advantages of one viral genome (for example, bigger payload or site-specifi c integration) with the tissue tropism of another virus. strategies to enhance the eff ectiveness of the lipoplexes used to deliver plasmids and other dna/rna oligonucleotides involve manipulation of the lipoplex lipid content and the use of targeting peptides. th e choice of lipid infl uences expression effi ciency by enhancing release of the genetic material within the target cell [ , ] . targeting peptides increases transfection effi ciency by directing the lipid to a particular cell membrane or cell type [ ] . physical methods of plasmid delivery such as electroporation [ ] and ultrasound can enhance gene transfer by bringing the plasmid dna into closer proximity with the cell membrane and/or causing temporary disruption of the cell membrane. other physical methods can also be used to increase in vivo gene transfer, including pressurized vascular delivery, laser, magnetic fi elds and gene gun delivery. th ese systems enable plasmid-based gene delivery to reach effi ciencies close to that achieved with viral vectors. successful gene therapy relies upon being able to target the injury site, and to control the duration and levels of gene expression. modifying the transgene dna to exclude nonmethylated cpg motifs, typical of bacterial dna, decreases the immune response and may increase transgene expression [ , ] . high-effi ciency tissue-specifi c promoters may improve the effi ciency and specifi city of transgene expression. lung-specifi c promoters include surfactant promoters [ ] such as the surfactant protein c promoter [ ] , a ciliated cell-specifi c promoter foxj [ ] , the cytokeratin promoter [ ] , and the clara cell -kda protein [ ] . promoters can also be used to target a specifi c phase of illness, switching on when required to produce an eff ect at the optimal time point. a related approach is the development of promoters that allow for transfected genes to be turned on and off . currently, the tetracycline-dependent gene expression vector [ ] is the most widely used regulated system as it has a good safety profi le. tetracycline is rapidly metabolized and cleared from the body, making it an ideal drug to control gene expression. however, the potential for an activator such as tetracycline to modulate the lung injury should be borne in mind. new-generation transactivators, with no basal activity and increased sensitivity, have now been developed [ ] . in an ards context, conditional regulation of gene expression by the combined use of a lung-specifi c promoter and the tetracycline-dependent gene expression system may be a useful approach [ ] . capsid protein modifi cation to reduce immunogenicity [ ] capsid protein modifi cation to enhance tissue specifi city [ ] envelope protein pseudotyping manipulation of lipoplex lipid content to enhance cellular uptake [ , ] use of targeting peptides on lipoplexes and polyplexes [ ] strategies to enhance gene transfer; for example, electroporation, ultrasound, gene gun delivery modifying transgene dna to eliminate bacterial motifs [ , ] development of high-effi ciency tissue-specifi c promoters [ ] [ ] [ ] [ ] development of promoters that regulate gene expression [ ] enhanced therapeutic targeting nebulization technologies [ ] strategies to target the pulmonary endothelium [ ] improved cellular uptake of vector surface active agents to enhance vector spread [ ] reduce ubiquitination of viral capsid proteins [ ] better therapeutic targets enhancement or restoration of lung epithelial and/or endothelial cell function [ ] strengthening lung defense mechanisms against injury [ ] speeding clearance of infl ammation and infection enhancement of the repair process following ali/ards [ ] . an advantage of gene-based strategies is the ability to target specifi c cells within an organ; for example, the epithelial cells of the lung. novel nebulization technologies, which facilitate the delivery of large quantities of undamaged vector to the distal lung, demonstrate considerable promise in this regard [ ] . alternative approaches to spatial targeting include targeting specifi c receptors that are plentiful on the target cell to increase transfection effi ciency. an interesting development in this regard is the targeting of systemically administered therapies to the pulmonary endothelium using antibodies to proteins expressed preferentially on these cells ( figure ) [ ] . in these studies, the antioxidant enzyme catalase was conjugated with antibodies to the adhesion molecule pecam, which is widely expressed on pulmonary endothelial cells, and to a nonspecifi c igg antibody. th e anti-pecam/catalase conjugate, but not the igg/catalase conjugate, bound specifi cally to the pulmonary endothelium and attenuated hydrogen peroxide injury. specifi c strategies have been developed to maximize uptake of vector into alveolar epithelial cells. it is possible to enhance lung transgene expression with the use of surface-active agents such as perfl urocarbon, which enhances the spread of vector and mixing within the epithelial lining fl uid [ ] . agents that reduce ubiquitination of aav capsid proteins following endocytosis, such as tripeptide proteasome inhibitors, dramatically augment (> , -fold) aav vector transduction in airway epithelia [ ] . ultimately, the success or failure of gene-based therapies for ali/ards is likely to rest on the identifi cation of better gene targets. ongoing advances in our understanding of the pathophysiology of ali/ards continue to reveal novel therapeutic targets for gene-based approaches. promising potential approaches include strate gies to enhance or restore lung epithelial and/or endothelial cell function [ ] , to strengthen lung defense mechanisms against injury [ ] , to speed clear ance of infl ammation and infection, and to enhance the repair process following ali/ards [ ] . ali/ards may be a particularly suitable disease process for gene-based therapies (table ). th is is supported by increasing evidence from relevant preclinical ards models for the effi cacy of gene-based therapies that enhance or restore lung epithelial and/or endothelial cell function, strengthen lung defense mecha nisms against injury, speed resolution of infl ammation and infection, and enhance the repair process following ali/ards. despite this promising preclinical evidence, the potential for gene based approaches to ali/ards in the clinical setting remains to be realized. multiple barriers exist to the successful use of gene-based therapies in the lung, which limit the effi cacy of these approaches. future research approaches should focus on overcoming these barriers, by developing more eff ective and less immunogenic vector delivery systems, developing strategies to focus gene expression on specifi c injury zones of the lung for defi ned time periods, and identifying better molecular targets that can take advantage of these potentially very powerful therapeutic approaches. abbreviations aav, adeno-associated virus; ali, acute lung injury; ards, acute respiratory distress syndrome; il, interleukin; nf, nuclear factor; shrna, small hairpin rna; sirna, small interfering rna. the authors declare that they have no competing interests. epidemiology of acute lung injury incidence and outcomes of acute lung injury one-year outcomes in survivors of the acute respiratory distress syndrome ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. the acute respiratory distress syndrome network pulmonary-artery versus central venous catheter to guide treatment of acute lung injury prone ventilation reduces mortality in patients with acute respiratory failure and severe hypoxemia: systematic review and meta-analysis elbourne d: effi cacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial the acute respiratory distress syndrome optimized aerosol delivery to a mechanically ventilated rodent pecamdirected delivery of catalase to endothelium protects against pulmonary vascular oxidative stress adenoviral augmentation of elafi n protects the lung against acute injury mediated by activated neutrophils and bacterial infection aerosol delivery of a β-galactosidase adenoviral vector to the lungs of rodents adenovirusmediated persistent cystic fi brosis transmembrane conductance regulator expression in mouse airway epithelium airway epithelial cftr mrna expression in cystic fi brosis patients after repetitive administration of a recombinant adenovirus analysis of risk factors for local delivery of low-and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of comorbid conditions modifi cation of nasal epithelial potential diff erences of individuals with cystic fi brosis consequent to local administration of a normal cftr cdna adenovirus gene transfer vector a phase i study of adenovirus-mediated transfer of the human cystic fi brosis transmembrane conductance regulator gene to a lung segment of individuals with cystic fi brosis aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fi brosis. i. methods, safety, and clinical implications recent developments in adeno-associated virus vector technology analysis of adeno-associated virus progenitor cell transduction in mouse lung adeno-associated virus type (aav ) but not aav binds to the apical surfaces of airway epithelia and facilitates gene transfer adeno-associated virus type (aav ) vectors mediate effi cient transduction of airway epithelial cells in mouse lungs compared to that of aav vectors repeated adeno-associated virus serotype aerosol-mediated cystic fi brosis transmembrane regulator gene transfer to the lungs of patients with cystic fi brosis: a multicenter, double-blind, placebo-controlled trial safety and biological effi cacy of an adeno-associated virus vector-cystic fi brosis transmembrane regulator (aav-cftr) in the cystic fi brosis maxillary sinus lentivirus vectors pseudotyped with fi loviral envelope glycoproteins transduce airway epithelia from the apical surface independently of folate receptor alpha gene therapy of human severe combined immunodefi ciency (scid)-x disease cavazzana-calvo m: insertional oncogenesis in patients after retrovirus-mediated gene therapy of scid-x rna interference for α-enac inhibits rat lung fl uid absorption in vivo eff ect of antisense oligonucleotides to nuclear factor-κb on the survival of lps-induced ards in mouse electroporation-mediated transfer of plasmids to the lung results in reduced tlr signaling and infl ammation a receptor-targeted nanocomplex vector system optimized for respiratory gene transfer cationic lipid-mediated cftr gene transfer to the lungs and nose of patients with cystic fi brosis: a double-blind placebo-controlled trial rna interference therapy in lung transplant patients infected with respiratory syncytial virus in vivo gene silencing (with sirna) of pulmonary expression of mip- versus kc results in divergent eff ects on hemorrhage-induced, neutrophil-mediated septic acute lung injury silencing cd gene expression results in the inhibition of latent-tgf-β activation and suppression of silica-induced lung fi brosis in the rat prevention of lung ischemia-reperfusion injury by short hairpin rna-mediated caspase- gene silencing nuclear factor-κb decoy oligodeoxynucleotides prevent acute lung injury in mice with cecal ligation and puncture-induced sepsis eff ects of intratracheal administration of nuclear factor-κb decoy oligodeoxynucleotides on long-term cigarette smokeinduced lung infl ammation and pathology in mice prevention of lpsinduced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin cell-based angiopoietin- gene therapy for acute lung injury stem cells and cell therapies in lung biology and lung diseases calculating expected lung deposition of aerosolized administration of aav vector in human clinical studies repeated aerosolized aav-cftr for treatment of cystic fi brosis: a randomized placebo-controlled phase b trial aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fi brosis. ii. transfection effi ciency in airway epithelium inhibition of lung tumor growth by complex pulmonary delivery of drugs with oligonucleotides as suppressors of cellular resistance gene transfer to the lung: lessons learned from more than decades of cf gene therapy acute lung injury does not impair adenoviral-mediated gene transfer to the alveolar epithelium fatal systemic infl ammatory response syndrome in a ornithine transcarbamylase defi cient patient following adenoviral gene transfer adenoviral gene transfer of a mutant surfactant enzyme ameliorates pseudomonas-induced lung injury gene therapy for oxidant injury-related diseases: adenovirus-mediated transfer of superoxide dismutase and catalase cdnas protects against hyperoxia but not against ischemiareperfusion lung injury angiopoietin- increases survival and reduces the development of lung edema induced by endotoxin administration in a murine model of acute lung injury enhanced expression of -kilodalton heat shock protein limits cell division in a sepsis-induced model of acute respiratory distress syndrome human apoa-i overexpression diminishes lps-induced systemic infl ammation and multiple organ damage in mice protection against pseudomonas aeruginosa pneumonia and sepsisinduced lung injury by overexpression of β-defensin- in rats overexpression of the na-k-atpase α -subunit improves lung liquid clearance during ventilation-induced lung injury interleukin- β causes acute lung injury via αvβ and αvβ integrin-dependent mechanisms adenoviral-mediated overexpression of socs enhances igg immune complex-induced acute lung injury dose-dependent improvements in outcome with adenoviral expression of interleukin- in a murine model of multisystem organ failure pretreatment with cationic lipid-mediated transfer of the na + k + -atpase pump in a mouse model in vivo augments resolution of high permeability pulmonary oedema electroporation-mediated gene transfer of the na + ,k + -atpase rescues endotoxin-induced lung injury interleukin- gene transfer: prevention of multiple organ injury in a murine cecal ligation and puncture model of sepsis nonviral gene delivery with indoleamine , -dioxygenase targeting pulmonary endothelium protects against ischemia-reperfusion injury silencing of fas, but not caspase- , in lung epithelial cells ameliorates pulmonary apoptosis, infl ammation, and neutrophil infl ux after hemorrhagic shock and sepsis caveolin- sirna increases the pulmonary microvascular and alveolar epithelial permeability in rats interleukin- delivery via mesenchymal stem cells: a novel gene therapy approach to prevent lung ischemia-reperfusion injury bone marrow stem cells expressing keratinocyte growth factor via an inducible lentivirus protects against bleomycin-induced pulmonary fi brosis cell-based gene transfer of vascular endothelial growth factor attenuates monocrotaline-induced pulmonary hypertension microvascular regeneration in established pulmonary hypertension by angiogenic gene transfer tetracycline-inducible transgene expression mediated by a single aav vector effi cient transfection of non-proliferating human airway epithelial cells with a synthetic vector system tailoring the aav vector capsid for gene therapy artifi cial evolution with adeno-associated viral libraries analysis and optimization of the cationic lipid component of a lipid/ peptide vector formulation for enhanced transfection in vitro and in vivo stabilized integrin-targeting ternary lpd (lipopolyplex) vectors for gene delivery designed to disassemble within the target cell cpg-free plasmids confer reduced infl ammation and sustained pulmonary gene expression toll-like receptor expression reveals cpg dna as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with cd ligand to induce high amounts of il- targeting type ii and clara cells for adenovirus-mediated gene transfer using the surfactant protein b promoter development of lentiviral vectors with regulated respiratory epithelial expression in vivo expression of cftr from a ciliated cell-specifi c promoter is ineff ective at correcting nasal potential diff erence in cf mice a human epithelium-specifi c vector optimized in rat pneumocytes for lung gene therapy tight control of gene expression in mammalian cells by tetracycline-responsive promoters use of a new generation reverse tetracycline transactivator system for quantitative control of conditional gene expression in the murine lung construction of an rtta (s)-m / tts(kid)-based transcription regulatory switch that displays no basal activity, good inducibility, and high responsiveness to doxycycline in mice and non-human primates adenoviral vector transfection into the pulmonary epithelium after cecal ligation and puncture in rats ubiquitination of both adeno-associated virus type and capsid proteins aff ects the transduction effi ciency of recombinant vectors gp -stat regulates epithelial cell migration and is required for repair of the bronchiolar epithelium spatial and temporal expression of surfactant proteins in hyperoxia-induced neonatal rat lung injury intrapulmonary tnf gene therapy reverses sepsis-induced suppression of lung antibacterial host defense clinical review: gene-based therapies for ali/ards: where are we now? the present work was supported by funding from the health research board key: cord- -idffrnac authors: xiang, meng; fan, jie title: pattern recognition receptor–dependent mechanisms of acute lung injury date: - - journal: mol med doi: . /molmed. . sha: doc_id: cord_uid: idffrnac acute lung injury (ali) that clinically manifests as acute respiratory distress syndrome is caused by an uncontrolled systemic inflammatory response resulting from clinical events including sepsis, major surgery and trauma. innate immunity activation plays a central role in the development of ali. innate immunity is activated through families of related pattern recognition receptors (prrs), which recognize conserved microbial motifs or pathogen-associated molecular patterns (pamps). toll-like receptors were the first major family of prrs discovered in mammals. recently, nacht–leucine-rich repeat (lrr) receptors and retinoic acid–inducible gene–like receptors have been added to the list. it is now understood that in addition to recognizing infectious stimuli, both toll-like receptors and nacht-lrr receptors can also respond to endogenous molecules released in response to stress, trauma and cell damage. these molecules have been termed damage-associated molecular patterns (damps). it has been clinically observed for a long time that infectious and noninfectious insults initiate inflammation, so confirmation of overlapping receptor-signal pathways of activation between pamps and damps is no surprise. this review provides an overview of the prr-dependent mechanisms of ali and clinical implication. modification of prr pathways is likely to be a logical therapeutic target for ali/acute respiratory distress syndrome. (tlrs - and tlrs - ) in mice have been defined ( ) . tlrs , , and are expressed intracellularly, whereas tlrs , , , , and are expressed on the cell surface. tlrs are expressed on a range of immune cells including macrophages, dendritic cells, b cells and certain types of t cells, as well as on certain nonimmune cells, such as endothelial cells, smooth muscle cells and epithelial cells that lie at potential sites of entry, including the skin and the respiratory, intestinal and genitourinary tracts. the expression of tlrs is modulated by activation, matu-ration or differentiation of the different cell types ( , ) . tlr proteins are a family of type i transmembrane receptors characterized by an nh -terminal extracellular leucine-rich repeat (lrr) domain, which mediate the recognition of their respective pamps, and a cooh-terminal intracellular tail containing a conserved region called the toll/interleukin (il- ) receptor (tir) homology domain. the tir domain is the defining motif of the tlr/il- superfamily, and it is likely to be one of the earliest signaling domains to have evolved ( ) . tlrs can recognize a diverse range of pamps, generate inflammatory signals to coordinate innate immune responses and modulate adaptive immune responses. the list of tlr ligands is growing. however, the ligand for tlr and mouse tlr remains unknown at present. activation of tlrs initiates two major pathways: the myd -dependent pathway, which is used by all tlrs except tlr , resulting in the activation of nuclear factor (nf)-κb and activator protein- (ap- ); and the trif-dependent pathway, which is initiated by tlr and tlr , resulting in the activation of type i interferons (ifns) ( , , ) . expression of numerous proinflammatory cytokines, such as tumor necrosis factor (tnf)-α, il- , il- and ifns, is one of the major outcomes of the activation of the pathways ( ) . tlr signaling is summarized and shown in figure . rlrs as dexd/h-containing rna helicases are expressed in the cytoplasm in a variety of cells, including immune and nonimmune cells. unlike membranebond tlr , tlr and tlr , which are localized on the endosome and recognize viral double stranded rna, singlestranded rna and dna, respectively, rlrs are cytoplasmic proteins that recognize viral rna produced as a consequence of viral replication ( , ) . rlrs consist of three family members: rig-i, melanoma differentiation-associated gene (mda ) and laboratory of genetics and physiology (lgp ) ( , ) . role of prrs in mediating inflammation and organ injury. infection causes pamp release, but also causes tissue and cell damage and subsequent damp release. similarly, injury caused by trauma or various other factors not only leads to damp release but also renders the patient more susceptible to infection and therefore pamp release. in turn, the pamps and damps act through prrs, which include tlrs, nlrs and rlrs, to activate the innate immune system, yet they can also contribute to persistent and deleterious systemic inflammation and organ injury, including ali. structurally, rig-i and mda contain a dexd/h box rna helicase domain and two caspase-recruiting domain (card)like domains required for eliciting downstream signaling pathways ( , ) . the c-terminal region of rig-i contains a repressor domain (rd), which inhibits downstream signaling. the mda c-terminal region is similar to the rd of rig-i; however, its function is not clear. lgp contains a dexd/h helicase domain and an rd, but lacks the card-like region. lgp was suggested to play an inhibitory role in virus-induced response, because the lgp rd binds the rig-i rd and suppresses signaling as a consequence of interfering with the self-association of rig-i ( , , ) . rig-i is essential for the recognition of a series of rna viruses, which include sendai virus, newcastle disease virus, influenza virus, vesicular stomatitis virus and japanese encephalitis virus ( ) . mda is required for the recognition of other rna viruses, including picornaviruses such as encephalomyocarditis virus, mengo virus and theiler virus ( , ) . thus, rig-i and mda have specificities in their detection of rna viruses, presumably through recognition of distinct structures of viral rna ( , ) . recent studies revealed a pathway of rlr regulation of nf-κb. rig-i/mda card domains, through a card-containing adaptor protein, ifnβ promoter stimulator , also known as mitochondrial antiviral signaling protein, and card adaptor inducing ifnβ, ultimately activate irf and nf-κb ( , ) . the role of rlrs in the mechanism of ali has not been elucidated. the nlr family is a group of recently identified cytoplasmic prrs that contain more than members in humans ( ) . the major role of nlrs is to recognize cytoplasmic microbial pamps and/or endogenous danger signals and initiate immunological responses, although the physiological function of most nlrs is poorly understood at present ( ) . members of the nlr family are categorized into at least five subfamilies according to their n-terminal structure, including nods (nucleotidebinding oligomerization domain- ), nalps (nacht-, lrr-and pyrindomain-containing proteins), ipaf (ice-protease activating factor), naips (neuronal apoptosis inhibitor factors) and class ii transactivator (ciita) ( ) . the nlr family shares a domain organization consisting of a c-terminal lrr domain, a central nucleotide-binding nacht domain, and an n-terminal protein-protein interaction domain composed of a card, pyrin domain (pyd) or baculovirus inhibitor of apoptosis repeat (bir) domain ( ) . nods and ipaf contain card effector domains, whereas nalps have pyd domains, and naips possess bir domains. the functions of nod , nod , ipaf and nalp are more studied. nod and nod are the first nlrs that are reported (tlr in association with tlr or tlr ), tlr , tlr and tlr are localized on the cell surface for ligand recognition. tlr , tlr and tlr are localized in the endosome for ligand recognition in the lumen of endosome. all tlrs, except tlr , recruit myd , and tlr , tlr , tlr and tlr recruit the additional adaptor tirap, which links the tir domain with myd . tlr and tlr recruit trif. tlr requires the additional linker adaptor tram, which links the tir domain of tlr with trif. stimulation of the cells with tlr , tlr , tlr , tlr and tlr ligands initiates the myd -dependent pathway whereas tlr ligands initiate the trif-dependent pathway. tlr activates both myd -dependent and trif-dependent pathways. in the myd dependent pathway, myd recruits the irak family of proteins and traf . in turn, traf activates tak . the activated tak activates the ikk complex, which activates nf-κb subunits. the activated tak also activates the mapk pathway. in the trif-dependent pathway, trif interacts with rip and traf . activated traf and rip activate nf-κb and mapks. trif also interacts with traf and activates tbk /ikki, which activate irf and irf . cells stimulated with tlr and tlr ligands activate nf-κb and mapks via the myd -dependent pathway. to induce type i ifns, myd associates with the irak family of proteins. irak and ikkα activate irf . irak also interacts with traf and activates irf . the activated nf-κb subunits and irfs are translocated to the nucleus. nf-κb and mapks initiate the transcription of inflammatory cytokine genes whereas irfs initiate the transcription of type i interferons. (the figure is adapted from [ ] and used with permission from elsevier.) to have a direct function as prrs in the recognition of peptidoglycan (pgn)derived peptides. nod senses γ-d-glutamyl-meso-diaminopimelic acid (that is,-dap) that is derived from gramnegative bacteria ( ) , whereas nod senses muramyl dipeptide, which is from both gram-positive and gram-negative bacteria ( , ) . when nods bind with pgn-derived peptides, they rapidly form oligomers, which lead to the recruitment of the receptor-interacting protein (rip ) kinase through card-card interactions ( ) . this complex of nod -rip or nod -rip then recruits the inhibitor of nk-κb kinase complex (ikk), leading to the activation of nf-κb. activation nod and nod can also initiate an mapk pathway that leads to the activation of p and erk. in addition, nod signaling can activate jnk as well ( ) . nalp proteins are characterized by the presence of an n-terminal pyrin effector domain ( ) . several nlrs, namely nalp , nalp and nalp , have an important role in activation of proinflammatory caspases through formation of inflammasome ( , ) . the inflammasome is a multiprotein complex of more than kda that is responsible for the activation of caspases and , leading to the processing and secretion of the proinflammatory cytokines il- β and il- ( , ) . martinon et al. have presented different caspase activation platforms in which different components constitute the various inflammasomes ( ) . two types of nalp inflammasome are better studied: the nalp inflammasome that is composed of nalp , the adaptor protein asc, caspase- and caspase- ( ) , and the nalp /nalp inflammasome that contains nalp or nalp cardi-nal, asc and caspase- ( ) . tlrs - are expressed in lung tissue ( ) , and individual tlrs are differentially regulated in specific lung cell populations in response to microbial stimula-tion. tlr , tlr , tlr and tlr are the most likely to be involved in recognition of bacteria in the lungs ( ) ( ) ( ) . a study by bernard et al. has shown that ali/ ards induced by lipopolysaccharide (lps) is a major cause of mortality among humans ( ) . the lps membrane receptor complex is composed of several accessory molecules, which include phosphatidylinositol-anchored cd , tlr , md and md ( ) . the lpsbinding protein (lbp) enhances the binding of lps to its receptor ( ) . absence of cd , md or lbp abrogates most lps responses ( , ) . expression of functional tlr has been found in many cell types in the lung ( ) , and lps-induced lethal shock and ali have been shown to be tlr dependent ( ) ( ) ( ) . thus, tlr plays a critical role in the mechanism of infection-related ali. a recent study has shown that respiratory infections in the human lung initiated by tlr agonist lipoteichoic acid (lta, a component of gram-positive bacteria) and tlr agonist lps (a component of gram-negative bacteria) exhibit different inflammatory responses ( ) . the study was performed on healthy subjects with lps or lta instillation into the contralateral lung. alveolar macrophages (amφ) isolated from bronchoalveolar lavage fluid were analyzed by multiplex ligation-dependent probe amplification. the results show that whereas both lps and lta elicited neutrophil recruitment, only lps instillation was associated with activation of neutrophils (pmn) (cd b surface expression and degranulation) and consistent rises of chemokine/cytokine levels. moreover, lps but not lta activated am, as reflected by enhanced expression of proinflammatory mediators and increased spontaneous cytokine release upon incubation ex vivo. remarkably, only lta induced c a release. these data suggest that stimulation of tlr or tlr results in differential pulmonary inflammation, which may be of relevance for understanding the differences during gram-positive and gram-negative respiratory tract infection ( ) . pulmonary endothelium is a major component of the alveolar-capillary unit, and is susceptible to injury from noxious agents that are either inhaled or delivered to the lung through the pulmonary circulation ( ) . in ali, pulmonary endothelium plays a major role by (a) altering metabolic activity to affect pulmonary and systemic homeostasis, (b) mediating polymorphonuclear pmn adhesion to promote pmn infiltration, (c) changing pmn barrier permeability to cause pulmonary edema and (d) secreting cytokines and chemokines to induce lung inflammation ( ) . a recent study by andonegui and colleagues has shown that endothelial cells (ecs) are the key sentinel cells for detecting infection by gram-negative bacteria and recruiting pmn to peripheral tissues ( , ) . indeed, previous studies have shown that direct activation of circulating pmn with lps is not sufficient to induce their sequestration within the lung ( ) . interactions of pmn with ecs seems important for the process of pmn sequestration into the lung ( , ) . lps stimulates the cd and tlr complex, which in turn activates nf-κb ( ) and increases the expression of adhesion molecule e-selection, intercellular adhesion molecule- (icam- ) and vascular cell adhesion molecule ( , ) . the tlr signaling also leads to production and release of various bioactive molecules, including il- β, il- , tnf-α, chemokines and nitric oxide ( ) , all of which are actively involved in the development of ali. more importantly, tlr signaling can also upregulate other tlrs, such as tlr , and thus amplify the inflammatory responses. although tlr is predominantly expressed in the first-line host defense cells (monocytes, macrophages, dendritic cells and pmn) ( , ) , its expression is low in ecs and epithelial cells ( ) . studies from our laboratory showed that lps through tlr -and myd -dependent signaling induced tlr upregulation in ecs ( ) . we have demonstrated that tlr signaling, through activating nf-κb, upregulates tlr expression in ecs, and this process is enhanced by oxidant signaling generated by pmn nad(p)h oxidase. the functional relevance of nad(p)h oxidase in mediating tlr -induced tlr expression in ecs is evident by markedly elevated and stable icam- expression as well as augmented pmn migration in response to sequential challenge with lps and peptidoglycan ( ) . thus, tlr activation, signaled by tlr and as regulated by pmn nad(p)h oxidase, is an important mechanism responsible for amplifying pmn transmigration to sites of infection ( figure ). the tlr -tlr interaction suggests a highly coordinated, oxidant-mediated upregulation of tlr in response to lps. when one considers the interactions of the innate immune system as microbes are first encountered, the value of such temporal organization is significant. for example, gram-negative bacteria persist in tissues and, if they are not immediately killed through the activation of pmn, complement and other antimicrobial factors, they may spill out systemically and result in septic shock. survival in the face of such infections depends on the innate immune system, which must be able to monitor and respond to pathogens over a prolonged period of time. given the need for a prolonged response to bacterial infection, it has always seemed somewhat surprising that response to lps is temporally finite. this endotoxin tolerance means that within hours after exposure to lps, innate immune cells are incapable of responding again to a rechallenge ( ) . but it is now clear that as lps sensitivity wanes, the immune system has at its disposal the capability of marshaling responses via oxidative metabolites and their ability to upregulate other tlrs ( ) . the subsequent means of responding to bacteria depend on the ability of the innate immune system to destroy microbes and enhance the release of alternative immune stimuli. the tlrs that are utilized are the ones that bind the constituents of degrading bacteria, such as lipopeptides, pgn, heat shock proteins and cpg dna. it seems plausible that activated pmn may even alter the phenomenon of lps tolerance, at least in a localized context, by setting into action a positive feedback loop at sites to which pmn are chemoattracted ( ) . this would enhance inflammatory responses locally and help fight infection. however, in a setting of posttrauma sirs or ali, the primed pmn activation serves as an amplifier to cause enhanced pmn infiltration and organ injury. extensive pmn influx into the lungs is one of the characteristics of ali. studies have shown that excessive induction of proinflammatory cytokines in pmn and delayed pmn apoptosis are associated with higher mortality and more severe organ dysfunction in sepsis patients ( , ) . human pmn express all tlr mrna except tlr . tlr are more abundant than tlr on pmn ( , ) . pmn recruitment to the lung after lps inhalation is primarily dependent on tlr -nf-κb signaling ( , ) . the pi k/akt pathway was also reported to be involved in tlr -induced expression of il- β, tnf-α and chemokine macro phage inflammatory protein (mip)- ( ) . we have reported that tlr , through regulating g-protein-coupled receptor kinases (grks), promotes pmn migration. we demonstrated that mip- induces grk and grk expression in pmns through phosphoinositide- -kinase (pi k)-γ signaling, and lps-activated signaling through the tlr pathway transcriptionally downregulates the expression of grk and grk in response to mip- . the reduced expression of grks lowers chemokine receptor desensitization and markedly augments the pmn migratory response. these data indicate that tlr modulation of pmn surface chemokine receptor expression after the downregulation of grk and grk expression is a critical determinant of pmn migration ( ) (figure ) . a recent study explored a novel role of mtor complex (mtorc ) in tlr and tlr -induced pmn activation ( ) . administration of rapamycin, an inhibitor of mtorc , decreased the severity of lung injury after intratracheal lps or pam (a tlr ligand) administration, as determined by diminished neutrophil accumulation in the lungs, reduced interstitial pulmonary edema, and diminished levels of tnf-α and il- in bronchoalveolar lavage fluid. these results indicate that mtorc activation is essential in tlr -and tlr -induced pmn activation, as well as in the development and severity of ali. the e ubiquitin ligase cblb has a crucial role in the prevention of chronic inflammation and autoimmunity. however, a recent study showed that cblb also has an unexpected function in acute lung inflammation ( ) . cblb attenuates the sequestration of pmn in the lungs after administration of lps. in a model of polymicrobial sepsis in which acute lung inflammation depends on tlr , the loss of cblb expression accentuates acute lung inflammation and reduces survival. cblb controls the association between tlr and the intracellular adaptor myd . expression of wt cblb, but not expression of a cblb mutant that lacks e ubiquitin ligase function, prevents the activity of a reporter gene for nf-κb in monocytes that have been challenged with lps. the downregulation of tlr expression on the cell surface of pmn is impaired in the absence of cblb. these data reveal that cblb regulates the tlr mediated acute inflammatory response that is induced by sepsis ( ) . pmn apoptosis is a crucial injurylimiting mechanism of inflammatory resolution. circulating pmn undergo constitutive apoptosis that results in the shutdown of secretary capacity and allows pmn recognition and removal by macrophages ( , ) . several inflammatory agents, such as lps, tnf, il- , il- , il- and granulocyte colony-stimulating factor (g-csf), can delay apoptotic response, providing pmn with a longer life span, which in turn allows the pmn to accumulate at local tissue sites of inflammation/infection ( , ) . protein (p ) is a transcription factor that is important in multicellular organisms, where it regulates the cell cycle and promotes apoptosis. modulation of p by nutlin- α diminished the response of pmn and macrophages to stimulation through tlr or tlr as well as attenuated lps-induced ali. nf-κb has been reported as a modulator of apoptosis in inflammatory cells ( ) . p can negatively regulate nf-κb activity by decreasing binding of nf-κb to the promoters of genes for proinflammatory cytokines. in p -/mice, the inflammatory process and severity of ali in response to lps are enhanced ( ) . amφ account for approximately % of airspace leukocytes ( ) . tissue damage induced by lps is mediated mainly by inflammatory products released from amφ ( , ) , thus activated amφ play a critical role in the development of ali ( ) . lps inhalation induces amφ to produce and release inflammatory mediators tnf-α, il- β and mip- in a tlr dependent manner ( ) , which further result in the recruitment of pmn into the lower respiratory tract and activate other cell types, including epithelia and endothelia ( ) . tlr is constitutively expressed in amφ, and tlr can be induced in response to lps or proinflammatory cytokines. the inducible tlr expression might be important in responding to other bacterial components from grampositive bacteria ( ) . the role of cd in the regulation of lps-tlr signaling in macrophages has recently been reported ( ) . cd is a transmembrane adhesion molecule and hemopoietic cd has an essential role in hyaluronan clearance and resolution of noninfectious lung injury. following intratracheal lps treatment, cd -/mice demonstrated an exaggerated inflammatory response characterized by increased inflammatory cell recruitment, elevated chemokine expression in bronchoalveolar lavage fluid and a marked increase in nf-κb dna-binding activity in lung tissue in vivo and in macrophages in vitro. furthermore, cd -/mice were more susceptible to lps-induced shock. the study further found that the induction of the negative regulators of tlr signaling il- r-associated kinase-m, toll-interacting protein and a by intratracheal lps in vivo and in macrophages in vitro was significantly reduced in cd -/mice. collectively, these data suggest that cd plays a role in preventing exaggerated inflammatory responses to lps by promoting the expression of negative regulators of tlr- signaling ( ). impairment of the alveolar epithelial barrier is important in the development of ali. under physiologic conditions the epithelial barrier is less permeable than the endothelial barrier; thus, destruction of epithelial barrier integrity prompts a progressive influx of protein-rich fluid into the alveoli ( ) . on the other hand, the loss of epithelial integrity represents an impairment of physiologic transepithelial fluid transport and further inhibits the reabsorption of alveolar edema ( ) . tlrs are critical for airway epithe-lial cell recognition of inhaled pathogens and for innate immune signaling. in cultured human lung epithelial cells, mrna of all tlrs has been detected ( , ) . tlr , tlr , tlr and tlr have the highest expression, and the ligands for these tlrs increased il- and vascular endothelial growth factor (vegf) production in normal human bronchial epithelial cells ( ) . tlr is a heterodimer with tlr and tlr , and each of these is present on the airway epithelial surface. tlr , which recognizes dsrna or poly(i:c), is located in endosomes in unstimulated human bronchial epithelial cells ( ) . tlr is also present on the airway epithelial surface where it can interact with epidermal growth factor receptor (egfr). studies have shown that tlr ligands stimulated il- and vegf production via egfr and the downstream signaling that might include map kinases and nf-κb ( , ) . interestingly, heat shock proteins (hsp), such as hsp and hsp , appear to be intimately involved in the recognition of lps. extracellular hsp released from virally infected airway epithelial cells induces il- express in human bronchial epithelial cells, resulting in the recruitment and activation of pmn via tlr ( , ) . type ii alveolar epithelial cells can also be activated by lps mediated through tlr signaling and in turn promote pulmonary inflammatory processes ( , ) . a study by togbe et al. of whether tlr gene dosage contributes to infection has demonstrated that overexpression of tlr augmented an lps-induced bronchoconstrictive effect, as well as tnf-α and cxc chemokine ligand (keratinocyte-derived chemokine) production ( ) . the study further showed that pmn recruitment, microvascular and alveolar epithelial injury with protein leak in the airways, and damage of the lung microarchitecture were dependent on tlr gene dose. therefore, the tlr expression level determines the extent of acute pulmonary response to inhaled lps, and tlr may thus be a valuable target for immunointervention in acute lung inflammation as a result of infection. in addition to the tlrs, nlrs are critically involved in the sensing of bacterial pathogens. nod senses diaminopimelic acid-containing peptidoglycan present in gram-negative bacteria, whereas nod senses the muramyl dipeptide present in most organisms. because of the apparent lack of direct effects on cell signaling induced by activators of nlrs, it is suggested that their role in pathogen sensing is one of cooperation with the tlrs ( ) . however, studies suggested that the actions of nod vary between cell types and, unlike those seen with lps, the in vivo effects may be independent of leukocyte activation. for instance, cartwright and colleagues have shown that although selective activation of nod in macrophages has no apparent effect, in vascular cells nod activation results in the profound induction of nosii and shock in vivo ( ) . nod is thought to be important in the maintenance of a healthy gut barrier because individuals who carry a defective nod have an increased risk of crohn disease and other intestinal disorders ( ) . although the importance of tlr family in sensing pathogens is well recognized, it is also plausible that they may function in noninfectious diseases because tlr expression is also regulated in conditions other than infection. indeed, growing evidence has shown that prrs play a central role in the mechanisms of noninfectious ali. several tlrs not only have the ability to recognize more than one ligand, but often recognize ligands with completely different chemical structures. such tlr activation does not occur under normal circumstances but only when there is a change in the environment that either leads to the release of endogenous ligands from a cellular compartment, or leads to the modification of endogenous mediator that gives them the ability to activate tlrs ( , ) . because of the association of many endogenous ligands with tissue injury, the nomenclature of damps has been suggested. the best characterized damps include those products released from cells in response to stress or undergoing abnormal death, including hsp , hsp , the extra domain a of fibronectin, oligosaccharides of hyaluronic acid and high-mobility group box (hmgb ). most of these ligands act as agonists of tlr or tlr , or both receptors ( ) . resuscitated hemorrhagic shock (hs) often promotes the development of lung injury by priming the immune system for an exaggerated inflammatory response to a second, often trivial, stimulus, the so-called "two hit hypothesis" ( ) . we used a simplified animal model of the two-hit paradigm to address the mechanisms of hs-primed pmn migration and lung inflammation ( ) . in this model, animals are subjected to a nonsevere resuscitated hs (hypotension at mmhg for hour, followed by a small dose of intratracheal lps. although neither shock nor lps alone induces injury, the combination caused lung pmn accumulation and increased i-albumin transpulmonary flux. findings from this model have suggested that the mechanisms underlying the priming of pmn and inflammation involve a complicated receptor cross-talk process and interaction between pmn and amφ, which is described below. we demonstrated that lps-tlr signaling upregulates tlr expression in amφ, and hs-activated pmn play a critical role in the mechanism of tlr upregulation ( ) . this cross-talk between tlr and tlr in amφ results in the amplification of expression of cytokines and chemokines in response to the bacterial products lps and pgn, and subsequently leads to enhanced pmn sequestration in the lung. these findings reveal a novel mechanism underlying hsprimed lung injury, namely that hs-activated pmn that were initially sequestered into the alveoli can instruct amφ to upregulate tlr , thereby sensitizing amφ to tlr ligands and promoting enhanced lung inflammation. how does hs-activated pmn enhance tlr upregulation of tlr in amφ? we found that reactive oxygen species (ros) derived from pmn nad(p)h oxidase play an important role in amplifying the tlr upregulation ( ) . studies have also shown that lack of endogenous nad(p)h oxidase in the amφ caused a decrease in tlr expression in response to lps stimulation; however, the decrease was restored when the amφ was coincubated with pmn isolated from wild-type mice subjected to hs. these results indicate that although the endogenous nad(p)h oxidase in amφ is also involved in the signaling, the exogenous oxidants from pmn nad(p)h oxidase are essential for inducing amplified tlr expression in amφ in response to lps. the tlr gene promoter contains multiple binding sites for transcriptional factors, which include nf-κb, ccaat/enhancer binding protein, camp response element-binding protein and stat (signal transducer and activator of transcription) ( ) . of these, nf-κb has been reported to regulate tlr expression in response to cytokines and mycobacterial infection ( , ) . it has been demonstrated that lps-tlr -induced tlr upregulation in amφ is largely mediated through the nf-κb signaling pathway, because the nf-κb inhibitor ikk-nbd significantly decreased lps-induced tlr expression in amφ ( ) . although oxidants are involved in the nf-κb signal transduction pathway ( ) ( ) ( ) , their molecular targets have not yet been defined. the contribution of redox regulation and location of potential redox-sensitive sites within the nf-κb activation pathway are the subjects of controversy ( ) . recently we reported that hmgb /tlr signaling mediates the hs-induced increase in tlr surface expression and decrease in tlr surface expression in the lung as well as in mouse lung vascular ecs (mlvec) ( ) . these alterations in tlr and tlr expression result in hmgb -mediated activation of nad(p)h oxidase and expression of icam- in mlvec that is tlr dependent in the early phase and switches to being tlr dependent in the late phase following hs. more importantly, the hs-induced surface expression of tlr contributes to an enhanced activation of mlvec and augmented pulmonary pmn infiltration in response to the tlr agonist pgn. thus, the study demonstrates a novel mechanism underlying hs-augmented lung inflammation, namely that induction of increased tlr surface expression in lung endothelial cells, which is induced by hs/r and mediated by hmgb activation of tlr signaling, is an important mechanism responsible for ec-mediated inflammation and organ injury following hs ( ) . we have shown that hs-induced pmn nad(p)h oxidase activation is mediated by hmgb -tlr signaling. hmgb was originally defined as a nuclear protein that functions to stabilize nucleosome formation, and it also acts as a transcription factor that regulates the expression of several genes ( ) . hmgb can be secreted by innate immune cells in response to microbial products or other inflammatory stimuli ( , ) . hmgb is also released by injured cells and is known as one of the main prototypes of the emerging damps ( ) ( ) ( ) . hmgb was initially identified as an inflammatory cytokine that is a late mediator of lethality in sepsis ( , ) . however, recent studies suggest that hmgb acts as an early mediator of inflammation, contributing to the development of ali after hemorrhage ( ) , and hepatic injury after liver ischemia-reperfusion ( ) . we found in our study that hs/r activates the tlr -myd -irak signaling pathway through hmgb , and further activates p mapk and akt pathways to initiate pmn nad(p)h oxidase activation. pmn nad(p)h oxidase-derived oxidants, in turn, mediate tlr -tlr cross-talk in amφ and sensitize amφ response to tlr ligands, which act in a positive feedback manner to amplify pulmonary pmn infiltration and inflammation ( ) . we have also addressed a fundamental question regarding how hs globally regulates pmn infiltration in the lungs. we have shown that hs, through alarmin hmgb , induced il- secretion from macrophages in an autocrine and tlr signaling-dependent manner. in turn, il- , through an il- -g-csf-mediated mechanism, induced pmn egress from bone marrow. therefore a sustained and hs-primed migration of pmn was maintained. we have also shown that β-adrenergic-receptor activation by catecholamine of macrophages mediated the hs-induced release of hmgb . these data indicate that hs, a global ischemia/ reperfusion stimulus, regulates pmn mobilization through a series of interacting pathways that include neuroendocrine and both innate and acquired immune systems ( ) . hyaluronan (ha) is a massive sugar polymer in the extracellular matrix. under physiologic conditions, ha exists as a high-molecular-weight polymer (> d) and undergoes dynamic regulation resulting in accumulation of lower molecular weight species ( - kd) after tissue injury. ha fragments can trigger innate immune responses in a manner that overlaps with both grampositive and gram-negative organism recognition pathways ( ) . it has been demonstrated that fragmented ha accumulates during tissue injury ( ) ( ) ( ) . cd is required to clear ha during tissue injury, and impaired clearance of ha results in unremitting inflammation. additionally, fragmented ha stimulates the expression of inflammatory genes by inflammatory cells at the injury site ( ) . recently, jiang et al. demonstrated that ha fragments require both tlr and tlr to stimulate mouse macrophages to produce inflammatory chemokines and cytokines. in a noninfectious lung injury model, mice deficient in both tlr and tlr showed an impaired transepithelial migration of inflammatory cells, increased tissue injury, elevated lung epithelial cell apoptosis and decreased survival ( ) . lung epithelial cell overexpression of high molecular mass ha protected mice against ali and apoptosis, in part through tlr-dependent basal activation of nf-κb. the exaggerated injury in tlr -and tlr -deficient mice appears to be due to impaired ha-tlr interactions on epithelial cells. these studies demonstrate that host-matrix component ha and tlr interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from ali ( ) . mechanical ventilation (mv) provides life-saving support for many patients with respiratory failure ( ) . however, mechanical stresses produced by mv can induce lung injury, termed ventilator-induced lung injury ( ) . evidence from animal experimental studies has demonstrated that mv per se can induce inflammatory responses ( ) ( ) ( ) . a recent report has demonstrated that tlr , but not tlr , played a role in development of the inflammatory response after shorttime mv ( ) . mv not only causes ventilator-induced lung injury in healthy animals, but also exacerbates damage in the injured lung ( ) . tlr blockade reduces pulmonary inflammation caused by the combination of lps and mv ( ) . in the mechanism of hyperoxia-induced ali, tlr expression and activation seems impotent. exposure of human epithelial cells to hyperoxia in the absence of an exogenous viral pathogen significantly increased tlr expression. in vivo studies showed that both the absence of tlr via gene deletion in mice and the presence of an anti-tlr antibody in wild-type mice conferred significant protection in a hyperoxia-mediated lung injury model ( ) . the inflammasome is a multiprotein complex that mediates the activation of caspase- , which promotes secretion of the proinflammatory cytokines il- β and il- , as well as pyroptosis, a form of cell death induced by bacterial pathogens. members of the nlr family, including nlrp , nlrp and nlrc , and the adaptor asc are critical components of the inflammasome that link microbial and endogenous danger signals to caspase- activation. the role of nalp in mediating noninfectious ali has been revealed by two recent studies. gasse and colleagues reported that uric acid locally produced in the lung upon bleomycin (blm)-induced dna damage and degradation triggers nalp inflammasome activation, and in turn causes lung injury ( ) . reduction of uric acid levels using the inhibitor of uric acid synthesis allopurinol or uricase leads to a decrease in blm-induced il- β production, lung inflammation, repair and fibrosis. local administration of exogenous uric acid crystals recapitulates lung inflammation and repair, which depend on the nalp inflammasome, myd and il- r pathways and tlr and tlr for optimal inflammation but are independent of the il- receptor ( ) . babelova et al., however, reported the role of biglycan, a ubiquitous lrr proteoglycan of the extracellular matrix, in mediating ali through interacting with tlr and tlr on macrophages ( ) . the study showed that in macrophages soluble biglycan induces the nlrp /asc inflammasome and subsequent activation of caspase- and release of mature il- β without need for additional costimulatory factors. this is caused by the interaction of biglycan with tlr / and purinergic p × /p × receptors, which induces receptor cooperativity. furthermore, ros formation is involved in biglycan-mediated activation of the inflammasome. by signaling through tlr / biglycan stimulates the expression of nlrp and pro-il- β mrna. these results provide evidence for direct activation of the nlrp inflammasome by biglycan and suggest a fundamental paradigm of how tissue stress and injury are monitored by innate immune receptors detecting the release of the extracellular matrix components and turning such a signal into a robust inflammatory response ( ) . susceptibility and response to infectious disease is, in part, heritable. potential associations between clinical outcome from sepsis and many inflammatory cytokine gene polymorphisms, innate immunity pathway gene polymorphisms and coagulation cascade polymorphisms have been observed. we may yet be able to tease out the complex influence of genetic variation on susceptibility and response to infectious disease ( ) . in , arbour and colleagues reported that two polymorphisms of the tlr gene were present in a higher proportion of individuals who are hyporesponsive to inhaled lps ( ) . this finding led to a number of studies investigating the potential impact of these tlr polymorphisms on the course of infectious diseases and the development of septic shock and tlr polymorphisms ( ) ( ) ( ) . these polymorphisms do not seem to confer sus-ceptibility to all gram-negative infections, because other groups ( , ) have shown no correlation in other infectious diseases such as meningococcal disease, and again one should be mindful that very significant hypofunctioning tlr alleles are likely to have a very strong negative selection pressure across the generations ( ) . tlr- polymorphisms have also been linked to susceptibility to staphylococcal infection ( ) and lepromatous leprosy ( , ) . although some of these studies are still relatively small in scale, they reinforce the important role of tlrs in pathogen recognition and immune response in humans. cd polymorphisms, key accessory molecules for tlr signaling, have been associated with increased prevalence of positive bacterial culture findings and sepsis attributed to gramnegative infections in a critically ill population ( ) , as well as the susceptibility to chronic chlamydia pneumoniae infection in patients with coronary artery disease ( ) . the discovery of the importance of prrs in the pathogenesis of sirs and organ injury, including ali, has led to a therapeutic strategy targeting prrs. tlrs are the most extensively studied family of prrs, and thus recently developed new drugs mainly target tlrs and are either agonists of tlrs to enhance immune responses against infectious agents or antagonists designed to reduce inflammation due to infection or autoimmune responses ( ) . the approaches to modulating tlr activity have focused on the following aspects: (a) ligands or analogues such as eritoran (e ) from eisai (woodcliff lake, nj, usa), a synthetic analogue of bacterial lipid a that inhibits lps from activating cells through the tlr /cd /md complex ( ) ( ) ( ) ; (b) monoclonal antibodies, soluble receptors and other accessory proteins, such as a natural soluble form of tlr , found in mouse plasma and breast milk, which acts to block tlr ligand stimulation ( ) , and a member of the tlr/il- receptor family (tir or sigirr) that inhibits nf-κb signaling and may be an endogenous inhibitor of the tlr system ( ); (c) signal transduction blockers; many of the key molecules in the signaling pathways for each tlr have been identified and are considered potential drug targets ( ) ( ) ( ) . the structural bases of tir domain interactions between tlrs and adapters such as myd , mal, tram and trif have been modeled, and small peptidic sequences based on the tir domain bb loop or peptidomimetics of this region have been made that can block the interactions ( , , ) . (d) sirna and antisense; studies with knockout mice suggest that deficiency in individual tlrs has limited consequences for animals under normal conditions, but exhibits impact under conditions of specific infectious challenge ( , , ) . however, sirna sequences themselves may be ligands for intracellular tlrs ( ) ; thus attention to the design of appropriate sequences is necessary. drugs targeting tlrs have not yet been clinically applied to the treatment of ali, but because of the critical role of prrs in the development of ali, targeting of prrs has opened up a productive area for the therapy of ali. current pharmacotherapy has not been highly successful in increasing patient survival in cases of ali/ards. since prrs were recognized, their significance in the mechanisms of ali has been quickly identified. the combined activation of these different receptors may result in complementary, synergistic or antagonistic effects that modulate the process of ali. therefore, modification of prr pathways is likely to be a logical therapeutic target for ali/ards. however, a complete understanding of the role of prrs in the mechanism of ali requires further "decoding" of these multiple receptor interactions. this work was supported by the national institutes of health grant r -hl- , national institutes of health center grant p -gm- , and a va merit award. the authors declare that they have no competing interests as defined by molecular medicine, or other interests that might be perceived to influence the results and discussion reported in this paper. neutrophils and adult respiratory distress syndrome: two interlocking perspectives in early predictors of postinjury multiple organ failure acute respiratory distress in adults multiple organ failure in polytrauma patients adult respiratory distress syndrome: risk with common predispositions the formation of a structure with the features of synovial lining by subcutaneous injection of air: an in vivo tissue culture system recent 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inflammation and fibrosis biglycan: a danger signal that activates the nlrp inflammasome via toll-like and p x receptors bench-tobedside review: association of genetic variation with sepsis tlr mutations are associated with endotoxin hyporesponsiveness in humans relevance of mutations in the tlr receptor in patients with gram-negative septic shock human toll-like receptor mutations but not cd polymorphisms are associated with an increased risk of gram-negative infections polymorphisms in tolllike receptor (tlr ) are associated with protection against leprosy a functional polymorphism of toll-like receptor is not associated with likelihood or severity of meningococcal disease variation in toll-like receptor and susceptibility to group a meningococcal meningitis in gambian children reducing the toll of inflammatory lung disease a novel polymorphism in the toll-like receptor gene and its potential association with staphylococcal infection cutting edge: a toll-like receptor polymorphism that is associated with lepromatous leprosy is unable to mediate mycobacterial signaling toll-like receptor (tlr ) polymorphisms are associated with reversal reaction in leprosy polymorphisms in cd , mannose-binding lectin, and toll-like receptor- are associated with increased prevalence of infection in critically ill adults cd promoter polymorphism - c>t is associated with susceptibility to chronic chlamydia pneumoniae infection in peripheral blood monocytes targeting toll-like receptors for drug development: a summary of commercial approaches lipoprotein distribution of a novel endotoxin antagonist, e , in plasma from human subjects with various lipid levels continuous pharmacodynamic activity of eritoran tetrasodium, a tlr antagonist, during intermittent intravenous infusion into normal volunteers tlr antagonists for endotoxemia and beyond soluble forms of tolllike receptor (tlr) capable of modulating tlr signaling are present in human plasma and breast milk si-girr inhibits interleukin- receptor-and tolllike receptor -mediated signaling through different mechanisms identification of critical residues of the myd death domain involved in the recruitment of downstream kinases pivotal advance: inhibition of myd dimerization and recruitment of irak and irak by a novel peptidomimetic compound peptide-mediated interference of tir domain dimerization in myd inhibits interleukin- -dependent activation of nf-{kappa}b the family of five: tir-domain-containing adaptors in tolllike receptor signalling cutting edge: role of toll-like receptor in mediating immune response to microbial lipoproteins cutting edge: tlr -deficient and myd -deficient mice are highly susceptible to staphylococcus aureus infection activation of the mammalian immune system by sirnas key: cord- - bzllo n authors: adrover, jose m.; aroca-crevillén, alejandra; crainiciuc, georgiana; ostos, fernando; rojas-vega, yeny; rubio-ponce, andrea; cilloniz, catia; bonzón-kulichenko, elena; calvo, enrique; rico, daniel; moro, maría a.; weber, christian; lizasoaín, ignacio; torres, antoni; ruiz-cabello, jesús; vázquez, jesús; hidalgo, andrés title: programmed ‘disarming’ of the neutrophil proteome reduces the magnitude of inflammation date: - - journal: nat immunol doi: . /s - - - sha: doc_id: cord_uid: bzllo n the antimicrobial functions of neutrophils are facilitated by a defensive armamentarium of proteins stored in granules, and by the formation of neutrophil extracellular traps (nets). however, the toxic nature of these structures poses a threat to highly vascularized tissues, such as the lungs. here, we identified a cell-intrinsic program that modified the neutrophil proteome in the circulation and caused the progressive loss of granule content and reduction of the net-forming capacity. this program was driven by the receptor cxcr and by regulators of circadian cycles. as a consequence, lungs were protected from inflammatory injury at times of day or in mouse mutants in which granule content was low. changes in the proteome, granule content and net formation also occurred in human neutrophils, and correlated with the incidence and severity of respiratory distress in pneumonia patients. our findings unveil a ‘disarming’ strategy of neutrophils that depletes protein stores to reduce the magnitude of inflammation. n eutrophils are endowed with highly specialized features to combat infections. they accumulate receptors, proteases, antimicrobial peptides and proteins inside cytoplasmic granules that mediate the production of reactive oxygen species or dna-based extracellular traps (nets), both of which are highly toxic and allow for effective containment of pathogens , . several types of granules coexist in the neutrophil cytoplasm , including primary (azurophilic) granules that store myeloperoxidase (mpo), secondary (specific) and tertiary (gelatinase) granules and secretory vesicles, each of which carry distinct types of cargo , . the different granules form at different stages of granulopoiesis in the bone marrow , , and the synthesis of granule proteins declines as neutrophils differentiate , . in contrast, receptors needed for sensing, migration and phagocytosis are mostly synthesized once they reach the circulation , . after only - h in the circulation , neutrophils rapidly disappear from blood , . however, this does not imply immediate elimination, as they can be found within the vessels or parenchyma of multiple organs, with a preference for the spleen, bone marrow and lungs . the homeostatic infiltration of granule-laden neutrophils, a process enabled by a cell-intrinsic program that relies on the chemokine receptor cxcr and the core clock protein bmal (ref. ), can be potentially damaging to highly vascularized tissues such as the lungs. indeed, lungs are highly susceptible to neutrophil-mediated damage, which can cause respiratory dysfunction and death, as shown in murine models of lung injury [ ] [ ] [ ] . in humans, neutrophil-mediated acute vascular damage followed by pulmonary edema and distress is common in patients with underlying pneumococcal infections or other pre-existing conditions , . here, we investigated whether specific mechanisms prevent neutrophil-inflicted toxicity in tissues. we found that the neutrophil proteome spontaneously 'disarms' as the neutrophils lose granule content during their lifetime in circulation. this resulted in a blunted capacity to produce nets, rendering the neutrophils less toxic before they reached tissues, and protected the lungs from injury both in mice and in patients with pneumonia. the neutrophil proteome changes during the day. neutrophils undergo transcriptional changes while in the circulation . however, because transcriptional changes take a relatively long time (in the range of hours) and diurnal transcriptional programs in mouse neutrophils favor their migration into tissues, we examined alternative mechanisms that might protect the tissues from the toxic activity of neutrophils. we undertook a proteomic approach to explore the changes in protein content in neutrophils at different times after their release from the bone marrow. because the number of neutrophils that can be purified from the blood of naïve mice at night (when neutrophils are released from the bone marrow) or at noon (when neutrophils have spent several hours in the circulation) is low, we increased the number of night-like neutrophils released from the bone marrow by a one-time intraperitoneal injection at zeitgeber time (zt , or h after lights on) of amd , an antagonist for cxcr that induces the rapid mobilization of neutrophils from the bone marrow . to increase the number of day-like neutrophils in circulation we injected blocking antibodies against endothelial selectins ( mg kg − , intravenously at zt at days − , − and − before collection) , (extended data fig. a) . the proteomic analysis of these night-and day-like neutrophils isolated at zt from the blood of wild-type mice yielded , proteins (supplementary table ). a total of proteins were differentially detected when comparing the night-like versus the day-like group, with proteins involved in regulation of the cytoskeleton (arghdib, rufy ), adhesion (thbs , itgam), inflammasome (nlrp , nlrp e) and vesicular transport (vps b) (fig. a) enriched in night-like neutrophils compared to day-like neutrophils. the differential expression of identified proteins in night-versus day-like neutrophils, such as vegfr , cd , cd / , cd or cd , was confirmed by flow cytometry (extended data fig. b) . gene ontology analyses (supplementary table ) indicated involvement of the differentially expressed proteins in immune defense, cytokine signaling and angiogenesis (extended data fig. c ). night-like neutrophils were enriched for pathways linked to c-kit signaling and platelet production, possibly reminiscent of their time in the bone marrow, as well as gtpase rho signaling (fig. b) , consistent with the enhanced migratory capacity of 'younger' neutrophils . day-like neutrophils showed upregulation of protein synthesis, pathogen sensing and respiration (fig. b) , suggesting specialization for effector functions and enhanced tlr signaling, as previously suggested , . comparison of the neutrophil proteome with transcriptional datasets associated with diurnal time showed poor correlation (extended data fig. d) , suggesting that the proteomic changes are not driven by direct transcriptional regulation. these results therefore identify prominent changes in the neutrophil proteome that are associated with the time spent in the circulation. circulating neutrophils degranulate in the steady state. based on the proteomic data, proteins stored in the various types of granules (azurophilic, specific and tertiary/gelatinase granules) were enriched in night-like neutrophils compared to day-like neutrophils ( fig. c and supplementary table ). enrichment in granule proteins was also detected in the proteome of circulating neutrophils from amd -mobilized mice compared to daytime neutrophils (extended data fig. e ,f and supplementary table ). further analysis in the reactome database indicated that proteins differentially detected in the night-versus day-like neutrophils were associated with degranulation (extended data fig. a ), altogether suggesting a progressive loss of granules as neutrophils circulated. cytometric analysis of wild-type circulating neutrophils purified every h (zt , zt , zt (daytime); zt , zt , zt (nighttime)) revealed circadian oscillations of side-scatter properties (extended data fig. b ), a feature that reflects cell granularity. to assess if these variations in side scatter were caused by degranulation, we stained cytospins of ly g + blood neutrophils purified at the same times as for mpo, a protein of azurophilic granules, and analyzed granule content by high-resolution confocal microscopy (fig. d) . the maximal abundance of mpo + granules was observed at zt (midnight, when neutrophils leave the marrow), while the valley was at zt (fig. e and extended data fig. c ), indicating progressive loss of primary granules. mpo + granule content in neutrophils was in antiphase with elastase activity in plasma (fig. f) , suggesting the release of granule content into blood. a -h shift in the animal light cycle for three weeks completely readjusted the pattern of granule content in neutrophils (extended data fig. d ), indicating that degranulation was circadian in nature. finally, ly g + neutrophils from lungs, spleen and liver had a lower mpo + granule content compared to ly g + neutrophils in the blood (extended data fig. e,f) . thus, neutrophils undergo homeostatic degranulation in the circulation before they enter tissues. neutrophils progressively lose net-forming capacity. proteases stored in the azurophilic granules degrade the nuclear histones and promote chromatin decondensation during net formation and granule proteins have been identified in the net proteome . among the proteins found in the proteomic analyses, those associated with nets (supplementary table ) were enriched in the proteome of night-like neutrophils (fig. a) , suggesting that net release is more prevalent at this time. to directly examine whether the formation of nets varied during the course of a day, we treated ly g + neutrophils purified at zt or zt from the blood of wild-type mice with phorbol myristate acetate (pma), a net-inducing compound, and analyzed for the presence of extracellular dna decorated with mpo protein and citrullinated histone (cit-h ). we found that neutrophils at zt (night) formed more nets than at zt (daytime) (fig. b) , revealing a correlation between diurnal azurophilic granule content and net-forming capacity in blood ly g + neutrophils. next, we assessed the presence of nets by whole-mount immunostaining with the same markers in cremaster muscles of wild-type and lyz cre mcl fl/fl mice, which are neutropenic , after ischemia-reperfusion. many more nets were detectable in the muscles of mice at zt than at zt , while no nets were detected in the lyz cre mcl fl/fl mice (fig. c) . thus, proteomic changes and degranulation of circulating neutrophils temporally coincide with loss of net-forming ability. proteome changes are driven by cxcr and bmal . diurnal changes in neutrophil transcription and migration are controlled by a cell-intrinsic mechanism, in which the expression of the chemokine cxcl is regulated by the molecular clock protein bmal and leads to the cell-autonomous, diurnal activation of neutrophils by signaling through cxcr (ref. ). we therefore tested whether this mechanism caused the proteome changes. treatment with cxcl induced degranulation in isolated wild-type ly g + , circulating neutrophils (fig. a) . analysis of the granule content of blood neutrophils from mrp cre cxcr fl/fl (referred to as cxcr Δn hereafter) , which have a neutrophil-specific deficiency in cxcr , at zt and zt , showed no differences in mpo + granule content or in sidescatter properties in blood neutrophils, compared with neutrophils from wild-type mice ( fig. b and extended data fig. b ). cxcr Δn neutrophils degranulated in response to pma and lipopolysaccharide (lps) (extended data fig. a,b) , indicating that degranulation could be induced in these cells. we also found a loss of diurnal oscillations in granule content in blood neutrophils from cxcl −/− mice (fig. b) . neutrophils from cxcr Δn and cxcl −/− mice also showed no differences in net formation between zt and zt (fig. c) . in chimeric mice transplanted with equal mixes of bone marrow from dsred + wild-type and cxcl −/− mice, cxcl −/− neutrophils had a higher granule content than wild-type neutrophils analyzed at the same time (zt ; fig. d ), suggesting that cxcl signaled in neutrophils in an autocrine fashion to control granule content. circadian expression of cxcl in neutrophils is controlled by the transcription factor bmal (ref. ). mrp cre arntl fl/fl mice, which have a neutrophil-specific deletion of arntl (which encodes bmal , referred hereafter as bmal Δn ) showed no circadian differences in mpo + granule content between zt and zt (extended data fig. a ) and net formation (extended data fig. b ) in blood ly g + neutrophils compared to neutrophils from wild-type controls, suggesting that bmal controlled the changes in the neutrophil proteome. proteome analysis in ly g + neutrophils purified at zt (day) or zt (night) from the blood of bmal Δn mice (extended data fig. c and supplementary table ) indicated that bmal Δn neutrophils did not show circadian changes in granule proteins or in net-associated proteins (extended data fig. d,e) . these data indicated that bmal and signaling through cxcl -cxcr controlled the changes in granule content and the loss in the capacity to form nets in neutrophils. the severity of lung injury varies during the day. to test whether the diurnal changes in the neutrophil proteome influenced the outcome of inflammatory responses in tissues, we used a model of endotoxin and antibody-induced acute lung inflammation (ali) , , which is dependent on neutrophils and nets [ ] [ ] [ ] , and recapitulates the pulmonary injury observed in transfused patients . we used ali-prone balb/c mice, which displayed normal diurnal variations in neutrophil number and phenotype (extended data fig. a-c) . lungs from balb/c mice treated to induce ali (by intraperitoneal injection of . mg kg − lps h before intravenous injection of . mg kg − of an anti-h d antibody) showed evidence of nets, defined by staining for mpo, dna and cit-h ( fig. a and supplementary videos and ) , . to assess the kinetics of net formation in vivo during ali, we performed high-speed multichannel intravital imaging of the lung microcirculation . neutrophils were identified by ly g staining, while net-like structures where identified by the rapid extrusion of sytox-green-labeled dna from ly g + neutrophils, as a result of intravenous injection of the the sytox-green + extrusions were inhibited by the pad inhibitor chloramidine injected intravenously h before induction of ali (fig. c) , indicating that they represented nets. continuous tracking of neutrophil behavior for min showed higher release of sytox-green + extrusions at zt compared with zt ( fig. c and supplementary video ). the enhanced net production at night was detectable minutes after induction of ali and was sustained for the rest of the experiment (fig. c) . in contrast, the number of ly g + neutrophils was similar at zt and zt in untreated and in chloramidine-treated mice (extended data fig. a -c). because platelets have been involved in neutrophil activation and net formation during ali , , we assessed their possible involvement in the observed differences between zt and zt . we found a sharp increase in the number of platelets in the pulmonary vessels in mice with ali; however, their numbers were comparable at zt and zt (extended data fig. b) , suggesting that the number of neutrophils or platelets did not influence the diurnal changes in pulmonary net formation. to evaluate the effects of the diurnal changes in net formation in the inflamed lungs, we measured the kinetics of edema formation, which is caused by infiltration of plasma in the alveolar space. intravenous injection with evans blue dye after ali induction at zt indicated a marked increase in pulmonary vascular permeability during ali, compared to lps-only treated mice (extended data fig. a) , which was specific to the lungs. we then used computerized tomography (ct) to track the in vivo dynamics of edema in mice in which we induced ali at zt (day) or at zt (night). we found a rapid and sharp increase in water content of the lungs, which occurred earlier and was higher in zt -compared with zt -induced mice (fig. d,e) . this correlated with reduced survival of mice in which ali was induced at zt compared to those induced at zt (fig. f) . finally, treatment with chloramidine h before induction at zt protected mice from edema and death (fig. e,f) , indicating that the release of nets was a primary cause of ali. thus, the severity of lung injury during ali displays diurnal patterns that correlate with the granule content and the capacity of neutrophils to form nets. , and enrichment of these proteins in nighttime neutrophils. b, ex vivo net-formation assay. neutrophils sorted at zt (nighttime) or zt (daytime) were stimulated with pma or vehicle to induce nets and were stained for cit-h and dna (left). colocalization of both markers was used to quantify net formation, as shown in the bar graph (right); n = mice per time point. c, representative images (left) and quantification (right) of in vivo net formation in the cremaster muscle subjected to ischemia/reperfusion at nighttime (zt ) or at daytime (zt ). triple colocalization of mpo, dna and cit-h was used to define and quantify the area of nets (red; arrowheads). neutropenic mcl ∆n mice (lyz cre ;mcl fl/fl mice) were used as controls and showed no nets. dotted areas are shown magnified in the bottom panels; n = mice per condition. scale bars, μm. bars show mean ± s.e.m. *p < . ; ns, not significant, as determined by unpaired two-tailed t-test analysis. to assess whether neutrophil-intrinsic variations in granule content and net formation caused lung inflammation, we used bmal Δn and mrp cre cxcr fl/fl mice, which lacked cxcr , a negative regulator of cxcr signaling , specifically in neutrophils (hereafter cxcr Δn ). neutrophils from bmal Δn and cxcr Δn mice share transcriptional and migratory properties with night and day neutrophils, respectively . mpo immunofluorescence and transmission electron microscopy (tem) imaging of blood ly g + neutrophils purified at zt revealed higher azurophilic granule content in bmal Δn neutrophils compared with wild-type neutrophils, and comparable with that of zt wild-type neutrophils (fig. a,b) . in contrast, cxcr Δn neutrophils at zt had strong reductions in azurophilic granule content compared with wild-type neutrophils at any time point (fig. a,b) . net formation in response to pma was elevated in zt bmal Δn neutrophils, and strongly reduced in cxcr Δn neutrophils, relative to zt wild-type neutrophils (fig. c) . thus, variations in granule content and net formation are driven by a neutrophil-intrinsic program. to test whether the diurnal changes in the susceptibility to ali were caused by changes in neutrophil granularity, we analyzed the responses of bmal Δn and cxcr Δn mice to induction of ali at zt . intravital microscopy analyses showed faster and elevated net formation in the lungs of bmal Δn mice compared with lungs of cxcr Δn mice, which were similar to the differences found at zt and zt , respectively, in wild-type mice (fig. d ). intravital microscopy and flow cytometry showed similar numbers of neutrophils and platelets, as well as the number of interactions with each other, within the microvasculature of wild-type, bmal Δn and cxcr Δn mice (extended data fig. d-g) , indicating an otherwise normal behavior of intravascular neutrophils in these mice. we found marked elevation in pulmonary edema and reduced survival in bmal Δn mice during ali induced at zt compared with cxcr Δn mice ( f, survival curves for bmal ∆n (n = mice) and cxcr ∆n (n = mice) mice subjected to ali at zt . data are shown as mean ± s.e.m. *p < . ; **p < . ; ***p < . ; ns, not significant, as determined by one-way anova with dunnet's multiple comparison test (a-d), two-way anova (e) or two-sided log-rank (mantel-cox) test (f). next, we tested the causal relationship between the diurnal proteome changes in neutrophils and the diurnal susceptibility to inflammation in the bmal Δn mice. neutrophils from bmal Δn mice had a constitutively high mpo + granule content and net-forming capacity (extended data fig. a,b) and reduced survival during ali at both zt or zt (extended data fig. a ). cxcr Δn neutrophils, in turn, had a similar loss in circadian granule content and net formation (extended data fig. b ,c) and higher survival during ali at both zt or zt (extended data fig. a ). of note, the number of circulating neutrophils in bmal Δn mice oscillated during the day, similar to wild-type mice (extended data fig. d) , whereas cxcr Δn mice display constitutively high neutrophil counts , indicating that the granule content, but not the number of neutrophils, was associated with the severity of pulmonary inflammation. these observations indicated that impaired diurnal changes in the proteome and the granule content in neutrophils were related to the magnitude of inflammation. human neutrophils undergo proteome disarming. clinical manifestations of inflammation display circadian oscillations in humans, and are associated with time-of-day variations in both the incidence and the severity of cardiovascular disease [ ] [ ] [ ] . we tested whether neutrophil 'disarming' in humans could underlie the circadian changes in vascular inflammation. we measured neutrophil counts in blood and performed proteomic analysis, granule content and net-formation assays in neutrophils isolated at : , : and : (extended data fig. a ), when diurnal patterns in neutrophil number and phenotype are prominent in humans , from ten healthy volunteers. human neutrophils display a night-like phenotype (similar to murine zt ) early in the morning, and progress toward a day-like phenotype (similar to murine zt ) over the next - h (ref. ). in our cohort, neutrophil counts increased over time and peaked at : - : (fig. a) , while the granularity of neutrophils, indicated by the side scatter by flow cytometry, decreased from : to : (fig. b) . direct examination of granules in human neutrophils stained for mpo (fig. c,d) , as well as by tem imaging (fig. e) , indicated a diurnal reduction in granule content, which peaked at : and showed a trough at : . proteomic analyses of neutrophils isolated in density gradients at : and : from the blood of five healthy volunteers (supplementary table ) indicated that, from a total of , proteins detected, around . % were differentially enriched between : and : (p < . ; fig. f ). gene ontology analyses revealed that the differentially detected proteins were in pathways related to vesicle-mediated transport, secretion, exocytosis or degranulation (extended data fig. b and supplementary table ) , consistent with a loss of granule content in human neutrophils over time. many of the individual proteins identified in the mouse analyses were not detected or did not show diurnal changes in the human samples (not shown), probably indicating a high variability among humans compared with co-housed and strain-matched mice. however, numerous granule proteins, such as mpo, mmp or ltf, were more abundant at : than at : in human neutrophils (fig. g) . similar to mouse neutrophils, there was a poor correlation between transcript and protein amounts in human neutrophils (extended data fig. c) , suggesting that the proteomic changes were largely independent of transcriptional regulation. ex vivo assays of human neutrophils purified using density gradients and treated with vehicle or pma indicated a marked reduction in net-forming capacity between : and : (fig. h,i) , corresponding with more netrelated proteins at : compared with : (extended data fig. d) . these observations suggested that diurnal degranulation also occurred in circulating human neutrophils. finally, we tested whether the changes in proteome, granule content and net-forming activity in human neutrophils were associated with variations in the susceptibility to develop inflammation and/or its severity. we focused on an existing cohort of , patients with community-acquired pneumonia, who are at risk of developing acute respiratory distress syndrome (ards) . retrospective analysis of the human cohort indicated that, among the patients hospitalized with community-acquired pneumonia, ( . %) developed ards. assessment of the temporal patterns of disease onset and severity (using the pneumonia severity index), by plotting the times of admission against the pneumonia index and death in this subset of patients, revealed consistent diurnal oscillations, with a peak around : and a trough at : - : for both parameters (fig. j) . these observations indicate that proteomic disarming occurs in human neutrophils and may influence the incidence and severity of inflammation (extended data fig. and supplementary video ). here, we describe the existence of a cell-intrinsic program in neutrophils that induced the progressive loss of granule proteins involved in inflammation and the formation of nets, and provided a previously unrecognized layer of protection against the toxic activity of these leukocytes. the phenomenon of proteome disarming described here shares key features with the process of neutrophil 'aging' , in terms of reliance on cxcr signaling, regulation by bmal and cxcr in a cell-intrinsic manner and similar temporal patterns (that is, peak at noon). this suggested that neutrophil aging and disarming might represent distinct manifestations of a core circadian program in neutrophils that allows anticipation of potential threats , but also protects from excessive inflammation, vascular damage and death. beyond regulating the magnitude of inflammation, changes in the neutrophil proteome displayed temporal oscillations that aligned neutrophil activity with circadian cycles. the circadian transcriptional regulation of migration-associated genes by bmal controls the migratory properties of neutrophils, and allows the infiltration of naïve tissues in anticipation of potential infections . in addition to these transcription-mediated mechanisms, we uncovered here that the neutrophil proteome is also subject to circadian regulation, which blunts the ability of neutrophils to release toxic mediators and to produce nets. this level of immune regulation may be particularly relevant for granulocytes, a group of innate immune cells that synthesize and store the majority of their early-response mediators inside dedicated organelles. because the formation of granules and their content occurs as neutrophils differentiate in the bone marrow , , it is unlikely that changes of the granule proteome that occur in the circulation are transcriptionally regulated, as illustrated by the poor correlation between the proteome and the transcriptome in these cells. our data, however, suggested a model in which upstream control of transcription of cxcl by bmal (ref. ) allowed indirect regulation of the neutrophil proteome by the canonical circadian machinery. conceptually, the temporal nature of this process in blood aligns with the evolutionary value of circadian oscillations in segregating mutually antagonistic processes, in this case potent antimicrobial responses and protection of the host against immunemediated damage. the circadian patterns in the granule content and elastase activity in plasma suggested the progressive discharge of granules while in the circulation, such that neutrophils that migrate into tissues during the day would have lost part of their antimicrobial arsenal, and would be, as such, less likely to elicit organ damage, as shown in the mouse model of ali. because neutrophil proteases are also important for the activation of many biological mediators, including cytokines, chemokines, growth factors and adhesion and pattern-recognition receptors , , and even modulate signaling cascades in other cells , , the programmed proteome changes reported here may have pleiotropic consequences in physiology. for instance, they predict that neutrophils that are newly released from the bone marrow in conditions of stress, such as obesity or cancer , , may be better suited to regulate physiological aspects of target organs than the aged neutrophils that enter the tissues at the end of their life cycle and have lost part of their cargo. our observations shed light into the particular susceptibility of the lungs to diurnal inflammation. large numbers of neutrophils accumulate within the pulmonary microcirculation in mice, rabbits and nonhuman primates after stimulation or in the steady state , , , and the lungs can provide a niche for effective antimicrobial responses . the homeostatic accumulation of neutrophils in the lungs at the end of the light cycle in mice would predict higher susceptibility to inflammatory stimuli at this time. however, we found that neutrophils recovered from naïve lungs had reduced granularity, and that mutant mice with constitutive low levels of granules in neutrophils displayed permanent protection from ali, suggesting a dominant contribution of the disarming process in tissue protection. it has long been recognized that multiple inflammatory processes in humans display circadian periodicity , and experimental models and retrospective studies have corroborated that not only the onset of inflammation, but also the severity of the inflammatory events manifest diurnal oscillations . these events are often caused by neutrophil activation and by thrombosis, which in turn can be exacerbated in the presence of neutrophils or nets , , , . thus, our current findings in the context of ali may extend to other inflammatory and thrombotic conditions affecting multiple organs, and suggest that interventions aimed at inducing controlled degranulation may effectively 'disarm' neutrophils and protect organs from catastrophic inflammation. any methods, additional references, nature research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/ . /s - - - . all experiments were performed in - -week-old male c bl/ or balb/c mice kept in a specific pathogen-free facility at centro nacional de investigaciones cardiovasculares (cnic) under a h light/ h dark schedule (lights on at : , off at : ), with water and chow available ad libitum. bmal ∆n , cxcr ∆n and cxcr ∆n mutants in the c bl/ background have been described . for the ali experiments, bmal ∆n and cxcr ∆n mice were back-crossed into the balb/c background for at least six generations and we confirmed susceptibility to antibody-induced lung injury at this stage (not shown). all procedures with back-crossed mice were controlled using cre-negative littermates. for bone marrow transplant experiments, wt donor bone marrow came from mice expressing dsred under the control of the β-actin promoter (dsred tg ). no specific randomization method was followed in this study. all experimental procedures were approved by the animal care and ethics committee of cnic and the regional authorities. neutrophils were obtained from the blood of c bl mice using different strategies. for night-like neutrophils, mice were treated with amd ( . mg kg − intraperitoneal) h before collection. for day-like neutrophils, mice were treated with anti-p/e-selectin antibodies ( mg kg − intravenous) for d, every other day, before collection. blood was obtained by collection in edta tubes to prevent coagulation. red blood cells (rbcs) were precipitated using methylcellulose, and leukocytes in the top phase were collected and washed before purification by negative selection. negative selection was performed by incubation with biotinylated antibodies against cd e, ter , b , cd , cd , cd , cd b and cd , then incubated with streptavidin-coated beads and applied in a magnet for negative selection of neutrophils. we obtained neutrophils with a purity over %. a total of million neutrophils from each source were obtained for analysis. after in-gel trypsin digestion, the 'night-like' samples were equally mixed with the 'day-like' samples, which were labeled with oxygen- . the resulting tryptic peptide mixtures were subjected to nanoliquid chromatography coupled to mass spectrometry (ms) for protein identification. peptides were injected onto a c- reversed-phase nanocolumn ( μm internal diameter and -cm length, acclaim pepmap , thermo scientific) and analyzed in a continuous acetonitrile gradient consisting of - % b in min, - % b in min (b is % acetonitrile, . % acetic acid). a flow rate of ~ nl min − was used to elute peptides from the reversed-phase nanocolumn to an emitter nanospray needle for real-time ionization and peptide fragmentation on a q exactive mass spectrometer (thermo fisher). an enhanced fourier transform-resolution spectrum (resolution was , ) followed by the tandem mass spectra (ms/ms) from the most intense parent ions were analyzed along the chromatographic run ( min). dynamic exclusion was set at s. for protein identification, ms/ms spectra were extracted and the charge state deconvoluted by proteome discoverer v. . . . (thermo fisher). all ms/ms samples were analyzed using sequest (thermo fisher) with a fragment ion mass tolerance of ppm and a parent ion tolerance of ppm. the carbamidomethyl of cysteine was specified in sequest as a fixed modification. oxidation of methionine was specified in sequest as a variable modification. peptide quantification from fullscan spectra and labeling efficiency calculation were performed as described using quixot, a custom software written in c#. statistical analysis was performed as reported . the standardized variable z q describes the log ratio for each protein corrected for the corresponding systematic error, in each experiment. finally, data were plotted using r (v. . . ). the code will be provided on request. statistical data on all proteomics analysis are presented in supplementary table . for proteomic analysis of human neutrophils, two million isolated cells were pelleted in . -ml eppendorf tubes and snap-frozen at − °c. on analysis of purity, the best five samples ( % mean purity) were used for multiplexed proteomic analysis at : and : . for mouse neutrophils, , cells were sorted from bmal ∆n mice at zt or zt (see cytometry and cell sorting). total protein extracts from each sample were treated with mm iodoacetamide and digested overnight at °c with trypsin (promega) at a : protein:trypsin (w/w) ratio using the filter aided sample preparation digestion kit (expedeon) according to the manufacturer's instructions. the resulting peptides were desalted with c- oasis cartridges (waters corporation) using % acetonitrile (v/v) in . % trifluoroacetic acid (v/v) as eluent, and vacuum-dried. the peptides were tandem mass tag (tmt)-labeled following the manufacturer's instructions, desalted, separated into four fractions using the high ph reversedphase peptide fractionation kit (thermo fisher) and analyzed using a proxeon easy nanoflow hplc system (thermo fisher) coupled via a nanoelectrospray ion source (thermo fisher) to a q exactive hf orbitrap mass spectrometer (thermo fisher). a c- -based reverse phase separation was used with a -cm trap column and a -cm analytical column (easy column, thermo fisher) in a continuous acetonitrile gradient consisting of - % a for min, - % b for min (a is . % formic acid; b is % acetonitrile, . % formic acid) at a flow rate of nl min − . mass spectra were acquired in a data-dependent manner, with an automatic switch between ms and ms/ms using a top method. ms spectra in the orbitrap analyzer were in a mass range of - , m/z and , resolution. higher-energy collisional dissociation fragmentation was performed at ev of normalized collision energy and ms/ms spectra were analyzed at , resolution in the orbitrap. all database searches were performed with proteome discoverer (v. . , thermo fisher) using sequest-ht (thermo fisher) against a uniprot database containing all sequences from human (accessed july ; , entries). for database searching, parameters were selected as follows: trypsin digestion with two maximum missed cleavage sites, precursor mass tolerance of da, fragment mass tolerance of ppm. methionine oxidation (+ . ) was set as a variable modification. lysine and peptide amino-terminal modification of + . da, as well as cysteine carbamidomethylation of + . da, were set as fixed modifications. the same ms/ms spectra collections were also searched against inverted databases constructed from the same target databases. peptide identification from ms/ms data was performed using the probability ratio method. false discovery rates (fdr) of peptide identifications were calculated using the refined method. a % fdr was used as the peptide identification criterion. each peptide was assigned only to the best protein proposed by the proteome discoverer algorithm. quantitative information from tmt reporter intensities was integrated from the spectrum level to the peptide level, and then to the protein level based on the wspp model using the generic integration algorithm (gia) , . briefly, for the human experiment, for each sample i, the values x qps ¼ log a i =c i i were calculated, where a i is the intensity of the tmt reporter of the : sample from the individual i in the ms/ms spectrum, s, coming from peptide, p, and protein, q, and c i is the intensity of the tmt reporter from the : sample from the same individual. protein quantifications where further integrated among the five comparisons in the human experiment, obtaining the averaged value x q À Á i with their corresponding weights w q À Á i (ref. ) for every protein. in the mouse tmt experiments, for each sample i, the values x qps = log a i /c i were calculated, where a i is the intensity of the tmt reporter from the individual i in the ms/ms spectrum, s, coming from peptide, p, and protein, q, and c i is the averaged intensity of the tmt reporters from all the samples in the given tmt experiment. the log ratio of each peptide (x qp ) was calculated as the weighted mean of its spectra, the protein values (x q ) were the weighted mean of its peptides and the grand mean ( x i ) was calculated as the weighted mean of all the protein values . the statistical weights of spectra, peptides and proteins (w qps , w qp and w q , respectively) and the variances at each one of the three levels (σ in all cases, protein abundance changes were then expressed in standardized units (z q ), from which statistical significance of the changes in terms of the p value were calculated. p values were corrected for multiple hypothesis testing by controlling for the fdr. abundance changes of proteins in the compared samples were considered significant at % fdr. data were analyzed using r (v. . . ). the analysis code will be provided on request. statistical data on all proteomics analysis is presented in supplementary table . to analyze the correlation between proteomics and transcriptomics data for fresh and aged neutrophils, we compared our proteomics data with previously generated rna-sequencing data on fresh (sorted at zt from blood) and aged neutrophils (sorted at zt from blood) available in gene expression omnibus under accession number gse (ref. ). to this end we obtained a set of common proteins and genes from both datasets, compared the proteomics z score and the sequencing log fc (both inform about the relative enrichment in fresh neutrophils) and calculated the regression (spearman) statistics using r (v. . . ). we performed similar analyses for paired transcriptome and proteome data from human samples. cytometry and cell sorting. cytometric analyses were performed using a sony sp spectral analyzer (sony biotechnology). analysis was performed using flowjo v.x (tree star). cell sorting experiments were performed using a facs aria cell sorter (bd biosciences). all analyses were conducted at the cellomics unit of the cnic. the following antibodies and streptavidin conjugates were used in this study: for neutrophils in blood versus tissues, we sorted cells and cytospun them as described. before sorting, the tissues were extracted and kept in cold pbs (except liver, which was kept at room temperature in hbss) and processed immediately after. to avoid activation of neutrophils, we did not perform digestion of the tissues, but mechanically dissociated them into single-cell suspensions by straining in -μm pore cell strainers (falcon). enrichment of leukocytes from liver was performed by % percoll (ge healthcare) in hbss (invitrogen) gradient centrifugation. single-cell suspensions were incubated with antibodies against cd , cd b and ly g before sorting of cd + , cd b + and ly g + neutrophils. finally, cytospun neutrophils were fixed as described above. for the light cycle inversion experiments, wt mice were housed in light cabinets with inverted light cycle for weeks before blood was withdrawn for analysis. then, whole blood extracted in edta tubes was rbc-lysed and smears were performed as described above. in all cases, after washing, cells were permeabilized for min with . % triton, % fbs in pbs and stained with biotinylated anti-mpo at °c overnight. afterwards, cells were washed and incubated with a -conjugated streptavidin for h at room temperature. finally, cells were washed, stained with , -diamidino- -phenylindole (dapi) and mounted in mowiol. imaging was performed using a leica sp x confocal microscopy system coupled to a dmi inverted microscope, with × (hc pl apo cs ×/ . oil) magnification objective. granule contents were analyzed using the imaris software. ex vivo net-formation assays. neutrophils were sorted as previously described and × neutrophils were plated with rpmi medium on poly-l-lysine-covered -well μ-slides (ibidi), and left min to adhere. subsequently, cells were incubated for h with nm pma or vehicle. cells were then fixed using % pfa for min, permeabilized with pbs with . % triton x- , % goat serum plus % bsa and stained with antibodies against cit-h , dna (sytox-green, molecular probes) and mpo. whole-slide z stack tilescan images were acquired with a nikon a r confocal system coupled to a nikon eclipse-ti inverted microscope or a leica sp confocal microscope, and analyzed using imaris. a two-event model of transfusion-related ali was adopted for our studies as described . male balb/c mice ( - -week-old) were injected intraperitoneally with . mg kg − lps. at h later, mice received an intravenous injection of . mg kg − anti-h d (clone - - s; bioxcell). some mice were treated h before ali induction with mg kg − of chloramidine (cayman chemical) to block net formation . for survival experiments, mice were observed for h during the acute phase of ali. neutrophil elastase activity assay. neutrophil elastase activity in plasma was measured using a commercially available kit (neutrophil elastase activity assay kit (fluorometric), nak - kt, sigma-aldrich) according to the manufacturer's instructions. we analyzed μl of plasma, with an excitation wavelength of nm and emission wavelength of nm in a fluoroskan ascent plate reader (thermo labsystems). standard curve goodness-of-fit had an r of . in linear regression. intravital imaging of the lung. intravital microscopy of the lung was performed as reported . briefly, mice were anesthetized and mechanically ventilated through the trachea using a small-animal ventilator model (harvard apparatus). then, right lateral thoracotomy was applied and the lung was positioned under the window of a custom-built fixation device. a mild vacuum was applied to hold the lung in position during microscopy. mice were injected with af -conjugated ly g antibody to stain neutrophils, pe-conjugated cd antibody to stain platelets and sytox-green (molecular probes) to stain extracellular dna. fourdimensional imaging was performed for each mouse in five random fields inside the visualization window, using the vivo system . analysis was performed in imaris software. for net-like structures, we quantified the number of spots in the neutrophil extracellular dna colocalization channel over time in all random fields. we found two types of net-formation events in lungs during ali. flowing nets were defined as events in which dna was rapidly extruded out of the neutrophil and, most commonly, was washed away by the flow. adherent nets, which were defined as dna being deployed slowly around the uropod region of the neutrophil as it crawled, stuck to the vasculature and were more resistant to blood-flow washing. to quantify the relative frequency of each of these events, we visually inspected every net-formation event in our time-lapse footage and ascribed each event to one of the two categories, or to none if it was unclear. whole-mount immunostaining and tissue clearing. to confirm the presence and abundance of nets in the lungs of mice after ali induction we performed wholemount immunostaining and tissue clearing of excised lungs. mice were subject to ali and euthanized with co min after ali induction. mice were then perfused with ml of saline through the left ventricle of the heart, and the lungs were collected in cold pbs. afterwards, lungs were fixed at °c overnight in pbs with % pfa and % sucrose. after three washes of h with pbs at room temperature, tissues were permeabilized in methanol gradients in pbs for min (pbs > % meoh > % meoh > % meoh). then, tissues were bleached with dent's bleach ( % h o , . % dmso in meoh) for h at room temperature, and then were rehydrated through descending methanol gradients in pbs ( % meoh > % meoh > pbs). then tissues were incubated with blocking buffer containing pbs with . % triton x- , . % bsa, % dmso, . % azide and % fbs overnight at °c with shaking. afterwards, lungs were stained with antibodies against cit-h , mpo and cd for d at °c with shaking. after washing for h in washing buffer (pbs with . % triton x- and % nacl), the tissues were stained with secondary antibodies anti-rabbit-af , anti-hamster-af and streptavidin-af for h at °c with shaking. after h, tissues were washed for h in washing buffer and were dehydrated in meoh gradients in dh using glass containers for min in each step ( % meoh > % meoh > % meoh > × % meoh). finally, tissues were cleared for min in meoh with % babb (benzyl alcohol:benzyl benzoate, : ) and afterwards in % babb and imaged in a leica sp x confocal microscopy system coupled to a dmi inverted microscope. ct for in vivo edema quantification. in vivo ct imaging was performed on a nanopet/ct small-animal imaging system (bioscan) equipped with a micro-focus x-ray source and a high-resolution detector ( , × , pixels; μm pixel size). acquisition parameters were kvp and µas. mice were intraperitoneally anesthetized and positioned onto a thermoregulated ( °c) mouse bed until the scan was completed. an ophthalmic gel was administered in their eyes to prevent retinal drying. six studies were acquired per animal: one prior the retroorbital injection of anti-h d to induce ali, and five postinjection over min approximately. data were acquired in helical mode obtaining projections per rotation with μm pixel size. reconstruction was performed with an fdkbased method included in the proprietary nucline software (medisoy). pulmonary edema was quantified using horos software (the horos project, distributed under lgpl license at horosproject.org) as the change of the mean value, in hounsfield units, within the region of interest of the lungs obtained by thresholding (− , and − lower and upper thresholds, respectively) followed by a closing morphological operation. intravital imaging of the cremaster muscle to quantify platelet-neutrophil interactions. intravital microscopy of the cremaster muscle after tnf-α stimulation (r&d systems, . μg intrascrotal injection) was performed as previously reported , using the vivo system. a total of - venule segments per mouse were analyzed - min after tnf-α treatment in multiple fluorescence channels (cy / for pe, fitc/ for fluorescein isothiocyanate (fitc) and cy / for allophycocyanin (apc)) and bright-field images with × or × binning with a -s interval for min on each field of view. for double staining with phycoerythrin (pe)-and fitc-conjugated antibodies, acquisition was facilitated in single (fitc) and quadrant (pe) filters to avoid bleed-through of fluorescent signals between channels. for the visualization of leukocytes, fluorescently labeled anti-ly g-apc and anti-cd l-fitc were injected, and to visualize platelets, anti-cd -pe was injected intravenously at μg per mouse. quantification of neutrophil-platelet interactions in either the leading edge or trailing edge was performed using slidebook ( i). vascular permeability assay. a . % solution of evans blue in sterile pbs was prepared and μl of the solution was injected intravenously into mice min before transfusion-related ali induction (or just lps injection as control, see antibody-induced ali for the protocol). mice were sacrificed min later, and tissues extracted and weighed. then, tissues were submerged in . ml formamide and incubated at °c for h. tissues were removed and the tubes centrifuged for min at g. finally, supernatants were measured for absorbance at nm using an xmark microplate spectrophotometer (biorad) plate reader. human studies. we performed human proteomics, rna-sequencing, netformation assays and granule quantification (see individual methods sections) in neutrophils from blood samples of heathy volunteers obtained at : , : and : at hospital de octubre. the study complied with all ethical regulations and was approved by the ethical research committee of hospital de octubre in madrid (ceim no. / ), and informed consent was obtained from all volunteers. human neutrophil granule quantification. diurnal blood samples were extracted from healthy volunteers at : , : and : . blood ( μl) was processed as indicated for mouse neutrophils. human neutrophil isolation. freshly obtained blood ( ml) extracted from healthy volunteers at the indicated times were used to isolate human neutrophils using histopaque- and - (sigma) gradients. in a -ml falcon tube, ml of histopaque- was added and then ml of histopaque- was carefully layered on top. whole blood was carefully layered on top of the low-density solution, and tubes were centrifuged for min at g at room temperature, with no brakes or acceleration. neutrophils layering between the and interphase were carefully aspirated and the isolated neutrophils washed twice in pbs. we obtained over % purity as assessed by flow cytometry. rna-sequencing of human neutrophils. total rna was prepared with the rna extraction rneasy plus mini-kit (qiagen). total rna ( ng) was used to generate barcoded rna-seq libraries as previously described . libraries were sequenced on a hiseq (illumina) to generate -base single reads. fastq files for each sample were obtained using bcl fastq v. . software (illumina). the rna-sequencing experiments were performed at the genomics unit at cnic. human ex vivo net-formation assay. human neutrophils ( × ) isolated as previously described were plated with rpmi medium on poly-l-lysinecovered -well μ-slides (ibidi) and left for min to adhere. adherent cells were incubated for h with ng ml − pma or vehicle and then fixed using % pfa for min, permeabilized with pbs and . % triton x- , % goat serum plus % bsa and stained with antibodies against cit-h (abcam), dna (sytox-green, molecular probes) and mpo (r&d systems). whole-slide z stack tilescan images were acquired with a leica sp confocal microscope. analysis of extranuclear dna was performed using imagej with an automated method for extranuclear dna quantification made available at https://doi.org/ . / m .figshare. .v . retrospective analysis of human ards. data from ards were extracted from a prospective observational cohort study of , consecutive adult patients with community-acquired pneumonia admitted to the intensive care unit who developed ards between and (ref. ), in the hospital clinic of barcelona. the time of admission, severity and survival were determined for the patients who developed ards. patients were excluded if they had severe immunosuppression or active tuberculosis. ards was identified on the basis of the berlin definition. only time points with three or more data points were included in subsequent analyses. the study was approved by the ethics committee of hospital clinic of barcelona (no. / ). tem imaging of neutrophils. neutrophils from mouse and human blood were isolated as previously described (facs sorting for mouse neutrophils, gradient isolation for human neutrophils), fixed in % pfa for min and washed with pbs. samples were then postfixed in distilled water containing % osmium tetroxide for h at room temperature. after washing in distilled water, samples were stained in a block with . % uranyl acetate in water for min. then, samples were dehydrated in ascending gradients of ethanol in water ( %, %, %, % and %) with a last step in acetone. samples were then included in epoxy resin (durcupan) in ascending steps of resin:acetone ( : , : ) and then pure resin. samples included in the resin were polymerized for h at °c and -nm slices were obtained in a leica ultracut s ultramicrotome in -mesh copper gratings. finally, samples were counterstained with uranyl acetate and lead citrate. images were acquired in a -kv jeol jem- tem microscope with a gatan sc camera coupled to the system. quantification of granule-covered cytoplasmic area was performed in imagej using a custom-made macro that allows calculation of cell, nucleus and granule areas, and which is made available at https://doi. org/ . /m .figshare. .v . statistical analysis. unless otherwise indicated, data are represented as mean values ± s.e.m. paired or unpaired two-tailed t-test was used when two groups were compared, and comparison of more than two datasets was done using one-way analysis of variance (anova) with turkey's posttest. where applicable, normality was estimated using the d' agostino-pearson or shapiro-wilk normality test. log-rank analysis was used for kaplan-meier survival curves. sample exclusion was not performed unless evident signs of disease were found in a mouse. for determination of diurnal patterns, we performed cosinor fitting of circadian curves . to determine whether a diurnal curve displayed an oscillating pattern we used the cosinor-calculated amplitudes and compared them with a hypothetical zero-amplitude curve (that is, with no circadian behavior) assuming that both curves have identical standard deviations. we finally compared the amplitudes of the two curves using unpaired two-tailed t-test analyses. all statistical analyses were performed using prism v. (graphpad software), except proteomics and sequencing analysis, which were performed using r (see individual methods). a p value below . was considered statistically significant; nonsignificant differences are indicated accordingly. reporting summary. further information on research design is available in the nature research reporting summary linked to this article. proteomics data for mouse and human neutrophils are available in the peptide atlas with accession number pass . proteomics data for bmal ∆n mice at zt and zt and wt vehicle versus amd -treated mice are also available in the peptide atlas with the accession number pass . mouse circadian transcriptomics used for the correlation analysis are available at the gene expression omnibus with accession number gse . human sequencing data used for the correlation analysis are available at geo with accession number gse . all other data are available upon request. imagej macros for tem granule quantification and extracellular dna quantification are available in figshare (see the relevant section of methods for specific links). all other code is available upon request. fig. | graphical abstract. neutrophils are released from the bone marrow into the bloodstream enriched in granule-held antimicrobial, cytotoxic and net-forming proteins. as they spend time in the circulation, they undergo a homeostatic process of proteome 'disarming' that is regulated by the clock gene bmal and signaling through cxcr . this process causes a reduction in granule content and their ability to form nets, ultimately reducing their toxicity towards host tissues. during acute lung injury, the presence of granule-poor neutrophils at specific times of day or in cxcr mutants protects the lungs and increases survival. disabling homeostatic degranulation in bmal mutants, in contrast, increases organ damage and death at all times of day. granulopoiesis and granules of human neutrophils neutrophils: new insights and open questions neutrophil granules: a library of innate immunity proteins proteome profiling of human neutrophil granule subsets, secretory vesicles, and cell membrane: correlation with transcriptome profiling of neutrophil precursors neutrophils, from marrow to microbes targeting of proteins to granule subsets is determined by timing and not by sorting: the specific granule protein ngal is localized to azurophil granules when expressed in hl- cells developmental analysis of bone marrow neutrophils reveals populations specialized in expansion, trafficking, and effector functions dynamics of transcription regulation in human bone marrow myeloid differentiation to mature blood neutrophils what's your age again? determination of human neutrophil half-lives revisited neutrophils instruct homeostatic and pathological states in naive tissues circadian expression of migratory factors establishes lineagespecific signatures that guide the homing of leukocyte subsets to tissues a neutrophil timer coordinates immune defense and vascular protection contribution of neutrophils to acute lung injury neutrophils and their fcγ receptors are essential in a mouse model of transfusion-related acute lung injury neutrophils scan for activated platelets to initiate inflammation the acute respiratory distress syndrome: pathogenesis and treatment rhythmic modulation of the hematopoietic niche through neutrophil clearance neutrophil mobilization via plerixafor-mediated cxcr inhibition arises from lung demargination and blockade of neutrophil homing to the bone marrow neutrophil ageing is regulated by the microbiome neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against candida albicans myeloid-specific deletion of mcl- yields severely neutropenic mice that survive and breed in homozygous form platelet depletion and aspirin treatment protect mice in a two-event model of transfusion-related acute lung injury platelets induce neutrophil extracellular traps in transfusion-related acute lung injury heterotypic interactions enabled by polarized neutrophil microdomains mediate thromboinflammatory injury extracellular dna traps are associated with the pathogenesis of trali in humans and mice pathophysiology of transfusionrelated acute lung injury stabilized imaging of immune surveillance in the mouse lung circadian variation in the frequency of onset of acute myocardial infarction circadian control of the immune system circadian variations of infarct size in acute myocardial infarction acute respiratory distress syndrome in mechanically ventilated patients with community-acquired pneumonia neutrophil granules and secretory vesicles in inflammation neutrophil-derived serine proteases modulate innate immune responses neutrophil serine proteases: specific regulators of inflammation neutrophil elastase-mediated degradation of irs- accelerates lung tumor growth neutrophils mediate insulin resistance in mice fed a high-fat diet through secreted elastase il- -producing γδ t cells and neutrophils conspire to promote breast cancer metastasis obesity alters the lung myeloid cell landscape to enhance breast cancer metastasis through il and gm-csf the role of cd -mediated adhesion in neutrophil sequestration induced by infusion of activated plasma in rabbits the lung is a host defense niche for immediate neutrophilmediated vascular protection circadian variation and cardiovascular disease thrombosis as an intravascular effector of innate immunity host dnases prevent vascular occlusion by neutrophil extracellular traps publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations general statistical framework for quantitative proteomics by stable isotope labeling a novel systems-biology algorithm for the analysis of coordinated protein responses using quantitative proteomics sanxot: a modular and versatile package for the quantitative analysis of high-throughput proteomics experiments pad is essential for antibacterial innate immunity mediated by neutrophil extracellular traps experimental strategy for proteomic analysis of day-like (from p-and e-selectin treated mice) and night-like (from amd -treated mice) neutrophils isolated by negative selection (see methods) from blood. b, intracellular staining of proteins from the proteomics dataset for validation in fresh (blue) and aged (violet) neutrophils obtained as indicated in a. all the proteins analyzed correlated with the proteomics data; n = mice per condition. c, go terms of the differentially expressed proteins (fdr< . , see methods section for o proteomics) in the proteomics dataset, showing terms with p < . (from single samples of million neutrophils pooled from mice (night) and mice (day)). bubble size represents overlap of query vs. the go term. d, scatterplot, correlation coefficient and significance level (pvalue) of the spearman's correlation of the direction of change of common proteins and genes from our proteomic analysis of fresh and aged neutrophils (this paper) and circadian rna-sequencing data previously reported (adrover et al. , from mice at zt and mice at zt ), showing poor correlation of rna and protein content. e, venn-diagram showing the number of differentially detected proteins heatmap showing levels of granule proteins in this dataset, note increased detection of most granule proteins in neutrophils from amd -treated mice. data in (b) are shown as mean ± sem. * , as determined by unpaired two-tailed t-test analysis reactome pathway analysis of the proteome of night and day neutrophils (see methods section for o proteomics, from single samples of million neutrophils pooled from mice (night) and mice (day)) showing pathways with p-value < . . b, light-scattering properties (a measure of granularity) of blood neutrophils during a full diurnal cycle, measured as side-scatter in flow cytometry. data are for wt, cxcr -, cxcr -or bmal -deficient neutrophils, showing that cell-intrinsic disruption of clock regulators blunts diurnal fluctuation in granularity. curves are repeated for two cycles ∆n ) mice per time point. c, kinetics of total (top) or aged (bottom) neutrophils in blood indicating times of release of young or accumulation of aged neutrophils; n = mice (zt , zt , zt , zt and zt ), n = mice (zt ). d, shift of the light cycle alters the pattern of granule content in neutrophils. left, representative confocal images of sorted neutrophils (mpo, green; dapi, blue; scale, μm); right, granule content per cell at the indicated times and light regime representative confocal images (scale, μm) and f, quantification of granule content in neutrophils from the blood or tissues of wt mice n = (blood, lung and spleen), n = (liver) cells from mice. data are shown as mean ± sem. * not significant, as determined by one-way anova with dunnet's multiple comparison test (d), or using the amplitude vs representative confocal images (scale, μm) and b, quantification of granule content (top) and mpo intensity (bottom) in cxcr -deficient neutrophils upon lps or pma stimulation. granule loss indicates that cxcr -deficient neutrophils are responsive to inflammatory stimuli; n = cells per group representative confocal images (left) and quantification of granule content (right) in bmal -deficient neutrophils at zt (night) and zt (day). n = - cells from mice; scale μm. b, ex vivo net formation by bmal -deficient neutrophils at zt and zt . note that bmal -deficient neutrophils fail to display circadian oscillations in both granule and net formation. n = mice per time point. c, experimental design of circadian proteomic analysis of bmal -deficient neutrophils. d, granule proteins (left) and net-associated proteins (right) in the circadian bmal ∆n neutrophil proteome (n = mice at zt and n = at zt ). black dots show all granule or net-associated proteins, respectively, none of which reached significance in differential expression between night and day heatmap of granule proteins in the circadian proteome of wild-type (same as in fig. ) and bmal ∆n neutrophils. note that the diurnal pattern is lost in bmal -deficient neutrophils. data in (a-b) are shown as mean ± sem extended data fig. | normal circadian oscillations in balb/c mice. a, total (left) and cd lo aged (right) neutrophil counts in the blood of balb/c mice circadian oscillations in cd l and cxcr expression in neutrophils from balb/c mice, measured as median fluorescence intensity (mfi) by flow cytometry n = mice (zt , zt , zt , zt and zt ), n = mice (zt ). c, side scatter values plotted together with surface levels of cd l in neutrophils all curves are repeated for two cycles (dashed line) to better appreciate the circadian pattern. data are shown as mean ± sem. p values were determined by the amplitude vs quantification of neutrophil a, and platelet b, numbers per field of view over time in wild-type mice subject to ali at night (zt , blue line) or during daytime (zt , red line), in the intravital imaging experiments shown in fig neutrophil numbers in the lungs of naïve, lps-only and wild-type mice in which ali was induced at zt (n = mice) or zt (n = mice), or at zt in the presence of cl-amidine (n = mice), as determined by flow cytometry. neutrophils d, and platelets e, numbers in mutant mice insets show area under the curve (auc) values. f, neutrophil numbers in the lungs of lps-only control mice (n = ) or during ali in bmal Δn (n = mice) or cre-control (n = mice); and cxcr Δn (n = mice) or cre-control mice (n = mice), as determined by flow cytometry. g, interactions between platelets and the uropod (u) or leading edge (le) of adherent neutrophils, in the inflamed cremasteric microvessels of wild-type (n = from mice), bmal Δn (n = from mice) and cxcr Δn mice not significant, as determined by unpaired two-tailed t-test analysis (a-d) or oneway anova with dunnet's multiple comparison test (e-g) vascular leakiness in a, wild-type, b, bmal ∆n , and c, cxcr ∆n mice after induction of ali (lps + antibody) or in control mice treated with lps only. wt and bmal ∆n mice displayed increased leakiness only in lungs upon ali induction, while cxcr ∆n mice were protected; n = (lps only) and (ali) mice per genotype; d, time-lapse images showing examples of flowing and adherent nets (asterisks) as observed by intravital imaging of the lung microvasculature during ali, representative of n = independent experiments. see also supplementary movie . e, relative frequency of net types in wt, bmal ∆n and cxcr ∆n mice during ali, n = fields from mice (wt), fields from mice (bmal ∆n ) and fields from mice (cxcr ∆n ) not significant, as determined by two-way anova (a-c; unless otherwise specified, comparisons did not reach significance extended data fig. | loss of circadian patterns in bmal ∆n and cxcr ∆n mice. a, survival of wild-type, bmal ∆n and cxcr ∆n mice subjected to ali at night (zt , solid line) or daytime (zt mice (zt ) for wild-type, n = mice per time point for bmal ∆n ; n = mice (zt ) and mice (zt ) for cxcr ∆n . b, representative confocal images (top) and quantification of granule content (bottom) in cxcr -deficient neutrophils at zt and zt . note the loss of diurnal fluctuations compared with wild-type mice n = cells (from mice) per time point ex vivo net formation after pma stimulation by cxcr ∆n neutrophils analyzed at zt (n = mice) and zt (n = mice). note the loss of diurnal changes in net-formation compared with wild-type cells (see fig neutrophil counts in blood at zt and zt in wild-type (n = mice at zt and mice at zt ) and bmal ∆n mice (n = mice per time point). data are shown as mean ± sem . ; n.s., not significant, as determined by two-sided log rank (mantel-cox) test (a) or unpaired two-tailed t-test (b-d) neutrophils were purified for proteomic analysis, granule quantification and net-formation assays. b, go terms of the differentially expressed proteins between am and pm in human neutrophils. c, correlation analysis (spearman) of the direction of change of common proteins and genes from paired human proteomic (n = per time) and rna sequencing (n = per time) analysis, showing poor correlation of rna and protein content. d, volcano plot of the human neutrophil proteome highlighting proteins found in nets. red dots and labels show proteins that are significantly different among samples we thank members of the comparative medicine unit and advanced microscopy unit at cnic. this study was supported by intramural grants from the severo ochoa program (igp-so), a grant from fundació la marató de tv ( /c/ - ), grant saf - -r from ministerio de ciencia, investigacion y universidades (mciu) with cofunding from fondo europeo de desarrollo regional, grant rti - -b-i from mciu and hr _ from fundación la caixa ( the authors declare no competing interests. extended data is available for this paper at https://doi.org/ . /s - - - .supplementary information is available for this paper at https://doi.org/ . / s - - - .correspondence and requests for materials should be addressed to a.h. ioana visan was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.reprints and permissions information is available at www.nature.com/reprints. key: cord- -mu u bvj authors: wiesen, jonathan; komara, john j; walker, esteban; wiedemann, herbert p; guzman, jorge a title: relative cost and outcomes in the intensive care unit of acute lung injury (ali) due to pandemic influenza compared with other etiologies: a single-center study date: - - journal: ann intensive care doi: . / - - - sha: doc_id: cord_uid: mu u bvj background: critical illness due to h n influenza has been characterized by respiratory complications, including acute lung injury (ali) or acute respiratory distress syndrome (ards), and associated with high mortality. we studied the severity, outcomes, and hospital charges of patients with ali/ards secondary to pandemic influenza a infection compared with ali and ards from other etiologies. methods: a retrospective review was conducted that included patients admitted to the cleveland clinic micu with ali/ards and confirmed influenza a infection, and all patients admitted with ali/ards from any other etiology from september to march . an itemized list of individual hospital charges was obtained for each patient from the hospital billing office and organized by billing code into a database. continuous data that were normally distributed are presented as the mean ± sd and were analyzed by the student’s t test. the chi-square and fisher exact tests were used to evaluate differences in proportions between patient subgroups. data that were not normally distributed were compared with the wilcoxon rank-sum test. results: forty-five patients were studied: in the h n group and in the noninfluenza group. mean ± sd age was similar ( ± and ± years, respectively, p = . ). h n patients had lower apache iii scores ( ± vs. ± , p = . ) and had higher pplat and peep on days , , and . hospital and icu length of stay and duration of mechanical ventilation were comparable. sofa scores over the first weeks in the icu indicate more severe organ failure in the noninfluenza group (p = . ). hospital mortality was significantly higher in the noninfluenza group ( vs. %, p = . ). the noninfluenza group tended to have higher overall charges, including significantly higher cost of blood products in the icu. conclusions: ali/ards secondary to pandemic influenza infection is associated with more severe respiratory compromise but has lower overall acuity and better survival rates than ali/ards due to other causes. higher absolute charges in the noninfluenza group are likely due to underlying comorbid medical conditions. the spread of a novel h n strain of the influenza a virus represents the first pandemic of the st century and the first influenza pandemic since [ ] . compared with seasonal influenza, this strain was more prevalent in younger-aged individuals, obese patients, and pregnant women [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . severe cases of pandemic h n resulted in respiratory failure thought to be secondary to direct cell damage and systemic cytokine release resulting in acute lung injury (ali) or acute respiratory distress syndrome (ards) requiring prolonged ventilatory assistance and the frequent use of rescue therapies [ , , , [ ] [ ] [ ] [ ] [ ] . limited data exist that compare the clinical differences between ali in h n patients and ali arising from other etiologies. furthermore, whereas a number of studies have assessed different aspects of the economic impact of the recent pandemic [ ] [ ] [ ] [ ] , few have focused on the health care cost of the pandemic, particularly the utilization of limited icu resources. we report the severity, clinical outcomes, and hospital charges of ali/ards secondary to pandemic influenza a infection compared with ali/ards from other etiologies during a similar period of time. based on clinical bedside observations and published reports [ , , ] , we hypothesize that ali/ards secondary to pandemic influenza is associated with similar icu outcomes but increased resource utilization and higher hospital charges due to the frequent need for rescue interventions and prolonged ventilatory assistance. the study was approved by the human investigation committee of the cleveland clinic foundation (ccf) (institutional review board approval # - ) as a retrospective, single-center study at the ccf medical icu. patients were identified from a unit-based acute lung injury screening database (cleveland clinic is one of the centers participating in the ardsnetwork) and the h n patient log maintained during the fall-winter season of - . patients were included if they met criteria for ali (pao /fio ≤ ; acute bilateral infiltrates; positive pressure ventilation via endotracheal tube; and no clinical evidence of left atrial hypertension or congestive heart failure) between the months of september to march -the time that influenza infection was most prevalent. diagnostic methods for influenza a virus detection consisted of rapid antigen testing, polymerase chain reaction (rtpcr), and viral culture from nasopharyngeal swabs, tracheal aspirates, and bronchioalveolar lavage specimens. the patients were grouped into two categories: those with laboratoryproven h n infection; and those in whom h n was not clinically suspected. only patients with confirmed infection were included in the influenza group to ensure that the clinical course of the disease was accurately captured. patients were excluded from the study if they did not meet the above criteria for ards, or if clinical suspicion pointed to a likely pandemic viral infection with negative diagnostics. a research electronic data capture (redcap) database was constructed with a complete listing of the patient's demographic and clinical information, including age, gender, height, weight, body mass index (bmi), presenting symptoms, past medical history, primary reason for admission to the icu, vital signs, presence of vasopressors, laboratory values, ventilator settings and respiratory parameters, acute physiology and chronic health evaluation (apache) iii and sequential organ failure assessment (sofa) scores on admission to the micu, number of intubated days, duration of icu and hospital stay, mortality, and rescue therapies (namely inhaled nitric oxide, proning, high-frequency oscillatory ventilation, and extracorporeal membrane oxygenation [ecmo]) [ ] . the data collection was de-identified and collected in accordance with hipaa guidelines. as part of the routine micu respiratory therapy protocol, mechanical ventilation parameters are recorded every hours. all patients are managed according to a mechanical ventilation protocol that incorporates the use of nonconventional modes when a lung protective strategy on conventional modes failed to provide adequate oxygenation. the following criteria were used to define the analyzed parameters: ) mode of ventilation: the mode of ventilation that was used for the longest time for a given day; ) pao /fio : worst daily ratios were recorded; ) plateau pressure (pplat): for patients on volume control ventilation the airway pressure was measured after a -second inspiratory hold without concomitant active inspiratory efforts, and for patients on pressure control ventilation (pcv) the highest total system pressure (peep + inspiratory pressure) was recorded; ) positive end expiratory pressure (peep): the value corresponding to the highest peep for the day was recorded; ) tidal volume (vt): the largest daily volume was recorded. respiratory data were captured on the first day of intubation (day ) and then on subsequent days , , and of mechanical ventilation. there were no differences in ventilator protocols or management between the two groups. an itemized bill of individual charges for each patient was obtained from the hospital billing office and was organized by billing code into the following categories: room/board, pharmacy, supplies, laboratory, radiology, surgical (including procedures performed under general anesthesia), blood products, respiratory services, dialysis, and miscellaneous (which included some professional fees, nonsurgical procedures and phlebotomy, and diagnostics not included in the other categories, such as electroencephalograms, electrocardiograms, echocardiograms, cardiac catheterizations, and vascular studies). the values represent the hospital charges for the aforementioned services rather than the actual reimbursement, which may be subject to more variability. the single-center nature of the study removes interfacility differences in clinical and billing practices. continuous data that were normally distributed are presented as the mean ± sd and were analyzed by the student's t test. the chi-square and fisher exact tests were used to evaluate differences in proportions between patient groups. in instances where the data were not normally distributed, the groups were compared with the wilcoxon rank-sum test. differences were considered statistically significant if the p value was < . . fifty-one patients were identified in the acute lung injury screening database between september and march . twenty-two met criteria for ali and did not have confirmed or suspected h n infection and were thus included in the noninfluenza group (ali/ards secondary to noninfluenza etiologies). thirty-six patients in the h n patient log had confirmed influenza a testing. of those, had ali requiring mechanical ventilation (mv) during their micu stay and were included in our analysis. demographics, presenting symptoms, past medical history, and acuity on admission are shown in table . patients in the influenza group tended to be younger with a higher bmi. patients in the influenza group presented more often with lower respiratory infection ( vs. %, p = . ) and had increased requirement for mechanical ventilation on admission to the icu ( vs. %, p = . ). on the other hand, the noninfluenza group had a higher propensity to present with shock requiring vasopressors ( vs. %, respectively, p = . ). the primary cause of ali in the h n group was pneumonia (n = ), whereas in the noninfluenza group the etiologies were more varied, including pneumonia (n = ), sepsis (n = ), aspiration of gastric contents (n = ), transfusion reaction (n = ), and other (n = ). whereas seven patients ( %) in the h n group were considered healthy, only one patient ( %) in the noninfluenza group had no comorbid medical conditions on admission to the icu (table ) . this difference is reflected in the lower mean apache iii score on admission to the icu in the h n group ( ± vs. ± , p = . ), despite similar sofa scores ( . ± . and . ± . , p = . ). there were no statistically significant differences between the two groups for initial laboratory data, including white blood cell count, platelets, serum creatinine, bilirubin, and creatinine kinase. the number of patients who developed acute renal failure that required dialysis throughout their icu stay was the same (n = ) in both groups. sofa scores on days , , , and of mechanical ventilation indicate that patients in the noninfluenza group had more severe organ failure during their icu stay (p = . ; table ). table shows oxygenation index and mechanical ventilation related parameters on days , , , and . there was a nonsignificant trend toward worsening hypoxia in the h n group, despite significantly higher peep and pplat on days , , and . tidal volumes were comparable throughout. plateau pressures in the h n group were high due to the relative decrease in pulmonary compliance in h n -related lung injury. four patients in both groups were ventilated with airway pressure release ventilation (aprv). more patients in the influenza group required rescue therapies on day of mechanical ventilation ( vs. , respectively, p = . ); however, similar numbers of patients in both groups required rescue therapies over the duration of mv ( and patients, respectively). rescue therapies in the h n group included inhaled no (n = ), ecmo (n = ), prone ventilation (n = ), and high-frequency ventilation (n = ), and in the noninfluenza group only inhaled no (n = ) and prone ventilation (n = ). mechanical ventilation days were comparable between groups ( ± vs. ± days for groups i and ii, respectively, p = . ) as were -day ventilator-free days ( ± . and . ± , p = . ). four patients in the h n group and seven in the noninfluenza group underwent a tracheostomy procedure. hospital and icu los were comparable (median ± iqr: ± vs. . ± . and ± vs. ± . days for the influenza group and ii, respectively, wilcoxon p = . and . ). mortality was significantly higher for patients in the noninfluenza group ( vs. %, p = . ). interestingly, a kaplan-meier curve of icu mortality (figure ) indicates that patients in the h n group were more likely to be discharged alive from the icu when the length of stay was greater than days, despite a trend toward higher mortality within the first weeks. even though all charges were higher in the noninfluenza group, only the difference in blood products utilized in the icu was significant ( ± vs. ± thousands of u.s. dollars, wilcoxon p < . ; table ). differences in icu charges in pharmacy (p = . ), supplies (p = . ), radiology (p = . ), and miscellaneous (p = . ) were large but not significant due to considerable variation. the proportion of charges in each of the major categories was similar between the groups (figure ). the average total icu cost per patient ( ± vs. ± thousands of u.s. dollars, wilcoxon p = . ) and the average icu cost per patient per day ( ± vs. ± thousands of u.s. dollars, wilcoxon p = . ) tended to be higher in the noninfluenza group. the fall of heralded the influx of patients suffering from severe hypoxic respiratory complications secondary to the pandemic h n influenza to icus across the country. due to the severity of pulmonary disease that many of these patients experienced, perception among treating clinicians was that these patients would have a all values expressed as mean ± sd. using mixed models, the overall p value comparing the influenza and noninfluenza groups is . . the trend over time was not significant (p = . ). worse outcomes and consume more resources, as measured by hospital charges, than patients who developed ali from other etiologies. we demonstrated that, contrary to what was perceived, pandemic influenza a ali/ ards was associated with a lower acuity and, consequently, lower hospital mortality that ali/ards from other etiologies, and had a similar icu and hospital los. icu and total hospital charges reflected a trend toward higher overall charges for room and board, blood products, pharmacy, and overall charge per patient in the noninfluenza group. in accordance with other descriptive reports of pandemic influenza [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , patients who tested positive for h n infection, tended to be young (no patients > years old), obese ( had bmi > kg/m ), and in relatively good health ( % with no comorbid medical conditions). there were no pregnant patients in either group. compared with other studies of pandemic influenza patients who required mechanical ventilation, sofa scores (mean . ) were similar, although apache ii ( ± ) scores were higher [ ] [ ] [ ] [ ] , , , ] . the degree of respiratory compromise in our patients was more severe than other reports judging by the higher peep requirements and longer duration of mechanical ventilation, which was roughly double that reported in other studies [ ] [ ] [ ] , , , , , ] . plateau pressures in these studies were not consistently reported. however, despite significantly longer ventilation duration and prolonged icu and hospital stays, the mortality in our cohort was not higher than that seen in other studies, which ranged from - % in patients who required mechanical ventilation [ ] [ ] [ ] , , , , , ] . looking at the different patient characteristics between groups, it may be tempting to postulate that the higher rate of patients with pulmonary ards in the h n group, in contrast to prevalent nonpulmonary ards in the noninfluenza group, would correlate with a higher peep response among the latter [ ] . our findings suggest the contrary. patients in the h n group had higher mean plateau pressure, likely indicative of lower compliance. the similarity of pao /fio ratios in the two groups may be a reflection of higher peep values used in the h n group for lung recruitment, rather than being indicative of comparable degrees of lung injury. although assessing recruitability from this retrospective analysis is difficult and may be inaccurate, the higher peep used and the implication of lower compliance observed are predictors of potentially recruitable lung [ ] . these observations support the recent call for a reevaluation of the ali and ards criteria to account for this heterogeneity in the patient population [ ] . a number of important differences between the two cohorts emerged as well. as expected, the noninfluenza group was older, had more comorbid medical conditions, and less often presented to the icu with respiratory failure. the degree of ventilator support was significantly higher in the h n group on days , , and , and there was a trend to more severe hypoxemia during that time as well. nevertheless, the use of use of aprv and rescue therapies was comparable in both groups. despite more severe respiratory compromise, h n patients did not have longer time on the ventilator, longer icu or hospital stays, or higher mortality. although sofa scores were similar, the noninfluenza group had significantly higher apache iii scores, likely secondary to points assigned to comorbid medical conditions. the high acuity of illness, as well as the presence of severe comorbidities, such as solid and hematologic oncologic conditions ( patients), chronic renal insufficiency ( patients), and cirrhosis of the liver ( patients), likely contributed to the poor outcomes in the noninfluenza group. conversely, despite more severe respiratory compromise, patients in the h n group were more likely to recover due to their younger age and better overall health histories. the % mortality in the noninfluenza group was much higher than typically reported in clinical trials, with one notable exception [ ] . however, reports from tertiary care centers involving patient cohorts with similar underlying comorbid conditions have reported equally high mortality rates [ ] . our observation brings up an interesting point, namely the difference between the reported mortality in clinical trials and the observed mortality in a similar clinical condition affecting patients that would have been excluded from such trials due to coexisting comorbidities. a kaplan-meier plot of icu mortality (figure ) indicates that although patients in the h n group were less likely to survive the first days of icu care, those that did survive past day were more likely to be discharged alive from the hospital. patients in the noninfluenza group were unlikely to survive if their icu length of stay exceeded weeks. ards is among the most expensive conditions encountered in the icu [ ] . in , bellamy and oye described the charges of patients with ards, with the most expensive being room and board ( %), clinical laboratory ( %), pharmacy ( %), and inhalation therapy and ventilation ( %) [ ] . twenty-five years later, our study indicates that the aforementioned categories continue to represent the most expensive charges incurred by ards patients in the icu. the overall similarity of charges in room and board and respiratory therapy between the two groups is likely indicative of the comparative durations of hospitalization and mechanical ventilation. interestingly, despite higher ventilatory requirements and more severe hypoxemia in the h n group, respiratory charges were similar between the two groups, suggesting that the high cost of maintaining a patient on mechanical ventilation is independent of the degree of ventilator support necessary. thus, respiratory charges are more likely a reflection of duration of mechanical ventilation rather than the degree of ventilator support necessary. absolute icu charges for room and board, blood products, pharmacy, radiology, average daily charge, and overall charge per patient were larger in the noninfluenza group. icu charges for blood products in the noninfluenza group were greater by a factor of four, and pharmacy charges double that of the h n group. this finding is likely a reflection of the higher prevalence of underlying comorbid medical conditions in the noninfluenza group, such as malignancy and cirrhosis, which require expensive medications and predispose to anemia. moreover, the high mortality in this cohort likely precluded even higher hospital charges. nevertheless, the h n cohort amassed charges of similar magnitude to the most ill and expensive patients in the icu, indicating the abundant health care resources consumed by severe pandemic influenza infection. there are a number of limitations to our study. as a retrospective chart review rather than a prospective investigation, the information was culled from sources that were at times incomplete. second, the study contained a relatively small number of patients, and measures taken to ensure internal validity of each group, such as limiting the influenza group to confirmed h n infection and the noninfluenza group to the duration of the influenza season, further limited its size. additionally, whereas our study provides descriptive information relevant to the patient population of our institution and tertiary referral centers with similar acuity, other icus may be exposed to a different cohort of patients. on the other hand, as a single-center study, potential differences in clinical and billing practices could be minimized. although a comprehensive charge profile of each patient was generated, trends in the timing of charges could not be obtained. finally, the hospital charge data were mined from an extensive database divided by charge coding, and therefore, some charges may have been mislabeled or inappropriately categorized. our study provides interesting observations about the clinical course, outcomes, and cost of the h n influenza pandemic. although patients with severe pulmonary complications of pandemic influenza infection have poor oxygenation and require significant ventilatory support and rescue therapies, their younger age and tendency to have fewer comorbid medical conditions contribute to their improved prognosis compared with patients with ali from other causes. both groups of patients consume enormous amounts of hospital resources, and physicians and policy makers must be aware of this when future pandemics arise. world now at the start of influenza pandemic california pandemic (h n ) working group: severe h n influenza in pregnant and postpartum women in california pediatric hospitalizations associated with pandemic influenza a (h n ) in argentina critical care services and h n influenza in australia and new zealand extracorporeal membrane oxygenation for influenza a(h n ) acute respiratory distress syndrome critically ill patients with influenza a(h n ) in mexico intensive care adult patients with severe respiratory failure caused by influenza a (h n )v in spain critically ill patients with influenza a(h n ) infection in canada pandemic (h n ) : epidemiological, clinical and prevention aspects hospitalized patients with h n influenza in the united states pandemic (h n ) influenza writing committee of the who consultation on clinical aspects of pandemic (h n ) influenza, bautista e, chotpitayasunondh t: clinical aspects of pandemic influenza a (h n ) virus infection clinical management of pandemic influenza a(h n ) infection h n : viral pneumonia as a cause of acute respiratory distress syndrome ventilator management for hypoxemic respiratory failure attributable to h n novel swine origin influenza virus hospitalized patients with h n influenza infection: the mayo clinic experience clinical findings and demographic factors associated with icu admission in utah due to novel influenza a(h n ) infection the macroeconomic impact of pandemic influenza: estimates from models of the united kingdom, france, belgium and the netherlands cost-effectiveness analysis of hospital infection control response to an epidemic respiratory virus threat economic consequences to society of pandemic h n influenza -preliminary results for sweden effectiveness and cost-effectiveness of vaccination against pandemic influenza (h n ) research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support severe refractory hypoxaemia in h n ( ) intensive care patients: initial experience in an asian regional hospital lung recruitment in patients with the acute respiratory distress syndrome epidemiology and outcomes of acute lung injury effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome adult respiratory distress syndrome: hospital charges and outcome according to underlying disease variable costs of icu patients: a multicenter prospective study relative cost and outcomes in the intensive care unit of acute lung injury (ali) due to pandemic influenza compared with other etiologies: a single-center study the authors declare that they have no competing interests.authors' contributions jw and jk were responsible for the data input. jw and jg composed the manuscript. hw provided editorial assistance. ew provided the statistical analysis. all authors read and approved the final manuscript. submit your manuscript to a journal and benefi t from: convenient online submission rigorous peer review immediate publication on acceptance open access: articles freely available online high visibility within the fi eld retaining the copyright to your article submit your next manuscript at springeropen.com key: cord- -aki lhp authors: chen, qi xing; song, sheng wen; chen, qing hua; zeng, cong li; zheng, xia; wang, jun lu; fang, xiang ming title: silencing airway epithelial cell-derived hepcidin exacerbates sepsis-induced acute lung injury date: - - journal: crit care doi: . /s - - - sha: doc_id: cord_uid: aki lhp introduction: the production of antimicrobial peptides by airway epithelial cells is an important component of the innate immune response to pulmonary infection and inflammation. hepcidin is a β-defensin-like antimicrobial peptide and acts as a principal iron regulatory hormone. hepcidin is mostly produced by hepatocytes, but is also expressed by other cells, such as airway epithelial cells. however, nothing is known about its function in lung infections and inflammatory diseases. we therefore sought to investigate the role of airway epithelial cell-derived hepcidin in sepsis-induced acute lung injury. methods: acute lung injury was induced by polymicrobial sepsis via cecal ligation and puncture (clp) surgery. adenovirus-mediated short hairpin rna specific for the mouse hepcidin gene hepc and control adenovirus were intratracheally injected into mice. the adenovirus-mediated knockdown of hepcidin in airway epithelial cells was evaluated in vivo. lung injury and the seven-day survival rate were assessed. the levels of hepcidin-related iron export protein ferroportin were measured, and the iron content and function of alveolar macrophages were evaluated. results: the hepcidin level in airway epithelial cells was upregulated during polymicrobial sepsis. the knockdown of airway epithelial cell-derived hepcidin aggravated the polymicrobial sepsis-induced lung injury and pulmonary bacterial infection and increased mortality ( . % in ad-shhepc -treated mice versus . % in ad-shneg-treated mice, p < . ). the knockdown of hepcidin in airway epithelial cells also led to reduced ferroportin degradation and a low intracellular iron content in alveolar macrophages. moreover, alveolar macrophages form the airway epithelial cell-derived hepcidin knockdown mice showed impaired phagocytic ability than those from the control mice. conclusions: airway epithelial cell-derived hepcidin plays an important role in clp-induced acute lung injury. the severe lung injury in the airway epithelial cell-derived hepcidin knockdown mice is at least partially related to the altered intracellular iron level and function of alveolar macrophages. acute lung injury (ali) and its severe form, acute respiratory distress syndrome (ards), are common clinical disorders and present substantial health problems worldwide [ ] [ ] [ ] . the incidence of ali has been reported to be , cases per year in the united states, and sepsis is one of the most commonly encountered conditions underlying the development of ali [ , ] . through advances in supportive care, the mortality rate for patients with ali/ards has decreased over time but still remains high [ , ] . therefore, elucidating the pathogenesis of ali/ards is necessary and may help with the development of novel therapeutic targets for ali/ards. the lung is exposed to a large number of potentially pathogenic microorganisms that are inhaled. to protect this vital organ against infection and inflammation, many defense mechanisms have been evolved in the lung to prevent the development of pulmonary infections. the secretion of endogenous cationic antimicrobial peptides by epithelial cells onto the airway surface represents an important component of immune defense in the lung [ ] [ ] [ ] . recent studies have demonstrated the importance of these antimicrobial peptides, such as defensins and cathelicidin, in the pulmonary immune system and in pulmonary diseases [ ] [ ] [ ] . hepcidin is a β-defensin-like antimicrobial peptide that is mainly produced by the liver. hepcidin not only shows antimicrobial activity against gram-positive bacteria, gram-negative bacteria and yeasts, but also functions as a principal iron regulatory hormone [ ] [ ] [ ] . hepcidin binds to the iron export protein ferroportin and induces its internalization and degradation, which leads to decreased cellular iron export and increased intracellular iron retention [ , ] . because iron is an essential nutrient for all organisms, hepcidin also restricts the iron available to invading microbes, thereby enhancing the host defense against pathogens [ ] [ ] [ ] . furthermore, hepcidin can modulate the lipopolysaccharide (lps)-induced acute inflammatory response via the suppression of cytokine expression [ , ] . the multiple functions of hepcidin suggest its importance in the host immune response to infection and inflammation. recent studies have reported that in addition to being produced mainly by hepatocytes, hepcidin is also expressed by other cells, such as airway epithelial cells (aecs) [ ] . however, the role of aecderived hepcidin in pulmonary immune defense against infection and inflammation remains unknown. in the present study, we generated a mouse model of aec-derived hepcidin knockdown and investigated the impact of interfering with aec-derived hepcidin on the pathophysiology of sepsis-induced ali. we also explored the possible mechanisms involved. adenovirus-mediated short hairpin rna (shrna) as the mouse hepcidin gene hepc performs similar biological actions as human hepcidin [ , ] , we investigated hepc in the present study. small interfering rna specific to the mouse hepc (acagaugagacagacuacadt dt) was described previously [ ] , and we used this template to design the corresponding shrna (accgac agatgagacagactacattcatgagatgtagtct gtctcatctgtcttttt) with minor modifications (invitrogen, shanghai, china). an adenovirus that expresses the hepc shrna or a nonspecific control oligonucleotide (gcctaaggttaagtcgccctcgcgaac gaaggcgagggcgacttaaccttaggtt) was prepared. briefly, the double-strand shrna oligo was cloned into pentr/u vector using the block-it u rnai entry vector kit (life technologies, grand island, ny, usa). the expression clone was then generated by recombination of pentr/u entry construct and pad/block-it-dest vector using block-it adenoviral rnai expression system (life technologies) according to the manufacturer's instructions. after digested by endonuclease paci (new england biolabs, ipswich, ma, usa), the recombinant adenoviral plasmid was transfected into a cells. the harvested adenovirus was concentrated and purified by cscl gradient centrifugation. viral titer was determined using adeno-x rapid titer kit (clontech, mountain view, ca, usa). the recombinant virus and control virus were named ad-shhepc and ad-shneg, respectively. to generate a mouse model of aec-derived hepcidin knockdown, ad-shhepc or ad-shneg (approximately . × viral particles) was intratracheally injected into the mice after anesthesia with chloral hydrate ( mg/kg, intraperitoneally (i.p.)). to improve the knockdown efficiency, a second administration of the adenovirus was performed hours later. twelve days after the first injection, the mice were ready used in the experiments. ali was induced by polymicrobial sepsis with cecal ligation and puncture (clp) surgery as described previously [ ] . briefly, after the abdominal wall was prepared with a % povidone-iodine solution, a -cm midline abdominal incision was made. the cecum was then exposed, isolated, and ligated with a - silk ligature just distal to the ileocecal valve to avoid intestinal obstruction. the puncture was performed twice with a -gauge needle. then, the cecum was repositioned, and the incision was closed with a - sterile suture. sham-operated mice were underwent the same procedure but without ligation and needle perforation of the cecum. at the end of the operation, the mice were resuscitated immediately by the subcutaneous administration of saline ( ml/mouse) and allowed free access to food and water after awakening. in the experiment to evaluate the survival rate, the mice were monitored for survival every six to twelve hours until seven days post-clp. twenty-four hours after clp, the mice were sacrificed via cervical dislocation. the chest cavity was opened via a midline incision. to perform the bronchoalveolar lavage, the lung was lavaged with . ml of chilled phosphate-buffered saline three times. in all cases, more than % of the total lavage volume was recovered. a . -ml aliquot of balf was used for the total cell counts and bacteriologic culture. the remaining balf was centrifuged at g for five minutes, and the cell-free supernatant was stored at - °c for further analysis. the total cell count in the balf was determined using a hemocytometer. the balf was serially diluted in phosphate-buffered saline and inoculated on luria broth agar plates. after incubation at °c for hours, visible colonies were counted and calculated as colony-forming units/ml of balf. the total protein in the cell-free supernatant of the balf was determined by using the bca protein assay kit (pierce, rockford, il, usa). the interleukin (il)- level in the balf was determined using an enzyme-linked immunosorbent assay kit according to the manufacturer's instructions (abcam, san francisco, ca, usa). in separate groups the lung was excised, weighed, and then placed in an oven at °c for hours to achieve the dry weight. the ratio of lung wet weight to dry weight was then calculated. the lung tissues from the mice were fixed in buffered % paraformaldehyde solution (ph = . ) for hours, embedded in paraffin, and sectioned at a thickness of μm. the histological examination was conducted in a blinded fashion after staining with hematoxylin and eosin. for rna extraction, alveolar macrophages were isolated by adhering bronchoalveolar lavage cells to plastic for one hour at °c in % co [ ] . total rna of the lung and liver tissues as well as the alveolar macrophages was extracted using the trizol™ reagent. reverse transcription was performed using μg of total rna with the reverse transcription system (promega, madison, wi, usa). the transcriptional level of hepcidin was quantified by real-time pcr using a standard sybr™ green pcr protocol on a cfx real-time pcr detection system (bio-rad laboratories inc., hercules, ca, usa). the housekeeping gene β-actin served as an internal control, and the relative expression level of hepcidin was calculated using the −ΔΔct method. immunohistochemical staining was performed following standard procedures. the formalin-fixed paraffin-embedded tissues were sliced into -μm-thick sections, deparaffinized, and rehydrated. for the hepcidin measurement in the alveolar macrophages, the cells were isolated by adhering bronchoalveolar lavage cells to coverslips at °c in % co . then, the cells were fixed with % ethanol for minutes. antigen retrieval was performed in mmol/ l citric acid buffer (ph . ) for minutes using a -w microwave. endogenous peroxidase activity was blocked with % hydrogen peroxide in methanol for minutes. after incubation with rabbit anti-mouse hepcidin antibody ( : dilution; abcam) overnight at °c, the sections were washed in phosphate-buffered saline and incubated with a polymer horseradish peroxidase-conjugated secondary antibody (zsgb-bio, beijing, china) for one hour. the sections were further incubated with dako liquid dab large-volume substrate-chromogen system (dako, glostrup, denmark) and counterstained with hematoxylin. negative controls were included in all assays by replacing the rabbit anti-mouse hepcidin antibody with nonimmune rabbit antiserum. the immunostaining was evaluated using an olympus bx- light microscope (olympus, tokyo, japan). the stain density was analyzed using the image pro-plus . analysis system (media cybernetics inc., silver spring, md, usa), and the level of hepcidin was measured as the integral optical density. the protein concentrations in the lung homogenate or lysate of alveolar macrophages were detected using a bca protein assay kit (pierce). the proteins ( μg) were denatured by heating at °c for minutes in × nupage lds sample buffer (life technologies) and separated by nupage bis-tris gel electrophoresis (life technologies). then, the proteins were blotted onto polyvinylidene fluoride membrane (millipore, billerica, ma, usa). the membranes were blocked with % nonfat milk in tris-buffered saline with . % tween- and incubated overnight with goat anti-ferroportin antibody (santa cruz biotechnology, dallas, tx, usa). the membranes were then washed with tris-buffered saline with . % tween- three times for five to ten minutes each. after incubation with the related horseradish peroxidaseconjugated secondary antibody (jackson immunoresearch laboratories, inc., west grove, pa, usa), the membranes were visualized with the supersignal west pico chemiluminescent substrate (pierce). the signals were quantified using the image j software by wayne rasband (national institute of health, bethesda, maryland, md, usa). β-actin served as a protein control. the iron content in alveolar macrophages and spleen was determined by prussian blue staining using a commercially available kit according to the manufacturer's instructions (shanghai yuanye bio-technology co., shanghai, china). the stain density in the spleen was analyzed using the image pro-plus . analysis system as described above. for the iron measurement in the alveolar macrophages, the cells were isolated by adhering bronchoalveolar lavage cells to coverslips at °c in % co . then, the cells were fixed with % ethanol for minutes. the cells were considered positive with the presence of bluecolored granules within intact alveolar macrophages under a microscope. at least random macrophages were counted, and the results are presented in terms of the percentage of positive cells. the serum iron concentration was measured using atomic absorption spectroscopy [ ] . to investigate the phagocytosis function of alveolar macrophage, alveolar macrophages were isolated as described above. the cells were then incubated with fluorescent escherichia coli (life technologies) for two hours. fluorescence from the extracellular bacteria was quenched with . % trypan blue. after three washes with phosphatebuffered saline, the fluorescence was observed under a fluorescence microscope (olympus). the phagocytic index was quantified as number of fluorescent e.coli internalized by one macrophage cell counted in random fields. the data are expressed as the mean ± standard deviation (sd) or median with range where applicable. the differences between the two groups were analyzed by the t test or mann-whitney u test. the survival curves were analyzed by the kaplan-meier log-rank test. the statistical analyses were performed using graphpad prism software . (graphpad software inc., la jolla, ca, usa) and spss . for windows (spss inc., chicago, il, usa). a twotailed p value of less than . was considered to be statistically significant. hepcidin expression is modulated in response to infectious and inflammatory stimuli [ , ] . we first investigated whether the hepcidin expression level in the lung tissue changed during polymicrobial sepsis. twenty-four hours after clp surgery, the hepcidin level was significantly increased in the lung tissue, especially in the aecs (figure ). this finding indicates that hepcidin derived from aecs may play an important role in sepsis-induced ali. to confirm the role of hepcidin in ali, ad-shhepc or ad-shneg was administered to the mice via intratracheal instillation. twelve days after administration of the adenovirus-mediated shrna, the mice were subjected to an ali model, and the hepcidin level in lung and liver were assessed at hours after induction of ali. as shown in figure a and b, both the mrna and protein levels of hepcidin in aecs was significantly reduced, whereas the hepcidin expression in alveolar macrophages ( figure c and d) and hepatocytes ( figure e and f) was not affected. these results demonstrated that in the current study the intratracheal administration of ad-shhepc only silenced the hepcidin gene transcription in aecs, which was in accordance with previous studies that adenovirus-mediated intratracheal gene delivery specifically inhibited targeted gene expression in lung epithelial cells but not in alveolar macrophages and other organs [ , ] . the lung injury was evaluated both in wild-type mice and adenovirus-treated mice at hours after challenge with clp or a sham operation. histological characteristics of lung injury, including diffuse alveolar damage, infiltration of numerous leukocytes and interstitial edema, were observed both in the wild-type mice and ad-shnegtreated mice after clp challenge. knockdown of hepcidin in aecs led to more severe lung damage in the ad-shhepc -treated mice after clp challenge ( figure a ). lung wet/dry weight ratios from the experimental mice further confirmed the histological findings ( figure b ). the balf analysis showed that the hepcidin knockdown mice had significantly higher cell counts and protein concentrations ( figure c and d ). of note, pulmonary bacterial colonization was much more severe in the ad-shhepc -treated mice ( figure e ). however, the balf il- level in the hepcidin knockdown mice was not significantly different from that in the control mice ( figure f) . moreover, the influence of disruption of the hepcidin gene in aecs on the outcome of ali was further studied. clp challenge caused a seven-day mortality of . % in the wild-type mice, comparable to a mortality of . % in the ad-shneg-treated mice. however, knockdown of hepcidin in aecs significantly increased the seven-day mortality after clp surgery ( . % versus ad-shneg-treated mice, p < . ; figure g ). hepcidin regulates iron metabolism by binding to ferroportin and causing its internalization and degradation. we therefore investigated the ferroportin levels in both whole lung tissue and alveolar macrophages. as expected, the ferroportin in the control mice was almost totally degraded, whereas a higher ferroportin level was observed in the hepcidin knockdown mice ( figure a and b) . we further asked whether hepcidin gene modification in aecs had an impact on local and systemic iron metabolism. of note, the knockdown of hepcidin in aecs resulted in less iron retention in the alveolar macrophages ( figure a ), whereas the iron contents in the spleen macrophages and serum iron concentration between the two groups showed no significant differences ( figure b and c) . moreover, to link the intracellular iron content to the function of alveolar macrophages, we assessed the phagocytosis of alveolar macrophages, and found that the alveolar macrophages from ad-shhepc -treated mice showed less phagocytic ability than those from the control animals ( figure d ). in the current study, we found that the pulmonary hepcidin level was upregulated during polymicrobial sepsis. the knockdown of hepcidin in aecs aggravated the polymicrobial sepsis-induced lung injury and pulmonary bacterial infection and increased mortality. these pathophysiologic changes are at least partially related to the altered intracellular iron level and function of alveolar macrophages in the hepcidin knockdown mice. hepcidin is produced predominantly by hepatocytes. its hepatic expression can be upregulated by iron overload and inflammation and suppressed by hypoxia and anemia [ , ] . a recent study reported that hepcidin is expressed in aecs in response to interferon-γ [ ] . in the current study, an increased hepcidin level in aecs was observed during polymicrobial sepsis. because the lung is the first vital organ that is adversely affected at the onset of sepsis [ ] , the elevated expression of hepcidin may protect the mice against lung injury. to support this hypothesis, knockdown of hepcidin in aecs exacerbated sepsisinduced lung injury (figure ). considering that disruption of hepcidin has a deleterious effect, a low severity of clp model was used in the current study. since the underlying pathophysiology of sepsis is based on the severity grade of the clp model, the increase in lung injury parameter such as wet/dry weight ratio was minuscule but significantly different. hepcidin is a master regulator of iron metabolism via its interaction with ferroportin. hepcidin can trigger ferroportin polyubiquitination and induce ferroportin endocytosis [ , ] . in the lung, aecs express high levels of hepcidin and are the main source of local hepcidin production. alveolar macrophages express ferroportin and are therefore target cells for hepcidin [ ] . in the present study, knockdown of hepcidin in aecs impacted its interaction with ferroportin, and prevented degradation of the ferroportin protein in both alveolar macrophages and the figure the expression of hepcidin in airway epithelial cells (aec) was upregulated after cecal ligation and puncture (clp) surgery. hepcidin expression was examined by immunohistochemistry. a representative image is presented (magnification: × ) and quantified data are shown. n = mice/group. *p < . . lung, which consequently caused intracellular iron export into the pulmonary microenvironment. the elevated iron in the lung can be used not only for invading pathogen growth and replication, resulting in more virulent and persistent infections [ ] , but also to generate reactive oxygen species, leading to cell damage and lung injury [ , ] . on the other hand, a recent study showed that iron status could impact cytoskeleton rearrangement, which is important for the phagocytic process in macrophages [ ] . indeed, the alveolar macrophages from ad-shhepc -treated mice showed less phagocytosis ability than those from the control animals. since liver is the major source of systemic hepcidin, in the current study liver hepcidin levels were not affected and circulating iron concentrations were comparable between the ad-shneg-and ad-shhepc -treated mice. therefore, the function of circulating leukocytes should not be influenced. although the inciting injury (clp) is remote, when the bacteria circulating in the blood stream invaded the lung after clp, the decreased phagocytosis function of the alveolar macrophages from the ad-shhepc -treated mice could result in bacterial accumulation in the lung. in addition, as hepcidin exhibits broad spectrum antimicrobial properties [ ] , the knockdown of hepcidin may contribute to more severe pulmonary infections and lung injuries. using primary human aecs and alveolar macrophages, frazier et al. reported that the treatment with exogenous hepcidin did not affect ferroportin expression in the aecs and did not alter iron accumulation in both aecs and alveolar macrophages [ ] . the contradictory findings between the current study and frazier's study may result from the difference between the in vivo model and ex vivo model used and the nature of hepcidin peptide. moreover, limited by the sensitivity of the prussian blue staining method, the iron content in the aecs was undetectable in this study. because the role of aecs in pulmonary iron metabolism is much less than that of alveolar macrophages [ ] , the iron status in aecs may not play a major role in the pathophysiology of lung injury. the production of cytokines and other inflammatory mediators at the site of injury is a feature of the pathogenesis of ali, with il- being one of the hallmarks [ ] . however, the balf il- level in the aec-specific hepcidin knockdown mice was not significantly different from that in the control mice. previous studies found that low intracellular iron in macrophages could impair the translation of specific inflammatory cytokine transcripts, such as il- [ , ] . because alveolar macrophages are a major source of il- production 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cycle and oxidative stress in the lung efficacy and toxicity of intravenous iron in a mouse model of critical care anemia murine macrophages response to iron leap- , a novel highly disulfide-bonded human peptide, exhibits antimicrobial activity identification of oxidative stress and toll-like receptor signaling as a key pathway of acute lung injury selective modulation of tlr -activated inflammatory responses by altered iron homeostasis in mice attenuated inflammatory responses in hemochromatosis reveal a role for iron in the regulation of macrophage cytokine translation submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by the national science fund for distinguished young scholars (no. ), a key program (no. ) and program (no. ) from the national natural science foundation of china, zhejiang provincial program for the cultivation of high-level innovative health talents, and zhejiang provincial natural science foundation of china (y ). the funding agencies did not have any role in design, collection, analysis, and interpretation of data, as well as in the writing of the manuscript and in the decision to submit it for publication. the current study explored the role of aec-derived hepcidin in polymicrobial sepsis-induced ali, which is at least partially related to the altered intracellular iron level and function of alveolar macrophages. these observations provide new insight into the pathogenesis of ali/ards and might have therapeutic implications for ali/ards. knockdown of airway epithelial cell-derived hepcidin aggravated the polymicrobial sepsis-induced lung injury and pulmonary bacterial infection and increased mortality. knockdown of hepcidin in airway epithelial cells led to reduced ferroportin degradation in lung and a low intracellular iron content in alveolar macrophages.these findings provide new insight into the pathogenesis of ali/ards and might have therapeutic implications for ali/ards. the authors declare that they have no competing interests.authors' contributions qxc contributed to the study design, data analysis and drafting of the manuscript. sws carried out the animal studies, analyzed the data and drafted the manuscript. qhc participated in the animal studies and performed the quantitative pcr experiment. clz carried out the immunoblot and phagocytosis assays. xz participated in the iron determination and balf analysis. jlw contributed to the study design and coordination, and helped to draft the manuscript. xmf conceived of the study and critically revised the manuscript for important intellectual content. all authors read and approved the final manuscript. key: cord- -tsk pakb authors: jesmin, subrina; gando, satoshi; zaedi, sohel; sakuraya, fumika title: differential expression, time course and distribution of four pars in rats with endotoxin-induced acute lung injury date: - - journal: inflammation doi: . /s - - - sha: doc_id: cord_uid: tsk pakb the hypothesis that the expression of protease-activated receptors (pars) protein is regulated at the level of transcription and that par isoforms, par- , par- , par- , and par- , in lung tissue show different patterns of expression in lipopolysaccharide (lps)-induced acute lung injury (ali) was tested. male wistar rats were rendered endotoxemic by intra-peritoneal injection of lps ( mg/kg body weight). we examined the expression of protein and mrna and the immunohistochemical localization of par isoforms in lung tissues , , , and h after lps administration. induction of ali by lps was confirmed based on histopathological changes. lps administration induced significant increases in the expression of par isoforms (protein) at the level of transcription in ali. while the time course of par- and - expressions were different, those of par- and - were almost similar. an immunohistochemical analysis showed localization of par isoforms in the vascular endothelium, alveolar epithelium, and alveolar macrophages. however, the cellular distribution patterns of par isoforms were different. we conclude that lps induces increase in protein expression of par isoforms at the level of transcription in rats with ali. the differential expression patterns (over a time course) and distribution of par isoforms suggests a distinct role for each isoform in the pathogenesis of lps-induced ali. intra-alveolar and -vascular fibrin deposition is common in acute lung injury (ali) and acute respiratory distress syndrome (ards) [ , ] . the up-regulation of procoagulant pathways, impairment of physiological anticoagulant systems, and depression of the fibrinolytic pathway, collectively lead to florid alveolar fibrin deposition [ ] . fibrin deposits, in turn, enhance the inflammatory response by increasing vascular permeability, and by activating neutrophils and endothelial cells to produce proinflammatory cytokines [ , ] . these processes are the hallmarks of ali/ards and contribute to its pathogenesis. recent evidence suggests that progressive ali/ards is closely linked to the activation of inflammation and coagulation [ , ] , and that protease-activated receptors (pars) are important candidates in this interaction [ ] . to date, four distinct par isoforms, namely par- , - , - , and - have been described. thrombin activates par- , - , and - , whereas, trypsin and mast cell tryptase activate par- [ y ]. recent evidence suggests that tissue factor/factor viia (fviia) and ternary tissue factor/fviia/fxa complexes activate par- , and par- and - , respectively [ , ] . although the distribution patterns of some of the par isoforms have been determined in several tissues, it still remains unclear in the lungs of ali/ards. we have previously demonstrated the pulmonary expression of proinflammatory cytokine tumor necrosis factor-! (tnf) and key procoagulant molecules of tissue factor, plasminogen activator inhibitor- (pai- ), and fibrin in rabbits with endotoxin-induced ali [ ] . in addition, we were able to observe increased levels of pars protein, and to co-localize par- and procoagulant molecules in the alveolar epithelium and vascular endothelium. collectively, these results suggest that increase in expression of pars, together with proinflammatory cytokine and procoagulant molecules may underlie the development of ali during endotoxemia. the aims of the present study were to complement the data of a previous study by testing the hypothesis that: ( ) the pulmonary expression of par isoforms (protein) is regulated at the level of transcription and that ( ) the distribution pattern of each of the four par isoforms is distinct in the lung. to test this hypothesis, we performed experiments using a rat model of lipopolysaccharide (lps)-induced acute lung injury. male wistar rats ( y g, weeks old) were used in all experiments. endotoxemia was induced by administration of bacterial lps from escherichia coli :b ( mg/kg), dissolved in sterile saline, via i.p injection. at this dose, lps induces lung injury, as well as the expression of inflammatory cytokines. groups of animals (n= ) were killed using sodium pentobarbital ( mg/kg bw, i.p.) at different time-points after lps administration ( , , , and h). the control group received an equal volume of sterile saline ( ml/body), without lps. at the indicated time, the blood samples were collected by cardiac puncture for blood gas analysis, and lung tissue specimens were harvested with care, frozen immediately in liquid nitrogen, and then stored at j -c. for paraffin sections, lung tissue specimens were postfixed in % paraformaldehyde overnight and then embedded in paraffin. all the experimental procedures were approved by the animal care and use committee of hokkaido university graduate school of medicine animal care and use committee. in order to determine the arterial blood pressure and heart rate of rats, a microtip pressure transducer catheter (spc- , millar instruments, houston, tx, usa) was inserted into the left carotid artery of anaesthetized animals (sodium pentobarbital ( mg/ kg body weight, i.p.)). then, the arterial blood pressure (bp) and heart rate (hr) were monitored using a pressure transducer (model sck- , gould, ohio, usa) and recorded (bp and hr) using a polygraph system (amplifier, ap- g, nihon kohden, tokyo, japan; tachometer, at- g, nihon kohden; thermalpen recorder, wt- g, nihon kohden). lung tissues were harvested, blotted dry and weighed in order to determine the weight of the lung in the wet state and calculate the wet-to-dry weight ratio, as follows: the lung tissues were weighed; wrapped loosely in aluminum foil; placed in a drying oven overnight; and weighed again. immediately after harvest, the specimens were fixed in % buffered formalin solution, dehydrated, embedded in paraffin, and then sliced into -mm-thick sections. after deparaffinization, tissue sections were stained using standard hematoxylin and eosin (he) staining method. morphological injury in lung was semi-quantified by two pathologists blinded to the experimental design by analyzing, from each section, about randomly selected images. the average score was then determined or calculated (n= rat per each group). for determining the cellular distribution of proteins of interest, tissue specimens were fixed in % buffered formalin solution, dehydrated, embedded in paraffin, and then sliced into -mm-thick sections. the sections were then deparaffinized and treated for min with citrate buffer ( mm citric acid, ph . ) in a microwave oven ( w) before immunostaining. in some cases, frozen sections were fixed in acetone and air dried. endogenous peroxidase activity was quenched by incubation in % hydrogen peroxide for min. one percent bovine albumin in tris was used for min at room temperature to block non-specific staining caused by secondary antibodies. the sections were then incubated with primary antibodies overnight at -c, rinsed in phosphate buffer solution and then exposed to the fluorescence secondary antibody, rhodamine-conjugated affinipure or fluorescein-conjugated affinipure anti-sheep, anti-rabbit, anti-goat or anti-mouse igg (jackson immuno research laboratories), for h according to the manufacturer_s instructions. the specificity of immunoreactivity was confirmed by negative controls where nonimmune igg was used instead of primary antibodies. the coverslips were mounted with immunon (thermo shandon). immunofluorescent images were observed using a laser scanning confocal imaging system (mrc- , bio-rad laboratories). immunofluorescence staining was semi-quantitated using a scale (+ to ++++) and the average score of randomly selected images was calculated. two pathologists blinded to the experimental design evaluated each slide. the same set of experiments was repeated at least three times. the immunoblotting procedure used in the present study has already been described in our previous report [ ] . briefly, ice-cold lung tissues were minced with scissors, homogenized, and centrifuged, followed by determination of protein concentration of the supernatant using the bicinchoninic acid protein assay (pierce biotechnology). samples were then boiled in reducing sds sample buffer for min, loaded onto an sdsypage ( y % polyarylamide) gel under reduced conditions, subjected to electrophoresis, and electrophoretically transferred to polyvinylidine difluoride filter membrane. to reduce non-specific binding, the membrane was blocked for h at room temperature with % non-fat milk in pbs ( mm nacl, . mm kcl, . mm na hpo , . mm kh po ) containing . % tween , incubated overnight at -c with primary antibodies in pbsytween buffer, washed three times with pbsytween buffer, and then the membrane was incubated with a suitable secondary antibody coupled to horseradish peroxidase for min at room temperature. the blots were washed five times in pbsytween buffer and subsequently visualized with an enhanced chemiluminescence detection system (amersham), exposed to x-ray film (fuji photo film). intensity of total protein bands per lane was evaluated by densitometry. negligible loading/transfer variation was observed between samples. in each experiment, ßactin was used as the loading control. for immunological-based detections, the following antibodies were used: anti-human par- rabbit polyclonal antibody, anti-human par- goat polyclonal antibody, anti-mouse par- goat polyclonal antibody and anti-mouse par- goat polyclonal antibody (santa cruz biotechnology); anti-rabbit fibrinogen sheep polyclonal antibody (cedarlane laboratories); anti-human fibrin mouse monoclonal antibody (chemicon international), anti-rabbit inducible nitric oxide (no) synthase (inos) mouse monoclonal antibody (affinity bioreagents, golden, co, usa), and anti-xenopus laevis ßactin mouse monoclonal antibody (abcam). in most cases, the specificity of each antibody was initially confirmed by blocking its expression using a competing peptide against which the antibody was raised. it should also be noted that no positive immunoreactivity was observed when non-immune igg was used instead of the primary antibodies. for par- and - , rat liver tissue was used as positive control for both immunofluorescence staining and the immunoblot analysis, according to the manufacturer_s instructions (santa cruz biotechnology) [ ] , whereas, for par- and - immunoreactivities, rat liver tissue and rat uterus were used as positive controls, respectively [ ] . please note that each of the anti-par antibodies showed no cross-reactivity with other par isoforms. anti-human fibrin mouse monoclonal antibody and anti-rabbit fibrinogen sheep polyclonal antibody recognize fibrin and fibrinogen, respectively, but can also detect each other_s target peptide. the immunogen of the anti-fibrin antibody is fibrin-like ß-peptide gly-his-arg-pro-leu-asp-lys-cys. total rna samples were prepared from lung tissue specimens using the guanidinium thiocyanatephenol-chloroform single-step extraction method with isogen (nippon gene, toyama, japan), which is routinely used in our laboratory [ ] . after isolation, the rna was processed as follows: treated with dnase i; quantified and then reverse transcribed to cdna by omniscript reverse transcriptase using a first-strand cdna synthesis kit (qiagen). the reverse transcription reaction was performed at -c for min. the expression of par isoform mrnas were analyzed by real-time quantitative pcr with taqman probe using an abi prism sequence detector (perkinyelmer applied biosystems, foster, ca, usa), as previously described [ ] . gene-specific primers and taqman probes were synthesized from primer express v. . software (perkinyelmer applied biosystems), according to published cdna sequences of each gene. the sequences of the oligonucleotides were as follows: tnf levels in the plasma and lung tissues were detected using an enzyme-linked immunosorbent assay (elisa) kit for screening rat tnf (pierce biotechnology, rockford, il). for inos, we used a human inos immunoassay kit (r and d systems, minneapolis, mn). the results were expressed as meantsd (n=total number of animals in each group). the means were compared by a one-way factorial analysis of variance, followed by scheffé_s test for multiple comparisons. differences were considered to be significant at a value of p< . . as shown in table , levels of both systolic and diastolic blood pressure decreased significantly after lps administration, in comparison to control rats. the peak levels of plasma tnf and its (tnf) concentration in the lung were significantly elevated at h after lps administration. similarly, levels of plasma inos and its (inos) protein and mrna expression in the lung increased after lps administration. table summarizes the values for blood gases and lactate concentrations in rats before and after lps was given. arterial pao was significantly reduced from control animals at all time points after lps administration. as a quantitative measure of fluid clearance in lungs, wet-to-dry weight ratios were evaluated in lungs removed from rats killed at specified times after lps administration. the effect of lps on the ratios (wet-to-dry weight ratios) occurred in a time-dependent manner, leading to a significant (p< . ) increase from baseline ( . t . ) to peak value ( . t . ) after lps administration. the lungs from control rats (untreated) showed no detectable injury, based on the histological analysis. in contrast, h after lps administration, the lungs of treated animals showed congestion (+), neutrophil infiltration (+) and thickening of alveolar septum (+). these changes were also observed at and h after lps administration. at h following the administra- tion of lps, the features of lung injury became more evident. the lungs showed congestion (+), infiltration of inflammatory cells in the alveoli (++) and a thickening of alveolar septum (++), which together are called glanulomatous changes. these changes are shown in fig. . the relative amounts of immunodetectable fibrinogen/fibrin increased steadily following induction of sepsis with lps. relative levels of fibrinogen/fibrin vs. control ( . ) at , , , and h after lps administration were . t . / . t . , . t . / . t . , . t . / . t . , and . t . / . t . , respectively (p< . ). fibrin was poorly detectable in control lungs (data not shown). at h after lps administration, fibrin deposition was evident in the intra-and extra-vascular spaces, in the alveoli and in the bronchial epithelium (data not shown). the par- specific antibody reacted with one band of õ kda that is consistent with the predicted molecular mass of this receptor [ , ] . its expression increased in a time-dependent manner (fig. ) , whereas, that of par- was observed at all time points after lps administration, peaking ( . -fold) h after lps administration (fig. ) . peak expressions of par- and - proteins were seen h after lps administration (fig. ) . par- was detected as a band of õ kda, consistent with the manufacturer_s information. in addition, the molecular weights of the bands believed to be those of par- and - were comparable to those of the genbank sequences. the mrna expression of the par isoforms, as determined by real-time pcr, corresponded to levels of their respective proteins in the lung (fig. ) . immunohistochemical staining of par- (fig. ) in the control lung were essentially nil. however, h after lps administration, par- was predominantly expressed in the endothelium of small-to micro-sized blood vessel, and vascular smooth muscle cells of medium-to large-sized blood vessel, while only a modest expression was observed in vascular smooth muscle cells of small-micro-sized blood vessels. the endothelium of medium-to large-sized blood vessels only exhibited moderate staining of par- . the alveolar epithelium and bronchial epithelium exhibited strong positive staining of par- , with only modest staining observed in the alveolar macrophages. similar to the pattern of par- immunoreactivity, par- (fig. ) immunoreactivity was essentially nil in the control lungs. however, h after lps administration, it (par- ) was localized in the endothelium and vascular smooth muscle cells of small to micro size blood vessels, at comparable intensities. in the vascular endothelium of medium-to large-sized blood vessels, par- immunoreactivity was strong, but was weakly stained in the vascular smooth muscle cells. the intensity of par- immunoreactivity in the alveolar and bronchial epithelia and alveolar macrophages were similar. unlike par- and - , there was some slight immunostaining observed for par- in the control lung (fig. ) , and by h after lps administration, it (par ) was intensively expressed in both the endothelium and vascular smooth muscle cells of small-to medium-sized blood vessels. in contrast, only moderate immunoreactivity was localized in the vascular smooth muscle cells of large-sized blood vessel, but nonetheless, strong staining in the endothelium. the intensity of par- immunoreactivity in the alveolar and bronchial epithelia, and alveolar macrophage, was similar to par- and - . immuno-expression of par- in control lungs was similar to par- and - (fig. ) , and h after lps administration, strong signals were localized in the vascular smooth muscle cells of small-to medium-sized blood vessels, and moderately expressed in the vascular endothelium. this was in contrast to the vascular endothelium of large-sized blood vessels, which exhibited strong par- immunoreactivity, with modest staining in vascular smooth muscle cells. strong positive staining of par- is localized in alveolar epithelium, while moderate staining is observed in alveolar macrophages and the bronchial epithelium. these results are summarized on table . here, we test the hypothesis that levels of par isoforms in lungs are regulated at the level of transcription, but each (isoform) with a distinct pattern of tissue distribution. following lps administration, we observed a significant reduction in blood pressure and an increased expression of plasma inos, which occurred in parallel to inos immunoreactivity in the lung [ ] . in addition, he staining demonstrated a potent infiltration of inflammatory cells and thickening of alveolar septum called glanuloma [ ] . collectively, these findings support the notion that lps (via i.p.) induces sepsis and the development of ali in a rat model. the present study observed increased expression of tnf protein in both lung tissue and plasma immediately after lps administration. tnf is known to induce the expression of tissue factor, which leads to the activation of the extrinsic coagulation pathway, and increases in levels of fxa and thrombin, and ultimately, enhancement of coagulation [ ] . tnf is also known to increase levels of pai- , which, in turn, inhibit fibrinolysis [ ] . following increase in tnf levels, we found a marked elevation of fibrin/fibrinogen protein levels in the lung tissues. this observation was confirmed by immunohistochemical analysis that demonstrated clear fibrin deposition in both intra-vascular and -alveolar spaces. although, in the present study, we did not measure levels of tissue factor or the pai- , in our previous investigation these molecules were highly expressed in the lung of lps-treated rats, as revealed by western blot and immunohistochemical analyses [ ] . collectively, these data suggest that enhanced inflammation, coagulation activation, and the inhibition of fibrinolysis lead to fibrin deposition in acutely injured lung, following lps administration. these results are consistent with the clinical and experimental evidence for ali and ards [ y ] . here, we also found that lps induces increases in the protein expression of pars isoforms to in the lung of rats. the parallel changes observed in levels of both protein and mrna of par isoforms suggests that lps may exert its influence at the transcription level. the disparity in the timing of pars expression between our previous and the present studies may be due to species differences [ ] . indeed, nysted et al. [ ] demonstrated that stimulation with the cytokines tnf and interleukin- (il- ), as well as bacterial lps resulted in a five to ten fold elevation of par- gene expression in a dose-dependent manner using huvec. in addition, the exogenous treatment of human skeletal muscle cell and monocyte with proinflammatory cytokines increased expression of par- and - [ , ] . lan et al. [ ] demonstrated that inflammatory stimuli induced by influenza a virus expressed par- , - , - , and - mrna in the lung of intact mice. it is interesting to note that the occurrence of peak levels for plasma and lung tnf ( h after lps administration), coincided with the point at which significant elevations of pars- ,- , and- were observed, compared to control. these results suggest that tnf, together with, or as well as, lps rapidly induce expression of pars gene expression through transcription factors in the injured rat lungs. nuclear factor-kappab (nf-kb) is a transcriptional factor that plays a critical role in sepsis by regulating gene expression of many inflammatory cellular mediators and receptors involved in immune recognition [ ] . the nf-kb activators include an extensive list of grampositive and -negative bacteriae and their products such as lps; viruses and their components; protozoan parasites; cytokines (e.g., tnf, il- ); free radical; and oxidants. although determination of factors likely to regulate the transcription of pars is important, it goes beyond the aims of the present study. we believe that nf-kb may be one of the most important transcriptional factors regulating pars expression in lps-induced ali and, as such, plan study it in our future projects. although little is known about the cellular distribution of par- and - in lung tissue, pars- and - have been demonstrated in the airway epithelial and smooth muscle cells within the respiratory tract, as well as endothelial and vascular smooth muscle cells [ ] . par- and - are also present in the terminal bronchial epithelium and type ii epithelial cells of the peripheral lung, and lung macrophages, mast cells, granulocytes, and lymphocytes [ ] . while our previous study demonstrated the immunolocalization of par- in these cells and tissues in lps-treated rabbits, the present study showed strong immunoreactivities for all isoforms of pars in the endothelium, alveolar epithelium, and lung macrophages using a rat model of ali [ ] . in addition to increased immunoreactivities of tissue factor and pai- in the endothelium and alveolar epithelium, as reported in the previous study, we also confirmed here a strong fibrin deposition in the alveolar epithelium, and intra-alveolar and -vascular spaces. both the endothelium and alveolar epithelium are known to play important roles in the pathogenesis of ali. the co-localization of the key procoagulant molecules and the four isoforms of par in these tissues imply that these molecules may also play a pivotal role in inducing permeability in endothelial and epithelial cells, as observed in lps-induced ali. in contrast to the distinct patterns in onset and course of expression, and localizations of par- and - , par- and - had almost a similar pattern. it has been suggested that, in certain instances, binding of protease to one receptor can facilitate cleavage of another receptor. this appears to be the case for par- and - on mouse platelets [ ] . other studies suggest that par- facilitates activation of par- in the presence of low thrombin concentration [ , ] . some data indicate that par- is a cofactor for par- in mouse platelets: the hirudin-like site of par- binds and concentrates thrombin at the cell surface, and, thereby, promote cleavage of thrombin to par- [ ] . while the pathological significance of these phenomena has not been elucidated, the co-localization and co-expression of par and - , as observed in the present study, may indicate a similar cofactor relationship. a recent study provides strong evidence that neutrophil elastase-mediated apoptosis plays a pivotal role in the pathogenesis of ali, through the par- dependent pathway [ ] . vogel et al. [ ] demonstrated abrogation of thrombin-induced increase in pulmonary microvascular permeability in par- knockout mice. furthermore, the absence of par- signaling appears to attenuate bleomycin-induced lung inflammation and fibrosis [ ] . indeed, using par- knockout mouse, su et al. [ ] demonstrated that par- activation induces lung inflammation and pulmonary edema. in addition to these studies, we found expression of par- and - and their localization in endothelium, alveolar epithelium and macrophages in the lung. taken together, these data suggest that par- and - may play a critical role in the pathogenesis of ali. however, the physiological and or pathological significance of par- and - expressions in the injured lung is currently unclear. the limitations of the present study include: ( ) a narrow window of time after administration of lps was investigated to study the pulmonary expression of pars; ( ) the single moderate dose of lps (and not higher dose), although able to induce endotoxemia in rat, could not induce severe endotoxemia. thus, the current investigation could not provide insights on pars expression patterns associated with severe endotoxemia. for this reason, future studies should investigate a possible correlation between the degree of severity of induced endotoxemia and the pattern of pars expression. in addition, a more extended and prolonged time course study should be undertaken that will enable examination of the pulmonary pars expression possible. in summary, we demonstrate expression of par- , - , - , and - proteins, which occurred at the level of mrnas for up to h after lps administration in a rat model of ali. the timing in expression, i.e., in onset and over the course of time, for the four isoforms of pars were distinct. they were also localized in diverse cells, including vascular endothelium, alveolar epithelium, and alveolar macrophages, a similar localization pattern as tissue factor and pai- , observed in our previous study. in addition, we also found tnf expression, as well as intra-alveolar and -vascular fibrin deposition in the lung. collectively, these results suggest that each par isoform plays a distinct important role in the pathogenesis of lps-induced ali. coagulation, fibrinolysis, and fibrin deposition in acute lung injury coagulation abnormalities in acute lung injury and sepsis bronchoalveolar coagulation and fibrinolysis in endotoxemia and pneumonia coagulation and inflammation in acute lung injury thrombin signaling and protease-activated receptors proteinase-activated receptors: novel mechanisms of signaling by serine proteases proteinase-activated receptors nonhemostatic activity of coagulation factor xa: potent implications for various diseases tissue factorand factor x-dependent activation of protease-activated receptor by factor viia temporal changes in pulmonary expression of key procoagulant molecules in rabbits with endotoxin-induced acute lung injury: elevated expression levels of protease-activated receptors diminished penile expression of vascular endothelial growth factor and its receptors at the insulin-resistant stage of a type ii diabetic rat model: a possible cause for erectile dysfunction in diabetes protease-activated receptor isoform expression in pregnant and nonpregnant rat myometrial tissue effects of exercise training on expression of endothelin- mrna in the aorta of aged rats thrombin and protease-activated receptor- agonists promote lipopolysaccharide-induced hepatocellular injury in perfused liver role of expression of thrombin receptor par- in muscle cells and neuromuscular junctions during the synapse elimination period in the neonatal rat immunochemical localization of inducible nitric oxide synthase in endotoxin-treated rats acute respiratory distress syndrome. a comprehensive clinical approach pathogenesis of disseminated intravascular coagulation in sepsis the proteinase-activated receptor is induced by inflammatory mediators in human endothelial cells. comparison with the thrombin receptor thrombin receptor induction by injury-related factors in human skeletal muscle cells thrombin receptor expression and responsiveness of human monocytic cells to thrombin is linked to interferon-induced cellular differentiation altered expression and in vivo lung function of protease-activated receptors during influenza a virus infection in mice nuclear factor-kb role of proteaseactivated receptors in airway function: a target for therapeutic intervention? par is a cofactor for par activation by thrombin protease-activated receptors in hemostasis, thrombosis and vascular biology proteaseactivated receptor- mediates elastase-induced apoptosis of human lung epithelial cells abrogation of thrombin-induced increase in pulmonary microvascular permeability in par- knockout mice absence of protease-activated receptor- signaling affords protection from bleomycin-induced lung inflammation and fibrosis protease-activated receptor- activation induces acute lung inflammation by neuropeptide-dependent mechanisms key: cord- -hx lkuj authors: morty, rory e.; eickelberg, oliver; seeger, werner title: alveolar fluid clearance in acute lung injury: what have we learned from animal models and clinical studies? date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: hx lkuj background: acute lung injury and the acute respiratory distress syndrome continue to be significant causes of morbidity and mortality in the intensive care setting. the failure of patients to resolve the alveolar edema associated with these conditions is a major contributing factor to mortality; hence there is continued interest to understand the mechanisms of alveolar edema fluid clearance. discussion: the accompanying review by vadász et al. details our current understanding of the signaling mechanisms and cellular processes that facilitate clearance of edema fluid from the alveolar compartment, and how these signaling processes may be exploited in the development of novel therapeutic strategies. to complement that report this review focuses on how intact organ and animal models and clinical studies have facilitated our understanding of alveolar edema fluid clearance in acute lung injury and acute respiratory distress syndrome. furthermore, it considers how what we have learned from these animal and organ models and clinical studies has suggested novel therapeutic avenues to pursue. acute lung injury (ali), and its more severe form, acute respiratory distress syndrome (ards), are important causes of morbidity and mortality in critically ill patients [ ] . one of the hallmarks of ali/ards is the accumulation of protein-rich edema fluid in the alveolar compartment of the lung [ ] , caused by increased fluid influx into the airspaces, and decreased fluid transport out of these airspaces. in the case of the latter, impaired alveolar fluid clearance (afc) is a key underlying cause of alveolar edema persistence [ , ] , and the ability of ali/ards patients to clear edema fluid is correlated with a shorter stay in the intensive care unit, and reduced mortality [ ] . much effort has therefore been focused on identifying pathogenic mechanisms underlying perturbed afc in ali/ards patients, and how we can potentiate afc, which may form the basis of novel or improved therapeu-tic strategies. the accompanying review by vadász et al. (http://dx.doi.org/ . /s - - - ) details our current understanding of the signaling mechanisms that facilitate afc. to complement that report this review focuses on how intact organ and animal models and clinical studies have facilitated our understanding of afc in ali/ards. we also discuss how some of these studies have led to novel therapeutic approaches. edema fluid is transported out of the alveolar airspaces into the interstitium, where it is cleared by the lymphatic drainage. alternatively, fluid can also be transported into the vasculature, where it is cleared by the circulation. maintenance of an optimum alveolar fluid volume results from a finely balanced influx of fluid into the lung, and fluid clearance out of the lung; therefore, endothelial and epithelial barrier integrity is essential for optimal fluid balance [ ] . in patients with ali/ards, the integrity of both the endothelial and epithelial barriers may be compromised, leading to accelerated fluid influx into the alveolar compartment and impaired afc [ ] . in healthy lungs the alveolar epithelium is considerably less permeable than the endothelium [ ] . approximately % of the alveolar epithelium surface area is composed of flat alveolar type i cells, with the remaining % accounted for by cuboidal type ii cells which produce surfactant and are progenitor cells that regenerate the epithelium after injury. historically, type ii cells are accredited with a key role in afc. it is widely accepted that afc is driven by sodium transport across the airway epithelium, which is affected by the concerted action of two sodium transport systems: the epithelial sodium channel (enac) [ ] and the na + , k + transporting adenosine- -triphosphatase (na,k-atpase) [ ] as well as the cystic fibrosis transmembrane conductance regulator (cftr; a chloride channel) [ ] and other, as yet uncharacterized channels [ ] . impaired function of any one of these ion transport systems can perturb afc. diffuse alveolar damage, including severe alveolar epithelial damage, is one of the hallmarks of ali/ards [ , ] , where extensive type i cell necrosis occurs, leaving an intact but denuded basement membrane which is repopulated by hyperplastic type ii cells that regenerate the alveolar epithelium [ ] . given the established importance of the type ii cell in afc [ ] and the emerging importance of the type i cell in afc with the recent discovery that type i cells also contain functional sodium and chloride channels [ ] , this epithelial damage fig. factors that cause impaired alveolar fluid clearance in ali/ards that have been investigated in animal and organ models and in clinical studies. anf, atrial natriuretic factor; enac, epithelial sodium channel; na,k-atpase, na + , k + transporting adenosine- triphosphatase, tgf-β, transforming growth factor β is likely to massively impact afc in ali/ards patients. this idea is strengthened by the observation that in patients with hydrostatic pulmonary edema, which may result from congestive heart failure or acute myocardial infarction, both the alveolar epithelial barrier and afc remain intact [ ] . animal and organ models have proved particularly useful to study afc, because the three-compartment (alveolar airspace-interstitium-vasculature) structure of the lung is preserved [ ] [ ] [ ] . studies in humans have relied heavily on the assessment of extravascular lung water (evlw) [ ] . more recently, nasal potential difference (npd) has been employed as a surrogate measurement for transepithelial ion transport [ ] , although this measurement is clearly limited in its usefulness when studying diseases that exclusively impair alveolar ion transport. the aspects of impaired afc in ali/ards that have been examined in animal or organ models and clinical studies are summarized in fig. . studies in live, anesthetized, ventilated sheep provided the first evidence that alveolar fluid relied on active ion transport, where clearance of salt and water occurred against an increase protein concentration in the alveolar lining fluid [ ] . supporting these data, fluid clearance was impeded at low temperature in in situ perfused goat lungs (maintained at °c) [ ] , and in isolated, perfused liquid-filled rat lungs [ ] consistent with the inhibition of an active transport process. active sodium transport in particular was implicated in afc, since amiloride, a potent and specific inhibitor enac inhibited - % of basal fluid clearance in sheep, rabbits, rats, mice, and in the human lung [ ] . o'brodovich and colleagues [ ] have further demonstrated that application of amiloride to newborn guinea pig lungs caused respiratory distress, hypoxemia and elevated extravascular lung water, implicating active sodium transport in afc in neonates. consistent with this idea, targeted deletion of both enacα alleles in mice caused death within h of birth as a consequence of impaired afc [ ] . transgenic overexpression of the enacα gene in an enacα -/null mouse background rescued this pulmonary phenotype [ ] . conversely, overexpression of enac in the mouse lower airways accelerated sodium absorption, and depleted the volume of fluid coating the airway epithelium [ ] . together, these data implicate both active transepithelial sodium transport and enac itself in afc. using small interfering rna directed against enacα applied to the airways of live rats, other non-enac type ion channels are also likely to play an important role in transepithelial sodium transport and fluid reabsorption, particularly baseline fluid reabsorption [ ] . while enac located on the apical surface of alveolar epithelial cells acts as a sodium channel, investigations in live animals and isolated organ models have revealed that the driving force behind enac-mediated sodium uptake is the na,k-atpase [ ] . ouabain, a potent and specific inhibitor of the na,k-atpase, inhibited afc in isolated, perfused fluid-filled mouse lungs [ ] . furthermore, adenoviral-mediated gene transfer of the na,k-atpase β subunit into the lungs of rats augmented afc [ ] . furthermore, overexpression of the na,k-atpase β subunit in the alveolar epithelium restored active transepithelial sodium transport and afc in a rat model of acute hydrostatic pulmonary edema [ ] . gene knockout strategies have also addressed a role for the two na,k-atpase α subunits in afc, where mice heterozygous for each of the na,k-atpase α subunits (α +/-/α +/-) exhibit reduced cyclic-adenosine monophosphate (camp) dependent afc [ ] . in addition to a role for sodium transport, it has recently emerged that chloride transport via the cftr channel also plays a role in afc. using glibenclamide, an inhibitor of potassium and cftr channels, fluid clearance from in situ perused and unperfused mouse lungs and ex vivo human lungs was impaired [ ] . these data were supported using a cystic fibrosis (∆f ) mouse in a model of acute hydrostatic edema, where chloride transport by the cftr channel was demonstrated to play a major role in afc [ ] . further supporting this idea, adenoviral-mediated transfer of the cftr gene into the lungs of live rats and mice significantly increased afc, an effect that was blocked in the presence the chloride channel blockers -nitro- -( phenylpropylamino) benzoic acid, bumetanide, and gliben-clamide [ ] . animal and organ models have thus confirmed invaluable in the identification of transepithelial active sodium transport as the basis of afc, and in the validation of enac and other channels and the na,k-atpase, as key components that drive this process. mechanisms of impaired fluid clearance in ali/ards: what we have learned from intact organ and animal models, and clinical studies hypoxia is a feature of both ards and high-altitude pulmonary edema (hape). in isolated, ventilated rat lungs perfused at constant pressure (therefore independent of pressure changes caused by adaptation to hypoxic pulmonary vasoconstriction), exposure to hypoxia ( . . these and other observations introduced the possibility of using gene therapy to augment afc and treat ali/ards. patients with ali/ards frequently require mechanical ventilation to facilitate breathing. however, mechanical ventilation can directly induce ali (ventilator-induced lung injury, vili), by promoting alveolo-capillary barrier permeability [ , ] , inducing proinflammatory cytokines [ ], or by directly inhibiting afc. supporting the latter idea, lecuona et al. [ ] demonstrated that ventilation of rats with high tidal volumes (v t ) of ml/kg and a peak airway pressure of cmh o rapidly impaired afc, accompanied by a significant decrease in activity (but not expression) of na,k-atpase. thus high v t ventilation can directly block afc by inhibiting na,k-atpase-driven sodium transport, and hence afc. mechanical ventilation can also worsen preexisting ali (ventilator-associated lung injury, vali). in a canine model of acid aspiration-induced ali corbridge et al. [ ] demonstrated that ventilation with high v t ( ml/kg) but a fixed, low positive end-expiratory pressure (peep; cmh o) increased evlw to a greater extent than did ventilation with lower v t ( ml/kg) but a fixed, higher peep ( cmh o). similar trends were observed in an acid aspiration-induced model of ali in rats, where a progressive reduction in v t from to to ml/kg, keeping peep fixed at to cm h o, was accompanied by a progressive decrease in evlw accumulation [ ] . in both instances a low v t /high peep strategy proved beneficial. thus in addition to reducing both permeability and proinflammatory cytokine release, a low v t /high peep ventilation strategy might also promote afc and has formed the basis of important advances in ventilation strategies for ali/ards patients that are in routine use today. hyperventilation and hypocapnic alkalosis are often found together in patients with ali/ards [ ] . in an isolated, buffer-perfused ventilated rabbit lung, exposure to hypocapnia (> h) increased vascular permeability, and hence promoted alveolar edema [ ] . further to this, using an isolated, perfused, fluid-filled rat lung, myrianthefs et al. [ ] demonstrated that hypocapnia dramatically impaired afc. this block was reversible upon restoration of normal co levels and was not induced by metabolic alkalosis. the afc block was attributed to impaired sodium transport resulting from decreased membrane abundance of the na,k-atpase in rat lungs [ ] . hypocapnia can therefore promote both the formation of alveolar edema and impair the resolution of this edema by blocking transepithelial sodium transport, and hence afc. procoagulant pathways are upregulated in ali/ards, while fibrinolysis is suppressed [ ] . coagulation proteases such as thrombin have well documented roles in the development of ali/ards where thrombin can directly increase vascular permeability and promote alveolar flooding [ ] . recent work by vadász et al. [ ] demonstrated that thrombin applied to the vascular compartment of isolated, ventilated and perfused rabbit lungs also impaired afc. this was attributed to a block in transepithelial sodium and hence fluid transport across the alveolocapillary barrier. it was subsequently demonstrated that thrombin promoted the endocytosis of the na,k-atpase, thereby reducing na,k-atpase activity (see the accompanying review by vadász et al. (http://dx.doi.org/ . /s - - - ). thus coagulation proteases play a dual role in impaired afc. in addition to causing a permeability edema, thrombin, by virtue of its signaling properties, directly blocks fluid reabsorption. in contrast to the thrombin-signaling effects on na,k-atpase trafficking, serine proteases can directly enhance β-adrenergic-stimulated enac activity in alveolar epithelial cells and enhance β-adrenergic-stimulated afc in live mice [ ] . thus serine proteases appear to have opposing effects on different components of the sodium transport machinery. infection and purified endotoxin promote vascular permeability and pulmonary edema by inflammatory mechanisms involving granulocytes [ ] . interestingly, afc was upregulated after intratracheal administration of endotoxin to live rats and in a rat model of septic shock [ ], possibly as a protective mechanism induced during septic shock to overcome alveolar flooding. however, both cell-surface enac expression and afc were downregulated in an experimental mycoplasma pneumoniae infection in live mice, an effect mediated by reactive oxygen-nitrogen intermediates [ ]. this phenomenon is not limited to bacterial pathogens, since in live rats, the influenza virus can directly impair enac activity by reducing the open probability (p o ) of the channel, and thus downregulate afc, independently of viral entry into the epithelium [ ] . thus it appears that lung infections can impair afc, although there appear to be multiple underlying causes. acute lung injury may develop rapidly in recipients of transfused whole plasma [ ] . this transfusion-associated acute lung injury (trali) is caused by biologically active lipids, and antileukocyte antibodies, which cause a pronounced vascular leak [ ] . in a new mouse model of trali looney et al. [ ] have demonstrated that afc is also impaired in trali, although the molecular mechanism has not been described. levels of proinflammatory factors are elevated in bronchoalveolar lavage fluids from ali/ards patients, including interleukin (il) β, il- , il- , tumor necrosis factor (tnf)-α, and transforming growth factor (tgf) β [ ]. based on studies in rats fukuda et al. [ ] demonstrated that different domains of tnf-α have opposing effects on sodium transport and afc, where interaction with the tnf-α receptor caused inflammation and increased permeability, while the tnf-α lectin-like domain directly activated enac and potentiated afc. in contrast, atrial natriuretic factor applied to the vasculature of isolated, perfused lungs [ ], and tgf-β applied intratracheally to live rats [ ] impaired transepithelial sodium transport and blocked afc. it was subsequently demonstrated that tgf-β downregulated enac gene expression [ ] . together these data indicate new and emerging roles for polypeptide growth factors and other hormones in the regulation of afc. targeting fluid clearance in ali/ards: what we have learned from intact organ and animal models, and clinical studies studies in animal models demonstrated that low v t and high peep ventilation strategies potentiated afc in acid aspiration-induced ali [ , ], suggesting a therapeutic benefit of this ventilation strategy in the management of ali/ards. several small phase iii clinical trials addressing the potential benefit of low v t (which is lung protective) vs. traditional higher v t (providing better oxygenation) yielded conflicting results (reviewed in [ ] ). a subsequent multicenter, randomized, controlled trial of patients conducted under the auspices of the national heart, lung and blood institute ards network demonstrated that a ml/kg v t ventilation strategy yielded a significant reduction in mortality compared with a ml/kg v t ventilation strategy (with a plateau pressure of < cmh o) [ ] . while evlw was not assessed in this trial, the ml/kg v t ventilation strategy also increased the number of ventilator-free days, indicative of improved lung function. to date this remains the only intervention with a confirmed benefit on clinical outcome, and clearly demonstrates how studies on ali/ards in animal and organ models have translated into a successful therapeutic strategy. catecholamine-stimulated transepithelial sodium transport is the most intensely explored possibility of manipulating afc in a therapeutic context. it was demonstrated in that epinephrine stimulated afc from the airspaces of newborn mammals [ ] , implicating β-adrenergic receptors in afc. both epinephrine and terbutaline stimulated afc in anesthetized sheep [ ] , and terbutaline stimulated transepithelial sodium transport in an isolated, buffer-perfused rat lung [ ] and dramatically enhanced alveolar fluid clearance in a resected human lung [ ] . β-adrenergic agonists also reduce high vascular pressureinduced vascular permeability in isolated rat lungs [ ] , indicating that β-adrenergic agonsist may influence both reduce barrier permeability and potentiate afc. the ability of β-adrenergic agonists to stimulate afc is attributed in part to recruitment from intracellular pools (a) of na,k-atpase to the basolateral membrane [ ] and (b) of enac to the apical membrane [ ] of alveolar epithelial cells (reviewed in [ ] ). furthermore, β-adrenergic stimulation can clear edema in hypoxia-induced ali [ ] and ventilator-associated lung injury [ ] in animals, suggesting a potentially exciting therapeutic potential for β-adrenergic agonists in the treatment of ali/ards. this idea was further supported by the observations that adenovirus-mediated transfer of β-adrenergic receptor genes to live rats improved afc due to increased sensitivity to endogenous catecholamines and consequent upregulation of na,k-atpase activity and enac protein expression the lung [ ] . more recently a role for the cftr channel in camp-stimulated afc has been proposed since afc in cystic fibrosis (∆f ) mice, which lack cftr activity was not stimulated with β-adrenergic agonists [ ] . furthermore, administration of a cftr inhibitor to live mice blocked camp-stimulated afc [ ] . these studies provide strong evidence of an important role for the cftr channel, along with the transepithelial sodium transport system, in β-adrenergic-stimulated afc. among the neurotransmitter catecholamines, dopamine stimulated afc in isolated, perfused rat lungs, by activation of the dopamine d receptor, which stimulated exocytosis of the na,k-atpase [ ] , and by activation of the d receptor, which induced na,k-atpase gene expression [ ] . dopamine also promoted edema clearance in rats when ali was induced either by hyperoxia or mechanical ventilation [ ] . although not assessed in animal or organ models, dopamine also exerted an enhancing effect on enac activity [ ] , where dopamine increased the p o of enac, without altering enac density on the apical surface of l cells. thus, separate from the β-adrenergic system, the dopamine system provides an alternative opportunity for the pharmacological manipulation of afc. some β-adrenergic agonists, for example, albuterol, can be deposited into the lungs of patients with pulmonary edema at therapeutic concentrations by aerosolization [ ] . in a double-blind, randomized, placebo-controlled study sartori et al. [ ] assessed the effect of inhalation of the β-agonist salmeterol ( µg every h) on the incidence of pulmonary edema in hape-prone subjects at high altitude. prophylactic salmeterol significantly reduced the incidence of hape by more than %, without any change in pulmonary hemodynamics. furthermore, this study demonstrated that transepithelial sodium transport in the nasal epithelium was reduced in hape-prone subjects. together, these data suggest a therapeutic benefit of inhaled β-agonists in the treatment of hape. the β-agonist lung injury trial (balti), a singlecenter, double-blind, randomized controlled trial, assessed the effect of sustained infusion of a β-agonist (in this case, µg/kg salbutamol per hour) on the resolution of pulmonary edema in ali/ards patients [ ] , using evlw as a primary endpoint. the balti trial demonstrated that sustained treatment with intravenous β-agonists was generally well tolerated, although patients receiving salbutamol exhibited a trend towards higher heart rates, and patients in the salbutamol group exhibited higher incidence ( % vs. % in the placebo group) of supraventricular arrhythmias. patients receiving salbutamol demonstrated a significant reduction in evlw in comparison to placebo-treated patients. the trial was not, however, powered to detect a mortality benefit. therefore a second multicenter, randomized, double-blind, placebo-controlled trial (balti- ; international standard randomized controlled trial number ) is currently underway to assess the influence of intravenous salbutamol in ards patients on -day mortality. although glucocorticoids have been employed in the management of ards to reduce inflammation (in the early phase), and fibrosis (in the late phase) [ ] , their use is controversial. the potential beneficial effects of glucocorticoid use are attributed in part to the ability of glucocorticoids to influence afc, by upregulating protein expression of the sodium transporting machinery. preterm infants with respiratory distress exhibit reduced expression of enac relative to healthy, full-term infants [ ] . administration of dexamethasone to these infants upregulated enac expression, restoring enac expression to levels observed in healthy infants [ ] . working in adult rats, noda et al. [ ] demonstrated that a single intraperitoneal injection of dexamethasone dramatically increased afc and reversed hypoxemia induced by an intratracheal fluid challenge, - h after dexamethasone administration. this was attributed to an increase in enac mrna levels. while na,k-atpase mrna levels were not altered, na,k-atpase activity was increased. these data suggest that glucocorticoids may be of use in the treatment of ards by potentiating afc. a number of clinical trials have addressed the use of glucocorticoids in early and late phase ards. notable among these, in a randomized, double-blind, placebocontrolled trial involving patients with unresolving ards, meduri et al. [ ] demonstrated that prolonged methlyprednisolone administration significantly improved lung function and reduced mortality. a larger randomized, multicenter, placebo-controlled trial of patients with unresolving ards (the "late steroid rescue study", lasrs) that used -day mortality as the primary endpoint did not support the use of methylprednisolone for unresolving ards [ ] . however, methylprednisolone did increase the number of ventilator-free days during the first days, accompanied by improved compliance and oxygenation. the evlw was not assessed in either study. several polypeptide growth factors can potentiate afc and protect against ali induced in experimental animal models, notable among them, keratinocyte growth factor (reviewed in [ ] ). keratinocyte growth factor upregulated alveolar fluid clearance in anesthetized, ventilated rats [ ] , and upregulated transepithelial sodium transport in isolated, ventilated, and perfused lungs from healthy rats as well as in rats in which ali has been induced with α-naphthylurea [ ] . this protective effect has been attributed to the ability of keratinocyte growth factor (kgf) to upregulate expression of components of the sodium transporting machinery, primarily the na,k-atpase, as well as its mitogenic properties on alveolar type ii cells, since the alveolar epithelium is may be denuded in ali/ards. an upregulation of afc attributable to elevated na,k-atpase levels has also been reported for epidermal growth factor (egf) instilled into lungs of live rats, where egf was proposed to upregulate expression levels of na,k-atpase [ ] . other nonpeptide hormones can also influence afc. for example, , , -triiodo-lthyronine upregulated afc in live adult rats, apparently by upregulation of transepithelial sodium transport [ ] . while no clinical trial has yet been initiated, recombinant human kgf and liposome-mediated kgf gene delivery into mouse lungs afforded significant protection against oleic acid induced lung injury by improving arterial oxygenation and lung compliance in comparison to the vehicle-treated group [ ] . it was not reported in that study, however, whether afc was improved in the kgf-treated group. however, given the ability of kgf upregulate afc in an isolated organ model, and to repop-ulate denuded areas of the alveolar epithelium by virtue of its mitogenic properties on type ii cells, further evaluation of the therapeutic potential of kgf appears warranted. the lectin-like domain of tnf-α enhanced afc in ventilated rats [ ] . mouse tnf-α promoted fluid reabsorption in wild-type mice and in mice deficient in both tnf-α receptors, indicating that the effect of tnf-α on afc was independent of both tnf-α receptors [ ] . intratracheal application of a synthetic peptide that was based on the lectinlike domain sequence to an isolated, ventilated, autologous blood-perfused rat lung model caused a significant reduction in lung water and improvement in lung compliance, in comparison to lungs treated with a scrambled peptide of the same length [ ] . thus at least two growth factors, kgf and the lectin-like domain of tnf-α, present us with novel opportunities for augmentation of afc. granulocyte-derived reactive oxygen and nitrogen species are believed to play an important role in ali/ards (reviewed in [ ] ), and increased levels of the stable byproducts of nitric oxide decomposition, nitrite, and nitrate are observed in edema fluid from patients with ali/ards [ ] . these and other data have suggested a therapeutic benefit of antioxidants, including nacetylcysteine (nac), procysteine, and albumin [ ] . antioxidant therapy could also augment afc. oxidants such as hydrogen peroxide suppress glucocorticoidinduced enac gene transcription, an effect reversed by reactive oxygen scavengers, including thioredoxin. in a rat model of hemorrhagic shock modelska et al. [ ] demonstrated that catecholamines were ineffective at upregulating afc. however, after either intravenous administration of nac, intratracheal administration of reduced glutathione or neutrophil depletion with vinblastine, the effect of catecholamines was restored. these data provided in vivo evidence that neutrophil-mediated oxidative injury impaired afc in ali. building on this study, lee et al. [ ] demonstrated that the induction of hemoxygenase i (ho- ), a potent antioxidant, restores normal afc after hemorrhagic shock by blocking inos-mediated no release. therefore with regard to edema resolution antioxidant therapy would most likely augment glucocorticoid or catecholamine-based efforts to upregulate afc. clinical trials involving antioxidants in ards have yielded equivocal results. in one randomized, placebocontrolled, double-blind trial of ards patients suter et al. [ ] demonstrated that intravenous nac ( mg/kg per day) improved systemic oxygenation and reduced the need for ventilatory support. in contrast, domenighetti et al. [ ] in a comparable trial (although employing a higher dose: mg/kg per day) demonstrated no improvement in systemic oxygenation or a reduction in the need for ventilatory support. neither study reported a survival benefit, nor was evlw assessed. clearly additional trials are warranted, although a recent, much larger phase iii double-blind, placebo-controlled, clinical trial evaluating procysteine in ali/ards was prematurely discontinued due to mortality concerns in the intervention group [ ] . to date, a single animal study has explored the effect of anticoagulation on afc, where the natural anticoagulant, activated protein c (apc) was applied in a pseudomonasinduced ali model in rats [ ] . in that study afc was potentiated in a tnfα-dependent manner; however, activated protein c did not enhance afc. given the recent report of vadász et al. [ ] which demonstrated that thrombin impaired afc by blocking transepithelial sodium transport mediated by na,k-atpase, it appears likely that anticoagulant therapy would improve edema resolution by upregulating na,k-atpase-mediated afc. to date, no randomized, placebo-controlled trials have been conducted with anticoagulants in ali/ards patients, although a randomized, phase ii clinical trial of apc is currently in progress at the university of california in san fransisco (clinicaltrials.gov identifier nct ), employing the number of ventilator-free days measured at day as the primary end-point. the lung is an organ that is particularly amenable to local delivery of dna, making it an attractive target for gene therapy studies. in the context of ali/ards this possibility is particularly attractive since ali/ards does not impair virus-mediated gene delivery to the alveolar epithelium [ ] . several studies have highlighted the potential benefit of augmenting afc by local delivery of genes into the lungs of live animals. the potential therapeutic benefit of upregulating na,k-atpase and cftr channels in the lung as well as the delivery of kgf and ho- to the lung have already been discussed. all four systems have been explored in the context of gene therapy for ali. adenovirus-mediated transfer of na,k-atpase genes to lungs of live rats increased afc [ ] . upregulation of na,k-atpase expression in the alveolar epithelium by gene transfer improved edema resolution in rat models of ali induced by elevated left arterial pressure [ ] and hyperoxia [ ] . furthermore, transfer of β -adrenergic receptor genes to the lungs of adult rats increased their sensitivity to exogenous catecholamines, and upregulated afc due to increased delivery of both enac and na,k-atpase to epithelial cell membranes [ ] . transfer of the ctfr gene to mouse and rat lungs also augmented afc and appeared to affect the expression and function of components of the sodium transport machinery [ ] . these data together suggest that the sodium transporting and β-adrenergic signaling systems in the lung are candidate targets for intervention by gene therapy in ali/ards. clearly, enough epithelial cells would have to be present in the damaged epithelium for gene therapy to be effective, therefore, regeneration of the damaged epithelium with growth factors such as kgf has also been addressed by gene therapy. liposome-mediated kgf gene delivery into mouse lungs afforded protection in an oleic-acid induced lung injury model [ ] . similarly, in the case of antioxidants adenovirus-mediated gene delivery of ho- into the lungs of live mice afforded significant protection against influenza virus-and lipopolysaccharide-induced methods of potentiating alveolar fluid clearance in ali/ards that have been validated in animal and organ models or in clinical studies. interventions involving gene therapy are indicated in the shaded box. cftr, cystic fibrosis transmembrane conductance regulator; d , dopamine d receptor; d , dopamine d receptor; egf, epidermal growth factor; enac, epithelial sodium channel; kgf, keratinocyte growth factor; na,k-atpase, na + , k + transporting adenosine - -triphosphatase; t , , , -triiodo-l-thyronine; tnf-α, tumor necrosis factor α ali [ ] . while afc was not specifically addressed in the kgf or ho- studies, the documented role of kgf and ho- in augmenting afc suggests that this phenomenon contributed to the beneficial effect observed. organ and animal models of ali together with clinical studies have helped us understand the contribution of impaired afc to the development and persistence of ali/ards (fig. ) . perturbations to the alveolo-capillary barrier can both promote the formation and prevent the resolution of alveolar edema associated with ali/ards. indeed, edema resolution is critical for the ali/ards patient to survive. animal and organ models of edema resolution and ali have proved irreplaceable in the development of novel therapeutic strategies that augment afc, the most obvious case being the low tidal volume ventilation strategy for the management of ards, a strategy that has now been validated in a large multicenter, randomized, controlled clinical trial. due to our focus on afc several other important ali/ards candidate therapies currently under evaluation have been omitted from this review, including other ventilation strategies, surfactant replacement, fluid management, inhaled vasodilators, and nonglucocorticoid anti-inflammatory agents. the reader is referred to other excellent reviews on these topics [ , ] . animal and organ models and clinical studies will no doubt continue to prove instrumental in the further development of several new and emerging ideas designed to augment afc in ali, notable among these are anticoagulation, stimulation of the β-adrenergic system, and local gene delivery to the alveolar epithelium. the acute respiratory distress syndrome alveolar fluid clearance is impaired in the majority of patients 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administration of activated protein c in pseudomonas-induced lung injury: impact on lung fluid balance and the inflammatory response acute lung injury does not impair adenoviral-mediated gene transfer to the alveolar epithelium adenovirus-mediated transfer and overexpression of heme oxygenase cdna in lungs attenuates elastaseinduced pulmonary emphysema in mice integrating acute lung injury and regulation of alveolar fluid clearance acknowledgements. the authors are supported by the deutsche forschungsgemeinschaft (sfb "kardiopulmonales gefäßsystem" and klifo "lungenfibrose") and the european commission sixth european framework programme "pulmonary hypertension". key: cord- - s ytfed authors: zeng, ling; yang, xue-tao; li, hai-sheng; li, yong; yang, ce; gu, wei; zhou, yin-han; du, juan; wang, hai-yan; sun, jian-hui; wen, da-lin; jiang, jian-xin title: the cellular kinetics of lung alveolar epithelial cells and its relationship with lung tissue repair after acute lung injury date: - - journal: respir res doi: . /s - - -y sha: doc_id: cord_uid: s ytfed background: organ regeneration in mammals is hypothesized to require a functional pool of stem or progenitor cells, but the role of these cells in lung regeneration is unknown. methods: based on the fact that postnatal regeneration of alveolar tissue has been attributed to alveolar epithelial cells, we established a hemorrhagic shock and lipopolysaccharide (lps) lung injury model. using this model, we analyzed the cellular kinetics of lung alveolar epithelial cells. results: the results showed that alveolar epithelium type cells (aec s) are damage resistant during acute lung injury, they might be the main cells involved in lung injury and repair. then we observed the relationship between the expression of hgf, c-met following ali in rat lung and proliferation of aec s. the proliferation of aec s was inhibited when isolated primary aec s were co-cultured with c-met inhibitor su . furthermore, the numbers of aec s was significantly decreased when ali rats were administrated with su in vivo. it provided further evidence that the hgf/c-met signaling plays a vital role in ali-induced aec s proliferation. conclusions: aec s are damage resistant during acute lung injury and the hgf/c-met signaling pathway is of vital importance in the proliferation of aec s after ali. lung is a highly quiescent tissue, particularly, compared with other adult organs such as the intestine and liver. but now, it's widely accepted that lung has a remarkable reparative capacity [ ] [ ] [ ] . when the epithelial cells lining the interior of the lung are damaged by infection with influenza virus, a rare stem-cell population distal airway stem cells (dascs) is induced to proliferate and migrate to the damaged site. they can differentiate into alveolar epithelium type and type cells (aec / s) [ ] . bronchioalveolar stem cell (bascs) is a regional pulmonary stem cell population, identified at the bronchioalveolar duct junction [ ] . it is resistant to bronchiolar and alveolar damage and proliferate during epithelial cell renewal in vivo [ ] . vaughan et al proposed a lineage-negative epithelial progenitor (lnep) as the major source of induced krt + cells and it mobilize to regenerate lung epithelium after major injury [ ] . using a microfluidic magnetic activated cell sorting system, our previous study has isolated mouse lung multipotent stem cells (mlscs) which play an important role in bronchiolar and alveolar epithelial cells injury repair [ ] . to our interest, bascs, dascs, lnep and mlscs are all rare stem cells which play their role in regeneration through differentiation into lung progenitor cells, especially aec s. aec s are widely accepted as progenitor cells of lung and contribute to the lung repair and regeneration process. during development, aec s and aec s arise from a bipotent progenitor cell lineage, whereas after birth, aec s can undergo long-term self-renewal and give rise to aec s during homeostasis [ , ] . but the mechanisms that regulate aec renewal are incompletely understood. more recently, genetic lineage tracing experiments showed evidence that aec s were capable of long-term self-renewal and the generation of aec in both alveolar regeneration and homeostasis. therefore aec s are stem cells, as a population, proliferate in vivo and give rise to aec s [ , ] . acute lung injury (ali) and acute respiratory distress syndrome (ards) are frequently seen in traumatically injured patients. they remain significant contributing factors to morbidity in the traumatically injured patient. survivors of ards often have a lower functional ability and lower than normal health related quality of life [ ] . during the course of ali, multi-factors such as the activation of inflammatory cells and release of inflammatory factors lead to damage of air-blood barrier (abb). however, present treatments such as infection control and mechanical ventilation are supportive therapies, promoting the regeneration of lung itself maybe an ideal therapy. in this study, we established a hemorrhagic shock and lps lung injury model. using this model, we analyzed the cellular kinetics of lung alveolar epithelial cells. in terms of mechanism research, we explored the role of hgf/c-met signaling pathway in aec s proliferation after ali. accordingly, we first examined the proliferation of aec s, expression of hgf after ali, and phosphorylation of c-met following ali in rat lung. western blotting using the c-met inhibitor su provided further evidence for the involvement of hgf/c-met signaling in ali-induced aec s proliferation. male sprague-dawley rats, - g, were anaesthetized with pentobarbital sodium ( mg/kg). immediately after induction of anaesthesia. we inserted a catheter into the femoral artery and registered the blood pressure. then, the blood pressure was decreased to - mmhg in min and lasted for . h by drawing blood from the femoral artery. after recovery, lps ( . - . mg/kg) were instilled intratracheally in ul phosphate-buffered saline (pbs). the sham operation control group was given femoral artery cannulation but without hemorrhagic shock and lps instillation. su treated group was administrated with su ( mg/kg) by intraperitoneal injection for consecutive days post ali. rats were sacrificed on days , , , and after injury to study time-specific proliferation of lung epithelial cells (n = for each group). animals were purchased from spf laboratory animal room (chongqing, china). the rats were housed in a temperature and humidity-controlled, pathogen-free facility with a h light-dark cycle ( l: d). the institutional animal care and use committee of the institute of zoology, the third millitary medical university approved all the procedures. all experiments were performed in accordance with the institutional animal care and use committee guidelines. five-micrometer sections of adult rat lung were fixed with paraformaldehyde ( %) and embedded in optimal cutting temperature medium were incubated in blocking buffer ( h, % wt/vol bsa, % skim milk, . % triton x- in pbs). sections were then incubated overnight with rabbit anti-prospc antibody (millipore) or mouse monoclonal pcna (abcam). and then washed in pbs ( . % tween ). sections were washed and then incubated with donkey anti-rabbit or anti-mouse conjugated to alexafluor (invitrogen) and alexafluor labeled tyramide (invitrogen) for h and then washed. nuclei were stained with dapi, followed by rinsing and mounting in vectashield mounting medium (vecta laboratories). for stereological analysis, random lung tissue sections stained by he were observed from every lobe ( lobes in the right lung and lobe in the left) in each rat lung tissue ( animals per group). the area of photograph is non-hemorrhage area adjacent to the hemorrhage area. the number of pro-spc positive cells was determined by counting positively stained cells per high power field (magnification x ). five even distributed areas of stained lung sections from five blocks of lung tissue in each group ( animals per group) were counted. nine consecutive images were taken in each area. cell numbers in fig. d are the mean ± sd for at cell numbers per high power field (the area of the field at this magnification was . mm ) in sham group, post acute lung injury group and su treated group. alveolar epithelial cells were identified by pro-spc positive staining (aec s) and aqp positive staining (aec s). proliferated cells were identified by pcna positive staining. the length of basement membrane of alveoli was measured by image j (version . ). the right lower lobes of right lung were prepared for flow cytometry analysis. in brief, ml dispase i ( u/ml, bd) was injected through the bronchi. subsequently, the lungs were incubated in a °c shaking incubator for min in ml of dispase( u/ml), ml of . % dnase (sigma), and ml of μg/ml collagenase/dispase (roche). the bronchi were removed, and the lungs were minced and incubated for min. this suspension was filtered by μm filter, centrifuged, and depleted of red blood cells by incubation in rbc lysis buffer (sigma). primary antibodies including rabbit anti-prospc, rabbit anti-aqp were added to incubate cells. these antibodies were detected following incubation with fitc conjugated donkey anti-rabbit. dead cells were discriminated by -amino-actinomycin d ( -aad) staining. tissue or cells were lysed in lysis buffer ( mm tris-hcl, ph . , % triton x- , mm edta, and mm phenylmethylsulfonyl fluoride, g/ml aprotonin, and g/ml leupeptin). the protein concentration was determined by the bca protein assay kit (genstar, beijing, china). ug of protein was separated on % sdspolyacrylamide gels, transferred to a nitrocellulose membrane using the semidry transfer apparatus (bio-rad) at ma for min. the membrane was stained with ponceau s to ensure proper transfer and blocked overnight with % dry skim milk powder in mm trisbuffered saline plus . % tween (tbs-t). the membranes were incubated with antibodies overnight at °c. after being washed in tbs-t times, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse, -goat, or -rabbit iggs ( : ) for h. the blots were washed again. the individual target proteins were visualized using the enhanced chemilumi-nescence detection system. vascular endothelial growth factor (vegf), epidermal growth factor (egf), keratinocyte growth factor (kgf) and hepatocyte growth factor (hgf) in the lung homogenate from acute lung injury were detected by sandwich enzyme linked immunosorbent assay (elisa), according to the manufacturer's instructions (takara, japan). the detection limits of the assay were pg/ml. we optimized a protocol for isolating alveolar epithelial cells on the basis of immunomagnetic enrichment. the isolation mainly includes two parts. first, rat igg panning to deplete immunocytes expressing fcr to enrich for alveolar epithelial cells. second, immunomagnetic capture using magnetic beads conjugated to monoclonal antibody against specific membrane markers-t α (sigma, usa) to purify aec s and epcam (abcam, usa) to purify aec s. the pneumocytes plate was subjected to macs immunomagnetic separation according to the manufacturer's specifications (miltenyi biotec). briefly, cells were incubated with rabbit anti-rat t α antibodies (sigma, usa) for min at °c. cells were then incubated with goat anti-rabbit micro-bead solution at °c for min. then they were centrifuged for min and resuspended with ml of the separation buffer. the cell suspension was applied on a macs separation column subjected to a magnetic field provided by the macs separator. the column was washed three times with μl separation buffer and then released from the magnetic field, allowing the t α-expressing cells to be eluted into a separate tube. the isolated t α-expressing epithelial cells were termed aec s. to gain high purity of aec s, collect t α-negative cells, incubated with mouse anti-rat epcam antibodies (abcam, usa) for min at °c, and then handle cells as above with rat anti-mouse microbead solution. thereafter isolated epcam-expressing epithelial cells were aec s. sorted aec cells were cultured with dulbecco's modified eagle's medium/ % fbs/penicillin/ streptomycin. the results are presented as mean ± sem; statistical analysis was performed using either one-way analysis of variance followed by student-newman-keuls multiple comparisons post-hoc analysis or kaplan-meier survival analysis as appropriate, with a p value of less than . considered significant. rat hemorrhagic shock and lps lung injury model was established, we found that rats exposed to . mg/kg lps instilled intratracheally exhibited % survival, the area of pulmonary hemorrhage is only about %- % (the hemorrhage area was quantified by he staining and calculated by the proportion of hemorrhagic alveoli area). in contrast, the . mg/kg group exhibited % survival rate, the area of pulmonary hemorrhage is about - % (fig. a) . we found . mg/kg lps resulted in % survival rate, the area of pulmonary hemorrhage is about % (a mild to moderate acute lung injury which can stimulate the endogenous repair of lung tissue), so the proper lps dose is . mg/kg. rat after ali or sham surgery were sacrified on days , , , and after ali to study time-specific proliferation of lung epithelial cells. according to the he staining of rat lungs of different days after ali, most alveolar walls are fractured on the first day after injury. on day and , alveolar spaces are filled with a mixed neutrophilic and monocytic infiltrate, and alveolar wall capillaries are congested. alveolar hemorrhage is visible. on days and , the structure of alveolar wall is recovered (fig. b) . the relative numbers of aec s were measured at various time points after acute lung injury using immunofluorescence and flow cytometry analysis. immunofluorescence staining for aqp (aec specific marker) showed that the first days after ali, the staining of aqp is discontinuous. a large number of aec s were fractured into fragments, and normal alveolar structure is destroyed. but from the th day, aec s began to repair and returned to a normal level on the th day (fig. c) . the flow cytometry analysis also showed that from the th day, the number of aec s gradually returned. on the th day the number returned to a normal level (fig. e) . immunofluorescence staining for aqp was used to analyze the percentage of aec s covering the surface of alveoli (fig. e) , the result showed that, the kinetics of aec s was in concordance with the results of flow cytometry analysis, a decline on the first days (about % of the normal level) and then gradually return to normal. immunostaining for prospc (aec specific marker) was performed on lung tissue from , , , and days after injury (fig. d) and aec s number after ali were measured. there was an obvious increase of aec s number on the nd day, it reached a summit on the rd day and improved gradually after th day (figs. d, d) . the rat whole lung cell suspensions were incubated with fitc conjugated to anti-prospc antibody, the percentage of aec s was analyzed by flow cytometry analysis (fig. e) , in sham-operated mice, there were no differences in numbers of aec s at various time points. however, the kinetics of aec s was in concordance with the results of quantitative analysis. the relative numbers of proliferated cells and proliferated aec s were measured at different time points using immunofluorescence. double-immunofluorescence staining for pro-spc and proliferating cell nuclear antigen (pcna) was used to monitor aec s proliferation after ali. in sham group, there were no differences between proliferated cells and proliferated aec s on each day after acute lung injury. but there was a significant increase of proliferated cells and proliferated aec s after ali, especially on the nd and rd day. most alveolar walls were thickened, pulmonary alveoli were slightly shrinks, cubic or round proliferated aec s lined in the alveolar wall. on the th day to th day the number of proliferated cells and aec s returned to a normal level. the results of quantitative stereological analysis showed that there was a significant increase in proliferated cells and proliferated aec s in ali vs. sham was first detected on day ( . ± . and . ± . cells per high power field), followed by further increases on day ( . ± . and . ± . cells per high power field). the percentage of proliferated aec s increased to . % on the nd day and . % on the rd day. after a period of active lung regrowth (days - ), the percentage of aec s dropped to baseline levels by day - ( fig. d) . previous studies from simian and rodent models suggested that aec s function as progenitor cells in the alveoli and proliferate and differentiate into aec s [ , ] . furthermore, barkauskas et al. verified that aec s are stem cells in adult lung [ ] . in our study, to determine whether acute lung injury affected the biological behavior of aec s, their number was quantitatively analyzed at various time points after ali. although significant aec s loss were observed by h after ali, the number of aec s did not significantly decrease at any time point. immunofluorescence staining revealed that there was a significant increase of proliferated aec s in alveolar on day and day ( fig. a and d) . consistent with if, facs analysis showed that the abundance of aec s did not change during day and day (fig. e) . the result of he staining also shows that alveolar walls are lined with cuboidal epithelial cells which are proliferating aec s on day and day . on day and , the structure of alveolar wall recovered. these results proved that aec s are damage resistant during acute lung injury, and they might be the main cell involved in lung injury and repair. the milieu of acute lung injury promoted aec s growth and accelerated aec s cell cycle we obtained bronchoalveolar lavage fluid (bal) and lung tissue homogenate (lth) from acute lung injury rats, to represent the acute lung injury in vivo damage environment, and compare their effects with bal and lth from normal rats when added to primary cultured, attached, non-confluent aec s (attached for h). aec s were isolated by immunomagnetic separation. after the preparation of rat lung single cell suspension, rat igg panning was used to deplete immunocytes expressing fcr to enrich for alveolar epithelial cells. t α-pos cells (aec s) (purity ± %) were firstly separated from rat lung single cell suspension, then epcam-pos cells (aec s) were isolated (purity ± %). freshly isolated aec s are essentially non-proliferative, with greater than % of the population in g phase of the cell cycle, and remain quiescent in culture [ , ] . the rd day's damaged lung tissue homogenates ( ug/ml) were added to -h cultured, adherent aec s. the bal had no effect on the proliferation of aec s. but the rd day's acute lung lth significantly increased the cell numbers when stimulated for h (p = . , fig. b ) and more aec s were in the s phase of cell cycle (fig. c) . the cell cycle distribution was measured using propidium iodide (pi) staining and detected by flow cytometry assay. the proportion of aec s in s phase of cell cycle stimulated with rd day's lth was approximately . ± . %, while it was only . ± . % of aec s stimulated with normal lung lth (fig. c) , suggesting that the milieu of acute lung injury is conducive to aec growth. in contrast, normal lung lth and the th day's lth did not significantly affect aec proliferation (fig. b) . the molecular mechanism of acute lung injury milieu induced aec proliferation increased hgf level are detected in in acute lung injury milieu to observe important cytokines' expression in lung, we detected vegf, egf, kgf and hgf in lung homogenate, and found that hgf was the only cytokine fig. a the immunofluorescence of proliferated aec cells after acute lung injury. dual immunostaining (arrowhead) for prospc (red) and pcna (green) was used to monitor aec s proliferation in alveolar after ali. sham d -d : day to day post sham operation. d -d : day to day post acute lung injury. su d -d : day to day post su treated. scale bar = μm. b the ali milieu influenced the proliferation of aec s. the rd day after ali's lung tissue homogenate promotes the proliferation of aec s significantly, *p = . (normal: co-cultured with normal rat lth; d : co-cultured with the rd day after ali's lth; d : co-cultured with the th day after ali's lth). c the milieu of acute lung injury accelerates aec cell cycle. the rd day after ali's lung tissue accelerates aec cell cycle significantly (*p = . ). d cellular kinetics of alveolar epithelial cells after acute lung injury. aec numbers per high power field (hpf, . mm ) (left), proliferated aec s per hpf (middle) and the percentage of aec s (%) were indicated (*p = . , **p = . for the association of sham group vs. ali group; # p = . , ## p = . for the association of ali group vs. su treated group) significantly elevated after acute lung injury, at levels > . -fold higher than sham control on the nd and rd day after acute lung injury (fig. b) , although there was not a significant difference by the first day post injury. we next examined whether hgf can accelerate aec cell cycle in vitro. figure c presents the cell cycle analysis of primary aec s and hgf stimulated aec s in vitro. and ng/ml hgf can significantly accelerate aec cell cycle (the percentage of aec s in s phase increased significantly), indicating that the proliferation of aec after acute lung injury may be induced by the elevated hgf. to clearly establish the relationship between hgf/c-met activation and aec s proliferation, we observed the expression level of the key proteins. immuno-magnetic separated aec s were co-cultured with normal or the rd day's milieu of ali in vitro. we found that the rd day's milieu of ali can induce the significant higher expression of phospho-c-met (p-c-met) while there is no difference of the expression of total c-met (p = . , fig. d ), which indicated that the regulation of high fig. a the concentration of hgf, kgf, vegf and egf in rat lung homogenate at different days post injury using enzyme linked immunosorbent assay (elisa). *p = . , **p = . . b hgf accelerates aec cell cycle in vitro. *p = . , **p = . . c the effect of ali on the expression of key protein of the hgf/c-met signaling. image-j was used in the comparing of the intensity of bands of western blot. the rd day after ali's lth can promote the significant higher expression of p-c-met, *p = . . d the effect of the rd day after ali's lth on the proliferation of aec 's in vitro. aec 's had an increased proliferation rate when co-cultured with the rd day after ali's lth ( ug/ml, h). when co-cultured with su , the proliferation rate of aec s co-cultured with the rd day after ali's lth was inhibited (left). the quantitative analysis of aec proliferation with or without su (right), *p = . expression of p-c-met was not due to the high expression of total c-met. su is a selective, atp-competitive inhibitor of met receptor tyrosine kinase, it reduced the rd day ali's lung tissue homogenates-increased aec s cell proliferation significantly (p = . ) (fig. e) . other important tyrosine kinase pathways (akt and mapk) had been detected, but there was no difference when treated with the rd day ali's lung tissue homogenates (fig. d) . furthermore, in order to observe the function of hgf/c-met signaling pathway in the proliferation of aec s in vivo, we treated rats with su every day after ali. the results of quantitative stereological analysis showed that the proliferated aec s number was . ± . per high power field after su treated, while it is . ± . in ali group on the nd day post ali ( # p = . , fig. d ). the percentage of proliferated aec s is also lower after su treated on the nd day post ali ( ## p = . , fig. d ). in the rodent lung, there are several stem cell niches that are the key in maintaining the epithelial layers of lung tissue. kim and colleagues describe a niche in the bronchioalveolar duct junction of adult mouse lung, which enriched, propagated, and differentiated these stem cells in vitro [ ] . kumar et al. infect the mouse airways with influenza a (h n ) virus, they found p + krt + basal-like cells expanded locally, organized into growing spheres with a lumen, and subsequently assumed expression of alveolar specific proteins, all indicative of proper differentiation and regeneration in vivo [ ] . vaughan et al. proposed that rare lneps residing in the bronchiolar airways are activated after influenza virus infection to expand and give rise to induced krt- + cells in the alveolar parenchyma and more differentiated lineage-committed bronchiolar and alveolar epithelial cells [ ] . desai et al. showed that, during development, although at and at cells arisen directly from a bipotent progenitor, after birth of new at cells derived from rare, self-renewing, long-lived, mature at cells that produce slowly expanding clonal foci of alveolar renewal [ ] . these stem cells are all rare cells (from . to . %) and play their role in regeneration through differentiation into aecs, especially aec s. many lineage-tracing experiments had established that spc+ aec s can proliferate and give rise to aec s in vivo [ ] . furthermore, new evidence shows that the same spc+ cells can maintain the aec population over the long term, an important criterion for defining the population as containing stem cells. in the unperturbed lung, lineage-labeled aec s give rise to only small clones of daughter cells, and there is a low rate of differentiation into aec s [ ] . but there is hardly any study examined the cellular kinetics of aec s and aec s post acute lung injury systematicly. using the hemorrhagic shock and lps lung injury rat model, we found that the number of aec s began to decline on the st day and declined to a bottom on the rd day post injury, then gradually returned to a normal level. the kinetics of aec s was different to aec s, a significant increase in aec s number was detected on the rd day, followed by a further increase from the th day to th day, then aec numbers gradually returned to normal. doubleimmunofluorescence staining for pro-spc and pcna showed that the percentage of proliferated aec s was significantly increased on the rd day. after a period of active lung repair, aec numbers dropped to baseline levels by day . it means that aec significantly proliferated after acute lung injury. they are injury resistant cells in acute lung injury and may have important roles in the regeneration of damaged lung. the local environment of a cell dictates cell fate, and bioengineering of complex organs like the lung will require a detailed knowledge of regional microenvironments to faithfully recapitulate regeneration. similarly, the effectiveness of stem cell therapy to ameliorate tissue damage could likely be optimized if the specific damage niche was well characterized. after acute lung injury, the milieu of the damaged tissue will determine the rate and nature of alveolar epithelial repair and is therefore of great interest. we speculate that the milieu of acute lung injury can promote the proliferation of aec s due to the cytokines released so we observed whether the milieu of acute lung injury had biological effect on aec s. the cytokine release of the lung homogenate was investigated. hgf was the only cytokine significantly elevated on the nd and rd day after acute lung injury. it can significantly accelerate aec cell cycle in vitro, indicating that the proliferation of aec after acute lung injury may be induced by the elevated hgf. hgf is a potent mitogen and motogen for various epithelial cells [ , ] . it is produced following hcl-or bleomycin-induced acute lung injury and plays a role in pulmonary epithelial cell regeneration [ , ] . studies have also demonstrated that hgf is responsible for most growth-promoting activity for aec s cells, compared with egf, tgf-a, acidic fibroblast growth factor, and keratinocyte growth factor [ ] . all biological effects of hgf are mediated by a single tyrosine kinase receptor (c-met), which is mainly expressed in cells with an epithelial or endothelial origin [ , ] . incubation with hgf results in the activation of c-met and increase of proliferation in lung adenocarcinoma cells and isolated type ii cells and stimulation of erk / phosphorylation in lung adenocarcinoma cells [ ] . to clearly establish the relationship between hgf/c-met activation and aec proliferation, we observed the key proteins' expression. we found that the rd day's milieu of ali can promote significant higher expression of p-c-met while there is no difference of the expression of total c-met. the met kinase inhibitor su significantly reduced rd day's damaged lung tissue homogenates-increased aec s cell proliferation. furthermore, we verified the role of c-met in the proliferation of aec s in vivo by treated of c-met inhibitor su . hgf/c-met signaling is likely a major factor responsible for the pulmonary epithelial cell proliferation after acute lung injury. in conclusion, our present findings support the hypothesis that the milieu of acute lung injury can stimulate pulmonary epithelial cell proliferation after acute lung injury. the hgf/c-met signaling is likely a major factor responsible for the pulmonary epithelial cell proliferation. these data have significant implications with regard to the role of hgf/c-met signaling in injury repair. extension of this work into the regulation of growth factor balance is required for alveolar epithelial cell maintenance and repair could have important translational clinical and bioengineering applications for patients with alveolar damage or disease. distal airway stem cells yield alveoli in vitro and during lung regeneration following h n influenza infection evidence for human lung stem cells matrix modulation of compensatory lung regrowth and progenitor cell proliferation in mice p (+) krt (+) distal airway stem cells are essential for lung regeneration identification of bronchioalveolar stem cells in normal lung and lung cancer lung stem cell differentiation in mice directly by endothelial cells via a bmp -nfatc- -thrombospondin- axis lineage-negative progenitors mobilize to regenerate lung epithelium after major injury isolation of lung multipotent stem cells using a novel microfluidic magnetic activated cell sorting system surfactant phospholipid metabolism lung regeneration: mechanisms, applications and emerging stem cell populations type alveolar cells are stem cells in adult lung multiple stromal populations contribute to pulmonary fibrosis without evidence for epithelial to mesenchymal transition mechanisms and clinical consequences of acute lung injury repir and regeneration of the respiratory system: complexity, plasticity, and mechanisms of lung stem cell function evidence for lung epithelial stem cell niches induction of a-and d-type cyclins and cdc kinase activity during recovery from short term hyperoxic lung injury the milieu of damaged alveolar epithelial type cells stimulates alveolar wound repair by endogenous and exogenous progenitors alveolar progenitor and stem cells in lung development, renewal and cancer scatter factor is a fibroblastderived modulator of epithelial cell mobility hepatocyte growth factor ameliorates mucosal injuries leading to inhibition of colon cancer development in mice relationship of keratinocyte growth factor and hepatocyte growth factor levels in rat lung lavage fluid to epithelial cell regeneration after bleomycin hepatocyte growth factor (hgf), an endogenous pulmotrophic regulator, for the rescue of acute and chronic lung disease hepatocyte growth factor isoforms in tissue repair, cancer, and fibrotic remodeling met signalling: principles and functions in development, organ regeneration and cancer the discovery of hepatocyte growth factor (hgf) and its significance for cell biology, life sciences and clinical medicine h pulmonary epithelial cell mitogenic effects and signaling pathways in response to hgf and tgf-alpha this work was supported by dr. yong tang in chongqing medical university. no commercial benefits of any kind have been or will be received from institutions related directly or indirectly to the subject of the manuscript. availability of data and materials not applicable. authors' contributions lz collected clinical data and wrote the manuscript; xy, hl and yl performed animal model, immunofluorescence, flow cytometry analysis and most of the in vitro assays; cy and wg supervised the whole project; yz prepared figures and helped revise the manuscript; jd, hw, js and dw performed stereological analysis and helped revise the manuscript; jj designed the project and analyzed data; all authors reviewed the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -z hh c authors: cotogni, paolo; trombetta, antonella; muzio, giuliana; brizzi, maria felice; canuto, rosa angela title: polyunsaturated fatty acids and cytokines: their relationship in acute lung injury date: journal: diet and nutrition in critical care doi: . / - - - - _ sha: doc_id: cord_uid: z hh c acute lung injury (ali) and acute respiratory distress syndrome (ards) are inflammatory diseases whose clinical severity depends on the grade of inflammatory response. inflammatory cytokines are key elements in the pathogenesis of ali/ards, and the occurrence of an imbalance between pro- and anti-inflammatory cytokines leads to additional non-pulmonary organ dysfunction which contributes to excess mortality rates. treatment of these patients includes nutrition support with lipids, usually soybean oil-based lipid emulsions, which are rich in omega (n)- polyunsaturated fatty acids (pufas) and deficient in n- pufas; however, too much n- pufas are detrimental due to their pro-inflammatory effects. conversely, a large amount of experimental studies and some randomized clinical trials showed the benefits of the n- pufa administration in the context of ali because of their anti-inflammatory properties. based on these data, several scientific societies recommended in their guidelines, with an a or b grade of recommendation, the use of n- pufas in ali/ards patients. however, at present, the issue of lipid therapy in ali/ards is still controversial due, at least in part, to inconclusive or contradicting results in several recent clinical trials using n- pufas. acute lung injury (ali) and acute respiratory distress syndrome (ards) are inflammatory diseases whose clinical severity depends on the grade of inflammatory response. inflammatory cytokines are key elements in the pathogenesis of ali/ards, and the occurrence of an imbalance between pro-and antiinflammatory cytokines leads to additional non-pulmonary organ dysfunction which contributes to excess mortality rates. treatment of these patients includes nutrition support with lipids, usually soybean oil-based lipid emulsions, which are rich in omega (n)- polyunsaturated fatty acids (pufas) and deficient in n- pufas; however, too much n- pufas are detrimental due to their pro-inflammatory effects. conversely, a large amount of experimental studies and some randomized clinical trials showed the benefits of the n- pufa administration in the context of ali because of their anti-inflammatory properties. based on these data, several scientific societies recommended in their guidelines, with an a or b grade of recommendation, the use of n- pufas in ali/ards patients. however, at present, the issue of lipid therapy in ali/ards is still controversial due, at least in part, to inconclusive or contradicting results in several recent clinical trials using n- pufas. acute respiratory distress syndrome (ards) is considered a form of acute diffuse lung injury (ali). according to the berlin definition (ferguson et al. ) , each subcategory of ards (mild, moderate, and severe) is defined by mutually exclusive ranges of the ratio between arterial oxygen partial pressure (pao ) and fractional inspired oxygen (fio ) ( mm hg<pao /fio mm hg, mm hg<pao /fio mm hg, and pao / fio mm hg, respectively). the acute phase of this syndrome is manifested by the early onset of respiratory failure (characterized by hypoxemia refractory to oxygen supply within week of a known clinical insult or new/worsening respiratory symptoms) related to several triggers and clinical disorders generally distinguished in pulmonary [pneumonia (bacteria or virus), microbial products (e.g., endotoxins), pulmonary aspiration of gastric contents] and extrapulmonary (trauma/hemorrhagic shock, extensive burns, ischemia/reperfusion injury, acute pancreatitis, cardiopulmonary bypass, transfusions of blood products) (leaver and evans ) . ards is frequently progressive and it is characterized by distinct phases with different clinical, radiographic, and histopathological findings. radiographic findings are similar to those observed in the case of cardiogenic pulmonary edema, mainly bilateral infiltrates (irregular and asymmetric) and sometimes pleural effusions (fig. ) . histopathological findings of ali include diffuse alveolar epithelium damage (principally, denudation of the basement membrane of the alveolus and necrosis of type i cells) with activated neutrophils, alveolar macrophages, hyaline membranes, and protein-rich edema fluid in the alveolar spaces (fig. ) . the pathophysiology of ali is complex and multifaceted. the lung inflammatory response involves various interactions among many cell types, and it is characterized by the production and release of several chemical mediators. figure is a simplified graphic description of how the anatomy and physiology of alveolar space, the main site of inflammatory reactions occurring in ali, change. the lung is a complex organ with many different cell types, i.e., pneumocytes (alveolar type i and ii), fig. anteroposterior chest radiograph from a patient with the acute respiratory distress syndrome who was receiving mechanical ventilation. irregular and asymmetric infiltrates are present throughout both lung fields pulmonary capillary endothelial cells, alveolar macrophages, and fibroblasts. several studies suggest that the lung itself can be an important cytokine-producing organ. cytokines are a diverse group of inflammatory mediators, produced by a wide variety of cell types, which initiate and orchestrate the host response to different injuries. intercellular communication through cytokine networking plays a key role for homeostasis. cytokines seem to have concentration-dependent effects. at lower concentrations, they modulate tissue response to injuries; at higher concentrations, they induce proportionally more severe local and systemic inflammatory responses. if this cytokine overproduction is not regulated, it may remarkably amplify the release of further pro-inflammatory mediators and, at least, the overall inflammatory response. both ali and ards are characterized by an intense inflammatory response. in ards early phase, the alveolar space is the site of significant inflammatory reactions (pugin et al. ) . ali/ards are characterized by alveolar infiltration with neutrophils and macrophages, both able to release inflammatory cytokines. however, recent theories suggest that it is not the neutrophil and macrophage number but their the normal alveolus and the alveolus in acute lung injury. neutrophil is shown migrating through a gap in the capillary endothelium into the alveolar space (activated neutrophil). in the alveolar space, an alveolar macrophage is secreting cytokines: tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , il- , and il- . the alveolar type i cells are damaged or necrotic, while alveolar type ii cells are intact and release cytokines. the tissue architecture is disrupted with the formation of hyaline membranes on the denuded basement membrane. the alveolar space is filled by protein-rich edema fluid fig. alveolar damage in acute lung injury/acute respiratory distress syndrome. the tissue architecture is disrupted, alveoli are collapsed, alveolar type ii (atii) cells are hyperplastic, and alveolar type i cells are necrotic. alveoli contain protein-rich edema fluid and macrophages. collagen deposition in the interstitium has begun. hematoxylin and eosin staining, Â magnification. the picture was kindly given by luisa delsedime, md, anatomia patologica, aou città della scienza e della salute, turin, italy pro-inflammatory cytokine release that produces lung injury. in this setting, cytokines have a central position as signaling mediators that initiate, increase, and maintain inflammatory responses on a local basis. indeed, in ali/ards, an accumulation of both pro-and anti-inflammatory cytokines within the alveolar spaces is demonstrated. many cytokines were detected at elevated levels in bronchoalveolar lavage (bal) fluid in patients with ards, i.e., tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , il- , and il- . tnf-α is the main pro-inflammatory cytokine and it is important in driving the initial lung inflammatory response, principally indirect by the stimulation of other cytokines (i.e., il- and il- ). il- occupies a crucial place in the ards inflammatory response. chemokine il- is the major neutrophil chemotactic factor into the alveolar space, and it is an early marker for the development of ards. finally, il- , which has anti-inflammatory effects, plays a pivotal role in closing down the inflammatory response once it achieves its primary purpose. the pivotal role of a hyper-inflammatory response, characterized by overproduction of tnf-α, il- , and il- , in the evolution of the disease from ali to ards is documented. indeed, many studies report a higher mortality in patients who have increased pro-inflammatory cytokine concentrations in the bal at the onset of ards or persistent increased concentrations in days - . physiologically, the critical dependency on the presence of the inflammatory ligand (e.g., platelet-activating factor) and free arachidonic acid (aa) may help to confine the outbreak of lipid-mediator production to sites of initial inflammatory process. conversely, compartmentalization of alveolar cytokines can be lost in the case of a damaged alveolar barrier. cytokines generated in the alveoli may go into the systemic circulation leading to the beginning of a systemic inflammatory response syndrome. the reason why a patient with ali develops ards while another recovers is still not completely clear. in ards-developing patients, it is hypothesized that, at some level, regulation of the inflammatory response must be inadequate, but why or when this regulation is insufficient is a matter of debate. the study of the balance between pro-and anti-inflammatory cytokines is of greater importance to understand the pathophysiology of ali than a single cytokine concentration, because the degree of cytokine imbalance is a contributing element to disease severity (park et al. ) . persistently high levels of pro-inflammatory cytokines, as well as a reduced production of anti-inflammatory cytokines, have been demonstrated to be correlated with the severity of lung injury. the degree of this cytokine imbalance has a pivotal role in inducing other non-pulmonary organ dysfunctions and increasing mortality rates in ards patients. in particular, the pro-inflammatory activity depends on the balance between the cytokine and its inhibitor(s). in ards early phase, the finding of low levels of anti-inflammatory cytokines (i.e., il- and il- receptor antagonist) in the bal is associated with an increase in patient mortality. the ratio tnf-α to il- in the bal fluid was significantly higher ( . vs. . ) in patients with ards than in those at risk of developing ards. among ali ventilated patients, malnutrition has been associated with adverse outcomes due to increased infection risk, prolonged mechanical ventilation dependency, prolonged intensive care unit (icu) length of stay (los), and higher mortality. in general, critically ill patients receive nutrition support including lipids. administration of lipid emulsions offers many advantages in patients with acute respiratory failure, because compared with glucose they provide a richer source of calories in a small volume with a lower carbon dioxide load and less hyperglycemia. moreover, lipid supply is mandatory to avoid essential fatty acid (fa) deficiency. accepted guidelines recommend providing - % of nonprotein calories as lipids in patients with ali/ards (mizock ) . lipids were introduced into parenteral nutrition (pn) formulations in the s. however, there is a lack of consensus about the optimal source and composition of lipids (long-vs. medium-chain triglycerides, soybean oil vs. olive oil and fish oil) both in parenteral and enteral formulas for the patient with ali/ards. commonly used parenteral lipid emulsions (i.e., soybean oil-based fat emulsions) contain large amounts of n- polyunsaturated fatty acids (pufas) (i.e., more than % of linoleic acid) that form aa [omega (n)- pufa], while only % α-linolenic acid can be metabolically converted to eicosapentaenoic acid (epa, n- pufa) and docosahexaenoic acid (dha, n- pufa). therefore, intravenous fat emulsions have a very low n- /n- pufa ratio, ranging between : and : . likewise, the n- /n- ratio in enteral products may be as low as : . thus, ali/ards patientseither on standard enteral nutrition (en) or pnare generally exposed to a large quantity of linoleic acid. however, administration of high quantities of linoleic acid is not desirable in ali/ards patients because it causes a production of high amounts of aa, whose main functional role is to be a substrate for the synthesis of bioactive pro-inflammatory mediators known as eicosanoids [above all, prostaglandin (pg)-e , thromboxane (tx)-a , and leukotriene (lt)-b ] and platelet-activating factor, which modulates severity and duration of inflammatory responses. the impact of parenteral lipid supply on the lung function of patients with acute respiratory failure has been contradicting. potential harmful effects have been related to type and/or administration rate of lipid emulsions. in particular, some authors thought that these negative effects might correlate to the infusion of soybean oil-based lipid emulsionsbecause they are rich in linoleic acidand suggested that linoleic acid-derived synthesis of eicosanoids may worsen gas exchange and pulmonary hemodynamics in critically ill patients. eicosanoids are major pathogenetic factors of ards, and their negative effects on pulmonary function (e.g., increased permeability, edema formation, polymorphonuclear activation, thrombocyte aggregation, vasoconstriction, and bronchoconstriction) have been widely investigated. as early as , the immunomodulatory properties of lipids were displayed. over the past years, there has been an improved understanding of pufa pathophysiology, and several mechanisms for the interaction between pufas and inflammation or immune response have been demonstrated. indeed, after pufa supply (diet, as well as en or pn), many cell properties and related functions are modified, mainly the inflammatory and immunity responses (calder ) . briefly, n- pufas are more regarded as anti-inflammatory, whereas n- pufas as proinflammatory. more recent studies suggested that n- pufas may be involved in the resolution of inflammation (calder a) . since a network of factors regulates the relationship between n- and inflammation, the mechanisms underlying the effects of n- pufas on cell event modulation are complex. to summarize the mechanisms of n- pufa modulation of the inflammatory response, it can be said that the main and well-demonstrated anti-inflammatory actions of n- pufas include the following: (a) a decreased production of aa-derived mediators such as -series pg and ltb as a result of a reduction in aa content of cell membranes and of inhibition of aa metabolism. (b) an increased production of eicosanoid mediators from epa (e.g., -series pg and ltb ), since these mediators have weak biological potency compared with those produced from aa; for example, ltb is -to -fold less potent as a neutrophil chemotactic agent than ltb , or pge is a less potent inducer of cyclooxygenase- gene expression in fibroblasts and of il- production by macrophages compared with pge . (c) a decreased t-cell proliferation and production of il- induced by epa and dha providing. (d) a decreased expression of some adhesion molecules on the surface of monocytes, macrophages, lymphocytes, and endothelial cells induced by epa and dha providing. (e) a decreased production of pro-inflammatory cytokines, due to decreased activation of pro-inflammatory transcription factors such as nuclear factor κ-b and increased activation of anti-inflammatory transcription factors such as peroxisome proliferator-activated receptor-γ. (f) an increased production of resolvins (e-series from epa and d-series from dha) and protectins (from dha) having potent antiinflammatory or inflammation-resolving properties. pufas and lung injury: nutrients or drugs? over the last years, the discovered ability of n- pufas to downregulate many different responses of the inflammatory process has suggested to several researchers that these fas might have a key role in determining the grade of severity of some inflammatory diseases. consequently, n- pufas have been widely used not only as nutrients but mainly as pharmacological agents. briefly, the rationale of the therapeutic use of n- pufas was based on several well-established issues: ( ) pufas are key structural and functional elements of the phospholipids in cell membranes; ( ) cells involved in the immunity and inflammation (neutrophils, monocytes, macrophages, dendritic cells, and endothelial cells) are richer in n- pufas than n- , but the contents of aa, epa, and dha can be modified by n- pufa (i.e., epa and dha) administration (oral, enteral, and parenteral); ( ) pufa challenge, as well as the fa composition of cell membrane of human inflammatory cells, influences the functions of those cells; and, last but more important, ( ) n- pufas and n- pufas have opposing influences on inflammation. ali/ards are inflammatory diseases too; therefore, numerous studies have been carried out to evaluate strategies (drugs or interventional procedures) to reduce the severity of lung inflammatory processes by lowering the production of pro-inflammatory mediators (eicosanoids and cytokines). nonetheless, few studies have found a significant improvement on mortality in those patients. since , many editorials have stressed the possibility to manipulate the inflammatory response in ali/ards patients using n- pufas as drugs (the so-called pharmaconutrition) (mayer and seeger ) . the research on the influence of fas on immunity started in the s. subsequently, a great number of works with cell or animal models have been carried out with the aim to demonstrate efficacy in modifying the inflammatory responses of fish oil or their main active components (i.e., epa and dha). likewise, there have been some clinical trials of enteral or parenteral administration of fish oil-enriched nutrition formulas in critically ill patients with ali/ards. the experimental models used were animal isolated lungs or, mainly, cell line models. frequently, a human lung carcinoma cell line (a cells) was used. a are alveolar epithelial cells with type ii pneumocyte properties that retain many of the characteristics of normal human type ii epithelial cells and could synthesize lecithin and phosphatidylcholine with a high percentage of desaturated fas. several authors used alveolar type ii cells because it was shown to have a central position in the pathophysiology of the alveolar space. after in vitro administration, fas were rapidly incorporated in the phospholipid spectrum of alveolar cell membranes; in fact, as pulmonary surfactant (the surface-active phospholipoprotein complex) producers, alveolar type ii cells have a higher lipid metabolism, e.g., a significant increase in dha concentration in the lung phospholipid fraction was observed h after dha infusion. several experimental studies with isolated lungs and culture of different pulmonary cells clearly showed a pivotal role of aa and its metabolites as mediators of injury. in contrast, supplementation of n- pufas demonstrated to reduce alveolar release of pro-inflammatory mediators and to reduce organ failure in animal lung models. rapid changes in cell membrane phospholipid fa composition and lipid-derived inflammatory mediator generation were observed both after short cell exposure to n- pufas and short-term intravenous administration of n- pufas. it was observed that pg was produced within min of a cell exposure to epawith a peak at hand this rapid increase of pg was correlated with the metabolism of epa by cyclooxygenases in the a cells. experimental data suggested that epa and dha are both able to inhibit pge production. a short-term n- pufa supplementation induced a significant increase in the n- pufa composition of perfusate (after min) and lung tissue (after h). after as little as h of lung perfusion with an n- pufa-enriched emulsion, significant alterations in the spectrum of eicosanoid generation were found in the isolated lung model. likewise, after min in a model of septic lung injury, it was found that the vascular leakage provoked by bacterial exotoxin in rabbit lungs was differentially influenced by aa versus epa, related to the generation of -versus -series lts. early studies demonstrated that epa and dha inhibited endotoxin-stimulated production of il- and il- by cultured human endothelial cells and n- pufas inhibited endotoxin-induced tnf-α production by cultured monocytes. the differential impact of manipulation of cell membrane phospholipid composition due to n- pufa versus n- pufa administration on cytokine production provoked by pro-inflammatory mediators was demonstrated in a cells. although dha is not able to give rise to lts, it has a pivotal role since it can be incorporated unchanged into membrane lipids or retroconverted into epa. evidence suggests that the antiinflammatory and immunosuppressive effects of n- pufa supply may be attributed mainly, or even exclusively, to dha supplementation. in the past two decades, many authors have highlighted that the n- /n- pufa ratio in nutrition support may influence inflammation since optimal n- administration was not only dose related but was also independently affected by the n- /n- pufa ratio. different n- /n- pufa ratios from : to : have been proposed, but the question of the most favorable n- /n- pufa ratio in ali/ards patients is still to be defined. the impact of the n- /n- pufa ratio on cytokine release by a cells exposed to an endotoxin (i.e., lipopolysaccharide -lps) challenge was investigated in a recent experimental study (cotogni et al. ) . as in many studies, the inflammatory process was elicited by administration of lpsa component of gram-negative bacteria cell wallbecause lps is an important mediator in the pathogenesis of ards and a cells are able to produce the acute phase protein lps-binding protein. in particular, it was determined the time-and dose-dependent effect of lps on the tnf-α response in a cells (fig. ) . the fa composition in phospholipids of a cell membranes was also analyzed, and the n- /n- pufa ratio was reported to be : , with aa as the most prevalent pufa (table ) . the administration of different ratios of aa and dha on the lps-induced cytokine response from a cells showed that the supply of : and tnf-α concentrations were measured in cell culture media by elisa assay at the time points indicated. the results were expressed as picograms of released cytokines per adherent cells (pg/ cells) and as mean ae sd (n = experiments). lps lipopolysaccharide, tnf-α tumor necrosis factor-α. * p < . lps and versus baseline, at and h; ** p < . lps versus baseline, at h; *** p < . lps versus baseline, at h; # p < . lps h versus lps h : dha/aa ratios reversed the baseline predominance of n- over n- in the n- /n- pufa ratio of cell membranes. particularly, the release of pro-inflammatory cytokines (tnf-α, il- , and il- ) was reduced by : and : dha/aa ratios; on the contrary, it was further increased by : and : dha/aa ratios if compared to lps challenge alone (fig. ) . moreover, the : and : dha/aa ratios were effective in increasing antiinflammatory il- release and in modifying the balance between pro-and anti-inflammatory cytokines (fig. ). this finding confirmed previous studies showing that il- protects against the lethal effects of lps by significantly reducing the production of pro-inflammatory cytokines in table percentage content of fatty acids in phospholipids of a cell membranes fatty acid c : (saturated) . c : (saturated) . c : (monounsaturated) . c : (saturated) . c : (monounsaturated) . c : (linoleic acid, n- ) . c : (arachidonic acid, aa, n- ) . c : (docosahexaenoic acid, dha, n- ) . n- /n- pufa ratio lipopolysaccharide-stimulated tnf-α, il- , il- , and il- release from a cells treated with different dha/aa ratios. interleukin concentrations were measured in cell culture media by elisa assay at h. the results were expressed as picograms (pg) of released cytokines per adherent cells and as mean ae sd (n = experiments). aa arachidonic acid, bl baseline, c control, dha docosahexaenoic acid, il interleukin, tnf-α tumor necrosis factor-α. * p < . versus control; ** p < . versus all; # p < . versus control and : human monocytes and murine peritoneal macrophages. these data suggested that inflammatory cytokine release was dependent on the proportion of n- in the n- /n- pufa ratio of alveolar cell membranes, being reduced with the supply of dha and increased with a high proportion of aa. according to several other data, shifting the pufa supply from n- to n- with a ratio of : may be a good means to dampen pro-inflammatory responses in ali. experimental studies with animal models demonstrated that production of aa-derived eicosanoids and cytokines decreases with a diet containing fish oil in ali and septic murine models. macrophages isolated from mice fed with a diet containing fish oil produced less pge and il- in response to lps stimulation than those isolated from mice fed with other lipid-enriched diets. feeding rats with an n- pufa-enriched diet, as compared with an n- pufa-enriched one, was associated to: ( ) a reduced severity of lung microvascular protein permeability and hypotension in a model of endotoxin-induced ali, ( ) a reduced synthesis of pro-inflammatory eicosanoid (ltb , pge , and txb ) released from stimulated alveolar macrophages, and ( ) a decrease in aa and an increase in epa and dha in cell membrane phospholipids of alveolar macrophages. after intraperitoneal injection of lps, peak plasma tnf-α, il- β, and il- levels were more reduced in mice fed with a fish oil diet than in those fed with a safflower oil diet (i.e., a diet rich in n- pufas). using either a model of ali, in which lps was instilled in the trachea, or a model of intraperitoneal inflammation, in which lps was injected intraperitoneally, it was demonstrated that a -day course of intravenous lipid emulsion infusions was sufficient to modify inflammatory responses. fish oil-based lipid emulsions reduced alveolar leukocyte transmigration, protein leakage, and cytokine production, as well as cytokine concentrations in the intravascular compartment. conversely, soybean oil-based lipids lead to a further increase in the inflammatory response. tnf-α /il- (log pg/ cells) il- /il- (log pg/ cells) il- /il- (log pg/ cells) clinical data showing benefits associated with the administration of nutrition formulas enriched with n- pufas in ali/ards patients came initially from three randomized controlled trials (rcts) using the enteral route. these rcts showed that en with a study formula containing epa, γ-linolenic acid, and antioxidants (vitamins c and e, β-carotene, and taurine), as compared with high-fat formula (control formula), reduced alveolar inflammatory mediators and improved clinical outcomes in patients with ali, ards, or sepsis. the first rct showed the ability of an enteral formula with a high n- /n- pufa ratio ( : ) to reduce pulmonary inflammation and improve clinical outcomes, i.e., better oxygenation, shorter requirement for mechanical ventilation, shorter icu-los, and less incidence of new organ failure; however, no difference in mortality was observed in ards patients (gadek et al. ) . similarly, an rct in ali/ards mechanically ventilated patients showed that an n- pufaenriched enteral diet may be more beneficial for gas exchange, respiratory dynamics, and length of mechanical ventilation if compared with a control formula, but no difference both in icu or hospital los and mortality was found between the two formulas (singer et al. ) . another rct was able to demonstrate that the administration of the same enteral diet contributed to guarantee an improved oxygenation and an independence from mechanical ventilation, less incidence of new organ dysfunction, and more icu-free days and, mainly, was associated with lower mortality rates in ards mechanically ventilated patients with severe sepsis and septic shock (pontes-arruda et al. ) . in , a meta-analysis of the pooled outcomes from these three rcts concluded that the n- pufa-enriched enteral formula could reduce mortality, rate of new organ failure, icu-los, and length of ventilation in ali/ards patients (pontes-arruda et al. ) . nevertheless, in , three further rcts were published that addressed the issue of the potential positive effects of an enteral n- pufa-enriched nutrition in ali/ards patients showing mixing results. the first rct analyzed the effect of an enteral n- pufa-enriched diet in septic patients with ali or ards showing that the administration of the study formula, compared to a control formula with less lipids than in the previous three studies, was associated to a shorter icu-los but not to an improvement in gas exchange or in a lower incidence of novel organ failures (grau-carmona et al. ) . a phase placebocontrolled rct in ali patients investigating an enteral administration of fish oil for up to days did not demonstrate a decrease in cytokine or other inflammatory marker concentrations both in bal fluid from baseline to day or and in plasma, as well as a decrease in ventilator-free days, icu-free days, organ failure score, or -day mortality (stapleton et al. ). the last rct tested the effects of enteral feeding with n- pufas, γ-linolenic acid, and antioxidants on clinical outcomes in ali patients in a phase trial (omega trial) using a twice-daily bolus administration (rice et al. ) . contrary to all other rcts, the authors reported that enteral administration of this formula was associated to significantly fewer ventilator-free days, icu-free days, and non-pulmonary organ failure-free days, as well as more days with diarrhea. the trial was stopped early for futility at the first interim analysis. the interpretation of the omega trial presents some difficulties (lev and singer ) . firstly, it was used a different approach of twicedaily bolus administration of enteral formulas instead of continuous enteral infusion. secondly, the enteral diets administered to the study group (i.e., megadoses of n- pufas and lower protein content) and to the control group (i.e., higher protein and carbohydrate content) are notably different from those used in the first three rcts. enteral administration of n- pufas might require more time before effectively influencing the cellular fa composition and the lipidmediated inflammatory response. moreover, the daily volume of enteral formula prescribed is calculated such as to meet patients' requirement of calories rather than a scheduled "pharmacological" dosage of n- pufas. it is well known that icu patients exhibit different degrees of tolerance to en, as it is well documented that the more patients are critically ill, the less they tolerate en. thus, an unpredictable number of critical patients might not receive the "pharmacological" dosage of n- pufas. an alternative approach to provide n- pufas to icu patients unable to meet their nutrition requirements by oral or enteral route is to administer a parenteral formula prepared from fish oil. differently to oral/enteral feeding, parenteral administration of pufas can induce more rapid changes in lipid-derived responses. indeed, in ali, there is the need of promptly active treatments to early modulate the net inflammatory response in the alveolar spaces. in septic patients, intravenous infusion of n- pufas modified lipid mediator synthesis and reduced endotoxin-stimulated monocyte proinflammatory cytokine production, while cytokine production was markedly amplified by n- pufa administration (mayer et al. ) . conversely, pn administered for days with a : mixture of medium-chain triglycerides and longchain triglycerides, with the n- /n- pufa ratio of : compared with a same mixture supplemented with fish oil with the n- /n- pufa ratio of : , did not affect inflammatory marker production or measures of clinical outcomes in an rct in unselected critically ill medical patients (friesecke et al. ) . in the last years, three rcts have investigated the effects of parenteral administration of n- pufa-enriched lipid emulsions in ali/ards patients. the first rct investigated the effects of an n- pufa-enriched lipid emulsion in ards patients with en intolerance showing no significant changes in hemodynamic and gas exchange parameters (sabater et al. ) . previously, the same authors had showed that parenteral administration of an n- -enriched lipid emulsion was accompanied by the synthesis of a smaller amount of pro-inflammatory lipid mediators compared to long-chain triglyceride % emulsion in ards patients. in a second trial, patients with systemic inflammatory response syndrome or sepsis were randomized into two groups to receive an intravenous lipid emulsion enriched or not in fish oil (barbosa et al. ) . the authors reported that the pao :fio ratio at day was significantly higher and plasma il- concentration significantly decreased in the group treated with fish oil; however, fish oil administration did not improve the length of ventilation or icu-los and mortality. the last rct investigated the effects of an intravenous administration of n- pufas with the n- /n- pufa ratio of : as supplementation of en for days in ards patients finding that fish oil administration alone did not improve ventilation, icu-los, or survival (gupta et al. ). ards is the most severe life-threatening manifestation of ali. both remain a significant health burden for intensivists and critical care physicians because of their considerable morbidity and mortality. in , in the usa, the crude incidence of ali was . per , person-years; each year, there are , cases of ali associated with , deaths and . million hospital days. in , in icus of ten european countries, ali occurred in ( %) out of , admissions, and in the % of the whole mechanically ventilated patients, % cases occurred on icu admission. pathologically, ali and ards are inflammatory diseases; clinically, their severity depends on the grade of inflammatory response. thus, numerous studies have been carried out investigating new strategies to reduce the grade of this inflammatory response, mainly by reducing the release of pro-inflammatory mediators. however, pharmacological therapies such as corticosteroids, ketoconazole, antioxidants, nitric oxide, prostacyclins, exogenous surfactants, and β agonists failed to show positive results in human studies. supplementation with n- pufas was demonstrated to exert beneficial effects in a number of inflammatory diseases. the rationale for their use in ali/ards patients is consistent and based on several issues. firstly, a large amount of experimental studies in lung models supports the antiinflammatory properties of n- pufas. secondly, patients at risk for ards and with established ards have plasma epa concentrations about % and %, respectively, as compared with normal, hypothesizing a potential benefit for n- pufa administration in those patients. finally, in three level i studies involving patients with ali, ards, and sepsis, the use of an enteral diet enriched with n- pufas (in the form of epa), γ-linolenic acid, and antioxidants was shown to significantly reduce icu-los, mechanical ventilation length, organ failure incidence, and mortality if compared to the use of a standard enteral formula. based on these data, the european society for clinical nutrition and metabolism, american society of parenteral and enteral nutrition, society of critical care medicine, and canadian guidelines recommended, with an a or b grade of recommendation, in their guidelines published between and , the use of n- pufas in ali/ards patients. however, nowadays, the clinical application of these recommendations has been lagging behind in critical or intensive care, which is due, at least in part, to inconclusive or contradicting results in several recent clinical trials using n- pufas. over the last years, the data coming out from a great number of studies have left us an improved knowledge of how n- pufas and their derivate mediators modulate inflammation/resolution and immunity. evidence that supports both the antiinflammatory and antitumor effects of n- pufas has been gathered; moreover, the molecular pathways underlying these effects have been clarified both in vitro and in vivo. in particular, there is now a better understanding of various mechanisms generating the production of inflammatory cytokines in several inflammatory, metabolic, and neurodegenerative diseases, as well as in inflammation-related cancers. the capacity of n- pufas to determine a shift from a strongly pro-inflammatory environment to one of reduced inflammation supports the hypothesis that these fas might be useful as a component of the therapy in various inflammatory diseases. thus, epa and/or dha have been evaluated in some inflammatory conditions. for the potential or in-use applications of n- pufas to clinical conditions different from ali, see a recent review (calder b) . briefly, fish oil or its derived epa and dha have demonstrated the efficacy in animal models of rheumatoid arthritis, inflammatory bowel diseases, and asthma. there have been a number of clinical trials evaluating the effects of administration of n- pufas in those diseases with contradicting results. for example, there is some pretty good evidence of the efficacy of n- pufas in rheumatoid arthritis. conversely, despite positive results in some studies, there is only weak evidence that n- pufas have clinical benefits in inflammatory bowel diseases. finally, clinical studies on fish oil in adult patients with asthma do not show benefit. the "european society for clinical nutrition and metabolism guidelines on enteral nutrition: intensive care" (kreymann et al. ) stated that "patients with ards should receive en enriched with n- fatty acids and antioxidants" as a b grade recommendation. however, this recommendation was not evaluated in the "espen guidelines on parenteral nutrition: intensive care" (singer et al. ), although the authors stated that "addition of epa and dha to lipid emulsions has demonstrable effects on cell membranes and inflammatory processes; fish oil-enriched lipid emulsions probably decrease length of stay in critically ill patients" both as b grade recommendations. the american society for parenteral and enteral nutrition "guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient" (mcclave et al. ), also being co-published by the society of critical care medicine, stated that "patients with ards and severe ali should be placed on an enteral formulation characterized by an antiinflammatory lipid profile (i.e., n- fish oil, borage oil) and antioxidants" (a grade recommendation). this recommendation was based on the three positive trials published at that time. the canadian guidelines (updated june ) stated that "based on level studies and level studies, the use of an enteral formula with fish oils, borage oils and antioxidants in patients with ali and ards should be considered. when pn with intravenous lipids is indicated, lipids that reduce the load of n- fa/soybean oil emulsions should be considered. however, there are insufficient data to make a recommendation on the type of lipids to be used that reduce the n- fa/soybean oil load in critically ill patients receiving pn." ali/ards patients are at high risk of malnutrition; therefore, they need nutrition support, including lipids. soybean oil-based lipid emulsions, rich in n- pufas and commonly used for these patients, may aggravate the inflammatory response. in contrast, fish oil-based lipid emulsions, rich in n- pufas, may exert an anti-inflammatory effect. these data suggest that shifting the pufa supply from n- to n- could be an important element affecting the alveolar cytokine and eicosanoid release and consequently ameliorates the clinical outcome in ali/ards patients. in summary, there are good experimental evidence and convincing rationale according to the n- pufas used in ali/ards patients. however, recent rcts using n- pufas in these patients did not confirm these positive effects. further studies are necessary to offer more precise indications, timing, and modalities for n- pufa administration. • acute lung injury (ali) and acute respiratory distress syndrome (ards) are inflammatory diseases whose clinical severity depends on the grade of inflammation. • cytokines are key elements in the pathogenesis of ali/ards. • soybean oil-based lipid emulsions, rich in omega (n)- polyunsaturated fatty acids (pufas), may aggravate the inflammatory response. in contrast, fish oil-based lipid emulsions, rich in n- pufas, may exert an antiinflammatory effect. • experimental and clinical data suggested that shifting the pufa supply from n- to n- decreases the alveolar cytokine and eicosanoid release and consequently ameliorates the clinical outcome in ali/ards patients. • based on these data, several scientific societies recommended in their guidelines the use of n- pufas in ali/ards patients. effects of fish oil containing lipid emulsion on plasma phospholipid fatty acids, inflammatory markers, and clinical outcomes in septic patients: a randomized, controlled clinical trial n- polyunsaturated fatty acids, inflammation, and inflammatory diseases n- fatty acids, inflammation and immunity: new mechanisms to explain old actions omega- polyunsaturated fatty acids and inflammatory processes: nutrition or pharmacology? canadian clinical practice guidelines for nutrition support in mechanically ventilated impact of the ω- to ω- polyunsaturated fatty acid ratio on cytokine release in human alveolar cells the berlin definition of ards: an expanded rationale, justification, and supplementary material fish oil supplementation in the parenteral nutrition of critically ill medical patients: a randomised controlled trial effect of enteral feeding with eicosapentaenoic acid, gammalinolenic acid, and antioxidants in patients with acute respiratory distress syndrome. enteral nutrition in ards study group effect of an enteral diet enriched with eicosapentaenoic acid, gamma-linolenic acid and antioxidants on the outcome of mechanically ventilated, critically ill, septic patients efficacy and safety of parenteral omega- fatty acids in ventilated patients with acute lung injury espen guidelines on enteral nutrition: intensive care acute respiratory distress syndrome n- fatty acids and γ-linolenic acid supplementation in the nutritional support of ventilated patients with acute lung injury or acute respiratory distress syndrome fish oil in critical illness parenteral nutrition with fish oil modulates cytokine response in patients with sepsis guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: society of critical care medicine (sccm) and american society for parenteral and enteral nutrition nutritional support in acute lung injury and acute respiratory distress syndrome cytokine balance in the lungs of patients with acute respiratory distress syndrome effects of enteral feeding with eicosapentaenoic acid, gammalinolenic acid, and antioxidants in mechanically ventilated patients with severe sepsis and septic shock the use of an inflammation-modulating diet in patients with acute lung injury or acute respiratory distress syndrome: a meta-analysis of outcome data the alveolar space is the site of intense inflammatory and profibrotic reactions in the early phase of acute respiratory distress syndrome enteral omega- fatty acid, γ-linolenic acid, and antioxidant supplementation in acute lung injury effects on hemodynamics and gas exchange of omega- fatty acidenriched lipid emulsion in acute respiratory distress syndrome (ards): a prospective, randomized, double-blind, parallel group study benefit of an enteral diet enriched with eicosapentaenoic acid and gammalinolenic acid in ventilated patients with acute lung injury espen guidelines on parenteral nutrition: intensive care a phase ii randomized placebo-controlled trial of omega- fatty acids for the treatment of acute lung injury key: cord- -ls l mlg authors: tindle, courtney; fuller, mackenzie; fonseca, ayden; taheri, sahar; ibeawuchi, stella-rita; beutler, nathan; claire, amanraj; castillo, vanessa; hernandez, moises; russo, hana; duran, jason; crotty alexander, laura e.; tipps, ann; lin, grace; thistlethwaite, patricia a.; chattopadhyay, ranajoy; rogers, thomas f.; sahoo, debashis; ghosh, pradipta; das, soumita title: adult stem cell-derived complete lung organoid models emulate lung disease in covid- date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: ls l mlg sars-cov- , the virus responsible for covid- , causes widespread damage in the lungs in the setting of an overzealous immune response whose origin remains unclear. we present a scalable, propagable, personalized, cost-effective adult stem cell-derived human lung organoid model that is complete with both proximal and distal airway epithelia. monolayers derived from adult lung organoids (alos), primary airway cells, or hipsc-derived alveolar type-ii (at ) pneumocytes were infected with sars-cov- to create in vitro lung models of covid- . infected alo-monolayers best recapitulated the transcriptomic signatures in diverse cohorts of covid- patient-derived respiratory samples. the airway (proximal) cells were critical for sustained viral infection, whereas distal alveolar differentiation (at →at ) was critical for mounting the overzealous host immune response in fatal disease; alo monolayers with well-mixed proximodistal airway components recapitulated both. findings validate a human lung model of covid- , which can be immediately utilized to investigate covid- pathogenesis and vet new therapies and vaccines. graphic abstract highlights human lung organoids with mixed proximodistal epithelia are created proximal airway cells are critical for viral infectivity distal alveolar cells are important for emulating host response both are required for the overzealous response in severe covid- in brief an integrated stem cell-based disease modeling and computational approach demonstrate how both proximal airway epithelium is critical for sars-cov- infectivity, but distal differentiation of alveolar pneumocytes is critical for simulating the overzealous host response in fatal covid- . sars-cov- , the virus responsible for covid- , causes widespread inflammation and injury in the lungs, giving rise to diffuse alveolar damage (dad) (arrossi and farver, ; borczuk et al., ; damiani et al., ; li et al., ; roden et al., ) , featuring marked infection and viral burden leading to apoptosis of alveolar pneumocytes (hussman, ) , along with pulmonary edema (bratic and larsson, ; carsana et al., ) . dad leads to poor gas exchange and, ultimately, respiratory failure; the latter appears to be the final common mechanism of death in most patients with severe covid- infection. how the virus causes so much damage remains unclear. a particular challenge is to understand the out-of-control immune reaction to the sars-cov- infection known as a cytokine storm, which has been implicated in many of the deaths from covid- . although rapidly developed pre-clinical animal models have recapitulated some of the pathognomonic aspects of infection, e.g., induction of disease, and transmission, and even viral shedding in the upper and lower respiratory tract, many failed to develop severe clinical symptoms (lakdawala and menachery, ) . thus, the need for pre-clinical models remains both urgent and unmet. to address this need, several groups have attempted to develop human pre-clinical covid- lung models, all within the last few months (duan et al., ; mulay et al., ; salahudeen et al., ) . while a head-to-head comparison of the key characteristics of each model can be found in table , what is particularly noteworthy is that none recapitulate the heterogeneous epithelial cellularity of both proximal and distal airways, i.e., airway epithelia, basal cells, secretory club cells and alveolar pneumocytes. also noteworthy is that models derived from ipscs lack propagability and/or cannot be reproducibly generated for biobanking; nor can they be scaled up in cost-effective ways for use in drug screens. besides the approaches described so far, there are a few more approaches used for modeling covid- -(i) d organoids from bronchospheres and tracheospheres have been established before (hild and jaffe, ; rock et al., ; tadokoro et al., ) and are now used in apical-out cultures for infection with sars-cov- (suzuki et al., ); (ii) the most common model used for drug screening is the air-liquid interphase (ali model) in which pseudo-stratified primary bronchial or small airway epithelial cells are used to recreate the multilayered mucociliary epithelium (mou et al., ; randell et al., ) ; (iii) several groups have also generated d airway models from ipscs or tissue-resident stem cells (dye et al., ; ghaedi et al., ; konishi et al., ; mccauley et al., ; miller et al., ; wong et al., ) ; (iv) others have generated at cells from ipscs using closely overlapping protocols of sequential differentiation starting with definitive endoderm, anterior foregut endoderm, and distal alveolar expression (chen et al., ; gotoh et al., ; huang et al., ; jacob et al., ; jacob et al., ; yamamoto et al., ) . (v) finally, long term in vitro culture conditions for pseudo-stratified airway epithelium organoids, derived from healthy and diseased adult humans suitable to assess virus infectivity (sachs et al., ; van der vaart and clevers, ; zhou et al., ) have been pioneered; unfortunately, these airway organoids expressed virtually no lung mesenchyme or alveolar signature. what remains unclear is if any of these models accurately recapitulate the immunopathologic phenotype that is seen in the lungs in covid- . we present a rigorous transdisciplinary approach that systematically assesses an adult lung organoid model that is propagable, personalized and complete with both proximal airway and distal alveolar cell types against existing models that are incomplete, and we cross-validate them all against covid- patient-derived respiratory samples. findings surprisingly show that cellular crosstalk between both proximal and distal components are necessary to emulate how sars-cov- causes diffuse alveolar pneumocyte damage; the proximal airway mounts a sustained viral infection, but it is the distal alveolar pneumocytes that mount the overzealous host response that has been implicated in a fatal disease. to determine which cell types in the lungs might be most readily infected, we began by analyzing a human lung single-cell sequencing dataset (gse ) for the levels of expression of angiotensin-converting enzyme-ii (ace ) and transmembrane serine protease (tmprss ), the two receptors that have been shown to be the primary sites of entry for the sars-cov- (hoffmann et al., ) . the dataset was queried with widely accepted markers of all the major cell types (see table ). alveolar epithelial type (at ), ciliated and club cells emerged as the cells with the highest expression of both receptors (fig a; fig s a) . these observations are consistent with published studies demonstrating that ace is indeed expressed highest in at and ciliated cells (jia et al., ; mulay et al., ; zhao et al., ) . in a cohort of deceased covid- patients, we observed by h&e (fig s b) that gas-exchanging flattened at pneumocytes are virtually replaced by cuboidal cells that were subsequently confirmed to be at -like cells via immunofluorescent staining with the at -specific marker, surfactant protein-c (sftpc; fig b upper panel; fig s c; top). we also confirmed that club cells express ace (fig s c; bottom) , underscoring the importance of preserving these cells in any ideal lung model of covid- . when we analyzed the lungs of deceased covid- patients, the presence of sars-cov- in alveolar pneumocytes was also confirmed, as determined by the colocalization of viral nucleocapsid protein with sftpc (fig b; lower panel; fig s d) . immunohistochemistry studies further showed the presence of sars-cov- virus in alveolar pneumocytes and in alveolar immune cells (fig s e) . these findings are consistent with the gathering consensus that alveolar pneumocytes support the interaction between the epithelial cells and inflammatory cells recruited to the lung; via mechanisms that remain unclear, they are generally believed to contribute to the development of acute lung injury and acute respiratory distress syndrome (ards), the severe hypoxemic respiratory failure during covid- (hou et al., ; spagnolo et al., ) . because prior work has demonstrated that sars-cov- infectivity in patient-derived airway cells is highest in the proximal airway epithelium compared to the distal alveolar pneumocytes (at and at ) (hou et al., ) , and yet, it is the at pneumocytes that harbor the virus, and the at pneumocytes that are ultimately destroyed during diffuse alveolar damage, we hypothesized that both proximal airway and distal (alveolar pneumocyte) components might play distinct roles in the respiratory system to mount the so-called viral infectivity and host immune response phases of the clinical symptoms observed in covid- (chen and li, ) . because no existing lung model provides such proximodistal cellular representation (table ) , and hence, may not recapitulate with accuracy the clinical phases of covid- , we first sought to develop a lung model that is complete with both proximal and distal airway epithelia using adult stem cells that were isolated from deep lung biopsies. lung organoids were generated using the protocol outlined in fig c and methods. organoids grown in d cultures were subsequently dissociated into single cells to create dmonolayers (either maintained submerged in media or used in ali model) for sars-cov- infection, followed by rna seq analysis. primary airway epithelial cells and hipsc-derived alveolar type-ii (at ) pneumocytes were used as additional models (fig d; left panel) . each of these transcriptomic datasets was subsequently used to cross-validate our ex-vivo lung models of sars-cov- infection with the human covid- autopsy lung specimens (fig d; right panel) to objectively vet each model for their ability to accurately recapitulate the gene expression signatures in the patient-derived lungs. three lung organoid lines were developed from deep lung biopsies obtained from the normal regions of lung lobes surgically resected for lung cancer; both genders, smokers and non-smokers were represented (fig s a; table ). three different types of media were compared (fig s b) ; the composition of these media was inspired either by their ability to support adult-stem cell-derived mixed epithelial cellularity in other organs (like the gastrointestinal tract (miyoshi and stappenbeck, ; sato et al., ; sayed et al., ) , or rationalized based on published growth conditions for proximal and distal airway components (gotoh et al., ; sachs et al., ; van der vaart and clevers, ) . a growth condition that included conditioned media from l-wrn cells which express wnt , r-spondin and noggin, supplemented with recombinant growth factors, which we named as 'lung organoid expansion media' emerged as superior compared to alveolosphere media-i and ii (jacob et al., ; yamamoto et al., ) (details in the methods), based on its ability to consistently and reproducibly support the best morphology and growth characteristics across multiple attempts to isolate organoids from lung tissue samples. three adult lung organoid lines (alo - ) were developed using the expansion media, monitored for their growth characteristics by brightfield microscopy and cultured with similar phenotypes until p and beyond (fig s c-d) . the d morphology of the lung organoid was also assessed by h&e staining of slices cut from formalin-fixed paraffin-embedded (ffpe) cell blocks of histogel-embedded alo - (fig s e) . to determine if all the major lung epithelial cells (illustrated in fig a) are present in the organoids, we analyzed various cell-type markers by qrt-pcr (fig b-h) . all three alo lines had a comparable level of at cell surfactant markers (compared against hipsc-derived at cells as positive control) and a significant amount of at , as determined using the marker aqp . alos also contained basal cells (as determined by the marker itga ), ciliated cells (as determined by the marker foxj ), club cells (as determined by the marker scgb a ) and stem cells (as determined by marker p -ngfr). as expected, the primary human bronchial epithelial cells (nhbe) had significantly higher expression of basal cell markers than the alo lines (hence, served as a positive control), but they lacked stemness and club cells (hence, served as a negative control). the presence of all cell types was also confirmed by assessing protein expression of various cell types within organoids grown in d cultures. two different approaches were used-(i) slices cut from ffpe cell blocks of histogel-embedded alo lines (fig i-j) or (ii) alo lines grown in -well chamber slides were fixed in matrigel (fig k) , stained, and assessed by confocal microscopy. such staining not only confirmed the presence of all cell types in each alo line but also demonstrated the presence of more than one cell type (i.e., mixed cellularity) of proximal (basal-krt ) and distal (at /at markers) within the same organoid structure. for example, at and basal cells, marked by sftpb and krt , respectively, were found in the same d-structure (fig j, interrupted curved line). similarly, ciliated cells and goblet cells stained by ac-tub and muc , respectively, were found to coexist within the same structure (fig j, interrupted box; fig k, arrow) . the presence of heterogeneous cellularity was documented in all three alo lines (see multiple additional examples in fig s ) . to model respiratory infections such as covid- , it is necessary for pathogens to be able to access the apical surface. many groups have modeled such access by dissociating d organoids into single cells and plating them as d-monolayers (duan et al., ; han et al., ; huang et al., ; mulay et al., ; sachs et al., ; zhou et al., ) . because the loss of dimensionality can have a major impact on cellular proportions and impact disease-modeling in unpredictable ways, we assessed the impact of the d-to- d conversion on cellularity by rna seq analyses. two commonly encountered methods of growth in d-monolayers were tested: (i) monolayers polarized on trans-well inserts but submerged in growth media, and ii) monolayers were grown at the air-liquid interface (popularly known as the 'ali model '(dvorak et al., ; prytherch et al., ) ) for -days to differentiate into the mucociliary epithelium (see fig a; fig s a-e) . the submerged d-monolayers had several regions of organized vacuolated-appearing spots (fig s a, d; arrow), presumably due to morphogenesis and cellular organization even in d. the ali-monolayers appeared to be progressively hazier with time after air-lift, likely due to the accumulation of secreted mucin (fig s e) , and formed an intact epithelial barrier, as determined by trans-epithelial electrical resistance (teer) (fig s c) . rna seq datasets were analyzed using the same set of cell markers, as we used in fig a ( table ). cell-type deconvolution of our dataset using cibersortx (https://cibersortx.stanford.edu/runcibersortx.php) showed that cellular proportions in the human lung tissues were also relatively well-preserved in organoids grown in d over several passages (fig b; left) ; both showed a mixed population of simulated alveolar, basal, club, ciliated and goblet cells. when d organoids were dissociated and plated as d monolayers on transwells, the at signatures were virtually abolished with a concomitant and prominent emergence of at signatures, suggesting that growth in d-monolayers favor differentiation of at cells into at cells (shami and evans, ) (fig b; middle) . a compensatory reduction in proportion was also observed for the club, goblet and ciliated cells. the same organoids, when grown in long-term d culture conditions in the ali model, showed a strikingly opposite pattern; alveolar signatures were almost entirely replaced by a concomitant increase in ciliated and goblet cells (fig b; right). these findings are consistent with the well-established notion that ali conditions favor growth as pseudo-stratified mucociliary epithelium (dvorak et al., ; prytherch et al., ) . as an alternative model for use as monolayers for viral infection, we developed hipsc-derived at cells and alveolospheres (fig c) , using established protocols (huang et al., ) . because they were grown in the presence of chir (an aminopyrimidine derivate that is a selective and potent wnt agonist) (abdelwahab et al., ; jacob et al., ; yamamoto et al., ) , which probably inhibits the at →at differentiation, these monolayers were enriched for at and devoid of at cells (fig d) . the multicellularity of lung organoid monolayers was also confirmed by immunofluorescence staining and confocal microscopy of the submerged and ali monolayers, followed by the visualization of cell markers in either max-projected z-stacks (fig e; left) or orthogonal views of the same (fig e; right) . as expected, markers for the same cell type (i.e., sftpb and sftpc, both at markers) colocalize, but markers for different cell types do not. submerged monolayers showed the prominent presence of both at (aqp -positive) and at cells. compared to the submerged monolayers, the ali model showed a significant increase in the ciliated epithelium (as determined by ac tub; compare ac tub stained panels in fig e with f) . this increase was associated with a concomitant decrease in both krt -stained basal and sftpc-positive at cells (fig f) . taken together, the immunofluorescence images are in agreement with the rna seq dataset; both demonstrate that the short-term submerged monolayer favors distal differentiation (at →at ), whereas the day ali model favors proximal mucociliary differentiation. it is noteworthy that these distinct differentiation phenotypes originated from the same d-organoids despite the seeding of cells in the same basic media composition (i.e., pneumacult tm ) prior to switching over to an ali-maintenance media for the prolonged growth at air-liquid interface; the latter is a well-described methodology that promotes differentiation into ciliated and goblet cells (rayner et al., ) . because the lung organoids with complete proximodistal cellularity could be differentiated into either proximalpredominant monolayers in submerged short-term cultures or distal-predominant monolayers in long-term ali cultures, this provided us with an opportunity to model the respiratory tract and assess the impact of the virus along the entire proximal-to-distal gradient. we first asked how viral infectivity varies in the two models. because multiple groups have shown the importance of the ciliated airway cells for infectivity (i.e., viral entry, replication and apical release (hou et al., ; hui et al., ; milewska et al., ; zhu et al., ) ), as positive controls, we infected monolayers of human airway epithelia (see fig s f-i) . at cells, which express high levels of viral entry receptors ace and tmprss (fig a, fig s a) have been shown to be proficient in viral entry, but are least amenable to sustained viral release and infectivity (hou et al., ; hui et al., ) . to this end, we infected monolayers of hipsc-derived homogeneous cultures of at cells as secondary controls (see fig s j- l). infection was carried out using the washington strain of sars-cov- , usa-wa / [bei resources nr- (rogers et al., ) ]. as expected, the d-lung monolayers we generated, both the submerged or the ali models, were readily infected with sars-cov- (fig s a) , as determined by the presence of the viral envelope gene (e-gene; fig g) ; however, the kinetics of viral amplification differed. when expressed as levels of e gene normalized to the peak values in each model (fig g) , the kinetics of the ali-monolayer model mirrored that of the primary airway epithelial monolayers; both showed slow beginning ( - hpi) followed by an exponential increase in e gene levels from to hpi. the submerged monolayer model showed sustained viral infection during the - hpi window (fig s a; left) . in the case of at cells, the - hpi window was notably missing in monolayers of hipsc-derived at cells (fig g; fig s a; right). when we specifically analyzed the kinetics of viral e gene expression during the late phase ( - hpi window), we found that proximal airway models [human bronchial airway epi (hbepc)] were more permissive than distal models [human small airway epi (hsaepc) and at ] to viral replication (fig s b) ; the alo monolayers showed intermediate sustained infectivity (albeit with variability). all models showed extensive cell death and detachment by hrs and, hence, were not analyzed. confocal imaging of infected alo monolayers with anti-sars-cov- nucleocapsid protein antibody showed that submerged alo monolayers did indeed show progressive changes during the to h window after infection ( taken together, these findings show that sustained viral infectivity is best simulated in monolayers that resemble the proximal mucociliary epithelium, i.e., d-monolayers of lung organoids grown as ali models and the primary airway epithelia. because prior studies conducted in patient-derived airway cells (hou et al., ) mirror what we see in our monolayers, we conclude that proximal airway cells within our mixed-cellular model appear to be sufficient to model viral infectivity in covid- . next, we asked if the newly generated lung models accurately recapitulate the host immune response in covid- . to this end, we analyzed the infected alo monolayers (both the submerged and ali variants) as well as the airway epithelial (hsaepc) and at monolayers by rna seq and compared them all against the transcriptome profile of lungs from deceased covid- patients. a publicly available dataset (gse ) (nienhold et al., ) , comprised of lung transcriptomes from victims deceased either due to non-infectious causes (controls) or due to covid- , was first analyzed for differentially expressed genes (fig a-b) . this cohort was chosen as a test cohort over others because it was the largest one available at the time of this study with appropriate postmortem control samples. differentially expressed genes showed an immunophenotype that was consistent with what is expected in viral infections (fig c; table ; fig s ) , and showed overrepresentation of pathways such as interferon, immune, and cytokine signaling (fig d; table ; fig s ) . differentially expressed gene signatures and reactome pathways that were enriched in the test cohort were fairly representative of the host immune response observed in patient-derived respiratory samples in multiple other validation cohorts; the signature derived from the test cohort could consistently classify control (normal) samples from covid- samples (roc auc . to . across the board; fig e) . the most notable finding is that the patient-derived signature was able to perfectly classify the epcam-sorted epithelial fractions from the bronchoalveolar lavage fluids of infected and healthy subjects (roc auc . ; gse -epithelium (liao et al., ) , suggesting that the respiratory epithelium is a major site where the host immune response is detected in covid- . when compared to existing organoid models of covid- , we found that the patient-derived covid- -lung signature was able to perfectly classify infected vs. uninfected late passages (> ) of hipsc-derived at / monolayers (gse ) (han et al., ) and infected vs. uninfected liver and pancreatic organoids (fig f) . the covid- -lung signatures failed to classify commonly used respiratory models, e.g., a cells and bronchial organoids, as well as intestinal organoids (fig f) . a similar analysis on our own lung models revealed that the covid- lung signature was induced in submerged monolayers with distal-predominant at →at differentiation, but not in the proximal-predominant ali model (roc auc . and . , respectively; fig g) . the ali model and the small airway epithelia, both models that mimic the airway epithelia (and lack alveolar pneumocytes; see fig b) , failed to mount the patient-derived immune signatures (fig h; left) . these findings suggested that the presence of alveolar pneumocytes is critical for emulating host response. to our surprise, induction of the covid- -lung signature also failed in hipsc-derived at monolayers (fig h; right), indicating that at cells are unlikely to be the source of such host response. these findings indicate that both proximal airway and at cells, when alone, are insufficient to induce the host immune response that is encountered in the lungs of covid- patient. next, we analyzed the datasets from our alo monolayers for differentially expressed genes when challenged with sars-cov- (fig a-b) . genes and pathways upregulated in the infected lung organoidderived monolayer models overlapped significantly with those that were upregulated in the covid- lung missing components, the model-derived deg signature was sufficient to consistently and accurately classify diverse cohorts of patient-derived respiratory samples (roc auc ranging from . to . ; fig f) ; the modelderived deg signature was significantly induced in covid- samples compared to normal controls (fig g-h ). most importantly, the model-derived deg signature was significantly induced in the epithelial cells recovered from bronchoalveolar lavage (fig i) . taken together, these cross-validation studies from disease to model (fig ) and vice versa (fig ) provide an objective assessment of the match between the host response in covid- lungs and our submerged alo monolayers. such a match was not seen in the case of the other models, e.g., the proximal airway-mimic ali model, hsaepc monolayer, or hipsc-derived at models. because the submerged alo monolayers contained both proximal airway epithelia (basal cells) and promoted at →at differentiation, findings demonstrate that mixed cellular monolayers can mimic the host response in covid- . a subtractive analysis revealed that the cell type that is shared between models which showed induction of host response signatures (i.e., alo submerged monolayers and gse (han et al., ); fig f) but is absent in models that do not show such response (hu bronchial organoids, small airway epi, ali-model of alo) is at . we conclude that distal differentiation from at →at , a complex process that is comprised of distinct intermediates (choi et al., ), is essential for modeling the host immune response in covid- . both proximal and distal airway epithelia are required to mount the overzealous host response in we next asked which model best simulated the overzealous host immune response that has been widely implicated in fatal covid- . to this end, we relied upon a recently described artificial intelligence (ai)-guided definition of the nature of the overzealous response in fatal covid- (sahoo et al., ) . using ace as a seed gene, a -gene signature was identified and validated as an invariant immune response that was shared among all respiratory viral pandemics, including covid- (fig a) . a subset of genes within the -gene signature was subsequently identified as a determinant of disease severity/fatality; these genes represented translational arrest, senescence, and apoptosis. these two signatures referred to as vip ( -gene) and severe vip ( -gene) signatures, were used as a computational framework to first vet existing sars-cov- infection models that have been commonly used for therapeutic screens (fig b-d) . surprisingly, we found that each model fell short in one way or another. for example, the vero e , which is a commonly used cultured cell model, showed a completely opposite response; instead of being induced, both the -gene and -gene vip signatures were suppressed in infected vero e monolayers (fig b) . similarly, neither vip signature was induced in the case of sars-cov- challenged human bronchial organoids(suzuki et al., ) ( fig c) . finally, in the case of the hipsc-derived at / organoids, which recapitulated the covid- -lung derived immune signatures (in fig f) , the -gene vip signature was induced significantly (fig d; top) , but the -gene severity signature was not (fig d; bottom) . these findings show that none of the existing models capture the overzealous host immune response that has been implicated in a fatality. our lung models showed that both the -and -gene vip signatures were induced significantly in the submerged alo-derived monolayers that had distal differentiation (fig e; left), but not in the proximal-mimic ali model (fig e; right) . neither signatures were induced in monolayers of small airway epithelial cells (fig f) or hipsc-derived at cells (fig g) . taken together with our infectivity analyses, these findings demonstrate that although the proximal airway epithelia and at cells may be infected, and as described by others (dye et al., ; hou et al., ) , may be vital for mounting a viral response and for disease transmission, these cells alone cannot mount the overzealous host immune response that is associated with the fatal disease. similarly, even though the alveolar pneumocytes, at and at cells, are sufficient to mount the host immune response, in the absence of proximal airway components, they too are insufficient to recapitulate the severe vip signature that is characterized by cellular senescence and apoptosis. however, when both proximal and distal components are present, i.e., basal, ciliated and at cells, the model mimicked the overzealous host immune response in covid- (fig h) . the most important discovery we report here is the creation of adult lung organoids that are complete with both proximal airway and distal alveolar epithelia; these organoids can not only be stably propagated and expanded in d cultures but also used as monolayers of mixed cellularity for modeling viral and host immune responses during respiratory viral pandemics. furthermore, an objective analysis of this model and other existing sars-cov- -infected lung models against patient-lung derived transcriptomes showed that the model which most closely emulates the elements of viral infectivity, lung injury, and inflammation in covid- is one that contained both proximal and distal alveolar signatures (fig h) , whereas, the presence of just one or the other fell short. there are three important impacts of this work. first, successful modeling of the human lung organoids that are complete with both proximal and distal signatures has not been accomplished before. the multicellularity of the lung has been a daunting challenge that many experts have tried to recreate in vitro; in fact, the demand for perfecting such a model has always remained high, not just in the current context of the covid- pandemic but also with the potential of future pandemics. we have provided the evidence that the organoids that were created using our methodology retain proximal and distal cellularity throughout multiple passages and even within the same organoid. although a systematic design of experiment (doe) approach (bukys et al., ) was not involved in getting to this desirable goal, a rationalized approach was taken. for example, a wnt/rspondin/noggin-containing conditioned media was used as a source of the so-called 'niche factors' for any organoid growth (sato and clevers, ) . this was supplemented with recombinant fgf / ; fgf is known to help cell proliferation and differentiation and is required for normal branching morphogenesis (padela et al., ) , whereas fgf helps in cell maturation (rabata et al., ) and in alveolar regeneration upon injury (yuan et al., ) . together, they are likely to have directed the differentiation toward distal lung lineages (hence, the preservation of alveolar signatures). the presence of both distal alveolar and proximal ciliated cells was critical: proximal cells were required to recreate sustained viral infectivity, and the distal alveolar pneumocytes, in particular, the ability of at cells to differentiate into at pneumocytes was essential to recreate the host response. it is possible that the response is mediated by a distinct at -lineage population, i.e., damageassociated transient progenitors (datps), which arise as intermediates during at →at differentiation upon injury-induced alveolar regeneration (choi et al., ) . although somewhat unexpected, the role of at pneumocytes in mounting innate immune responses has been documented before in the context of bacterial pneumonia (wong and johnson, ; yamamoto et al., ) . in work (huang et al., ) that was published during the preparation of this manuscript, authors used long-term ali models of hipsc-derived at monolayers (in growth conditions that inhibit at →at differentiation, as we did here for our at model) and showed that sars-cov- induces iat -intrinsic cytotoxicity and inflammatory response, but failed to induce type interferon pathways (ifn α/β). it is possible that prolonged culture of iat pneumocytes gives rise to some datps but cannot robustly do so in the presence of inhibitors of at differentiation. this (spatially segregated viral and host immune response) is a common theme across many lung infections (including bacterial pneumonia and other viral pandemics (chan et al., ; hou et al., ; taubenberger and morens, ; weinheimer et al., ) and hence, this mixed cellularity model is appropriate for use in modeling diverse lung infections and respiratory pandemics to come. second, among all the established lung models so far, ours features key properties that are desirable whenever disease models are being considered for their use in htp modes for rapid screening of candidate therapeutics and vaccines -(i) reproducibility, propagability and scalability, (ii) cost-effectiveness, (iii) personalization, and (iv) modularity, with the potential to be used in multi-dimensional complex lung models with other cell types if/when needed. we showed that the protocol we have optimized supports isolation, expansion and propagability at least up to - passages (at the time of submission of this work), with documented retention of proximal and distal airway components up to p (by rna seq). feasibility has also been established for scaling up for use in -well htp assays. we also showed that the protocols for generating lung organoids could be reproduced in both genders and regardless of the donor's smoking status, consistency in outcome and growth characteristics were observed across all isolation attempts. the alos are also cost-effective; the need for exclusive reliance on recombinant growth factors was replaced at least in part with conditioned media from a commonly used cell line (l-wrn/ atcc® crl- cells). such media has a batch to batch stable cocktail of wnt, r-spondin, and noggin and has been shown to improve reproducibility in the context of gi organoids in independent laboratories (vandussen et al., ) . the use of conditioned media may not only have made our model more cost-effective than others but also likely improved the rigor and reproducibility in lung modeling and research. because the model is propagable, repeated ipsc-reprogramming (another expensive step) is also eliminated, further cutting costs compared to many other models. as for personalization, our model is derived from adult lung stem cells from deep lung biopsies; each organoid line was established from one patient. by avoiding ipscs or epscs, this model not only captures genetics but also retains organ-specific epigenetic programming in the lung, and hence, potentially additional programming that may occur in disease (such as in the setting of chronic infection, injury, inflammation, somatic mutations, etc.). the ability to replicate donor phenotype and genotype in vitro allows for potential use as pre-clinical human models for phase ' ' clinical trials. as for modularity, by showing that the d lung organoids could be used as polarized monolayers on transwells to allow infectious agents to access the apical surface (in this case, sars-cov- ), we demonstrate that the organoids have the potential to be reverse-engineered with additional components in a physiologically relevant spatially segregated manner: for example, immune and stromal cells can be placed in the lower chamber to model complex lung diseases that are yet to be modeled and have no cure (e.g., idiopathic pulmonary fibrosis, etc). third, the value of the alo models is further enhanced due to the availability of companion readouts/ biomarkers (e.g., vip signatures in the case of respiratory viral pandemics, or monitoring the e gene, or viral shedding, etc.) that can rapidly and objectively vet treatment efficacy based on set therapeutic goals. of these readouts, the host response, as assessed by vip signatures, is a key vantage point because an overzealous host response is what is known to cause fatality. recently, a systematic review of the existing pre-clinical animal models revealed that most of the animal models of covid- recapitulated mild patterns of human covid- ; no severe illness associated with mortality was observed, suggesting a wide gap between covid- in humans (spagnolo et al., ) and animal models (ehaideb et al., ) . the model revealed here, in conjunction with the vip signatures described earlier (sahoo et al., ) , could serve as a pre-clinical model with companion diagnostics to identify drugs that target both the viral and host response in pandemics. milewska, a., kula-pacurar, a., wadas, j., suder, a., szczepanski, a., dabrowska, a., owczarek, k., marcello, a., ochman, m., stacel, t., et al. ( ) . replication of severe acute respiratory syndrome coronavirus in human respiratory epithelium. j virol . sayed, i.m., suarez, k., lim, e., singh, s., pereira, m., ibeawuchi, s.r., katkar, g., dunkel, y., mittal, y., chattopadhyay, r., et al. ( ) . host engulfment pathway controls inflammation in inflammatory bowel disease. zhu, n., wang, w., liu, z., liang, c., wang, w., ye, f., huang, b., zhao, l., wang, h., zhou, w., et al. ( ) . morphogenesis and cytopathic effect of sars-cov- infection in human airway epithelial cells. nat commun , . whisker plots display relative levels of ace expression in various cell types in the normal human lung. the cell types were annotated within a publicly available single-cell sequencing dataset (gse ) using genes listed in table s . p values were analyzed by one-way anova and tukey post hoc test. b. ffpe sections of the human lung from normal and deceased covid- patients were stained for sftpc, alone or in combination with to generate adult healthy lung organoids, fresh biopsy bites were prospectively collected after surgical resection of the lung by the cardiothoracic surgeon. before collection of the lung specimens, each tissue was sent to a gross anatomy room where a pathologist cataloged the area of focus, and the extra specimens were routed to the research lab in human transport media (htm, advanced dmem/f- , mm hepes, x glutamax, x penicillin-streptomycin, m y- ) for cell isolation. deidentified lung tissues obtained during surgical resection, that were deemed excess by clinical pathologists, were collected using an approved human research protocol (irb# ; pi: thistlethwaite). isolation and biobanking of organoids from these lung tissues were carried out using an approved human research protocol (irb# : pi ghosh and das) that covers human subject research at the uc san diego humanoid center of research excellence (core). for all the deidentified human subjects, information including age, gender, and previous history of the disease, was collected from the chart following the rules of hipaa and described in the table. a portion of the same lung tissue specimen was fixed in % zinc-formalin for at least hrs followed by submersion in % etoh until embedding in ffpe blocks. the lung specimens from covid- positive human subjects were collected through autopsy (the study was irb exempt). all donations to this trial were obtained after telephone consent followed by written email confirmation by the next of kin/power of attorney per california state law (no in-person visitation could be allowed into our covid- icu during the pandemic). the team member followed the cdc guidelines for covid and the autopsy procedures ((cap), ; (cdc), )). lung specimens were collected in % zinc-formalin and stored for hrs before processing for histology. patient characteristics are listed in the table. autopsy # was a standard autopsy performed by anatomical pathology in the bsl autopsy suite. the patient expired and his family consented for autopsy. after hrs, the lungs were removed and immersion fixed whole in % formalin for hrs and then processed further. lungs were only partially fixed at this time (about % fixed in thicker segments) and were sectioned into small - cm chunks and immersed in % formalin for further investigation. autopsy # and # were collected from rapid post-mortem lung biopsies. the procedure was performed in the jacobs medical center icu (all of the icu rooms have a pressure-negative environment, with air exhausted through hepa filters [biosafety level (bsl )] for isolation of sars-cov- virus). biopsies were performed - hrs after patient expiration. the ventilator was shut off to reduce the aerosolization of viral particles at least hr after the loss of pulse and before sample collection. every team member had personal protective equipment in accordance with the university policies for procedures on patients with covid- (n mask + surgical mask, hairnet, full face shield, surgical gowns, double surgical gloves, booties). lung biopsies were obtained after l-thoracotomy in the th intercostal space by the cardiothoracic surgery team. samples were taken from the left upper lobe (lul) and left lower lobe (lll) and then sectioned further. a previously published protocol was modified to isolate lung organoids from human subjects (sachs et al., ; zhou et al., ) . briefly, normal human lung specimens were washed with pbs/ x penicillinstreptomycin and minced with surgical scissors. tissue fragments were resuspended in ml of wash buffer (advanced dmem/f- , mm hepes, x glutamax, x penicillin-streptomycin) containing mg/ml collagenase type i (thermo fisher, usa) and incubated at °c for approximately hr. during incubation, tissue pieces were sheared every min with a ml serological pipette and examined under a light microscope to monitor the progress of digestion. when - % of single cells were released from connective tissue, the digestion buffer was neutralized with ml wash buffer with added % fetal bovine serum; the suspension was passed through a -µm cell strainer and centrifuged at rcf. remaining erythrocytes were lysed in ml red blood cell lysis buffer (invitrogen) at room temperature for min, followed by the addition of ml of wash buffer and centrifugation at rcf. cell pellets were resuspended in cold matrigel (corning, usa) and seeded in µl droplets on a well tissue culture plate. the plate was inverted and incubated at °c for min to allow complete polymerization of the matrigel before the addition of ml lung expansion medium per well. lung expansion media was prepared by modifying the gi-organoid media ( % conditioned media, prepared from l-wrn cells with wnt a, r-spondin, and noggin, atcc-crl- tm ) (ghosh et al., ; sayed et al., a; sayed et al., b; sayed et al., c) with a proprietary cocktail from the humanoid core containing b , tgf-β receptor inhibitor, antioxidants, p mapk inhibitor, fgf , fgf and rock inhibitor. the lung expansion media was compared to alveolosphere media i (imdm and f as the basal medium with b , low concentration of kgf, monothioglycerol, gsk inhibitor, ascorbic acid, dexamethasone, ibmx, camp and rock inhibitor) and ii (f as the basal medium with added cacl , b , low concentration of kgf, gsk inhibitor, tgf-β receptor inhibitor dexamethasone, ibmx, camp and rock inhibitor) modified from previously published literature (jacob et al., ; yamamoto et al., ) . neither alvelosphere media contain any added wnt a, r-spondin, and noggin. the composition of these media was developed either by fundamentals of adultstem cell-derived mixed epithelial cellularity in other organs (like the gastrointestinal tract (miyoshi and stappenbeck, ; sato et al., ; sayed et al., c) , or rationalized based on published growth conditions for proximal and distal airway components (gotoh et al., ; sachs et al., ; van der vaart and clevers, ) . organoids were maintained in a humidified incubator at °c/ % co , with a complete media change performed every days. after the organoids reached confluency between - days, organoids were collected in pbs/ . mm edta and centrifuged at rcf for min. organoids were dissociated in ml tryple select (gibco, usa) per well at °c for - min and mechanically sheared. wash buffer was added at a : , tryple to wash buffer ratio. the cell suspension was subsequently centrifuged, resuspended in matrigel, and seeded at a : ratio. lung organoids were biobanked and passage - cells were used for experiments. subculture was performed every - days. lung-organoid-derived monolayers were prepared using a modified protocol of gi-organoid-derived monolayers (ghosh et al., ; sayed et al., a; sayed et al., b; sayed et al., c) . briefly, transwell inserts ( . mm diameter, . um pore size, corning) were coated in matrigel diluted in cold pbs at a : ratio and incubated for hr at room temperature. confluent organoids were collected in pbs/edta on day and dissociated into single cells in tryple for - min at °c. following enzymatic digestion, the cell suspension was mechanically sheared through vigorous pipetting with a µl pipette and neutralized with wash buffer. the suspension was human ipsc-derived alveolar epithelial type cells (ihaepc ) were obtained from cell applications inc. and cultured in growth media (i k- , cell applications inc.) according to the manufacturer's instructions. all the primary cells were used within early passages ( - ) to avoid any gradual disintegration of the airway epithelium with columnar epithelial structure and epithelial integrity. lung organoid-derived monolayers or primary airway epithelial cells either in well plates or in transwells were washed twice with antibiotic-free lung wash media. e pfu of sars-cov- strain usa-wa / (bei resources nr- ) in complete dmem was added to the apical side of the transwell and allowed to incubate for , , and hrs at °c and % co . after incubation, the media was removed from the basal side of the transwell. the apical side of the transwells was then washed twice with (antibiotic-free lung wash media) and then twice with pbs. trizol™ reagent (thermo fisher ) was added to the well and incubated at ˚c and % co for min. the trizol™ reagent was removed and stored at - ˚c for rna analysis. organoids and monolayers used for lung cell type studies were lysed using rna lysis buffer followed by rna cov studies were lysed in tri-reagent and rna was extracted using zymo research direct-zol rna miniprep. organoid and monolayer cell-type gene expression was measured by qrt-pcr using x sybr green qpcr master mix. cdna was amplified with gene-specific primer/probe set for the lung cell type markers and qscript cdna supermix ( x). qrt-pcr was performed with the applied biosystems quantstudio real-time pcr system. cycling parameters were as follows: °c for s, followed by cycles of s at °c and s at °c. all samples were assayed in triplicate and eukaryotic s ribosomal rna was used as a reference. itga itga f 'cgaaaccaaggttctgagccc' itga r 'cttggatctccactgaggcagt' goblet muc ac muc ac f ' ggaactgtggggacagctctt' muc ac r ' gtcacattcctcagcgaggtc' cilia foxj foxj f 'actcgtatgccacgctcatctg' foxj r 'gagacaggttgtggcggattga' club cell scgb a scgb a f ' caaaagcccagagaaagcatc' scgb a r ' cagttggggatcttcagcttc' alveolar type aqp , pdpn, p rx aqp f ' tacggtgtggcaccgctcaatg' aqp r ' agtcagtggaggcgaagatgca' pdpn f ' gtgccgaagatgatgtggtgac' pdpn r ' ggactgtgctttctgaagttggc' p rx f ' gtggcggattatgtgataccagc' p rx r ' cacacagtggtcgcatctggaa' alveolar type abca , sftpa , sftpc, lamp abca f ' cttgacagtcgcagagcacctt' abca r ' ctccgtgagttccacttgtcct' sftpa f ' cacctggagaaatgccatgtcc' sftpa r ' aagtcgtggagtgtggcttgga' sftpc f ' gtcctcatcgtcgtggtgattg' sftpc r ' agaaggtggcagtggtaaccag' lamp f ' tgggagcctatttgaccgtctc' lamp r ' gctgacaactggaggctctgtt' assessment of sars-cov- infectivity test was determined by qpcr using taqman assays and taqman universal pcr master mix as done before (corman et al., ; lamers et al., ) . cdna was amplified with gene-specific primer/probe set for the e gene and qpcr was performed with the applied biosystems rrna. cycling parameters were as follows: °c for s, followed by cycles of s at °c and s at °c. all samples were assayed in triplicate and gene eukaryotic s ribosomal rna was used as a reference. organoids and lung organoid-derived monolayers were fixed by either: ( ) % pfa at room temperature for min and quenched with mm glycine for min, ( ) ice-cold % methanol at - °c for min, ( ) ice-cold % methanol on ice for min. subsequently, samples were permeabilized and blocked for hrs using an inhouse blocking buffer ( mg/ml bsa and . % triton x- in pbs); as described previously (lopez-sanchez et al., ) . primary antibodies were diluted in blocking buffer and allowed to incubate overnight at °c; secondary antibodies were diluted in blocking buffer and allowed to incubate for hrs in the dark. antibody dilutions are listed in the supplementary key resource table. prolong glass was used as a mounting medium. # thick coverslips were applied to slides and sealed. samples were stored at °c until imaged. ffpe embedded organoid and lung tissue sections underwent antigen retrieval as previously described in methods for immunohistochemistry staining. after antigen retrieval and cooling in di water; samples were permeabilized and blocked in blocking buffer and treated as mentioned above for immunofluorescence. images were acquired at room temperature with leica tcs spe confocal and with dmi b microscope using the leica las-af software. images were taken with a × oil-immersion objective using -, -, -nm laser lines for excitation. z-stack images were acquired by successive z-slices of µm in the desired confocal channels. fields of view that were representative and/or of interest were determined by randomly imaging different fields. zslices of a z-stack were overlaid to create maximum intensity projection images; all images were processed using fiji (image j) software. organoids were seeded on a layer of matrigel in -well plates and grown for - days. once mature, organoids were fixed in % pfa at room temperature for min and quenched with mm glycine for min. organoids were gently washed with pbs and harvested using a cell scraper. organoids were resuspended in pbs using wide-bore µl pipette tips. organoids were stained using gill's hematoxylin for min for easier ffpe embedding and sectioning visualization. hematoxylin stained organoids were gently washed in pbs and centrifuged and excess hematoxylin was aspirated. organoids were resuspended in °c histogel and centrifuged at °c for min. histogel embedded organoid pellets were allowed to cool to room temperature and stored in % ethanol at °c until ready for ffpe embedding by lji histology core. successive ffpe embedded organoid sections were cut at a µm thickness and fixed on to microscope slides. for sars cov-nucleoprotein (np) antigen retrieval, slides were immersed in tris-edta buffer (ph . ) and boiled for min at °c inside a pressure cooker. endogenous peroxidase activity was blocked by incubation with % h o for minutes. to block non-specific protein binding . % goat serum was added. tissues were then incubated with a rabbit sars cov-np antibody (sino biological, see supplementary key resource table) for . hrs at room temperature in a humidified chamber and then rinsed with tbs or pbs times, for min each. sections were incubated with horse anti-rabbit igg secondary antibodies for min at room temperature and then washed with tbs or pbs times for min each. sections were incubated with dab and counterstained with hematoxylin for sec. annotations were computed using tximport and biomart r package. a custom python script was used to organize the data and log reduced using log (tpm+ ). the raw data and processed data are deposited in gene expression omnibus under accession no gse , and gse . publicly available covid- gene expression databases were downloaded from the national center for biotechnology information (ncbi) gene expression omnibus website (geo) (barrett et al., ; barrett et al., ; edgar et al., ) . if the dataset is not normalized, rma (robust multichip average) (irizarry et al., a; irizarry et al., b) is used for microarrays and tpm (transcripts per millions) (li and dewey, ; pachter, ) is used for rnaseq data for normalization. we used log (tpm+ ) to compute the final log-reduced expression values for rnaseq data. accession numbers for these crowdsourced datasets are provided in the figures and manuscript. all of the above datasets were processed using the hegemon data analysis framework (dalerba et al., ; dalerba et al., ; volkmer et al., ) . deseq (love et al., ) was applied to uninfected and infected samples to identify up-and down-regulated genes. a gene signature score is computed using both the up-and down-regulated genes which are used to order the sample. to compute the gene signature score, first, the genes present in this list were normalized according to a modified z-score approach centered around stepminer threshold (formula = (expr -sthr)/ *stddev (fabregat et al., ) . reactome identifies signaling and metabolic molecules and organizes their relations into biological pathways and processes. violin, swarm and bubble plots are created using python seaborn package version . . . single cell rnaseq data from gse was downloaded from geo in the hdf feature barcode matrix format. the filtered barcode data matrix was processed using seurat v r package (stuart et al., ) and scanpy python package (wolf et al., ) . pseudo bulk analysis of gse data was performed by adding counts from the different cell subtypes and normalized using log (cpm+ ). epithelial cells were identified using sftpa , sftpb, ager, aqp , sftpc, scgb a , krt , cyp f , ccdc , and tppp genes using scina algorithm (zhang et al., ) . pseudo bulk datasets were prepared by adding counts from the selected cells and normalized using log (cpm+ ). cibersortx (https://cibersortx.stanford.edu/runcibersortx.php) was used for cell-type deconvolution of our dataset (which was normalized by cpm). as reference samples, we first used the single cell rnaseq dataset (gse ) from gene expression omnibus (geo). next, we analyzed the bulk rna seq datasets for the identification of cell types of interest using relevant gene markers (see table (ace ,tmprss ) were identified using scina algorithm. then, normalized pseudo counts were obtained with the cpm normalization method. the cell-type signature matrix was derived from the single cell rnaseq dataset, cell-types, and gene markers of interest. it was constructed by taking an average from gene expression levels for each of the markers across each cell type. all experiments were repeated at least three times, and results were presented either as one representative experiment or as average ± s.e.m. statistical significance between datasets with three or more experimental groups was determined using one-way anova including a tukey's test for multiple comparisons. for all tests, a p-value of . was used as the cutoff to determine significance (*p < . , **p < . , ***p < . , and ****p < . ). all experiments were repeated a least three times, and p-values are indicated in each figure. all statistical analyses were performed using graphpad prism . . a part of the statistical tests was performed using r version . . ( - - ). standard t-tests were performed using python scipy.stats.ttest_ind package (version . . ). marker used in this work for immunofluorescence (if) marker used in this work for qpcr obscure markers (not a lot of research relative to lung) brca cybb krt c qb fcgr a il il cd cd xage b ccr ccr alox b cmklr mx tnfrsf ccr cxcr cdk gbp hla-g ido isg lag mad l cxcl mki snai ifitm gzmb cd cd bst bub ccl ccnb cxcl ifi ifi tdo gzma oas pou af cxcl gnly dmbt ddx tnfaip lamp kiaa melk slamf il foxm ifih ifi pdcd lg ifit ifit cxcl irf psmb ccl tnfsf isg cdkn c qa oas oas ifit top a lilrb herc tnfsf b ifi l stat collection and submission of post-mortem specimens from deceased persons with known or suspected covid- ncbi geo: mining millions of expression profiles--database and tools ncbi geo: archive for functional genomics data sets--update detection of novel coronavirus ( -ncov) by real-time rt-pcr single-cell dissection of transcriptional heterogeneity in human colon tumors cdx as a prognostic biomarker in stage ii and stage iii colon cancer gene expression omnibus: ncbi gene expression and hybridization array data repository the reactome pathway knowledgebase the stress polarity signaling (sps) pathway serves as a marker and a target in the leaky gut barrier: implications in aging and cancer generation of alveolar epithelial spheroids via isolated progenitor cells from human pluripotent stem cells summaries of affymetrix genechip probe level data exploration, normalization, and summaries of high density oligonucleotide array probe level data derivation of self-renewing lung alveolar epithelial type ii cells from human pluripotent stem cells sars-cov- productively infects human gut enterocytes rsem: accurate transcript quantification from rna-seq data with or without a reference genome giv/girdin is a central hub for profibrogenic signalling networks during liver fibrosis moderated estimation of fold change and dispersion for rna-seq data with deseq in vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture models for transcript quantification from rna-seq long-term expanding human airway organoids for disease modeling single lgr stem cells build crypt-villus structures in vitro without a mesenchymal niche the dna glycosylase neil suppresses fusobacterium-infection-induced inflammation and dna damage in colonic epithelial cells helicobacter pylori infection downregulates the dna glycosylase neil , resulting in increased genome damage and inflammation in gastric epithelial cells host engulfment pathway controls inflammation in inflammatory bowel disease comprehensive integration of single-cell data three differentiation states risk-stratify bladder cancer into distinct subtypes scanpy: large-scale single-cell gene expression data analysis long-term expansion of alveolar stem cells derived from human ips cells in organoids scina: a semi-supervised subtyping algorithm of single cells and bulk samples differentiated human airway organoids to assess infectivity of emerging influenza virus name pvalue fdr interferon signaling . e- . e- interferon alpha/beta signaling . e- . e- cytokine signaling in immune system . e- . e- immune system . e- . e- interleukin- signaling . e- . e- interferon gamma signaling . e- . e- chemokine receptors bind chemokines . e- . e- signaling by interleukins . e- . e- insulin-like growth factor- mrna binding proteins (igf bps/imps/vickzs) bind rna ifi epsti amigo ifitm slc a cmpk wars faap apol oasl ifi isg oas ifi l cd slc f ifit ifi samd l ifit parp srp p a. whisker plots display relative levels of ace expression in various cell types in the normal human lung. the cell types were annotated within a publicly available single-cell sequencing dataset (gse ) using genes listed in table s . a. schematic lists the various markers used here for qpcr and immunofluorescence to confirm the presence of all cell types in the d lung organoids here and in d monolayers later (in fig ) . b-h. bar graphs display the relative abundance of various cell type markers (normalized to s) in adult lung organoids (alo), compared to the airway (nhbe) and/or alveolar (at ) control cells, as appropriate. p-values were analyzed by one-way anova. error bars denote s.e.m; n = - datasets. i. d organoids grown in -well chamber slides were fixed, immunostained and visualized by confocal microscopy, as in fig k. scale bar = µm. c. monolayers of alo - were challenged with sars-cov- for indicated time points prior to fixation and staining for krt (red) and viral nucleocapsid protein (green) and dapi (blue; nuclei) and visualized by confocal microscopy. representative images are shown, displaying various cytopathic effects. scale bar = µm. publicly available rna seq datasets (gse ) of lung autopsies from patients who were deceased due to covid- or noninfectious causes (normal lung control) were analyzed for differential expression of genes and displayed as a heatmap. figure . reactome pathway analysis of differentially expressed genes in lung autopsies (normal vs. covid- ). reactome-pathway analysis of the differentially expressed genes shows the major pathways upregulated in covid- -affected lungs. top: visualization as flattened (left) and hierarchical (right, insets) reactome. bottom: visualization of the same data as tables with statistical analysis indicative of the degree of pathway enrichment. figure . differential expression analysis of rna seq datasets from lung organoid monolayers, infected or not, with sars-cov- . adult lung organoid monolayers infected or not with sars-cov- were analyzed by rna seq and differential expression analysis. differentially expressed genes are displayed as a heatmap. figure . reactome pathway analysis of differentially expressed genes in lung organoid monolayers infected with sars-cov- . reactome-pathway analysis of the differentially expressed genes shows the major pathways upregulated in sars-cov- -infected lung organoid monolayers. top: visualization as flattened (left) and hierarchical (right, insets) reactfoam. bottom: visualization of the same data as tables with statistical analysis indicative of the degree of pathway enrichment. key: cord- - tq p z authors: wang, ting; yegambaram, manivannan; gross, christine; sun, xutong; lu, qing; wang, hui; wu, xiaomin; kangath, archana; tang, haiyang; aggarwal, saurabh; black, stephen m. title: rac nitration at y is involved in the endothelial barrier disruption associated with lipopolysaccharide-mediated acute lung injury date: - - journal: nan doi: . /j.redox. . sha: doc_id: cord_uid: tq p z acute lung injury (ali), a devastating illness induced by systemic inflammation e.g., sepsis or local lung inflammation e.g., covid- mediated severe pneumonia, has an unacceptably high mortality and has no effective therapy. ali is associated with increased pulmonary microvascular hyperpermeability and alveolar flooding. the small rho gtpases, rhoa and rac are central regulators of vascular permeability through cytoskeleton rearrangements. rhoa and rac have opposing functional outcome: rhoa induces an endothelial contractile phenotype and barrier disruption, while rac stabilizes endothelial junctions and increases barrier integrity. in ali, rhoa activity is increased while rac activity is reduced. we have shown that the activation of rhoa in lipopolysaccharide (lps)-mediated ali, is dependent, at least in part, on a single nitration event at tyrosine (y) . thus, the purpose of this study was to determine if the inhibition of rac is also dependent on its nitration. our data show that rac inhibition by lps is associated with its nitration that mass spectrometry identified as y , within the switch i region adjacent to the nucleotide-binding site. using a molecular modeling approach, we designed a nitration shielding peptide for rac , designated nipr (nitration inhibitor peptide for the rho gtpases ), which attenuated the lps-induced nitration of rac at y , preserves rac activity and attenuates the lps-mediated disruption of the endothelial barrier in human lung microvascular endothelial cells (hlmvec). using a murine model of ali induced by intratracheal installation of lps we found that nipr successfully prevented rac nitration and rac inhibition, and more importantly attenuated pulmonary inflammation, reduced lung injury and prevented the loss of lung function. together, our data identify a new post-translational mechanism of rac inhibition through its nitration at y . as nipr also reduces sepsis induced ali in the mouse lung, we conclude that rac nitration is a therapeutic target in ali. the current pandemic of covid- has caused over a hundred and sixty thousand deaths in usa, mainly due to a severe form of lung inflammation and edema, called acute lung injury (ali) or its severe form acute respiratory distress syndrome (ards) [ ] . ali and ards represent the same disease spectrum characterized by hypoxemic respiratory insufficiency from pulmonary edema [ ] . the acute lung inflammation and increased vascular hyperpermeability associated with ali can be caused by systemic inflammation such as sepsis, or lung inflammation such as severe pneumonia induced by covid- or other bacteria or viruses [ ] . besides supportive care with low tidal volume mechanical ventilation, general anti-inflammatory steroids, and extracorporeal membrane oxygenation (ecmo) in the intensive care unit (icu), there are no specific drug therapies for ali/ards, which still has an unacceptably high mortality of ~ % [ ] . thus, a devastating demand for effective therapies for ali/ards exists. respective primary antibody in blocking buffer, and the secondary antibody in blocking buffer after three time of wash in tbst. reactive bands were visualized using chemiluminescence (pierce) using either a kodak cf image station or a li-cor biosciences odyssey imaging system. band intensity was quantified using either kodak d image processing software or image studio software version . hlmvec were homogenized in pierce ip lysis buffer (thermo-fisher). after centrifugation, the supernatant was collected and mixed with a/g agarose beads (emd/calbiochem) with the pull-down antibody for h at ºc. the beads were collected after centrifugation at , rpm for min and washed times with ip wash buffer (thermo-fisher). the beads were then boiled with x laemmli buffer for min. proteins were separated on % gels and transferred to pvdf membrane and analyzed using western blot methods described above. in another experiment, immunoprecipitated proteins in gel stained with coomassie blue dye were digested with chymotrypsin and used for mass spectrometry. rac activity was measured using the rac pull-down activation assay biochem kit (cytoskeleton inc.). briefly, hlmvec and mouse lung tissue were lysed using the cell lysis buffer and the lysate with pak-pbd beads was incubated at °c on a rotator for h. the pak-pbd beads were centrifuged at , g and washed with wash buffer. equal j o u r n a l p r e -p r o o f volume of x laemmli sample buffer was added to each tube and boiled for min. samples were analyzed by sds-page and western blot analysis as described above. all spectra were taken on an ab sciex maldi-tof-tof mass spectrometer in positive reflector mode ( kv) with a matrix of chca. masses were calibrated to sciex nitration, we exposed nipr to authentic peroxynitrite ( μm) for min. samples were desalted using a c- ziptip, mixed with chca matrix and were spotted directly onto a maldi plate for further ms and ms/ms analysis. the ability of nipr or nipr f peptides to bind to rac was also analyzed by mixing ng/ml of peptide with ng/ml of human recombinant rac protein for min at rt and using maldi ms linear mode acquisition with sinapinic acid as a matrix. a nitro-y rac specific antibody (rac -y -no ) was raised against a synthetic peptide antigen (llisyttnafpgey no iptvfd), where y-no represents -nitrotyrosine as previously described [ ] . the peptide was used to immunize rabbits. tyrosine nitration-reactive rabbit antiserum was first purified by affinity chromatography. further purification was carried out using immunodepletion using non-nitrated peptide (llisyttnafpgeyiptvfd) resin chromatography, after which the resulting eluate was tested for antibody specificity by immunoblotting and immunoprecipitation followed by mass spectrometry. the rhoa-y -no antibody was generated as previously described [ ] . j o u r n a l p r e -p r o o f his-tagged rac recombinant protein was expressed in e. coli as previously described [ ] . briefly, isopropyl-beta-d-thiogalactopyranoside (iptg, mm) was added and the cells were incubated for - h at °c. bacteria were then harvested by centrifugation and the pellet was immediately lysed in mm tris-hcl, % glycerol, mg/ml lysozyme, mm nacl, protease inhibitor cocktail, ribonuclease a (sigma), and deoxyribonuclease i (sigma). the pellet was gently rocked for minutes, sonicated and subjected to ultracentrifugation. the supernatant was loaded onto a hisprep ff / column (sigma) using binding buffer ( mm tris-hcl, mm nacl, % glycerol, mm imidazole) at . ml/min flow. the column was washed with mm tris-hcl, mm nacl, % glycerol, mm imidazole using a flow rate of . ml/min. elution of the histidine-tagged protein was accomplished using elution buffer ( mm tris-hcl, mm nacl, % glycerol, mm imidazole) at . ml/min flow. collected fractions were loaded for size-exclusion gel filtration on a hiload / superdex column (sigma) using gel filtration buffer ( mm tris-hcl, mm nacl, % glycerol) at . ml/min flow. fractions were collected and analyzed by coomassie blue staining and western blot. all purification steps were performed at °c, and purified protein was stored at - °c. recombinant human rhoa protein was also purified using a bacterial expression system as previously described [ ] . j o u r n a l p r e -p r o o f pulmonary arterial endothelial cells (paec) were cultured as described [ ] . ovine paec, isolated as previously described [ ] , were transduced with a his-tagged rac adenovirus (moi = ) and incubated for h at °c. cells were lysed and the histagged rac protein was purified using hispur ni-nta columns (thermo scientific) and stored at - °c. fractions were analyzed by coomassie blue staining and western blot analysis. peptides containing amino acids (fpgeyiptvf) from aa - of rac fused with the cell permeable tat sequence were synthesized by peptide . inc (chantilly, va). this peptide was designated nipr (nitration inhibitory peptide for the rho gtpases ) and designed to interact with the flap region of the rac protein. a similar phenylalanine-substituted peptide (nipr f) was also synthesized harboring a y f mutation. the electrical resistance of the endothelial cell monolayer was measured with the electrical cell impedance sensor (ecis) technique [ ] . hlmvec were cultured on gold plated electrodes ( e +) until % confluence. the change in electric resistance across the monolayer was measured continuously. the data was normalized to the initial values of basal resistance. in the transwell permeability assay, hlmvec were seeded on a semi-permeable membrane in the upper chamber of the transwell ( μm pore size, bd biosciences, san jose, ca). after appropriate treatments, , mw fitc-dextran j o u r n a l p r e -p r o o f ( μg/μl) (sigma-aldrich) was added to the upper chamber. the amount of fitc-dextran infiltrating into the lower chamber was determined using a fluoroskan ascent fluorometer. the committee on animal research at georgia regents university and university of arizona approved all animal protocols and procedures. in the pre-injury model, male c bi/ mice ( weeks) received vehicle (saline) or mntmpyp ( mg/kg body weight) via an intraperitoneal injection min before intratracheal installation of e. coli :b lps, . x endotoxin units/g body weight, sigma-aldrich). mice were examined h after lps challenge. at the end of the treatment, animals were anesthetized, and the lungs were flushed with ml of ice-cold pbs (to remove blood) gently, and excised. a portion of the lung was quickly snap-frozen in liquid nitrogen and stored at - ºc. in the post-injury model, male c bi/ mice ( - weeks) received vehicle ( . % saline) or e. coli :b lps ( . mg/kg body wt, sigma-aldrich) prepared in . % saline via an intratracheal injection days before administration of nipr or nipr f peptide (intraperitoneal injection, mg/kg). on day (post lps) mice were euthanized, lungs were flushed with ml of ice-cold pbs (to remove blood) gently, and excised. a portion of the lung was quickly snap-frozen in liquid nitrogen and stored at - ºc. the remaining lung tissue was fixed in pbs buffered formalin for histology analysis. j o u r n a l p r e -p r o o f balf was obtained by instilling and withdrawing ml of ice-cold hanks buffer (sigma) via a tracheal cannula. bal fluid was then centrifuged g for min to collect pelleted cells. bal cells were then resuspended in red cell lysis buffer (rclb) for s, centrifuged g for min to remove rclb, and resuspended in µl of pbs. total cell count was then determined using a haemocytometer. cell suspension ( µl) was also separated by cytospin centrifuge (thermo-fisher) g for min onto glass slides. after air dry, the slides were stained with dif-quik staining (vwr) for differential cell count. lungs were inflated with % formalin under cm h o pressure and immersed in the same solution before tissue processing into paraffin-embedded blocks and μm sections were then cut stained with h&e. histopathological assessment was conducted by two researchers who were masked to treatment group. h&e stained sections were scored for the presence of neutrophil in the alveolar space, neutrophils in the interstitial space, the existence of hyaline membranes, proteinaceous debris filling the airspaces and alveolar septal thickening as described previously [ ] . mice were remaining anesthetized with isoflurane ( % with oxygen), tracheostomized with a metal . mm (internal diameter) cannula and connected to a flexi vent (scireq inc) ventilator. ventilation was initiated at a tidal volume of ml/kg and a rate of /min. a tlc maneuver was performed, followed by sec later, by a sinusoidal hz j o u r n a l p r e -p r o o f oscillation. subsequently, an sec forced oscillatory signal ( . - . hz) was applied, the mechanical input impedance of the respiratory system was calculated, and a model containing a constant phase tissue compartment was fit to input impedance in order to evaluate tissue elastance. dynamic pressure-volume maneuvers were performed by stepwise increasing the airway pressure to cm h o and then reversing the process. transcutaneous oxygen saturation were monitored via a small animal pulse oximeter (mouseox plus, starr life sciences corporation, oakmont, pa, usa) by placing the non-invasive sensor on the neck, as previously described [ ] . statistical analysis was performed using graphpad prism version . (graphpad). the mean ± sd or sem was calculated for all samples, and the significance was determined either by the unpaired t-test (for groups) and anova (for > groups). for the anova analyses, newman-kuels post-hoc testing was employed. a value of p < . was considered significant. in cultured hlmvec, lps challenge ( eu/ml) induced significant decrease in rac gtpase activity and this was attenuated by the peroxynitrite scavenger, mntmpyp ( figure a) . utilizing immunoprecipitation analyses we demonstrated that rac is subject to protein nitration and again this is attenuated by mntmpyp ( figure b&c ). these findings suggest that lps-mediated rac nitration is associated with reduced rac gtpase activity. to further confirm the nitration on rac peptide and identify the j o u r n a l p r e -p r o o f nitration site, we first utilized recombinant human rac protein purified using an expression purification system based in e.coli [ ] . purity of rac was determined to be > % using both sds page with coomassie staining (figure a ) and western blot analysis ( figure b ). the purified recombinant human rac protein was exposed to authentic peroxynitrite ( µm, min) and then digested using chymotrypsin endoproteinase. the resulting peptide fragments were subjected to maldi-tof ms and ms/ms analysis. a nitrated peptide ( . da) with sequence ttnafpgey was identified with a single nitration site present on the tyrosine residue. a ms peak with + da in molecular weight difference (equal to nitro group addition) was observed with the nitrated rac peptide fragment, within the region of aa - ( figure c ). further ms-ms analysis confirmed the peptide sequence and the position of the nitro-group as y ( figure c ). we next investigated the nitration of rac in cultured pulmonary arterial endothelial cells (paec). in order to achieve a quantifiable intracellular level of rac , paec were transduced with an adenovirus construct containing a his-tagged human wildtype rac . we confirmed that human rac protein could be purified utilizing a hispur ni-nta column ( figure d ). rac purification was confirmed by sds page with coomassie staining ( figure d ) and the identification of a single band by western blot analysis ( figure e ). transduced paec were then exposed to the peroxynitrite donor, sin- ( µm for h) and western blot analysis utilized to confirm that peroxynitrite did not alter total rac levels in the cells ( figure f ). however, utilizing a nitrotyrosine antibody which specifically binds nitrated tyrosine we were able to see a strong band in western blot at . kda indicating a nitrated rac protein in transduced paec exposed to sin- ( figure g ). the nitrated rac protein was then purified using hispur j o u r n a l p r e -p r o o f ni-nta column and the peptide fragments generated by overnight chymotrypsin digestion were subjected to maldi-tof ms and ms/ms analysis. after exposure to sin- , the same nitration site y was detected in the rac protein isolated from paec ( figure h ). these results indicate that peroxynitrite is capable of inducing rac nitration at a specific site, y both in vitro and in vivo. next, using the known rac protein structure and a previously established computational modeling method [ ] , we simulated the structural impact of y nitration on rac . this modeling predicts that the y containing region (the "flap" next to the switch i region of the catalytic domain) leads to a "close" state in the flap region ( figure i ), compared to the "open" state induced by nitration of y in the flap region of rhoa [ ] ( figure j ). these computational modeling data, coupled with our rac -gtpase activity measurements, ( figure a ) clearly demonstrate that lps-mediated peroxynitrite stress induces rac nitration at y and that this is inhibitory for rac gtpase activity. in order to increase the detection sensitivity for y nitrated rac , we next generated a polyclonal antibody (rac -y -no ) that specifically recognizes y nitrated rac following the protocol from the previous study which used an antibody (rhoa-y -no ) that recognized y nitrated rhoa [ ] . we confirmed that the rac -y -no antibody selectively detects y nitrated rac in recombinant protein exposed to authentic peroxynitrite in-vitro ( figure a ) and in-vivo hlmvec exposed to sin- ( figure b ). further, we validated that rac -y -no and rhoa-y -no antibodies are preferential for nitrated rac and nitrated rhoa respectively ( figure c ). using rac -y -no j o u r n a l p r e -p r o o f antibody we were next able to confirm that there was significant increase in rac nitration in the mouse lung h after i.t. lps challenge, and that again rac nitration was significantly suppressed by mntmpyp ( figure d ). these changes in rac nitration correlated with change in rac gtpase activity ( figure e ). these data demonstrate that nitration of rac at y , is intimately involved in the loss of rac activity in the mouse lung during sepsis-mediated ali. we next aimed to design a peptide which specifically targets the flap region of the rac protein to prevent its nitration at y . docking simulations ( figure a ) were used to design a nitration shielding approach that would bind to the flaps region of rac similar to that used earlier for rhoa [ ] . this resulted in the design of a peptide with sequence hrkkrrqrrrqfpgeyiptvf designated nitration inhibitory peptide for rho gtpases (nipr ). the molecular mass of the nipr peptide of . da was confirmed by maldi ms ( figure b ) and peptide sequence was confirmed by ms/ms ( figure c ). the nipr peptide was then exposed to authentic peroxynitrite ( µm, figure e ). as expected, when the nipr f peptide was exposed to peroxynitrite ( µm, min) there was no mass difference observed in ms and ms/ms spectrum because of absence of the target tyrosine residue required for nitration ( figures d and e) . using maldi ms linear mode acquisition, with sinapinic acid as a matrix, we also confirmed that both nipr and nipr f peptides we able to bind efficiently to intact human recombinant rac ( figure f ). to evaluate the therapeutic potential of nipr , we utilized a mouse model of ali figure a ). as expected, nipr , but not nipr f, significantly attenuated lps-mediated rac nitration ( figure b ) and restored rac gtpase activity ( figure c ). mice were also used to perform additional physiological, biochemical and morphological studies to evaluate the efficacy of the nipr peptide in attenuating symptoms of ali. cell infiltration into the balf was significantly increased in lps treated animals ( figure d ). nipr , but not nipr f, reduced this increase ( figure d ). we also assessed histopathological changes in the lungs. lps induces severe alveolar damage that includes the presence of large numbers of neutrophils and red blood cells in the alveolar and interstitial space, formation of hyaline membranes, septal thickening and debris accumulation in the j o u r n a l p r e -p r o o f alveoli ( figure e ). again, nipr , but not nipr f, reduced these pathological changes ( figure e &f) . we next assessed the effect of nipr on lung function in lps challenged mice. nipr restored oxygen saturation from ~ % to ~ % ( figure a ). using flexivent technology, we also performed respiratory pressure-volume loop (pv loop) measurement and analysis. lps induced a characteristic downward shift of the pv loop, compared to control mice ( figure b ). consistent with the oxygen saturation findings, nipr , but not nipr f, restored the pv loop, indicative of an improvement in respiratory mechanical function. specifically, nipr increased the respiratory maximal volume at highest pressure ( figure c ). nipr also prevented the lps-mediated attenuation of total respiratory compliance ( figure d ) and the increase in respiratory elastance ( figure e ). together these data demonstrate that nipr significantly improved lung function in this murine model of acute lung injury. ali is a complex syndrome with an unacceptable mortality. ali pathogenesis involves disruption of the epithelial and endothelial barriers leading to an increased permeability and decreased edema fluid clearance [ ] . lung barrier permeability is tightly regulated by adherens junctions, tight junctions, and gap junctions. junctional integrity is vital for cell-cell adhesion, actin cytoskeleton remodeling, intercellular signaling, and transcriptional regulation [ ] . rac regulates lung endothelial integrity via cytoskeleton j o u r n a l p r e -p r o o f rearrangements that determine cell shape and junctional integrity of the lumen [ ] . interestingly, within the rho-family small gtpases, rac and rhoa are antagomirs [ ] . rac is involved in the maintenance and stabilization of microvascular endothelial barrier functions (maintains junctional formation and cell relaxation), whereas rhoa primarily acts antagonistically to impair barrier properties [ ] . post-translational modification can directly influence the structure, function and stability of the rho gtpases [ ] . this particular study confirms, for the first time, that rac can be nitrated at y , which leads to a persistent inhibition of gtpase activity. crystal structure analysis of rac indicates that y nmr and x-ray crystal structures for rho family gtpases propose that the cys thiol in the gxxxxgk(s/t)c motif is accessible for solvent and suggest reactive oxygen species and reactive nitrogen species possibly target the cys thiol [ ] [ ] [ ] . further, we have shown that s-nitrosylation of rhoa attenuates the activity of rhoa [ ] . the effect of s-nitrosylation on rac is unresolved but we speculate that as nitration activates while s-nitrosylation inhibits rhoa that s-nitrosylation may stimulate rac activity. however, further studies will be required to test this possibility. besides nitration a number of other post translational modifications have been reported that have the potential to alter its activity/function. these include ubiquitination [ ] [ ] [ ] , phosphorylation [ , ] , and adenylylation (ampylation) [ ] , all of which have all been shown to play important roles in the regulation of rac . interestingly, it has been reported that y in rac is a target for ampylation [ ] . therefore, we speculate that nitration might inhibit phosphorylation and/or ampylation of y in rac which could permanently alter its activity. thus, it is likely that the competition among these various potential modifications likely has both physiological and pathological implications. again, further studies will be required to investigate this. during sepsis-like conditions, lps exposure stimulates the production of peroxynitrite which leads to protein nitration of tyrosine residues, a nonreversible event which often affects protein structure and function. previously we had reported that peroxynitrite j o u r n a l p r e -p r o o f stress induced by lps challenge causes rhoa nitration at y which facilitates its persistent activation [ ] . rac and rhoa, as a balanced control mechanism of lung endothelial integrity, is disrupted by peroxynitrite stress. even though rhoa and rac share structural homology, rhoa is involved in endothelial barrier disruption during the development of ali, while rac is involved in endothelial barrier recovery during the resolution phase of ali. the fact that each protein acts in a different phase of ali could provide a therapeutic window to target rac , instead of rhoa, in the patients in icu, most of whom have passed the earlier phase of endothelial disruption and are most likely in the slow recovery phase of this devastating disease. this possibility is supported by our animal studies in which we have utilized our nipr peptide to target rac nitration in a "reversal"-, instead of "prevention"-, strategy to effectively "treat" experimental ali. our study reveals a new mechanism by which endothelial integrity loss is maintained through nitration-mediated rac inhibition during sepsis-associated ali. importantly, we developed two new research tools in this study, a polyclonal antibody which specifically detects y -nitrated rac , and nipr , a synthetic peptide which prevents rac nitration at y . since rac is wildly expressed in multiple tissues with multidisciplinary function, our nitration specific rac antibody has significant potential as a research tool in a number of pathologic states. further, we validated the nitration shielding potency nipr in both cultured cells and a murine model of sepsis induced ali. thus, nipr , or its next generation, may have clinical utility given the fact that ali still does not have a selective drug therapy. however, this possibility should be tempered by the fact that previous ali drug clinical trials have all failed. we propose that this is due to the fact that ali is a syndrome that results from different insults (e.g., j o u r n a l p r e -p r o o f covid- induced ali is clearly different from trauma induced ali), and the involvement of multiple systems failure such as endothelial barrier disruption, macrophage activation, and leukocyte infiltration. in this study, we report that nipr selectively targets rac nitration and effectively blocks murine lung injury, but the therapeutic efficacy in ali patients is hard to predict. we anticipate that a successful clinical efficacy of nipr or similar product might require: ) precision medicine approach to identify patients in the sub-group with satisfactory responsiveness of rac nitration blockade, as not all triggers of ali (e.g., trauma) will lead to endothelial oxidative stress and peroxynitrite generation; ) combination therapy with other effective reagents, including suppressor of the cytokine storm and/or neutrophil eliminators; ) selective tissue delivery vehicle, to increase therapeutic index. further, we propose that conditional genetically modified in-vivo models will be needed to fully elucidate the biological functions of the rho gtpases in different physiological environments and pathological conditions, and this information will likely be required to develop precise pharmacological inhibitors. lps is a known activator of endothelial ros generation [ , ] . interestingly the generation of ros might be "designed" a result of detoxification of oxidative species in oxygen-rich environment, and sometimes as a signaling cascade, since it can be generated and removed quickly. however, these protective or regulatory signaling pathways can be overwhelmed, leading to "oxidative stress". in the lungs, multiple cell types, including endothelial cells, macrophages, epithelial cells, and infiltrated inflammatory leukocytes, are good ros generators. lps can activate several j o u r n a l p r e -p r o o f endogenous machineries to generate ros including nadph oxidase (nox), uncoupled nos, dysfunctional mitochondria, and xanthine oxidase, all of which have been reported to be involved with the oxidative stress associated with ali [ ] [ ] [ ] [ ] [ ] . the link between increased oxidative stress and lung injury, as well as the success of antioxidant therapy in preclinical models makes ros generation an attractive target in ali. however, the complex roles of ros in ali development and recovery have restricted the efficacy of antioxidant therapy. ros is involved in tissue injury [ ] , barrier disruption [ ] , proinflammatory cytokine production [ ] . while it is also involved in endothelial barrier recovery [ ] , and endothelial adherens junction re-assembly [ ] . endothelial cells have abundant no which is mainly produced by enos, which, upon lps-challenge mediated uncoupling, also starts to generate ros. the downstream effector of nos uncoupling is likely peroxynitrite, formed from the interaction of no with superoxide generated from different sources. we propose that targeting adverse events downstream of ros, such as rac nitration, will be more selective and effective to terminate ros associated adverse outcome, and reduce ali. nitration unlike phosphorylation, which is selectively mediated by kinases and phosphatases, and modulated by signaling cascades regulating these enzymes, is not dependent on enzymes to facilitate the post-translational modification. peroxynitrite mediated tyrosine nitration is a covalent modification that adds a nitro group (-no ) to one ortho carbon of tyrosine's phenolic ring to form -nitrotyrosine. protein tyrosine nitration introduces a net negative charge to the nitrated tyrosine at physiological ph, thus altering structural properties. previously, we had reported that like rac , its antagomir rhoa can also be nitrated at y , a similar location in the flexible flap region, leading to its persistent j o u r n a l p r e -p r o o f activation. we speculate that nitration will only occur at accessible tyrosine residues that are already sites for other post-translational modifications such as phosphorylation. indeed, nitration is very similar to phosphorylation in terms of charge, and thus could lead to a similar structural/functional outcome to that obtained by phosphorylation. alternatively, the presence of a -nitrotyrosine modification could potentially prevent the tyrosine residue being available for phosphorylation events. since no enzyme has been identified as being responsible for the removal of nitration sites, mimicking the counter part of phosphatase for phosphorylation, the structure/function effect is persistent and thus deleterious as it is not balanced and countered. this might also explain the opposite functional outcome between rac and rhoa nitration at a similar domain. in conclusion, for the first time, we have used mass spectrometry as well as cell and molecular biology methodologies to identify rac as being susceptible to nitration. this single nitration site located at y leads to rac gtpase inhibition and enhances lpsmediated barrier disruption. we have also demonstrated that rac nitration plays an important role in sepsis-mediated ali and that blocking rac 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experimental acute lung injury in animals dimethylarginine dimethylaminohydrolase ii overexpression attenuates lps-mediated lung leak in acute lung injury altered expression of tight junction molecules in alveolar septa in lung injury and fibrosis ve-cadherin: at the front, center, and sides of endothelial cell organization and function role of gtpases in control of microvascular permeability rho gtpases and the regulation of endothelial permeability rac and rhoa: networks, loops and bistability rho gtpases, their post-translational modifications, diseaseassociated mutations and pharmacological inhibitors crystal structure of human rhoa in a dominantly active form complexed with a gtp analogue the crystal structure of human rac , a member of the rho-family complexed with a gtp analogue nitric oxide-induced inhibition of smooth muscle cell proliferation involves s-nitrosation and inactivation of rhoa rhoa s-nitrosylation as a regulatory mechanism influencing endothelial barrier function in response to g(+)-bacterial toxins the tumour suppressor hace controls cell migration by regulating rac degradation iaps regulate the plasticity of cell migration by directly targeting rac for degradation scf e ligase f-box protein complex scf(fbxl ) regulates cell migration by mediating rac ubiquitination and degradation tyrosine phosphorylation of rac : a role in regulation of cell spreading akt protein kinase inhibits rac -gtp binding through phosphorylation at serine of rac the fic domain: regulation of cell signaling by adenylylation irak- contributes to lipopolysaccharide-induced reactive oxygen species generation in macrophages by inducing nox- transcription and rac activation and suppressing the expression of antioxidative enzymes in vivo lipid-derived free radical formation by nadph oxidase in acute lung injury induced by lipopolysaccharide: a model for ards oxidative stress contributes to lung injury and barrier dysfunction via microtubule destabilization role of nadph oxidase in lipopolysaccharide-induced proinflammatory responses by human aortic endothelial cells mechanisms of nitric oxide synthase uncoupling in endotoxin-induced acute lung injury: role of asymmetric dimethylarginine lipopolysaccharide induces mitochondrial dysfunction in rat cardiac microvascular endothelial cells upregulation of xanthine oxidase by lipopolysaccharide, interleukin- , and hypoxia. role in acute lung injury signaling in the pathogenesis of acute lung injury (ali) and acute respiratory distress syndrome (ards) autophagy maintains the integrity of endothelial barrier in lps-induced lung injury superoxide potentiates nf-kappab activation and modulates endotoxin-induced cytokine production in alveolar macrophages regulation of endothelial barrier function by reactive oxygen and nitrogen species reactive oxygen species mediate rac-induced loss of cell-cell adhesion in primary human endothelial cells • endotoxin exposure induces site specific nitration of rac at y via peroxynitrite stress.• rac nitration at y leads to persistent rac gtpase inhibition and endothelial barrier disruption. • novel rac nitration shielding peptide, nipr blocks rac nitration and rescues endotoxin induced lung inflammation. • nipr is potentially an effective therapy for sepsis induced lung injury by targeting rac y nitration. the authors have no conflict of interest to declare related to this research article . key: cord- -p joajvn authors: liu, zhicheng; yang, zhengtao; fu, yunhe; li, fenyang; liang, dejie; zhou, ershun; song, xiaojing; zhang, wen; zhang, xichen; cao, yongguo; zhang, naisheng title: protective effect of gossypol on lipopolysaccharide-induced acute lung injury in mice date: - - journal: inflamm res doi: . /s - - - sha: doc_id: cord_uid: p joajvn objective: gossypol has been reported to have anti-inflammatory properties. the purpose of this study was to evaluate the effect of gossypol on acute lung injury (ali) induced by lipopolysaccharide (lps) in mice. methods: male balb/c mice were pretreated with gossypol h before intranasal instillation of lps. then, h after lps administration, the myeloperoxidase in histology of lungs, lung wet/dry ratio and inflammatory cells in the bronchoalveolar lavage fluid (balf) were determined. the levels of tumor necrosis factor-α (tnf-α), interleukin- (il- ) and interleukin- β (il- β) in the balf were measured by elisa. the extent of phosphorylation of iκb-α, p nf-κb, p –p jnk, p –p erk, and p were detected by western blot. results: gossypol markedly attenuated the lps-induced histological alterations in the lung and inhibited the production of tnf-α, il- β and il- . additionally, gossypol reduced the inflammatory cells in balf, decreased the wet/dry ratio of lungs and inhibited the phosphorylation of iκb-α, p nf-κb, p –p jnk, p –p erk, and p caused by lps. conclusion: the data suggest that anti-inflammatory effects of gossypol against the lps-induced ali may be due to its ability of inhibition of the nf-κb and mapks signaling pathways. gossypol may be a promising potential therapeutic reagent for ali treatment. acute lung injury (ali), the basis of acute respiratory distress syndrome (ards), is characterized by severe hypoxemia, pulmonary edema and neutrophil accumulation in the lung [ , ] . lipopolysaccharide (lps), a main component of outer membrane of gram-negative bacteria, has been referred to be an important risk factor of ali [ , ] . intratracheal administration of lps has gained wide acceptance as a clinically relevant model of severe lung injury. in the clinical cases, ali is a major problem that has a high mortality rate of - % and there are still few effective measures or specific medicines to treat it [ , ] . therefore, the development of novel therapies for ali is urgently needed. gossypol, a yellow polyphenolic compound extracted from cottonseed ( fig. ) , has long been used as a male contraceptive drug [ , ] . in recent years, gossypol has been shown to exhibit a variety of other pharmacological activities, including anti-tumor, anti-oxidant, anti-virus and anti-inflammatory activities [ ] [ ] [ ] . gossypol was found to inhibit the activation of human t-lymphocytes stimulated with polyclonal activators, to suppress nf-jb activity and nf-jb-related genes expression in human leukemia u cells [ ] . although a number of studies have addressed the therapeutic potential of gossypol, its ability to protect against bacterial endotoxin-induced ali remains poorly understood. in this study, we sought to assess the preventive effects of gossypol in a lps-induced mouse ali model and elucidated the potential anti-inflammatory mechanism. male balb/c mice, weighing approximately - g, were purchased from the center of experimental animals of jilin university (changchun, china). and this study was approved by the ethical committee on animal research at the university of jilin (approval id: - ). the mice were housed in a room maintained at ± °c with - % humidity. all animals received food and water ad libitum. all animal experiments were performed in accordance with the guide for the care and use of laboratory animals established by the us national institutes of health. gossypol (purity:[ %) was purchased from the national institute for the control of pharmaceutical and biological products (beijing, china), dexamethasone (dex, purity: [ . %) was purchased from changle pharmaceutical co. (xinxiang, henan, china). mouse tnf-a, il- and il- b enzyme-linked immunosorbent assay (elisa) kits were purchased from biolegend (ca, usa). the myeloperoxidase (mpo) determination kit was provided by the jiancheng bioengineering institute of nanjing (nanjing, jiangsu, china). mouse monoclonal phospho-specific p antibody, mouse monoclonal phospho-specific p -p erk antibody, mouse monoclonal phospho-specific p -p jnk antibody, mouse mab phospho-nf-jb p , mouse mab phospho-ijb-a and rabbit mab ijb-a were purchased from cell signaling technology inc (beverly, ma). hrp-conjugated goat anti-rabbit and goat-mouse antibodies were provided by ge healthcare (buckinghamshire, uk). all other chemicals were of reagent grade. mice were randomly divided into eight groups: blank control group, lps group, gossypol ( . , , , and mg/kg) ? lps group, dex ? lps group. before inducing acute lung inflammation, gossypol ( . , , , and mg/kg) was given by intraperitoneal injection (i.p.), while dex, . mg/kg, was administrated intra-gastrically as a positive control. blank control and lps group mice were given an equal volume of distilled water by i.p. h later, mice were slightly anesthetized with an inhalation of diethyl ether, lg of lps in ll pbs was instilled intranasal (i.n.) to induce lung injury. blank control group mice were given a ll pbs by i.n. without lps. all the mice were alive after h lps treatment. the mice were killed by exsanguination at h after the administration of lps. collection of bronchoalveolar lavage fluid (balf) was performed three times through a tracheal cannula with autoclaved pbs, instilled up to a total volume of . ml. mice were randomly divided into six groups: blank control group, gossypol ( . , , , and mg/kg). gossypol ( . , , , and mg/kg) was given by intraperitoneal injection (i.p.). h after injection of gossypol, ll of peripheral blood was collected and mixed with the anticoagulant na -edta. automated hematological analysis was performed using a mek- k automated hematology analyzer (nihon kohden, japan). the following blood components were determined: white blood cell (wbc) count, the number of neutrophils and lymphocytes. lung wet-to-dry weight (w/d) ratio after the mice were euthanized, the lungs were removed and the wet weight recorded. the lungs were then placed in an incubator at °c for h to obtain the 'dry' weight. the ratio of wet lung to dry lung was calculated to assess tissue edema. the balf samples were centrifuged ( °c, , rpm, min) to pellet the cells. the cell pellets were resuspended in pbs for the total cell counts using a hemacytometer, and cytospins were prepared for differential cell counts by staining with the wright-giemsa staining method. determination of tnf-a, il- b and il- levels inflammatory cytokines of tnf-a, il- b and il- in balf were measured using specific elisa kits according the accumulation of neutrophils in the lung tissue was assessed by mpo activity. briefly, h after lps administration, mice under diethyl ether anesthesia were killed, and the right lungs were excised. one hundred milligrams of lung were homogenized and fluidized in extraction buffer to obtain % of homogenate. the sample including . ml homogenate and . ml of reaction buffer was heated to °c in water for min, on which occasion, the enzymatic activity was determined by measuring the change in absorbance at nm using a -well plate reader. histopathologic examination was performed on mice that were not subjected to balf collection. lungs were fixed with % buffered formalin, imbedded in paraffin and sliced. after hematoxylin and eosin (h&e) stain, pathological changes of lung tissues were observed under a light microscope. the lung injury score was quantificated by a scoring system as described elsewhere [ ] . the lung injury score was assessed as follows: no oedema, mild oedema, moderate oedema, severe oedema. for leucocyte or other cell infiltration, the grading system was that used to determine the extend of oedema: no cellular infiltration, mild cellular infiltration, moderate cellular infiltration, and severe cellular infiltration. each one gave a score for each from to . at h after the injection of lps, lung tissues were harvested and frozen in liquid nitrogen immediately until homogenization. proteins were extracted from the lungs using t-per tissue protein extraction reagent kit (thermo) according to the manufacturer's instructions. protein concentrations were determined by bca protein assay kit and equal amounts of protein were loaded per well on a % sodium dodecyl sulphate polyacrylamide gel. subsequently, proteins were transferred onto polyvinylidene difluoride membrane. the resulting membrane was blocked with tris-buffered saline containing . % tween- (tbs-t), supplemented with % skim milk (sigma) at room temperature for h on a rotary shaker, and followed by tbs-t washing. the specific primary antibody, diluted in tbs-t, was incubated with the membrane at °c overnight. subsequently, the membrane was washed with tbs-t followed by incubation with the peroxidase-conjugated secondary antibody at room temperature for h. the immunoactive proteins were detected by using an enhanced chemiluminescence (ecl) western blotting detection kit. all values are expressed as mean ± sem. differences between mean values of normally distributed data were analyzed using one-way anova (dunnett's t test) and twotailed student's t test. statistical significance was accepted at p \ . . to test if gossypol treatment effected the blood leukocytes of mice, we detected the blood leukocytes by routine blood test. the results showed gossypol treatment did not effect the blood leukocytes (table ) . gossypol inhibited lps-induced lung w/d ratio lps caused a significant increase in lung w/d ratio (p # \ . ) compared to the control group (fig. ) . gossypol ( and mg/kg) and dex significantly decreased the lung w/d ratio (p* \ . ) compared to those in the lps group (fig. ) . gossypol inhibited the inflammatory cell count in the balf of lps-induced ali mice the number of inflammatory cells, such as neutrophils and macrophages, in balf were analyzed at h after lps challenge. as shown in fig. , lps challenge significantly increased the number of total cells, neutrophils and macrophages compared with the control group (p # \ . ). meanwhile, pretreatment with gossypol ( . , , , and mg/kg) and dex ( . mg/kg) was found to significantly decrease the number of total cells (p \ . ), neutrophils (p \ . ), and macrophages (p \ . ). gossypol suppressed the production of cytokines in the balf of lps-treated ali mice the effect of gossypol on tnf-a, il- b and il- production was analyzed at h after lps challenge by elisa. as shown in fig. , the concentrations of tnf-a, il- , and il- b in balf were significantly increased after lps administration. gossypol ( . , , , and mg/kg) and dex significantly reduced tnf-a (p* \ . ), il- (p* \ . ), and il- b (p* \ . ) production compared to those in the lps group. the mpo activity (fig. ) was determined to assess the neutrophil accumulation within pulmonary tissues. lps challenge resulted in significantly increased lung mpo activity compared with the control group (p \ . ). however, this increase was apparently reduced by gossypol ( . , , , and mg/kg) (p \ . ) or dex (p \ . ). lung tissues, harvested at h after injection of lps, were subjected to he staining. as shown in fig. , lung tissues from the control showed a normal structure and no histopathologic changes under a light microscope (fig. a) . lung sections obtained from mice in lps group showed characteristic histological changes, including areas of inflammatory infiltration, focal areas of fibrosis with collapse of air alveoli and emphysematous, as well as thickening of the alveolar wall and pulmonary congestion (fig. b) . however, lps-induced pathological changes were significantly attenuated by gossypol ( , , and mg/kg) (fig. e-h) and dex ( . mg/ kg) treatment (fig. c) . evaluation of the lung injury score revealed that gossypol significantly attenuated lps-induced ali ( table ) . western blot analysis showed that nf-jb and mitogenactivated protein kinase (mapk) signaling pathways were activated h after lps treatment. pretreatment with gossypol ( mg/kg) inhibited the phosphorylation of ijb-a, p nf-jb (fig. ) , p , jnk and erk (fig. ) in all, these results showed that gossypol ( mg/kg) could simultaneously inhibit nf-jb and mapk signaling pathways efficiently in a mouse model of ali. lps-induced ali was characterized by the disruption of endothelial and epithelial integrity, lung edema, the release of inflammatory mediators, and extensive neutrophil infiltration [ ] . though several candidate therapies have been applied to reduce lung injury, there are still few effective measures or specific medicines to treat it. gossypol is a yellow polyphenolic compound isolated from cottonseed and has been shown to exhibit anti-inflammatory effects recently. in the present study, we observed the effect of gossypol on ali induced by lps in mice. the results showed that pretreatment with gossypol attenuated lung damage induced by lps and decreased the w/d ratio, proinflammatory cytokine production, inflammatory cell migration into the lung, protein leakage, the activation of nf-jb and mapk. this suggests that gossypol may be a promising potential therapeutic reagent for ali treatment. pulmonary edema is one of the major characteristics of ali [ ] . in this study, we evaluated the w/d ratio of the lung to quantify the magnitude of pulmonary edema. our experiments showed that gossypol significantly inhibits edema of the lung, as shown by a w/d ratio in the gossypol group that was significantly lower than the lps group. mpo activity, reflecting the parenchymal infiltration of neutrophils and macrophages, lps-induced ali is characterized by the infiltration of neutrophils in the lung, exhibiting increased mpo activity [ , ] . in this study, we found that lps administration significantly increased the mpo activity and pretreatment with gossypol decreased lps-induced increases in mpo activity in the lungs. this indicated that the protective effect of gossypol in ali is related to attenuation of neutrophil influx into the lung tissue. pro-inflammatory cytokines including tnf-a, il- b and il- participated in the development of ali [ ] [ ] [ ] . some reports have shown that lps-induced ali can lead to the overproduction of these cytokines. tnf-a is the earliest and primary pro-inflammatory factor produced when infection [ ] . il- b is a crucial mediator in ali. it plays an important role in the progression multiple organ failure in lps-induced endotoxic shock [ , ] . il- is also a marker of the acute inflammatory response in lpsinduced ali mode [ , ] . in the present study, gossypol significantly inhibited the production of tnf-a, il- b and il- induced by lps. these results indicate that the protective effects of gossypol on ali induced by lps may be attributed to the compound's inhibition of inflammatory factors. lps induces its inflammatory reaction through the activation of both nf-jb and mapks signaling pathways to regulate the release of pro-inflammatory cytokines [ ] . nf-jb is normally present in the cytoplasm as a heterodimer and is linked to the inhibitory proteins ijbs. once activated, nf-jb units p dissociates from its inhibitory protein ijb-a and translocates from the cytoplasm to the nucleus where they may trigger the transcription of specific target genes such as tnf-a, il- b and il- [ ] . in this study, we tested the effects of gossypol on nf-jb activation and ijb-a degradation. the results showed that lps stimulation dramatically increased the phosphorylation of ijb-a and nf-jb p protein. however, lps-induced ijb-a degradation and nf-jb p activation were significantly blocked by pretreatment with gossypol. mapks also play an important role in inducing cytokine production [ , ] . the lps stimulation of murine macrophages has been known to induce phosphorylation and activation of erk / , jnk, and p mapks [ ] . therefore, we investigated the effect of gossypol on activation (phosphorylation) of three mapks induced by lps in the mice of ali. the results showed that gossypol ( . , , and mg/kg) inhibited the phosphorylation of p -p erk, p , and p -p jnk in lps-stimulated mice. taken together, these results indicate that gossypol may exert its anti-inflammatory action by inhibition of the nf-jb and mapks signaling pathways activation. in conclusion, the present study demonstrated that gossypol has a protective effect against lps-induced ali, which may be related to its suppression of nf-jb and mapks activation, and subsequently leads to the reduction the inflammatory cell infiltration and proinflammatory cytokine expression in lung tissues. these findings suggest that gossypol may be an agent for preventing and treating lps induced ali. epidemiology of acute lung injury protective effect of florfenicol on acute lung injury induced by lipopolysaccharide in mice the pulmonary physician in critical care. : acute lung injury and the acute respiratory distress syndrome: definitions and epidemiology incidence and outcomes of acute lung injury the acute respiratory distress syndrome neutrophil activation and acute lung injury gossypol: a contraceptive for men a combined regimen of gossypol plus methyltestosterone and ethinylestradiol as a contraceptive induces germ cell apoptosis and expression of its related genes in rats induction of apoptosis by (-)-gossypol-enriched cottonseed oil in human breast cancer cells gossypol inhibits phosphorylation of bcl- in human leukemia hl- cells induction of neutrophil mac- integrin expression and superoxide production by the medicinal plant extract gossypol gossypol suppresses nf-kappab activity and nf-kappab-related gene expression in human leukemia u cells inhibition of inducible nitric oxide synthase attenuates acute endotoxin-induced lung injury in rats acute lung injury induces cardiovascular dysfunction: effects of il- and budesonide/formoterol antiinflammatory effects of hydrogen peroxide in neutrophil activation and acute lung injury role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome stem cells in sepsis and acute lung injury cytokine-mediated inflammation in acute lung injury local stimulation of alpha cholinergic receptors inhibits lps-induced tnf-alpha release in the mouse lung the role of nuclear factor-kappa b in pulmonary diseases tnf: a key neuroinflammatory mediator of neurotoxicity and neurodegeneration in models of parkinson's disease il- cytoprotection in hyperoxic acute lung injury occurs via pi k/akt-mediated bax phosphorylation phosphoinositide-mediated adaptor recruitment controls toll-like receptor signaling introduction to nf-kappab: players, pathways, perspectives map kinase activation in macrophages kinetics of mitogenactivated protein kinase family in lipopolysaccharide-stimulated mouse kupffer cells and their role in cytokine production isoeugenol suppression of inducible nitric oxide synthase expression is mediated by down-regulation of nf-kappab, erk / , and p kinase key: cord- -bz ui a authors: hans-peter, kapfhammer title: posttraumatic stress disorder in survivors of acute respiratory distress syndrome (ards) and septic shock date: - - journal: psychosom konsiliarpsychiatr doi: . /s - - -x sha: doc_id: cord_uid: bz ui a acute lung injury (ali) and acute respiratory distress syndrome (ards) define medical conditions of acute respiratory insufficiency deriving from direct and indirect damage of the alveolar parenchyma and often associated with multiorgan dysfunction (mods). as a rule, intensive care is based on mechanical ventilation often requiring high doses of sedatives and narcotics. despite major progress in intensive care medicine the rate of mortality is still very high. whereas in the past the level of medical progress has been rated based on the mortality rate alone, the many negative somatic and psychological sequelae in long-term-survivors of ards are only now being appreciated. from a perspective of c/l psychiatry persisting cognitive dysfunctions, anxiety and mood disorders, posttraumatic stress disorders (ptsd) in their negative impact on health-related quality of life are intensively investigated. in the etiopathogenesis of ptsd associated with ali/ards, many influences have to be discussed, e.g., increases in co( ) triggering panic affects, a mismatch of norepinephric overstimulation and cortisol insufficiency, negative effects of high doses of benzodiazepines resulting in oversedation, prolonged phases of weaning and more frequent states of delirium. consolidation and retrieval of traumatic memories of the icu stay are influenced by complex factors. from a clinical point of view prophylactic stress doses of hydrocortisone may reduce the major risk of ptsd associated with ali / ards. abstract acute lung injury (ali) and acute respiratory distress syndrome (ards) define medical conditions of acute respiratory insufficiency deriving from direct and indirect damage of the alveolar parenchyma and often associated with multiorgan dysfunction (mods). as a rule, intensive care is based on mechanical ventilation often requiring high doses of sedatives and narcotics. despite major progress in intensive care medicine the rate of mortality is still very high. whereas in the past the level of medical progress has been rated based on the mortality rate alone, the many negative somatic and psychological sequelae in long-term-survivors of ards are only now being appreciated. from a perspective of c/l psychiatry persisting cognitive dysfunctions, anxiety and mood disorders, posttraumatic stress disorders (ptsd) in their negative impact on health- in the etiopathogenesis of ptsd associated with ali/ ards, many influences have to be discussed, e.g., increases in co triggering panic affects, a mismatch of norepinephric overstimulation and cortisol insufficiency, negative effects of high doses of benzodiazepines resulting in oversedation, prolonged phases of weaning and more frequent states of delirium. consolidation and retrieval of traumatic memories of the icu stay are influenced by complex factors. from a clinical point of view prophylactic stress doses of hydrocortisone may reduce the major risk of ptsd associated with ali / ards. [ ] . in den letzen bis jahren gab es geradezu einen explosionsartigen wissenszuwachs zur pathophysiologie und differenzialtherapie von mods bis hin zur aufdeckung molekularer mechanismen. neben untersuchungen zu akuten krankheitsstadien von ali und ards zeichnen sich die mittel-und langfristigen probleme eines Überlebens aber ebenfalls immer deutlicher ab. auf einer somatischen ebene sind vor allem ein reduziertes körpergewicht, eine eingeschränkte körperliche belastungsfähigkeit, persistierende schmerzsyndrome, neuropathien, heterotrophe ossifikationen, kosmetisch störende narben von tracheostomien, fixierte deformationen an fingern und schulter hervorzuheben [ ] . hiermit assoziierte bedeutsame einbußen in der gesundheitsbezogenen lebensqualität sind im langzeitverlauf zu beachten [ ] . in einer konsiliarpsychiatrischen perspektive sind die vielfältigen psychopathologischen komplikationen, die eine schwerwiegende somatische erkrankung wie ards oder septischer schock sowie deren intensivmedizinische therapiemodalitäten während des aufenthalts auf einer intensivstation begleiten können, seit langem bekannt [ ] . die langfristigen psychosozialen und psychologischen probleme als konsequenzen aus dieser erkrankung und dem notwendigen intensivmedizinischen behandlungskontext werden in studien erst in den letzten jahren zunehmend stärker beachtet. diskutiert wird vor allem eine erhöhte psychiatrische komorbidität hinsichtlich neurokognitiver dysfunktionen, angst-und stimmungsstörungen und speziell posttraumatischer belastungsstörungen. negative interferenzen sowohl mit der gesundheitsbezogenen lebensqualität als auch mit der somatischen morbidität werden erkennbar. einflussfaktoren auf diese komplexen somatopsychischen und psychosomatischen zusammenhänge zeichnen sich erst allmählich ab. dies gilt auch für die erprobung therapeutischer und präventiver interventionsstrategien. Überlebende einer akuten respiratorischen insuffizienz im rahmen eines ali oder ards weisen ein signifikant erhöhtes risiko für anhaltende neurokognitive dysfunktionen im langzeitverlauf auf. hierauf machte bereits eine frühere neuropsychologische studie aufmerksam [ ] . mittlerweile existieren mehrere untersuchungen von unterschiedlichen arbeitsgruppen zu diesem thema. sie bestätigen, dass eine subgruppe von früheren ards-patienten in der tat persistierende kognitive leistungseinbußen zeigt [ ] . die prävalenzzahlen schwanken zwischen einem drittel und ca. drei viertel der Überlebenden eines ards. zahlreiche methodologische probleme erschweren aber die interpretation dieser stark divergierenden häufigkeitsangaben. nicht selten ist die unterscheidung von daten zur prävalenz und zur inzidenz unmöglich, da in den studien nur ausnahmsweise informationen zur prämorbiden kognitiven performanz enthalten sind. dies ist von bedeutung, da in einigen bedingungskonstellationen wie z. b. einer vorbestehenden alkoholabhängigkeit nicht nur ein erhöhtes risiko zu einem ards selbst besteht, sondern auch eigenständig kognitive dysfunktionen assoziiert sein können. ebenso bleibt unklar, ob eher spezifische kognitive leistungsdomänen wie aufmerksamkeit, merkfähigkeit oder exekutivfunktionen oder überwiegend das globale kognitive leistungsvermögen diffus durch den somatischen krankheitsprozess und/oder interferierende therapiemaßnahmen negativ beeinflusst werden. auch wenn zahlreiche variable wie hypoxie, delir, glukosedysregulation, metabolische entgleisung, inflammation, medikamenteneffekte von sedativa und narkotika mögliche und auch wahrscheinliche mechanismen einer vermittlung dieser kognitiven beeinträchtigungen andeuten, ist eine differenzielle ätiopathogenetische bewertung noch nicht möglich. der sich in einigen studien andeutende spezielle zusammenhang von deliranten zuständen während der intensivmedizinischen behandlung und kognitiven defiziten in der langzeitperspektive stellt sich wiederum in anderen untersuchungen nicht so klar dar [ ] . von großer klinischer relevanz allerdings erscheint, dass diese dauerhaften neurokognitiven defizite mit signifikanten einschränkungen der gesundheitsbezogenen lebensqualität, der beruflichen rehabilitation sowie mit beachtlichen ökonomischen kosten einhergehen [ , , ] . nach intensivmedizinischen behandlungen wegen eines ards liegt die inzidenz einer neu auftretenden major depression bei ca. % [ ] , nach sars (severe acute respiratory syndrome im kontext einer infektion mit dem sars-coronavirus) in einem ähnlich hohen umfang [ , ] . die rate an angststörungen, vor allem an panikstörungen ist ebenfalls deutlich erhöht und bewegt sich zwischen bis % [ , ] . in einer konsiliarpsychiatrischen perspektive überwiegen angststörungen eher schon während der unmittelbaren intensivmedizinischen behandlung, während depressive störungen sich erst allmählich gegen ende des aufenthalts auf intensivstation und in der weiteren folge darstellen. nicht selten kann bei letzteren auch bereits prämorbid eine depressive vulnerabilität nachgewiesen werden [ ] . in einer allgemeinen ätiopathogenetischen betrachtung darf nicht allein auf die bedingungen von ali/ ards und intensivmedizinische interventionen fokussiert werden, sondern ist eine multifaktorielle betrachtungsweise zu fordern. somatische folgezustände nach überlebtem ali/ards bedeuten für viele patienten erhebliche funktionsbehinderungen (s. oben). sie können pessimismus, resignation und demoralisierung fördern. sowohl angst als auch depression bewirken im verlauf sehr häufig eine subjektive befundverschlimmerung, ohne dass hiermit auch objektivierbare verschlechterungen der lungenfunktionsparameter einhergehen müssen. sie führen zu einer erhöhten inanspruchnahme von medizinischen einrichtungen und zu einer unnötig intensivierten medikamentösen therapie. die gesundheitsbezogene lebensqualität ist oft gerade infolge persistierender angst und depressivität dramatisch reduziert [ ] . im kontext einer betrachtung von affektiven und vor allem von angst-und panikstörungen nach ali und ards ist in den letzten jahren eine klinische und wissenschaftliche diskussion auch um ein erhöhtes risiko einer posttraumatischen belastungsstörung als möglicher langzeitfolge entstanden. in einer ersten retrospektiven untersuchung wiesen schelling et al. [ ] bei insgesamt patienten ( patienten nach ards und nach septischem schock) ca. jahre nach der erkrankung auf eine prävalenz von ca. % an schweren posttraumatischen stresssyndromen hin. prävalenz und schweregrad der in einem selbstfragebogen (ptss- ) erfassten posttraumatischen stresssymptome korrelierten in dieser studie nicht mit dem schweregrad von ards/septischem schock oder dem ausmaß der assoziierten organdysfunktionen sondern mit der von den patienten nach intensivbehandlung jeweils erinnerten anzahl traumatischer erlebnisse (definiert als angst / panikreaktionen, atemnot, schmerz und alpträume / halluzinationen). patienten mit multiplen (> ) traumatischen erfahrungen während der intensivmedizinischen behandlung zeigten eine signifikant schlechtere gesundheitsbezogene lebensqualität, wobei insbesondere die psychosoziale, weniger die körperliche funktionsfähigkeit der patienten eingeschränkt war. kapfhammer et al. [ ] bestätigten an derselben patientenpopulation in einer nachfolgenden konsiliarpsychiatrischen studie, die sich methodisch auf ein standardisiertes klinisches interview mittels scid sowie auf verschiedene psychometrische tests stützte, im wesentlichen diese zusammenhänge. zum zeitpunkt der entlassung von der intensivstation hatten , % dieser patienten das vollbild einer ptsd, , % wiesen eine sub-ptsd auf. zum follow-up termin acht jahre später zeigte sich bei noch , % das vollbild eines ptsd und bei , % ein sub-ptsd. kein patient ohne posttraumatische symptome bei der entlassung hatte eine ptsd mit verzögerter manifestation entwickelt. bei patienten mit ptsd-vollbild zum zeitpunkt der entlassung persistierte diese störung über die gesamte follow-up zeit und schwächte sich im günstigeren fall in richtung eines sub-ptsd ab. in der psychometrischen testung erzielten die patienten mit dem vollbild einer ptsd durchwegs ungünstigere resultate. die deutlichsten einbussen zeigten sich in der gesundheitsbezogenen lebensqualität (sf- ), der situationsangst (stai-x ) sowie der somatisierung (soms). das ausmaß an koexistenter depressivität (madrs) erschien in dieser gruppe vergleichsweise nur moderat auffällig. kognitive dysfunktionen (skt) waren zwar in einer subgruppe nachweisbar, diskriminierten aber nicht hinsichtlich des ptsd-status. als risikofaktoren für die entwicklung eines ptsd konnten nicht die schwere der somatischen erkrankung (apa-che ii score, lung injury score), aber die anzahl der tage der intensivmedizinischen therapie sowie multiple subjektive traumatische erinnerungen (> alpträume, angst/panik, respiratorischer distress, erstickungsgefühle oder unzureichend behandelte schmerzen) auf intensivstation identifiziert werden. mittlerweile existiert eine reihe weiterer studien aus unterschiedlichen arbeitsgruppen, deren ergebnisse in mehreren systematischen reviews detailliert dargestellt sind [ , , ] . in einer zusammenfassenden beurteilung scheint wenig zweifel daran zu bestehen, dass persistierende symptome eines ptsd mögliche langzeitfolgen nach ali/ards sein können und hiermit erhebliche einschränkungen in der gesundheitsbezogenen lebens-qualität einhergehen. ebenso klar muss aber festgehalten werden, dass große unterschiede in den designs der einzelnen studien, ihr überwiegend retrospektiver charakter, meist nur sehr kleine sample-größen, heterogene messzeitpunkte im hinblick auf den zeitabstand zur intensivmedizinischen behandlung, ein erheblicher verlust von patienten in der perspektive des follow up und damit fragliche generalisierbarkeit der gefundenen ergebnisse hinsichtlich der definierten ausgangsstichprobe, eine häufig unzureichende psychiatrische diagnostik, eine nichtbeachtung von zwischenzeitlichen einflussfaktoren eine realistische einschätzung des ausmaßes eines ptsd nach ali/ ards etwa im vergleich nach exposition gegenüber anderen traumatischen ereignissen noch nicht erlauben. diese zurückhaltung ist auch im hinblick auf diskutierte risikovariablen wie länge des aufenthalts auf intensivstation und krankenhaus, beatmungsdauer, sedierungsgrad, weibliches geschlecht, lebensalter, prämorbide psychopathologie, anzahl traumatischer erinnerungen, verfügbare psychosoziale unterstützung angezeigt [ , ] . Über neurobiologische mechanismen der traumatisierung und der entwicklung eines ptsd jenseits der oft beeindruckenden subjektiven berichte von patienten, an welche traumatische erfahrungen sie sich während einer intensivmedizinischen behandlung erinnern und sowohl in intrusiven tagesbildern als auch in wiederkehrenden alpträumen oft über viele jahre wiedererleben, kann vorläufig nur in ersten ansätzen diskutiert werden. einige aspekte sollen aufgenommen werden. nach der prominenten hypothese von klein [ ] ist das auftreten von panik pathophysiologisch auf einen falschen erstickungsalarm zu beziehen. panikattacken resultieren demnach aus einer abnorm sensitiven reagibilität des medullären chemorezeptorensystems, dem entscheidenden atmungskontrollsystem im hirnstamm auf ein ansteigendes arterielles carbondioxid (co health-related quality of life stress doses of hydrocortisone reverse hyperdynamic septic shock: a prospective, randomized, double-blind, single center study indications and 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ards: results of a psychiatric follow-up study and psychological tests false suffocation alarms, spontaneous panics, and related conditions: an integrative hypothesis the long-term psychological effects of daily sedative interruption on critically ill patients daily interruption of sedative infusions in critically ill patients undergoing mechanical ventilation quality of life and psychological status in survivors of severe acute respiratory syndrome at months postdischarge acute respiratory distress syndrome ahl ström g ( ) severely ill icu patients recall events and unreal experiences of hospital admission and icu stay -- and months after discharge intensive care unit drug use and subsequent quality of life in acute lung injury patients controlled sedation with alphaxolonealphadolone factual memories of icu: recall at two years postdischarge and comparison with delirium status during icu admission --a multicentre cohort study the relationship between cognitive performance and 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distress syndrome the effect of stress doses of hydrocortisone during septic shock on post-traumatic stress disorder and health-related quality of in life in survivors (eds) handbook of liaison psychiatry epidemiology and treatment of psychiatric conditions that develop after critical illness epidemiology of depression and antidepressant therapy after acute respiratory failure medical post-traumatic stress disorder. catching up with the cutting edge in stress research post-icu consequences of patient wakefulness and sedative exposure during mechanical ventilation stress doses of hydrocortisone reduce chronic stress symptoms and improve healthrelated quality of life in high-risk patients after cardiac surgery: a randomized study posttraumatic stress, anxiety, and depression in survivors of severe acute respiratory syndrome (sars) post-traumatic stress disorder key: cord- -e s racp authors: wu, xiaojing; kong, qian; zhan, liying; qiu, zhen; huang, qin; song, xuemin title: tipe ameliorates lipopolysaccharide-induced apoptosis and inflammation in acute lung injury date: - - journal: inflamm res doi: . /s - - - sha: doc_id: cord_uid: e s racp objective: tumour necrosis factor-α-induced protein -like (tipe ) has strong anti-inflammatory properties. however, it is unknown whether increased tipe is protective against lipopolysaccharide (lps)-induced ali. in the current study, we aimed to investigate whether increased tipe can exert protective effects in a mouse model of ali induced by lps. methods: we administered tipe adeno-associated virus (aav-tipe ) intratracheally into the lungs of mice. three weeks later, ali was induced by intratracheal injection of lps into balb/c mice. twenty-four hours later, lung bronchoalveolar lavage fluid (balf) was acquired to analyse cells and protein, arterial blood was collected for arterial blood gas analysis and the determination of pro-inflammatory factor levels, and lung issues were collected for histologic examination, transmission electron microscopy (tem), tunel staining, wet/dry (w/d) weight ratio analysis, myeloperoxidase (mpo) activity analysis and blot analysis of protein expression. results: we found that tipe overexpression markedly mitigated lps-induced lung injury, which was evaluated by the deterioration of histopathology, histologic scores, the w/d weight ratio, and total protein expression in the balf. moreover, tipe overexpression markedly attenuated lung inflammation, as evidenced by the downregulation of polymorphonuclear neutrophils (pmns) in the balf, lung mpo activity, and pro-inflammatory cytokine levels in the serum. moreover, tipe overexpression not only dramatically prevented lps-induced pulmonary cell apoptosis in mice but also blocked lps-activated jnk phosphorylation and nf-κb p nuclear translocation. conclusions: our study shows that the increased expression of aav-mediated tipe in the lungs of mice inhibits acute inflammation and apoptosis and suppresses the activation of nf-κb and jnk in a murine model of ali. acute lung injury (ali) and acute respiratory distress syndrome (ards) are life-threatening medical conditions with high morbidity and mortality rates, and they are triggered by common pathologies such as sepsis, trauma and pneumonia [ ] . ali pathophysiology is characterised by the increased permeability of the alveolar-capillary barrier, interstitial edema, neutrophil recruitment, and inflammatory stress-induced cell apoptosis [ , ] . the pathophysiology of sepsis-induced ali is characterised by complex mechanisms that involve cell inflammation, cytokine production, and abnormal apoptosis [ ] . to date, there is no effective pharmacological approach to treat ali. apoptosis, known as programmed cell death, is essential for the selective elimination of cells. however, the dysregulation of apoptosis pathways has been demonstrated to contribute to epithelial and endothelial injury, which are characteristic of ali [ ] . the inhibition of apoptosis increases the animal survival rate in an lps-induced ali model [ ] . lipopolysaccharide (lps), a main component of the outer membrane of gram-negative bacteria, is widely used to induce animal models of ali through intratracheal instillation [ ] . after binding with toll-like receptor (tlr ), lps induces the activation of downstream signalling pathways responsible for the infiltration of inflammatory cells (i.e., neutrophils) into the lungs and the production of proinflammatory cytokines [ ] . the binding of lps to tlr also induces iκb-α phosphorylation and degradation, promotes the nuclear translocation and activation of nf-κb, and subsequently leads to the excessive release of pro-inflammatory cytokines [e.g., tumour necrosis factor-α (tnf-α), interleukin (il)- β, il- ] [ ] . moreover, lps can activate jnk, a member of the mapk family. activated jnk can phosphorylate numerous mitochondrial proteins, including bcl- and bcl-xl [ , ] . subsequently, cytochrome c is released from the mitochondria, which leads to the activation of death signals [ ] . jnk activation is essential for lps-induced macrophage apoptosis during sepsis [ ] . tumour necrosis factor-α-induced protein- (tnfaip )like (tipe ), which is an essential negative regulator of tlr and tcr function, has been confirmed to inhibit caspase-mediated apoptosis [ ] . tipe has been reported to be a negative regulator of the activating protein (ap)- , nf-κb, jnk, and p pathways [ ] . whether tipe has a therapeutic effect on lps-induced ali has not been reported. the current study was designed to test the hypothesis that tipe attenuates lps-induced ali through the inhibition of lung inflammation and apoptosis, which may be associated with suppressing nf-κb and jnk activation. reagents lps (e. coli :b ) was obtained from sigma-aldrich (st. louis, mo). rabbit polyclonal bax, caspase , tipe and lamin a antibody were sourced from abcam limited. rabbit polyclonal jnk, rabbit monoclonal p-jnk and β-actin antibody were from cell signaling technology (boston, ma). rabbit polyclonal bcl- , nf-κb p , and caspase antibodies were from proteintechgroup, inc (wuhan, china). hrpconjugated secondary antibody was obtained from boster biological technology co. ltd (wuhan, china). enzymelinked immunosorbent assay (elisa) kits were purchased from abclonal technology (usa). mpo kit was obtained from nanjing biohelper co., ltd. (nanjing, china). adult male babl/c mice ( - weeks) weighing - g were purchased from the wuhan institute of biological products co., ltd. (wuhan, china). the mice were housed under specific pathogen-free (spf) conditions that provide relative humidity ranging between % and %, temperature of ± °c, a : h light-dark cycle, with free access to food and water. the animals were adapted to this environment for week before the experiment. this study was approved by medical ethics committee of renmin hospital of wuhan university and was performed in accordance with the national institutes of health guidelines for the care and use of laboratory animals. a recombinant adeno-associated virus containing the mouse tipe gene was purchased from hanheng company (hanheng biotechnology co., ltd., shanghai, china). an adeno-associated virus expressing no transgene was used as a negative control (paav-ires-zsgreen). twenty-one days before lps instillation, balb/c mice were anaesthetized using sodium pentobarbital and given × vector genomes (vg) of raav -flag-mtipe ( × vg/ml) in μl of pbs via intratracheal (i.t.) administration to induce the overexpression of pulmonary tipe . control mice were treated with the control adeno-associated virus. the efficacy of the fusion protein was evaluated by western blotting. forty mice were randomly divided into four groups (n = per group): the control group (pbs); the lps group (lps); the tipe + pbs group (aav-tipe + pbs); and the tipe + lps group (aav-tipe + lps). as previously described by matute-bello et al. [ ] , to establish the ali model, mice were anaesthetized with an intraperitoneal (i.p.) injection of pentobarbital sodium ( mg/kg), orally intubated with a sterile plastic catheter and subsequently intratracheally injected with lps (escherichia coli : b ; sigma, st. louis, mo, usa) at a dose of mg/kg body weight. control mice were intratracheally administered μl of sterile phosphatebuffered saline (pbs). twenty-four hours after lps treatment, the mice were sacrificed by an i.p. injection of pentobarbital ( mg/kg; sigma). arterial blood was collected, and then a median sternotomy was performed to expose the lungs. in each mouse, after the hilum of the right lung was ligated, the left lung was lavaged to obtain the balf. the right upper lobe of the lung was excised to calculate the lung wet/dry weight ratio. lung tissues from part of the right middle lobe of the lung were taken for he staining, immunohistochemical staining, transmission electron microscopy, and tunel staining. lung tissues from part of the right lower lobe of lung were taken for mpo activity detection and western blotting. lung tissues were snap-frozen in liquid nitrogen and stored at − °c for later analysis. lung tissues were harvested for observing morphologic alterations at h after lps or pbs administration. the right middle lobe of lung were excised, washed and fixed with % (v/v) paraformaldehyde for h at °c. lung tissues were embedded in paraffin, sectioned at μm thickness, dewaxed and rehydrated, and stained with hematoxylin and eosin (h&e) solution (hematoxylin, mhs ; eosin, ht ; sigma-aldrich, usa) to estimate inflammation in alveolar and peribronchial lesions. the stained slides were then observed with the light microscope and the digital micrographs were taken for analyzing. histologic changes were evaluated by a pathologist blinded to the experiment. the degree of lung injury was graded using a histologic ali scoring system based on histologic features, including neutrophils infiltration, hyaling membranes, proteinaceous debris, and alveolar septal thickening [ ] . sections of paraffin-embedded tissue were subjected to immunohistochemical staining. the rabbit polyclonal tipe antibody ( - -ap, proteintech group, inc. usa) was used at : . sections were incubated with primary antibody ( °c, h), followed with a poly-horseradish peroxidase anti-rabbit secondary antibody (dilution ratio : , ; a , thermo fisher scientific inc. usa) incubated ( °c, min), and diaminobenzidine (dab) was used to visualize the complex. subsequently, the sections were counterstained with hematoxylin, dehydrated, and mounted. the expression of tipe was evaluated using a light microscope (bx ; olympus corporation, tokyo, japan). the lungs were isolated and cut into - -mm cubes. lung tissue samples were fixed by immersion in . % glutaraldehyde buffer for h at °c, washed with pbs solution three times, post-fixed for h in % osmium tetroxide, dehydrated in graded solutions of ethyl alcohol ( %, %, %, % and %), and embedded in epoxy resin. ultrathin sections ( nm) that were double-stained with uranyl acetate and lead citrate were examined under a transmission electron microscope (hitachi h- , hitachi, tokyo, japan). apoptosis was detected and quantified by the tunel assay using the in situ cell death detection kit (roche diagnostics gmbh, mannheim, germany.) according to the manufacturer's protocol. apoptotic cells exhibited brownish staining in the cell nuclei. ten random sections of the lung from each mouse were analysed without knowledge of the group of mice from which the lung tissue was taken, and the apoptosis index was expressed as a percentage of tunel-positive cells. the examination was performed by two pathologists blinded to the experimental design. to obtain the balf, the lungs were lavaged three times with ice-cold pbs ( . ml) and withdrawn each time using a tracheal cannula (a total volume of . ml). the collected balf was centrifuged at ×g for min at °c, and the supernatants were collected and frozen at − °c for subsequent assays. the cell pellet was resuspended in pbs, and after excluding the dead cells by trypan blue staining, the total number of inflammatory cells in the balf was determined by counting the cells with a haemocytometer (beckman coulter, inc). to analyse the cell numbers, μl of balf was centrifuged onto slides by a cytospin (thermo fisher scientific, waltham, usa). after the slides were dried, the cells were fixed and stained using wright stain solution ( , sigma, usa) according to the manufacturer's instructions. the number of polymorphonuclear neutrophils (pmns) was classified by a laboratory technologist blinded to the experimental design to determine the percentage of neutrophils. the frozen balf supernatant was thawed and thoroughly mixed, and the total protein concentration was determined by the bca (bicinchoninic acid) method. after mice were anesthetized, the arterial blood sample was collected with a heparinized syringe from the carotid artery. the arterial blood samples were immediately injected into an abl radiometer (radiometer america, usa) to measure ph value, partial gas pressures of oxygen (pao ), pao /fraction of inspired oxygen (fio ), and carbon dioxide (paco ). the magnitude of pulmonary edema was determined by calculating the lung wet/dry weight ratio. the right upper lobe of the lung was excised, washed with phosphate-buffered saline (pbs), blotted and then weighed to obtain the "wet" weight. the lung was then placed in an oven for h at °c and weighed to obtain the "dry" weight. the wet/dry ratio was calculated to quantify the degree of pulmonary edema. ripa lysis buffer was used for lysing the lung tissues, and mg of tissue was used for each test sample. after washing with cold pbs, the tissues were resuspended in four volumes of mpo assay buffer and then centrifuged ( , ×g for min, °c). the supernatant was collected and transferred to clean tubes, which were placed on ice. the mpo activity was assayed using a myeloperoxidase activity assay kit (abcam, ab ) by measuring the absorbance of the sample at nm using a microplate reader (bio-rad laboratories, hercules, ca, usa). the specific mpo activity in the lungs is expressed as unit/mg protein. blood was collected from the carotid artery, and serum was obtained following centrifugation ( ×g for min). the levels of tnf-α, il- and il- β in serum were determined using enzyme-linked immunosorbent assay (elisa) kits according to the manufacturer's instructions (r&d systems, minneapolis, mn, usa). the absorbance was measured at nm using an elisa reader (biotek instruments, inc., usa). h after the injection of lps, the lung tissues were harvested and snap-frozen in liquid nitrogen until homogenization. the lung tissues were homogenized using a homogenizer with tissue nuclear and cytoplasmic extraction reagents (sigma-aldrich, usa), according to the manufacturer's instructions. protein concentrations were determined using the bca protein assay kit (invitrogen; thermo scientific). equal amounts of protein ( μg) were loaded per well on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page) and transferred onto polyvinylidene difluoride membranes. the resulting membranes were blocked by incubation with % skim milk in tbst at room temperature for h on a rotary shaker, followed by washing with tbst. subsequently, the membranes were incubated with specific primary antibody overnight at °c. the membranes were washed with tbst followed by incubation with horseradish peroxidase (hrp)-conjugated secondary antibody at room temperature for h. the blots were washed tbst and detected using an enhanced chemiluminescence (ecl) western blotting detection kit. the proteins bands were observed using an ecl western blotting analysis system (bio-rad laboratories, inc., usa) and quantified by densitometry (image lab software version . . ). the data are expressed as the mean ± sem. the statistical analysis was performed using graphpad prism (version . ; graphpad software, inc., la jolla, ca, usa) by one-way analysis of variance (anova) followed by dunnett's least significant difference post hoc test. p < . was considered statistically significant. in the present study, we first investigated the effects of tipe overexpression on lung histopathology and function in mice challenged with lps (fig. ) . the lung tissues were harvested h after lps stimulation and subjected to h&e staining. there were no obvious histological changes in the lung tissues of the mice group a and group c. significant pathological changes, including pulmonary capillary congestion, pulmonary interstitial edema, mass inflammatory cell infiltration into the alveolar space and lung interstitium, and alveolar wall thickening, were observed in the lung tissues of lps-challenged mice. a subsequent western blot assay further demonstrated that tipe protein levels were significantly increased in the lung tissues of aav-tipe infected mice. treatment with aav-tipe significantly attenuated the histopathological changes induced by lps (fig. a) . in addition, a scoring system was used to assess the degree of lung injury. as shown in fig. b , the quantitative scoring of histological lung injury in the ali mice was markedly increased compared with that in the control group h after lps challenge. however, recombinant adenoassociated virus-mediated tipe overexpression markedly decreased the pathological scores compared with those of the lps group. transmission electron microscopy was used to examine the ultrastructural changes of lung tissues (fig. ) . in the control group, there were abundant mitochondria with regularly arranged mitochondrial cristae and homogeneous matrix. lungs from lps-treated mice exhibited a disordered arrangement of mitochondrial cristae, and the number of lamellar bodies was decreased. in the tipe + lps group, the pathologic damage was significantly alleviated compared with that in the lps group. we investigated the effects of tipe overexpression on lung cell apoptosis in lps-challenged mice by tunel staining. tunel staining revealed few apoptotic cells in the lungs of the control group. after the administration of intratracheal lps, unlike in control mice, numerous lung cells were strongly positive for tunel staining. however, in the lung tissues of aav-tipe -treated mice, a few of the lung cells were tunel-positive (fig. a) . for quantitative measurement, the percentage of tunel-positive lung cells was analysed for each specimen (fig. b) . the results showed that lps-challenged mice showed a significant increase in the number of apoptotic cells, which was reduced by aav-tipe treatment. these data indicated that tipe overexpression inhibited apoptosis in the lung after lps challenge. the lung w/d ratio and balf protein concentration are two commonly used indicators of pulmonary vascular permeability, which is an important characteristic of ali/ards. lps-challenged mice showed a significant increase in the lung w/d ratio (fig. a) and balf protein concentration (fig. b) when compared with those of the control group, and these levels were decreased by aav-tipe treatment. we also detected the ratio of the number of pmns relative to the number of total cells in the balf and the activity of mpo, an indicator of neutrophil infiltration, in the lung h after lps administration. compared with those in the control group, the pmn/total cell ratio in the balf (fig. c) and lung mpo activity (fig. d) in lps-challenged mice were dramatically increased, and these levels were inhibited by aav-tipe treatment. these results suggest that tipe overexpression attenuates lung edema and inflammation in lps-challenged mice. as shown in fig. , the arterial blood gas analysis of mice that received lps treatment showed significant changes compared with that of the control group, with the ph (fig. a) , partial pressure of arterial oxygen (pao ) (fig. b) and pao /fio (fig. c) decreasing and the partial pressure of arterial carbon dioxide (paco ) (fig. d) increasing. the pao /fio of the mice in the lps group however, tipe overexpression effectively mitigated the change in arterial oxygenation. compared with those in the lps group, the ph and pao were increased, and paco was decreased in the tipe + lps group. the pao /fio in the tipe + lps group recovered to normal levels and was higher than that in the lps group. as depicted in fig. , we found that the levels of the proinflammatory cytokines tnf-α (fig. a) , il- ( fig. b) and il- β (fig. c) in the serum were significantly increased h after lps challenge in mice. aav-tipe treatment significantly downregulated the levels of pro-inflammatory cytokines in the serum of lps-challenged mice. previous studies have shown that tipe is a negative regulator of the nf-κb, jnk, and p mapk pathways in macrophages [ ] . the effect of tipe on nf-κb and jnk activation was assessed in mice after lps challenge. to evaluate the effect of tipe overexpression on lung cell apoptosis in lps-challenged mice, the protein expression levels of anti-apoptotic proteins (bcl- ) and proapoptotic proteins (bax, cleaved caspase- , cleaved caspase- ) in the lung tissues were analysed by western blotting. as shown in fig. , compared with control mice, mice with lps-induced ali exhibited decreased bcl- expression and increased bax, bax/bcl- , cleaved caspase- , and cleaved caspase- protein expression in lung tissue samples, whereas the changes in the expression of the proteins were reversed by aav-tipe treatment. in addition, lps stimulation markedly increased nuclear nf-κb p and phosphorylated jnk (p-jnk) expression and decreased tipe expression in the lungs compared with that in the control group. however, adeno-associated virus-mediated tipe overexpression suppressed the increase in the expression of nuclear nf-κb p and p-jnk induced by lps. the expression of the tipe protein was significantly increased in the alveolar epithelium after aav-tipe administration (fig. ) . the expression of tipe was decreased h after lps challenge. the tipe + lps group exhibited higher expression of tipe compared with that in the lps group. ali and ards are the two main causes of acute lung failure, which is characterised by high morbidity and mortality and for which effective therapeutic strategies are lacking [ ] . thus, identifying novel therapeutic treatments for ali is urgently needed. in the current study, we found that tipe overexpression attenuated lps-induced ali in mice via its anti-inflammatory and anti-apoptotic effects. first, tipe overexpression significantly improved lps-induced lung injury, as evidenced by changes in histopathology, the lung w/d weight ratio, balf protein concentration, and arterial blood gas. second, tipe overexpression decreased inflammatory cell infiltration into the lungs and pulmonary cell apoptosis. third, the levels of pro-inflammatory cytokines in the serum of lps-challenged mice were reduced by aav-tipe treatment. fourth, tipe overexpression inhibited lps-induced nf-κb p nuclear translocation and jnk phosphorylation in the lungs. finally, tipe overexpression prevented lung cell apoptosis by downregulating the expression of pro-apoptotic proteins (bax, cleaved caspase- , and cleaved caspase- ) and upregulating the expression of an anti-apoptotic protein (bcl- ) in the lungs of lps-challenged mice. in conclusion, these results demonstrate that tipe overexpression ameliorates lps-induced ali via reducing fig. tipe overexpression reduced lps-induced proinflammatory cytokine levels in mice. the mice were treated as described in fig. . blood sample was collected for measuring the pro-inflammatory cytokines levels at h after lps or pbs administration. a tnf-α; b il- ; c il- β. the data are presented as mean ± sem. n = /group, * p < . versus pbs group; # p < . versus lps group lung inflammation and apoptosis, which may be associated with decreased nf-κb and jnk activity. the characteristics of ali/ards can be reproduced by the intracerebral administration of lps [ ] , which acts via tlr to induce the production of inflammatory cytokines, resulting in damage to microvascular and epithelial integrity and increased alveolar and interstitial edema [ ] . in this study, we successfully produced a mouse model of ali by the intratracheal administration of lps. we found that acute lung injury, which was characterised by pathological changes in lung tissue (by h&e staining and tem), increased lung water content, the infiltration of inflammatory cells, and pulmonary dysfunction was present h after lps administration. however, aav-tipe treatment markedly reduced lps-induced lung injury. tipe is a critical regulator of immune homeostasis that negatively regulates t cell receptor (tcr) and toll-like receptor (tlr) signalling [ , ] . tipe deficiency in mice induces foetal inflammatory diseases, and tipe downregulation in humans causes systemic autoimmunity [ , ] . moreover, tipe has also been identified as an apoptosis regulator that contains a death effector domain (ded) and is able to inhibit the activities of the apoptotic enzymes caspase- and caspase- [ ] . sun et al. demonstrated that the deletion of tipe amplifies jnk and p mapk phosphorylation and nf-κb activation, suggesting that tipe is a negative regulator of jnk, p mapk and nf-κb [ ] . in lps-induced ali, lps is recognized by tlr and subsequently promotes the activation of nf-κb [ ] , which is a pivotal transcription factor in the pathogenesis of ali. when activated, nf-κb p translocates to the nucleus, where it triggers the transcription of inflammatory cytokines, such as tnf-α, il- , and il- β [ ] . the inactivation of nf-κb p inhibits inflammation-induced inflammatory cell infiltration, edema, and pro-inflammatory cytokine production in the lungs [ ] . in the current study, tipe fig. tipe overexpression suppressed the activation of jnk and nf-κb as well as the protein expression of genes involved in apoptosis and lung injury induced by lps. the mice were treated as described in fig. . lung tissues were collected for western blotting analysis at h after lps or pbs administration. a western blotting analysis of protein expression of bax, cleaved caspase- , cleaved cas-pase- , bcl- , jnk, p-jnk, nuclear nf-κb p , and tipe ; b-h the relative ratio of bax, cleaved caspase- , cleaved caspase- , bcl- , p-jnk, nuclear nf-κb p , and tipe protein expression. the data are presented as mean ± sem. n = /group, * p < . versus pbs group; # p < . versus lps group overexpression significantly inhibited the lps-induced levels of tnf-α, il- , and il- β in the serum-induced and concomitantly decreased nf-κb activation. therefore, the inhibitory effect of tipe overexpression on lps-induced increases in the levels of pro-inflammatory cytokines may be ascribed to its suppression of nf-κb activation. liu mw et al. found that, after lps challenge, the increased expression of tipe is associated with the inhibition of nf-κb expression, the production of tnf-α and il- and decreased ros activity in raw . cells [ ] . in another study by liu et al., melilotus extract was shown to have protective effects against clp-induced lung injury, possibly by upregulating the expression of tipe [ ] . thus, tipe may have a protective effect against sepsis-induced lung injury and lps challenge in cells. in our present study, aav-tipe treatment markedly reduced lps-induced lung injury. pulmonary cell apoptosis also plays a critical role in the pathogenesis of ali [ ] . lps-induced cell apoptosis may be partly dependent on the mitochondria pathway [ ] . jnk, a member of the mapk family, is critical for lps-induced apoptosis in macrophages [ ] . lps triggers jnk phosphorylation, which subsequently mediates the phosphorylation of anti-apoptotic proteins (bcl- /bcl-xl) and upregulates proapoptotic bax. when the bax/bcl- ratio is elevated, the mitochondrial membrane potential changes, causing the release of cytochrome c into the cytosol from the mitochondria [ ] and resulting in the activation of caspase- and then caspase- to induce apoptosis [ ] . several previous studies have shown that tipe contains a death effector domain (ded) and that the overexpression of tipe is associated with enhanced survival and the inhibition of caspasemediated apoptosis through the inhibition of the activities of the apoptotic enzymes caspase- and caspase- [ ] [ ] [ ] . in addition, the depletion of tipe enhances cell death [ , ] . in the current study, we found that aav-tipe administration remarkably inhibited the expression of pro-apoptotic proteins (bax, cleaved caspase- , and cleaved caspase- ) and jnk phosphorylation and restored the expression of the antiapoptotic protein bcl- . these findings suggest that epithelial tipe drives the protective effects against lps-induced injury and that the inhibitory effect of tipe overexpression on lps-induced cell apoptosis may be attributed to its suppression of jnk activation. together, tipe exerts a protective effect against lpsinduced ali possibly through its anti-inflammatory and antiapoptotic activities, and it may be a potential therapeutic target for ali. in conclusion, our study demonstrated that tipe expression was significantly decreased in lung tissues in ali mice after lps challenge. moreover, adeno-associated virus-mediated tipe overexpression remarkably inhibited inflammation and cell apoptosis induced by lps. we also provided evidence that the anti-inflammatory and anti-apoptotic effects of tipe might at least partly involve the inhibition of nf-κb and jnk activation, respectively. therefore, our results indicate that tipe might be useful as a potential therapeutic target for sepsis-induced ali. fig. immunohistochemical staining of tipe protein expression in the lung. the mice were treated as described in fig. . lung tissues were collected for immunohistochemical staining at h after lps or pbs administration. representative images of lung tissues under light microscope (immunohistochemical staining, magnification, × ) the acute respiratory distress syndrome lfg- , a newly synthesized flavonoid, attenuates lipopolysaccharide-induced acute lung injury and inflammation in mice tylvalosin exhibits anti-inflammatory property and attenuates acute lung injury in different models possibly through suppression of nf-kappab activation incidence and outcome of acute lung injury and acute respiratory distress syndrome in the surgical intensive care unit acute lung injury: apoptosis in effector and target cells of the upper and lower airway compartment protection from lethal apoptosis in lipopolysaccharide-induced acute lung injury in mice by a caspase inhibitor preventive and therapeutic effects of thymol in a lipopolysaccharide-induced acute lung injury mice model macrophages and myeloid dendritic cells, but not plasmacytoid dendritic cells, produce il- in response to 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in septic mice in vivo antioxidant treatment suppresses nuclear factor-kappa b activation and neutrophilic lung inflammation the role of apoptosis in the pathophysiology of acute respiratory distress syndrome (ards): an up-to-date cell-specific review glutathione supplementation attenuates lipopolysaccharide-induced mitochondrial dysfunction and apoptosis in a mouse model of acute lung injury the release of cytochrome c from mitochondria: a primary site for bcl- regulation of apoptosis identification of a novel tumor necrosis factor-alpha-inducible gene, scc-s , containing the consensus sequence of a death effector domain of fas-associated death domain-like interleukin- beta-converting enzyme-inhibitory protein nuclear factorkappa b-inducible death effector domain-containing protein suppresses tumor necrosis factor-mediated apoptosis by inhibiting caspase- activity expression of scc-s , an antiapoptotic molecule, correlates with enhanced proliferation and tumorigenicity of mda-mb cells novel tumor necrosis factor alpha-regulated genes in rheumatoid arthritis role of scc-s in experimental metastasis and modulation of vegfr- , mmp- , and mmp- expression publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgements this work was supported by research grants from the national natural science foundation of china (no. ). conflict of interest the authors declare no conflict of interest. key: cord- -amo e h authors: yang, zhongwei; deng, yuxiao; su, diansan; tian, jie; gao, yuan; he, zhengyu; wang, xiangrui title: tlr as receptor for hmgb -mediated acute lung injury after liver ischemia/reperfusion injury date: - - journal: lab invest doi: . /labinvest. . sha: doc_id: cord_uid: amo e h acute lung injury (ali) frequently occurs after liver transplantation and major liver surgery. proinflammatory mediators released by damaged liver after liver ischemia/reperfusion (i/r) injury might contribute to this form of ali, but the underlying mechanisms have not been well characterized. high-mobility group box protein (hmgb ), a recently identified proinflammatory cytokine, was found to be significantly higher in the serum after liver i/r injury. this study investigated whether hmgb was involved as a stimulating factor, and whether its downstream toll-like receptor (tlr ), p mitogen-activated protein kinase (p mapk), and activator protein- (ap- ) signaling pathways act as mediators in the development of liver i/r injury-induced ali. extensive ali and lung inflammation was induced in a rat model of liver i/r injury. serum hmgb was significantly higher after liver i/r injury, and more importantly, expression of hmgb mrna and protein in the lung tissue was also significantly increased. we further found that liver i/r injury enhanced the expression of tlr mrna and protein, and the activity of p mapk and ap- in the lung tissue. inhibition of tlr expression in the lung tissue by infection with pgcsil-gfp-lentivirus-expressing small hairpin rnas targeting the tlr gene (tlr -shrna lentivirus) significantly attenuated ali, lung inflammation, and activity of p mapk and ap- in the lung tissue. these findings indicate that hmgb might contribute to the underlying mechanism for liver i/r injury-induced ali and that its downstream tlr , p mapk, and ap- signaling pathways are potentially important mediators in the development of ali. acute lung injury (ali) and its more severe form, the acute respiratory distress syndrome (ards), are common complications of liver transplantation and major liver surgery, and thereby significantly contributed to perioperative morbidity and mortality. , liver i/r injury may account for this form of ali, but the underlying mechanisms have not been fully elucidated. since the lungs are the first capillary bed that is reached by the blood after leaving the hepatic circulation, one proposed mechanism is that ali may be induced by proinflammatory mediators released from the injured liver into system circulation. [ ] [ ] [ ] [ ] [ ] high-mobility group box protein (hmgb ), originally identified as a dna-binding protein, is a proximal trigger that is sufficient to induce the release of other cytokines classically associated with mediating inflammatory responses, including tumor necrosis factor-a (tnf-a), interleukin- b (il- b), and interleukin- (il- ). interestingly, hmgb was rapidly mobilized and released into system circulation by hepatocytes in the setting of liver i/r injury. , hmgb has also been reported to participate in ali and lung inflammatory response induced by endotoxin, ventilator, and hemorrhage. [ ] [ ] [ ] for example, hmgb expression in the lung was found to be increased within h of hemorrhage, and then remained elevated for more than h after blood loss. blockade of hmgb by the administration of neutralizing antibodies prevented hemorrhage-induced increases in pulmonary levels of proinflammatory cytokines, including keratinocyte-derived chemokine, il- , and il- b. however, whether hmgb participated in liver i/r injuryinduced ali is still unclear. extracellular hmgb functions as a damage-associated molecular pattern molecule and activates proinflammatory signaling pathways by activating pattern recognition receptors, including toll-like receptor (tlr ) and the receptor for advanced glycation end-products (rage) , . while rage has only a minor role in macrophage activation by hmgb , the triggering of the tlr signaling pathway by hmgb leads to the activation of p mitogen-activated protein kinase (p mapk) through the accessory protein myd , which subsequently activates activator protein- (ap- ), and thus regulates inflammatory cytokine expression. , excessive cytokine-mediated inflammation has a fundamental role in the pathogenesis of ali. , in addition, tlr is widely distributed in alveolar and bronchial epithelial and vascular endothelial cells in the lung. [ ] [ ] [ ] we have previously demonstrated that the activation of tlr and its downstream intracellular signal-transduction pathways is crucial to lipopolysaccharide (lps)-induced ali. [ ] [ ] [ ] other studies also reported that tlr mutant (c h/hej) mice developed less ali after unresuscitated hemorrhagic shock or being challenged with lps. , conversely, the overexpression of tlr in transgenic mice aggravated ali and pulmonary inflammation, endothelial cell damage, and the recruitment of neutrophils to the lung. the hmgb -tlr signaling pathway has been strongly implicated in the pathogenesis of small intestinal damage induced by nonsteroidal anti-inflammatory drugs and other forms of ali. , thus, we hypothesized that hmgb may be involved in ali and lung inflammation, and its downstream tlr , p mapk, and ap- signaling pathways may act as potentially important mediators in the development of ali. in this study, a rat liver i/r injury model was used to induce ali. the levels of serum and pulmonary hmgb were examined. to study the role of tlr and its downstream p mapk and ap- signaling pathways in the pathogenesis of liver i/r injury-induced ali, tlr -small hairpin rna (shrna) lentivirus were used to inhibit tlr expression in rat lung tissue. all animal work was approved by the animal care and use committee of the shanghai jiaotong university school of medicine. all animals received humane care in accordance with the guidelines for animal care published by the united states' national institutes of health (nih) for animal care (guide for the care and use of laboratory animals, department of health and human services, nih publication no. - , revised ). shrna lentivirus vector of the tlr gene shrna targeting rattus norvegicus tlr gene (shtlr ; genbank accession no.: nm_ ) and non-targeting shrna (shnt) sequences were designed, chemically synthesized, and inserted into the pgcsil-gfp by shanghai genechem co. ltd (shanghai, china). the targeted sequences of tlr and non-targeting negative control were -aacctagaacatgtggatctt- and -ttctccgaa cgtgtcacgt- , respectively. then, the recombinant virus was packaged using lentivector expression systems (shanghai genechem co. ltd). the correct clone was identified by agei and ecori restriction digestion and sequenced. transfection efficiency of lentivirus was investigated by bioluminescence imaging, and best transfection efficiencies were observed at days after lentivirus infection (data not shown). in this study, a total of male sprague-dawley rats ( - g), purchased from sino-british sippr/bk lab (shanghai, china), were randomly divided into six groups (n ¼ per group): liver i/r injury were performed according to the method described by yamamoto et al with minor modification. briefly, rats were anesthetized with pentobarbital sodium ( mg/kg) and the depth of anesthesia was checked by monitoring the pedal withdrawal reflexes and the corneal reflex. when the mouse was sufficiently anesthetized, a -cm midline abdominal incision was made. vascular structures to the left and median lobe liver were identified and clamped for min using a vascular clamp. after min of warm ischemia, the clamps were removed to allow reperfusion for h. the rats received approximately ml of ns intraperitoneally before closure of the incision. , , and, the rats were allowed to regain consciousness during tlr -mediated ali after liver i/r injury z yang et al reperfusion period. the sham procedure was performed identically without vascular occlusion. as a physiologic measure of lung function, gas exchange was calculated as pao /fio (p/f ratio), where pao is the partial pressure of oxygen in the arterial blood and fio is the fraction of inspired oxygen and is equal to % by experimental design. briefly, the rats were sufficiently anesthetized and breathing spontaneously of room air at baseline and h after liver reperfusion. then, the arterial blood was obtained ( . ml) in heparinized syringes from femoral artery. to get pao values, the sample was measured with a gem premier gas analyzer (instrumentation laboratory, milan, italy) immediately. changes in the lung tissue were examined morphologically as described previously. briefly, the lung tissue were immersed in a % solution of paraformaldehyde in pbs and were fixed for h. sections of mm thick were cut, deparaffinized, rehydrated, and stained with hematoxylin and eosin. the degree of microscopic injury was graded on a scale of - ( , absent and appears normal; , light; , moderate; , strong; , intense) for interstitial edema and neutrophil infiltration. moreover, a total lung injury score was calculated as the sum of the two components (three sections from each lung). evaluations were performed by a pathologist blind to experimental groups using an olympus ch microscope. the levels of serum hmgb were measured with commercial hmgb enzyme-linked immunosorbent assay (elisa) kit & (type b; shino-test corporation, tokyo, japan). blood samples ( ml) were collected from femoral artery before liver ischemia and h after liver reperfusion, and centrifuged at g for min at c to collect serum. the serum was kept at À c until analyzed. the levels of il- b in the lung tissue were measured with commercial elisa kits (r&d systems, minneapolis, mn, usa). lung tissue were perfused with ice-cold pbs at h after liver i/r injury and washed three times in pbs, homogenized, centrifuged at g at c for min, and the supernatant was obtained. the supernatant fraction was kept at À c until analyzed. before elisa analysis, protein was quantified with a bca protein assay kit (tiangen, beijing, china). protein extraction and concentration determination were the same as described in elisa. samples of mg were run on % sds-page. the proteins were then electrotransferred onto nitrocellulose filter membranes. the membranes were incubated in pbs containing % non-fat dry milk for h at c. the blots were then incubated for h at c with primary antibodies for hmgb ( : ; abcam, usa), tlr ( : ; cell signaling technology, usa), phospho-p ( : , abcam) or b-actin ( : ; epitomics, usa), and then incubated with irdye cw-conjugated goat antirabbit secondary antibody ( : ; rockland, usa) for h at c. the infrared fluorescence image was obtained using odyssey infrared imaging system (li-cor bioscience, lincoln, ne, usa), and the bands were quantified using image-pro plus . software. experiments were repeated at least three times and the relative expression of the target protein was normalized to the level of b-actin in the same sample. total rna was extracted from the lung tissue (n ¼ each group) using the trizol method (invitrogen, carlsbad, ca, usa). with the use of mg total rna, the first strand of cdna was synthesized by the amv enzyme in a -ml reaction mixture (takara, otsu, japan). utilizing ml of reverse transcriptase products, real-time quantitative pcr was performed in a final volume of ml using the gene-specific primers. the following primers designed with primer express software were used: rat hmgb - -tcccctactaaagac ctgagaatg- (sense) and -tttatccgctttccttgt atctg- (antisense); rat tlr - -cagggagcacgagg cttctaacc- (sense) and -cttgtgccctgtgaggtc gttga- (antisense); and rat gapdh- -agacctctat gccaacacagtgc- (sense) and -gagccaccaatcca cacagagt- (antisense). amplification was processed as follows: c, s, cycle; c, s and c (hmgb ) or c (tlr ), s for cycles, and then the melting curve was determined. gene transcripts were quantified with sybr premix ex taq kit (takara). data were calculated by the À ddct method and presented as fold change of transcripts for the hmgb and tlr gene in the lung tissue of other groups compared with sham-operated rats (defined as . -fold). rat gapdh was used as an internal control. the relative expression of the target gene was normalized to the level of gapdh in the same cdna. nuclear extracts of the lung tissue were prepared with the pierce nuclear and cytoplasmic extraction reagent kit (pierce) according to the manufacturer's instructions and quantified using the bca kit (pierce). electrophoretic mobility shift assay (emsa) was performed as described previously with minor modification. in brief, the ap- consensus oligonucleotide probe ( -cgcttgatgactcag ccggaa- ) was end-labeled with dig (roche applied science, indianapolis, in, usa). after incubation of the nuclear protein ( mg) with the dig-labeled oligonucleotide probe, the dna-protein complexes were resolved on an % non-denaturing polyacrylamide gel, electrophoresed, dried, and electrotransferred to nitrocellulose filter membranes. chemiluminescence detection of dig-labeled dna-protein complexes on the nylon membranes was detected using tlr -mediated ali after liver i/r injury z yang et al hyperfilm ecl (amersham, ge health care, piscataway, nj, usa). a quantitative densitometric analysis was performed using quantity one . , and then normalized to the control group to determine the fold change. all values are expressed as means±s.d. results of serum hmgb and p/f ratio were analyzed by paired student's t-test. all the other results were analyzed by anova, followed by tukey's method. two-sided po . was considered statistically significant. to investigate tlr expression in the lung tissue, real-time quantitative pcr and western blot analysis were used to detect tlr mrna and protein expression in the lung tissue at h after operation, respectively (n ¼ ). compared to lung tissue from the shnt group, tlr mrna expression decreased significantly in the lung tissue from the shtlr group, which indicated a valid inhibition of tlr in the lung tissue by tlr -shrna lentivirus ( . ± . vs . ± . ). at h after operation, liver i/r injury significantly increased tlr mrna expression in the lung tissue from i/r and shnt þ i/r groups compared to the control group. however, only a slight increase of tlr mrna was observed in the lung tissue from shtlr þ i/r after liver i/r injury, and the increase was not significant. when compared to the shnt þ i/r group, the expression of tlr mrna in the lung tissue from the shtlr þ i/r group were significantly lower (figure a) . similar results were found in protein expression of tlr (figure b) . to examine whether ali was induced by liver i/r injury, we performed lung histological analysis at h after operation (n ¼ in each group). as shown in figure a , normal lung structure was found in the control group. lung tissue from the i/r group displayed a feature of lung injury, including alveolar septal thickening, interstitial edema, and vascular congestion, as well as a mild neutrophil infiltration in the interstitium. no pathological changes were observed in the lung tissue from rats of shnt and shtlr groups. while lung tissue from rats receiving shnt þ i/r treatment displayed similar injury compared to those from the i/r group, coadministration of shtlr markedly attenuated the liver i/r injury-induced pulmonary neutrophil infiltration and interstitial edema. as shown in figure b , these results were also confirmed by histological scores. p/f ratio, one of the markers for lung injury, was determined at baseline and h after operation (figure c ; n ¼ in each group). all groups displayed normal p/f ratio at baseline. compared to baseline, p/f ratios were significantly lower after liver i/r injury in both i/r and shnt þ i/r groups, whereas not found in the shtlr þ i/r group. in addition, the levels of il- b in the lung tissue were used to describe the extent of lung inflammatory response. expression of il- b in the lung tissue from i/r, shnt þ i/r, and shtlr þ i/r groups increased significantly h after surgery. interestingly, il- b in the lung tissue from the shtlr þ i/r group displayed a lower level at h after liver i/r injury when compared to the shnt þ i/r group (n ¼ ; figure d ). figure expression of toll-like receptor (tlr ) in the lung tissue from rats at h after liver ischemia/reperfusion (i/r) injury or sham operation. tlr mrna (a, real-time pcr) and protein (b, western blot) expression levels in the lung tissue from rats at h after liver i/r injury or sham operation. *po . compared with the control group; w po . compared with the non-targeting short hairpin rna (shnt) group; # po . compared with the shnt þ i/r group. relative levels of tlr mrna to the control group are normalized to glyceraldehyde -phosphate dehydrogenase (gapdh), and relative levels of tlr protein to the control group are normalized to b-actin. data are expressed as means±s.d.. blots shown here are from a representative experiment repeated three times with similar results (n ¼ per group). control, control group; i/r, liver i/r injury group; shnt, negative control group; shnt þ i/r, positive control group; shtlr , tlr inhibition group; shtlr þ i/r, tlr inhibition group with liver i/r injury treatment. to investigate whether hmgb was released to the serum after liver i/r injury, serum hmgb levels were measured (figure a, n ¼ ) . at baseline, there were no significant differences among these groups. serum hmgb levels in i/r, shnt þ i/r, and shtlr þ i/r groups increased significantly at h after liver i/r injury when compared to its corresponding baseline. and, no significant differences were found between shnt þ i/r and shtlr þ i/r groups after liver i/r injury. sham operation did not increase serum hmgb levels in control, shnt, and shtlr groups. we also examined relative mrna and protein expression of hmgb in the lung tissue. as is shown in figure b , relative levels of hmgb mrna in the lung tissue from i/r, shnt þ i/r, and shtlr þ i/r groups increased significantly at h after liver i/r injury when compared to the control group, respectively. western blot analysis showed that protein levels of hmgb in the lung tissue at h after operation were in parallel with mrna expression (figure c ). both mrna and protein levels of hmgb in the lung tissue were similar between shnt þ i/r and shtlr þ i/r groups. relative expression of phospho-p in lung tissues at h after operation was also measured by western blot analysis (figure a ). compared to the control group, protein levels of phospho-p in the lung tissue from i/r and shnt þ i/r groups increased significantly. however, only a slight increase of phospho-p was observed in the lung tissue from shtlr þ i/r after liver i/r injury, and the increase was not significant when compared to the control group. in addition, the expression of phospho-p protein in the lung tissue (c) pao /fio (p/f) ratios were obtained at baseline and h after liver i/r injury; (d) interleukin (il)- b levels in the lung tissue from rats at h after liver i/r injury. *po . , **po . compared to the control group; # po . compared to non-targeting short hairpin rna (shnt) þ i/r group; y po . compared to baseline. data are expressed as means ± s.d. (n ¼ per group). control, control group; i/r, liver i/r injury group; shnt, negative control group; shnt þ i/r, positive control group; shtlr , tlr inhibition group; shtlr þ i/r, tlr inhibition group with liver i/r injury treatment. tlr -mediated ali after liver i/r injury z yang et al figure expression of high-mobility group box protein (hmgb ) in serum and lung tissue from rats at h after liver ischemia/reperfusion (i/r) injury or sham operation. serum hmgb levels at baseline and h after liver i/r injury (a); relative mrna (b) and protein (c, western blot) expression in the lung tissue at h after liver i/r injury were determined by real-time quantitative polymerase chain reaction (pcr) and western blot, respectively. *po . , **po . compared to the control group; y po . compared to baseline. relative levels of hmgb mrna to the control group are normalized to glyceraldehyde -phosphate dehydrogenase (gapdh), and relative levels of hmgb protein to the control group are normalized to b-actin. data are expressed as means ± s.d. blots shown here are from a representative experiment repeated three times with similar results (n ¼ per group). control, control group; i/r, liver i/r injury group; shnt, negative control group; shnt þ i/r, positive control group; shtlr , tlr inhibition group; shtlr þ i/r, tlr inhibition group with liver i/r injury treatment. activation of p mitogen-activated protein kinase (p mapk) and activator protein- (ap- ) in the lung tissue from rats at h after liver ischemia/reperfusion (i/r) injury or sham operation. (a) expression of phospho-p in the lung tissue from rats at h after liver i/r injury was determined by western blot. relative levels of phospho-p protein to the control group are normalized to b-actin. blots shown here are from a representative experiment repeated three times with similar results. (b) ap- activity was evaluated with electrophoretic mobility shift assay (emsa) with representative image shown. *po . , **po . compared to the control group; # po . compared to the shnt þ i/r group. data are expressed as means ± s.d. (n ¼ per group). control, control group; i/r, iver i/r injury group; shnt, negative control group; shnt þ i/r, positive control group; shtlr , tlr inhibition group; shtlr þ i/r, tlr inhibition group with liver i/r injury treatment. tlr -mediated ali after liver i/r injury z yang et al from the shtlr þ i/r group were significantly lower when compared to the shnt þ i/r group. the results from emsa showed that ap- activity was low in nuclear extracts of lung tissues from control, shnt, and shtlr rats. ap- activity increased significantly in the i/r group, shnt þ i/r group, and shtlr þ i/r group when compared to the control group. compared to the shnt þ i/r group, ap- was less activated in the lung tissue from the shtlr þ i/r group (figure b) . discussion ali and ards are common complications after liver transplantation and after major liver surgery, and thereby significantly contribute to perioperative morbidity and mortality. , this form of lung injury has been attributed to hepatic i/r injury, but the underlying mechanisms have not been fully elucidated. [ ] [ ] [ ] the aim of this study was to investigate whether hmgb was involved as a stimulating factor, and whether tlr , p mapk, and ap- signaling pathways act as mediators in the development of liver i/r injury induced ali. we found that levels of serum and lung hmgb increased significantly after liver i/r injury; that tlr , p mapk, and ap- signaling pathways were activated in the pathologic process of liver i/r injuryinduced ali; and that ali and lung inflammatory response can be attenuated by knockdown of tlr in the lung tissue. these results suggest that hmgb might be involved as a stimulating factor in liver i/r injury-induced ali, and its downstream tlr , p mapk, and ap- signaling pathways mediate this form of ali. to investigate whether liver i/r injury can induce lung inflammatory response and ali, we used a classical rat liver i/r injury model. features characteristic of lung injury were found after liver i/r injury by histological analysis, which was also confirmed by significantly decreased p/f ratios. pulmonary levels of il- b were also significantly higher after liver i/r injury compared to the control group, which indicated lung inflammatory response. similar results were found in previous studies; liver i/r injury induced lung edema, pulmonary neutrophil accumulation, and inflammatory chemokine expression. , , in this study, p/f ratio was used as an index to characterize the ali, and has not been reported previously. as stated above, our findings strongly suggested that liver i/r injury would ultimately lead to lung inflammatory response and ali. it has been demonstrated that liver i/r injury results in the activation of several inflammatory pathways, such as those for free radicals, cytokines, and neutrophil-mediated tissue damage. the lung is frequently damaged by proinflammatory mediators released from the injured liver after liver ir, as the lungs are the first capillary bed that is reached by the blood after leaving the hepatic circulation. [ ] [ ] [ ] [ ] [ ] was recently found to act as a potent proinflammatory cytokine and participated in the development of systemic inflammatory response, when it was released into the extracellular environment. , hmgb was also found to be involved in many other types of ali, such as endotoxin, ventilator, and hemorrhage-induced ali. [ ] [ ] [ ] interestingly, hmgb was reported to rapidly mobilize and release into system circulation from damaged liver in the setting of liver i/r injury. , previous studies also demonstrated that serum hmgb level remained elevated until h after reperfusion, and that the removal of excessive serum hmgb with adsorption column improved the lung injury score. whether or not hmgb was transported to the lung and whether downstream signaling pathways in lung were activated was not investigated. in this study, similar results were obtained; serum hmgb increased significantly after liver i/r injury. more importantly, hmgb protein was found to be significantly higher in the lung tissue, which suggests that hmgb might be transported to lung tissue by systemic circulation. in addition, hmgb was reported to be released from macrophages and monocytes after exposure to proinflammatory cytokines. [ ] [ ] [ ] [ ] given that proinflammatory cytokines might be transported to lung tissue after liver i/r injury, - lung cells might be activated by proinflammatory cytokines and then release hmgb . , for this reason, we examined hmgb mrna in the lung tissue, and found that hmgb mrna was significantly increased in the lung tissue after liver i/r injury. these results suggest that hmgb , both released passively from damaged liver and actively from lung cells, might be involved in activating lung inflammatory response in liver i/r injury-induced ali. future experiments using an hmgb -neutralizing antibody may help reveal its role in liver i/r injury-induced ali. the toll-like receptor family is one of the best-characterized pattern recognition receptor families and is responsible for sensing invading pathogens. hmgb activates inflammatory pathways by stimulating tlr in many types of tissue injuries. , our previous work, [ ] [ ] [ ] as well as the work of others, [ ] [ ] [ ] indicated that tlr mediates many forms of ali, such as hemorrhagic shock-induced ali and lps-induced ali. overexpression of tlr in the lung tissue amplifies the severity of lps-induced ali. in this study, tlr mrna and protein expression increased significantly in the lung tissue h after liver i/r injury. interestingly, liver i/r injury-induced lung damage, decrease of p/f ratio, and lung inflammatory response were significantly attenuated by knockdown of pulmonary tlr . to our knowledge, this is the first time to report that tlr was involved in the pathogenesis of liver i/r injury-induced ali. we further investigated whether pulmonary tlr activation leads to activation of its downstream p mapk and ap- signaling pathways, which are considered to be important in regulating inflammatory response. , in this work, activation of p mapk in the lung tissue enhanced significantly after liver i/r injury, and the increases were inhibited by tlr knockdown, which implies that p mapk tlr -mediated ali after liver i/r injury z yang et al signaling pathway might contribute to liver i/r injuryinduced ali. activation of ap- in the lung tissue also increased after liver i/r injury, which was suppressed by tlr knockdown, indicating the involvement of ap- in tlr -mediated lung inflammation after liver i/r injury. activation of p mapk by tlr was reported to upregulate the inflammation-related genes and stimulated the release of il- , tnf, cxcl , and cxcl protein in the lungs. tlr mediated-activation of p mapk was also contributed to hmgb -induced human lung endothelial cell cytoskeletal rearrangement and barrier disruption. moreover, ap- is a critical transcription factor required for the expression of many cytokines involved in the pathogenesis of ali. , considering the roles of hmgb , tlr , p mapk, and ap- signaling pathways in ali and lung inflammation, our findings implicated p mapk and ap- signaling pathways as potentially important mediators of the classic tlr / myd signal-transduction pathways involved in lung inflammatory injury after liver i/r injury. in addition, activation of tlr can also activate other intracellular signaling systems, including the extracellular signal-regulated kinase (erk), c-jun nh -terminal kinase (jnk), as well as the nuclear factor kb (nf-kb) pathways, all of which are pivotal regulators of inflammatory immune responses and cell survival and death. furthermore, studies are needed to investigate the involvements of erk, jnk, and nf-kb in liver i/r injury-induced ali. in conclusion, our study suggests that hmgb might be a stimulating factor in liver i/r injury-induced ali, and that its downstream tlr , p mapk, and ap- signaling pathways are potentially important mediators in the development of ali. thus, blocking hmgb or inhibiting tlr , p mapk, and ap- signaling pathways may be a useful therapeutic option against liver i/r injury-induced ali. pulmonary complications following adult liver transplantation pulmonary complications and mortality after 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of airway epithelial cells by toll-like receptor agonists inhibiting toll-like receptor signaling ameliorates pulmonary fibrosis during acute lung injury induced by lipopolysaccharide: an experimental study hydroxyethyl starch ( kd) inhibits tolllike receptor signaling pathways in rat lungs challenged with lipopolysaccharide lipopolysaccharide induces lung fibroblast proliferation through toll-like receptor signaling and the phosphoinositide -kinase-akt pathway distinct roles of pattern recognition receptors cd and toll-like receptor in acute lung injury tlr is essential in acute lung injury induced by unresuscitated hemorrhagic shock tlr gene dosage contributes to endotoxin-induced acute respiratory inflammation hemoperfusion with a high-mobility group box adsorption column can prevent the occurrence of hepatic ischemia-reperfusion injury in rats the effect of methylprednisolone on warm ischemia-reperfusion injury in the liver interleukin- exacerbates pulmonary injury after hepatic ischemia/reperfusion in mice tlr mediates lung injury and inflammation in intestinal ischemia-reperfusion hmg- as a late mediator of endotoxin lethality in mice release of chromatin protein hmgb by necrotic cells triggers inflammation hmgb release induced by liver ischemia involves toll-like receptor dependent reactive oxygen species production and calcium-mediated signaling interferon regulatory factor mediates acetylation and release of high mobility group box from hepatocytes during murine liver ischemia-reperfusion injury novel role of pkr in inflammasome activation and hmgb release inflammasome-dependent release of the alarmin hmgb in endotoxemia hmgb is a therapeutic target for sterile inflammation and infection the cytokine activity of hmgb effects of the tlr agonists malp- and pam cys in isolated mouse lungs hmgb induces human lung endothelial cell cytoskeletal rearrangement and barrier disruption a critical cysteine is required for hmgb binding to toll-like receptor and activation of macrophage cytokine release induction of in vitro reprogramming by toll-like receptor (tlr) and tlr agonists in murine macrophages: effects of tlr 'homotolerance' versus 'heterotolerance' on nf-kappa b signaling pathway components angiotensin ii type- receptor antagonist attenuates lps-induced acute lung injury toll-like receptors in ischemiareperfusion injury the authors declare no conflict of interest. key: cord- -ueccexxc authors: yang, ce; jiang, jianxin; yang, xuetao; wang, haiyan; du, juan title: stem/progenitor cells in endogenous repairing responses: new toolbox for the treatment of acute lung injury date: - - journal: j transl med doi: . /s - - - sha: doc_id: cord_uid: ueccexxc the repair of organs and tissues has stepped into a prospective era of regenerative medicine. however, basic research and clinical practice in the lung regeneration remains crawling. owing to the complicated three dimensional structures and above types of pulmonary cells, the regeneration of lung tissues becomes a great challenge. compelling evidence has showed that distinct populations of intrapulmonary and extrapulmonary stem/progenitor cells can regenerate epithelia as well as endothelia in various parts of the respiratory tract. recently, the discovery of human lung stem cells and their relevant studies has opened the door of hope again, which might put us on the path to repair our injured body parts, lungs on demand. herein, we emphasized the role of endogenous and exogenous stem/progenitor cells in lungs as well as artificial tissue repair for the injured lungs, which constitute a marvelous toolbox for the treatment of acute lung injury. finally, we further discussed the potential problems in the pulmonary remodeling and regeneration. with the occurrence and evolution of critical diseases (trauma, burn, infections, sepsis, hemorrhagic shock), lungs belong to the most easily injured organs. acute lung injury (ali) also constitutes the causative factor for the other organ chaos [ ] . thus, it is important to prevent and cure the respiratory dysfunction for the improvement of treatment in multiple organ dysfunctions (mods) [ ] . however, compelling evidence indicates that the remedy of ali and acute respiratory distress syndrome (ards) based on the ventilation function support and antiinflammatory treatment remains unsatisfied [ ] [ ] [ ] . actually, the key point to treat the ali and ards is to realize both the structural remodeling and functional repair, and recover the normal gas exchange. presently, the potential measures to realize the repair and regeneration of injured adult lung tissues is to activate the self repairing potential through an extra-or intra-pulmonary route [ , ] , and improve the local pulmonary microenvironment so as to promote the reconstruction of breathing function. during these complex courses, the principal biological event is that stem/progenitor cells are synergistically involved in the repair of injured lung tissues (fig. ). (g-csf) is known to induce mobilization of bmscs to peripheral blood, while their increased homing to sites of injury would improve tissue healing. also, the mobilizers could induce the increase of bone marrow-derived epcs in the murine model of emphysema [ ] , inducing angiogenesis in injured lungs through mobilizing epc [ ] . similarly, in the patients suffered from bacteria pneumonia and ali, the number of circulating epcs is obviously increased, which is even related to their prognosis. in turn, the mobilizing capacity of bone marrowderived epcs is impaired after ards [ ] , indicating the necessity of improvement of bone marrow mobilization so as to promote the pulmonary repair. meanwhile, mobilization of hspcs and colony formation capacity of peripheral blood mononuclear cells demonstrated great significance after ali [ ] [ ] [ ] . all these findings indicate fig. schematic illustration of the exogenous and endogenous stem/progenitor cells as well as the regular delivery routes in the repair and regeneration in acute lung injury that the bone marrow-derived stem/progenitor cells exhibit the mobilizing courses, and play a substantial role in the regression of excessive inflammatory responses and repair in injured lungs. in addition, recent researchers found that ali with endotoxin or no does not enhance development of airway epithelium from bone marrow [ ] , suggesting that the expansion and proliferation of endogenous bone marrow-derived stem/progenitor cells toward airway descendants are further required once their mobilization occurs. presently in the clinical stem cell therapy, mesenchymal stem cells (mscs) are widely used owing to the easy accessibility and low immunogenicity [ ] . the allograft of bone marrow mscs are easily tolerated for the acceptors due to the low expression of major histocompatibility complex (mhc) i, ii and co-stimulator molecules in t cells. thus, these theoretically reasonable cells are further stored until use without ethical disputation. in recent years above clinical trials of mscs have been registered and carried out. bone marrow mscs have been proved to efficiently alleviate the lung injury and promote the recovery courses [ ] , partly due to the immunoregulatory effects [ , ] . meanwhile, administration of mscs via the vein or trachea also reduces the lps-induced ali, alleviating the chest impact injury and hyperoxia-induced lung injury, reversing the pathological reduction of pulmonary surface area and the blunted breathing function in rodents [ , ] . bleomycininduced inflammation, collagen deposition and fibrosis were also attenuated after the mscs injection [ ] . the conditioned medium for mscs culture further showed similar therapeutic effects on pulmonary function [ ] . hence, bone marrows mscs possess the prospective clinical value for the repair and regeneration in ali. concerning the protective roles of bone marrow-and peripheral blood-derived epcs in ali, recent studies showed that their peripheral infusion could lead to homing in injured lung tissues [ ] , relieving the inflammatory injury [ , ] and promote the endothelial repair and recovery of immune function dissonance [ , ] , which may be enhanced by the treatment of simvastatin [ ] . also, inhaled no contributes to the repair of injured lungs in piglets via increasing circulating endothelial progenitor cells [ ] . the number of epcs is positively related to the animal survival. from the clinical point of view, the increased number of epcs in the pneumonia patients is the innocent representation of self repair in bodies. if the number of circulating epcs didn't increase in ali [ ] , these patients might have distressing outcome, indicating the great necessity for supplementing the epcs in combination with immune modulatory measures. presently, the understanding of hspcs efficacy in ali remains limited. interestingly, stem/progenitor cells derived from the circulation contribute to the repair of injured lungs in surgically generated parabiotic mice [ ] , indicating the potential contribution of hspc in ali. in fact, during the human prenatal development, the hspcs firstly appear in the fetal yolk sac. four months later. they transfer to the fetal livers for the further growth and differentiation. in the newborn phase, they finally locate in the bone marrow, followed with the appearance of varying stages of leukocytes, red cells and platelets. thus, the role of hspcs at least includes the immunoregulatory and repairing effects in ali. since the discovery of adscs by zuk et al. [ ] , their capacity in the repair and regeneration of injured lungs has been widely investigated. adscs exhibit large reserve quantity owing to the extensively distributed adipose tissues in bodies. as compared with the bmscs, adscs were easily harvested from dumped adipose end product via the regular adipose aspiration technique. the total volume of adult bone marrow extracts is ml while that of adult adipose can easily reach ml. moreover, the harvest of adscs is easy to adopt as well as no potential blood-derived contamination and immunologic rejection compared with the bone marrow allograft [ ] . the pickup rate of adscs is - times of that of bone marrow mscs. so, adscs can be abundantly harvested in the limited time without in vitro expansion. in a randomized, placebo-controlled pilot study, adscs showed significant protective effects on ali. optical imaging analysis further indicated that they promote the subacute airway remodeling, and ameliorates ventilator-induced lung injury in rats [ , ] . the main protective reasons referred to enos and enos-derived no [ ] . actually, adscs could secrete vascular endothelial growth factor, granulocyte colonystimulating factor (g-csf), hepatocyte growth factor (hgf), stromal derived factor- (sdf- ) when promoting the angiogenesis. also, they release collagen i and iii and laminin via a paracrine route [ , ] . all these factors may play a substantial role in pulmonary structural repair and functional reconstruction in ali. placenta-derived stem/progenitor cells come from placenta, umbilical cord and amniotic fluid and their contents. among them, the placenta is structurally complicated. human placenta consists of amnion, chorion and basal deciduas. the amnion and chorion are from fetus while basal deciduas are from precursor. the placenta is the reservoir of stem and progenitor cells during the fetal development. once the fetal disengagement is finished, it is easily acquired without ethical disputation. presently, placenta-derived stem/progenitor cells are positively involved in the repair of injured lungs including fetal membrane-derived mscs, umbilical cord mscs, umbilical cord blood-derived mscs, amniotic fluid stem cells, and amnion epithelial cells, etc. [ ] . previous studies have demonstrated that amniotic fluid stem cells could attenuate hyperoxia-induced ali in mice [ ] , inhibiting the progression of bleomycin-induced pulmonary fibrosis via ccl modulation in bronchoalveolar lavage [ ] . amnion epithelial cells could also act as a seed cells for the therapy of ali [ ] . meanwhile, human umbilical cord mscs reduced systemic inflammation and attenuated lps-induced ali in rats [ ] . human cd + progenitor cells from umbilical cord blood could also attenuate inflammatory lung injury following lps challenge [ ] . further, intratracheal administration of umbilical cord blood-derived mscs played a pleasing role for the patient with ards [ ] . therefore, placenta-derived stem/progenitor cells act as efficient candidates for the treatment of ali. human embryonic stem cell-derived progenitor cells could ameliorate sepsis-induced lung inflammatory injury via interaction with a specific population of with cd b+ cells [ ] . meanwhile, researchers could get functional airway epithelium from human embryonic stem cells through an expansive generation measure [ ] . however, owing to the ethical bottleneck and law disputation, they shouldn't be recommended to be an ideal seed cells in the repair and regeneration of injured lungs. induced pluripotent stem cells (ipscs) were firstly acquired from genetically engineered fibroblasts in skin, similar to the dedifferentiation findings by fu, et al. [ ] in . now, it has been confirmed that adscs are easier to be transferred to ipscs than fibroblasts [ ] . the embryonic stem cell-like functions of ipscs were proved occasionally since . ipscs were also got from a patient with ali [ ] , which could differentiate into alveolar epithelial cells in vitro for use in vivo [ ] . the therapeutic capacity of ipscs might be related to the inhibition of src, nf-κb and pi k/akt pathway as well as ip- -dependent paracrine regulation [ ] [ ] [ ] . in view of the initialization characteristics of ipscs from adult cells, they could pave the way to the structural remodeling and functional repair in ali without any ethical and law obstacles. meanwhile, stem/progenitor cells were widely used to act as the vectors of target genes to attenuate the inflammatory injury and promote the repair and regeneration of injured lung. recent studies have demonstrated that the genetically engineered stem cells with overexpression of cxcr [ ] , angiotensin-converting enzyme [ ] , il- antagonist soluble il- receptor-like- [ ] , keratinocyte growth factor, angiopoietin [ ] and dominant-negative inhibitor of ccl [ ] could greatly facilitate treatment of ali in rodents. all these results substantially indicate that the therapeutic efficacy of genetically engineered stem/progenitor cells boosted by the stably transfected target genes in the pulmonary repair. in the adult mammalian tissues and organs, there are still some endogenous stem/progenitor cells, which distribute the predetermined microenvironment named niche. the niche supplies the repairing cells for the homeostasis and repair of tissues and organs. concerning the endogenous pulmonary stem/progenitor cells, researchers reported that they exist in the adult respiratory tissues in rodents and humans (table ) . although there remains lack of the specific molecular markers for the lung stem/progenitor cells, their isolation and culture seems difficult, and the classification of these cells is also in controversial, they have been widely approved for the maintaining of pulmonary structural stability and functional repair. pulmonary stem/progenitor cells (trachea and bronchial stem cells, bronchiolar stem cells, bronchioloalveolar stem cells, alveolar stem cells and alveolar type ii cells, etc.) were shown to play substantial roles for the recovery of homeostasis and repair of injured tissues through molecular markers, lineage tracing and clonal analysis [ ] [ ] [ ] [ ] [ ] [ ] (fig. ) . the alveolar epithelium is composed of the flat alveolar type i (at ) cells comprising % of the gas-exchange surface area and less than % of cuboidal alveolar type ii (at ) cells comprising the rest. once ali occurs, at cells showed injury, necrosis or apoptosis. then, rare, long-lived, mature at cells could differentiate and substitute the disabled at cells in the injured area [ , ] . at this moment, some of at cells were found to become hypertrophy, which is easily discriminated in various injured lung tissues. meanwhile, the heterogeneity of at cells had been presented and classified with three subgroups including the alveolar renewal focus, alveolar repair focus and at replacement focus [ ] . further, the subgroup of stem cells were shown to exist within the at cells, which were found to widely distribute in the terminal bronchiole, bronchioalveolar epithelial progenitor cell isolation anti-inflammation, tissue repair [ ] hematopoietic stem/ progenitor cell isolation anti-inflammation, tissue repair [ ] engineering stem/ progenitor cell ipscs isolation and genetic manipulation anti-inflammation via nf-kb and src pathway, differentiation toward alveolar epithelial cells [ ] [ ] [ ] [ ] modified mesenchymal stem cell anti-inflammation, tissue repair [ , , ] duct junction (badj) and alveolus. in comparison, the contribution of at cells is more significant than bronchioalveolar stem cells (bascs) concerning the numerical preponderance and differentiation potential [ , ] . some murine at cells can also generate bascs unexpectedly [ ] . therefore, during the course of pulmonary remodeling after ali, the efficient shift of repair potential in at cells including the number, distribution and cellular transfer path should be undoubtedly weighted. the endogenous stem cells in the resident lung cells were further confirmed using the gfp-labeled chimera mice, which synergistically contribute to the regenerative alveolus. robust preclinical literature has showed that at cells could repair the injured alveolar epithelium. however, the potential of pulmonary endogenous stem cells to substituting the injured at cells remains unclear. to address this question, new findings suggested that the murine stem cell antigen (sca)- positive cell may be the endogenous lung stem cell although it could not found in rats [ ] . the cell number with stem cell marker (sca- , cd and c-kit) increased in the elastase-induced lung injury. the combination of hgf and elastase could synergistically increase the number of sca- +/spc+ cells. most of the sca- + belong to the endogenous lung stem cells while most of the c-kit+ cells come from the bone marrow [ , ] . therefore, the increase in the number of endogenous lung stem/ progenitor cells is in great need for the repair of injured lung tissues. in recent years, zhang and colleagues [ ] found that the excitation of wnt signal pathway could significantly increase the number of bascs. the pharmacological modulators, lithium may also promote the amplification and differentiation of specific stem cell group in the lung tissues [ ] , which supplies the new avenue for the endogenous repair of injured lungs on the basis of pulmonary stem cells. clara cells possess anti-inflammatory capacity and impacts the pulmonary innate immune response [ , ] . conditional depletion of clara cells induced peribronchiolar fibrosis, and potentiated lung inflammation and alveolar dysfunction, demonstrating its role of functional repair/regeneration in ali [ ] . concurrently, our studies further showed that the boosted expression of clara cells resulted in the transformation of cellular shape from sporadic cube to serried high prismatical, and the enhancement of anti-inflammatory effect of clara cell secreting protein (ccsp) after retinoic acid plus simvastatin treatment in shock-endotoxin-induced pulmonary damage [ ] . ccsp, an important lung derived protective factor, may play a substantial role on the pathogenesis of ali induced by endotoxemia [ ] . moreover, compelling evidence showed that the differentiation potential of clara cells towards at and at cells after severe lung injury [ , ] . so, the improved at and at cells maybe partly derived from facultative clara cells so as to keep the integrity of alveolar walls. presently, as compared with the boosting of murine lung stem cells, the investigation of human lung stem cells (hlscs) remains superficial since their presentation by kajstura et al in [ ] . the main reasons are as follows. first, there remains lack of the specific markers for hlsc. second, the acquisition of human lung tissues is limited. nonetheless, the hlscs researches proved their in vitro c-kit positive characteristics, which were further confirmed using the in vivo experimental models. also, some experimental preparations have been given for the potential clinical usage. but it remains a long way to go before the clinical engraftment for hlscs. first, how efficient is the hlscs engraftment. whether does the newborn lung tissues differentiated from hlscs possess the normal physiological function? second, how is the meanwhile, recent studies further acquired human alveolar progenitor cells (aepcs) [ ] , aepcs have the endothelial phenotype with mscs character. by using the chip analysis, aepcs were found to share many genes with mscs and at cells, indicating the phenotype overlapping between alveolar epithelial cells and mscs. in fact, apecs possess the capacity of phenotypic conversion between the mesenchymal and epithelium, indicating their potential in the pulmonary tissue repair. the mesenchymal characteristics, especially anti-apoptotic ability may benefit the functional epithelial progenitor cells. further investigation is necessary to elucidate their detailed pathophysiological role in the repair of injured lungs. the lung mscs have the ability of self renewal and differentiate into mesenchymal cell. given the varying characteristics in the different organs, the basic criteria for lung mscs include the adhesive ability on the plastic petri dishes, and the in vitro ability of osteogenesis, adipogenesis and cartilagenesis [ ] since there are no specific cellular markers on the surface of pulmonary mscs. the lung mscs can be isolated from the lung and bronchoalveolar lavage fluid. karoubi et al. [ ] isolated the mscs from the human lung tissues in the surgical operation, and successfully induced their differentiation toward the at cells expressing aqp and ccsp. although the action of mscs is not completely clear in the lung regeneration, the beneficial effects of mscs on the lung injuries have been extensively investigated. mscs can secrete diverse cytokines and growth factors. co-culture of lps-stimulated lung cells and mscs could result in the reduction of pro-inflammatory cytokines, indicating that the soluble mediators may inhibit excessive inflammatory responses, or the direct interactions of lung cells and mscs could produce the anti-inflammatory effects. similar results have showed that the immunoregulatory role of mscs on immune cells other studies reported that the intra trachea administration of pulmonary stem cells with the mscs phenotype attenuated the elastase-induced emphysema [ ] . the transplanted stem cells can reach the alveolar space besides some of them reserved in the alveolar wall. these results didn't support the idea of cell differentiation, but indicated their immunoregulatory effects in the injured lung tissues. in addition, new mechanisms included the mitochondria dna transmission between the mscs and other cytosolic components through intercellular bridges, which may regulate the cellular biological ability in the recipient cells [ ] . hence, the protective effects of mscs are rather the anti-inflammatory effects than the differentiation towards the lung cells. the protective role of stem/progenitor cells is to release the anti-injured and pro-reparative factors mainly via the paracrine/endocrine pathways, which is primarily due to their significant apoptosis and clearance by unknown innate immune mechanisms after their transplantation. likewise, the researches concerning their microenvironment regulation demonstrated that the stem/progenitor cell-derived conditioned medium possesses the similar efficacy, suggesting that the secreting factors (il- , il-rn, vegf, angiopoietin- ) act as the anti-inflammatory and pro-reparative mediators on the gas-blood barrier in ali [ ] [ ] [ ] [ ] . moreover, microvesicles containing anti-inflammatory mrna and mirna secreted by the stem/progenitor cells also possessed the therapeutic potential during the repairing courses [ ] [ ] [ ] . second, bmsc could protect against oxidative stress in escherichia coli-induced ali in mice [ ] , and ameliorate seawater-exposure-induced ali by inhibiting autophagy in lung tissues [ ] . concurrently, they could restore sodium transport and preserve epithelial permeability in an in vitro model of acute alveolar injury [ ] . third, bmsc could reduce inflammation while enhancing bacterial clearance in bacterial pneumonia [ , ] . fourth, the number of a population of clara cells possessing secreting capacity; named bronchioalveolar stem cells (bascs) were increased in ali [ ] . the ex vivo colony formation experiments proved that the proliferation of bascs was maybe due to mscs but not growth factors. the therapeutic effect of mscs on the chronic bronchopulmonary dysplasia showed that bascs help the reconstruction of pulmonary epithelial structure. so, the repairing effects of mscs maybe realize via the stimulation of bascs proliferation, demonstrating the peculiarly promoting effects of extra pulmonary stem cells on the lung stem cells during the repair and regeneration. the previous cell therapy on ali showed that the time courses of pulmonary remodeling and functional repair varies depending on the wound agents (live bacteria, oleic acid, bleomysin, etc.) [ ] , indicating the clinical therapeutic efficacy may be intrinsically related to the ali etiology. thus, it is important to make the sensible selection and stringent judgment for the initiating factors, contaminated pathogens (bacteria, virus, and fungus) and potential window phases in the stem/progenitor cell-mediated lung repair. the injured lungs may release large quantities of stressrelated neuroendocrine hormones, neuromediators and neuropeptides [ ] [ ] [ ] , which deeply influence the biological activities of stem/progenitor cells. steadily growing evidence has been shown that glucocorticoids, epinephrine and norepinephrine may be involved in the migrating or chemotaxis activities during the mobilizing courses [ ] . acetylcholine released via vagus nerve and postganglionic neurons of adrenergic nerve has also been proved to modulate these courses [ ] . recently, melatonin treatment was found to improve adscs therapy for acute lung ischemia-reperfusion injury [ ] . concurrently, the immune status are also related to the protective efficacy of mscs because they could reduce lung injury in immunocompromised but not immunocompetent mice [ ] . thus, the consonance of neuroendocrine immune network is of great importance for the lung repair and regeneration. the cell therapy of ali suggested that the prominent advantage of exogenous stem/progenitor cells is quantity-controllable although the procedures of harvest, purification and expansion are required in most conditions. hence, the potential contamination and biosafety should be carefully considered (table ) . once the transplantation procedure is completed, the turnover of these stem/progenitor cells should also be further controlled to lower the risk of graft rejection and teratoma formation [ ] [ ] [ ] . in comparison, the greatest advantage of endogenous stem/progenitor cells lies in the good safety only required to use the mobilizers or activating elements for their redistribution within the bone marrow, blood and lungs. however, the constraint of cell numbers especially in the blunted mobilization responses for some patients may deteriorate the therapeutic efficacy. in addition, other important findings have reported that there exist some differences between mscs from bone marrow, placenta and umbilical cord blood in terms of their immunosuppressive properties against t cells [ ] . first, the stem/progenitor cells in the same species is better than in the different species in the ali therapy [ ] . likewise, the autotransplantation is preferentially selected compared with allotransplantation. second, aging stem/progenitor cells were showed to have impaired migration and anti-inflammatory responses as well as abnormal immunosuppressive properties against t cells [ , ] , indicating the selection of young stem/ progenitor cells is in preference. third, the delivery routes for stem/progenitor cell transplantation are also comparable. the therapeutic effects via intraperitoneal route were slightly inferior to intravenous route in amiodarone induced lung injury in rats [ ] . so, the reasonable transplantation route (venous, trachea, intrapleural or intraperitoneal pathway) should be carefully considered for the ideal therapeutic efficacy (fig. ) . until now, few lungs are available for transplantation and the results have not been completely assentient. hence ways are being sought to either engraft stem/ progenitor cells or fetal lung cells that will form pulmonary structures to the severely injured lungs, or build bio-artificial lungs that completely replace the depleted. however, owing to the complicated three-dimensional tissue structure and above cell types, lungs are difficult to be artificially constructed perfectly. recently, several artificial lung models have been presented. the essential researches utilized the scaffold material that supports the development of alveolar-like epithelia and endothelia from fetal lung cells. meanwhile, the decellularized lungs were also repopulated with fetal lung epithelial cells delivered via the trachea and lung endothelial or human umbilical cord vein cells through the pulmonary artery. the constructs were then cultured in a bioreactor where the cells regenerated region-specific tissue with the characteristics of normal alveolar tissue before transplantation. although these cells need to be carefully investigated before the clinical usage, the integrity of lung stroma remains to be resolved, and the gas exchange capacity is limited, the concept of bio-artificial lungs may supply a candidate replacement measure for the severe lung injury owing to its low immune rejection and controlled organ origin, which might throw sunshine on the million end staged-patients with ali. to the end, the endogenous lung remodeling, repair and regeneration has become the new avenue for the refractory lung diseases. however, the following concerns remains to be added. first, most of successful ali experiments were accomplished with rodents [ ] . their lungs in thoracic cavities possess the energetic proliferating capacity in the whole life period which is quite different from human lungs because the transplantation of exogenous stem/progenitor cells into matured human lung tissues seems difficult at least owing to our limited insight in the hlsc. but the initiation of endogenous stem/ progenitor (self renewal, proliferation, migration, and differentiation) is safe and maybe efficient via some modulators (kgf, hgf, retinoid acid, etc.). second, in view of the bio-safety of endogenous stem/progenitor cells and the quantity constraint of exogenous stem/progenitor cells, it may be reasonable to consider the combination of these two types of cells in the ali therapy. third, the repair and regeneration of injured lungs is complicated. we should emphasize the microenvironmental regulation via the neuroendocrine immune network [ , ] . only in this way may the stem/progenitor cells possess the ideal biological capability (migration, mobilization, chemotaxis and homing, expansion, differentiation and proliferation). such good "soil" may benefit these "seed" cells in the remodeling and regeneration of injured lungs. fourth, regarding the complicated cell types in lungs, the integration of inflammatory modulation and pro-repair factors' increase may help inhibit their deleterious injury and promote the structural remodeling. the potential measures should consider the synergistic combination of statins plus retinoid acid, statins plus hgf, etc. fifth, concerning the stem/progenitor cell pool of the bone marrow, and the energetic reservoir of the stem/progenitor cell pool of placenta, umbilical cord and amnion during the perinatal period, the selection of compositive stem/ progenitor cell populations maybe benefit ali treatment more than a single stem/progenitor cell population, which has been partly confirmed by the previous studies of mononuclear cell populations in the bone marrow [ ] . sixth, concerning their robust protective capacity via paracrine/endocrine mediators released by stem/ progenitor cells, it is valuable to develop some stem/progenitor cell-derived therapeutic fluid similar to the conditioned medium of stem/progenitor cell in combined with the microvesicles for the ali therapy. finally, from the previous experience in the research of bio-artificial lungs, it is valuable to deeply emphasize the contribution of extracellular matrix while using stem/progenitor cells, which might pave the road for the pulmonary integrity in the lung remodeling and regeneration (fig. ). taken together, the remaining unknown issues include the protraction regularity of lung stem/progenitor cell lineage, the transition and turnover of extra pulmonary stem/ progenitor cell and the integration and docking between intra-and extra-pulmonary stem/progenitor cells. nonetheless, the ideal animal models, clinical samples as well as usage of intra-and extra-pulmonary stem/progenitor cells will undoubtedly contribute to the elucidation of pathophysiological mechanism of lung regeneration, and the pursuit of new measures for the refractory ali. adscs: adipose-derived stem cells; aepcs: alveolar progenitor cells; ali: acute lung injury; ards: acute respiratory distress syndrome; badj: bronchioalveolar duct junction; bascs: bronchioalveolar stem cells; bmscs: bone marrow derived mesenchymal cells; ccsp: clara cell secreting protein; epcs: epithelial progenitor cells; g-csf: granulocyte colony-stimulating factor; hgf: hepatocyte growth factor; hlscs: human lung stem cells; hspcs: hematopoietic stem/progenitor cells; ipscs: induced pluripotent stem cells; mhc: major histocompatibility complex; mods: multiple organ dysfunctions; mscs: mesenchymal stem cells; sca: stem cell antigen; sdf- : stromal derived factor- . authors'contributions cy, xy, hw and jd drafted the manuscript; cy and jj critically reviewed the manuscript. all authors read and approved the final manuscript. acute lung injury and the acute respiratory distress syndrome: a clinical review expressions of scavenger receptor, cd and protective mechanisms of carboxymethyl-beta- , -glucan in posttraumatic endotoxemia in mice preventing acute lung injury and acute respiratory distress syndrome: back to square one management of acute lung injury and 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human and mouse adiposederived stem cells in a murine model of lipopolysaccharide-induced acute lung injury aging mesenchymal stem cells fail to protect because of impaired migration and antiinflammatory response comparative study between intravenous and intraperitoneal stem cell therapy in amiodarone induced lung injury in rat concise review: current status of stem cells and regenerative medicine in lung biology and diseases bilateral regulatory action of corticotropin-releasing hormone on immune-mediated inflammation effects of hypothalamus destruction on the level of plasma corticosterone after blast injury and its relation to interleukin- in rats cell therapy demonstrates promise for acute respiratory distress syndrome-but which cell is best? the authors thank professor min zhao (university of california, davis) for his critical reading of this manuscript. we also apologize for the omission of any references due to the space constraints of this review and wish to thank members of their laboratories for helpful criticism. the authors declare that they have no competing interests. this • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -q jamg authors: hahka, taija m.; xia, zhiqiu; hong, juan; kitzerow, oliver; nahama, alexis; zucker, irving h.; wang, hanjun title: resiniferatoxin (rtx) ameliorates acute respiratory distress syndrome (ards) in a rodent model of lung injury date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: q jamg acute lung injury (ali) is associated with cytokine release, pulmonary edema and in the longer term, fibrosis. a severe cytokine storm and pulmonary pathology can cause respiratory failure due to acute respiratory distress syndrome (ards), which is one of the major causes of mortality associated with ali. in this study, we aimed to determine a novel neural component through cardiopulmonary spinal afferents that mediates lung pathology during ali/ards. we ablated cardiopulmonary spinal afferents through either epidural t -t dorsal root ganglia (drg) application or intra-stellate ganglia delivery of a selective afferent neurotoxin, resiniferatoxin (rtx) in rats days post bleomycin-induced lung injury. our data showed that both epidural and intra-stellate ganglia injection of rtx significantly reduced plasma extravasation and reduced the level of lung pro-inflammatory cytokines providing proof of principle that cardiopulmonary spinal afferents are involved in lung pathology post ali. considering the translational potential of stellate ganglia delivery of rtx, we further examined the effects of stellate rtx on blood gas exchange and lung edema in the ali rat model. our data suggest that intra-stellate ganglia injection of rtx improved po and blood acidosis days post ali. it also reduced wet lung weight in bleomycin treated rats, indicating a reduction in lung edema. taken together, this study suggests that cardiopulmonary spinal afferents play a critical role in lung inflammation and edema post ali. this study shows the translational potential for ganglionic administration of rtx in ards. respiratory failure due to acute respiratory distress syndrome (ards) is one of the major causes of mortality associated with acute lung injury (ali) including covid- . [ ] [ ] [ ] [ ] [ ] most forms of ali/ards are also associated with acute cytokine release, pulmonary edema and in the longer term, fibrosis. , however, the mechanisms underlying these pathological changes in the lungs during ali/ards are not fully understood. in particular, a neural component that mediates lung pathology during ali/ards has been less considered. sensory neurons innervating the heart and lung enter the central nervous system by one of two routes; through the vagus nerve into the brain stem (medulla) with cell bodies residing in the nodose ganglia and directly into the spinal cord where cell bodies reside in the dorsal root ganglia (drg). afferents are composed of elements that respond to a variety of sensory modalities including mechanical deformation, heat, cold, ph, and inflammatory mediators, just to name a few. the reflex effects following stimulation of these afferents depends on the type of stimulus and the neural pathway involved. activation of vagal afferent pathways tends to be sympatho-inhibitory and antiinflammatory. , on the other hand, activation of spinal afferents tends to be sympathoexcitatory and pro-inflammatory. [ ] [ ] [ ] [ ] [ ] [ ] it is well known that small diameter spinal transient receptor vanilloid (trpv )-positive afferent c-fibers contain neuropeptides such as substance p (sp) and calcitonin gene related peptide (cgrp). these peptides tend to dilate adjacent vasculature and increase microvascular permeability. in the lung, this can cause pulmonary edema resulting in reduced oxygen diffusion and promote immune cell infiltration resulting in neural inflammation. therefore, in the current study we hypothesized that ablation of lung afferent innervation (thoracic spinal) by application of an ultrapotent, selective afferent neurotoxin, resiniferatoxin (rtx) will modify the course of the pathology including lung edema and local pulmonary inflammation associated with progressive ali. committee of the university of nebraska medical center and performed in accordance with the national institutes of health's guide for use and care of laboratory animals and with arrive guidelines. , experiments were performed on adult, male, - g sprague-dawley rats purchased from the charles river laboratories. animals were housed on-site and given a one-week acclimation period prior to experimentation. food and water were supplied ad libitum, and rats were on -hour light/dark cycles. rats were randomized into three groups and evaluated at -week post-instillation as follows: sham rats, bleomycin (bleo)-exposed rats with saline (epidural or intrastellate injection), and bleo-exposed rats with rtx (epidural or intra-stellate injection). bleo ( . mg/kg, ~ . ml) was instilled intra-tracheally to the lungs under % isoflurane anesthesia. sham control rats underwent intra-tracheal instillation of saline. animals were treated with rtx or vehicle (phosphate buffered saline) by either the epidural t -t drgs route ( µg/ml, µl/per ganglia) or intra-stellate ganglia administration ( µg/ml, µl/per side) days following bleomycin delivery ( figure ). in a pilot experiment, the upper thoracic spinal afferents were ablated by epidural application of rtx as previously described. briefly, rats were anesthetized using %- % isoflurane:oxygen mixture. rats were placed in the prone position and a small midline incision was made in the region of the t -l thoracic vertebrae. following dissection of the superficial muscles, two small holes (approximately mm x mm) were made in the left and right sides of t vertebrae. a polyethylene catheter (pe- ) was inserted into the subarachnoid space via one hole and gently advanced about cm approximating the t level. the upper thoracic sympathetic afferent ganglia were ablated by injecting resiniferatoxin (rtx; sigma aldrich), an ultra-potent agonist of the trpv receptor into the aubarachnoid space via the catheter. rtx ( mg; sigma aldrich) was dissolved in a : : mixutre of ethanol, tween (sigma-aldrich), and isotonic saline. the first injection of rtx ( µg/ml, ul) was made at a very slow speed (~ minute) to minimize the diffusion of the drug. the catheter was then pulled back to t , t and t , respectively to perform serial injections ( ul/each) at each segment. the catheter was withdrawn and the same injections were repeated on the other side. silicone gel was used to seal the hole in the t vertebra. the skin overlying the muscle were closed with a - polypropylene simple interrupted suture, and betadine was applied to the wound. for post-procedure pain management, buprenorphine ( . mg/kg) was subcutaneously injected immediately after surgery and twice daily for days. rats were anesthetized using %- % isoflurane:oxygen mixture. after the trachea was cannulated mechanical ventilation was started (model , harvard apparatus, south natick, ma). the skin from the rostral end of the sternum to the level of third rib was incised. portions of the superficial and deep pectoral muscles and the first intercostal muscles were cut and dissected. to localize the left or right stellate ganglion, the left or right precava vein were separated with a hooked glass or steel rod laterally away from the brachiocephalic artery to expose the internal thoracic artery and the costocervical artery, which are descending branches of the right subclavian artery. stellate ganglia and ansa subclavia are located medially to the origins of the internal thoracic and costocervical arteries. then, rtx ( µl, mg/ml) was injected into the ganglia with a µl hamilton syringe (microliter # , hamilton, reno, nv, usa.) over s bilaterally. an image of this procedure is shown in figure . following these maneuvers, the thorax between the st and nd intercostal spaces was closed with continuous - dexon ii coated braided absorbable polyglycolic acid suture and the skin was closed with - polypropylene suture and the chest evacuated. betadine was applied to the wound and the rats were allowed to recover from the anesthesia. for post-procedure pain management, buprenorphine ( . mg/kg) was injected subcutaneously immediately after surgery and twice daily for days. the artery on the ventral aspect of the rat tail was used for the collection of small amounts of blood (~ . ml) for analyzing arterial blood gas at day post bleomycin. the animal was restrained with a commercial restrainer so that its tail was accessible. the tail was prepared aseptically by alternating alcohol prep pads and iodine prep pads three times and the artery was punctured using a g needle. a small volume of blood (~ . ml) was gently aspirated into the syringe for blood gas analysis (istat, abbott, chicago, il, usa). after sample collection, the needle was removed, and a gauze swab was pressed firmly on the puncture site to stop bleeding. rats were anaesthetized with pentobarbitone ( mg/kg). evans blue, mg/kg ( mg/ml, dissolved in saline + ie per ml heparin) was administered intravenously. right panel: at day , bleomycin or saline was given intra-tracheally; at day , resiniferatoxin or vehicle was given into epidural space or into stellate ganglia; at say , the rats were sacrificed. the procedure by which the stellate ganglia were exposed and injected in rats under anesthesia is shown in figure . plasma extravasation (evans blue) was used to assess vascular permeability after acute lung injury. as shown in figure , bleomycin-treated lungs exhibited a wide distribution of evans blue areas in both sides. the highest intensity of evans blue was shown at the medial aspect of each lung. the evans blue areas were largely reduced following epidural rtx treatment at the -day time point after bleomycin administration (figure ) . three pro-inflammatory tissue cytokines were prevalent in the lung following bleo treatment are shown in figure . il- , il- ß and ifný were markedly elevated following bleo treatment. these cytokine levels were normalized in epidural rtx treated rats. plasma extravasation in response to bleo was also reduced after stellate injection of rtx ( figure ) . as can be seen, there was a marked reduction in evans blue dye in the lung following stellate injection of rtx. arterial blood gas data were evaluated in rats treated with vehicle vs rtx intra-stellate. compared to sham rats, wet lung weight (wlw) as well as the ratio of wlw to bw was significantly higher in the bleo+veh rats, which was significantly reduced by intrastellate injection of rtx. these data suggest that intra-stellate injection of rtx reduces lung edema post bleo. the data from this study provides proof of principle and is highly suggestive of an important role for trpv -positive spinal afferent-mediated neuroinflammation in acute lung injury and progression to acute respiratory distress. the evidence provided demonstrates that ablation of trpv sensory afferents in the presence of acute lung injury using rtx delivered by either of two routes that target cardiopulmonary afferents leads to a rapid reduction in lung microvascular permeability and a reduction in tissue and plasma inflammatory markers. while pulmonary function per se was not directly measured in this series of experiments, arterial blood gas data strongly suggest an improvement in gas exchange. the improved body weight and reduced lung weight in rats with lung injury after receiving stellate ganglia administration of rtx suggest potential clinical benefits from reduced lung edema, and protective effects for nonpulmonary organs that would otherwise be impacted by the pulmonary triggered systemic inflammatory process. the lung is innervated by a dual sensory system including vagal and spinal afferents. both vagal and spinal afferent fibers are composed of a (high conduction velocity) and c-fiber (low conduction velocity) axons. these fibers and their sensory endings express a variety of membrane receptors that mediate ion channel function including traditional na + , k + and ca + channels (both voltage and ligand gated). importantly, non-specific cation channels that are highly permeable to calcium are expressed mostly in small diameter c-fibers. , these include at least members of the transient receptor potential family including transient receptor potential a (trpa) and trpv receptors. trpv receptors transduce sensations of heat and neuropathic pain in the periphery. estimates are that approximately percent of thoracic drg neurons are positive for trpv . upon activation, trpv channels are highly permeable to calcium. , high levels of intracellular calcium are toxic and thus damage or kill these specific afferent neurons. thus, a unique strategy has been developed to modulate the pathological effects of trpv afferent neurons. the ultrapotent neurotoxin, rtx binds avidly to the trpv receptor. after initial stimulation, high intracellular levels of calcium mediate inhibition of neuronal function. site-specific delivery of rtx can be used to intervene in various conditions to alleviate pain, inflammation, fibrosis and plasma extravasation. it has been shown that rtx-induced trpv sensory afferent deletion can block the afferent-contained neuropeptide release and reduce inflammatory pain. cardiopulmonary spinal afferents can also be targeted with rtx by either application into the epidural space at thoracic levels t -t (with some spread to higher and lower segments) or by injection into the stellate ganglia. while drgs are considered exclusively sensory in nature, the stellates contain soma for sympathetic efferent fibers and fibers of passage for thoracic afferents as they course through drgs and enter the spinal cord. it should be noted that in humans the stellate ganglia can be easily identified, and that this type of transcutaneous procedure can be performed with fluoroscopic or ultrasound guidance (intra-ganglionic or nerve 'block' approach). compared to epidural delivery that requires relatively larger injection volume (~ µl for bilateral injection) to sufficiently cover the t -t drgs, intra-stellate injection requires a much smaller volume ( µl for bilateral injection), which reduces the risk of systemic absorption of rtx and allows a higher dose of rtx to be used for local injection. the current data clearly show that intra-stellate ganglia injection of rtx markedly attenuated lung extravasation post ali, suggesting that a large proportion of thoracic afferents passing through the stellates innervate the lungs. taken together, we believe that intra-stellate ganglia delivery of rtx should be a clinically feasible intervention to treat acute lung injury compared to the epidural approach. the preliminary data presented here for epidural administration of rtx provides proof of principle that it reduces plasma extravasation in the lung. the main focus of this study was on the therapeutic effect of the stellate ganglia approach on lung pathology in our ali rat model. prior work from this laboratory has demonstrated that ablation of cardiac trpv positive afferents reduces sympathetic nerve activity and cardiac remodeling in a post myocardial infarction model of chronic heart failure. trpv -expressing cardiopulmonary afferents participate in a sympatho-excitatory reflex that has been termed the cardiac afferent sympathetic reflex (csar) and the pulmonary afferent sympathetic reflex (psar) . the csar is augmented in heart failure along with cardiac afferent discharge in response to bradykinin or capsaicin. epicardial administration of rtx reduces sympathetic outflow to the heart and kidneys and improves cardiac diastolic function while reducing fibrosis and cytokine content in the heart. furthermore, cardiac application of local anesthetic lowers sympathetic nerve activity in anesthetized vagotomized animals suggesting tonic input from these spinal afferents in heart failure. , on the other hand, it has been widely reported that activation of trpv expressing afferents causes secretion of neuropeptides such as substance p (sp) and calcitonin gene-related peptide (cgrp). [ ] [ ] [ ] [ ] [ ] released sp, but not cgrp, in sensory endings binds neurokinin (nk) receptors on blood vessels and causes vasodilation and increased vascular permeability that allows loss of proteins and fluid (plasma extravasation) thus promoting the regional accumulation of monocytes and leukocytes contributing to inflammation. [ ] [ ] [ ] in the lung, this process not only impairs alveolar gas exchange but may initiate and exacerbate a fulminant cytokine storm from adjacent cells and from circulating macrophages. the current study supports the idea that selective ablation of trpv afferents mitigates neuroinflammation in the lung by inhibiting trpv afferent-mediated plasma extravasation, at least in the bleomycin model of ali. importantly, although we did not directly measure respiratory parameters such as minute ventilation and respiratory rate in vehicle and rtx treated bleomycin rats, we observed significantly reduced lung weight and improved blood gas parameters including blood ph, po and so in bleomycin rats treated with intra-stellate ganglia injection of rtx, suggesting an improvement in lung function. combined with the results shown in the current model of ali, the potential of rtx to rescue lung function and protect multiple organs from collateral damage due to lung injury triggered inflammation is very encouraging and warrants additional studies as a rescue therapy for patients with lung injuries or infections resulting in inflammatory -mediated pneumonia. clinical assessments including potential acceleration for return to normal function or long-term protective effects on lung fibrosis should also be explored in upcoming studies. in the current study, we chose the bleomycin-induced lung injury model to study the role of pulmonary spinal afferent ablation in lung pathology after acute lung injury. intratracheal injection of bleomycin has been widely used to evoke pulmonary interstitial lesions in animal models. , bleomycin-induced lung injury is primarily mediated by alveolar epithelial damage resulting in the release of large number of inflammatory cells and cytokines. following pulmonary insult with bleomycin at day , inflammation progresses to peak levels around day three. pulmonary edema, respiratory distress, body and organ weight loss associated with systemic inflammation is observed up to day . the model was chosen as it closely reproduces important aspects of ards including local inflammation, cytokine storm, progression to respiratory distress, and multi-organ impact. the timing of therapeutic intervention (day ) was also carefully selected to coincide with high levels of inflammatory mediators and lung damage that would be found in infectious disorders including in covid- patients progressing to ventilatory support. we acknowledge a limitation that since the clinical etiology of lung injury is variable (e.g. viral/bacterial infection, chemical and surgical), the bleomycin model does not completely mimic all pathological characteristics of lung injury in humans. while we considered the lipopolysaccharide (lps)-induced lung injury model, lps has been shown to have a direct effect on sympathetic and parasympathetic afferent and efferent neurons in addition to its effect on the lung itself, , - and thus was less suitable for use in this study. as far as a viral infection model is concerned, we have not attempted to directly apply our findings to relevant diseases such as covid , although there may be some phenotypic overlap between the bleomycin lung injury and viral infection models. more work needs to be done to validate the efficacy of rtx in other lung injury models. our data suggest that pulmonary spinal afferent ablation by intra-stellate injection of rtx reduces plasma extravasation and local pulmonary inflammation post bleomycininduced lung injury which results in improved blood gas exchange. these findings suggests that local stellate application of rtx could be used as a potential clinical intervention to mitigate lung pathology after ali. this study was supported by sorrento therapeutics inc. dr. hanjun wang is also supported by margaret r. larson professorship in anesthesiology. dr. irving h. zucker is supported in part by the theodore f. hubbard professorship for cardiovascular research. the lung/rtx project is currently sponsored by sorrento therapeutics inc. lb: pathogenesis of acute respiratory distress syndrome clinical features of patients infected with novel coronavirus in uk: covid- : consider cytokine storm syndromes and immunosuppression sa: respiratory support for patients with covid- infection clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china cs: acute respiratory distress syndrome the trp superfamily of cation channels. science&# ;s stke d: vagal afferent activation suppresses systemic inflammation via the splanchnic antiinflammatory pathway anti-inflammatory properties of the vagus nerve: potential therapeutic implications of vagus nerve stimulation hj: trpv (transient receptor potential vanilloid ) cardiac spinal afferents contribute to hypertension in spontaneous hypertensive rat hj: sympatho-excitatory response to pulmonary chemosensitive spinal afferent activation in anesthetized, vagotomized rats the paradoxical role of the transient receptor potential vanilloid receptor in inflammation p: the insulin receptor is colocalized with the trpv nociceptive ion channel and neuropeptides in pancreatic spinal and vagal primary sensory neurons the innervation of the kidney in renal injury and inflammation: a cause and consequence of deranged cardiovascular control ih: cardiac sympathetic afferent reflex control of cardiac function in normal and chronic heart failure states chapter -nociceptive physiology dg: improving bioscience research reporting: the arrive guidelines for reporting animal research nr: guide for the care and use of laboratory animals role of calcium ions in the positive interaction between trpa and trpv channels in bronchopulmonary sensory neurons intrathecal resiniferatoxin in a dog model: efficacy in bone cancer pain hj: identification of cardiac expression pattern of transient receptor potential vanilloid type (trpv ) receptor using a transgenic reporter mouse model mj: deletion of vanilloid receptor -expressing primary afferent neurons for pain control ih: cardiac sympathetic afferent denervation attenuates cardiac remodeling and improves cardiovascular dysfunction in rats with heart failure hl: cardiac vanilloid receptor -expressing afferent nerves and their role in the cardiogenic sympathetic reflex in rats hj: sympathoexcitation in response to cardiac and pulmonary afferent stimulation of trpa channels is attenuated in rats with chronic heart failure w: interaction between cardiac sympathetic afferent reflex and chemoreflex is mediated by the nts at receptors in heart failure tc: impact of neuropeptide substance p an inflammatory compound on arachidonic acid compound generation hydrogen sulfide and substance p in inflammation sensory-nerve-derived neuropeptides: possible therapeutic targets the role of neuromodulators (substance p and calcitonin gene-related peptide) in the development of neurogenic inflammation in the oral mucosa h]resiniferatoxin autoradiography in the cns of wild-type and trpv null mice defines trpv (vr- ) protein distribution kl: nk- receptor mediation of neurogenic plasma extravasation in rat skin nw: desensitization of the neurokinin- receptor (nk -r) in neurons: effects of substance p on the distribution of nk -r, galphaq/ , g-protein receptor kinase- / , and betaarrestin- / je: pharmacologic differentiation of inflammation and fibrosis in the rat bleomycin model chronic interstitial pulmonary fibrosis produced in hamsters by endotracheal bleomycin: pathology and stereology w: lipopolysaccharide-mediated inflammatory priming potentiates painful post-traumatic trigeminal neuropathy cg: lipopolysaccharides and trophic factors regulate the lps receptor complex in nodose and trigeminal neurons novel pathway for lps-induced afferent vagus nerve activation: possible role of nodose ganglion gm: lipopolysaccharide induces substance p in sympathetic ganglia via ganglionic interleukin- production key: cord- - cqqj n authors: li, tiao; zou, chunbin title: the role of deubiquitinating enzymes in acute lung injury and acute respiratory distress syndrome date: - - journal: int j mol sci doi: . /ijms sha: doc_id: cord_uid: cqqj n acute lung injury and acute respiratory distress syndrome (ali/ards) are characterized by an inflammatory response, alveolar edema, and hypoxemia. ards occurs most often in the settings of pneumonia, sepsis, aspiration of gastric contents, or severe trauma. the prevalence of ards is approximately % in patients of intensive care. there is no effective remedy with mortality high at – %. most functional proteins are dynamic and stringently governed by ubiquitin proteasomal degradation. protein ubiquitination is reversible, the covalently attached monoubiquitin or polyubiquitin moieties within the targeted protein can be removed by a group of enzymes called deubiquitinating enzymes (dubs). deubiquitination plays an important role in the pathobiology of ali/ards as it regulates proteins critical in engagement of the alveolo-capillary barrier and in the inflammatory response. in this review, we provide an overview of how dubs emerge in pathogen-induced pulmonary inflammation and related aspects in ali/ards. better understanding of deubiquitination-relatedsignaling may lead to novel therapeutic approaches by targeting specific elements of the deubiquitination pathways. acute lung injury and acute respiratory distress syndrome (ali/ards) are a group of illnesses with features of lung inflammation, air-blood barrier disfunction, and hypoxemia. ali/ards are life-threatening with a severe public health concern, approximately , people per year develop into ali/ards in the united states, and the mortality rates are high at - % [ ] [ ] [ ] [ ] [ ] . it is believed that about~ % of patients in intensive care units eventually develop into ali/ards worldwide. etiologically, microbial pneumonia, sepsis, aspiration of gastric contents, or severe trauma are the major causes of ali/ards. approximately % of the ali/ards patients are linked with viral and bacterial pneumonia. the outbreak of severe acute respiratory syndrome coronavirus (sars-cov- ), which causes coronavirus disease (covid- ) , has become a pandemic disease. by now, millions of people have suffered from this disease with hundreds of thousands of deaths in almost all countries all over the world because of the pandemic, and the numbers of the diagnosed patients and the deaths due to this disease are climbing each day (https://coronavirus.jhu.edu/map). for severe covid- patients, ali/ards represent one of the major pathological changes; phenotypes include inflammatory infiltration and inflammatory storm, alveolar epithelial-capillary damage, lung embolism and hemorrhage, hypoxia, and poor prognosis with high mortality. the pathobiology of the disease is incompletely understood [ , [ ] [ ] [ ] . furthermore, no specific effective therapeutic method has been developed to treat the illness. thus, understanding the molecular mechanisms of ali/ards is of particular important in developing effective remedies against the illness. overwhelmed immune responses are believed to be a major contributing factor in the pathogenesis of ali/ards. in the initial pulmonary infection, invaded microbial pathogens including viruses and bacteria attract and activate residential microphages to release chemokines and cytokines, along with infiltration of leukocytes, particularly neutrophils and lymphocytes, into the alveolar sacs [ , ] . in ideal scenarios, host immune responses clear and exclude the invaded pathogens and repair the diseased tissues. however, host immune responses may be unable to achieve this goal due to the pathogenicity of the microbe or the compromised capacity of the host defense, such as in patients with cancer, organ transplantation, diabetes, or hiv infection. higher inflammatory responses may occur in these cases and an over-reacted inflammatory response eventually leads to an overwhelmed inflammatory response. an overwhelmed inflammatory response is increasingly noticed as one of the key contributors to the poor prognosis of ali/ards. a dysregulated high inflammatory response, also referred to as a "cytokine storm", increases mortality in ali/ards patients [ , ] . along with the process of cytokine storm, dysregulated molecular signaling may cause deleterious damage independent of microbial pathogens that increase mortality. however, a high inflammatory storm turns into low inflammation in the later stage due to immune paralysis that may lead to immunosuppression, which contributes to secondary infection and worsens the prognosis of the patients as well [ ] . in the meantime, the invasion of microbial pathogens causes airway epithelial and pulmonary endothelial cell death, destroys alveolar architecture, and damages the air-blood barrier [ , ] . these pathological changes impair effective air-blood exchange, which results in edema and hypoxemia. clinically, hypoxemia in patients with ards is caused by ventilation-to-perfusion mismatch, as well as right-to-left intrapulmonary shunting [ ] . in addition, impaired excretion of carbon dioxide is a major component of respiratory failure, resulting in elevated minute ventilation that is associated with an increase in pulmonary dead space (that is, the volume of a breath that does not participate in carbon dioxide excretion). elevation of pulmonary dead space and a decrease in respiratory compliance are independent predictors of mortality in ards [ ] . the pathophysiological mechanisms of ali/ards are yet to be fully understood. a large number of signal transduction pathways have been revealed to be involved in this process. signal transductions in control of protein stability and availability, including protein ubiquitination and degradation, are typical among the pathways. several review articles have introduced the role of ubiquitination and proteolysis in lung diseases [ ] [ ] [ ] . in this review, we summarize recent findings regarding the importance of deubiquitination and dubs in regulation of inflammation and related pathologies and highlight the role of dubs in ali/ards. proteins dynamically exert their diversified functions in life processes in response to different pathophysiological settings. in concert with gene transcription, ubiquitin proteasome degradation governs the abundance and availability of the protein in the cell. most of the proteins modified by a post translational modification called ubiquitination are deemed to be degraded [ , ] . ubiquitination involves the covalent attachment of the small conserved protein called ubiquitin (ub, amino acids in length) to a target protein, almost exclusively at a lysine residue. ubiquitination is an enzymatic cascade that requires the orchestrated interplay of three different enzymes ( figure ). e ub-activating enzymes bind to both atp and ubiquitin and expose a cysteine residue, the active site of ubiquitin, with the release of an amp. e ub-conjugating enzymes take over activated ubiquitin from e enzymes and cooperate with e ub-ligases. the e ub-ligases interact with e enzymes and recruit protein substrates to initiate conjugation of single ubiquitin or polymeric ubiquitin chains to the protein substrates. e ub-ligases recognize the protein substrates and determine the specificity of protein substrates [ , ] . in humans, there are two e ub-activating enzymes, e ub-conjugating enzymes, and approximately e ub-ligases [ ] . protein ubiquitin proteasomal degradation and deubiquitination. a protein destined for degradation unleashes a cascade of enzymatic activity involving ubiquitination and proteasomal degradation. e ub-activating enzymes activate ubiquitin and pass the ubiquitin to e -ub-conjugating enzymes. e ub-ligases recognize the protein substrates and couple e -ub-conjugating enzymes to covalently add the ubiquitin or ubiquitin moieties to the protein substrates. the ubiquitinated proteins are then degraded by the proteasome. deubiquitinating enzymes remove the mono-ubiquitin or polyubiquitin chains from the ubiquitinated protein to stabilize the protein from proteasomal degradation and recycle ubiquitin units. ub: ubiquitin; e : e ub-activating enzyme；e : e -ub-conjugating enzyme，e : e ub-ligases; dub: deubiquitinating enzyme. the process of ubiquitination is reversible, a group of enzymes called deubiquitination enzymes conduct the enzymatic process [ ] (figure ). deubiquitination is the reverse process of ubiquitination, that removes the mono-ubiquitin and poly-ubiquitin chains from the modified proteins to generate free ubiquitin, which terminates the function of ubiquitinated protein, and specifically, stabilizes the ubiquitinated protein from degradation. deubiquitination also replenishes the ubiquitin pool, and maintains homeostasis of the cellular ubiquitin [ ] . this process is performed by deubiquitinating enzymes (dubs), which are a large set of proteases. the number of dubs in humans is about , while ~ dubs exist in the yeast saccharomyces cerevisiae [ ] [ ] [ ] . a number of approaches are utilized in studying dubs and the related diseases. these approaches include conventional protein-protein interaction techniques such as immunoprecipitation, enzymatic assays, bioinformatics, proteomic, transcriptomic, and structure analysis techniques. based on the architecture of their catalytic domains, to date, six structurally distinct dub families have been described [ ] . five families of dubs are cysteine proteases, including members of usps(ubiquitin-specific proteases)in humans, four members of uchs(ubiquitin carboxy-terminal hydrolases), members of otus(ovarian tumor proteases), four members of mjds (machado-josephin disease protein domain protease) [ , ] , and four members of mindys (motif interacting with ubiquitin (miu)-containing novel dub family) [ ] . the sixth subfamily is jamms (zn-jab /mpn/mov domain protease), which includes a conserved zinc metallopeptidase [ , ] . all dub family members bear a catalytic domain that removes ubiquitin from the protein substrates [ ] . the catalytic domain of miu family sub-members is a new folding variant within the superfamily of cysteine protease and shows a remarkable selectivity for cleaving long lysine (k )-linked ubiquitin chains. in particular, cleavage selectivity of dubs is determined by catalytic the process of ubiquitination is reversible, a group of enzymes called deubiquitination enzymes conduct the enzymatic process [ ] (figure ). deubiquitination is the reverse process of ubiquitination, that removes the mono-ubiquitin and poly-ubiquitin chains from the modified proteins to generate free ubiquitin, which terminates the function of ubiquitinated protein, and specifically, stabilizes the ubiquitinated protein from degradation. deubiquitination also replenishes the ubiquitin pool, and maintains homeostasis of the cellular ubiquitin [ ] . this process is performed by deubiquitinating enzymes (dubs), which are a large set of proteases. the number of dubs in humans is about , while~ dubs exist in the yeast saccharomyces cerevisiae [ ] [ ] [ ] . a number of approaches are utilized in studying dubs and the related diseases. these approaches include conventional protein-protein interaction techniques such as immunoprecipitation, enzymatic assays, bioinformatics, proteomic, transcriptomic, and structure analysis techniques. based on the architecture of their catalytic domains, to date, six structurally distinct dub families have been described [ ] . five families of dubs are cysteine proteases, including members of usps(ubiquitin-specific proteases)in humans, four members of uchs(ubiquitin carboxy-terminal hydrolases), members of otus(ovarian tumor proteases), four members of mjds (machado-josephin disease protein domain protease) [ , ] , and four members of mindys (motif interacting with ubiquitin (miu)-containing novel dub family) [ ] . the sixth subfamily is jamms (zn-jab /mpn/mov domain protease), which includes a conserved zinc metallopeptidase [ , ] . all dub family members bear a catalytic domain that removes ubiquitin from the protein substrates [ ] . the catalytic domain of miu family sub-members is a new folding variant within the superfamily of cysteine protease and shows a remarkable selectivity for cleaving long lysine (k )-linked ubiquitin chains. in particular, cleavage selectivity of dubs is determined by catalytic domain alone, whereas a dub called mindy requires a motif interacting with ubiquitin (miu) as well as a catalytic domain for maximal dub activity [ ] . the physiological roles of dubs include controlling protein stability and quality, maintaining ubiquitin homeostasis, and regulating ubiquitin signals against the functions of e ub ligase [ ] . therefore, dubs regulate numerous cellular events such as the cell cycle, dna damage response, inflammatory signaling, and proliferation and cell death. mounting studies have focused on inflammation to dissect its underlying molecular mechanisms in the pathogenesis of ali/ards. deubiquitinating enzymes play crucial roles in modulation of inflammation by changing the protein stability of the critical molecules (table ) . several usps have proved to play emerging roles in the regulation of lung inflammation [ , ] . innate immunity provides the first line of host defense against pathogens. in lung inflammation, usp protein is over-expressed, reducing i-κb protein levels and thus increasing cytokine release in lung epithelial cells [ , ] . usp acts as a negative regulator of the nf-κb pathway by mediating the deubiquitination of nemo, traf and ikkγ, which leads to the retention of nf-κb in the cytosol, thus suppressing its activity [ , ] . pro and anti-inflammatory cytokines increase in bronchoalveolar lavage fluid and circulating plasma of patients at different stages of ards. tnf-α and il- β are important proinflammatory cytokines in the pathogenesis of ards [ ] . after their receptor activation, ciap-mediated k -ubiquitination of ripk and the traf proteins leads to the recruitment of linear ubiquitin chain assembly complex (lubac). the stability of lysophosphatidic acid receptor (lpa ) is up-regulated by ubiquitin-specific protease (usp ), which deubiquitinates lpa and enhances lpa -mediated proinflammatory effects [ , [ ] [ ] [ ] [ ] . furthermore, the deubiquitinating enzyme usp stabilizes the anti-inflammatory receptor il- r /sigirr to suppress lung inflammation [ ] [ ] [ ] . [ , ] regulates tlr signaling [ ] inhibits inflammation [ ] akt [ ] regulates tgf-β signaling [ ] pai- [ ] regulates acute lung injury [ ] usp- nlrp [ ] regulates nlrp inflammasome activation [ ] nf-κb [ ] , nemo [ ] regulates nf-κb signaling [ , ] vp [ ] involves in virus replication [ ] tat [ ] involves in virus production [ ] traf /traf [ ] modulates antiviral signaling [ ] traf /ikkγ [ ] regulates tlr signaling [ ] usp- cftr [ , ] epithelial mucosal clearance [ , ] nicd [ ] regulates notch signaling [ ] usp- e f [ ] regulates lung epithelia proliferation and wound healing [ ] lpa [ ] enhances inflammation [ ] usp- il- r /sigirr [ ] suppresses lung inflammation [ ] pten [ ] regulates cell apoptosis [ ] mcl [ ] regulates transformation of fibroblasts [ ] stat [ ] regulates ifn signaling [ ] sting [ ] negatively regulates antiviral responses [ ] usp- i-kb [ ] increases cytokine release [ ] cbp [ ] lung inflammation [ ] usp- iκbα [ ] nf-κb activation [ ] usp- hdac [ ] reverses glucocorticoid resistance [ ] traf /traf [ ] lung inflammation [ ] [ ] inhibits type i ifn signaling and antiviral response [ ] poh pro-il- β [ ] negatively regulates the immune response [ ] brcc nlrp [ ] promotes the inflammasome activation [ ] stambp nalp [ ] reduces pro-inflammatory stress [ ] alveolar residential macrophages are central to the development of the inflammatory response by recruiting neutrophils and circulating macrophages to the site of injury, their functions are modulated by deubiquitinating enzymes [ , ] . these cells secrete cytokines, chemokines, reactive oxygen species, proteases, and other mediators that modulate the inflammatory responses and injure the alveolocapillary barrier. gram-negative bacteria-derived endotoxin lipopolysaccharide (lps) promotes stability of a histone acetyltransferase hbo via the function of usp . hbo is believed to fire dna replication licensing at the s-phase of the cell cycle, however, it also regulates inflammatory gene transcription in settings of pulmonary infection. usp -stabilized hbo promotes inflammatory gene transcription in monocyte thp- cells [ ] . in addition, inhibition of usp and usp blocks the nlrp inflammasome by preventing apeck-like protein containing a card (asc) oligomerization and speck formation in macrophages [ ] . usp mediates macrophage-promoted inflammation and stemness in lung cancer cells by regulating traf /traf complex formation [ ] . the activity of deubiquitination regulates inflammasome assembly and function. deubiquitination of nlrp has been suggested to contribute to inflammasome activation. upon treatment with nlrp ligands after the priming step, abro , a subunit of the brisc deubiquitinating complex, is required for optimal nlrp -asc complex formation, asc oligomerization, caspase- activation, and il- β and il- production. this evidence indicates that efficient nlrp activation requires abro [ ] . protein kinase jnk catalyzes nlrp phosphorylation at s within nlrp , which is critical for nlrp deubiquitination and facilitates its self-association and the subsequent inflammasome assembly [ ] . another inflammasome component nalp is regulated by the deubiquitinating enzyme stam-binding protein (stambp), targeting the stambp with a small molecule that inhibits nalp inflammasome activity [ ] . the activities of deubiquitinating enzymes are involved in many aspects of the pathogenesis in ali/ard. lung epithelial cell death is a hallmark in ali/ards. massive lung epithelial cell death has been reported in ards patients. lung epithelial cell death is regulated by deubiquitinating enzymes. loss of dub cyld can activate nf-κb to inhibit apoptosis in lung epithelial cells [ ] . in lung infection, usp are aberrantly expressed, inhibition of usp reduces the abundance of anti-apoptotic protein mcl in the lung [ ] . on the other hand, recent mechanistic studies have reported that lung epithelial cells may defend from bacterial invasion through several mechanisms. usp may regulate the degradation of a deacetylation enzyme hdac to modulate cellular pseudomonas aeruginosa bacterial load, probably via interferon signaling in bronchial lung epithelial cells [ ] . otub interferes with bacterial uptake by modulating the rhoa level [ ] . furthermore, deubiquitination has been proposed to play an important role in alveolar epithelial dysfunction during ali. usp exerts an effect on mucociliary clearance by regulating the endocytic recycling of the cystic fibrosis transmembrane conductance regulator (cftr) in airway epithelial cells [ , ] . in addition, accumulating data suggest that deubiquitination may regulate structural components of the alveolar epithelial monolayer. structural integrity of epithelial cells and intercellular junctions plays an important role in the maintenance of alveolar epithelial barrier integrity. a study suggests that phosphorylated e f is stabilized by nuclear usp to drive peg gene expression and activate proliferation of lung epithelial cells [ ] . finally, airway barrier integrity is primarily maintained by intercellular junctions, which in turn control the paracellular transport of proteins, fluids, and small molecules. cell junction and junctional protein recycling and remodeling is pivotal in barrier integrity. deubiquitination and dubs have been shown to regulate adherence of junctional proteins [ ] . for example, usp regulates e-cadherin mrna levels through stabilizing the traf -jnk pathway in lung epithelial cells [ ] . this study exhibits an indirect effect of dubs on regulation of e-cadherin levels and lung epithelial barrier integrity. until now, the mechanism of covid- infection has not been well illustrated yet. from the biopsy or autopsy of covid- patients, diffuse damage of lung parenchyma has been shown [ , ] . experts hypothesized that sars-cov- invasion severely interrupts the integrity of the airway barrier, thus inducing aberrant inflammatory release ("cytokine storm") and further worsening the lung injury and microcirculation dysfunction, resulting in uncontrolled sepsis in severe cases [ ] . whether dubs participate in the mechanism of sars-cov- infection has not been reported. the coronavirus family contains six members. sars-cov and middle east respiratory syndrome coronavirus (mers-cov) are the two members that have brought an epidemic in recent years. sars-cov and mers-cov, containing the papain-like cysteine proteases (plpro), termed sars-covplpro and mers-covplpro respectively, are antagonists of the host antiviral immune response as they remove ubiquitin and its modifier interferon-stimulated gene (isg ) signals from host cell factors [ , ] . whether such a protease encoded by the sars-cov- genome exists has not been reported, which might expand the field of sars-cov- study. furthermore, human dubs might be potential targets for sars-cov- invasion. we scanned the related dataset of genes and proteins in covid- and the sars-cov- infected cells. data showed that a majority of dubs are decreased in human ipsc-cardiomyocytes infected with sars-cov- via rna-sequencing [ ] . in ace positive type ii pneumocytes, a number of usps including usp and usp are elevated compared to ace negative cells using next generation sequencing [ ] . sars-cov- spike (s) protein invades human tissue through binding angiotensin-converting enzyme (ace ), which reminds us that usps might play an important role in covid- development. however, in the sera of covid- patients, no dubs have been found through proteomics [ ] . in all, the above data revealed that dubs might be involved in the mechanism of sars-cov- infections, but further studies are still urged to explore the function of dubs in covid- . the usp subfamily contains the majority of dubs encoded by the human genome, which are the most diversified members within the dub family [ ] [ ] [ ] . the most studied dub family member in usps is cylindromatosis (cyld). cyld was originally identified as a tumor suppressor, where loss of which causes a benign human syndrome cyld [ ] . with sequence homology to the catalytic domain of ubiquitin carboxy-terminal hydrolases (uch), cyld cleaves k -linked polyubiquitin chains off its target proteins [ ] [ ] [ ] . cyld is proven to be induced by gram-negative and gram-positive bacterial pathogens or their products [ , , , ] . the transcription factor nf-κb activated by bacteria is essential for induction of cyld, in turn, induced cyld negatively regulates the bacteria induced nf-κb signaling [ , ] . cyld deubiquitinates traf and traf to negatively regulate peptidoglycan-induced toll-like receptor (tlr ) signaling and inflammation [ ] . cyld is also highly induced by pneumolysin (ply). cyld deficiency protects mice from acute lung injury in lethal streptococcus pneumoniae infections by inhibiting plasminogen activator inhibitor- (pai- ) expression [ , ] . furthermore, evidence shows that cyld negatively regulates the s. pneumoniae-induced nuclear factor of activated t cells (nfat)signaling pathway by deubiquitinating tgf-β-activated kinase (tak ) [ ] . in contrast, cyld(-/-) mice are hypersusceptible to escherichia coli pneumonia with enhanced nf-κb activation [ ] . perhaps different pathogens may use distinct mechanisms to promote lung inflammation. in the late stage of bacterial infection, cyld exhibits negative effects on injury-induced lung fibrotic response by inhibiting tgf-β-signaling [ ] . these discoveries indicated that cyld might possess a potential drug target for the treatment of bacterial infection pneumonia. usp (hausp)is originally identified as a viral binding protein that preferentially cleaves k -, k -and k -linked ubiquitin chains [ , ] . usp is involved in viral infection by targeting virus related protein to modulate virus replication and production [ ] [ ] [ ] . usp is reported to deubiquitinate and stabilize nf-κb to increase its transcriptional activity in tlr-induced inflammatory gene expression [ ] . furthermore, usp fine-tunes notch signaling in angiogenic sprouting by deubiquitinatingnotch intracellular domain (nicd ) to slow down its turnover of the short-lived form of the activated notch receptor [ ] . ups is also reported to regulate antiviral responses, however, its function is controversial. usp is considered to promote ifn signaling and play an antiviral role by stabilizing stat [ ] . nevertheless, usp deficiency enhances antiviral responses through deubiquitinating stimulator of interferon (sting) [ ] . during bacterial infection, usp loses its activity for iκbα deubiquitination by interacting with e ub-ligase hrd to promote tlr -induced inflammation [ ] . usp mediates deubiquitination and stabilization of hdac in cigarette smoke extract-induced inflammation [ ] . usp also preserves a negative effect on tnf-α-and il- β-triggered nf-κb activation by deubiquitinating tak [ ] . usp interacts with tir domain-containing adaptor inducing interferon-β (trif), and thus impairs its recruitment to tlr / [ ] . usp deficient mice produce exacerbated inflammatory cytokines and are more susceptible to septicemia death [ , ] . usp affects ddx /rig-i-mediated type i interferon signaling through ubiquitinating becn and promoting the formation of autophagosomes [ ] . usp plays a protective role in virus or bacterial infection. several studies showed that usp negatively regulates virus-induced type i ifn signaling by stabilizing traf , traf and traf [ , , , , [ ] [ ] [ ] [ ] . furthermore, usp inhibits tlr -activated innate immunity via removing k ubiquitination of traf [ ] . usp deficient mice have been shown to be more susceptible to virus infection and lps-induced septic shock [ , ] .il- -mediated inflammation is also attenuated by usp through traf and traf deubiquitination [ ] . the anti-malarial drug chloroquine is suggested to alleviate lps-induced inflammation by up regulatingusp in macrophages [ ] . the otu family dubs can be divided into four subfamilies, including otulins (otulin and fam a), otubs/otubains (otub and otub ), otuds (otud , otud /yod , otud , otud , otud /duba, otud a, otud b, alg , and hin l), and a s (a , cezanne, cezanne , trabid, and vcpip) [ ] . the majority of otu members are reported to regulate pathogen-induced cell signaling cascades. in innate and adaptive immunity, otulin is an essential negative regulator of lubac, which hydrolyzes lubac induced met- lineal ubiquitination to prevent nf-κb-or tnf-induced inflammation augmentation [ ] [ ] [ ] ] . otulin can also control antiviral signaling by regulating the lineal ubiquitination chain of stat [ ] . for the negative role of otulin in immune responses, otulin deficiency might cause auto-inflammatory syndrome [ ] . otub and otub regulate virus-triggered ifn inflammation by deubiquitinating traf and traf [ ] . otub suppresses the e ubiquitin-ligase by co-opting k ubiquitin recognition to regulate dna damage [ , [ ] [ ] [ ] [ ] . recent studies also show that otub augments nf-κb-dependent immune responses in dendritic cells in infection and inflammation by stabilizing ubc [ ] . otub recruits phosphorylated smad / and inhibits its ubiquitination by binding with e ub-conjugating enzyme to enhance tgf-β signaling [ ] . otub regulates the maturation and activation of nk and cd +t cells via inhibiting akt ubiquitination [ ] . furthermore, virus-induced otub degradation blocks the rig-i-dependent immune signaling cascade and antiviral response [ ] . several studies showed that otud plays an important role in inflammation regulation [ , ] . rna viruses induceotud to promote the degradation of the mavs/traf /traf signalosome to inhibit innate immunity [ ] . furthermore, otud inhibits type ifn induction after virus infection through cleaving noncanonical k -linked ubiquitination of irf [ ] . otud knockout mice show more resistance to virus infection and lps stimulation [ , ] . otud is a k -specific deubiquitinating enzyme that is previously been reported to maintain the stability of the alkylation repair enzyme alkbh for promoting dna damage repair [ ] . however, otud also preserves k -linked deubiquitinating activity, specifically targetingmyd to inhibit nf-κb signaling [ ] . a recent study shows the role of otud in innate antiviral immunity. otud is induced by virus infection and targets mavs ubiquitination, triggeringirf and nf-κb signaling to sustain antiviral responses [ ] .like most of the dubs, the family member a shows the negative effect on the activation of nf-κb signaling [ , , ] .myeloid-a -deficiency shows a higher inflammatory reaction and sustained nf-κb activation [ ] . a terminates tlr signals by targeting traf deubiquitination [ ] . similar to otub , a suppresses nf-κb signaling by conjugating to e ub-ligase [ ] . histone methyltransferase-enhanced a can also suppress the inflammatory response by modulation of nemo and deubiquitination of traf [ ] . due to its role in inflammation inhibition, a induced by tnfα participates in age-related macrophage dysfunction in the lung [ ] . otuds are newly discovered in antiviral immune responses, which reminds us of the potential drug target for the treatment of virus-induced lung injury. the jamms are the third largest subfamily in dubs, and it comprises members: cop signalosome subunit (csn) , s proteasome non-atpase regulatory subunit (poh ), brca /brca -containing complex subunit (brcc , also known as brcc in humans), mpn domain containing (mpnd, myb-like swirm and mpn domains (mysm ), eukaryotic translation initiation factor subunit (eif )h, csn , s proteasome non-atpase regulatory subunit (psmd ), eif f, anti-müllerian hormone (amsh), amsh-lp, and pre-mrna-processing-splicing factor (prpf ) [ , [ ] [ ] [ ] [ ] .stambp (also known as the associated molecule with the sh domain of stam or amsh), a metalloprotease and a member of the jab /mpn metalloenzyme (jamm) family of dubs, impedes the lysosomal degradation of nacht, lrr and pyd domain-containing protein (nalp )to inhibit inflammasome activity [ , ] . poh deubiquitinates pro-il- β and inhibits mature il- β production, thus restricting inflammasome activity and lps-induced inflammation [ ] . dubbrcc forms a multi-protein complex (brisc) with abro , nba , and bre that specifically cleaves k -linked ubiquitin in the cytoplasm [ ] . abro is important in efficient nlrp activation. abro deubiquitinates nlrp to promote nlrp inflammasome activation [ ] . brcc also targets nlrp to regulate inflammasome formation [ ] . the enzymes of the uch protein family includes four members, uchl /pgp . (protein gene product . ), uchl , uchl /uch , and brca associated protein- (bap ), which contain a conserved catalytic uch domain of~ amino acids [ , ] . the activities of these proteins have been associated with the occurrence and development of cancer [ ] . uchl /uch is suggested to play an anti-apoptotic role in lung epithelial cells through altering bax/bcl- , caspase , and caspase signals [ ] . uch /uch deubiquitinates both smad and smad to promote tgfβ- induced lung fibrosis [ ] . studies of uchs in lung injury and pathogen invasion are still lacking. the mjd family onlycontains four members: ataxin (atxn) , atxn l, josephin domain containing (josd), and josd [ ] . studies show that atxn andjosd are involved in antiviral responses. atxn enhances type ifn signaling during viral infection through deubiquitinating and stabilizing hdac [ ] . nevertheless, josd exhibits a negative role in antiviral activity. josd inhibits the ifn signal cascade via deubiquitinating and stabilizing socs [ ] . the mcpip, also known as zc h a (zinc finger ccch-type containing a) family includes mcpip - members [ ] [ ] [ ] . mcpip implicates a negative role in regulation of the cellular inflammatory responses [ ] . mcpip is the most studied in the mcpip family. acting as a deubiquitinating enzyme, mcpip inhibits nf-κb and c-jun n-terminal kinase (jnk) signaling pathways by removing the ubiquitin moieties from tnf receptor-associated factors (trafs), including traf , traf and traf [ ] .as an rnase, mcpip also regulates inflammatory cytokines like il- by regulating rna decay [ ] and innate defense via degrading viral rna [ ] . mcpip deubiquitinates traf to impede nf-κb signaling [ ] . like the recently identified dubs, the mindy family contains four members: mindy - [ ] , which are highly selective at hydrolyzing k -linked poly-ubiquitin. no data about mindys in ali/ards pathogenesis has been reported. the above dubs play essential roles in the initial development of cancer. however, their functions in lung injury are not fully elucidated. pathogen-related dubs are promising potential targets of drug discovery for human pathogen infection and associated inflammatory disorders. bacteria-encoded dubs might promote bacterial pathogenicity through inhibiting the human ubiquitin-proteasome system [ ] . furthermore, viruses with genes for dubs might inhibit the antiviral pathways using a dub strategy to modulate protein-protein interactions. the sars-covplpro and mers-covplpro papain-like cysteine proteases have been reported, showing a conserved similar structure to the usp family of dubs by x-ray structure, which shows the potential targets of dubs for antiviral drug discovery [ , ] . in addition, several rna virus-related proteases containing the out domain can also remove ubiquitin and isg- signals from host cellular proteins, which represents a potential promising domain for antiviral therapy [ ] . the above findings present great interest to explore a dub-associated anti-infective strategy for human pathogen invasions. however, despite the possibility of dubs as drug targets, the drug discovery for ali/ards is still challenging, with few dub inhibitors or activators having been explored. during the past decade, studies began to dissect the role of dubs in ali/ards. increasing evidence proved that immune responses, inflammation, cell death, air-blood barrier integrity, and invasiveness of the pathogens are fine-tuned by dubs in ali/ards (figure ). modulation of critical proteins via ups and dubs plays a central role in the pathogenesis of diseases such as cancer and autoimmune disease. furthermore, dubs are drawing increasing interest as therapeutic targets against these diseases. our understanding of dubs in ali/ards is limited, and the specific role of dubs remains largely unknown. particularly, the global outbreak of covid- has raised the demand for research on the pathological mechanisms of ali/ards. discovery of the role of dubs in ali/ards might bring valuable information on the pathogenesis of the illness and thereafter drug discovery. the diversified microbial pathogens may cause ali/ards via distinct molecular mechanisms, which increase the complexity of the whole picture that we are attempting to figure out. on the other hand, the current studies are mostly focused on the function of dubs on the regulation of protein degradation and stability. the functions of dubs other than protein stability are yet to be studied in the setting of ali/ards.as a post-translational modification, ubiquitinated proteins may exert a range of functions in life processes and in the pathogenesis of ali/ards, such as signaling transduced via ubiquitinated protein. it is hoped that more data on dubs might lead to identification of novel molecular mechanisms in ali/ards, thus allowing the development of specific dub inhibitors/agonists for the treatment of this acute and severe respiratory illness. funding: this work is supported with r grants (hl and hl ) from national institute of health at the united states to c.z. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. funding: this work is supported with r grants (hl and hl ) from national institute of health at the united states to c.z. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. proteasome non-atpase regulatory subunit prpf pre-mrna-processing-splicing factor psmd proteasome non-atpase regulatory subunit rig- retinoic acid-inducible gene i ripk receptor-interacting protein kinase sars-cov- severe acute respiratory syndrome coronavirus stambp stam-binding protein sting stimulator of interferon tak tgf-β-activated kinase tgfβ- transforming growth factor β- trif tir domain-containing adaptor inducing interferon-β tnf-α tumor necrosis factor-α traf tumor necrosis factor receptor-associated factor uba ub-activating enzymes ubc ub-conjugating enzymes uchs ubiquitin carboxy-terminal hydrolases usps ubiquitin-specific proteases the acute respiratory distress syndrome: from mechanism to translation acute respiratory distress syndrome: the berlin definition the acute respiratory distress syndrome in acute respiratory distress syndrome acute respiratory distress syndrome novel concepts of acute lung injury and alveolar-capillary barrier dysfunction mechanisms of pulmonary edema clearance during acute hypoxemic respiratory failure: role of the na,k-atpase healthcare disparities in patients with acute respiratory distress syndrome. toward equity early local immune defences in the respiratory tract the pathogenesis and treatment of the 'cytokine storm' in covid- into the eye of the cytokine 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deubiquitinases mechanisms of deubiquitinase specificity and regulation mindy- is a member of an evolutionarily conserved and structurally distinct new family of deubiquitinating enzymes toll-like receptor and rig-i-like receptor signaling innate immune recognition of viral infection overexpression of usp protease reduces i-kappab protein levels and increases cytokine release in lung epithelial cells regulation of the ubiquitylation and deubiquitylation of creb-binding protein modulates histone acetylation and lung inflammation hscarg downregulates nf-kappab signaling by interacting with usp and inhibiting nemo ubiquitination hsv icp recruits usp to modulate tlr-mediated innate response inflammatory mechanisms in the lung destabilization of lysophosphatidic acid receptor reduces cytokine release and protects against lung injury the deubiquitinating enzyme usp regulates the endocytic recycling of cftr in airway epithelial cells lopez-castejon, g. usp and usp deubiquitinases regulate nlrp inflammasome activation deubiquitination of nf-kappab by ubiquitin-specific protease- promotes transcription the deubiquitinase usp stabilizes the anti-inflammatory receptor il- r /sigirr to suppress lung inflammation down-regulation of usp mediates phenotype transformation of fibroblasts in idiopathic pulmonary fibrosis deubiquitinase usp dictates mcl stability and sensitivity to bh mimetic inhibitors tumor suppressor cylindromatosis acts as a negative regulator for streptococcus pneumoniae-induced nfat signaling cylindromatosis (cyld) inhibits streptococcus pneumonia-induced plasminogen activator inhibitor- expression via interacting with traf- the tumor suppressor cylindromatosis (cyld) acts as a negative regulator for toll-like receptor signaling via negative cross-talk with traf and traf tumor suppressor cyld acts as a negative regulator for non-typeable haemophilus influenza-induced inflammation in the middle ear and lung of mice cyld negatively regulates transforming growth factor-beta-signalling via deubiquitinating akt tumor suppressor cyld regulates acute lung injury in lethal streptococcus pneumoniae infections regulation of the ebola virus vp protein by sumo usp deubiquitinase controls hiv- production by stabilizing tat protein usp -dependent regulation of traf activation and signaling by a viral interferon regulatory factor homologue a pseudomonas aeruginosa toxin that hijacks the host ubiquitin proteolytic system deubiquitinase usp regulates notch signaling in the endothelium phosphorylated e f is stabilized by nuclear usp to drive peg gene expression and activate lung epithelial cells ubiquitin-specific protease regulates ifn signaling by stabilizing stat usp negatively regulates antiviral responses by deubiquitinating sting er-localized hrd ubiquitinates and inactivates usp to promote tlr -induced inflammation during bacterial infection usp -mediated deubiquitination and stabilization of hdac in cigarette smoke extract-induced inflammation usp mediates macrophage-promoted inflammation and stemness in lung cancer cells by regulating traf /traf complex formation usp inhibits tnf-alpha-and il- beta-triggered nf-kappab activation by deubiquitinating tak regulation of trif-mediated innate immune response by k -linked polyubiquitination and deubiquitination the becn -usp axis plays a role in the crosstalk between autophagy and antiviral immune responses ubiquitin-specific protease regulates tlr -dependent innate immune responses through deubiquitination of the adaptor protein traf ubiquitin-specific proteases negatively regulates virus-induced type i interferon signaling induction of usp by viral infection promotes innate antiviral responses by mediating the stabilization of traf and traf negative regulation of il- -mediated signaling and inflammation by the ubiquitin-specific protease usp lps promotes hbo stability via usp to modulate inflammatory gene transcription in thp- cells cigarette smoke extract modulates pseudomonas aeruginosa bacterial load via usp /hdac axis in lung epithelial cells the deubiquitinating enzyme usp stabilizes traf and reduces e-cadherin-mediated adherens junctions ubiquitin carboxyl-terminal hydrolase-l promotes tgfbeta- signaling by de-ubiquitinating and stabilizing smad /smad in pulmonary fibrosis otulin restricts met -linked ubiquitination to control innate immune signaling otulin antagonizes lubac signaling by specifically hydrolyzing met -linked polyubiquitin the deubiquitinase otulin is an essential negative regulator of inflammation and autoimmunity regulation of the linear ubiquitination of stat controls antiviral interferon signaling regulation of virus-triggered signaling by otub -and otub -mediated deubiquitination of traf and traf the deubiquitinase otub augments nf-kappab-dependent immune responses in dendritic cells in infection and inflammation by stabilizing ubc the deubiquitinase otub controls the activation of cd (+) t cells and nk cells by regulating il- -mediated priming post-translational modification of the deubiquitinating enzyme otubain modulates active rhoa levels and susceptibility to yersinia invasion otub is a key regulator of rig-i-dependent immune signaling and is targeted for proteasomal degradation by influenza a ns otub enhances tgfbeta signalling by inhibiting the ubiquitylation and degradation of active smad / induction of otud by rna viruses potently inhibits innate immune responses by promoting degradation of the mavs/traf /traf signalosome mutations of deubiquitinase otud are associated with autoimmune disorders otud negatively regulates type i ifn induction by disrupting noncanonical ubiquitination of irf otud is a phospho-activated k deubiquitinase that regulates myd -dependent signaling induction of otud by viral infection promotes antiviral responses through deubiquitinating and stabilizing mavs noncanonical regulation of alkylation damage resistance by the otud deubiquitinase the ubiquitin-modifying enzyme a is required for termination of toll-like receptor responses inhibition of nf-kappab signaling by a through disruption of ubiquitin enzyme complexes klf alleviates lipopolysaccharide-induced inflammation by inducing expression of mcp- induced protein to deubiquitinate traf atxn positively regulates type i ifn antiviral response by deubiquitinating and stabilizing hdac josd inhibits mitochondrial apoptotic signalling to drive acquired chemoresistance in gynaecological cancer by stabilizing mcl josd negatively regulates type-i interferon antiviral activity by deubiquitinating and stabilizing socs poh deubiquitinates pro-interleukin- beta and restricts inflammasome activity deubiquitination of nlrp by brcc critically regulates inflammasome activity targeting the deubiquitinase stambp inhibits nalp inflammasome activity diverse macrophage populations mediate acute lung inflammation and resolution the contributions of lung macrophage and monocyte heterogeneity to influenza pathogenesis abro promotes nlrp inflammasome activation through regulation of nlrp deubiquitination nlrp phosphorylation is an essential priming event for inflammasome activation loss of the cylindromatosis tumour suppressor inhibits apoptosis by activating nf-kappab the role of ubiquitination and deubiquitination in the regulation of cell junctions pathological findings of covid- associated with acute respiratory distress syndrome pulmonary pathology of early-phase novel coronavirus (covid- ) pneumonia in two patients with lung cancer sars-cov- and viral sepsis: observations and hypotheses sars hcov papain-like protease is a unique lys linkage-specific di-distributive deubiquitinating enzyme a noncovalent class of papain-like protease/deubiquitinase inhibitors blocks sars virus replication human ipsc-derived cardiomyocytes are susceptible to sars-cov- infection sars-cov- receptor ace is an interferon-stimulated gene in human airway epithelial cells and is detected in specific cell subsets across tissues proteomic and metabolomic characterization of covid- patient sera structural basis of ubiquitin recognition by the deubiquitinating protease usp . structure amino-terminal dimerization, nrdp -rhodanese interaction, and inhibited catalytic domain conformation of the ubiquitin-specific protease (usp ) structure and mechanisms of the proteasome-associated deubiquitinating enzyme usp identification of the familial cylindromatosis tumour-suppressor gene cyld: a deubiquitination enzyme with multiple roles in cancer the tumour suppressor cyld negatively regulates nf-kappab signalling by deubiquitination cyld is a deubiquitinating enzyme that negatively regulates nf-kappab activation by tnfr family members nf-kappab is essential for induction of cyld, the negative regulator of nf-kappab: evidence for a novel inducible autoregulatory feedback pathway cyld is a crucial negative regulator of innate immune response in escherichia coli pneumonia targeting ubiquitin specific proteases for drug discovery screening of dub activity and specificity by maldi-tof mass spectrometry critical role of traf in the toll-like receptor-dependent and -independent antiviral response specificity in toll-like receptor signalling through distinct effector functions of traf and traf regulation of antiviral responses by a direct and specific interaction between traf and cardif duba: a deubiquitinase that regulates type i interferon production chloroquine attenuates lipopolysaccharide-induced inflammatory responses through upregulation of usp . can otu deubiquitinases reveal mechanisms of linkage specificity and enable ubiquitin chain restriction analysis linear ubiquitination signals in adaptive immune responses otulin deficiency causes auto-inflammatory syndrome otub co-opts lys -linked ubiquitin recognition to suppress e enzyme function non-canonical inhibition of dna damage-dependent ubiquitination by otub evidence for bidentate substrate binding as the basis for the k linkage specificity of otubain the mechanism of otub -mediated inhibition of ubiquitination a (tnfaip ) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis histone methyltransferase ash l suppresses interleukin- production and inflammatory autoimmune diseases by inducing the ubiquitin-editing enzyme a elevated a contributes to age-dependent macrophage dysfunction in the lungs k -specific deubiquitination by two jamm/mpn+ complexes: brisc-associated brcc and proteasomal poh structural basis for specific cleavage of lys -linked polyubiquitin chains sequential ubiquitination and deubiquitination enzymes synchronize the dual sensor and effector functions of trim activation of the endosome-associated ubiquitin isopeptidase amsh by stam, a component of the multivesicular body-sorting machinery the lys -specific deubiquitinating enzyme brcc is regulated by two scaffold proteins localizing in different subcellular compartments up-regulation of expression of the ubiquitin carboxyl-terminal hydrolase l gene in human airway epithelium of cigarette smokers ubiquitin carboxyl-terminal hydrolases: involvement in cancer progression and clinical implications effect of ubiquitin carboxy-terminal hydrolase on apoptotic in a cells deubiquitylating enzymes and drug discovery: emerging opportunities monocyte chemotactic protein (mcp)- promotes angiogenesis via a novel transcription factor, mcp- -induced protein (mcpip) regulatory feedback loop between nf-kappab and mcp- -induced protein rnase mcpip negatively regulates toll-like receptor signaling and protects mice from lps-induced septic shock a novel ccch-zinc finger protein family regulates proinflammatory activation of macrophages mcp-induced protein deubiquitinates traf proteins and negatively regulates jnk and nf-kappab signaling zc h a is an rnase essential for controlling immune responses by regulating mrna decay mcpip ribonuclease exhibits broad-spectrum antiviral effects through viral rna binding and degradation the molecular basis for ubiquitin and ubiquitin-like specificities in bacterial effector proteases crystal structure of the middle east respiratory syndrome coronavirus (mers-cov) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression crystal structure of the papain-like protease of mers coronavirus reveals unusual, potentially druggable active-site features ovarian tumor domain-containing viral proteases evade ubiquitin-and isg -dependent innate immune responses this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -mwtqxwbw authors: zhang, leifang; xu, chenming; chen, xiaoming; shi, qiwen; su, weike; zhao, hang title: socs- suppresses inflammation through inhibition of nalp inflammasome formation in smoke inhalation-induced acute lung injury date: - - journal: inflammation doi: . /s - - -y sha: doc_id: cord_uid: mwtqxwbw smoke inhalation leads to acute lung injury (ali), a devastating clinical problem associated with high mortality rates. suppressor of cytokine signaling- (socs- ) is a negative regulator of proinflammatory cytokine signaling. we have found that adenoviral gene transfer of socs- ameliorates smoke inhalation-induced lung injury in c bl/ mice. we also found that the release of adenosine triphosphate (atp) was increased post smoke exposure, while oxidized atp, an inhibitor of purinergic p x receptor, suppressed smoke-induced nalp inflammasome assembly, caspase- activation, and k(+) efflux. similar to oxidized atp, high protein level of socs- dampened the formation of nalp inflammasome and the activation of caspase- and il- β induced by smoke exposure in mouse alveolar macrophages. in conclusion, socs- relieves smoke inhalation-induced pulmonary inflammation and injury by inhibiting nalp inflammasome formation. each year, burn-related injuries result in nearly , deaths globally and deaths in the usa. ninety percent of burn-related mortality can be partially attributed to smoke inhalation-associated acute lung injury (si-ali). despite advances in care and rehabilitation of patients with burns, si-ali-related mortality remains substantial at . % in [ ] . smoke inhalation is the leading cause of acute lung injury (ali), acute respiratory distress syndrome (ards), and serious respiratory failure. ali/ards is also a significant clinical problem with no drug treatment currently approved by the food and drug administration. because ali/ards patients entering the intensive care unit often have already developed clinical diseases, therapeutics targeting not only the initiation but also the propagation of ali/ards is necessary. therefore, understanding the molecular mechanisms of smokeinduced lung injury can help the treatment of ali/ards. yet, despite decades of intense research, the molecular mechanisms involved in the pathogenesis of smoke inhalation-induced ali are poorly understood. smoke directly destroys endothelial and epithelial barrier function and aggregates mucous with sloughed airway epithelium and cellular debris, leading to airway obstruction, change in regional blood flow, increased airway microvascular permeability, exfoliation of epithelial lining, enhanced surfactant denaturation, and toxin/ cytokine-mediated inflammation [ ] [ ] [ ] [ ] . the most abundant non-parenchymal cell in the lung is alveolar macrophage, which plays a central role in maintaining normal lung structure and function in both the maintenance of immunological homeostasis and host defense [ , ] . the inflammatory responses triggered by direct or indirect insults to the lung involve the recruitment and activation of tissue macrophages in the lung parenchyma of ali/ards. macrophages can be rapidly activated by burn injury to release proinflammatory cytokines, such as tumor necrosis factor-alpha (tnf-α) and interleukin- beta (il- β), and may therefore play important roles in burn-induced ali [ , ] . il- β is known as one of the most potent inflammatory initiating cytokines observed in patients suffering from ali [ ] . macrophage depletion improves alveolar barrier dysfunction and lung inflammatory response caused by severe burns [ ] . suppressor of cytokine signaling (socs- ) is a member of the cytokine signaling pathway inhibitor family, which is known as a potent negative regulator of proinflammatory cytokine signaling [ , ] . socs- is characterized by the presence of an n-terminal proline rich domain called the central src homology (sh ) domain and a c-terminal conserved motif named the socs box [ ] . the socs box can couple with sh domain-binding proteins to activate ubiquitin-proteasome pathway and thus function as a negative regulator of cytokine signaling [ , ] . socs- deletion causes fatal inflammation, which can be rescued by deletion of ifng [ ] . previously, findings indicate that socs- is critical in macrophages for preventing uncontrolled inflammation [ ] . in lpsinduced ali, downregulation of socs- by alveolar macrophages-derived mir- , which is a proinflammatory factor blinding to '-utr of socs- , remarkably exaggerates lung inflammation [ ] . increased expression of ad-socs- has been shown to protect lung from hyperoxic injury by suppressing il- β levels [ ] . ad-socs- -infected murine exhibits a lower degree of lymphocytic inflammation and pulmonary fibrosis as well as a diminished mortality after bleomycin treatment [ ] . however, the role of socs- in inflammation during smoke inhalation-induced ali has not been revealed. nalp inflammasome is a multiprotein oligomer formed by nalp , apoptosis-associated speck-like protein containing a card (asc), and pro-caspase- . nalp also participates in the pathogenesis of ali [ , ] . atp, an nalp agonist, acts on p x purinergic receptor (p rx ) to alter the permeability of the cell membrane, which results in cytosolic k + efflux and the collapse of cation gradient [ ] . a drop in cytosolic k + level is a common and sufficient trigger for nalp inflammasome assembly and caspase- activation [ ] . in response to the rising outflow of k + , the assembly of asc specks, which are large multimeric complexes predominantly composed of dimers and oligomers of asc, is activated. this allows procaspase- to be recruited and starts the autocatalytic event that ultimately leads to the formation of active caspase- heterotetramers and the conversion of inactive cytosolic pro-il- β to the functional form [ ] [ ] [ ] [ ] . recent in vitro experiment using alveolar macrophages (ams) indicates that ros-mediated nlrp inflammasome plays an essential role in burn-induced ali [ ] . other studies suggest that socs- may suppress the activity of nalp , resulting in attenuated encephalomyelitis (eae) pathogenicity [ ] . we therefore hypothesize that socs- exhibits important effects on smoke inhalation-induced ali through an anti-inflammatory mechanism. to test our hypothesis, c bl/ mice were infected with ad-socs- or ad-gfp by microsprayer. then, the degree of lung damage and the survival rates were examined after smoke inhalation. furthermore, to reveal the anti-inflammatory mechanism of socs- , we studied both the activation process of the nalp inflammasome assembly in response to smoke exposure and the inhibitory action of socs- on smokeinduced nalp inflammasome assembly, caspase- activation, and il- β expression. c bl/ male mice aged - weeks were obtained from the zhejiang academy of medical sciences. all animals were maintained under specific pathogen-free conditions. smoke inhalation was administered as previously described [ ] . after induction of anesthesia with % chloral hydrate . ml/g (tianjin dingshengxin chemical industry co., ltd., china), mice were briefly placed in the smoke chamber, which was filled with smoke from mg of smoldering cotton heated to °c, for min. during smoke exposure, the temperature in the smoke chamber did not exceed °c to prevent thermal injury to the airways. all mice were returned to the cages for recovery, which was recorded as day . during the experimental period, mice were allowed free access to food and water. mice were euthanized to collect lung macrophages. lungs were perfused at °c with ml of cold normal saline (ns) times. lung tissue was placed on a culture plate containing cold rpmi and then chopped. cell suspension was filtered twice by a -μm steel mesh. lung macrophages were centrifuged with sterile water to destroy red blood cells. cells were washed with rpmi . the suspension was then placed in lymphocyte separation medium and centrifuged to obtain lung macrophages. the experimental method is similar to smoke inhalation [ ] . briefly, after isolation of mouse lung macrophages, cells were cultured in six well plates overnight. during smoke exposure, the temperature in the smoke chamber was maintained at °c. the smoke was introduced to the cells at a flow rate ml/min using an air sampling pump for min. after completion of the exposure period, the cells were washed using × pbs, and added into the new culture medium with % fcs with % co in an incubator at °c for h. cells were digested and collected for subsequent experiments. twenty-four hours after smoke exposure, the mice were sacrificed and the lungs were collected. each lung was fixed in % formalin and embedded in paraffin. the fixed lungs were then sectioned at μm of thickness, and the tissue was dewaxed in ethanol, rehydrated, and stained with hematoxylin and eosin. adenoviral expression vector carrying the coding sequences of the mouse socs- gene (ad-socs- ) were from abm inc. (richmond, canada). primers were designed and synthesized by tiangen biotech (beijing) co., ltd. socs- sense: '-cacgcact tccgcacattcc- ′ ; socs- antisense: ' -tccagcagctcgaagaggca- ′. briefly, total rna was extracted from c bl/ mouse liver tissue by trizol rna extraction kit (invitrogen, us). rt-pcr (thermo fisher scientific, us) was used to obtain msocs- cdna by primescript®rt reagent kit with gdna eraser (takara, japan). the msocs- cdna was cloned into a multicloning site of phmcmv . the padhm -socs- was constructed by ligation of i-ceui/pi-scei-digested padhm and i-ceui/pi-scei-digested phmcmv -msocs- . to generate the virus, padhm -socs- was digested with paci and purified by phenol-chloroform extraction and ethanol precipitation. linearized dnas were transfected into hek- cells with lipofectamine™ (invitrogen, us) according to the manufacturer's instructions. the viruses were amplified in hek- cells. before virus purification, cell lysates were centrifuged to remove cell debris and digested for min at °c with μg/ml dnase i and μg/ml rnase a in the presence of mm mgcl . viruses were purified by cscl step gradient ultracentrifugation followed by cscl linear gradient ultracentrifugation. the purified viruses were dialyzed against a solution containing mm tris-hcl (ph . ), mm mgcl , and % glycerol. they were then stored at − °c. viral particle (vp) and biological titers were determined by a spectrophotometrical method and by using an adeno-x rapid titer kit (bd clontech, us), respectively. the ratios of the biological-to-particle titer were : and : for ad-socs- and ad-gfp (control), respectively. lung tissue lysates or cell extracts were electrophoresed on sds-page gels (bio-rad, china) and transferred to polyvinylidene difluoride (pvdf) membranes (millipore, us). the membranes were washed in × pbs tween- for min and blocked with % fat-free milk at room temperature for h. then they were incubated with socs- antibody, nalp , asc, caspase- , active caspase- , or gapdh antibody (abcam, china) in tbst containing % fat-free milk at °c overnight on a gentle shaker. after washing with × pbs tween- for min, the membranes were incubated with the appropriate secondary antibody (abcam, china) in tbst containing % fat-free milk at room temperature for h according to manufacturer's instruction. proteins were detected by ecl chemiluminescence (ge, healthcare, china). immunoprecipitation was performed using the mper™ immunoprecipitation kit (pierce biotechnology, us). five hundred micrograms of protein lysate from alveolar macrophages were incubated with anti-nalp or anti-asc antibody ( - μg) at °c overnight with rocking. the immune complexes were allowed to bind to μl of recombinant protein g agarose beads (invitrogen, usa) at °c for h, and the beads were washed three times with lysis buffer. after centrifugation, the supernatants were obtained as immunoprecipitates and dissolved in sds-page sample buffer for electrophoresis and immunoblot analysis. quantification of atp was determined by atp determination kit (thermo fisher, us). the concentration of extracellular atp was determined by the culture supernatant. one hundred microliters per well recombinant firefly luciferase (llr) was added, and the solution was shaken gently. the transmitted light was detected with a luminometer (biosystems / luminometer, turner), and fluorescence intensity was measured continuously for min [ ] . macrophages were seeded at a density of × cells per well in -well tissue culture dishes and maintained for h. the next day, media were collected at the indicated time points, and cells were washed once with phosphatebuffered saline. cells were lysed in ml of % nitric acid. the extracellular (medium) and the intracellular k + content was quantified using atomic absorbance spectroscopy [ ] . data are expressed as mean ± sem from three or more independent experiments. when comparing between three or more groups, differences were tested using oneway analysis of variance followed by the bonferroni test. when comparing between two groups, we used a student's paired two-tailed t test. ad-socs- or ad-gfp ( × pfu/mouse) was delivered to c bl/ mice lung by a microsprayer (penn-century, us). mice lungs and survival were examined after smoke exposure. the overexpression of socs- was confirmed in mice lungs h after ad-socs- infection by western blot (fig. a) . the results showed that transfection of socs- was successful, and the expression of socs- in the lung of the ad-socs- group was significantly higher than that in ad-gfp group. after min of smoke inhalation, mice survival was monitored for up to days. three days after smoke inhalation, all ad-gfp-administered mice died, while % of ad-socs- -administered mice still lived. twenty-five percent of ad-socs- -administered mice even survived up to days, at which point they were sacrificed (fig. b) . the results show that the high expression of socs- in vivo can significantly improve the survival rate of mice with smoke-induced lung injury. after exposure to smoke for min and a day of rest, mice were sacrificed to observe the degree of lung injury in each group. ad-gfp mice had obvious lung tissue injury after smoke exposure: lung tissues appeared darkly red with a volume expansion, and lung surface was moist with increased exudation; diffuse hyperemia and edema were visible, and congestion was obviously in some lung tissues. conversely, in ad-socs- mice, lung tissues appeared pink with a smooth and rosy surface; no blood, bleeding spots, congestion, or edema was observed. there was an obvious reduction in lung injury (fig. a) . the results showed that compared to ad-gfp-infected mice, ad-socs- -infected mice appeared to have a lower level of lung tissue damage. thus, socs- overexpression significantly reduces lung injury caused by smoke inhalation. to further demonstrate that socs- overexpression can significantly reduce lung injury caused by smoke inhalation, lungs were isolated and lung tissue sections were stained by h&e after infection with or without smoke exposure. mice were exposed to smoke for min and sacrificed day later. under optical microscope, in the ad-gfp mice, the capillaries in the alveolar walls showed swelling and were congested with many red blood cells. alveolar epithelial cells were edematous. there was visible neutrophil and monocyte infiltration in the pulmonary interstitium. hemorrhage in the alveolar spaces was found as well. on the contrary, in the ad-socs- mice, the lung tissues in the specimens were clear and integral. the alveolar septum was thin and the wall was smooth. there was rarely neutrophil and monocyte infiltration observed in spaces (fig. b) . the results showed that ad-socs mice exposed to toxic smoke had a marked decrease in immune cells infiltration. it is confirmed that socs- overexpression can significantly reduce smoke inhalation-caused lung injury. atp activates the nalp inflammasome through p x receptor mediated k + efflux [ , ] . to determine whether smoke exposure affects atp, an important activator of nalp , alveolar macrophages isolated from non-infected mice lungs were treated with or without smoke for min, and the extracellular concentration of atp was analyzed min after the end of smoke treatment. compared to non-treated macrophages, smoke-treated macrophages displayed a significant upsurge in atp release of approximately ± . nm/ cells (fig. a) . to determine whether smoke affects cytoplasmic k + level, we detected both extracellular and intracellular k + concentrations up to min after smoke exposure by atomic absorption flame photometry with or without oxidized atp (oxatp), a p rx antagonist, and observed that oxatp reduced smoke-induced k + efflux (fig. b, c) . before exposure to smoke, the extracellular k + baseline level of macrophages was nm/well (fig. b) , and the intracellular k + baseline level was nm/well (fig. c) . after exposure to smoke, there was about an % decrease in the intracellular k + concentration, while the extracellular k + level was surged to nm/well. the depletion of cytoplasmic k + was detected within min and was maintained for min after inhalation of smoke. these results show that smoke-induced k + efflux through interacting with p rx (fig. b, c) . to determine whether p rx is required for smokeinduced inflammasome activation, isolated macrophages were pretreated with oxatp and exposed to smoke or air for min. immunoblotting revealed that smoke significantly increased the protein level of nalp , asc, and cleaved caspase- . oxatp reduced smoke-mediated caspase- activation but not smoke-induced nalp and asc expression (fig. d) . immunoprecipitation of cell lysates with anti-asc antibody and the quantifications of relative protein expression analyzed by the intensity of the signals affirmed that oxatp suppresses the interaction between asc and nalp enhanced by smoke (fig. e) . this indicates that p rx is a necessary component of smoke-increased nalp inflammasome assembly (fig. d, e) . to determine whether socs- reduces atp and k + efflux induced by smoke, alveolar macrophages isolated from ad-socs- mice lungs were treated with or without smoke for min, and the extracellular concentration of atp was analyzed min after the end of smoke treatment. macrophages from the lungs of ad-socs- mice showed a significant decrease in atp release compared to ad-gfp mice alveolar macrophages (fig. a) . we also detected both extracellular and intracellular k + concentrations up to min after smoke and observed that macrophages from ad-socs- -infected mice exhibited a lower degree of k + efflux than those from ad-gfp-infected mice (fig. b, c) . to test whether socs- exerts anti-inflammatory effect through inhibition of nalp inflammasome formation and consequent activation of caspase- and il- β, ad-gfp, or ad-socs- -adminstered mice were exposed to smoke for min and euthanized day later to collect alveolar macrophages. immunoblotting i ndicat ed that overexpression of socs- helped macrophages resist smoke-enhanced caspase- cleavage. additionally, the amount of asc protein pulled down with nalp was detected after immunoprecipitation with anti-nalp antibody. the results indicate that socs- repressed smokeinduced interaction between nalp and asc (fig. a) . we also explored caspase- activity and il- β level by enzyme-linked immunosorbent assay (elisa) and found that compared to the macrophages in smoke-treated ad-gfpinfected mice, the macrophages in smoke-treated ad-socs- -infected mice had lower levels of both caspase- activity and il- β secretion (fig. b) . our study demonstrates that socs- obstructs smoke-activated nalp inflammasome assembly, caspase- cleavage, and il- β secretion. this study aims to identify the role of socs- in smoke inhalation-induced ali. the results reveal that socs- attenuates ali resulting from smoke exposure by suppressing macrophage-mediated inflammation. adenovirus-mediated transfer of socs- to mice attenuates smoke exposure-induced ali and improves the survival of mice. additionally, smoke-enhanced atp release promotes nalp inflammasome formation and caspase- activation through the stimulation of p rx -mediated k + efflux. finally, adenoviral gene transfer of socs- blocks atp and k + efflux to diminish nalp inflammasome assembly, caspase- activation, and il- β expression prompted by smoke in macrophages (fig. ) . previous reports indicate that socs- plays an important role in preventing inflammation. socs- is critical for preventing lethal inflammation in t cells and macrophages, and deficiency of socs- results in spontaneous inflammation [ ] . socs- in adaptive immune cells inhibits antiviral immunity, and its presence in innate/stromal cells is responsible for aggravated lung damage [ ] . socs- -deficient mice die within weeks of birth with severe inflammation and lung damage through infiltration fig. . reduced degree of smoke inhalation-induced lung injury in ad-socs- -treated mice. mice infected with ad-gfp or ad-socs- were exposed to smoke for min. after day, a the degree of lung injury was observed in each group, and b lung tissue sections stained by h&e were observed to study morphological alternation (magnification × ). of inflammatory cells [ ] . compared to control mice, mice with overexpression of socs- exposed to hyperoxia show a significant amelioration in inflammation and alveolar damage [ ] . as is shown in our results and other reports, smoke exposure can lead to ali and result in inflammation and alveolar damage [ , ] . mice with ad- fig. . the requirement of p rx in smoke exposure-induced nalp inflammasome assembly and caspase- cleavage. alveolar macrophages were isolated from c bl/ mice, treated with or without oxatp, exposed to smoke for min, and then analyzed for a atp release, b extracellular, and c intracellular k + levels, d expression of inflammasome components nalp , asc, and caspase- , and e the complex formed by nalp and asc. the intensity of the signals were quantified by imagej software for more strict and direct viewing to reflect the interaction between asc and nalp (e). fig. . negative regulation of socs- on smoke-induced atp and k + efflux. ad-gfp or ad-socs- -treated mice were exposed to smoke for min and allowed to rest for day. isolated alveolar macrophages were analyzed for a atp release and b intracellular and c extracellular k + levels.(p < . vs. the ad-gfp group). gfp exposed to smoke all died within days and the lung tissue showed lung tissue damage and immune cells invasion. however, survival rates and these pathological changes were significantly ameliorated in ad-socs- mice. galam et al. show that socs- overexpression inhibits ask- and suppresses il- β levels to restore lung tissue in hali [ ] . therefore, we speculate that ask- may participate in inflammation of lung injury induced by smoke and interact with socs- in this process. . negative regulation of socs- on smoke-induced nalp inflammasome assembly, caspase- activation, and il- β expression. ad-gfp or ad-socs- -treated mice were exposed to smoke for min and allowed to rest for day. isolated alveolar macrophages were analyzed for a nalp inflammasome assembly and b caspase- activity and il- β secretion. nalp inflammasome is activated and the inflammatory cascade is triggered with subsequent fibrosis in crystalline silica-induced lung injury [ ] . bleomycininduced inflammation and fibrosis of ali also require activation of nalp [ ] . using the mouse model of hemorrhagic shock (hs) in the lungs, hs activates lung endothelial nad(p)h oxidase through high mobility group box (hmgb ) to induce the production of reactive oxygen species (ros). ros then promotes association of thioredoxin-interacting protein (txnip) w i t h n a l p a n d s u b s e q u e n t a c t i v a t i o n o f inflammasome and il- β secretion [ ] . nalp inflammasome formation and il- β cleavage are induced by hyperoxia both in vivo and in vitro [ , , , ] . in the hyperoxia-induced lung injury mice model, hyperoxia can induce atp efflux. the elevated extracellular atp interacts with p x receptors, causing k + efflux and activation of nalp inflammasome [ ] . likewise, our results show that smoke-induced atp promotes inflammasome formation and caspase- activation through stimulation of p x receptorinduced k + efflux. previous study investigating the effect of nalp on an inflammatory disease, eae, demonstrates that ifnar (the shared receptor for ifnα and ifn-β) signaling in macrophages induces socs- -mediated ubiquitination and degradation to decrease the production of mitochondrial ros, resulting in inhibition of nalp inflammasome activity [ ] . our results indicate that socs- reduces nalp inflammasome formation through repression of the smoke-induced interaction between nalp and asc, which thereby inhibits caspase- and il- β activated by nalp . recent reports also indicate that cobalt protoporphyrin induces cox- expression by activation of p x receptors, ask- /map kinases, and stat- degradation signaling pathways [ ] . therefore, there may be a relationship between ask- and p rx that leads to the formation of nalp in smoke-induced ali, which we will further investigate in the future. in summary, socs- significantly ameliorates smokeinduced ali. the protective mechanism of socs- from smoke-induced ali is associated with a reduction in inflammation. these studies are the first to demonstrate that socs- is a critical regulator of smoke inhalation-induced ali and a key mediator in inflammation protection. socs- may lead to the discovery of anchor points for new treatment strategies for smoke-induced ali. funding information this study is supported by the national natural science foundation of china (no. ) and zhejiang natural science foundation of china (ly h ) for their financial supports. suffredini, and us critical illness and injury trials group: smoke inhalationassociated acute lung injury (si-ali) investigators (usciit-si-ali). . risk factors for in-hospital mortality in smoke inhalation-associated acute lung injury: data from united states hospitals imbalance of th /tregs in rats with smoke inhalation-induced acute lung injury pathophysiology, management and treatment of smoke inhalation injury bronchoscopy-derived correlates of lung injury following inhalational injuries: a prospective observational study high mw hyaluronan inhibits smoke inhalation-induced lung injury and improves survival inflammation and the acute respiratory distress syndrome ros-mediated nlrp inflammasome activity is essential for burn-induced acute lung injury acute lung injury review role of pulmonary alveolar macrophage activation in acute lung injury after burns and smoke inhalation ethanol intoxication prolongs post-burn pulmonary inflammation: role of alveolar macrophages molecular mechanism underlying the antiproliferative effect of suppressor of cytokine signaling- in nonsmall-cell lung cancer cells suppressors of cytokine signaling and immunity negative regulation of cytokine signaling pathways socs- and socs- block insulin signaling by ubiquitin-mediated degradation of irs and irs the suppressor of cytokine signaling socs promotes apoptosis of intestinal epithelial cells via p signaling in crohn's disease socs- /ssi- -deficient nkt cells participate in severe hepatitis through dysregulated cross-talk inhibition of ifn-gamma and il- signaling in vivo suppressor of cytokine signaling- in t cells and macrophages is critical for preventing lethal inflammation macrophage micro-rna- promotes lipopolysaccharide-induced acute lung injury in mice and rats socs- rescues il- beta-mediated suppression of epithelial sodium channel in mouse lung epithelial cells via ask- suppressor of cytokine signaling inhibits pulmonary inflammation and fibrosis. the journal of allergy and cytokine-mediated inflammation in acute lung injury melatonin alleviates acute lung injury through inhibiting the nlrp inflammasome the cytolytic p z receptor for extracellular atp identified as a p x receptor (p x ) k(+) efflux is the common trigger of nlrp inflammasome activation by bacterial toxins and particulate matter structure and assembly of the mouse asc inflammasome by combined nmr spectroscopy and cryo-electron microscopy caspase- mediated interleukin- activation in neutrophils promotes the activity of rheumatoid arthritis in a nlrp inflammasome independent manner atp induces il- beta secretion in neisseria gonorrhoeaeinfected human macrophages by a mechanism not related to the nlrp /asc/caspase- axis interferon-beta therapy against eae is effective only when development of the disease depends on the nlrp inflammasome nebulization with gammatocopherol ameliorates acute lung injury after burn and smoke inhalation in the ovine model socs- ameliorates smoke inhalation-induced acute lung injury through inhibition of ask- activity and disc formation physiological regulation of atp release at the apical surface of human airway epithelia differential requirement of p x receptor and intracellular k+ for caspase- activation induced by intracellular and extracellular bacteria liaisons dangereuses: p x( ) and the inflammasome expression of suppressor of cytokine signaling (socs ) impairs viral clearance and exacerbates lung injury during influenza infection adenovirus-mediated transfer of the socs- gene to mouse lung confers protection against hyperoxic acute lung injury lung injury from smoke inhalation pathophysiology of acute lung injury in combined burn and smoke inhalation injury il- r /myd signaling and the inflammasome are essential in pulmonary inflammation and fibrosis in mice hemorrhagic shock activation of nlrp inflammasome in lung endothelial cells the inflammasome activation of the nalp inflammasome is triggered by low intracellular potassium concentration the inflammasome mediates hyperoxia-induced alveolar cell permeability cobalt protoporphyrin upregulates cyclooxygenase- expression through a heme oxygenaseindependent mechanism conflict of interest. the authors declare that they have no conflict of interest. key: cord- -kagggou authors: liu, chang; yin, zhigang; feng, tingting; zhang, min; zhou, zhi; zhou, ying title: an integrated network pharmacology and rna-seq approach for exploring the preventive effect of lonicerae japonicae flos on lps-induced acute lung injury date: - - journal: j ethnopharmacol doi: . /j.jep. . sha: doc_id: cord_uid: kagggou ethnopharmacological relevance: lonicerae japonicae flos (ljf, the dried flower bud or newly bloomed flower of lonicera japonica thunb.), a typical herbal medicine, targets the lung, heart and stomach meridian with the function of clearing heat and detoxication. it ameliorated inflammatory responses and protected against acute lung inflammation in animal models. acute lung injury (ali) is a kind of inflammatory disease in which alveolar cells are damaged. however, a network pharmacology study to thoroughly investigate the mechanisms preventing ali has not been performed. aim of the study: in this study, we examined the main active ingredients in ljf and the protective effects of ljf on lps-induced ali in rats. materials and methods: first, the main active ingredients of ljf were screened in the tcmsp database, and the ali-associated targets were collected from the genecards database. then, we used compound-target and target-pathway networks to uncover the preventive mechanisms of ljf. furthermore, we assessed the preventive effects of ljf in an lps-induced rat model with the rna-seq technique to validate the possible molecular mechanisms of the effects of ljf in the treatment of ali. results: the network pharmacology results identified main active compounds in ljf, and eight chemical components highly related to the potential targets, which were potential active compounds in ljf. in all, potential targets were recognized, including il , tnf, ptgs , app, f , and grm . the pathways revealed that the possible targets of ljf involved in the regulation of the il- signalling pathway. then, in vivo experiments indicated that ljf decreased the levels of proinflammatory cytokines (tnf-, il- , and il- ) in serum and bronchoalveolar lavage fluid, decreased the levels of oxidative stress factors (mda and mpo) and increased the activities of sod and gsh-px in lung tissue. the rna-seq results revealed that , and differentially expressed genes (degs) in ctrl (control group), ali-ljf (lonicerae japonicae flos group) and ali-dxsm (dexamethasone group), respectively. kegg pathway analysis showed that the degs associated with immune response and inflammation signalling pathways and the il- signalling pathway were significantly enriched in ljf. compared with those in ali, the expression of cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, mmp and tnfaip , which are involved in the il- signalling pathway, were significantly decreased in the ljf group according to the qrt-pcr analyses. conclusions: in view of the network pharmacology and rna-seq results, the study identified the main active ingredient and potential targets of ljf involved in protecting against ali, which suggests directions for further research on ljf. f , and grm . the pathways revealed that the possible targets of ljf involved in the regulation of the il- signalling pathway. then, in vivo experiments indicated that ljf decreased the levels of proinflammatory cytokines (tnf-, il- , and il- ) in serum and bronchoalveolar lavage fluid, decreased the levels of oxidative stress factors (mda and mpo) and increased the activities obtain the aqueous extract, which was kept at - ℃ until use. prior to hplc analysis, the solution was filtered through a . μm membrane. the chromatographic fingerprinting of ljf showed main peaks, which were identified as (neochlorogenic acid), (chlorogenic acid), (chlorogenic acid), (secoxyloganin), (isochlorogenic acid b), (isochlorogenic acid a), and (isochlorogenic acid). the method could be used to identify the characteristics of ljf (fig. s ). for further analyses, the bronchoalveolar lavage fluid (balf), lung tissue and blood were collected and stored at − °c. after treatment, the levels of il- β, il- , and tnf-α in the serum and balf were determined with an enzyme-linked immunosorbent assay (elisa) kit, and an elisa kit was used to determine the levels of gsh-px, mpo, mda, and sod in the lung tissue. all data are presented as the mean ± sd. graphpad prism software was employed for one-way anova, and a p-value ≤ . was confirmed to be statistically significant. the rat lung tissues were fixed in % formaldehyde, embedded in paraffin and cut into μm sections. then, the sections underwent haematoxylin-eosin (h&e) staining. the changes in pulmonary histopathology were visualized under a microscope, and the pathological scores were obtained. the degree of pulmonary injury was evaluated according to a previous report (liu et al., ) . total rna was extracted with trizol reagent (tiangen, china) and detected on a % agarose gel. the purity, concentration, and integrity of the total rna samples were assessed prior to further analysis. after cluster generation, the library preparations were sequenced on the illumina hiseqtm platform by biomarker technologies (beijing, china), and the raw reads were generated. then, the raw reads were filtered by removing adapter and poly-n sequences and inferior quality reads from the raw reads. the clean reads were mapped to the rattus norvegicus (rnor_ . ) reference genome sequence by the hisat tools. the levels of quantitative gene expression were estimated by determining the fragments per kilobase of transcript per million fragments mapped. gene expression analysis of the different groups was performed by deseq . genes with a p-value < . were defined as differentially expressed genes (degs). then, we used the kobas . platform to perform the enrichment analysis of the degs (xie et al., ) . the volcano plot and heatmap were generated with the omicshare online tools the mrna expression of bcl a , cxcl , cxcl , cxcl , dnajc , fadd, kit, ifng, il r , il , il a, il f, il c, itga , mmp , nfkbia, nploc , ppp r a, thbs , tnfaip , tradd, and gapdh were detected by qrt-pcr in the lung tissues. the genes primer sequences (table s ) j o u r n a l p r e -p r o o f for qrt-pcr were designed the primer platform (http://frodo.wi.mit.edu/primer /). all qrt-pcr was repeated three times, the expression levels of candidate genes were determined using the -ΔΔct method. expression levels were normalized against the reference gene gapdh. data are represented as mean values ± sd, and graphpad prism software was employed for one-way anova, *p< . , ** p< . . in total, ingredients in ljf fulfilled the two criteria simultaneously (dl ≥ . and ob ≥ %). however, five ingredients (scolymoside, hederagenol, heriguard, hyperin, and luteolin- -o-glucoside) did not meet the criteria of ob ≥ %, but they were also presented as candidates. hence, main ingredients were obtained as potentially active constituents in ljf, including flavonoids and organic acids (table s ). these active components were used to identify the targets in swisstargetprediction and sea. after removing duplicates, we finally acquired targets (table s ). the ali-related genes were collected from the genecards database, and ali-related genes were identified (table s ). the shared targets of the active compounds and ali-related genes were identified by generating venn diagrams. ultimately, genes were identified as both the targets of active ingredients and ali-related genes. the identified genes were used to construct the compound-target network with cytoscape . . software. among the active compounds in ljf, eight active compounds demonstrated a higher number of connections and were connected with more than targets, including ethyl-linolenate, hederagenol, chrysoeriol, kaempferol, luteolin, mandenol, quercetin and zinc . the network analysis showed that one compound in the herbs can be linked with more than one target. in the compound-target network, alox (arachidonate -lipoxygenase) was simultaneously targeted by active ingredients, akr b (aldo-keto reductase family member b ) was targeted by active ingredients, and ptgs (prostaglandin g/h synthase ) was targeted by active ingredients (fig. .) . in the ppi network, eleven targets were linked ptgs might be identified as hub genes. the potential target proteins were subjected to enrichment analysis by kobas . . as a result, significant pathways related to ljf were identified by kobas . (table s ) . then, the target-pathway network was constructed (fig.s ), containing targets and corresponding pathways. it was obvious that the targets for ljf were mainly interlinked in three pathways, including metabolic pathways (degree = ), neuroactive ligand-receptor interaction pathways (degree = ), and pathways involved in cancer (degree = ). moreover, we identified the calcium signalling pathway (degree = ) and nod-like receptor signalling pathway (degree = ). fortunately, we also observed that these targets participated in pathways linked to immune and inflammatory signalling pathways, including human cytomegalovirus infection-associated pathways, the il- signalling pathway, the ppar signalling pathway, the pi k-akt signalling pathway, human t-cell leukaemia virus infection-associated pathways, pathways involved in inflammatory mediator regulation of trp channels, the jak-stat signalling pathway, natural killer cell-mediated cytotoxicity-associated pathways, t cell receptor signalling pathway, nf-kappa b signalling pathway, tnf signalling pathway and the toll-like receptor signalling pathway. to detect the anti-inflammatory effects of ljf, we examined the levels of il- , tnf-α, and il- β in serum and balf after treatment with lps, ljf and dxsm. the results showed that exposure of serum and balf to lps increased the production of these inflammatory cytokines (p< . ). however, production of these inflammatory cytokines (il- , tnf-α, and il- β) were significantly inhibited by ljf and dxsm (p< . ) ( fig. a and b) . then, the level of oxidative stress was measured, and lps-induced ali could increase the levels of mda and mpo and decrease the activities of sod and gsh-px in the lung tissue (p< . ) (fig. c) . interestingly, the levels of sod and gsh-px were significantly enhanced by ljf and dxsm (p< . ). h&e staining was used to detect the pathological changes in the lungs. after treatment with lps, the rat lung tissue showed increases in inflammatory cell infiltrates and alveolar histological structure damage compared with the lung tissue in the ctrl group. in our study, the ali group showed severe alveolar oedema fluid accumulation, alveolar capillary congestion and bronchial epithelial detachment (fig. b) . ljf was superior to dxsm in alleviating lps-induced ali ( (table s ) . to determine the differentially expressed genes (degs), a p-value < . was used as the cut-off value for gene expression in the ctrl, ali, ali-ljf, and ali-dxms groups, which was detected by pairwise comparisons between the ali group and the ctrl, ali-ljf, and ali-dxms groups. as a result, , degs in rat lung were identified after lps stimulation, whereas and , degs were identified in rat lung tissue treated with ljf and dxsm, respectively. overall, , upregulated and , downregulated degs were identified in the ali vs. ali-dxsm groups, and upregulated and downregulated degs were identified in the ali vs. ali-ljf groups ( fig. a-c) . in brief, after removing the duplicate genes, deg genes were associated with the ali group that were also affected in the ctrl, ali and ali-ljf groups; , genes were associated with the ctrl, ali and ali-dxsm groups; and only key deg genes were associated with the ctrl, ali, ali-ljf and ali-dxsm groups (fig. d) . to characterize these differentially expressed genes, trend analysis was applied to determine the expression patterns of the degs in the ctrl, ali and ali-ljf groups (table s ). in the j o u r n a l p r e -p r o o f ali vs. ctrl groups, the expression of genes displayed an initial increase, but there was a decrease in the ali vs ali-ljf groups; however, the expression of genes exhibited a reduction in the ali vs. ali-ljf groups, but there was a subsequent increase in the ali vs. ali-ljf groups (fig. ) . specifically, eight genes (dyrk a, ca , il , ptafr, arg , mgll, ltb r, and tymp) among the degs of the ali-ljf group were predicted as targets of active ingredients of ljf. to verify the reliability of the gene expression data obtained by rna-seq, eight genes (il r , dnajc , thbs , nploc , bcl a , ppp r a, kit, and itga ) were randomly selected for qrt-pcr detection. the qrt-pcr results showed that the tendency of gene expression was consistent with the rna-seq results. for each gene, the qrt-pcr expression results showed a similar tendency to the rna-seq results (fig.s ) . the results showed that the rna-seq results were credible in this study. to thoroughly investigate the potential pathways involved in the immune and inflammatory responses, the kobas . platform was employed for kegg pathway analysis of these degs. the kegg analysis showed that kegg pathways were significantly enriched for these degs (table s ; signalling pathway (fig. ) . within the four classic immune pathways (rno , rno , rno , and rno ), we identified candidate genes associated with ali: cxcl , cxcl , cxcl , il , lif, il rb , il , nfkbia, ifng, il a, il , birc , il a, il c, il f, cxcl , il r , tradd, mmp , ccnd , il , fadd, bcl a , and tnfaip . in this study, il- signalling pathway involved in thirteen degs, including cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, mmp , tnfaip , fadd and tradd. ljf significantly inhibited cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, mmp , and tnfaip mrna expression in lung tissue homogenates according to rna-seq, which indicates that the il- signalling pathway is critical for treatment of lps-induced ali with ljf (fig.s ) . interestingly, the involved il- family members, including il- a, il- c and il- f, played a significant role in the acute inflammatory responses (fig.s ) . consistent with the rna-seq data, the expression of cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, mmp and tnfaip in lung tissue was significantly decreased compared with that in the ali and ljf groups according to the qrt-pcr analyses (p< . ) (fig. ) . the expression of tradd and fadd was increased compared with that in the ali and ljf groups by the qrt-pcr analyses (fig. ) , and associated with apoptosis in the il- signalling pathway (fig.s ) . we employed network pharmacology to determine the potential active ingredients and targets of ljf. then, we performed compound-target and target-pathway network analyses to explore the mechanisms of ljf. furthermore, an lps-induced rat model was constructed to evaluate the effect of ljf in the treatment of ali. according to the degree of the nodes in the compound-target network, we identified eight compounds as potential active ingredients that might participate in the regulatory processes of ljf in ali. obviously, the active ingredients chrysoeriol (wei et qrt-pcr analysis showed that the trends for eight genes (il r , dnajc , thbs , nploc , bcl a , ppp r a, kit, and itga ) were consistent with the rna-seq results. these findings indicated that the rna-seq results were credible. by comparative analysis, we found that there were more differentially expressed genes in the ali, ali-ljf and ali-dxsm groups. in all, degs were unique to the ali-ljf group, and degs were shared among the three groups. through kegg enrichment analysis, we found that the il- signalling pathway was significantly enriched in the ali-ljf group. thirteen genes were found, such as cxcl , cxcl , cxcl , nfkbia, ifng, il , il a, il f, il c, tradd, mmp , kaempferol regulates mapks and nf-κb signaling pathways to attenuate lps-induced acute lung injury in mice eupatorium lindleyanum dc. flavonoids fraction attenuates lipopolysaccharide-induced acute lung injury in mice mast cell and macrophage chemokines cxcl /cxcl control the early stage of neutrophil recruitment during tissue inflammation mmp- and mmp- in bronchoalveolar lavage fluid is associated with acute lung injury after cardiopulmonary bypass in a swine model shuang-huang-lian attenuates lipopolysaccharide-induced acute lung injury in mice involving anti-inflammatory and antioxidative activities. evid based complement alternat med local stimulation of alpha cholinergic receptors inhibits lps-induced tnf-alpha release in the mouse lung enforced expression of mir- b attenuates lps-induced acute lung injury network pharmacology: the next paradigm in drug discovery puerarin inhibits inos, cox- and crp expression via suppression of nf-kappa b activation in lps-induced raw . macrophage cells il- cytokines in immunity and inflammation acute lung injury: epidemiology, pathogenesis, and treatment protective and immunomodulatory effect of flos lonicerae japonicae by augmenting il- expression in a murine model of acute lung inflammation hisat: a fast spliced aligner with low memory requirements myeloperoxidase: friend and foe lonicera japonica resolution of acute inflammation in the lung exerts anti-viral and anti-inflammatory activity against novel coronavirus (sars-cov- ) sini decoction alleviates e. coli induced acute lung injury in mice via equilibrating ace-angii-at r and ace -ang-( - )-mas axis can chinese medicine be used for prevention of corona virus disease (covid- )? a review of historical classics, research evidence and current prevention programs acute lung injury and the acute respiratory distress syndrome -four decades of inquiry into pathogenesis and rational management stlr /smd- complex alleviates lps-induced acute lung injury by inhibiting pro-inflammatory cytokines and chemokine cxcl expression chinese traditional patent medicine in treating a family case of covid- in wuhan kaempferol reduces k -linked polyubiquitination to inhibit nuclear factor-κb and inflammatory responses in acute lung injury in mice glycyrrhizic acid ameliorates lps-induced acute lung injury by regulating autophagy through the pi k/akt/mtor pathway tcmsp: a database of systems pharmacology for drug discovery from herbal medicines incidence and outcomes of acute lung injury luteolin attenuates acute lung injury in experimental mouse model of sepsis cytoscape: a software environment for integrated models of biomolecular interaction networks lonicera japonica thunb.: ethnopharmacology, phytochemistry and pharmacology of an important traditional chinese medicine kobas . : a web server for annotation and identification of enriched pathways and diseases trifunctional inhibition of cox- by extracts of lonicera japonica: direct inhibition, transcriptional and post-transcriptional down regulation anti-inflammatory and protective properties of daphnetin in endotoxin-induced lung injury systems pharmacology for investigation of the mechanisms of action of traditional chinese medicine in drug the rna sequencing results revealed differentially expressed genes in lung tissue. (b) differentially expressed genes were confirmed by qrt-pcr. data are represented as mean values ± sd key: cord- -zvc s authors: choudhary, ishita; vo, thao; bathula, chandra s.; lamichhane, richa; lewis, brandon w.; looper, jayme; jeyaseelan, samithamby; blackshear, perry j.; saini, yogesh; patial, sonika title: tristetraprolin overexpression in non-hematopoietic cells protects against acute lung injury in mice date: - - journal: front immunol doi: . /fimmu. . sha: doc_id: cord_uid: zvc s tristetraprolin (ttp) is a mrna binding protein that binds to adenylate-uridylate-rich elements within the ′ untranslated regions of certain transcripts, such as tumor necrosis factor (tnf) mrna, and increases their rate of decay. modulation of ttp expression is implicated in inflammation; however, its role in acute lung inflammation remains unknown. accordingly, we tested the role of ttp in lipopolysaccharide (lps)-induced acute lung injury (ali) in mice. lps-challenged ttp-knockout (ttp(ko)) mice, as well as myeloid cell-specific ttp-deficient (ttp(myeko)) mice, exhibited significant increases in lung injury, although these responses were more robust in the ttp(ko). mice with systemic overexpression of ttp (ttp(Δare)) were protected from ali, as indicated by significantly reduced neutrophilic infiltration, reduced levels of neutrophil chemoattractants, and histological parameters of ali. interestingly, while irradiated wild-type (wt) mice reconstituted with ttp(ko) hematopoietic progenitor cells (hpcs) showed exaggerated ali, their reconstitution with the ttp(Δare) hpcs mitigated ali. the reconstitution of irradiated ttp(Δare) mice with hpcs from either wt or ttp(Δare) donors conferred significant protection against ali. in contrast, irradiated ttp(Δare) mice reconstituted with ttp(ko) hpcs had exaggerated ali, but the response was milder as compared to wt recipients that received ttp(ko) hpcs. finally, the reconstitution of irradiated ttp(ko) recipient mice with ttp(Δare) hpcs did not confer any protection to the ttp(ko) mice. these data together suggest that non-hpcs-specific overexpression of ttp within the lungs protects against ali via downregulation of neutrophil chemoattractants and reduction in neutrophilic infiltration. acute lung injury (ali) and its severe form, acute respiratory distress syndrome (ards), are serious health concerns due to a high rate of mortality ( ) . ali is characterized by elevated levels of proinflammatory mediators, exaggerated neutrophil recruitment, and compromised pulmonary epithelial-endothelial barrier, resulting in increased vascular permeability ( ) . non-cardiogenic pulmonary edema, characterized by excessive accumulation of protein-rich edematous fluid and inflammatory cells in the alveolar spaces, results in hypoxemia in ards that requires aggressive clinical management including mechanical ventilation ( ) . despite significant health burdens posed by these diseases, the identity of key cellular and molecular players of host defense against ali remains unclear. zinc finger protein (zfp ), commonly known as tristetraprolin (ttp), is an mrna binding protein that binds to adenylate-uridylate-rich elements (ares) within the untranslated regions ( utrs) of target mrnas and increases their rate of decay ( ) . germline ttp-knockout (ttp ko ) mice exhibit the spontaneous development of a systemic inflammatory syndrome characterized by cachexia, erosive arthritis, myeloid hyperplasia, dermatitis, conjunctivitis, and autoimmunity ( ) . these phenotypes were shown to be essentially completely prevented in ttp ko mice with either tnf receptor deficiency, or when ttp ko mice were treated with anti-tnf antibodies ( ) . biochemical studies demonstrated that ttp binds to ares within the tumor necrosis factor (tnf ) mrna utr and results in tnf mrna degradation under normal conditions ( , ) . subsequent reports have shown that a number of other proinflammatory mediators including cxcl ( , ) , cxcl ( ), il- ( ), il- ( ) , ccl ( ) , and il- ( ) are also regulated by ttp ( ) . recently, using a systemic ttp overexpression (ttp are ) mouse model, we demonstrated protective effects of enhanced ttp levels in chronic immune-mediated inflammatory diseases including mouse models of arthritis, psoriasis, and autoimmune encephalomyelitis ( ) . ttp are mice lack ares in the utr of the endogenous ttp gene (zfp ) that results in increased stability of ttp mrna and, in turn, moderately increased expression of ttp protein in essentially all the tissues ( ) . together, these studies have indicated that ttp may be an endogenous anti-inflammatory protein and that enhancing its levels may be beneficial against various chronic inflammatory diseases. in the present study, we investigated the role of ttp in regulating lung inflammation in a mouse model of ali. using an oropharyngeal aspiration approach, lipopolysaccharide (lps)induced ali was modeled in adult mice, and the animals were monitored for signs of ali. to identify the protective role of ttp in a cell-specific manner, we performed bone marrow irradiation and reconstitution experiments in wild-type (wt), ttp ko ( ) , and systemic ttp overexpression (ttp are ) mice ( ) . our findings elucidate cell-specific roles of ttp in protection against ali, and indicate that ttp is an important modulator of endotoxin-induced ali. zfp floxed mice (zfp flox/flox ) ( ) were crossed with lysmcre recombinase expressing mice ( ) to generate mice for experimental (cre +/+ /zfp flox/flox ; ttp myeko ) and control (cre −/− /zfp flox/flox ; cre +/+ /zfp wt/wt ) groups. genotype status of progeny was determined by polymerase chain reaction (pcr) as described previously ( ) . ttp knockout mice (ttp ko ) and ttp overexpression mice (ttp are ) have been described before ( , ) . all the animal experiments were performed in accordance with principles and procedures outlined in the national institute of health guide for the care and use of laboratory animals and were approved by the louisiana state university animal care and use committee. both male and female adult ( - -week-old) mice were used for experiments. mice were anesthetized with isoflurane/oxygen followed by administration of µg lipopolysaccharide (lps) from escherichia coli o :b (l , sigma-aldrich) per mouse dissolved in sterile endotoxin-free saline ( µl total volume), or an equivalent volume of sterile endotoxin-free saline as a vehicle control, via oropharyngeal aspiration ( ) . mice were observed for signs of distress including anorexia, weight loss, hunched posture, ruffled haircoat, labored breathing, and dehydration every - h post lps challenge. mice exhibiting at least four of these clinical signs were humanely euthanized before the end of the study. following saline or lps treatment, mice were anesthetized with , , -tribromoethanol (sigma-aldrich, st. louis, mo, united states) at the indicated time points, and mid-line laparotomy was performed. briefly, bronchoalveolar lavage fluid (balf) was harvested from the right lung. recovered balf was processed and analyzed for total and differential cell counts by routine methods ( ) . unlavaged left lung lobes were fixed in % neutral buffered formalin (nbf) and used for preparation of slides for histopathological evaluation. right lung lobes were snap-frozen and stored at − • c. bronchoalveolar lavage fluid was harvested and centrifuged at × g for min, and the supernatant was stored at − • c for further analyses. the cell pellet was resuspended in µl of pbs and total cell counts were determined using a hemocytometer (brightline, horsham, pa, united states). cytospins were prepared using µl of cell suspension (statspin cytofuge ; hemocue, brea, ca, united states) followed by differential staining (modified giemsa kit; newcomer supply, middleton, wi, united states). mouse cytokine and chemokine levels were assayed in cell-free balf supernatant using luminex-xmap-based assay (mcytomag- k), according to the manufacturer's instructions (emd millipore, billerica, ma, united states). five micrometer sections of lung were stained with hematoxylin and eosin (h&e) for routine histology. histology: a semiquantitative histopathological scoring system was used to analyze the sections as follows: ( ) consolidation (percent of total surface area of lung section affected); ( ) bronchiolitis ( , no bronchioles affected; , one bronchiole affected; , between - bronchioles affected; , more than bronchioles affected); ( ) perivascular edema ( , minimal; , mild; , moderate; , severe); ( ) perivascular inflammation/inflammatory cells ( , absent; , minimal; , mild; , moderate; , severe); ( ) airspace edema ( , minimal; , mild; , moderate; , severe); ( ) airspace hemorrhages ( , absent, , patchy, mild; , extensive, moderate; , extensive, severe). slides were graded in a blinded manner without knowledge of sex and treatment groups. bone marrow transplantation experiments were performed as described previously ( ) . briefly, - -week old recipient mice were irradiated with megavolt x-rays from a linear accelerator (varian clinac ex) with two (dorsal and ventral) -rad ( cgy) doses. to prepare bone marrow cells for transplantation, femur bones of donor mice were flushed to collect bone marrows, and single cell suspensions were prepared. a total of × cells were injected into the tail vein of lethally irradiated recipient mice. reconstituted recipient mice were given . % neomycin sulfate dissolved in acidified water for the first weeks post-transplantation. lps-challenge experiments were performed weeks post bone marrow reconstitution, which has been previously shown to be an optimal period for repopulation of resident alveolar macrophages with donor cells following total body irradiation ( ) . lung tissue was lysed using pierce tm ripa buffer (thermo fisher scientific, waltham, ma, united states) supplemented with pierce tm protease inhibitor mixture (thermo fisher scientific, waltham, ma, united states) and phosphatase inhibitors ( mm sodium fluoride and mm sodium orthovanadate). tissues were mechanically homogenized using a bead beater (thermo fisher scientific, waltham, ma, united states). tissue lysates were centrifuged ( , × g, min, • c) to remove insoluble material and protein concentration of the supernatants was measured through bradford assay (bio-rad laboratories, hercules, ca, united states). equivalent amounts of denatured protein was separated on a - % bis-tris plus precast gels (invitrogen, carlsbad, ca, united states), transferred on to pvdf membrane (invitrogen, carlsbad, ca, united states) and probed with a : dilution of rabbit antiserum raised against a recombinant mouse ttp-maltose binding protein fusion ( ) followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit igg (bio-rad). signal was determined using supersignal west pico chemiluminescent substrate (pierce) on x-ray film. significant differences among groups were determined by oneway analysis of variance (anova) followed by tukey's post hoc test for multiple comparisons except for cytokine assays where two-way anova was used. measurements from two groups were compared using student's t-test assuming unequal variance. all data were expressed as mean ± sem. a p-value < . was considered statistically significant. statistical analyses were performed using graphpad prism . (graphpad software, la jolla, ca, united states). in order to explore the role of ttp in ali, ttp ko and littermate control wt mice were subjected to ali through oropharyngeal aspiration of endotoxin (lps) (single dose; µg lps/mouse) for a period of h. while saline-treated ttp ko and wt groups had comparable numbers of total immune cells in the balf (saline-treated wt; × ± × , saline-treated ttp ko , × ± × ), lps administration resulted in increased infiltration of immune cells in both the ttp ko and the wt groups. the total number of recovered immune cells in lps-challenged ttp ko mice ( × ± × ) were ∼ fourfold higher as compared to lps-challenged wt ( × ± × ) mice ( figure a) . increases in total cell counts in lps-challenged ttp ko mice were attributed to a significant increase in neutrophil ( figure b) , macrophage ( figure c) , and lymphocyte counts ( figure d ). these increases were associated with an increased injury to the pulmonary vascular barrier, as depicted by the presence of red blood cells in the cytospins prepared from the balf fluid of ttp ko mice ( figure e ; right panel, black arrow) versus control lps-challenged wt mice ( figure e ; left panel). histologically, the lungs of lps-challenged wt mice were characterized by mild to moderate consolidation (∼ % of total area of lung section), two-to fourfold increase in alveolar septal thickening (broken green arrow), moderate perivascular and airspace edema, and perivascular inflammation (figures f-h) . in contrast, the lung injury in lps-challenged ttp ko mice was characterized by severe consolidation (> % of total area of lung section) (figures f,g) that included infiltration of neutrophils, edema, fibrin, and airspace hemorrhage within the airway and alveolar lumen, multifocal loss of bronchiolar epithelium with infiltration of neutrophils and red blood cells within the bronchiolar lumen, and moderate to severe perivascular edema and inflammation (figures f-h) . of note, ∼ % lps-challenged ttp ko mice succumbed to lps challenge before -h and these had to be excluded from the analysis. these data suggest that systemic loss of ttp results in extreme susceptibility of mice to lpsinduced ali. in order to explore the role of myeloid cell-specific ttp on inflammatory response in ali, myeloid cell-specific ttp deficient mice (ttp myeko ; cre +/+ /zfp flox/flox ) and control (cre control; cre +/+ /zfp wt/wt and flox control; cre −/− /zfp flox/flox ) mice were challenged with lps. similar to saline-treated wt and ttp ko groups, saline-treated control and ttp myeko groups had comparable numbers of total . data are represented as mean ± sem. statistical analysis was performed by one-way anova followed by tukey's correction for multiple comparison test. *p < . ; **p < . ; ***p < . . n = each for wt and ttp ko saline controls; n = , wt group; n = , ttp ko group for lps-challenge group. three lps-challenged ttp ko mice succumbed to lps-challenge before h and were not lavaged for further analyses. representative photomicrographs of wright-giemsa stained balf cytospins of lps-challenged wt (e; left panel) and lps-challenged ttp ko (e; right panel) mice. neutrophil (red arrow), macrophage (green arrow), lymphocyte (blue arrow), red blood cell (black arrow) (original magnification × ). representative photomicrographs (f) from h&e-stained left lung lobe sections from adult lps-challenged wt (f; left panel) and lps-challenged ttp ko (f; right panel) mice. septal thickening (green broken arrow), intra-alveolar neutrophilic infiltrates (green arrow), intra-alveolar red blood cells (red arrow), bronchiolar lumen neutrophilic accumulation (blue arrow), and perivascular cellular infiltration (black arrow). asterisk represents alveolar space that is minimally affected (f; left) or severely consolidated with blood and neutrophils (f; right) (original magnification × ). semiquantitative histopathological scoring for consolidation (g) is shown as a percent of total surface area of the lung section affected in lps-challenged wt and lps-challenged ttp ko mice. semiquantitative histopathological scoring of lung sections for bronchiolitis, perivascular edema, perivascular inflammation, airspace hemorrhage, and airspace edema in lps-challenged wt and lps-challenged ttp ko mice (h). statistical analysis in g and h was performed using student's t-test. *p < . ; **p < . ; ***p < . ; ****p < . . immune cells (flox control; × ± × , ttp myeko ; × ± × ). lps administration resulted in increased numbers of immune cells in ttp myeko as well as both the control groups. this increase in total cell counts was, however, significantly greater in ttp myeko ( × ± × ) compared to cre control ( × ± × ) mice. the increase in cellular recovery did not reach statistical significance in lps-challenged ttp myeko when compared to the lps-challenged flox control group (p = . ) (figure a ). this effect was comparable in both sexes (data not shown). of note, the increase in cellular infiltration was ∼ threefold less in lps-challenged ttp myeko ( × ± × ) (figure a ) when compared to lps-challenged ttp ko mice ( × ± × ) ( figure a) . while neutrophil counts were comparable between lps-challenged ttp myeko and lpschallenged control mice (figures b,e) , macrophage counts were significantly elevated in the balf obtained from lps-challenged ttp myeko mice compared to the two groups of control mice (figures c,e) . lymphocyte counts did not differ between the lps-challenged ttp myeko and the two groups of control mice (figures d,e) . histopathological analysis revealed comparable levels of lung consolidation with widespread inflammatory cellular infiltrates within the airspaces of both lps-challenged ttp myeko and flox control mice; however, perivascular edema, perivascular inflammation, and airspace edema were somewhat exaggerated in lps-challenged ttp myeko mice compared to the flox control group (figures f-h) . unlike lps-challenged ttp ko mice, airspace hemorrhage was not observed in any of the groups. all the lps-challenged ttp myeko mice survived lps challenge, as compared to the lps-challenged ttp ko mice, in which ∼ % mortality was observed. these data indicate that myeloid cell-specific ttp is essential for protection against ali. mitigates lps-induced ali in mice during acute and sub-acute course of lung injury next, we examined whether the systemically ttp overexpressing (ttp are ) mice exhibit protection against ali. in experimental ali, while time points earlier than h of lps-challenge represent acute phases of ali, later time points represent somewhat sub-acute to chronic or resolution phases of endotoxin-induced ali in mice ( ) . therefore, we examined both lps-challenged wt and lps-challenged ttp are mice over time points representing acute to subacute phases, i.e., ± . × , . × ± . × , and . × ± . × cells at h, h, h, days, and days, respectively) ( figure a) . lipopolysaccharide administration resulted in increased numbers of total cells in balf from both wt and ttp are mice when compared to saline administration ( figure a) . total cell counts in balf from lps-challenged wt mice were . × ± . × , . × ± . × , × ± × , × ± . × , and . × ± . × at h, h, h, days, and days time points, respectively ( figure a) . interestingly, significantly reduced numbers of immune cells were recovered from the lungs of lps-challenged ttp are mice compared to lungs of lps-challenged wt mice at all time points examined post lpschallenge ( . × ± . × , . × ± . × , . × ± . × , . × ± . × , and . × ± . × at h, h, h, days, and days, respectively) ( figure a) . the decrease in total cell counts in lps-challenged ttp are mice was contributed by significantly reduced numbers of neutrophils at , , and h (figures b,e) . interestingly, however, the total numbers of macrophages were only significantly different between lps-challenged wt and lps-challenged ttp are mice at h and days time points (figures c,e) . lymphocyte counts were not significantly different in lps-challenged ttp are mice compared to lps-challenged wt mice (figures d,e) . cellular counts followed the same trend in both the sexes (data not shown). reduced cellular infiltration was also evident in cytological slides prepared from lps-challenged wt and lps-challenged figure | myeloid-ttp deficiency exacerbates lps-induced ali in mice. total cell counts (a) in the harvested balf of adult saline-(white bars; n = each for flox control and ttp myeko ) or lps-challenged cre control (cre +/+ /zfp wt/wt , gray bar; n = ), flox control (cre -/-/zfp flox/flox , purple bar; n = ), and ttp myeko (cre +/+ /zfp flox/flox , green bar; n = ) mice. differential cell counts are shown for neutrophils (b), macrophages (c), and lymphocytes (d). data are represented as mean ± sem. statistical analysis was performed by one-way anova followed by tukey's correction for multiple comparisons. ns, non-significant; *p < . ; ***p < . ; ****p < . . representative photomicrographs of wright-giemsa stained balf cytospins from lps-challenged flox control (e; left) and ttp myeko (e; right) mice. neutrophil (red arrow), macrophage (green arrow), lymphocyte (blue arrow) (original magnification × ). representative photomicrographs from h&e-stained left lung lobe sections from post- h lps-challenged flox control (f; left) and ttp myeko (f; right) mice (original magnification × ). asterisk represents alveolar spaces minimally obliterated in flox control (f; left) and severely obliterated in lps-challenged ttp myeko mice (f; right). bronchiolar lumen neutrophilic infiltrates (blue arrow), absent in lps-challenged flox control (f; left) but present in lps-challenged ttp myeko (f; right). semiquantitative histopathological scoring for consolidation (g) is shown as a percent of total surface area of the lung section affected in lps-challenged flox control and lps-challenged ttp myeko mice. semiquantitative histopathological scoring of lung sections for bronchiolitis, perivascular edema, perivascular inflammation, and airspace edema in lps-challenged flox control and lps-challenged ttp myeko mice (h). n = (flox control); n = (ttp myeko ). statistical analysis in g and h was performed using student's t-test. *p < . . ttp are bal fluid, which showed the sparsely present neutrophils, macrophages, and lymphocytes in lps-challenged ttp are mice compared to dense presence of these cells in lps-challenged wt mice ( figure e) . histologically, lpschallenged wt lungs exhibited ∼ % lung consolidation with significantly increased infiltration of immune cells within the airspaces, bronchiolitis, perivascular edema, and inflammation (figures f-h) . in contrast, bronchiolitis and perivascular edema were significantly attenuated in lps-challenged ttp are mice. lung consolidation, perivascular inflammation, and airspace edema were not significantly different between the two groups, and airspace hemorrhage was not observed in any group (figures f-h) . we next analyzed the changes in the protein expression levels of ttp over the course of ali in wt and ttp are mice whole lung tissue. as shown in figure i , basal expression levels of ttp were higher in ttp are versus wt lungs. upon lps administration, the expression levels of ttp increased in both wt and ttp are mice lungs by h post lps administration. this increase was significant in ttp are mice lungs as compared to unchallenged wt mice lungs. the levels of ttp started reducing at subsequent time points in both wt and ttp are lungs. while the ttp expression decreased to basal levels in wt mice, the ttp expression remained at relatively higher levels in the ttp are mice lungs at all subsequent time points. these data show that the presence of higher levels of ttp under basal conditions, combined with a significant increase at h post-lps challenge and maintenance of higher expression levels at later time points post lps challenge, protects ttp are mice from ali. cellular recruitment within the lung in response to lps challenge is mediated by the production of chemoattractants. therefore, next we sought to examine the levels of inflammatory cytokines and chemokines within the balf of lps-challenged wt and lps-challenged ttp are mice. of the cytokines/chemokines analyzed, four cytokines/chemokines, including g-csf, kc, il- , and il- p , were found to be significantly different between the lps-challenged wt and the lpschallenged ttp are mice. g-csf was significantly reduced in lps-challenged ttp are compared to lps-challenged wt mice at and h; kc and il- were significantly reduced at h; and il- (p ) was significantly reduced at h post-lps challenge (figures a-d) . interestingly, the levels of tnfα ( figure e ) and mip (figure f) , two known ttp targets, were not significantly different between the two groups. to determine the cell-specific role of ttp levels in ali, we modulated ttp levels in hematopoietic progenitor cells (hpcs) and non-hpcs. in order to test whether donor hpcs repopulate the recipient mouse lungs, we first made bone marrow chimeras in which total body irradiated wt mice were transplanted with hpcs from a mouse expressing green fluorescent protein (gfp) in their somatic cells. we found that greater than % of the cells recovered from the balf of these mice were gfp positive (data not shown). next, we generated three bone marrow chimeras in which irradiated wt recipient mice received hpcs from either wt (wt→wt), ttp are (ttp are →wt), or ttp ko (ttp ko →wt) mice ( figure a) . while saline-treated ttp ko →wt chimeras had no signs of cellular infiltration that included total cells (figure b) , neutrophils (figure c) , macrophages (figure d) , and lymphocytes (figure e) , lps-challenged wt→wt, ttp are →wt, and ttp ko →wt chimera mice had significant cellular recruitment, as indicated by balf total cellular counts (figure b) , neutrophils (figure c) , and macrophages ( figure d) . while lpschallenged ttp are →wt mice had somewhat reduced cellular infiltration as compared to lps-challenged wt→wt chimeras (figures b-d) , the lps-challenged ttp ko →wt chimera mice had remarkably higher number of bal cellular counts (figures b-d) . lymphocyte counts did not differ significantly between the three groups ( figure e) . histologically, ∼ , , and % of the lung parenchyma was consolidated in lps-challenged wt→wt, lps-challenged ttp are →wt, and lps-challenged ttp ko →wt group, respectively (figures a top panel, c) . consolidated parenchyma was characterized by the presence of large infiltrates of neutrophils and macrophages within the airspaces (alveolar and airway). other histological findings included the presence of edema and immune cells within the perivascular spaces, bronchial lumen cellular infiltrates, airspace edema, and occasional bronchoalveolar lymphoid aggregates (figures d-g) . these data suggest that while baseline expression of ttp in hpcs is essential for protection against exaggerated ali, its overexpression in these cells does not confer significant additional advantage. next, we generated bone marrow chimeras in which irradiated ttp are recipient mice received hpcs from either wt (wt→ttp are ), ttp are (ttp are →ttp are ), and ttp ko (ttp ko →ttp are ) mice ( figure a) . as compared to wt recipient chimera counterparts, lps-challenged wt→ttp are , lps-challenged ttp are →ttp are , and lps-challenged ttp ko →ttp are chimeras had significantly lower degrees of cellular recruitment, that included total cells ( figure b , blue solid bar, orange solid bar, green solid bar), neutrophils ( figure c , blue solid bar, orange solid bar, green solid bar), and macrophages ( figure d , blue solid bar, orange solid bar, green solid bar). lymphocyte counts were significantly reduced in lps-challenged ttp are →ttp are compared to the lps-challenged wt→wt chimeras ( figure e ). although the lps-challenged ttp ko →ttp are chimeras had significantly higher cellular recruitment as compared to lps-challenged wt→ttp are and lps-challenged ttp are →ttp are chimeras, the extent of recruitment was much diminished in lps-challenged ttp ko →ttp are chimera as compared to lps-challenged ttp ko →wt chimera ( figure b) . histologically, ∼ , %, % of the lung parenchyma was consolidated in lps-challenged wt→ttp are , lpschallenged ttp are →ttp are , and lps-challenged ttp ko →ttp are , respectively (figures a bottom panel, figure c ). histologically, mild increases in septal thickening with cellular infiltration, mild bronchiolitis, perivascular edema, inflammation, and airspace edema, and occasional balts were evident in all the three groups (figures d-g, solid blue, orange, and green bars). these data suggest that enhanced expression of ttp in lung non-hpc populations conferred significant protection against ali. further, this protection is somewhat compromised in the absence of baseline levels of ttp in hpcs. a tabular summary of differential cellular and ali responses in various chimeric mice is included in supplementary table . finally, we generated bone marrow chimeras in which irradiated ttp ko recipient mice received hpcs from ttp are (ttp are →ttp ko ). as expected, the cellular counts were significantly higher than any of the other lps-challenged chimeras (figures b-e, red solid bar) . however, total cellular counts in lps-challenged ttp are →ttp ko chimera were ∼ twofold lower than the lps-challenged ttp ko mice (figure ) . additionally, none of the lps-challenged ttp are →ttp ko chimeras succumbed to ali, as compared to % mortality in lps-challenged ttp ko mice (figure ) . these data suggest that complete loss of ttp in lung non-hpc populations significantly exaggerates ali, and that overexpression of ttp in hpcs may provide partial protection in severe ali. histologically, this group exhibited the most severe lung injury, characterized by ∼ % lung consolidation with neutrophils, macrophages, fibrin, and edema, severe bronchiolitis, and moderate to severe bronchiolar and alveolar hemorrhages (figures b-g) . tristetraprolin knockout (ttp ko ) mice exhibit a systemic inflammatory syndrome that is characterized by cachexia, polyarticular synovial arthritis, dermatitis, and myeloid hyperplasia ( ). however, the lungs of ttp ko mice exhibit very little spontaneous inflammation, characterized by the presence of rare foci of leucocytic infiltrates within the pulmonary parenchyma ( ) . these rare leucocytic infiltrates are abrogated upon combined deletion of ttp and tnf receptors, indicating the role for tnf activity in leucocytic infiltration ( ) . myeloid cell-specific loss of ttp (ttp myeko ) does not recapitulate the ttp ko phenotype, indicating that non-myeloid cells are required for the overall manifestation of the ttp deficiency syndrome ( ) . however, ttp myeko mice were found to be hypersensitive to endotoxin-induced systemic inflammation, particularly through exaggerated tnf production, delineating the critical role of myeloid cell-specific ttp in protection against systemic injury and inflammation ( ) . the role of ttp in localized lung inflammation, however, has remained unexplored. therefore, in this study we sought to explore the role of ttp in ali. we hypothesized that ttp modulates acute lung inflammation and that cell-specific modulation of ttp levels will differentially affect the outcome of acute lung inflammation. the unchallenged ttp ko mice display minor degrees of leukocytic infiltration in lung parenchyma that were thought to be contributed by tnf activity ( ) . here, in lps-challenged ttp ko mice, immune cell infiltration was ∼ fourfold higher as compared to lps-challenged wt mice. in fact, the susceptibility of ttp ko mice to lps-induced ali was so severe that ∼ % ttp ko mice succumbed to lps-challenge within - h post-lps administration, while no mortality was observed in lpschallenged wt mice. the surviving ttp ko mice displayed severe pulmonary pathology with exaggerated airspace and interstitial neutrophilic infiltration, exaggerated edema, vascular congestion, and lung injury that included epithelial and endothelial damage. it is likely that the increased production of inflammatory mediators, that are otherwise regulated by ttp, in lpschallenged ttp ko mice, contribute to their worsened pulmonary pathology. one such mediator, tnf, is already established to be highly secreted in ttp ko mice ( , ) . macrophages are the primary source of tnf in ali ( , ) although alveolar epithelial cells have also been suggested to release tnf in lps-induced lung inflammation ( ) . accordingly, we reasoned that, if macrophages are the primary source of tnf, deletion of ttp in myeloid cells would enhance its activity, leading to worsening of pulmonary pathology. although the lps-challenged ttp myeko mice had exaggerated pulmonary pathology as compared to lps-challenged wt mice, the extent of tissue damage and neutrophilic infiltration was not as severe as in lps-challenged ttp ko mice. these differences in the sensitivity of systemic versus myeloid cell-specific ttpdeficient mice indicate that ttp in cells other than myeloid-cells may play critical roles in modulating endotoxin-induced ali. these data suggest that while loss of myeloid cell-specific ttp worsens the ali, ttp expression in non-myeloid cells confers significant protection. clinically, the numbers of neutrophils within the balf of patients with ards have been shown to correlate with the severity of disease and poor outcome ( , ) . despite being the first line of defense against pathogenic insults, excessive recruitment and activation of neutrophils leads to lung tissue damage and loss of lung function ( , ) . therefore, targeting neutrophilic recruitment through suppressed release of key neutrophil chemoattractants may be an attractive therapeutic strategy against ali. we observed correlations between ttp deficiency or ttp overexpression and neutrophilic inflammation. for example, balf counts and tissue infiltration of neutrophils were overwhelming in the lps-challenged ttp ko mice. these outcomes were also exaggerated, but to a lesser degree, in lps-challenged ttp myeko mice. on the other hand, these outcomes were significantly attenuated in the lps-challenged ttp are mice. our findings are in line . data are represented as mean ± sem. statistical analysis in c-g was performed by one-way anova followed by tukey's correction for multiple comparisons within the three recipient groups and student's t-test between the three recipient groups. *p < . ; **p < . ; ***p < . ; ****p < . . with previous reports in other tissues. for instance, massive infiltration of neutrophils has been shown to occur in the skin of ttp ko mice subjected to psoriasis-like inflammation ( ) , whereas reduced airway neutrophilic infiltration was shown in mice genetically modified to express constitutively active endogenous unphosphorylated ttp following challenge with cigarette smoke ( ) . a number of studies have shown that ttp is phosphorylated at multiple sites by p -regulated kinase mapk-activated protein kinase (mapkapk ) and that ttp activity is significantly affected by its phosphorylation status ( ) ( ) ( ) . ttp phosphorylation has been shown to result in reduced ttp activity/reduced binding of ttp to ares, thus resulting in stabilization of its target mrnas ( ) . regulation of ttp activity by p mapk was in fact shown to result in a biphasic response of tnfα-induced il- expression in human bronchial smooth muscle cells ( ) . conversely, dephosphorylation of ttp resulted in reduced expression of il- and il- in a lung epithelial cells ( ) . since lps is an inducer of both ttp and p mapk, ttp would be expected to undergo phosphorylation and inactivation shortly after lps challenge. however, the effect of ttp phosphorylation is transient and would be expected to be reversed upon dephosphorylation when p is turned off. although we did not track the phosphorylation status of ttp in various lung cells during ali, we speculate that the p -mediated phosphorylation of ttp occurs well before h post lps challenge and is reversed by h time point. consistent with our speculation, we found differential effect of ttp overexpression on late phase cytokines (kc) versus early phase cytokines (tnf) (figure ) . tristetraprolin is a known regulator of key neutrophil chemoattractants including cxcl /kc ( , ) and cxcl /mip ( , ) . cxcl /kc is a central chemokine in neutrophil recruitment into the airspace in ards ( ) ( ) ( ) . clinically, increased concentrations of il- (cxcl /kc homolog in human), disease severity, and neutrophil migration into airspaces have been shown to be correlated ( ) ( ) ( ) . cxcl mrna utr contains ttp-binding sites and has been previously demonstrated to be a direct target of ttp ( , ) . consistent with this, the lps-challenged ttp are mice had significantly lower cxcl /kc concentrations. in contrast, the two well characterized ttp targets, tnf and mip /cxcl , were not significantly different between lps-challenged wt and lpschallenged ttp are mice. these observations are in line with our previous report, in which no differences were observed in the levels of tnf and mip /cxcl in the serum of wt and ttp are mice systemically challenged with lps ( ). these two inflammatory mediators peak early (before h in the serum) ( ) , and we speculate that overexpressed ttp may be less effective at mrna decay due to its phosphorylation status at these early time points. g-csf, another neutrophil chemoattractant, is also consistently detected within the balf of ali and ards patients and has been suggested to be associated with the accumulation and activation of neutrophils in ards ( ) . plasma g-csf levels have also been shown to correlate with clinical outcome in patients with ali ( , ) . g-csf has also been shown to exacerbate bleomycin induced lung injury in rats through marked infiltration of activating neutrophils ( ) . g-csf levels have been found to be increased in ttp ko mouse plasma ( ) . although g-csf has not been shown to be a direct ttp target, g-csf mrna utr in mouse possesses two ttp binding sites, uauuuau. the balf g-csf levels were significantly reduced in lps-challenged ttp are mice. g-csf levels were also found to be significantly reduced in ttp are mice in a previous study where we showed ttp are mice to be significantly protected from exhibiting collagen-antibody induced arthritis ( ) . lipopolysaccharide-challenged ttp myeko mice exhibited milder ali as compared to lps-challenged ttp ko mice, indicating that total loss of ttp expression in non-myeloid as well as myeloid cells contributes to severe ali. on the other hand, systemic ttp overexpression conferred significant protection against lps-induced ali. here, we addressed two logical questions: ( ) whether enhanced levels of ttp in non-hpcs would ameliorate endotoxin-induced ali, ( ) whether enhanced levels of ttp in hpcs would confer protection against endotoxin-induced ali. towards this, employing various bone marrow chimeras, we investigated cell-specific roles of ttp in protection against ali. in these experiments, we modulated ttp levels in hpcs by using bone marrow donors that were either wt, ttp are , or ttp ko . to modulate ttp levels in non-hpcs, wt, ttp are , or ttp ko recipients were used. one limitation of this study was that since hpcs also include various non-myeloid populations, we were not able to specifically investigate the effect of ttp modulation in myeloid cells. the bone marrow irradiation and reconstitution experiments revealed somewhat unexpected but interesting findings. as compared to wt→wt chimera, the ali in ttp are →wt chimeras was somewhat attenuated, whereas the ali in ttp ko →wt chimeras had worsened significantly. these data suggest that, when the ttp expression in non-hpcs is genetically unaltered (wt ttp in the recipient's non-hpcs), the ttp overexpression in hpcs is partially protective against lps-induced ali. however, when the ttp expression was ablated in non-hpcs [ttp depleted in the recipient's non-hpcs (ttp are →ttp ko chimera)], the overexpression of ttp in the hpcs was not sufficient to confer protection against ali. as compared to ttp are →ttp ko chimeras, in ttp ko →ttp are chimeras, the mere overexpression of ttp in non-hpcs (ttp overexpression in the recipient's non-hpcs) provided significant protection, even though the hpcs were ttp-deficient. however, this protection was further enhanced when the normal levels of ttp were restored in the hpcs (wt→ttp are chimera). based on these data, we conclude here that the ttp levels in non-hpcs are critical in protection against ali. further, while the wt levels of ttp in hpcs are essential for additional protection, the overexpression of ttp in hpcs is not significantly advantageous. since the non-hpcs population in lungs consists of a multitude of cell types including epithelial cells, endothelial cells, and fibroblasts, the cell-specific role of ttp still remains elusive. contribution of non-hpc-specific ttp toward modulation of inflammatory responses has also been suggested in previous reports. for instance, ttp regulation of tnf in keratinocytes, but not in myeloid or dendritic cells, was shown to protect mice from exacerbation of psoriasis-like pathology, development of spontaneous systemic inflammation, and dactylitis ( ) . another study suggested a role of intestinal epithelial cell-specific ttp in exacerbation of acute colitis ( ) . along similar lines, ttp depletion in myeloid cells did not replicate the ttp-deficiency phenotype ( ) , and the spontaneous reactive granulopoiesis seen in ttp ko mice was suggested to be caused by a non-cell autonomous mechanism likely contributed by liver cells ( ) . all these studies indicated an essential role of ttp in non-hpcs in regulating inflammation. among various non-hpc cell types in the lung, alveolar epithelial cells produce pro-inflammatory cytokines and chemokines ( ) . in in vitro conditions, lpschallenged pulmonary type ii epithelial cells have been shown to produce greater levels of neutrophil chemoattractants [il- , a human homolog for cxcl (kc), cxcl (mip ), and cxcl (lix)] indicating that ttp in lung alveolar epithelial cells may play an important role in regulating ali ( ) . consistent with these reports, while our data indicate critical roles of non-hpcs in lungs in mediating neutrophil recruitment to the airspaces, the exact identity of these non-hpcs remain unknown. in conclusion, our results show that (a) enhancing the levels of ttp is protective against endotoxin-induced ali; (b) the protective effect seen in ttp are mice is attributable to reduced neutrophilic recruitment and, in turn, reduced lung damage; (c) reduced neutrophilic recruitment is attributed to reduced secretion of chemoattractants, particularly kc and g-csf; and that, (d) ttp in non-hpcs plays an essential role in protection against endotoxin-induced ali. based on these results, we propose a model in which endotoxin damages epithelial cells within the lung, which then initiates a cascade of events leading to exaggerated release of proinflammatory mediators and neutrophilic chemoattractants, resulting in further lung damage and neutrophilic infiltration (figure ) . in this process, ttp acts as an intracellular regulator for the expression of proinflammatory mediators and neutrophilic chemoattractants. ttp expression in the non-hpcs, i.e., most likely epithelial and endothelial cells, confers protection against endotoxin-induced ali via suppression of mrnas encoding proinflammatory mediators and neutrophilic chemoattractants. hence, strategies to increase ttp expression or activity in non-hpcs together with hpcs population may prove beneficial for patients with ali/ards. in future studies, ttp expression within the lung could also be investigated as a prognostic indicator for the severity of ali/ards. our studies could also have implications for the lung hyper-inflammation and potentially life-threatening cytokine storms in the severe coronavirus disease (covid- ). the raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. the animal study was reviewed and approved by lsu iacuc. sp conceived and designed the study, maintained the animal colony, and performed histopathological analyses. ic, tv, and bl conducted animal necropsies. ys, cb, and rl performed balf cellularity assays. rl performed the intravenous injections. ys, tv, ic, and cb performed cytokine assays. jl performed the irradiations. sj provided technical expertise on bone marrow transplantations and reviewed the manuscript. pb provided various transgenic and knockout mice and edited the manuscript. sp and ys wrote and reviewed the manuscript for intellectual contents. all authors contributed to the article and approved the submitted version. the acute respiratory distress syndrome: 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reactive granulopoiesis by a non-cellautonomous mechanism without disturbing long-term hematopoietic stem cell quiescence tristetraprolin targets nos expression in the colonic epithelium differential regulation of cytokine release and leukocyte migration by lipopolysaccharide-stimulated primary human lung alveolar type ii epithelial cells and macrophages we thank thaya stoufflet for assistance with multiplex cytokine assays, sherry ring for histological tissue processing, and deborah stumpo for helping with the ttp mutant mice and their genotyping. the supplementary material for this article can be found online at: https://www.frontiersin.org/articles/ . /fimmu. . /full#supplementary-material conflict of interest: the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © choudhary, vo, bathula, lamichhane, lewis, looper, jeyaseelan, blackshear, saini and patial. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord- -b ui vx authors: zhang, rui; wang, xuebin; ni, leng; di, xiao; ma, baitao; niu, shuai; liu, changwei; reiter, russel j. title: covid- : melatonin as a potential adjuvant treatment date: - - journal: life sciences doi: . /j.lfs. . sha: doc_id: cord_uid: b ui vx abstract this article summarizes the likely benefits of melatonin in the attenuation of covid- based on its putative pathogenesis. the recent outbreak of covid- has become a pandemic with tens of thousands of infected patients. based on clinical features, pathology, the pathogenesis of acute respiratory disorder induced by either highly homogenous coronaviruses or other pathogens, the evidence suggests that excessive inflammation, oxidation, and an exaggerated immune response very likely contribute to covid- pathology. this leads to a cytokine storm and subsequent progression to acute lung injury (ali)/acute respiratory distress syndrome (ards) and often death. melatonin, a well-known anti-inflammatory and anti-oxidative molecule, is protective against ali/ards caused by viral and other pathogens. melatonin is effective in critical care patients by reducing vessel permeability, anxiety, sedation use, and improving sleeping quality, which might also be beneficial for better clinical outcomes for covid- patients. notably, melatonin has a high safety profile. there is significant data showing that melatonin limits virus-related diseases and would also likely be beneficial in covid- patients. additional experiments and clinical studies are required to confirm this speculation. coronaviruses (covs) are rna viruses infecting both human and animals; this infection involves the respiratory, gastrointestinal and central nervous system [ ] . severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov) are infectious and lethal, and have caused thousands of deaths in the past two decades. the recent outbreak was discovered in wuhan, china; this highly contagious disease has spread throughout china and other countries [ ] . although antiviral therapy, corticosteroid therapy and mechanical respiratory support have been applied, there is lack of a specific treatment for covid- [ ] . melatonin (n-acetyl- -methoxytryptamine) is a bioactive molecule with an array of health-promoting properties; melatonin has been successfully used to treat sleep disorders, delirium, atherosclerosis, respiratory disease and viral infections [ ] . previous research has documented the positive effects of melatonin in alleviating acute respiratory stress induced by virus, bacteria, radiation, etc. [ ] [ ] [ ] . herein, we review the evidence indicating that melatonin will have supportive adjuvant utility in treating covid- induced pneumonia, acute lung injury (ali) and acute respiratory distress syndrome (ards). patients with covid- (who were infected by sars-cov- ) are reported to present with fever, dry cough, myalgia, fatigue, and diarrhea, etc. with symptoms varying somewhat with the patients' age. in some cases, the severe progression of the disease results in ali/ards, respiratory failure, heart failure, sepsis, and sudden cardiac arrest within a few days [ , ] . the pathogenic examination of lung specimens from mild covid- patients (who were retrospectively found to have covid- at the time of lung cancer surgery) showed edema, proteinaceous exudate with globules, patchy inflammatory cellular infiltration and moderate formation of hyaline membranes [ ] . in a postmortem assessment of a covid- patient with severe ards, specimens of infected lungs demonstrated bilateral diffuse alveolar damage with edema, pneumocyte desquamation and hyaline membrane formation [ ] . though these pathological reports were reported in only a small number of cases, the findings do resemble the pathological features found in sars-and mers-induced pneumonia [ ] . sars-covs, mers-covs and sars-cov- are classified in beta-coronavirus family members [ ] . recent published research suggests that sars-cov- shares . % nucleotide identity to sars-cov and . % identity to mers-cov [ ] , indicating a high genetic homology among sars-cov- , mers-cov and sars-cov. in sars-cov and mers-cov infected animal model, marked inflammatory and immune responses may activate a "cytokine storm", and apoptosis of epithelial cells and endothelial cells; subsequently, vascular leakage, abnormal t cell and macrophages responses ensue and induce ali/ards or even death [ ] . based on genetic homology and pathologic features of the infected lung, we predicted that a cytokine storm also prevails in patients with covid- . in the blood of patients with covid- , there was a marked increase in interleukin β (il- β), interferon γ (ifn-γ), interferon-inducible protein (ip- ), and monocyte chemoattractant protein (mcp- ), as well as il- and il- when compared to that of sars patients. this suggests some potential difference from sars and mers in the pathogenesis of coronavirus [ ] . there is also a potential repressed immune function in covid- patients with the hypo-albuminemia, lymphopenia, neutropenia, and decreased percentage of cd + t cell [ , ] . recent reports suggest that in some covid- patients, although being negative for the viral nucleic acid test, still sometimes present with a high level of inflammation. a clinical trial using certolizumab pegol (a tnf blocker) along with other anti-virus therapies may have beneficial effects in covid- patients. collectively, the finding indicates that inflammation is a major feature in covid- patients. thus, we hypothesize that excessive inflammation, depressed immune system, and an activate cytokine storm substantially contribute to the pathogenesis of covid- . in the early stages of coronaviruses infection, dendritic cells and epithelial cells are activated and express a cluster of pro-inflammatory cytokines and chemokines including il- β, il- , il- , il- , both ifn-α/ β, tumor necrosis factor (tnf), cec motif chemokine (ccl ), ccl , ccl , and ip- , etc. these are under the control of immune system. thus, the overproduction of these cytokines and chemokines contributes to the development in disease [ ] [ ] [ ] . il- , produced by t-helper- (th ), is antiviral, with an infection of coronaviruses leading to a marked decrease in this agent [ , ] . interestingly, covid- patients sometimes have a significantly elevated level of il- [ ] . whether this is a feature of the covid- infection or the result of medical treatment is unknown. the amplification of the inflammatory response would promote cellular apoptosis or we postulated that lungs infected by sars-cov- , and a suppressed immune response, elevated inflammation and excessive oxidation stress proceed unabated, this results in the activation of the cytokine storm. ali/ards may ensue, accompanied by a series of complications, the outcomes of which vary according to the severity of the disease. melatonin may play a role of adjuvant medication in the regulation of immune system, inflammation and oxidation stress, and provide support for patients with ali/ards and related complications. ali: acute lung injury; ards: acute respiratory distress syndrome. necrosis of the affected cells, which would further fuel inflammation, followed by increasing permeability of blood vessels and the aberrant accumulation of inflammatory monocytes, macrophages and neutrophils in the lung alveoli [ ] . this vicious circle would intensify the situation as the regulation of immune response is lost and cytokine storm is further activated, resulting in dire consequences. this putative "cytokine storm" pathology associated with coronaviruses is also supported by experimental sars-cov models, one of which showed that the severity of ali was accompanied by an elevated expression of inflammation-related genes rather than increased viral titers. in another case, the ablation of ifn-α/β receptor or the depletion of inflammatory monocytes/macrophages caused a marked rise in the survival rate of coronaviruses host without a change in viral load [ , ] . both situations suggest a potential amplifying mechanism involved in cov-induced ali/ards regardless of the viral load. if a similar pathology also exists in covid- , the attenuation of the cytokine storm by targeting several key steps in the process could bring about improved outcomes. melatonin is not viricidal but it has indirect anti-viral actions [ ] due to its anti-inflammation, anti-oxidation and immune enhancing features [ ] [ ] [ ] [ ] . there are situations in which melatonin suppresses the features of viral infections. in mice whose central nervous system is infected by virus (e.g., encephalitis), the use of melatonin caused less viremia, reduced paralysis and death, and decreased virus load [ ] . in previous respiratory syncytial virus models, melatonin caused downregulation of acute lung oxidative injury, pro-inflammatory cytokine release and inflammatory cell recruitment. these findings, along with those recently summarized by reiter et al. [ ] , support a rationale for melatonin use in viral diseases. also, melatonin's anti-inflammation, anti-oxidation, immune enhancing actions supports its potential attenuation of covid- infection (fig. ). melatonin exerts anti-inflammatory effects through various pathways. sirtuin- (sirt ) may mediate the anti-inflammatory actions of melatonin by inhibiting high mobility group boxechromosomal protein (hmgb ), and thus down-regulating the polarization of macrophages towards the pro-inflammatory type [ ] . in sepsis-induced ali, the proper regulation of sirt attenuates lung injury and inflammation, in which the application of melatonin might be beneficial [ ] . nuclear factor kappa-b (nf-κb) is closely associated with pro-inflammatory and pro-oxidative responses while being an inflammatory mediator in ali. the anti-inflammatory effect of melatonin involves the suppression of nf-κb activation in ards [ , ] . melatonin reportedly down-regulate nf-κb activation in t cells and lung tissue [ , ] . the stimulation of nf-e -related factor (nrf ) is crucial in protecting lung from injury. in related studies, melatonin induces the up-regulation of nrf with therapeutic effects in hepatoprotection, cardioprotection, etc. [ ] . whether nrf is involved in the cov-induced ali remains unknown, but the close interaction of sirt , nf-κb and nrf suggests their participation in the cov-induced ali/ards. as such, the data support the potential anti-inflammatory action of melatonin. inflammation is commonly associated with an elevated production of cytokines and chemokines, while melatonin causes a reduction in the pro-inflammatory cytokines. tnf-α, il- β, il- , and il- , and an elevation in the level of anti-inflammatory cytokine il- [ , ] . there may be, however, some concerns about the potential pro-inflammatory actions of melatonin when used in very high doses or under suppressed immune conditions where it may induce an increase production of pro-inflammatory cytokines, il- β, il- , il- , il- , tnf-α, and ifn-γ [ ] . conversely, in ali infection models, melatonin presents with anti-inflammatory and protective action [ ] . the anti-oxidative effect of melatonin cooperates with its anti-inflammatory actions by up-regulating anti-oxidative enzymes (e.g. superoxide dismutase), down-regulating pro-oxidative enzymes (e.g. nitric oxide synthase), and it may also interact directly with free radicals, functioning as free radical scavenger [ , ] . viral infections and their replication constantly generate oxidized products. in a sars-induced ali model, the production of oxidized low density lipoprotein activates innate immune response by the overproduction of il- alveolar macrophages via toll-like receptor (tlr )/nf-kb signaling, thereby leading to ali [ ] . tlr is a receptor for the innate immune system, and it is also a therapeutic target for melatonin. in brain ischemia, gastritis and periodontitis disease models, melatonin has documented anti-inflammation actions via tlr signaling [ ] [ ] [ ] . the anti-oxidative effect of melatonin has also been confirmed in ali caused by radiation, sepsis and ischemia-reperfusion [ , , ] . in ali/ards patients, especially when the disease is advanced and in patients treated in intense care units (icus), severe inflammation, hypoxemia and mechanical ventilation with high oxygen concentrations inevitably increases oxidant generation locally and systematically [ , ] . accordingly, we speculate that excessive oxidation also is likely involved in covid- . the extensive studies of gitto et al. [ , ] , who used melatonin to treat newborn infants with respiratory distress, has documented the anti-oxidant and anti-inflammatory actions of melatonin in the lung. thus, it is likely that the application of melatonin would be beneficial in controlling the inflammation and oxidation in coronavirus infected subjects. when virus is inhaled and infects respiratory epithelial cells, dendritic cells phagocytose the virus and present antigens to t cells. effector t cell function by killing the infected epithelial cells, and cytotoxic cd + t cells produce and release pro-inflammatory cytokines which induce cell apoptosis [ ] . both the pathogen (cov) and cell apoptosis trigger and amplify the immune response. the exacerbation of cytokine production, excessive recruitment of immune cells and the uncontrollable epithelial damage generates a vicious circle for infection related ali/ards [ ] . the clinical characteristics of covid- suggest that a reduced level of neutrophils, lymphocytes and cd + t cells in peripheral blood [ , ] . melatonin exerts regulatory actions on the immune system and directly enhances the immune response by improving proliferation and maturation of natural killing cells, t and b lymphocytes, granulocytes and monocytes in both bone marrow and other tissues [ ] . in macrophages, antigen presentation is also augmented after the application of melatonin, where the up-regulation of complement receptor , mhc class i and class ii, and cd antigens were detected [ ] . nod-like receptor (nlrp ) inflammasome is part of the innate immune response during lung infection. the pathogen, including a virus (covs has not yet been tested), triggers nlrp activation to amplify the inflammation. there is probably a balance of the protective and damaging actions of nlrp in the lung. thus, in a mouse experiment, inhibition of nlrp in the early phase of infection increased mortality, whereas suppression of nlrp at the peak of infection allowed for a protective effect [ ] . this supports the use of melatonin in ali/ards when inflammation is most severe. inflammasome nlrp is correlated to lung diseases caused by infection, including influenza a virus, syncytial virus, and bacteria [ ] [ ] [ ] . the efficacy of melatonin in regulating nlrp has been proven in radiation-induced lung injury, allergic airway inflammation and oxygen-induced ali and lps-induced ali models, in which melatonin reduced the infiltration of macrophages and neutrophils into the lung in ali due to the inhibition of nlrp inflammasome [ , , , ]. although there is obviously no report related to the use of melatonin in covid- patients, in subjects with other diseases and an increased level of inflammation, the application of melatonin showed promising results regarding the attenuation of circulating cytokines levels. in a randomized controlled trial, -week oral intake of mg/d melatonin caused a significant decrease in serum levels of il- , tnf-α and hs-creactive protein (hs-crp) in patients with diabetes mellitus and periodontitis [ ] . in another trial of patients suffering with severe multiple sclerosis, orally mg/d of melatonin for months also promoted a significant reduction in serum concentrations of tnf-α, il- , il- β and lipoperoxides [ ] . in the acute phase of inflammation, including during surgical stress [ ] , brain reperfusion [ ] , and coronary artery reperfusion [ ] , melatonin intake of mg/d, mg/d and mg/d of melatonin for less than days induced a reduced level of pro-inflammatory cytokines. a recent meta-analysis of a total of randomized controlled trials suggested that a supplementary use of melatonin is associated with a significant reduction of tnf-α and il- level [ ] . this clinical evidence suggests that the use of melatonin as a supplement may effectively reduce the levels of circulating cytokines, and may potentially also lower pro-inflammatory cytokine levels in covid- patients. the integrity of the vascular endothelial barrier is crucial in the immunoregulation within alveoli. severe inflammation and immune responses induce epithelial and endothelial cell apoptosis, as well as increasing the production of vegf, which aggravates edema and the extravasation of the immune cells from blood vessels. experimental evidence suggests that melatonin mediates the suppression of vegf in vascular endothelial cells [ ] . based on clinical reports of covid- , patients with severe ali/ards may also have an increased risk of sepsis and cardiac arrest [ ] . published reports indicate that the application of melatonin may ameliorate the septic shock via the nlrp pathway [ ] . specifically, melatonin may a have preventive effect against sepsis-induced renal injury, septic cardiomyopathy and liver injury [ ] [ ] [ ] . it was also reported that melatonin had benefits in patients with myocardial infarction, cardiomyopathy, hypertensive heart diseases and pulmonary hypertension, and probably functions via the tlr /survivor activating factor enhancement pathway [ ] . moreover, melatonin exerts neurological protection by reducing the cerebral inflammatory response, cerebral edema and brain-blood barrier permeability under a number of experimental conditions [ ] . in the icu, deep sedation is associated with increased long-term mortality, and the application of melatonin reduces sedation use and the frequency of pain, agitation, anxiety [ , ] . also, a recent meta-analysis showed that melatonin improves sleep quality in patients in the icu [ ] . thus, the rationale for the use of melatonin in covid- patients not only focuses on the attenuation of the infection-induced respiratory disorders, but also on an overall improvement and prevention of patients' wellbeing and potential complications. when considering the use of melatonin to treat covid- , the safety of the melatonin is of utmost significance to consider. as reviewed previously, short-term use of melatonin is safe, even in those given high doses, and the reported adverse effects are limited to occasional dizziness, headache, nausea and sleepiness; in general melatonin's safety in humans is very high [ ] . in clinical trials, doses of mg, mg and mg of melatonin oral intake by patients in icu showed satisfactory safety when compared to placebo [ , , ] . also, even when melatonin was given to humans at dose of g/d for a month, there were no adverse reports of the treatment [ ] . finally, there were no adverse effects recorded after the use of melatonin in ali/ards animal studies [ , , ] . while the safety of melatonin has been verified in many human studies, its effect when given to covid- patients should be carefully monitored despite the very high safety profile of melatonin. the possible beneficial effects of melatonin as adjuvant use in covid- in anti-inflammation, anti-oxidation, immune response regulation has been repeatedly demonstrated in respiratory disorder models induced by infections and associated complications. melatonin has a high safety profile. although the direct evidence of melatonin application in covid- is unclear, both its use in experimental animal models and in studies on humans has continuously documented its efficacy and safety and its use by covid- patients predictably would be highly beneficial. origin and evolution of pathogenic coronaviruses clinical features of patients infected with novel coronavirus in treatment of ebola and other infectious diseases: melatonin "goes viral melatonin alleviates radiationinduced lung injury via regulation of mir- e/nlrp axis melatonin treatment improves adipose-derived mesenchymal stem cell therapy for acute lung ischemia-reperfusion injury inhibitory effect of melatonin on lung oxidative stress induced by respiratory syncytial virus infection in mice epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study pulmonary pathology of early phase sarscov- pneumonia, preprints (www.preprints.org) 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administered melatonin in critically ill patients the effects of oral melatonin on skin color and on the release of pituitary hormones we thank all the doctors, nurses, and researchers who have fought against the virus on the frontline of -ncov epidemic. we thank everyone who has given great and selfless support to the fight against this deadly infection. this research did not receive a specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors declare that there are no conflicts of interest. key: cord- - a w h authors: thomas, neal j.; dahmer, mary k.; quasney, michael w. title: genetic predisposition to critical illness in the pediatric intensive care unit date: - - journal: pediatric critical care study guide doi: . / - - - - _ sha: doc_id: cord_uid: a w h much progress has been made in the past decade in the understanding of the genetic contribution to the development of human disease in general, and critical care illness specifically. with the mapping of the human genome and on-going mapping of genetic polymorphisms and haplotypes in humans, the field of critical care is now in prime position to study the impact of genetics on common illnesses that affect children who require critical care, to examine how differences of the host defense response lead to variable outcomes in outwardly appearing similar disease states, and to study how genetic differences in response to therapy will help practitioners tailor therapeutic interventions to an individual child’s genetic composition. while we are still years away from true individualized medicine, we are now closer than ever to understanding why two might children respond to the same environmental insult in vastly different ways. much progress has been made in the past decade in the understanding of the genetic contribution to the development of human disease in general, and critical care illness specifi cally. with the mapping of the human genome and on-going mapping of genetic polymorphisms and haplotypes in humans, the fi eld of critical care is now in prime position to study the impact of genetics on common illnesses that affect children who require critical care, to examine how differences of the host defense response lead to variable outcomes in outwardly appearing similar disease states, and to study how genetic differences in response to therapy will help practitioners tailor therapeutic interventions to an individual child's genetic composition. while we are still years away from true individualized medicine, we are now closer than ever to understanding why two might children respond to the same environmental insult in vastly different ways. neal j. thomas , mary k. dahmer, and michael w. quasney before being able to appreciate the advances in research that have been accomplished in relation to the genetic impact on critical illness in children in recent years, it is important to understand the basics of human genetics, and become familiar with the terminology that is utilized to discuss these remarkable advances. once the genetic basics are clear, discussion can then proceed to genetic associations that have been determined in critical illness in children. the nucleus of all cells holds chromosomes that contain deoxyribonucleic acid (dna), the genetic material that is inherited from parents. dna is responsible for determining the structure of the cell, the function and activity of the cell in response to various stimuli, and the interaction the cell has with other cells and the extracellular environment. the dna molecule consists of two chains of deoxyribonucleotides held together by complementary base pairs. the deoxyribonucleotides contain the four nucleotide bases, adenine (a), thymine (t), guanine (g), and cytosine (c) that are covalently bound together by phosphodiesterase bonds linking the ¢ carbon of one deoxyribose group to the ¢ carbon of the next group. the two chains of deoxyribonucleotides are linked by hydrogen bonds between the a's of one strand and the t's of the other. likewise, the g's of one strand are linked by hydrogen bonds to the c's of the complementary strand. these two complementary strands form the dna double helix ( fig. - ) , with one strand running in the ¢ to ¢ direction while the other strand runs in the ¢ to ¢ direction. the order of nucleotides bases is termed the sequence and is read in the ¢ to ¢ direction. the genetic information of an individual is encoded by the precise positioning and order of these base pairs. the order of nucleotide bases is termed the sequence and is read in the ¢ to ¢ direction. the four nucleotides of the dna double helix (from national human genome research institute's talking glossary of genetics ( http://www.genome.gov/ glossary.cfm#s )) the entire dna content of an organism is their genome . every cell of an organism contains two copies of the dna, with the exception of red blood cells which lack a nucleus and dna and sperm and egg cells which contain one copy of the dna. overall, humans have chromosomes, including pairs of autosomal chromosomes and one pair of sex chromosomes. each chromosome is made up of a centromere and two telomeres (ends) (fig. - ). the two arms of the chromosome are the short arm (p) and long arm (q). the parts of the genome that contain nucleotide sequences that code for proteins are the genes and it is estimated that the human genome contains about , - , genes. the structure of genes is very complex and highly variable. genes are made up of a variable number of exons , which contain the actual coding sequence for the proteins, and introns , which are noncoding regions which separate the exons. while the function of the introns is unclear, some disease processes have been found to be associated with certain nucleotide variations located in these intron regions ( fig. - ). genes also have regulatory regions, including promoter sequences that generally reside at the ¢ end of the gene (referred to as upstream) and regions at the ¢ end associated with stability of the mrna. genetic recombination , which is the re-shuffl ing of genes from generation to generation, is the basis of genetic diversity in sexually reproducing organisms. the analysis of genetic recombination is a useful method of mapping genes in the genome. genetic recombination results in an exchange of genetic material between homologous chromosome pairs. this results in segments of dna being exchanged with the other chromosome of the pair, thereby shuffl ing the genetic material. the basic principal of linkage analysis , a method used to fi nd disease causing genes, relies on the genetic recombination frequency between two loci on a single chromosome. this allows the estimation of the relative distance between them, and is crucial for the mapping of genes in the genome. recombination frequencies can be measured by genotyping individuals in a family pedigree. the closer together two loci are on a chromosome, the lower the likelihood of recombination to occur between them. if loci are very close, they are said to be linked. the reliability of genetic linkage between loci is determined using the lod score , which is an estimate of whether two loci are likely to lie near each other on a chromosome and are therefore likely to be inherited together. a lod score of or more, which represents odds of : or greater in favor of linkage, is used to indicate statistically signifi cant linkage, and therefore concludes that the two loci of interest are close. the development of genetic maps has been very useful in fi nding genes which may cause human disease. genes may be mapped to a particular location in the genome based on being inherited with respect to a marker of known map location, and with the assumption of no the entire dna content of an organism is their genome. genes are made up of a variable number of exons, which contain the actual coding sequence for the proteins, and introns, which are noncoding regions which separate the exons. nucleus telomere telomere centromere the structure of a chromosome (from national human genome research institute's talking glossary of genetics ( http://www. genome.gov/glossary.cfm#s )) genetic recombination. there are a number of polymorphic markers which may be utilized for genetic map construction, including minisatellites, microsatellites, and single nucleotide polymorphism (snps). linkage disequilibrium (ld), often referred to as allelic association, is a measure of physical association between two alleles and occurs when closely linked alleles are inherited together during many generations. no signifi cant degree of genetic recombination occurs between them, and they continue to be passed along together throughout generations. therefore, knowledge of one marker can be used to study the other. there are many potential benefi ts of identifying genes/gene variants involved in disease. these include, but are not limited to, an improved understanding of the disease etiology, insight into the mechanisms of disease pathogenesis, an ability to develop an early disease risk assessment, the potential to discover novel therapeutic drug targets, the ability to estimate the therapeutic response to specifi c pharmacologic therapies, the possibility of targeted disease prevention strategies to be utilized in high-risk populations based on genetic predisposition, and the movement from the classic symptoms-based disease defi nition towards a true molecular defi nition of complex disease processes. genetic mutations are changes that occur in the sequence of dna. mutations can be classifi ed as somatic mutations, which occur in somatic cells and are not commonly passed on to offspring, and germ-line mutations which occur in the reproductive cells and are passed on linkage disequilibrium (ld), often referred to as allelic association, is a measure of physical association between two alleles and occurs when closely linked alleles are inherited together during many generations. the structure of a gene, including the exons (coding sequence) and introns (noncoding sequence) (from national human genome research institute's talking glossary of genetics ( http://www. genome.gov/glossary.cfm#s )) to offspring. there are several different types of mutations. translocations are large-scale mutations comprised of switching of chromosomal regions between one chromosome and another chromosome. mutations can also consist of single changes in the nucleotide bases and include substitution, deletion, or insertion of nucleotides. insertions and deletions can also involve hundreds of nucleotides. mutations that occur in the coding regions can have several consequences: they can change the amino acid of the protein at a single site, they can cause a premature stop codon resulting in early termination of translation, and, consequently, lead to a truncated protein, or they may have no effect at all if the mutation leads to a nucleotide substitution that does not alter the amino acid. likewise, mutations in noncoding regulatory regions (such as promoters) may also affect the expression of the gene by altering the quantity of mrna transcribed and, hence, the level of the protein. mutations in the intron/ exon boundary region may also lead to incorrectly spliced mrnas and result in signifi cantly different proteins or differences in levels of protein products. the sequencing of the human genome has revealed that most genes are polymorphic; that is, there are small differences in the nucleotide sequences. there are estimates that the human genome may contain over million of these types of variations. these differences in the nucleotide sequence are what give rise to our genetic variability; they account for inherited differences in our physical traits and the way we respond to environmental stimuli and medications. while the majority of these nucleotide variations do not cause a disease, some genetic variations may infl uence the development of certain diseases. the mutations discussed in the preceding paragraph are variations that occur in less than % of the population and are, thereby, rare. on the other hand, variations that occur at a frequency greater than % in the population are referred to as polymorphisms . if the polymorphism is a change in a single nucleotide, it is referred to as a single nucleotide polymorphism (snp) . these more common genetic variations, whether snps or small insertions or deletions of nucleotides, are the ones currently being examined in many studies for associations with susceptibility to and outcome from diseases seen in the intensive care unit setting. copy number variations (cnvs) are stretches of dna of greater than kb that show differences in the expected number of copies of the dna in greater than % of the human population. very recently it has become clear that cnvs are also common in human genomes and contribute significantly to human genetic variation. another important concept in genetics is a locus, which refers to the location in the genome of a specifi c gene or variant. keeping in mind the above discussion of genetic variations, a locus may contain two slightly different sequences for a specifi c gene. these alternative forms of a gene are termed alleles , or variants . the alleles for a specifi c individual at a genetic locus is that person's genotype . an example is the surfactant protein b (sp-b) + site. an individual's genotype at that site may either be tt, ct, or cc. individuals are heterozygous if they possess two different alleles at the locus of interest and homozygous if they possess two identical alleles at that locus. but we obviously do not contain only one genetic variation in our genome. a haplotype represents a combination of polymorphic alleles on a single chromosome delineating a pattern that is inherited together and transmitted from parent to offspring. haplotype analysis is a useful tool for analysis of disease gene discovery, as investigators may capitalize on the fact that many of the polymorphisms of interest are not transmitted independently of each other, and the presence of one gene variant can tag the presence of another polymorphism from the same chromosome. in some cases, haplotype assessment can provide a higher level of specifi city, sensitivity, and accuracy in "true" associations with disease risk or severity. by focusing on haplotypes as well as snps, researchers are now able to more accurately study genetic predisposition to various diseases of interest. with the recent report of the international hapmap consortium, and the identifi cation and cataloging of haplotypes now available, the utility of this type of study is brought into focus as an important tool to guide genetic association studies on complex human diseases. genetic polymorphisms, like the rarer mutations, may also infl uence the quantity of the mrna made if present in a regulatory region, or they may also infl uence the functional activity of the protein product. there has been an explosion of studies attempting to determine if these genetic polymorphisms may account for some of the clinical variability we as clinicians observe at the bedside in the picu. for example, can the difference in disease genetic mutations are rare changes that occur in the sequence of dna that occur in less than % of the population. polymorphisms are variations that occur at a frequency greater than % in the population; if the polymorphism is a change in a single nucleotide, it is referred to as a single nucleotide polymorphism (snp). a haplotype represents a combination of polymorphic alleles on a single chromosome delineating a pattern that is inherited together and transmitted from parent to offspring. c hapter • g en etic pr edis pos ition to c r itical i lln ess severity between two children with pneumonia be associated with variations in their genes coding for one of the surfactant proteins? gene expression is the process by which the information contained within genes is used to make proteins ( fig. - ) . this occurs by a combination of two distinct processes: transcription and translation. transcription is the process by which the genetic information in dna is transcribed into messenger ribonucleic acid (mrna). mrna differs from dna in that it is singlestranded, has a modifi ed sugar backbone, and contains uracil (u) instead of t. the process of transcription involves the unwinding of the two complementary strands of dna, the enzyme rna polymerase binding to the promoter region of a gene on a single strand of dna, and synthesizing the mrna molecule by adding ribonucleotides in an order that is complementary to the dna strand. the transcribed mrna thus contains all the genetic information between the transcriptional start and stop sites on the dna including exons and introns. the non-coding intron sequences are removed by a process referred to as splicing which connects all the exons together to form the fi nal mrna product. this mrna represents the coding dna sequences for a single gene. (it should be noted that splicing variations have been identifi ed that infl uence disease processes that impact the fi nal protein product by altering the mrna sequences that are spliced together.) the mrna is then transported to the cytoplasm, and translation occurs, in which the genetic information from mrna is utilized to guide the synthesis of proteins. proteins are composed of amino acids. there are different amino acids in humans, and each is encoded by a set of nucleotides in the mrna. these three nucleotides are called triplets or codons . the corresponding anticodon on the transfer rna (trna) links with a codon, presenting its unique amino acid in the process of translational protein synthesis. gene expression is the process by which the information contained within genes is used to make proteins. transcription is the process by which the genetic information in dna is transcribed into messenger ribonucleic acid (mrna). translation is the process by which the genetic information from mrna is utilized to guide the synthesis of proteins. since all cells that contain a nucleus carry the full set of genetic information, it is necessary for gene expression to be selective and tightly controlled, in a way that guarantees specifi c proteins are expressed in specifi c cells under appropriate conditions. this differential expression of genes ensures that cells develop correctly, can differentiate and function as specialized cells, and can mount various responses to external stimuli. in certain disease states, expression of specifi c genes may change, thereby providing a clue as to which genes may be important in that disease process. recent advances in technology have provided a valuable tool to evaluate the expression of genes during various diseases, including sepsis. these technologies include dna microarrays, in which the basic approach is as follows: small strands of dna probes representing the genes of interest, for example, tumor necrosis factor alpha (tnf-a ), are attached to a solid substrate such as a glass slide or silicon chip (in reality, thousands of probes are applied to the same micorarray chip). mrna is then isolated from, for example, a patient without acute lung injury (ali) and one with ali. these two samples are then separately converted to complementary dna (cdna) in a manner which incorporates different fl uorophores in the two samples of cdna (e.g., red into all the cdnas from the patient with ali which would also include cdna made from the mrna coding for tnf-a , and green into all the cdnas from the patient without ali including the cdna made from the mrna coding for tnf-a ). the cdnas from the two patients are then mixed and hybridized to the chip containing the dna probes. thus, if tnf-a is up-regulated in ali and there is signifi cantly more cdna in the ali sample than the non-ali sample, then more red fl uorophore -labeled cdna would be present. when the mixture of cdnas is hybridized to the chip containing the probes, the probe for tnf-a would light up red and represent increased expression of the tnf-a gene in ali. if tnf-a is expressed at a lower concentration in the patient with ali then when the samples are mixed, more green fl uorophore-labeled cdna would be present than red fl uorophore-labeled cdna, and the probe for tnf-a on the microarray chip would light up green indicating decreased expression of tnf-a gene in ali. finally, if there is no change in the expression of the tnf-a gene, the amounts of red and green fl uorophorelabeled cdnas would be equal, and the probe for the tnf-a gene would light up yellow. in this fashion, one can identify specifi c genes that are expressed in the development of ali. several examples of the use of this technology in the critically ill patient have been published which have aided our understanding of the pathophysiology of certain icu specifi c diseases. up to this point, we have discussed the structure of dna and the process of getting from the code in the dna to protein. it is the functional aspects of these proteins that give rise to the observed traits, whether it be the color of one's eyes, the rate of metabolism of a drug, or the effi ciency with which a protein receptor on a cell surface recognizes a pathogen. the observable characteristics of an individual defi ne that individual's phenotype . this may include common physical and biochemical characteristics, but can also describe a person's disease status (such as in cystic fi brosis). phenotypes caused by mutations in a single gene may show mendelian inheritance patterns . these patterns can be autosomal dominant (where a single copy of the gene causes the phenotype), autosomal recessive (where both copies of the gene are necessary for the phenotype), or sex linked (where the mutation occurs on the x chromosome). it is crucial to note that mendelian inheritance patterns are only seen for single gene disorders. critical care diseases and syndromes, such as sepsis and acute respiratory distress syndrome (ards), are complex disorders whose genetic predisposition to the development of the disease is due to multiple genes and other factors, such as environmental exposures. the multifaceted gene-gene and gene-environment interactions make the study of these diseases extremely complex. there are many common complex disorders that display obvious familial aggregation of cases, but have no clear mendelian inheritance patterns. the most commonly studied in medicine are cancer, diabetes, hypertension, and obesity, among others. the disease aggregation may be due to complex genetic factors, the interaction of multiple genes on the development the observable characteristics of an individual defi ne that individual's phenotype. c hapter • g en etic pr edis pos ition to c r itical i lln ess of the disease of interest, a host of environmental factors which place the individual at risk for the disease, or, most commonly, a combination of all of the above factors ( fig. - ). in other words, a person's genetic background may make them prone to a specifi c disease; this is called susceptibility gene variants or susceptibility genes. due to their inherited genetic make-up, the individual is at a higher inborn risk for developing the disease of interest, but only if they are exposed to the environmental stressor that is known to associate with the disease. the susceptibility gene variant will not directly lead to the disease, but will put that person at a higher risk if they are exposed to the environmental risk. for example, individuals may possess a susceptibility gene variant for the development of lung cancer, but this will only lead to the development of cancer if they are subject to a known environmental risk factor, such as smoking. alternatively, a newborn may possess one or more susceptibility gene variants for the development of bronchopulmonary dysplasia, but if they are not born prematurely and do not require mechanical ventilation in the newborn period (and therefore are not subject to the environmental stressors known to impact the development of this lung disease), they will never develop this disease despite being genetically susceptible. gene variants can also decrease the susceptibility, or increase the resistance against a disease. these are protective gene variants . to give an example, there are individuals who smoke their entire adult lives and yet never develop chronic obstructive pulmonary disease. it is likely that these individuals possess protective gene variants against the development of this disease, even in the face of a strong environmental insult. a person may possess both susceptibility and protective genetic variants for the same disease, and the mix of these variants will impact together the overall genetic risk of that person to the disease of interest. this resultant genetic risk also interacts with the environmental risk of the individual, leading to the overall risk of that person developing the disease. in critical care, it is unlikely that one gene will cause the diseases that are treated in the intensive care unit; it is more likely that multiple genes will interact with multiple environmental insults to predispose individuals to diseases processes resulting in an overall risk for an individual patient to develop a certain disease of interest. interestingly, certain populations seem to be immune to certain complex diseases. examples include the australian aborigines and inuits from greenland, in which both populations appear to be resistant to the development of type diabetes. it is plausible that the disease resistance observed in these populations is due to absence of susceptibility gene variants or presence of protective gene variants in the group's gene pool. when it is determined that a complex disease has familial aggregation, it is important to take into account that families may also share environmental or social factors that predispose to the disease of interest, and therefore the entire impact may not be genetic. one example would be radon gas present in a neighborhood leading to an increased incidence of lung cancer in families living in close proximity. while it may be assumed that the genetic impact is responsible for the development of the disease, environmental exposure is the likely source. twin studies and adoption studies are utilized to attempt to determine the relative weight of genetics and environment in the development of a certain disease. genomic medicine and the concept that an individual's genetic makeup may infl uence not only the severity of and outcome from their critical illness, but also their response to the therapies, has begun to makes it's way into the intensive care unit. this section will highlight disease aggregation may be due to complex genetic factors, the interaction of multiple genes on the development of the disease of interest, a host of environmental factors which place the individual at risk for the disease, or, most commonly, a combination of all of the above factors. the complex interactions that can occur between genes that may impact a disease process (gene-gene interaction) as well as between the genes of interest and environmental factors (gene-environment interaction) studies involving analysis of gene expression and genetic association studies in patients with critical illness. while it may appear that advances have been made in the fi eld, it is important to understand that we are not yet in the age of personalized medicine and much work needs to be done. the expression of specifi c genes in many cases represents the body's specifi c and complex response to environmental stimuli, such as in the case of a severe infection, trauma, or cardiopulmonary bypass. examining gene expression may, therefore, provide a clue as to which genes are important in a specifi c critical illness. many studies have examined gene expression in critically ill patients, but most examine the expression of only a few genes. with the advent of the dna microarray technology discussed above investigators have begun to explore gene expression patterns in thousands of genes in children with septic shock using mrna isolated from whole blood representing the gene expression response in circulating white blood cells. these studies have compared gene expression from blood samples obtained within day of admission to the picu with that observed in blood of healthy controls, examined longitudinal changes in gene expression in children with septic shock (days and ) and compared expression in patients with septic shock to expression in children with sepsis or systemic infl ammatory response syndrome (sirs). genes that were up-regulated, down-regulated, or unchanged between the groups were examined. the genes that were up-regulated when the septic shock group was compared to healthy controls included genes related to immunity and infl ammation as would be expected. unexpectedly the study demonstrated that many genes related to zinc biology and zinc homeostasis were down-regulated. the signifi cance of this fi nding was supported by the observation that children who did not survive septic shock had lower serum levels of zinc and the demonstration in a murine model that zinc depletion leads to increased mortality from sepsis. in addition, genes involved in t-cell receptor signaling and antigen presentation appeared decreased suggesting that septic shock may be associated with depression of the adaptive immune system. interestingly expression studies of adults with sepsis and septic shock did not identify a down-regulation of genes related to zinc biology although upregulation of genes related to immunity and infl ammation and down-regulation of genes related to the adaptive immune system was observed. the longitudinal study in children with septic shock demonstrated that in general the observed changes in up-regulated and down-regulated gene expression persisted over time. in addition, the study comparing expression in children with shock to those with sepsis or sirs indicated that while there were patterns of expression that were similar in all three groups (such as genes involved in innate immunity that were up-regulated) there were genes that were unique to the septic shock group with relation to the degree, and duration, of the response. examples include the fi nding that up-regulated genes that were involved in the il- signaling pathway had a greater signal which persisted for longer in the septic shock patients and down-regulation of genes for zinc biology and the adaptive immune system was greater and lasted longer than that seen in the other two groups. in addition to providing insight into the pathophysiology of sepsis and identifying potentially important proteins in early sepsis, the use of this technology may also provide physicians with a unique diagnostic tool. if the gene expression profi le of a patient who is in the early stages of sepsis is different from a patient who exhibits sirs but does not develop sepsis, then earlier therapies could be initiated before full blown sepsis is clinically evident. there is also some evidence that various subclasses of sepsis and septic shock may be able to be identifi ed using this technique. dna microarrays have also been used to investigate the expression profi le in adults with ali. mrna for pre-b-cell colony enhancing factor (pbef), a cytokine that is involved in the maturation of b-cell precursors, inhibition of neutrophil apoptosis, and perhaps regulation of endothelial cell calcium-dependent cytoskeletal arrangement was noted to be signifi cantly increased in adults with ali, a fi nding that was also consistent in both a canine and mouse model of ali. in addition to the elevated mrna levels, pbef protein in bronchoalveolar lavage fl uid was also elevated in adults with ali. it is also worth mentioning an important with the advent of the dna microarray technology, gene expression patterns in thousands of genes can lead to insight into disease pathogenesis, treatment, and outcome. c hapter • g en etic pr edis pos ition to c r itical i lln ess study in a canine model of lung injury to highlight the value of gene expression arrays. the use of mechanical ventilation is invariably needed to treat patients with ali though the use of positive pressure ventilation itself may exacerbate the lung injury. gene expression arrays in a canine model of ventilator associated lung injury have identifi ed a number of genes that are regulated during ali. many of the genes can be grouped into biological processes known to important in the pathophysiology of ali; these include infl ammation (e.g., il- b, il- , il- ra, mmif), coagulation (tissue factor, pai- ), and chemotaxis/cell motility (myosin light chain kinase, cell chemokine receptor ). several other genes also appeared to be expressed including pbef, heat shock protein (hsp ), and vascular endothelial growth factor (vegf). thus, the use of expression arrays has identifi ed a number of candidate genes that may play important roles in the development of ali. as will be discussed below, genetic variations that infl uence the activity or level of the protein in several of these candidate genes have been examined in gene association studies in patients with ali. as described above, a large amount of genetic variability exists throughout our genome. whether these differences infl uence the susceptibility to or outcome from diseases in the critical care setting is an area receiving a great deal of interest. perhaps the greatest amount of focus of genetic association studies on critical illnesses is in sepsis and ali. the general approach has been to compare the frequencies of polymorphisms in specifi c candidate genes between a cohort of patients with sepsis or ali and an at-risk cohort without sepsis or ali. this section will review some of these studies. individual variability in the susceptibility to and outcome from sepsis and lung injury has long been observed in critically ill patients. why one child with pneumococcal pneumonia has little consequence of their infection and can be treated as an outpatient while another child develops refractory septic shock and respiratory failure has been attributed to a number of factors. these have included virulence of the pathogen, length of time between onset of symptoms and appropriate treatment, and comorbid conditions. while all these certainly contribute to the severity of disease, a growing body of evidence suggests that genetic variations in the individual patient may also contribute to the severity of and outcome from critical disease. these genetic polymorphisms may not be of any consequence during normal healthy periods but their importance may only become evident during a severe stressor such as an infection, trauma, cardiopulmonary bypass, or other scenarios seen in the intensive care unit (table . ). a strong genetic infl uence on the outcome from infections was indicated by a family based study of adoptees. adoptees with a biological parent who died due to infection before the age of had a relative risk of death due to infection of . (ci = . - . ); a higher relative risk than that seen when risk related to early death of a biologic parent due to cardiovascular and cerebrovascular disease ( . ; . - . ) or cancer ( . ; . - . ) was examined. thus, an individual's genetic makeup may infl uence the severity of disease in infection and sepsis. given the tens of thousands of genes in the human genome and the millions of genetic polymorphisms, on which polymorphisms and in which genes should investigators focus? one approach in choosing the candidate gene is to examine the pathways by which pathogens lead to the clinical symptoms of sepsis. the body's response to infections involves recognition of pathogen-associated products followed by an infl ammatory response that involves a large number of cellular proteins. genetic variations that lead to alterations in the amount or functional activity of any of these proteins involved in the recognition of or response to pathogen-associated products may infl uence the individual's response. examples of the infl uence of genetic variations in proteins involved in recognition of pathogens on the severity of infections include polymorphisms in the genes coding for mannose binding individual variability in the susceptibility to and outcome from critical care diseases has long been observed, and advances in genomic medicine now gives an opportunity to understand these differences. the body's response to infections involves recognition of pathogenassociated products followed by an infl ammatory response that involves a large number of cellular proteins. genetic variations that lead to alterations in the amount or functional activity of any of the proteins involved in the recognition of or response to pathogenassociated products may infl uence the individual's response. lectin (mbl) , the receptor for fc g , and toll-like receptor (tlr) . the heterotrimeric mbl is involved in binding bacterial surface carbohydrates and the opsonization of bacteria. a helical domain in the tertiary structure of the protein is crucial for formation of the active heterotrimer. three genetic polymorphisms in the gene coding for mbl result in amino acid changes in the helical tails of the protein and result in increased degradation and decreased serum levels of mbl. genetic association studies have demonstrated associations between variant b, c, d variants associated with decreased levels and activity and increased risk of infection fc g riia h r r associated with decreased affi nity to igg and opsonization and increased risk of infection and septic shock tlr asp gly/thr ile gly/ile associated with decreased expression, increased risk of sepsis and mortality cd- − c/t t allele associated with increased levels and susceptibility to sepsis and sepsis-related mortality in adults md- − c/g − g allele associated with higher risk of sepsis and multiple organ dysfunction score in chinese adults tnfa − g/a, − g/a, lta + g/a a alleles for each polymorphism are associated with increased tnfa levels, increased mortality in sepsis and meningococcal disease, increased sepsis in adults with pneumonia, and increased mortality in bacteremia and sepsis il- − g/c g associated with increased il- levels in patients but c associated with increased levels in monocytes from neonates, sepsis in neonates but not adults, and severe sepsis and organ dysfunction in children il- ra variable -bp repeat a associated with increased levels of il- ra and variable results of association studies examining risk of sepsis and mortality il- − g/a, − c/t, − c/a gcc haplotype associated with increased levels and sepsis but not mortality irak- + t/c c associated with increased nf-kb translocation and presence of shock and higher -day mortality in adults with sepsis hsp a b − c/t + g/a − c/+ a associated with decreased hspa b and tnf a and + a associated with septic shock in adults with cap ace bp i/d dd associated with increased serum and tissue levels and more severe meningococcal disease in children; no association with sepsis related mortality in neonates or adults pai- g/ g g associated with increased levels and septic shock in meningococcal disease protein c − a/g and − c/t ac haplotype associated with increased mortality and organ dysfunction in adults with sepsis and with decreased protein c serum level; gc haplotype associated with more severe sepsis in children less than year of age with meningococcemia fibrinogenbeta − g/a, − g/a, + g/a gaa haplotype associated with higher levels of fi brinogen, lower day mortality and less severe organ dysfunction mbl mannose-binding lectin, ig immunoglobulin, tlr toll-like receptor, rsv respiratory syncytial virus, md myeloid differentiation, tnf tumor necrosis factor, lt lymphotoxin, il- ra interleukin receptor antagonist, il- interleukin- , irak- interleukin receptor-associated kinase , hsp heat shock protein, cap community acquired pneumonia, ace angiotensin converting enzyme, pai plasminogen activator inhibitor a terminology used for the various polymorphisms are the ones most commonly used in the literature and may refer to the nucleotide position, amino acid position, or name of the allele. this table is representative of polymorphisms examined in sepsis but does not include all such polymorphisms the mbl genetic polymorphisms and increased susceptibility to infections, hospitalizations due to infections, number of acute respiratory infections, and risk of meningococcal infections in children, and pneumonia and sepsis in neonates. in adults these polymorphisms have been associated with recurrent respiratory infections, invasive pneumococcal infections and viral coinfections with pneumococcal pneumonia. the family of leukocyte fc g receptors is also involved in the recognition of bacteria such as streptococcus pneumoniae , haemophilus infl uenzae type b, and neisseria meningitides. fc g receptors bind the fc portion of igg bound to bacteria, thereby facilitating phagocytosis and inducing the infl ammatory response. several polymorphisms have been described in the genes coding for the various fc g receptors that alter their binding affi nity to the various subclasses of igg. two such polymorphisms have been described in the genes coding for the fc g riiib receptor and the fc g riia receptor . in the case of the fc g riiib receptor, the genetic polymorphism results in a four amino acid substitution (allotypes fc g riiib-na or -na ) in the receptor that alters the opsonization effi ciency. in the case of the fc g riia receptor, the genetic polymorphism results in replacing a histidine for an arginine in the extracellular domain of the receptor at amino acid position . the variant fc g riia receptor containing the histidine binds the fc region of igg with a lower affi nity and results in reduced phagocytocytosis in vitro compared with the more common fc g riia receptor containing the arginine. in association studies, a higher frequency of individuals homozygous for the na allotype of the fc g riiib receptor or an arginine at position in the fc g riia receptor was found in patients with severe meningococcal disease or fulminant meningococcal sepsis. the fi nal examples of genetic variation in genes coding for pathogen recognition products infl uencing the severity of sepsis are the polymorphisms in the gene coding for the tlr receptor . this receptor is a component of a complex that includes cd- and myeloid differentiation (md)- that binds lipopolysaccharide (lps), one of the major cell wall components of gram negative bacteria. in addition, tlr recognizes the f protein of the respiratory syncytial virus (rsv). two genetic polymorphisms have been identifi ed in the gene coding for tlr that result in the change of a threonine for a glycine at amino acid position and a threonine for a isoleucine at amino acid position . the gly ile variant form of the receptor appears to be expressed on the cell surface in lower amounts and result in a lower systemic cytokine response to lps and rsv. genetic association studies have demonstrated an association between the tlr gly ile variant and gram negative bacterial infections and septic shock as well as mortality in patients with systemic infl ammatory response syndrome. however, a number of studies have also shown confl icting results. these tlr variants have also been reported to be associated with susceptibility to and severity of respiratory syncytial virus infections in children. future studies with more participants will be required to determine whether variations in the tlr gene are involved in infection and/ or severity of disease. thus far, the focus has been on genetic variations in genes coding for proteins involved in pathogen recognition, and in each case, the variation results in an inferior host response resulting in more severe disease. currently it is thought that severe sepsis and septic shock may be the result of an imbalance in the infl ammatory response. the mechanism by which this imbalance occurs is thought to be multi-factorial. one possibility that has attracted much interest is that the host may harbor genetic variations in the regulatory regions of genes involved in the response to noxious stimuli resulting in an imbalance between pro-and anti-infl ammatory cytokines. these variations can result in an over-expression of pro-infl ammatory cytokines, such as tnf-a and interleukin (il)- , or an under-expression of anti-infl ammatory cytokines, such as il- . in either case, the normal infl ammatory response is dysregulated. one of the pro-infl ammatory genes in which genetic polymorphisms infl uence expression is tnf-a . as a key pro-infl ammatory cytokine, tnf-a is responsible for the activation of the infl ammatory response and by itself can produce many of the clinical manifestations of sepsis such as capillary leak, hypotension, and multiple organ dysfunction syndrome. the regulatory region of the gene coding for tnf-a has several polymorphisms that alter transcription of tnf-a , thereby infl uencing the amount of tnf-a produced. several of these polymorphisms alter nucleotides which make up the recognition sequences of some of the genetic variation of toll-like receptor may be an important contributor to difference in the host response to infectious illness observed in children. transcription factors that regulate transcription. two of these polymorphisms in particular have been studied. the rarer a allele (tnf-a - ) at a location base pairs upstream from the transcriptional start site results in greater transcription than the more common g allele. a second rare a allele (tnf-a - ) at a location base pairs upstream from the transcriptional start site results in lower transcription than the g allele. in addition, another site located ~ , base pairs upstream from the transcriptional start site of the tnf-a gene and located in the gene coding for another gene, lymphotoxin (lt)-a , (also referred to as the tnfb allele, tnf-b + , and lt-a + ) also appears to regulate transcription of the tnf-a gene. in genetic association studies, the frequency of the tnf-a - a allele has been shown to be higher in children who died from meningococcal infections and adults who died with septic shock compared with controls. genetic association studies examining the infl uence of the polymorphic lt-a + site has shown a higher frequency of the a allele in adults with pneumonia presenting with the clinical symptoms of sepsis, in adults in post-operative and trauma intensive care units who develop sepsis and who have a higher mortality, and in bacteremic children who exhibit higher serum tnf-a levels and have a higher mortality. however other genetic association studies examining the tnf-a gene have reported confl icting results. recently a well designed, prospective study examining a number of polymorphisms in the gene for tnf-a (including those described above) in adults with trauma admitted to the icu demonstrated that the a allele of tnf-a - was associated with elevated tnf, sepsis syndrome and death in trauma patients both in their initial cohort and a replication cohort. the gene for il- (another pro-infl ammatory cytokine) also contains variations which multiple studies suggest are associated with the susceptibility to or outcome from sepsis. as mentioned above, the progression to severe sepsis is believed to be an imbalance in the pro-and anti-infl ammatory mediators. in addition to polymorphisms that result in increased levels of pro-infl ammatory cytokines, examples of polymorphisms that result in lower levels of anti-infl ammatory cytokines also exist. il- receptor antagonist (il- ra) is one of the body's mechanisms for keeping the infl ammatory reaction in check by binding to the il- receptor without activating the signal transduction pathway. the gene coding for il- ra contains a polymorphic region consisting of a variable number of base-pair tandem repeats. these different il- ra alleles have been associated with variable circulating levels of both il -ra and il- b (the two genes are located close to one another on chromosome ), and several association studies have suggested an infl uence of this variation on a variety of diseases in which infl ammation plays an important role, including the susceptibility to sepsis. il- is another anti-infl ammatory cytokine for which genetic polymorphisms appear to alter transcription levels. a number of studies have demonstrated an association between an increased susceptibility to sepsis and certain il- polymorphisms although confl icting results have also been reported. it is important to remember that the cytokines and their receptors mentioned above activate a complex signal transduction pathway composed of dozens of proteins with the end result of a well coordinated cellular response to the noxious stimulus. genetic variation in any of the proteins in the pathway may also infl uence the fi nal response. recent studies have begun to analyze components of various pathways involved in the development of sepsis. one example is il- receptor-associated kinase- (irak- ) that plays an important role in the signal transduction pathway initiated by the activation of the il- receptor. activation of irak- results in increased transcription of a variety of pro-infl ammatory genes modulated by nf-k b, a key transcription factor in the infl ammatory response. genetic variations in the gene coding for irak- have been shown to be associated with elevated nuclear levels of nf-k b as well as the presence of shock and a higher -day mortality in patients with sepsis. the association of a variant in irak-i with severity of septic shock has been independently replicated in a large multi-centered cohort of adult patients with septic shock. interestingly, this study also indicated that age might modify the relationship as this association was stronger for younger patients. many other proteins involved in these complex signaling pathways have yet to be investigated for the infl uence of genetic variations on critical illnesses. many of the genes discussed thus far have a role in infl ammation, a key component in the pathophysiology of sepsis. loss of homeostatic mechanisms regulating the coagulation/ fi brinolytic system also plays an important role in sepsis. plasminogen activator inhibitor the regulatory region of the gene coding for tumor necrosis factora (tnfa ) has several polymorphisms that alter transcription of tnfa , thereby infl uencing the amount of tnfa produced. il- receptor antagonist (il- ra) is a key mechanism for keeping the infl ammatory reaction in check by binding to the il- receptor without activating the signal transduction pathway. together with il- , another anti-infl ammatory cytokine, genetic polymorphisms appear to alter transcription levels of these proteins. (pai- ) inhibits fi brinolysis thereby favoring the formation of microthrombi in the capillaries. the pathophysiology of multiple organ dysfunction syndrome in patients with sepsis is thought to involve, in part, intravascular fi brin deposition. thus, elevated pai- activity could contribute to organ failure in sepsis and elevated plasma concentrations have been observed in patients with sepsis. a genetic variation in the gene coding for pai- consisting of either the presence of guanines or guanines at a specifi c location appears to infl uence the amount of pai- produced. individuals homozygous for the g genotype ( g/ g) produce more pai- than individuals homozygous for the g genotype ( g/ g) or individuals that are heterozygous ( g/ g). association studies have demonstrated that children with meningococcal disease who were g/ g at this site had an increased risk of death compared to children who were g/ g or g/ g. more recent studies in both children and adults have demonstrated higher mortality in individuals homozygous for the g allele in a number of infectious diseases. since fi brin deposition is thought to play a role in the multiple organ system failure in patients with sepsis, genetic variations that infl uence the production of fi brin might also infl uence the severity of disease in patients with sepsis. the production of fi brinogen, the precursor to fi brin, is dependent on the transcription of fi brinogen-beta . several polymorphisms in the promoter region have been associated with higher plasma levels of fi brinogen, and higher levels of fi brinogen have been associated with improved outcomes in sepsis. association studies have demonstrated that the gaa haplotype, consisting of the genotypes at the − , − , and + sites, is associated with higher levels of fi brinogen and with decreased mortality and less organ dysfunction. protein c has anticoagulant activity as well as anti-infl ammatory and anti-apoptotic effects suggesting that diminished activity of protein c may lead to increased fi brin deposition, infl ammation, and apoptosis. genetic polymorphisms located in the promoter region of the gene coding for protein c result in decreased levels. association studies in caucasian and han chinese adults have demonstrated increased mortality and organ dysfunction in adults with sepsis who carry the a allele at the − site and the c allele at the − site. this haplotype has also been reported to be associated with decreased protein c concentration. interestingly, an increased risk of more severe sepsis has been observed in children less than year of age with meningococcal disease who carry the g allele at the − site along with the c allele at the − site. this haplotype was not associated with severe sepsis in older children suggesting a potential developmental difference in these variations on the severity of sepsis. severe lung injury in both adults and children can be precipitated by a diverse array of causes and are classifi ed as either direct injury when the insult is from the alveolar side of the alveolar/capillary membrane or indirect injury when the insult is from the capillary side. major causes of direct lung injury include pneumonia, aspiration, pulmonary contusion, and inhalation while major causes of indirect injury include sepsis, trauma without pulmonary contusion, cardiopulmonary bypass, and multiple transfusions. despite these various causes, the central pathogenesis of ali involves derangements in multiple biological processes. these include activation of infl ammation, loss of coagulation and fi brinolytic homeostasis, disruption of vascular permeability, epithelial and endothelial cell apoptosis as well as proliferation, and derangements in surfactant. some of these processes, notably infl ammation and coagulation, play key roles in the pathophysiology of sepsis as well as ali. thus, it is not surprising to fi nd that the candidate genes examined in genetic association studies for ali are in many cases the same as those examined in sepsis (table . ). this section will review some of the genetic association studies examining the infl uence of genetic variations on the development of ali. pulmonary surfactant is synthesized by the type ii alveolar epithelial cells and is required for normal lung function. one of surfactant's primary functions is to lower the surface tension at the alveolar air-liquid interface. surfactant is composed of phospholipids and four proteins, surfactant protein (sp)-a, sp-b, sp-c, and sp-d. knockout models in mice have demonstrated that of these four proteins, only sp-b is absolutely required for post-natal genetic polymorphisms located in the promoter region of the gene coding for protein c result in decreased levels, and possibly an impact on mortality with sepsis. defi ciency in, or impaired activity of surfactant protein-b appears responsible for a number of interstitial pulmonary diseases in humans including ards. survival. defi ciency in, or impaired activity of sp-b appears responsible for a number of interstitial pulmonary diseases in humans including ards. several genetic variations exist in the genes coding for the surfactant proteins and two will be discussed here; the sp-b + t/c polymorphism and insertion/deletion polymorphism consisting of dinucleotide (ca) tandem repeats in intron . several studies have demonstrated an association between these polymorphisms and the need for mechanical ventilation in children (sp-b + t/c) and mechanical ventilation and ards in adults (sp-b + t/c polymorphism and insertion/ deletion of dinucleotide (ca) tandem repeats). the consequences of these variations are not fully known. the sp-b + t/c polymorphism results in an amino acid change in exon in a region of the amino terminal propeptide which is thought to play a role in targeting of sp-b to lamellar bodies. the resulting amino acid change alters glycosylation of sp-b and may affect the level of sp-b by altering its processing or stability. aberrant proteolytic processing of the sp-b product encoded by the c allele is supported by a recent report demonstrating that the c allele is associated with absence of a specifi c pro-sp-b cleavage product in neonates. the intron dinucleotide repeat length variation polymorphism is associated with incompletely spliced sp-b mrna. interestingly, in caucasians this length variation polymorphism in intron is in linkage disequilibrium with the sp-b + t/c polymorphism; the c allele of rs is associated with the deletion variants at the intron polymorphic site. further work is needed to not only defi ne the consequence of these genetic variations on surfactant function but also to further evaluate whether these genetic variations infl uence the development of ali. another study of interest involves the susceptibility to pneumonia, the leading cause of ali and ards in both children and adults. as discussed above, the g/ g genotype in the gene coding for pai- is associated with higher levels of pai- expression. in a large cohort of adults, those individuals with the g allele demonstrated a signifi cantly higher susceptibility to pneumonia. while pai- activity inhibits fi brinolysis leading to formation of microthrombi, it also demonstrates anti-infl ammatory activity, and in this fashion, may increase the susceptibility to infection. in patients with ali, plasma levels of pai- levels are elevated and protein c levels are diminished. in addition, alveolar levels of pai- are elevated suggesting a possible local activation of the fi brinolytic system. recent studies have demonstrated that the g allele of pai- is associated with increased mortality in patients with severe pneumonia and patients with ards. to date there are no reports indicating association of specifi c protein c variants with ali. infl ammation is one of the hallmarks of ali and as with sepsis, it is thought that one of the central components of ali is an imbalance between pro-and anti-infl ammatory cytokines in the lung. the infl uence of genetic variation in several genes involved with infl ammation on the development of ali has been examined. the tnf-a - a allele appears to be associated with increased mortality in adults with ards but not with the susceptibility to ards when compared with adults who were at-risk for the development of ards. the lt-a + polymorphism that appears to be associated with more severe sepsis did not infl uence the severity of ards. macrophage migration inhibitory factor (mif) plays a central role in regulating the infl ammatory response by directly increasing tnf-a and il- and countering the antiinfl ammatory actions of glucocorticoids. mif mrna has been demonstrated in cells from bronchoalveolar lavage of patients with ali and mif concentrations in serum are elevated in patients with ali compared with controls. haplotypes composed of polymorphisms in the ' end of the gene coding for mif are associated with the development of ali in both caucasian and african american populations. a number of studies have demonstrated association of specifi c haplotypes in another gene involved in the regulation of infl ammation, the gene for il- , with the development of ali. whether these haplotypes are associated with elevated levels of il- is still unclear. association studies have also been performed examining the genetic variants discussed earlier in the promoter region of the anti-infl ammatory cytokine il- . the − gg genotype results in higher levels of il- and is associated with the development of ards in younger adults. furthermore, the adults with ards who carried the gg genotype at this site demonstrated lower mortality and organ failure. the genetic variants in the gene coding for mbl have also been examined for their infl uence on ali. one of the variants described previously that results in decreased serum levels of mbl, variant b, is associated with the susceptibility to ards and a greater degree of organ dysfunction and higher mortality in patients with ards. while no reports have described the tlr polymorphisms specifi cally in ali or ards, the gly ile variant form of the receptor is associated with an increased risk of severe rsv bronchiolitis and increased risk for hospitalization for rsv in previously healthy infants suggesting a potential role for tlr in infl uencing the severity of lung injury. signal transduction pathways activated after stimulation of a variety of immune receptors including the tlrs and members of the family of il- and tnf receptors result in the upregulation of specifi c genes involved in the innate and adaptive immune responses. several transcription factors are involved in this process and genetic variation in any of these factors may infl uence the level of transcription. one such factor is nuclear factor k b (nf-k b) which under non-stimulated conditions is inhibited by the cytoplasmic inhibitor nf k bia . upon activation of cytokine-mediated signal transduction pathways, nf k bia is degraded allowing nf-k b to translocate to the nucleus. a number of polymorphisms located in the macrophage migration inhibitory factor (mif) plays a central role in regulating the infl ammatory response by directly increasing tnfa and il- and countering the anti-infl ammatory actions of glucocorticoids. haplotypes composed of polymorphisms in the ¢ end of the gene coding for mif may be associated with the development of ali. promoter region of the gene coding for nf k bia have been described but their functional consequence is unknown. when individual nf k bia promoter polymorphisms were examined to determine if they are associated with the development of ali, none by themselves demonstrated an association. however, the haplotype of − g/− t/− c was found in a higher frequency in adults who developed ards especially in males with direct lung injury. another transcription factor is nf-e related factor (nrf ) which, under nonstressed conditions, is located in the cytoplasm. under oxidative stress nrf translocates to the nucleus and results in transcription of several anti-oxidant enzymes. several polymorphisms within the promoter region of the gene coding for nrf have been identifi ed that reduce transcription. one such variant, − a allele, is associated with the development of ali in adult trauma patients. the role of angiotensin converting enzyme ( ace) in lung injury has recently attracted uch interest. ace is present in pulmonary endothelium and is responsible for converting ati to atii. ace levels are elevated in the bronchoalveolar lavage fl uid of adults with ards and higher levels are associated with mortality from ards. the key component is most likely atii, which has apoptotic effects on alveolar epithelial and endothelial cells in vitro . atii receptor antagonists block pneumocyte apoptosis in a model of meconium aspiration. another important component of this system is ace , a homologue of ace expressed in human lungs, which is a negative regulator of the renin-angiotensin system as well as the probable receptor for the sars virus in humans. lung injury models using knockout mice lacking the ace gene have higher atii levels and exaggerated lung injury compared to wild type mice. however, the lung injury is reversed if the ace gene is inactivated in the ace knockout mice. this suggests that ace induces lung injury through atii and that ace protects against lung injury. indeed, ace inhibitors and atii antagonists appear to decrease the severity of lung injury in animal models, the risk of aspiration pneumonia in some adult populations, and reduce the -day mortality in adults with pneumonia. several studies have demonstrated the d/d genotype appears associated with the susceptibility to and outcome from ards. as discussed previously, expression microarrays have been invaluable in identifying other potential mediators involved in the pathophysiology of lung injury. the expression of pre-b cell colony enhancing factor (pbef) was found to be signifi cantly elevated in both animal and human studies of ali using this approach. pbef is a lesser studied cytokine involved in the maturation of b-cells, inhibition of neutrophil apoptosis, and perhaps regulation of the endothelial cell calcium-dependent cytoskeletal arrangement. two genetic polymorphisms have been identifi ed in the promoter region, − t/g and − c/t which appear to infl uence the development of ali. carriers of the g allele at position − had a . -fold increased risk of ali and the g allele remained an independent risk factor after controlling for several other variables. the t allele at position − was found at a lower frequency in adults with ali. combining these two polymorphisms in a haplotype analysis demonstrated that adults with the − g/ − c haplotype had a higher risk of ali ( . fold). the consequence of these two polymorphisms remains to be elucidated though the − t allele may result in reduced expression. one fi nal gene to be discussed in this section is the myosin light chain kinase (mlck) gene. three isoforms of the protein exist, and one isoform is a key component in the cytoskeletal arrangement regulating vascular permeability, angiogenesis, endothelial cell apoptosis, and leukocyte diapedesis. several polymorphisms in the gene coding for mlck have recently been identifi ed. analysis was performed not only on the infl uence of single polymorphisms on the development of ali but also a number of haplotypes using a sliding window approach. several strong associations between various single nucleotide polymorphisms as well as haplotypes and the risk of ali and sepsis were identifi ed in adults. this included one haplotype, ggt, composed of markers mylk_ , mylk_ , and mylk_ spanning a region of base pairs between the ¢ untranslated region and the fi rst exon that appeared to be specifi cally associated with the risk of ali and not sepsis. the functional signifi cance of these haplotypes remains to be determined. specifi c variants in the mylk gene were also shown to be associated with trauma-induced ali, however association of specifi c genetic variants and lung injury were not observed in children or adults with pneumonia. two other areas should be mentioned in regards to the infl uence of genetic variations in the picu. the fi rst is in the area of coagulation. several examples of genetic polymorphisms in genes coding for proteins involved in coagulation and fi brinolysis were discussed above in relation to sepsis and ali. however, these and many other genetic variations that exist in other components of the coagulation cascade could also infl uence the development of thrombosis including deep venous thrombosis in critically ill patients. thrombosis of central venous catheters is a recurring problem in picus and while certain environmental factors play a role (eg, length of time catheter is in place, size of the patient and vessel), genetic polymorphisms in the patient favoring the formation of thrombi may also play a role. finally, the action of every drug that is used in the picu can potentially be infl uenced by genetic variation in the patient. whether it be inhaled b -agonists, or the array of intravenous vasoactive agents, sedatives, muscle relaxants, antibiotics, steroids, etc.; all bind to protein receptors and either activate or block specifi c signal transduction pathways, many bind protein carriers or transporters, and most are metabolized by various protein enzymes. every gene coding for each of these proteins has multiple genetic variations with the potential to infl uence the levels or activities of these proteins. the area of pharmacogenomics attempts to determine the infl uence of genetic variations in genes affecting these various aspects of drug action. however, while the list of genetic polymorphisms in genes affecting drug action is growing rapidly, there are few clinical examples of the degree of infl uence that these genetic variations have on the response to drugs in the picu. for example, warfarin is the most widely used oral anticoagulant for long-term prophylaxis and treatment of thromboembolic disorders and is used in many children and adults with mechanical valves. the metabolism of and response to warfarin involves several enzymes, two of which exhibit genetic variations that dramatically alter the levels of warfarin. for one of these genes, cyp c , the common allele is referred to as cyp c * and is consider the wildtype while cyp c * contains a c to t nucleotide change at position in exon and cyp c * contains an a to c nucleotide change in exon . cyp c * has approximately % of the metabolic activity of the wild type cyp c * while cyp c * contains only % of the wildtype activity. by also using genetic variation in a second gene, vitamin k epoxide reductase complex subunit or vkorc , one can account for more than % of the observed dosing variability. current practice in the use of warfarin usually involves starting at an age and weight specifi c dose and monitoring coagulation studies. however, because of the genetic variations in these two enzymes and perhaps others, different patients take different amounts of time to achieve the appropriate therapeutic dose. knowing the specifi c genotypes of patients prior to initiating warfarin may allow for more appropriate dose selection, less time to achieve therapeutic levels, and less risk of adverse events. recently, an algorithm using the patient's genotypes at these two sites has been developed that allows for more accurate dosing in some populations. although these algorithms are being developed and tested it should be kept in mind that they do not account for drug-drug interactions. the era of the study of the genetic impact on critical illness in children is present. clinicians must be prepared to deal with the growing body of literature related to genetic infl uence on critical disease development, treatment and outcome, and be able to critically review the literature in order to determine the impact on the patients they are caring for daily. for the results of these representative genetic association studies to take the leap into clinically impacting care, they must meet certain criteria. first and foremost, the phenotype must be well defi ned; that is, the enrolling patients with ali/ards or sepsis must meet strict and well accepted criteria. second, they must be high quality studies, utilizing highly sensitive and specifi c methods for genotyping. third, the studies must use a large sample size to assure that no type i or type ii errors are made based simply upon the number of individuals genetic polymorphisms in genes coding for proteins involved in coagulation and fi brinolysis may be very important in the risk of bleeding and thrombosis in critically ill children. the area of pharmacogenomics attempts to determine the infl uence of genetic variations in genes affecting the various aspects of drug action. ace i/d but not agt (− )a/g polymorphism is a risk factor for mortality in ards g g genotype of the plasminogen activator inhibitor- promoter polymorphism associates with disseminated intravascular coagulation in children with systemic meningococcemia mechanisms and regulation of the gene-expression response to sepsis comparison of two polymorphisms of the interleukin- gene family: interleukin- receptor antagonist polymorphism contributes to susceptibility to severe sepsis recent advances in genetic predisposition to clinical acute lung injury novel polymorphisms in the myosin light chain kinase gene confer risk for acute lung injury macrophage migration inhibitory factor in acute lung injury: expression, biomarker, and associations polymorphisms in the mannose binding lectin- gene and acute respiratory distress syndrome severity of meningococcal disease in children and the angiotensin-converting enzyme insertion/ deletion polymorphism polymorphisms of human sp-a, sp-b, and sp-d genes: association of sp-b thr ile with ards deletions within a ca-repeat-rich region of intron of the human sp-b gene affect mrna splicing lipopolysaccharide hyporesponsiveness as a risk factor for intensive care unit hospitalization in infants with respiratory syncitial virus bronchiolitis fibrinogen-beta gene haplotype is associated with mortality in sepsis sepsis syndrome and death in trauma patients are associated with variation in the gene encoding for tumor necrosis factor association of tnf , a tnf-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study variation in the tumor necrosis factor-alpha gene promoter region may be associated with death from meningococcal disease association between surfactant protein b + polymorphism and the risk of respiratory failure in adults with community-acquired pneumonia genome-level longitudinal expression of signaling pathways and gene networks in pediatric septic shock microarray analysis of regional cellular responses to local mechanical stress in acute lung injury genetic and environmental infl uences on premature death in adult adoptees the national human genome research institute's talking glossary of genetic terms irak functional genetic variant affects severity of septic shock genome-level expression profi les in pediatric septic shock indicate a role for altered zinc homeostasis in poor outcome pre-b-cell colony-enhancing factor as a potential novel biomarker in acute lung injury disease phenotype, preferably by a different group of investigators. finally, the impact of the genetic variant on the protein product must possess biologic plausibility as impacting the development or the outcome of the disease of study. only after all of these criteria are met should clinicians be comfortable moving to the arena of tailoring therapy based on genetic variations. . you are caring for two brothers with acute lung injury secondary to smoke inhalation from an apartment fi re. these two infants were apparently sleeping in the same crib when they were rescued simultaneously by fi re fi ghters. the fi rst infant was extubated within h of intubation and is doing well with a minimal oxygen requirement. the second has experienced a much more severe lung insult and remains intubated on high frequency oscillatory ventilation. in attempting to understand the difference in their clinical response to the seemingly identical insult, you suspect that it may be related to a polymorphism in one of the genes that codes for surfactant protein b. in considering this possibility, which of the following is true? a. although plausible, it is unlikely to be associated with a polymorphism because polymorphisms are rare occurring in less than one percent of the population. b. it is not plausible because variances in the translation of such a complex protein require differences in an entire haplotype, and not simply a single nucleotide polymorphism. c. it is plausible because genetic polymorphisms may infl uence the quantity of mrna transcribed and/or the functional activity of the surfactant protein b. d. it is unlikely as there are no reports of associations between surfactant protein gene polymorphisms and outcomes from pulmonary disease. e. it is unlikely because dysfunctional surfactant protein b demonstrates an x-linked pattern of inheritance. key: cord- - iwsx authors: gupta, kushagra; hergrueter, anja; owen, caroline a title: adipose-derived stem cells weigh in as novel therapeutics for acute lung injury date: - - journal: stem cell res ther doi: . /scrt sha: doc_id: cord_uid: iwsx acute lung injury is characterized by intense neutrophilic lung inflammation and increased alveolar-capillary barrier permeability leading to severe hypoxemia, and is associated with high mortality despite improvements in supportive care. there is an urgent need for effective therapies for acute lung injury. zhang and colleagues tested the efficacy of adipose-derived stem cells in acute lung injury in mice. when adipose-derived stem cells were delivered to mice that had been challenged with lipopolysaccharide, they potently limited acute lung inflammation and injury in the mice, indicating that adipose-derived stem cells have therapeutic potential in acute lung injury in humans. herein, we discuss the advantages and potential limitations of using adipose-derived stem cells as therapeutics for human acute lung injury. mscs include bone marrow-derived stem cells (bmscs), mscs in umbilical cord blood, and adipose-derived stem cells (ascs). in the fi eld of ali/ards cell-based therapeutics, most attention has focused on bmscs, which have therapeutic effi cacy in rodent and human tissue models of ali and sepsis [ , ] . however, a number of barriers that may limit the clinical usefulness of bmscs in human ali/ards have been identifi ed [ , ] ( table ) . like bmscs, ascs have anti-infl ammatory and immuno-suppressive activities. ascs inhibit immune cell activation and proliferation by inducing cell-to-cell contact and signaling and releasing mediators that limit tissue injury [ ] . ascs have greater anti-infl ammatory potential than bmscs because they secrete higher levels of bioactive mediators [ ] . th is and other properties of ascs (table ) make them an attractive alternative to bmscs as cell-based therapeutics for human ali/ards. consistent with this concept, recent studies have shown that transplantation of autologous ascs attenuates ischemia-reperfusion lung injury in rodents [ , ] . th e paper by zhang and colleagues [ ] builds upon this literature by examining whether ascs have effi cacy in a model of direct ali in mice. zhang and coworkers challenged mice with bacterial lipopolysaccharide (lps) by oropharyngeal route, and h later ascs isolated from syngeneic mice (mascs) or humans (hascs) were delivered to the mice by the same route, and ali severity was assessed and h later. th e lps-challenged and masc-or hasc-treated mice lost less body weight, and had decreased alveolar-capillary barrier injury as assessed by broncho-alveolar lavage fl uid albumin levels, and reduced alveolar septal thickening and exudates when compared with lps-challenged mice not treated with ascs. treatment of the lps-challenged mice with hascs and mascs also reduced polymorphonuclear neutro phil infl ux into the lungs, and suppressed lung levels of pro-infl ammatory mediators. murine ascs increased lung levels of anti-infl ammatory interleukin- in lps-challenged mice. a strength of the paper is its novel focus on ascs as a therapy for ali, and its approach to test ascs therapeutically (rather than prophylactically) in an ali model abstract acute lung injury is characterized by intense neutrophilic lung infl ammation and increased alveolar-capillary barrier permeability leading to severe hypoxemia, and is associated with high mortality despite improvements in supportive care. there is an urgent need for eff ective therapies for acute lung injury. zhang and colleagues tested the effi cacy of adipose-derived stem cells in acute lung injury in mice. when adipose-derived stem cells were delivered to mice that had been challenged with lipopolysaccharide, they potently limited acute lung infl ammation and injury in the mice, indicating that adipose-derived stem cells have therapeutic potential in acute lung injury in humans. herein, we discuss the advantages and potential limitations of using adiposederived stem cells as therapeutics for human acute lung injury. that induces robust neutrophilic lung infl ammation. additionally, the authors compared the therapeutic effi cacy of human versus murine ascs. while both hascs and mascs attenuated ali, it is noteworthy that for most ali parameters examined, mascs were more potent than hascs. however, it is possible that hascs have greater potency at restraining acute lung infl ammation in human subjects. th e study of zhang and colleagues has several limitations that need to be addressed before ascs can be advanced to human clinical trials. first, no single ali animal model can completely reproduce all the pathologic features of human ali/ards. while the lps model studied by zhang and coworkers induces robust neutrophilic lung infl ammation, it causes only modest alveolar-capillary barrier injury, which is a hallmark of ali/ards. th us, it will be important to test the effi cacy of ascs in ali models associated with severe ali (such as hyperoxia and acid-induced ali) and to assess the eff ects of ascs on physiologic readouts of ali, including lung compliance and hypoxemia. second, ali/ards in humans is often initiated by bacterial infections, but the model chosen (lps-mediated ali) causes sterile lung infl ammation. given that ascs suppress immune responses, it will be important to assess their eff ects on host responses to pathogens that can cause ali/ards. th ird, the mechanisms by which ascs produce their benefi cial eff ects in this model were not addressed. fourth, given that ards patients are often not treated within the fi rst hours of illness (the single time-point when ascs were delivered to mice in this study), future studies should determine how late in the disease course ascs can be delivered and still induce a protective eff ect and for how long this protective eff ect is sustained. moreover, the long-term safety of delivering ascs was also not assessed in this study. it is noteworthy in this respect that hascs promote the growth of tumor cells [ ] . th erefore, before ascs can be used to treat human subjects, it will be necessary to investigate their longterm safety in animals. th e study by zhang and colleagues provides evidence that ascs have potential as novel cell-based therapeutics for ali/ards. th e next logical step towards advancing ascs into human clinical trials for ali/ards will be to further test ascs for their effi cacy and safety in additional small and large animal models of ali, including models more clinically relevant than the lpsmediated ali model studied by zhang and colleagues. isolation of cells safe, relatively non-invasive and inexpensive procedure. adipose tissue is abundant, expendable, easily accessible, and can be a waste product of many therapeutic and cosmetic procedures the acute respiratory distress syndrome network: ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome immunomodulatory activity of mesenchymal stem cells intrapulmonary delivery of bone marrow-derived mesenchymal stem cells improves survival and attenuates endotoxin-induced acute lung injury in mice bone marrow stromal cells attenuate sepsis via prostaglandin e( )-dependent reprogramming of host macrophages to increase their interleukin- production comparison of immunomodulatory properties of mesenchymal stem cells derived from adult human tissues hepatitis b virus infection and replication in human bone marrow mesenchymal stem cells current status of human adiposederived stem cells: diff erentiation into hepatocyte-like cells ifats collection: in vivo therapeutic potential of human adipose tissue mesenchymal stem cells after transplantation into mice with liver injury autologous transplantation of adipose-derived mesenchymal stem cells markedly reduced acute ischemia-reperfusion lung injury in a rodent model melatonin treatment improves adipose-derived mesenchymal stem cell therapy for acute lung ischemiareperfusion injury comparison of the therapeutic eff ects of human and mouse adipose-derived stem cells in a murine model of lipopolysaccharide-induced acute lung injury adipose tissue-derived stem cells promote prostate tumor growth this work was supported by phs grants #hl and hl , and po hl , and the brigham and women's hospital-lovelace respiratory research institute consortium. these funding bodies had no role in the design or the writing of this manuscript or in the decision to submit the manuscript for publication. key: cord- - ijxilpb authors: xu, g.l.; yao, l.; rao, s.y.; gong, z.n.; zhang, s.q.; yu, s.q. title: attenuation of acute lung injury in mice by oxymatrine is associated with inhibition of phosphorylated p mitogen-activated protein kinase date: - - journal: j ethnopharmacol doi: . /j.jep. . . sha: doc_id: cord_uid: ijxilpb oxymatrine is one of the alkaloids extracted from chinese herb sophora japonica (sophora flavescens ait.) with activities of anti-inflammation, inhibiting immune reaction, antivirus, protecting hepatocytes and antihepatic fibrosis. however, the effect of oxymatrine on acute lung injury (ali) has not been known yet. in this study, the effect of oxymatrine on ali was investigated using an oleic acid-induced ali mouse model. morphological findings showed that the oleic acid group demonstrated a marked lung injury represented by prominent atelectasis, intraalveolar and interstitial patchy hemorrhage, edema, thickened alveolar septum, formation of hyaline membranes and the existence of inflammatory cells in alveolar spaces. while in the oxymatrine/dexamethasone group, these changes were less severe and in the vicinity of the control group. furthermore, pretreatment with oxymatrine significantly alleviated oleic acid-induced lung injury accompanied by reduction of lung index and wet-to-dry weight ratio, decreases in serum tnf-α level and inhibition of phosphorylated p mapk. these findings suggest that oxymatrine has a beneficial effect on acute lung injury induced by oleic acid in mice and may inhibit the production of proinflammatory cytokine, tnf-α, by means of the inhibition of p mapk. acute lung injury (ali) is characterized by an intense pulmonary inflammatory response, with neutrophil accumulation, interstitial edema, disruption of epithelial integrity and leakage of protein into the alveolar space (john et al., ) . acute respiratory distress syndrome (ards) is characterized by increased capillary endothelial permeability that leads to segmental accumulation of high protein content in interstitial edema. fibrin and platelet plugs occlude the microvasculature of the lung and neutrophil sequestration and activation in the interstitium leads to further segmental alveolar damage and flooding. these processes result in decreased pulmonary compliance, compromised gas exchange and extensive intra-pulmonary shunting with concomitant ventilation-perfusion mismatching. at present, ards is often classified as a continuum of injury ranging from the milder form 'acute lung injury (ali)' to the more severe form 'ards' (christopher et al., ) . ali/ards is associated with the development of multiple organ dysfunction syndrome (mods), which plays an important role in the death of patients with sepsis, pneumonia, aspiration of gastric contents, trauma, multiple transfusions and ischemia reperfusion. severe acute respiratory syndrome (sars) is a novel global infectious disease induced by a virus from the family coronaviridae. clinical investigation shows that pathological changes in sars patients are similar to those of acute lung injury, as revealed by alveolar cell collapse, severe exudation, acute inflammatory reaction and hyaline membrane formation (zhong, ; du et al., ) . currently, no corrective therapy is available for the it is generally accepted that the development of mods follows the gradual route of ali-ards-mods. during this pathway, the lung behaves as a central organ and is the first target subject to injury. because pulmonary dysfunction may lead to severe hypoxemia and further other multiple organ dysfunction or failure, the treatment of the above mentioned diseases should concentrate on the development of ali and take measures as soon as possible . the research and development of anti-sars drugs should also comply with this law. potential new therapeutic strategies for sars have been shown to include a wide search for drugs, which is conducive to reduction of lung injury and management of symptoms (zhong, ; du et al., ) . as a traditional chinese medicine, sophora japonica has been used for treatment of many diseases for thousands of years. matrine-like alkaloids have been found to be the chief active components of chinese herb sophora japonica including matrine, oxymatrine, sophocarpine, sophoramine, sophoridine et al. the content of oxymatrine in the composite of sophora japonica is up to . % (qi et al., ) . basic and clinical researches have shown that oxymatrine has the following pharmacological effects: anti-inflammation, inhibiting immune reaction, antivirus, protecting hepatocytes and antihepatic fibrosis (liu et al., (liu et al., , liu and chiou, ; chen et al., ; dong et al., ; xiang et al. ; yang et al., ) . chinese bureau of science and technology had announced during the outbreak of sars that the composite sophora japonica injection has distinct effects in the treatment of sars (zhong, ) . considering the pharmacological effects of oxymatrine, we speculate that oxymatrine may play a central role in the composite injection although the effect of oxymatrine on sars has not been reported to date. in order to test this hypothesis and because the pulmonary pathological changes in sars are similar to those of acute lung injury, the effect of oxymatrine on acute lung injury was investigated using an ali mouse model in this study. we also studied whether the p mapk intracellular signal pathway was involved in the development of ali and discussed whether oxymatrine could become a therapeutic candidate drug for ali, ards and sars. oxymatrine was obtained from yixin pharmaceutical co. ltd. (zhejiang, china) with hplc purity > %. oleic acid was purchased from linfeng chemical co. ltd. (shanghai, china). albumin was obtained from sigma. rabbit polyclonal antibodies for phosphorylated and nonphosphorylated p mapk were provided by cell signaling technology. tween and glycine were purchased from amresco co. polyvinylidene difluoride (pvdf) transfer membranes for western blotting were obtained from roche molecular biochemicals (quebec, canada). protein assay dye reagent was purchased from jiancheng bioengineering co. (nanjing, china) . hrp conjugate goat anti-rabbit igg and pipa lysis buffer were purchased from shengnengbocai biotech inc. (shanghai, china) . mouse tnf-␣ elisa assay kit was obtained from jingmei biotech co. (guangdong, china). all other reagents were of the highest grade available commercially. male kunming strain mice weighing - g were obtained from laboratory animal center, school of medicine, southeast university (nanjing , china). the mice were housed in climate-controlled quarters ( - • c at % humidity) with a h light/dark cycle and free access to food and water. all experiments were conducted according to the "guide for the care and use of laboratory animals", published by the national institutes of health (nih publication - , revised ). mice were randomly assigned into six groups. the oleic acid group received . ml/kg, i.v. of oleic acid (mixed with . % bovine serum albuman). the control group received . ml/kg, i.v. of saline. the three oleic acid + oxymatrine groups were treated with oxymatrine for three consecutive days before oleic acid injection (oxymatrine; . , and mg/kg, i.p.) (xiang et al., ) . the dexamethasone group received intraperitoneal injection of dexamethasone at dose of . mg/kg h before oleic acid injection. the oleic acid and control groups were treated with an equivalent volume of saline instead of oxymatrine. mice were sacrificed h after oleic acid injection. immediately after each animal had been sacrificed, the thorax was opened and the lung was removed en bloc by observers unaware of the nature of the experiment. the left lower lobe was excised and fixed in % buffered formalin. the lungs were embedded in paraffin, and the sections stained with hematoxylin and eosin were examined by light microscopy for evidence of lung injury, as described in the following: alveolar congestion, hemorrhage, edema, infiltration/aggregation of neutrophils in the airspace or vessel wall, thickness of the alveolar wall and hyaline membrane formation. electron microscopy of lungs was carried out on samples fixed in phosphate buffer (ph . ) containing . % glutaraldehyde and post-fixed in osmium tetraoxide. transmission electron micrographs were produced with a hitachi h- electron microscope. the wet weight of the whole lung was weighed on an automatic electric balance and the lung index was calculated according to the following formula: lung index (%) = the wet weight of the whole lung/body weight × %. subsequently, the right lung was excised and weighed to obtain the 'wet' weight. the lung was then dried in an oven at • c for days to obtain the 'dry' weight. to assess tissue edema, the ratio of wet weight to dry weight (w/d ratio) was calculated. the left lung was cut into pieces and subjected to histological examination and electron microscopy observation. blood samples were harvested from the eyes before mice were killed. the blood was allowed to clot for approximately h at room temperature and then centrifuged at × g for min to obtain the serum. the serum was stored at − • c until assayed. the tnf-␣ was quantitated according to the instructions available in elisa kits. the levels in mice samples were calculated from a standard curve generated from recombinant mice tnf-␣. the detection range of this assay for tnf-␣ is - ng/l. samples with a concentration exceeding the limits of the standard curve were repeated after dilution. three mice in each group were adopted min after oleic acid injection. the lungs were frozen in liquid nitrogen for measurements immediately after they were removed. before the assay, the specimens were cleared of fat and debris. all specimens had a wet weight of mg. phenylmethylsulfonylfluoride (pmsf; mmol) was added just before use. the samples were homogenized in l of pipa lysis buffer using a microhomogenizer on ice. all debris and nuclei were removed by centrifugation at × g at • c for min. the supernatant obtained was used as the whole cell lysate. protein concentrations were determined with bovine serum albumin as the reference standard using protein assay dye reagent. an g of proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (sds-page). the membranes were first incubated with % nonfat milk in tris-buffered saline (tbs). after washing three times in . % tween -tbs (tbst), the membranes were incubated with p mapk and phosphorylated p mapk antibodies separately ( : ) in tbst over night at • c, and then washed three times in tbst. they were incubated with horseradish peroxidaselinked goat anti-rabbit igg ( : ) for h at room temperature and detected with the tmb substrate for horseradish peroxidase. data from experiments are expressed as mean ± s.e.m. and statistically analyzed using the student's unpaired t-test. a value of p < . was considered significant. light microscopic findings in the lung at h after oleic acid injection demonstrated a marked lung injury resembling those seen in lung of patients with ali/ards, represented by prominent atelectasis, intraalveolar and interstitial patchy hemorrhage, edema, thickened alveolar septum, formation of hyaline membranes and the existence of inflammatory cells in alveolar spaces (fig. a) , which were not observed in the control group (fig. b) . in oleic acid + oxymatrine groups, these changes were less severe than in the oleic acid group and were in the vicinity of the control group (fig. c ). in the oleic acid + dexamethasone group, these changes were not pronounced and were close to the high dose oxymatrinetreated group (fig. d) . as shown in fig. , electron microscopic findings in the lung after oleic acid injection included epithelial cell swelling (endoplasmic reticulum dilation and mitochondria swelling), the existence of blood cells and inflammatory cells in alveolar spaces (fig. b-d) , which were not observed in the control group ( fig. a) . these changes were improved or not evident in oleic acid + oxymatrine/dexamethasone groups ( fig. e and f). values of the lung index and wet-to-dry lung weight ratio (w/d) in various groups of experimental animals were shown in fig. . oleic acid injection resulted in an increase in the lung index and w/d ratio of the lung, as compared to the control (p < . , p < . ). both were attenuated by mg/kg (p < . , p < . ) and mg/kg (p < . , p < . ) oxymatrine treatment. in contrast, . mg/kg oxymatrine did not diminish the above two values significantly although they were lower than the oleic acid group. the lowest level was found in animals receiving dexamethasone treatment. as tnf-␣ plays a pivotal role in mediating oleic acidinduced ali, we also assessed the regulation of tnf-␣ production by oxymatrine (fig. ) . the serum tnf-␣ level in the oleic acid group was significantly higher than that in the control group (p < . ). treatment with oxymatrine inhib- ited the increase in a dose dependent manner and the level at the highest dose reverted to the control level. serum level of tnf-␣ in the dexamethasone group was evidently lower than that in the oleic acid group and similar to the control group. this suggested that tnf-␣ increases induced by oleic acid could be suppressed by oxymatrine. as shown in fig. , the level of p phosphorylation in the oleic acid group was significantly higher than that in the control group. pretreatment with oxymatrine inhibited phosphorylation of p in a dose dependent manner. p phosphorylation in the highest oxymatrine group was similar to that of the control group. in contrast, the total p protein remained unchanged. xiang and colleagues used oxyimatrine to evaluate effect of oxymatrine on fulminant hepatitis and hepatocyte apoptosis in mouse models and oxymatrine was administered at dose of mg/kg intraperitoneally (i.d. × days) (xiang et al., ) . initially, we administered oxymatrine at the dose of mg/kg intraperitoneally twice a day for three consecutive days, resulting in a protective effect that could be easily monitored by macroscopic and/or microscopic observation. then we used, oxymatrine at doses of , . and mg/kg to investigate the relationship between dose and effect. results showed that no protective effects were observed at . mg/kg or below. so , and . mg/kg were ultimately selected as the test dosage. our results showed that a series of pathological changes were observed under light and electron microscopy after an intravenous administration of oleic acid in mice, which mimicked the pathological changes of clinical ali/ards satisfactorily. furthermore, the lung index and wet/dry weight ratio were greater in ali mice than in control group. these findings are in agreement with other reports (kenji et al., ) , demonstrating that the oleic acid-induced ali mouse model is reproducible and our ali model is successful. the underlying mechanism of ali induced by oleic acid is associated with cytokines releases such as tnf-␣, which stimulates monocytes to produce il- .as the core of the cytokine-network, tnf-␣ and il- play important roles not only in the production of other inflammatory cytokines, but also in the migration and adherence of neutrophils to endothelial cells (yoshimi et al., ) , which together with endothelial cell injury by cytokines result in the production of superoxide radicals and subsequently injure alveolar epithelial cells. all these cause ultimate pulmonary dysfunction. in the present study, our data revealed that serum tnf-␣ level was higher in oleic acid group than that in control group. oxymatrine evidently decreased serum tnf-␣ level, lung index as well as wet-to-dry ratio and reduced pulmonary injury induced by oleic acid. these findings not only corroborate the direct relationship between tnf-␣ and ali but also suggest that oxymatrine have a protective effect on oleic acid-induced ali. fig. . ultrastructure of the lung tissue in mice without or with oleic acid injection ( ×). representative photomicrographs showing: epithelial cell swelling (endoplasmic reticulum dilation and mitochondria swelling) (b), the existence of red blood cells (c) and inflammatory cells (d) in alveolar spaces were observed in oleic acid group. these changes were not observed in the control group (a) and improved or not evident in oleic acid + oxymatrine/dexamethasone groups (e, f): a, normal epithelial cell; b, swollen epithelial cell; c, red blood cell in alveolar spaces; d, inflammatory cell in alveolar spaces. fig. . effect of oxymatrine on lung index and wet-to-dry weight ratio (w/d) in mice with lung injury induced by oleic acid. the mice were given oxymatrine ( . , and mg/kg, i.p.) for three consecutive days and dexamethasone ( mg/kg, i.p.) h before injection of oleic acid. the control and oleic acid groups received normal saline. the mice were then sacrificed h after oleic acid administration and lung index, wet-to-dry weight ratio (w/d) were calculated. mean ± s.e.m., n = ; ** p < . when compared with control; # p < . and ## p < . when compared with oleic acid group. fig. . effect of oxymatrine on serum tnf-␣ level in mice with lung injury induced by oleic acid. the mice were given oxymatrine ( . , and mg/kg, i.p.) for three consecutive days and dexamethasone ( mg/kg, i.p.) h before injection of oleic acid. the control and oleic acid groups received normal saline. the mice were then sacrificed h after oleic acid administration and serum tnf-␣ level were determined by elisa assay. mean ± s.e.m., n = ; ** p < . when compared with control; ## p < . when compared with oleic acid group. bands of p map kinase were identified in the five groups, and mean density levels in the five groups were almost the same. phosphorylated p map kinase was augmented in the oleic acid group compared with the control group, whereas expression of phosphorylated p map kinase was markedly attenuated in the oxymatrine group compared with the oleic acid group. it has been reported that oxymatrine concentration is higher in lung and heart than in other organs. this signifies the anti-ali effect of oxymatrine has a pharmcokinetic basis (wang and wang, ) . additionally, dexamethasone as positive control exerted a significant preventive effect on oleic acid-induced ali injury. this might be explained by its potent anti-inflammation effect described as follows: diminution of alveolocapillary permeability; reduction of alveolar epithelial response to pathogen; stability of cell and lysosome membrane; enhancement of surfactant release; prevention of microthrombogenesis and blockade of neutrophil activation (wang et al., ; su et al., ) . under the stimuli of different ali/ards pathogens, a wide and complicated signal transduction process occurs in many different cells. although the detailed mechanism is still unknown, the p mitogen-activated protein kinase (mapk) has been paid special attention. p mapk is a cytokinesuppressive anti-inflammatory drug target first discovered by lee et al. ( ) . most reports demonstrate that p mapk is responsible for regulating inflammatory responses (nick et al., (nick et al., , fang et al., ) . on the other hand, arcaroli et al. ( ) reported that p did not have a central role in the development of ali after either hemorrhage or endotoxemia. thus, the role of p mapk in the development of ali is still uncertained. as a result, p mapk has not been proposed as a valid target for clinical management of ali/ards. in order to examine the role of p mapk in ali, we then analyzed the expression of p mapk and evaluated the effects of oxymatrine. the expression of p map kinase was shown using western blotting analysis. results showed that phosphorylated p map kinase was augmented in the oleic acid group compared with the control group, whereas expression of phosphorylated p map kinase was attenuated in the oxymatrine group compared with the oleic acid group in a concentration-dependent manner. this suggested that the administration of oxymatrine before ali markedly attenuate the activation of p map kinase during lung injury. it has been reported that tnf-␣ is augmented via activation of the p map kinase-related intracellular signal pathway (johnson et al., ; lee et al., ) . oxymatrine markedly attenuated the phosphorylation of p map kinase and disturbed the mechanism for the production of tnf-␣, thus resulting in the lower serum tnf-␣ level. from these findings, we conclude that oxymatrine ameliorates ali by attenuating the production of proinflammatory cytokines, and that this attenuation is associated with suppression of p map kinase activation. in addition, our results confirm that p map kinase does play an important role in the development of oleic acid-induced ali. based on our results and previous reports, we expect that p map kinase may become a promising target for clinical management of ali, although it needs to be supported by further animal experiments and clinical data. reports have shown that there are at least three types of map kinase: extracellular signal-regulated protein kinase (erk / ; p /p ); c-jun n-terminal protein kinase (jnk); p map kinase (davis, ; kyriakis et al., ; bogoyevitch et al., ; han et al., ; raingeaud et al., ; xia et al., ) . the nf-b pathway is also reported to be involved in the process of ali. together with the three types of mapk, nf-b may create a network to regulate inflammatory responses in ali. in this study, we only studied the effect of oxymatrine on p mapk. further study is needed to elucidate whether oxymatrine has modulative effects on other pathways. in china, pure oxymatrine injection has been available in hospital for treatment of hepatitis and tumor for many years. however, it has not been used for ali/ards/sars in clinic. since our results indicate that oxymatrine prevents mice from oleic acid-induced ali, we hope that oxymatrine can be used to treat ali/ards/sars although further research should be carried out on more animal experiments before clinical trials. in this paper, we found oxymatrine had a beneficial effect on ali in mice for the first time. although details of the mechanism of oxymatrine remain to be unraveled, the present results suggest that oxymatrine improves acute lung injury by attenuating the production of tnf-␣, and that this attenuation is associated with suppression of p map kinase activation. role of p map kinase in the development of acute lung injury cellular stresses differentially activated c-jun n-terminal protein kinase and extracellular signal-regulated protein kinase in cultured ventricular myocytes the inhibitory effect of oxymatrine on hepatitis c virus in vitro continuous dose furosemide as a therapeutic approach to acute respiratory distress syndrome (ards) the mitogen-activated protein kinase signal transduction pathway effects of oxymatrine on the serum levels of t helper cell and cytokines and the expression of the s gene in hepatitis b virus s gene transgenic mice: a study on the anti-hepatitis b virus mechanism of oxymatrine targets and studies on anti-sars drugs effect of p mapk on mechanical ventilation-induced lung injury a map kinase targeted by endotoxin and hyperosmolarity in mammalian cells role of p map kinase in the development of acute lung injury human recombinant tumor necrosis factor alpha infusion mimics endotoxemia in awake sheep effect of methylprednisolone on phospholipase a activity and lung surfactant degradation in acute lung injury in rabbits the stress activated protein kinase subfamily of c-jun kinase a protein kinase involved in the regulation of inflammatory cytokine biosynthesis discussion on acute lung injury and acute respiratory distress syndrome the effect of chinese hepatoprotective medicines on experimental liver injury in mice medicinal herbs for hepatitis c virus infection: a cochrane hepatobiliary systematic review of randomized trials effects of chinese herbal products on mammalian retinal functions selective suppression of neutrophil accumulation in ongoing pulmonary inflammation by systemic inhibition of p mitogenactivated protein kinase role of p mitogen-activated protein kinase in a murine model of pulmonary inflammation progress in studies of matrine and oxymatrine pro-inflammatory cytokines and environmental stress cause p mitogen-activated protein kinase activation by dual phosphorylation on tyrosine and threonine inhibition of inflammatory responses by ambroxol, a mucolytic agent, in a murine model of acute lung injury induced by lipopolysaccharide pharmacological effects and clinical application of matrine and oxymatrine effect of dexamethasone on humoral immunity in mice of acute lung injury induced by oleic acid effect of oxymatrine on murine fulminant hepatitis and hepatocyte apoptosis opposing effects of erk and jnk-p map kinase on apoptosis prophylactic and therapeutic effect of oxymatrine on d-galactosamine-induced rat liver fibrosis the effect of fr on pulmonary ischemia-reperfusion injury in rats consensus for the management of severe acute respiratory syndrome key: cord- -sg n hs authors: suri, h. s.; li, g.; gajic, o. title: epidemiology of acute respiratory failure and mechanical ventilation date: journal: intensive care medicine doi: . / - - - - _ sha: doc_id: cord_uid: sg n hs acute respiratory failure, and the need for mechanical ventilation, remains one of the most common reasons for admission to the intensive care unit (icu). the burden of acute respiratory failure is high in terms of mortality and morbidity as well as the cost of its principal treatment, mechanical ventilation. very few epidemiologic studies have evaluated the prevalence and outcome of acute respiratory failure and mechanical ventilation in general. most of the published literature has focused on specific forms of acute respiratory failure, particularly acute lung injury (ali) and acute respiratory distress syndrome (ards). in this chapter, we provide a brief review of the pathophysiology of acute respiratory failure, its definition and classification, and then present the incidence and outcomes of specific forms of acute respiratory failure from epidemiologic studies. acute respiratory failure, and the need for mechanical ventilation, remains one of the most common reasons for admission to the intensive care unit (leu). the burden of acute respiratory failure is high in terms of mortality and morbidity as well as the cost of its principal treatment, mechanical ventilation. very few epidemiologic studies have evaluated the prevalence and outcome of acute respiratory failure and mechanical ventilation in general. most of the published literature has focused on specific forms of acute respiratory failure, particularly acute lung injury (ali) and acute respiratory distress syndrome (ards) . in this chapter, we provide a brief review of the pathophysiology of acute respiratory failure, its definition and classification, and then present the incidence and outcomes of specific forms of acute respiratory failure from epidemiologic studies. normal respiration requires the integrated function of several components of the respiratory system (fig. ) . dysfunction of any component results in the impairment of normal gas exchange and may lead to acute respiratory failure and the need for mechanical ventilation. according to the underlying pathophysiologic mechanism, acute respiratory failure is usually divided into four patterns: types i-iv ( ta ble ). type i and type ii respiratory failure are also referred to as hypoxemic and hyper- capnic respiratory failure, based on a predominant gas exchange abnormality. in many disease states however, more than one pathophysiologic mechanism is operational and clinical criteria that incorporate setting, acuity, and severity are used more often ( table ) . acute episodes (exacerbations) of respiratory failure in patients with chronic compensated respiratory insufficiency are usually referred to as 'acute on chronic' respiratory failure. a consensus definition of acute respiratory failure is not available and most studies have used the combination of mechanical ventilation (of variable duration) with or without evidence of severe hypoxemia on arterial blood gas analysis. while some studies utilized a more strict definition than others, the essential component in all has been the need for mechanical ventilation. the indications for mechanical ventilation , however, are mostly based on clinical observations (increased respiratory rate, use of accessory muscles, paradoxical chest wall movements, changes in mental state), none of which has sufficient accuracy or precision. therefore, the epidemiology of acute respiratory failure has so far been restricted to 'treated' acute respiratory failure, possibly explaining the wide variations in the reported incidence and outcomes of acute respiratory failure and associated clinical syndromes. since the availability of intensive care and mechanical ventilation vary greatly in different parts of the world, the burden of acute respiratory failure may be severely underestimated depending on the access to leu services. the incidence of acute respiratory failure varies according to the definition used and the population studied ( table ) . two european studies , one conducted in germany [i] and the other in sweden, denmark, and iceland [ ] , estimated very similar incidences, . and . cases per , person-years. both studies used an identical definition (intubation and mechanical ventilation for > h regardless of arterial blood gas findings) and employed a multicenter approach with large patient cohorts over a short period ( weeks). on the other hand, behrendt reported a much higher incidence of acute respiratory failure in the usa, . per , patient-years. this incidence was estimated based on the icd- -cm disease codes for diagnoses and treatment in patients > yrs old observed over a l-year period [ ] . the significant variation between the us and european incidences may in part be explained by the differences in study design ( table ) and in part by incons istent indications and access to mechanical ventilation in different countries. acute respiratory failure is often accompanied or followed by a failure of other vital organs, and death most often occurs becau se of multiorgan failure (mof) and the withdrawal of mechan ical ventilati on when the chances for a meaningful recovery of the patient's qual ity of life are deemed to be exceedingly low. imprecision of clinical prognostic criteria, variations in available resources, and patient and provider preferences limit the interpretation of mortality data from different epidemiologic studies. reported mortality rates for acute respir ator y failure from the s are remarkably similar, approximately % in spite of differences in study designs and the definit ions applied ( table ). lewandowski and coworkers [ ] studied patients from icus in berlin, germany and reported mortality rates of - % depending on the lung injury score (lis). in a large prospective study from scandinavia, luhr and coworkers reported an all-cause -day mortality of % [ ] . in a large, prospective international cohort involving icus, esteban and coworkers reported an icu mortality of . %. mortality increased significantly in patients with sepsis, shock, ards, or liver failure [ ] . vincent and coworkers used sequential organ failure assessment (sofa) score criteria and the need for mechanical ventilation to define acute respiratory failure and estimated an overall icu mortality of %. the mortality was much lower ( %) when the lung was the only organ involved [ ] . recently, flaatten and coworkers reported the mortality from acute respiratory failure at different time points after disease onset . mortality was again the lowest in single organ acute respiratory failure and rose with each add itional organ failure. higher mortality rates were found days after the onset compared to at icu or hospital discharge [ ] . mof following an icu admission , presence of circulatory shock on icu admission , older age, and pre-existing comorbidities (cirrhosis, malignancy and chronic renal failure) were independent risk factors for the mortality rate reported in several studies [ , [ ] [ ] [ ] . acute lung injury (ali) ali and its more severe form, ards, are clinical syndromes defined as an acute onset of hypoxemic respiratory failure with diffuse pulmonary infiltrates in the absence of left atrial hypertension as the principal cause of acute pulmonary edema. ali is a major cause of acute respiratory failure in the icu and is associated with high morbidity and mortality. since it was first described by ashbaugh and colleagues [ ] and than redefined in [ ] , there have been significant advances in the understanding of etiology, pathophysiology, and the epidemiology of ali. clinical risk factors for ali are usually divided into direct (pulmonary) and indirect (extrapulmonary). pneumonia, aspiration, lung contusion; and inhalation injury are the principal pulmonary risk factors, while sepsis, shock, trauma, pancreatitis, and multiple transfusions represent the most important extrapulmonary risk factors. in recent years, transfusion-related ali (trali) and novel viral pathogens (severe acute respiratory syndrome [sarsj) have emerged as important risk factors for ali. the reported incidence of all has varied significantly. the report of the national heart and lung institute task force on respiratory diseases, estimated , cases of ards per year yielding the annual incidence of per , person-years . subsequent studies reported an incidence of all ranging from to cases per , person-years in european countries [ , ] and australia [ ] , and a much higher incidences of ali in the usa, per , persons-years ( , cases per year) [ ] [ ] [ ] . while a significant minority of patients with all is treated with non-invasive ventilation (niv), the majority of studies included only patients treated with invasive ventilation. a recently completed, retrospective, community cohort study in olmsted county, minnesota included patients treated with niv and found an even higher incidence of ali, per , person-years (personal communication, rodrigo cartin -ceba), mortality from ali varies greatly depending upon the age of the patient, underlying chronic illnesses, ali risk factors, and non-pulmonary organ dysfunctions [ ] . two decades ago, the mortality rate from ali ranged from - % [ , , , ] , but has since declined and more recently has been estimated to be about - % [ , , , ] . advances in general supportive care [ ] and the use of new mechanical ventilation strategies [ ] may account for most of the change. both the incidence of and mortality from all increase exponentially with age [ - , , ] . mof [ , , ] , liver failure, severe sepsis [ , , , ] , aspiration [ ] , presence of infection and neurological failure on icu admission [ ] , and pree-xisting cirrhosis [ , , , ] , bone marrow transplantation, human immunodeficiency virus (hiv) [ ] , hematologic [ , ] or active malignancy [ , ] , and charlson comorbidity score [ ] have been associated with a higher mortality. persistent severe hypoxemia and cardiovascular failure also predict poor outcomes [ , ] . non-survivors of ali die predominantly of moe a landmark study published in reported that only % of deaths were caused by respiratory failure [ ] . similar results ( % and %) were reported by two stud ies conducted in recent years [ , ] . mof, septic shock , and underlying comorbidities are the most common causes of death in patients with ali. survivors of ali often have a prolonged recovery and significant short and longterm disability. while lung function usually returns to normal within several months [ ] , neuromuscular and neurocognitive sequelae may persist much longer [ , ] . the most important predictors of prolonged disability are the use of systemic steroids during the icu stay, presence of a complicating illness acquired during the icu stay, and the rate of resolut ion of ali and mof [ ] . neuropsychological sequelae are also common and about % of long-term survivors develop posttraumatic stress disorder [ ] . with a decline in mortality from ali, more survivors are at risk of prolonged morbidity ('chronic critical illness') contributing to substantial increases in the utilization of health care resources. cardiogenic pulmonary edema is a common cause of acute respiratory failure. in about % of the mechanically ventilated patients in an international cohort study, cardiogenic pulmonary edema was the principal reason for instituting mechanical ventilation [ ] . other epidemiologic studies reported similar rates of cardiogenic pulmonary edema [ , ] with mortality ranging from - % [ , ] . in the past two decades, niv, both continuous positive airway pressure (cpap) and bilevel positive airway pressure (bipap) ventilation, have received a great deal of interest in the management of patients presenting with acute cardiogenic pulmonary edema . randomized trials comparing either cpap or bipap with standard medical therapy, found similar improvements in arterial blood gases and breathing rates, reduced need for intubation, and improved outcome [ ] . according to the world health organization, chronic obstructive pulmonary disease (copd) ranks fourth among all causes of death with an age-adjusted mortality rate of . per , person-years. the th century pandemic of cigarette smoking is taking its toll, evident by the increase in the annual hospitalization rate for acute exacerbation of copd from . in to . % in . moreover, about % of all hospitalizations are directly or indirectly attributable to copd [ ] . many patients with acute exacerbation of copd require admission to the icu for acute respiratory failure. in an international cohort study [ ] , acute exacerbation of copd was a principal indication for initiating mechanical ventilation in % of patients with acute respiratory failure. the hospital mortality rate of copd patients admitted with acute exacerbation varies between . - %, depending on the methodology of the data collection and the patient population. seneff et al. [ ] reported a hospital mortality rate of % in admissions for acute exacerbation of copd selected from the acute physiology chronic health evaluation (apache) iii database of , admissions in a prospective multicenter trial. mortalities rose to % at hospital discharge and doubled to % at the l-year follow-up. invasive mechanical ventilation was instituted in of patients with a mortality rate of %. after controlling for the severity of illness , mechanical ventilation at icu admission was not asso ciated with either hospital mortality or subsequent survival. development of non-respiratory organ dysfunction was the most important predictor of hospital mortality, while the abnormalities in gas exchange (pac , ph , pa ) indicative of advanced dysfunction were strongly associated with six month mortality. esteban et al. [ ] reported a hospital mortality of % in patients receiving mechanical ventilation for acute exacerbation of copd. liu et al. [ ] retrospectively studied a cohort of patients with copd requiring invasive mechanical ventilation for acute respiratory failure. the cause of acute respiratory failure was acute exacerbation of copd in % and pneumonia in % of patients. the hospital mortality rate was . % in all patients and . % in the acute exacerbation of copd subgroup. respiratory acidosis was corrected (ph> . ) in . % of survivors but only in . % of non-survivors. in a recent study, conse cutive patients hospitalized with acute exacerbation of copd were followed prospectively for years [ ] . the in-hospital mortality rate was . %. the overa l -month mortality was %, with -, -, and -year mortality rates of %, %, and %, respectively. more severe gas exchange abnormalities and longer hospital stays were associated with the hospital mortalities. long-term mortality was associated with longer disease duration, lower serum albumin, low body mass index, and lower pa ' mof and sepsis were the most common immediate causes of death in patients with acute exacerbation of copd admitted to the icu. in another prospective study of patients with acute exacerbation of copd [ ] , invasive mechanical ventilation was started in % and niv was tried in % of patients and was successful in % of them. median duration of ventilation was days. after several clinical tr ials reported improved outcomes [ ] , niv has become the principal init ial mode for providing mechanical ventilation to patients with acute exacerbation of copd [ ] . since the indications for niv are more liberal than those of invasive ventilation, it is difficult to directly compare the outcomes of mechanically ventilated patients treated with the two modes. in a study by girou et al., however, adjusted odds of death ( . ; % confidence interval [ci], . - . ) suggested that the mortality in patients with similar severity of illness treated with niv was significantly lower. severe status asthmaticus is a rare cause of acute respiratory failure requmng mechanical ventilation ( . % of patients in the international cohort study) [ ] . patients in status asthmaticus who require invasive mechanical ventilation are at high risk of severe complications (pneumothorax, cardiopulmonary arrest) and mortality. afessa et al. reported the incidence and outcomes of status asthmaticus in a us inner city hospital, from to [ ] . forty-eight out of hospital admissions required mechanical ventilation ( %). mechanically ventilated patients had significant mortality ( %) and high complication rates. sixteen patients developed non-pulmonary organ failure and four developed pneumothorax requiring chest tube drainage. interestingly, all patients who died in this study were female . pneumonia is a common cause of hypoxemic acute respiratory failure. approximately - % of acute respiratory failure episodes requiring mechanical ventilation are due to pneumonia. icu mortality rates from acute respiratory failure due to pneumonia range from - % [ , , , ] . in many patients with pneumonia, however, complications such as septic shock and ali, or acute worsening of underlying chronic lung disease (copd) are the principal reasons for instituting mechanical ventilation . compared to other ali risk factors, pneumonia is associated with higher mortality (see ali paragraph above). the majority of patients with interstitial lung disease and acute respiratory failure admitted to the icu require invasive mechanical ventilation . interstitial lung disease is, however, an uncommon cause of acute respiratory failure (less than % of patients in the international cohort study [ ] ). in a retrospective review [ ] of patients with interstitial lung disease who were mechanically ventilated at mayo clinic from to , acute respiratory failure was the most common cause of icu admiss ion ( %), followed by sepsis ( %) and cardiopulmonary arrest ( %). seventeen patients were initially treated with niv but eventually all patients required invasive mechanical ventilation. hospital mortality was %. patients with idiopathic pulmonary fibrosis tended to have a higher mortality rate than non -idiopathic pulmonary fibrosis forms of interstitial lung disease. conventional lung protective mechanical ventilation was not associated with improved outcome. worsening hypoxemia and higher positive end-expiratory pressure (peep) settings were associated with increased mortality. in an earlier study, saydain and coworkers observed the clinical course of patients with idiopathic pulmonary fibrosis admitted to the icu. acute respiratory failure was the most common reason for icu admission. while % of the patients survived to hospital discharge, of survivors ( %) died within months after hospital discharge [ ] . patients with neuromuscular disease are frequently treated with both acute and chronic mechanical ventilation. neuromuscular disease accounted for % of patients receiving mechanical ventilation in the international cohort study [ ] . compared to other causes of acute respiratory failure, patients with neuromuscular disease had higher costs and length of icu stay and % required tracheostomy [ ] . hospital mortality was %. epidemiologic studies looking at the outcomes of acute respiratory failure due to specific forms of neuromuscular disease are scarce. recently, ali et al. [ ] reported on the outcomes of patients with guillain-barre syndrome who required mechanical ventilation. all but six patients ( %) required tracheostomy. forty-six patients ( %) survived to hospital discharge, and ( %) were alive at the l-year follow-up according to the international cohort study [ ] , in . % of patients mechanical ventilation was employed because of trauma. hospital mortality for these patients was %. in a retrospective incident study of acute respiratory failure in the usa [ ] , acute respiratory failure related to trauma was more common in the younger age group and trauma without mof was associated with a very low mortality rate. in a scandinavian study, approximately % of cases of acute respiratory failure were caused by trauma [ ] . of the cases of acute respiratory failure in the berlin study, were due to trauma with mortality of % [ ] . complicating coma, shock, and ali are common indications for mechanical ventilation in patients with trauma. about - % of cases of ali are related to trauma [ , , ] with mortality lower than that for other ali risk factors ( %, see above) [ ] . shock is characterized by global hypoperfusion leading to lactic acidosis, hyperventilation and hypoperfusion of respiratory muscles, resulting in type iv respiratory failure. up to % of oxygen consumption in shock may be used by the respiratory muscles contributing to a global imbalance between oxygen delivery and consumption . pulmonary edema, ali, and anemia often contribute towards respiratory distress. work of breathing may ultimately overcome respiratory reserve leading to the development of acute respiratory failure. early use of mechanical ventilation in severe shock may be justified to limit the work of breathing and decrease oxygen consumption by respiratory muscles. septic shock, in particular, is commonly associated with acute respiratory failure and ali. in the international cohort study, septic shock was a primary indication for mechanical ventilation in % of patients with mortality of % [ ] . coma is a non-specific syndrome of widespread central nervous system impairment resulting from various metabolic and structural etiologies. it usually results in type ii respiratory failure due to upper airway dysfunction and hypoventilation. intubation and invasive mechanical ventilation are usually required to protect the airway and maintain gas exchange. in the study by esteban et al. 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systematic review and meta -analysis factors affecting survival of hospitalized patients with copd hospital and i-year survival of patients admitted to intensive care units with acute exacerbation of chronic obstructive pulmonary disease analysis of risk factors for hospital mortali ty in patients with chronic obstructive pulmonary diseases requiring invasive mechanical ventilation prognostic factors, clinical course, and hospital outcome of patients with chronic obstructive pulmonary disease admitted to an intensive care unit for acute respiratory failure non-invasive positive pressure ventilation for treatment of respiratory failure due to exacerbations of chronic obstructive pulmonary disease secular trends in nosocomial infections and mortality associated with noninvasive ventilation in patients with exacerbation of copd and pulmonary edema clinical course and outcome of patien ts admitted to an icu for status asthmaticus hospital survival rates of patients with acute respiratory failure in modern respiratory intensive care units. an international, multicenter, prospective survey gajic ( ) ventilator settings and outcome in pat ients with interstitial lung disease requiring mechanical ventilation in the intensive care unit outcome of patients with idiopathic pulmonary fibrosis admitted to the intensive care unit gajic ( ) mechanical ventilation in patients with guiilain-barre syndrome key: cord- - yfs ve authors: flores, carlos; del mar pino-yanes, maria; villar, jesús title: a quality assessment of genetic association studies supporting susceptibility and outcome in acute lung injury date: - - journal: crit care doi: . /cc sha: doc_id: cord_uid: yfs ve introduction: clinical observations and animal models provide evidence that the development of acute lung injury (ali), a phenomenon of acute diffuse lung inflammation in critically ill patients, is influenced by genetic factors. association studies are the main tool for exploring common genetic variations underlying ali susceptibility and/or outcome. we aimed to assess the quality of positive genetic association studies with ali susceptibility and/or outcome in adults in order to highlight their consistency and major limitations. methods: we conducted a broad pubmed literature search from to june for original articles in english supporting a positive association (p ≤ . ) of genetic variants contributing to all-cause ali susceptibility and/or outcome. studies were evaluated based on current recommendations using a -point quality scoring system derived from criteria, and the gene was considered as the unit of replication. genes were also categorized according to biological processes using the gene ontology. results: our search identified a total of studies reporting positive findings for genes involved mainly in the response to external stimulus and cell signal transduction. the genes encoding for interleukin- , mannose-binding lectin, surfactant protein b, and angiotensin-converting enzyme were the most replicated across the studies. on average, the studies had an intermediate quality score (median of . and interquartile range of . to . ). conclusions: although the quality of association studies seems to have improved over the years, more and better designed studies, including the replication of previous findings, with larger sample sizes extended to population groups other than those of european descent, are needed for identifying firm genetic modifiers of ali. critical illness in adults often is followed by acute lung injury (ali). ali and its most severe form, the acute respiratory distress syndrome (ards), are currently defined as a phenomenon of acute diffuse lung inflammation pathologically characterized by an acute onset of non-cardiogenic pulmonary edema resulting from increased capillary-alveolar permeability. both are clinically manifested by hypoxemia under mechanical ventilation (arterial partial pressure of oxygen/fraction of inspired oxygen [pao /fio ] of less than or equal to mm hg for ali and pao /fio of less than or equal to mm hg for ards), diffuse bilateral pulmonary infiltrates on chest radi-ographs, and reduced lung compliance [ ] . pneumonia and sepsis are the main and most common risk conditions associated with the development of both disorders [ ] . ali and ards remain a major health problem worldwide: it has been estimated that each year in the us there are , cases of ali, which are associated with , deaths and . million hospital days [ ] . our understanding of the pathogenesis of ali and ards has improved in recent years with the appreciation that inflammation is a fundamental component of the pathophysiology of these two clinical manifestations of the same syndrome. ali: acute lung injury; ards: acute respiratory distress syndrome; ci: confidence interval; fio : fraction of inspired oxygen; il- : interleukin- ; iqr: interquartile range; ld: linkage disequilibrium; ncbi: national center for biotechnology information; pao : arterial partial pressure of oxygen. clinicians have long recognized that all critically ill patients with ali are not alike. it is becoming apparent that the diversity of clinical manifestations and the response to treatment and outcome among patients with the same disease process are influenced by genetic factors [ ] [ ] [ ] . the first piece of evidence supporting a role for genetic differences in infection risk and outcome came from an epidemiological study reporting a strong association between death from infection in adoptees and their biological, but not adoptive, parents [ ] . for ali, this is further strengthened by the mortality rate disparities across the different ethnic groups in the us [ ] . in addition, ali models in inbred rodents have demonstrated differences for susceptibility and severity traits, allowing the identification of several loci and pinpointing the multigenic nature of the condition [ ] [ ] [ ] . in our attempt to better define patients at risk, recent trends have turned our attention to the search for common genetic variation underlying ali susceptibility and/or outcome. based on the extensive evidence that common genetic variation with modest effects underlies susceptibility to common complex diseases [ ] and on the impossibility of linkage analysis to detect such signals [ ] , association studies have constituted the main tool for improving our understanding of the genetic factors affecting ali susceptibility and outcome. association studies compare two groups of samples (cases and controls) for statistical differences in the frequency of variants at one or more sites of the genome. although the international hapmap project and the development of genotyping technologies have made possible the testing of more than one million of these variants in a single experiment [ ] , they have been available for a short period of time [ ] . thus, currently, association studies in ali have exclusively used a candidate gene approach, in which one or several genes -known to be etiologically involved in the disease -are studied for relevant variant sites. in general, the inconsistency of findings across association studies [ ] -partially attributed to inappropriate designs, implementations, and/or interpretations of studieshas motivated the formulation of standards to improve their quality and to help perform meta-analysis [ ] under the premise that the replication of previous findings most likely reflects interesting biological processes rather than methodological quirks. here, we aimed to examine studies reporting positive findings with all-cause ali susceptibility and/or outcome in adults in order to evaluate their relative merits and caveats based on actual recommendations. we conducted a broad pubmed literature search from to june for original articles by querying for 'polymorphism and acute lung injury' and 'polymorphism and ards'. the retrieved references were then manually curated, and those reporting genetic association studies and published in english were sought. studies were considered if a positive association (p ≤ . ) was reported with either susceptibility or outcomes of all-cause ali or ards. since the current tendency to perform association analysis at the individual variant level may be problematic (for example, there may be important differences in allele frequency or linkage disequilibrium [ld] structure across different populations), we instead considered the gene as the unit of replication [ ] . the gene ontology was used to categorize associated genes according to biological processes [ ] . among reports with positive associations, study qualityrather than significance value -was reviewed based on current recommendations. since performing a checklist of all issues to consider in association studies would require more than a single article, we have focused on the most relevant criteria from a checklist suggested recently [ ] . all together, criteria were considered and each of them was scored as if present or if absent. scoring was performed independently by two authors. studies were divided into case-control or cohort studies based on the design in which the authors reported the positive association. if a case-control study reported a positive association with an outcome in the case series, the positive finding of the study was also considered as found in a cohort design. a final quality score was obtained by adding up scores from all criteria (see below). a reported association could have a maximum score of points for casecontrol studies if more than one polymorphism was analyzed, a maximum of points if reporting a case-control study for a single polymorphism (multiple testing adjustment not needed) or for a cohort with more than one polymorphism analyzed (definition of the control group not needed), or a maximum of points for cohorts analyzing a single locus (definition of the control group and the multiple testing adjustment are not needed). to facilitate comparison across study designs, scores were then transformed to a -to -point scale. criteria that were evaluated in relation to the study design included power calculation, characterization of cases and controls or the cohort, and whether the study considered common gene-wide variation. power calculation was scored as present only if it was explored prospectively or retrospectively as part of the original study. controls were considered to be adequate if obtained from the same population as cases and described in such a way that could be replicated. this criterion was not scored in the cohort studies. adequacy of case groups was considered if demographical and clinical data were reported in sufficient detail in the text and/or a table. mentioning accepted international guidelines for phenotype definition [ ] as the sole description of cases was not considered to be acceptable. to cover the adequacy of exploring gene-wide variation in the association, ld must have been explored for polymorphism selection and/or for the interpretation of results. to evaluate study reproducibility, unambiguous identification of polymorphisms by means of national center for biotechnology information (ncbi) reference numbers or flanking sequences was scored as present. the sole description of amplification primer pairs and/or a reference to a previous publication that reported the assay was not considered to be acceptable. the three other criteria evaluated as part of study reproducibility relate to genotyping quality control measures. duplicate genotyping of a portion of individuals by means of the same or alternative genotyping techniques to calculate an error rate was considered to be adequate and scored as present. testing of hardy-weinberg equilibrium was scored as present even when significant p values were reported for any of the groups as long as a duplicate genotyping was performed. finally, adequate studies performed an interpretation of results blind to the clinical status of samples. to evaluate the statistical analyses, we considered the presence of multiple testing adjustments to be adequate. however, note that this category was not scored if a single polymorphism was assessed since we did not consider an adjustment for the multiple explored phenotypes or outcomes for the adequacy of the study to be necessary. three other categories scored as adequate included an evaluation of other recorded risk factors by means of regression models, reporting major findings in terms of risks (as hazard or odds ratios) and their % confidence intervals (cis), and an empirical assessment or adjustment for population stratification by means of an independent set of polymorphic markers. finally, we scored as adequate additional support from studies performing a validation in at least a second independent sample as part of the original study. studies designed to confirm previously associated polymorphisms were not considered to be acceptable for this category. studies that also included experiments providing evidence of functionality for associated variant(s) were scored as adequate. the sole reference to previous publication(s) providing the functional evidence of the associated polymorphism was scored as absent. searching for 'polymorphism and acute lung injury' or 'polymorphism and ards', we retrieved and original articles, respectively. this allowed us to identify a total of articles [ - ] on genes that showed a positive association with susceptibility and/or outcomes of all-cause ali or ards in at least one study (table ) . although we used broad terms for this search, the possibility for missing additional studies with positive findings might still exist. nevertheless, a complementary search querying for the disease name in the hugenet navigator [ ] gave completely overlapping results, showing studies for additional genes, albeit reporting negative findings. most studies ( . %) were carried out exclusively in populations of european descent (defined as 'whites' or caucasians). a minority of studies were performed in east asians ( %) and the remaining . % of studies included populations of both european and african descent. among the genes that showed a positive association in at least one study, four genes were replicated in at least a second article, three genes were replicated in at least three studies, and one gene was replicated in four studies (figure ). since with only two exceptions [ , ] none of these studies attempted to validate the association results in an independent sample, all studies were counted as a single contribution for the purpose of this assessment. ontology analysis of these genes showed that the majority of them were involved in the response to external stimulus ( . %) and cellular signal transduction ( %). there was also a prominent representation of genes implicated in cell proliferation ( . %), inflammatory response ( . %), immune response ( %), and chemotaxis ( %). seventeen studies ( . %) reported positive findings using a case-control design and ( . %) using a cohort. median sample sizes among studies were of cases (interquartile range [iqr]: to ) and controls (iqr: to ), whereas the median sample size for cohort studies was patients (iqr: to ). overall median quality score was . (iqr: . to . ) and maximum and minimum scores were . and . , respectively. when studies were classified by design, the median quality score in case-controlled studies ( . ; iqr: . to . ) was significantly higher than in cohort studies ( . ; iqr: . to ) (p = . , mann-whitney u test). when studies were explored by the year of publication, there was an improvement trend of association studies over time (spearman rho = . , p = . ), but this was due mostly to case-controlled studies (spearman rho = . , p = . ) since no significant trend was observed for cohort studies (spearman rho = . , p = . ). genes that showed positive association with either susceptibility and/ or outcome with all-cause acute lung injury or acute respiratory distress syndrome genes that showed positive association with either susceptibility and/ or outcome with all-cause acute lung injury or acute respiratory distress syndrome. ace, angiotensin-converting enzyme; cxcl , chemokine cxc motif ligand ; f , coagulation factor v; il- , interleukin- ; il- , interleukin- ; mbl , mannose-binding lectin- ; mif, macrophage migration inhibitory factor; mylk, myosin light-chain kinase; nfkb , nuclear factor kappa light polypeptide gene enhancer in b cells; nfk-bia, nuclear factor kappa light polypeptide gene enhancer in b cells inhibitor alpha; nrf , nuclear factor erythroid-derived factor; pbef, pre-b cell-enhancing factor; plau, plasminogen activator urokinase; sftpb, surfactant pulmonary-associated protein b; tnf, tumor necrosis factor; vegf, vascular endothelial growth factor. almost two thirds of the studies ( . %) did not explore their power to detect positive findings. nearly all studies ( %) fulfilled the internationally accepted definition criteria for ali and ards [ ] , and most studies ( . %) appropriately described demographical and clinical data from cases ( figure ). more heterogeneity was found for the criteria to select a control group: although most studies used healthy subjects or population-based controls ( %), a great proportion of studies preferred icu patients as controls ( %). in any case, . % of studies fulfilled the required criteria to have an adequate control group. most studies ( . %) analyzed a few variants per gene ( . % analyzed a single variant with anticipated functionality) without providing appropriate coverage or discussion to other untyped common variation by means of ld-based methods. in almost half of the studies ( . %), we were not able to identify the associated polymorphism(s) in ncbi databases straightforwardly and unambiguously since flanking sequences or genetic reference numbers were lacking. less than half of the studies reported genotyping error checks ( . %) or a blinding strategy ( . %) to avoid biased results ( figure ). however, hardy-weinberg equilibrium was assessed separately in cases and controls or in the cohort in . % of studies. remarkably, three of these studies reported a positive finding for polymorphisms that nominally deviated from hardy-weinberg expectations in control samples. adjustments for multiple testing were lacking in most studies since only . % of them made adjustments during statistical interpretation. conversely, regression analyses to adjust for covariates were used in a high proportion of studies ( . %). likewise, the magnitude of effects has been appropriately reported in terms of hazard or odds ratios and their % cis in most studies ( . %). by contrast, adjustments for the underlying population stratification were nearly absent as part of the statistical toolbox of the studies ( . %). as few as studies ( . %) supported the association in an independent validation sample [ , ] . only of studies ( . %) explored functional significance of variants associated with disease, either by evaluating the functionality of the associated polymorphism using gene reporter assays [ , ] or by its correlation with serum protein levels [ , , , ] . this quality assessment of genetic association studies with positive findings in susceptibility or outcome of ali and ards identified a total of articles and genes. due to our limited knowledge of the pathogenesis of these conditions and given that it is likely that many common genes and pathways contribute to the onset, course, or severity of these two forms of the same disease process, for the purpose of genetic susceptibility and outcome in this systematic review, we considered ali and ards as a single entity. the top gene ontologies represented in current association studies fit within the major biological processes underlying ali development on the basis of different microarray experiments among several studies using diverse animal models of the disease and cellular models of stretch-induced injury [ ] . overall, the paucity and quality of association data in ali/ ards call for more and better designed studies with larger sample sizes with unambiguous identification of the studied variants and procedures that allow monitoring of genotyping quality for a consistent replication and with better statistical a names are those originally reported in the corresponding reference. ins/del, insertion-deletion polymorphism. ace, angiotensin-converting enzyme; ali, acute lung injury; ards, acute respiratory distress syndrome; cap, community-acquired pneumonia; cxcl , chemokine cxc motif ligand ; f , coagulation factor v; il- , interleukin- ; il- , interleukin- ; mbl , mannose-binding lectin- ; mif, macrophage migration inhibitory factor; mv, mechanical ventilation; mylk, myosin light-chain kinase; nfkb , nuclear factor kappa light polypeptide gene enhancer in b cells; nfkbia, nuclear factor kappa light polypeptide gene enhancer in b cells inhibitor alpha; nrf , nuclear factor erythroid-derived factor; pbef, pre-b cell-enhancing factor; plau, plasminogen activator urokinase; sars, severe acute respiratory syndrome; sftpb, surfactant pulmonaryassociated protein b; sirs, systemic inflammatory response syndrome; snp, single-nucleotide polymorphism; tnf, tumor necrosis factor; tr, tandem repeat (polymorphism); vegf, vascular endothelial growth factor. positive genetic association studies with acute lung injury/acute respiratory distress syndrome susceptibility and/or outcome (by year of publication) percentage of studies scored as adequate for criteria (x-axis) used for the quality assessment of genetic association studies supporting susceptibility and/or outcome in acute lung injury percentage of studies scored as adequate for criteria (x-axis) used for the quality assessment of genetic association studies supporting susceptibility and/or outcome in acute lung injury. ld, linkage disequilibrium; pop. stratification adjust., population stratification adjustment. analyses. some of the reported associations, mostly in populations of european descent, have already set the bar high in the field with 'high-quality' studies, either with well-powered studies [ , ] or with a functional correlation of the associated polymorphism [ ] . however, most of those association studies examining the functional effects of polymorphisms have reported the plasma levels of the gene product (protein) at one time point during the development or evolution of the disease process, so the role of those protein levels in the natural history of ali or ards remains to be defined. additionally, positive association studies on ali/ards have focused essentially on exploring genetic risk effects of candidate gene variants in european populations. thus, future studies must try to fill this gap by extending the association analysis to other populations that might give us an overall picture of cosmopolitan and population-specific genetic risks. this also requires authors to give a more appropriate interpretation of results in light of power estimates since genetic effects are expected to be weak and sample sizes will rarely increase to the extent considered necessary [ ] . the current evidence also encourages more replication studies, especially of those genes that have been positively associated in at least two studies [ ] . a strong candidate would be the gene encoding the pro-inflammatory cytokine interleukin- (il- ). extensive cross-species gene expression pattern comparisons in experimental models of ali have shown that il- is highly upregulated [ ] and at increased circulating concentrations in ali patients [ ] . however, undisputed evidence supporting the association of il- gene variants with ali/ards susceptibility or outcome is still lacking, even though positive results have been found in four studies. one of the major reasons is that the predicated association has been explored in a single polymorphism of the il- gene (g/c at position - from the transcription start site). association studies using a gene-wide coverage of common variation may reveal more robust patterns of variation associated with the disease [ , ] . in this sense, a (nearly) full coverage of common variation of the candidate gene in association studies of ali is especially important since no association is yet definitive and our understanding of the functional elements of our genome is incomplete [ ] . classification and characterization of ali/ards across reviewed studies were highly concordant. however, another face of the problem is that ali/ards is still ill defined and the problem is further confounded by the diversity of etiological mechanisms such as sepsis, pneumonia, trauma, and massive transfusion that predispose patients to the condition. furthermore, it has been recently shown that patients meeting current american-european consensus conference ards criteria may have highly variable levels of lung injury and outcomes [ ] . we believe that the development of novel diagnostic tools to precisely characterize the ali and ards phenotypes or the risk factors underlying disease development might result in associations that are more reproducible. as a result of the progress of our understanding of this disease and the use of high-throughput methodologies [ ] , it is expected that robust well-replicated associations between genetic polymorphisms and ali/ards susceptibility and outcome will become a reality in the near future. to reach this point, guidelines to report genotype data fulfilling minimum quality standards need to be implemented to improve our understanding of the genetic architecture of this disease. in addition, statistical methodologies such as multiple testing and population stratification adjustments, which to date have been almost completely absent in these studies, need to be routinely employed as well. since all studied candidate genes await validation in independent studies using larger samples, the search for genetic variants determining susceptibility and outcome in ali or ards still needs to grow and continue improving for the identification of true associations between genotype and clinical outcomes important in the care of ali/ards patients. integration of data across studies (for example, gene expression profiling, association studies, and proteomics) may reveal novel insights into ali development which may allow us to identify cellular pathways specific to the disease. this knowledge will speed up the development of better and increasingly efficient tailored therapies for ali/ards patients admitted to the intensive care unit. tions, mechanisms, relevant outcomes and clinical trial coordination the help network: an early peep/fio trial identifies different degrees of lung injury in patients with acute respiratory distress syndrome incidence and outcomes of acute lung injury understanding genetic predisposition to sepsis injury research in the genomic era. lancet genetic determinants of phenotypic diversity in humans genetic and environmental influences on premature death in adult adoptees race and gender differences in acute respiratory distress syndrome deaths in the united states: an analysis of multiple-cause mortality data ( - ) genetic analysis of ozone-induced acute lung injury in sensitive and resistant strains of mice use of consomic rats for genomic insights into ventilator-associated lung injury reciprocal congenic lines of mice capture the aliq effect on acute lung injury survival time genetic association studies the future of genetic studies of complex human diseases the international hapmap consortium: a second generation human haplotype map of over . million snps what can genome-wide association studies tell us about the genetics of common disease? what makes a good genetic association study? meta-analysis of genetic association studies supports a contribution of common variants to susceptibility to common disease the future of association studies: genebased analysis and replication gene ontology: tool for the unification of biology. the gene ontology consortium working group on replication in association studies: replicating genotype-phenotype associations polymorphisms of human sp-a, sp-b, and sp-d genes: association of sp-b thr ile with ards genetic polymorphisms associated with susceptibility and outcome in ards angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome association between surfactant protein b + polymorphism and the risk of respiratory failure in adults with community-acquired pneumonia polymorphism in the surfactant protein-b gene, and the risk of direct pulmonary injury and ards pre-b-cell colony-enhancing factor as a potential novel biomarker in acute lung injury mannose-binding lectin in severe acute respiratory syndrome coronavirus infection the association of interleukin haplotype clades with mortality in critically ill adults functional genomic insights into acute lung injury: role of ventilators and mechanical stress and tnfb polymorphisms in acute respiratory distress syndrome vascular endothelial growth factor gene polymorphism and acute respiratory distress syndrome novel polymorphisms in the myosin light chain kinase gene confer risk for acute lung injury interleukin- polymorphism in position - and acute respiratory distress syndrome polymorphism of the angiotensin-converting enzyme gene affects the outcome of acute respiratory distress syndrome macrophage migration inhibitory factor in acute lung injury: expression, biomarker, and associations pre-bcell colony-enhancing factor gene polymorphisms and risk of acute respiratory distress syndrome functional polymorphisms in the transcription factor nrf in humans increase the risk of acute lung injury a cxcl polymorphism is associated with better outcomes in patients with severe sepsis polymorphisms in the mannose binding lectin- gene and acute respiratory distress syndrome ace i/d but not agt (- )a/g polymorphism is a risk factor for mortality in ards inhibitor kappab-alpha haplotype gtc is associated with susceptibility to acute respiratory distress syndrome in caucasians insertion/deletion polymorphism in the promoter of nfkb influences severity but not mortality of acute respiratory distress syndrome genotypes and haplotypes of the vegf gene are associated with higher mortality and lower vegf plasma levels in patients with ards association between urokinase haplotypes and outcome from infection-associated acute lung injury variation in the mylk gene is associated with development of acute lung injury after major trauma mannose-binding lectin and mannose-binding lectinassociated serine protease in susceptibility, severity, and outcome of pneumonia in adults an il gene-wide haplotype is associated with susceptibility to acute lung injury the - interleukin- polymorphism is associated with acute respiratory failure after major trauma: a prospective cohort study factor v leiden mutation is associated with improved -day survival in patients with acute respiratory distress syndrome a navigator for human genome epidemiology microarray-based analysis of ventilator-induced lung injury the complex interplay among factors that influence allelic association angiotensin-converting enzyme insertion/deletion polymorphism is not associated with susceptibility and outcome in sepsis and acute respiratory distress syndrome orthologous gene-expression profiling in multi-species models: search for candidate genes persistent elevation of inflammatory cytokines predicts a poor outcome in ards. plasma il- beta and il- levels are consistent and efficient predictors of outcome over time encode project consortium: identification and analysis of functional elements in % of the human genome by the encode pilot project highly parallel genomic assays this work was supported in part by ministerio de ciencia (spain) (saf / ) and funcis ( / ). cf was supported by a specific agreement between instituto de salud carlos iii and funcis (emer / ) under the encyt framework. the authors declare that they have no competing interests. all authors contributed equally in the assessment design and the literature search and read and approved the final manuscript. • current evidence suggests that acute lung injury (ali) and its most severe form, the acute respiratory distress syndrome, are influenced by genetic factors.• association studies, the main tool for the exploration of common genetic variation underlying ali, have thus far reported a total of genes associated with ali susceptibility and/or outcome.• these genes are involved mainly in the response to external stimulus and cell signal transduction.• more studies with improved designs, as well as replication of previous findings with larger sample sizes, are needed to definitely identify genetic factors predisposing patients to ali. key: cord- -mgsuwft authors: machado, roberto f.; garcia, joe g. n. title: genomics of acute lung injury and vascular barrier dysfunction date: - - journal: textbook of pulmonary vascular disease doi: . / - - - - _ sha: doc_id: cord_uid: mgsuwft acute lung injury (ali) is a devastating ­syndrome of diffuse alveolar damage that develops via a variety of local and systemic insults such as sepsis, trauma, ­pneumonia, and aspiration. it is interestingly to note that only a subset of individuals exposed to potential ali-inciting insults develop the disorder and the severity of the disease varies from complete resolution to death. in addition, ali susceptibility and severity are also affected by ethnicity as evidenced by the higher mortality rates observed in african-american ali patients compared with other ethnic groups in the usa. moreover, marked differences in strain-specific ali responses to inflammatory and injurious agents are observed in preclinical animal models. together, these observations strongly indicate genetic components to be involved in the pathogenesis of ali. the identification of genes contributing to ali would potentially provide a better understanding of ali pathobiology, yield novel biomarkers, identify individuals or populations at risk, and prove useful for the development of novel and individualized therapies. genome-wide searches in animal models have identified a number of quantitative trait loci that associate with ali susceptibility. in this chapter, we utilize a systems biology approach combining cellular signaling pathway analysis with population- based association studies to review established and suspected candidate genes that contribute to dysfunction of endothelial cell barrier integrity and ali susceptibility. acute lung injury (ali) is a devastating syndrome of diffuse alveolar damage that develops via a variety of local and systemic insults such as sepsis, trauma, pneumonia, and aspiration [ ] . deranged alveolar capillary permeability, profound inflammation, and extravasation of edematous fluids into the alveolar spaces are critical elements of ali, reflecting the substantial surface area of the pulmonary vasculature needed for alveolar gas exchange. ali, together with its severest form, acute respiratory distress syndrome (ards), afflicts approximately , patients per year in the usa and has a mortality rate of - % [ , ] . it is interestingly to note, however, that only a subset of individuals exposed to potential ali-inciting insults develop the disorder and the severity of the disease varies from complete resolution to death. in addition, ali susceptibility and severity are also affected by ethnicity as evidenced by the higher mortality rates observed in african-american ali patients compared with other ethnic groups in the usa [ ] . moreover, marked differences in strain-specific ali responses to inflammatory and injurious agents are observed in preclinical animal models [ ] . together, these observations strongly indicate genetic components to be involved in the pathogenesis of ali. the role that genetics plays in determining ali risk or the subsequent severity of the outcome is one of the many unanswered questions regarding ali pathogenesis and epidemiology. the identification of genes contributing to ali would potentially provide a better understanding of the pathogenic mechanisms of ali, yield novel biomarkers, identify individuals or populations at risk, and prove useful for the development of novel and individualized therapies. however, a traditional genetic approach to studies using family linkage mapping is not feasible given the sporadic nature of ali and the necessity of an extreme environmental insult. further, genetic studies of ali are challenging owing to the substantial phenotypic variance in critically ill patients, diversity in the lung injury evoking stimuli, presence of varied comorbid illnesses common in the critically ill patient, complex gene-environment interactions, and potentially incomplete gene penetrance [ , ] . despite these inherent challenges, the unrivaled progress made in the post-human genome era combined with the utilization of sophisticated bioinformatics and high-throughput methods have allowed significant advances to be made. for example, these tools are now linked to escalating knowledge of the molecular mechanisms of lung endothelial permeability, a hallmark of ali and an attractive target for the design of novel therapies, to identify candidate genes whose variants are potentially involved in ali susceptibility. genome-wide searches in animal models have identified a number of quantitative trait loci that associate with ali susceptibility [ ] . in this chapter, we utilize a systems biology approach combining cellular signaling pathway analysis with population-based association studies to review established and suspected candidate genes that contribute to dysfunction of endothelial cell barrier integrity and ali susceptibility. the integration of high-throughput gene expression profiling in preclinical models of ali with bioinformatics has led to the identification of differentially expressed genes in response to ali whose variants are potentially involved in ali susceptibility and severity. this approach confirmed long-suspected ali-associated candidate genes, but more importantly, identified novel genes not previously implicated in ali. increasing knowledge of the molecular mechanisms of endothelial-barrier-regulatory pathways has also enhanced the ability to find novel ali candidate genes. the analysis of the molecular pathways involving the cytoskeletal scaffolding and the dynamic cytoskeletal changes driving cell shape alterations, a key feature of vascular permeability, has identified additional genes contributing to the development and severity of ali, thereby providing novel therapeutic targets in this devastating illness. genes encoding proinflammatory cytokines, growth factors and mediators, receptors for barrier-regulatory agonists, and mechanical-stress-sensitive genes expressed in endothelium which regulate inflammatory responses also serve as attractive ali candidate genes and are representative of the diverse but fertile areas of exploration for candidate snps affecting ali susceptibility and severity. angiotensin-converting enzyme (ace) is a member of the rennin-angiotensin system (ras), balancing the levels of angiotensin i and angiotensin ii, with significant expression in lung vascular endothelium as compared with other vascular beds [ ] . the ras is considered to be an important regulator of inflammation that contributes to ali by altering vascular permeability, vascular tone, fibroblast activation, and endothelial-epithelial cell survival [ ] [ ] [ ] . for example, angiotensin ii activates inflammatory processes by upregulating proinflammatory cytokines and chemokines via type i and type ii angiotensin ii receptors that subsequently activate the nuclear factor kb (nf-kb) pathway [ , ] . the ras is also involved in the fibrotic response to ali via induction of transforming growth factor expression [ ] . the most compelling evidence for ras involvement in ali has come from the effective attenuation of ali pathobiology by ace inhibitors or angiotensin receptor blocking drugs [ , ] and ace knockout mice in preclinical models of ali [ ] . an intronic insertion (i) or deletion (d) of a -bp alu repeat sequence in the human ace gene, located on chromosome q , has been associated with ace levels and activity in serum [ , ] . the d allele possesses a higher enzyme activity which parallels the higher gene expression in individuals with dd genotype [ ] . the initial association of the dd genotype in the ace gene with increased ali mortality provided the impetus for subsequent studies to more firmly establish a genetic basis of ali and to identify ali candidate genes [ ] . caucasian patients with ards show significantly higher frequencies of the dd genotype and the d allele as compared with ventilated intensive care unit (icu) patients without ards, patients after coronary artery bypass surgery, or healthy controls. moreover, ards patients with dd genotype show markedly higher mortality ( %) in comparison with the ii genotype ( %) or strike '' '' id genotype ( %) [ ] . the higher mortality rate in ards patients with dd or id genotype as compared with ii genotype was subsequently confirmed in han chinese patients in taiwan, although the frequency of the d allele is significantly lower in the chinese population as compared with western populations [ ] . compared with caucasians, a higher frequency of d allele has been reported among africans (nigerian and african-american populations) [ , ] , potentially contributing to the observed disparity in ali-associated higher mortality rates in african-americans [ ] . however, to date, no association study of ace polymorphisms and lung injury has been performed in african-americans. in contrast, mexican and amerindian populations have slightly lower allelic frequencies of the d allele [ ] . thus, ace represents a highly viable endothelial candidate gene and attractive target in acute inflammatory lung disease. tumor necrosis factor (tnf) a, an early mediator of ali development, is a potent proinflammatory cytokine which dramatically increases endothelial cell permeability, cytokine production, and a variety of cytotoxic and proinflammatory compounds which lead to subsequent vascular leakage and disturbed lung water balance. both tnfa and tnfb subtypes appear in the circulation, in bronchoalveolar lavage (bal) fluid and in pulmonary edema fluid during the onset of lung injury. as such, the elevated levels of tnf and its soluble receptors are commonly used as markers of inflammation and are associated with morbidity and mortality in ali patients [ ] . both the tnfa and tnfb genes lie in close proximity within the major histocompatibility complex, with several polymorphisms described in this region. the - g/a promoter polymorphism in the tnfa gene and the ncoi restriction fragment length polymorphism in the tnfb gene appear to influence the expression of tnfa. the carriers of the - a allele and homozygotes for the tnfb allele exhibit increased tnfa expression and have increased susceptibility and mortality to sepsis [ , ] . in patients with ards, the - a allele is also associated with increased -day mortality, with the strongest association found among younger individuals [ ] . however, in ards patients with direct or indirect pulmonary injury, these snps are associated with alterations in ali susceptibility (tnfa - g/a snp only in the direct pulmonary injury group, and tnfb ncoi only in the indirect pulmonary injury group). owing to the extent of linkage disequilibrium in the region, it remains unclear as to whether these are regulatory snps or if the tnf protein level is modulated by a third locus or a haplotype [ ] . promoter snps within the tnfa gene (- g/a, - c/t) have been associated with inflammatory bowel disease along with the - g/a snp [ ] . thus, the role of tnf variants in inflammatory disorders is apparent and indicates a need for further study of other tnf variants in association with ali. interleukin- (il- ) is an acute-phase response cytokine that plays a key role in the activation of b and t cells. inflammatory cytokines, including il- , are essential for the immune system homeostasis; however, when il- production is exaggerated as observed in inflammatory lung disorders including ali [ , ] , clearly detrimental outcomes are observed. ali-related increased levels of il- have been established in the bal fluid of critically ill patients with ards, sepsis, and trauma [ , ] in association with ali adverse outcome [ ] and development of multisystem organ failure [ ] . in prior reports, we observed significantly higher expression of il- and the il- receptor genes across multiple-species ali models and in human lung endothelium exposed to ventilator-induced mechanical stress as well as in differential region-specific expression in lungs of the canine ali model [ ] [ ] [ ] . on the basis of these data, the il- gene constitutes an excellent candidate gene to understand the genetic basis underlying ali. a functional polymorphism in the il- gene promoter region at the - position (g- c) has been associated with alterations in both gene expression and il- levels and lower circulating il- concentrations and lower mortality rates in patients with acute respiratory failure admitted to the icu [ ] . the contrasting correlation between g- c alleles and circulating il- levels has also been reported [ ] . the haplotype involving - g/c, c/g, and g/c is associated with higher mortality (and other secondary clinical outcomes) in a cohort of septic patients of european descent [ ] . we further evaluated il- gene tagging snps covering the entire gene for potential association in sepsis and ali patients of european descent [ ] . no single snp was identified as significantly associated with ali; however, a common haplotype (comprising - g/- g/- g/ a/ a/ c) with a frequency of % in cases and % in controls showed a significant association with ali susceptibility. in addition, homozygote carriers of the risk haplotype are twice as frequent in ali cases ( . %) than in controls ( . %), yielding a highly significantly increased odds ratio for developing ali (odds ratio . ; % confidence interval, . - . ; p = . ). this haplotype spans the entire il- gene including the g allele at position - , i.e. the risk allele for susceptibility to ali noted above. these data support the association of the il- gene with ali susceptibility and illustrate the value of haplotype analysis as a robust approach in association studies. vascular endothelial growth factor (vegf) is an endothelialcell-specific mitogen that regulates angiogenesis, migration, and cell permeability [ ] . vegf plays an important role in several organs by directly regulating vascular permeability to water and proteins. lung overexpression of vegf induces increased pulmonary vascular permeability, resulting in marked pulmonary edema [ ] , and plasma vegf levels are significantly elevated in ali patients [ ] . several studies have reported the association of low levels of vegf with the severity of ards and elevated levels with the recovery from ards, indicating a role for vegf in the repair process of lung injury [ ] . several polymorphisms have been described in the vegf gene, primarily in association with cancer susceptibility and severity. the c/t snp at position of the ¢ untranslated region (utr) of the gene has been associated with higher vegf plasma levels in healthy subjects [ ] . recently, the c t snp in the vegf gene has been associated with ards susceptibility and severity (increased mortality) in subjects of european descent [ , ] . the haplotype tct at position c- t, c + g, and c + t was significantly associated with a higher rate of mortality in ards patients and higher plasma levels of vegf [ ] . these studies highlight the vegf gene as an attractive barrier-regulatory ali candidate gene and molecular target in ali therapeutic strategies. chemokine receptor (cxcr ) is an a-chemokine receptor specific for stromal-derived factor (sdf- ; also known as cxcl ) that plays an important role in cell migration, inflammation, b lymphocyte development, angiogenesis, and human immunodeficiency virus (hiv) infection (hiv coreceptor) [ ] [ ] [ ] . chemokine receptors are g-protein-coupled receptors, which trigger diverse signaling cascades including activation of g proteins and the phosphatidylinositol -kinase, janus kinase/signal transducer and activator of transcription, rho-p rho kinase, and mitogen-activated protein kinase signaling pathways [ ] . the activation of these signaling pathways is often accompanied by the internalization of chemokine receptors and their trafficking back to the plasma membrane. this intracellular turnover determines the leukocyte responsiveness to chemokines [ ] . nonmuscle myosin ii a is a molecular motor that binds with the cytoplasmic tail of cxcr and ccr and participates in the sdf- -dependent endocytosis of cxcr via dynamic interaction with a-arrestin, a key component of the cxcr internalization pathway [ ] . the cxcr gene was identified as a novel candidate gene in ali as it survived two filtering strategies dedicated to identifying ali-susceptibility genes associated with elevated levels of mechanical stress as observed in mechanical ventilator-associated lung injury (vali). our orthologous gene approach determined ali-specific gene ontologies -coagulation, inflammation, chemotaxis/cell motility, and immune response [ ] -involving recognized genes likely to participate in ali pathogenesis [il- , aquaporin (aqp- ), plasminogen activator inhibitor type i (pai- )], as well as novel genes not previously known to be mechanistically involved in ali, including cxcr [ ] ( table ) . we subsequently utilized a consomic rodent approach with introgression of rat chromosomes , , , and , which contained the highest density of vali-responsive genes [ ] . introgression of the vali-sensitive brown norway (bn) rat chromosome , containing several genes, including cxcr- , into the vali-resistant dahl salt-sensitive (ss) rat resulted in conversion of the ss consomic rats to a valisensitive phenotype [ ] . surface expression of cxcr is downregulated by interleukin- , interleukin- , and granulocyte-macrophage colony-stimulating factor and upregulated by interleukin- and transforming growth factor-b (tgfb) [ ] , suggesting that cxcr may also play a role in the fibrotic response to ali via tgfb signaling. polymorphisms in the cxcr gene have not yet been reported; however, a snp in the ¢ utr of the sdf- gene (g a), is associated with susceptibility to aids and type diabetes [ , ] . we are currently exploring cxcr as a potential ali-associated candidate gene as suggested by the density of pubmatrix citations relating cxcr to inflammation ( , published papers), endothelium ( published papers), ali ( published papers) and endothelial permeability (eleven published papers). pubmatrix is a web-based tool that allows simple text-based mining of the ncbi literature search service pubmed using any two lists of keywords terms, resulting in a frequency matrix of term co-occurrence. the advent of high-throughput gene sequencing and expression technologies, and complete genome sequencing of model organisms, now provides the tools to perform largescale analyses of the genome in complex disorders such as ali. whole genome scans, in silico approaches, utilization of consomic rats, and a candidate gene approach involving expression profiling and pathway analysis are proving exceptionally useful in identifying novel candidate genes and genetic variations (fig. ). high-throughput whole genome scanning technology has recently emerged as a powerful tool, particularly in detecting disease-susceptibility genes with modest effects. the haplotype mapping project [ ] , which identified blocks of snps associated with each other, has allowed selection of the most informative snps for further disease association studies [ ] . currently, the most commonly used high-throughput snp platforms involve assessment of over one million snps spanning the genome, i.e. genome-wide association studies (gwas). gwas platforms are effective and have been successfully used in diverse disorders such as agerelated macular degeneration [ ] , inflammatory bowel disease [ ] , type diabetes [ ] , and stroke [ ] . although this approach has yet to be employed in either sepsis or ali, the application of gwas to the disease is clearly imminent. another method to identify ali candidate genes is an orthologue gene in silico approach. the basis of this approach is the hypothesis that patients with ali and preclinical animal models of ali would exhibit commonality in expression of evolutionarily conserved genes across species. for example, profiling results from more than affymetrix microarray chips obtained from ventilator-associated ali models (human, rat, mouse, canine) identified , genes whose expression was altered across all four species in response to ventilator-associated high-throughput gene sequencing and expression technologies, and complete genome sequencing of model organisms, now provide the tools to perform large-scale analyses of the genome in complex disorders such as ali. genome-wide association study (gwas) platforms are effective and have been successfully used in diverse disorders, but although this approach has yet to be employed in either sepsis or ali, the application of gwas to the disease is clearly imminent. the differential gene expression between lung apex/base regions as well as between gravitationally dependent/nondependent regions of the lung base in a canine model of ventilator-associated lung injury (vali) identified aliimplicated lung genes in response to local mechanical stress within the lung. this approach identified the already established ali gene macrophage migration inhibitory factor and novel genes such as growth arrest dna damage inducible (gadd ) and pre-b cell colony enhancing factor (pbef). our multispecies orthologous gene approach in human (endothelial cells), rat, mouse, and canine models of vali exhibits expression of common ali-implicated evolutionarily conserved genes (orthologues) across the species. the genes with a unidirectional . -fold change (p > . ) are found to reside in high density on rat chromosomes and , the chromosomal loci used to develop the consomic rodent model. together, these approaches identified novel ali genes such as pbef, chemokine receptor (cxcr- ) gadd . interrogating the prospective pathways involved in endothelial permeability and correlation with these differentially expressed genes in vali models identified the most putative ali genes such as myosin light chain kinase (mylk), sphingosine -phosphate receptor , cmet, and vascular endothelial growth factor (vegf) mechanical stress [ , ] . filtering these results for a unidirectional change in gene expression with greater than . fold change in expression refined the list to genes, reflecting specific ali-associated gene module/ontology categories: coagulation, inflammation, chemotaxis/cell motility, and immune response. this approach identified multiple genes already recognized as ali genes (such as il- , aqp- , and pai- ), but also identified several novel genes that were not previously known to be mechanistically involved in ali [ ] . complementing the in silico approach described above, a consomic rat approach can also be utilized to identify novel ali gene candidates. in an experimental study, two strains of inbred rodents were determined to have differing susceptibility to vali ( ml/kg, h): vali-sensitive bn rats and the valiresistant dahl ss rats. using microarray analysis and a bioinformatic-intense candidate gene approach, we identified differentially expressed potential vali genes with ontologies such as transcription, chemotaxis, and inflammation. because chromosomes , , , and were found to contain the highest number of vali-response genes, consomic ss rats containing substituted bn chromosome were exposed to vali mechanical stress, resulting in conversion of the resistant ss rat to vali sensitivity [ ] . extensive expression profiling across preclinical ali models can extend the identification of ali gene candidates to determination of allelic frequencies of gene polymorphisms (snps) that may confer ali risk or severity. this "candidate gene approach" has identified several candidates with hypothesized significant mechanistic roles in lung injury, inflammation, or repair in the setting of ali and vali [ ] . further, given the availability of sophisticated bioinformatic methods and increasing knowledge of the molecular and cellular mechanisms of lung injury, candidate genes can also be identified via analysis of cellular pathways involved in ali pathogenesis [ , ] . the application of the novel techniques described in the previous section is proving to be exceptionally useful in identifying novel candidate genes and genetic variations in the study of the pathobiology of ali. these novel gene and biomarkers are discussed in this section. myosin light chain kinase (mlck) is an enzyme that phosphorylates regulatory myosin light chains, which allows myosin cross-bridging interactions with f-actin. in endothelial cells, the contraction of the actomyosin complex generates a stronger centripetal force that overcomes the force keeping the adjacent endothelial cell tethered, leading to endothelial retraction, decreased intercellular adhesion, and increased vascular permeability [ , ] . this phenomenon is physiologically relevant as evidenced by nonmuscle mlck (nmmlck) isoform knockout mice [which retain the smooth muscle mlck (smmlck) isoform] that are less susceptible to lipopolysaccharide (lps)-and ventilator-induced ali [ , ] . further, treatment with a mlck inhibitor prior to lps exposure in the wild-type mice attenuates endothelial cell barrier dysfunction and inflammation [ ] . thus, the myosin light chain kinase gene (mylk), which encodes for mlck, is an excellent ali candidate gene. since initial cloning of the highly expressed nmmlck in endothelium in our laboratory [ ] , we have identified substantial roles of nmmlck in cytoskeleton rearrangement of endothelial cells regulating vascular barrier function [ , ] , angiogenesis, and leukocyte diapedesis [ ] , consistent with a potential mechanistic role for mlck in the genesis of ali. the human mylk gene is located on chromosome q and encodes three proteins, including nmmlck, smmlck, and telokin. we sequenced exons, exon-intron boundaries, and kb of the ¢ utr of mylk in healthy individuals, patients with sepsis alone, and patients with sepsis-associated ali, all of european and african-american descent [ ] , and identified snps (ten exonic, intronic, nine in the ¢ utr, and one in the noncoding exon ), of which were chosen for further linkage disequilibrium studies. five of the ten coding mylk snps confer an amino acid change (pro his, pro ser, val ala, ser pro, and arg gln) in mlck. subsequently, association analysis of both single snps and haplotypes demonstrated very strong associations in both ethnic groups [ ] . in european americans, the rs a/ mylk_ c haplotype was associated with more than a fivefold increase in the risk of developing ali and sepsis. in contrast, the haplotype mylk_ g/mylk_ g/ mylk_ t conferred specific risk for ali but not sepsis [ ] . the ¢ haplotype of the mylk gene also conferred alispecific risk in both european-and african-descent subjects; however, the ¢ region haplotype was associated with ali only in african-descent subjects. in african-americans, the haplotype hcv c/mylk_ a/rs g is substantially more prevalent in ali ( %) as compared with sepsis ( %). this cag haplotype is not found in european americans, suggesting a potential genetic contribution to the observed ethnicity-specific differences in ali/ards prevalence and susceptibility [ ] . we noted similar findings in association studies involving a cohort with trauma-induced ali [ ] . we have also evaluated the association of mylk genetic variants with severe asthma in both european american and african-american populations and identified a snp highly associated with severe asthma in african-americans [ ] consistent with data linking this chromosomal locus (mylk, q . ) to asthma and asthmarelated phenotypes [ ] . taken together, these data strongly implicate mylk genetic variants as risk variants in inflammatory lung disorders, such as ali and asthma. macrophage migration inhibitory factor (mif) is an ali candidate gene and recognized biomarker, initially discovered as a soluble product of activated t cells and named for its role in inhibiting random macrophage migration [ ] . mif is a proinflammatory cytokine which binds to cd and cd and is produced by many cell types, including monocytes/macrophages, pituitary cells, vascular endothelium, and respiratory epithelium [ , ] . mif may serve as a delicate regulator of the cytokine balance between immunity and inflammation as mif counterregulates the immunosuppressive effects of glucocorticoids [ ] . the role of mif as an endogenous prosurvival factor has been demonstrated in vitro. lps-mediated induction of flice-like inhibitory protein (flip) by mif confers resistance to lps-mediated endothelial cell death [ ] . suppression of mif by rna interference induces cell death and sensitivity to apoptotic stimuli [ ] . in addition, mif interacts with the multidimensional nmmlck [ ] isoform which regulates tnf-mediated apoptosis in addition to its potent effects on endothelial cell barrier dysfunction as discussed earlier [ , ] . together, these findings implicate the role of mif in regulation of nonmuscle cytoskeletal dynamics and vascular pathophysiology, which is evident from the enhanced mif levels in the serum, bal fluid, and alveolar endothelium of patients with ards as compared with other critically ill patients [ , , ] . we found significant increases in mif transcript and protein levels in murine and canine models of ventilator-induced lung injury (vili) (using high mechanical ventilation and endotoxin exposure, respectively) [ ] and in human lung endothelium cells exposed to h of cyclic stretch [ ] . mif deficiency or immunoneutralization appears to protect mice or rats from fatal endotoxic shock or other inflammatory diseases [ ] although these results are not without controversy [ ] and our own studies in - -week-old mice failed to demonstrate a vili/ali-related phenotype which was different from controls (data not shown). mif also upregulates the expression of aqp- , the water channels expressed in alveolar endothelial and epithelial cells, and a candidate gene we identified in models of vili-associated mechanical stress [ ] . mif may serve to modulate fluid movement into alveolar spaces, a cardinal feature of ali [ ] . to extend the likelihood that mif serves as a putative candidate gene in ali and sepsis, we studied the association of eight mif polymorphisms, including the most studied mif promoter g/c snp at position - , in a sepsis-induced ali cohort (n = ) of african-and european-descent cases [ ] . no individual snp showed a significant association with either ali or sepsis; however, the carriers of the cc genotype (rs ) and the carriers of the tt genotype (rs ) showed more than twofold increased risk of developing sepsis and ali, respectively. this association was lost, however, after age and gender adjustment in a logistic regression model. in contrast, mif haplotypes at the ¢ region of the gene display strong association with ali and sepsis, conferring both protection as well as susceptibility to ali, in european and african populations [ ] . furthermore, the haplotype at the ¢ promoter region of the gene involving a short tandem repeat at position - (catt) and the - g allele show significant association with both ali and trauma [ ] ; however, no association was found between promoter region haplotypes and mif levels. rheumatoid arthritis patients with the - c allele have higher levels of mif in the serum and synovial fluid than the carriers of the g allele and have a higher probability of developing idiopathic arthritis [ ] . thus, given these diverse mif functions, mif remains an attractive target in inflammatory diseases including the lung. the bioactive sphingolipid metabolite sphingosine -phosphate (s p) is an important lipid mediator that enhances endothelilal cell barrier function in vivo and in vitro by ligating s p receptor (s p ), which is encoded by an endothelial differentiation gene (edg or s p ) [ , ] . s p is a pertussis-toxin-sensitive, g i -coupled receptor which induces rac gtpase-dependent substantial increases in cortical actin polymerization critical to endothelial cell barrier enhancement [ , ] . s p activation enhances the organization and redistribution of vascular endothelial cadherin and b-catenin in junctional complexes in endothelium by phosphorylation of cadherin as well as p catenin and inducing the formation of cadherin/catenin/actin complexes [ ] . understanding the role of s p in enhancing endothelial cell barrier function underscores its importance as a therapeutic target in reversing loss of endothelial cell barrier integrity. in vivo administration of selective s p competitive antagonists induces a dose-dependent disruption of barrier integrity in pulmonary endothelium [ , ] , whereas s p agonists, sew and fty , promote vascular endothelial barrier function [ ] [ ] [ ] . a compelling argument for s p as an attractive ali candidate gene is not only its ability to transduce signals which restore barrier integrity but also that s p is the target for transactivation by receptors for other potent barrier-protective agonists. these include epcr (receptor for activated protein c) [ ] , c-met [receptor for hepatocyte growth factor (hgf)] [ ] , cd (receptor for high molecular weight hyaluronan) [ ] , and the atp receptor [ ] . we recently resequenced the s p gene ( african-americans and european americans) to search for common variations in the edg gene and identified snps in the edg gene, with several promoter snps associated with asthma, another inflammatory lung syndrome [ ] . receptor) the role of hgf and its tyrosine kinase receptor c-met has been investigated in lung development, inflammation, and repair [ ] as well as in neoplastic processes such as cellular transformation, neoplastic invasion, and metastasis [ , ] . snps causing underexpression of c-met have been associated with autism and c-met snps/mutations appear to be linked to lung cancer disparities in different ethnic groups. these include an n s mutation in the hgfbinding domain of c-met, an r c snp/mutation in the juxtamembrane domain, and an activating m t mutation in the tyrosine kinase domain (exon ), all linked to development of solid tumors such as lung cancer, renal cancer, gastric cancer, and hepatocellular carcinoma [ ] . hgf influences morphogenesis in epithelial cells from a variety of organs, including lungs, where hgf antisense oligonucleotides block alveolar and branching morphogenesis [ ] . hgf expression and activity increase after - h of lung injury with intratracheally administered hydrochloric acid, suggesting that hgf plays a role in reparative responses to lung injury [ ] . c-met expression on type ii pneumocytes is likely involved in increased type ii pneumocyte proliferation and restoration of an intact alveolar epithelium [ ] . c-met is composed of a -kda extracellular a subunit and a -kda transmembrane b subunit [ ] which contains tyrosine kinase domains, tyrosine phosphorylation sites, and tyrosine docking sites [ ] . we demonstrated that hgf-mediated c-met phosphorylation and c-met recruitment to caveolin-enriched microdomains (cems) protects against the lps-induced pulmonary vascular hyperpermeability that is regulated by high molecular weight hyaluronan (cd ligand) [ ] . our novel findings indicate that hgf/c-met-mediated, cd -regulated cem signaling promotes tiam (a rac exchange factor)/dynamin dependent rac activation, and peripheral recruitment of cortactin (an actin cytoskeletal regulator), processes essential for endothelial cell barrier integrity. understanding the mechanism(s) by which hgf/c-met promotes increased endothelial cell barrier function may lead to novel treatments for diseases involving vascular barrier disruption, including inflammation, tumor angiogenesis, atherosclerosis, and ali. however, on the contrary, the higher mortality rate in ali patients with increased levels of hgf in bal fluids [ ] and in pulmonary edematous fluids [ ] indicates severer injury and inflammation in response to increased hgf levels. it has now become increasingly clear that hgf plays an important role in normal and injured lung and may have a therapeutic potential in lung diseases. pre-b cell colony enhancing factor (pbef), was first identified by samal and colleagues in as a protein secreted by activated lymphocytes in bone marrow stromal cells that stimulate early stage b cell formation in conjugation with stem cell factor and interleukin- . a large body of work has now highlighted the power of a systems biology approach in the search for novel disease-susceptibility genes and potentially novel biomarkers, with pbef serving as an excellent example of this approach (fig. ) . we first identified marked upregulation of pbef via microarray analyses of murine and canine models of vili/ali with increased gene/ protein expression in bal fluid and serum samples from critically ill icu patients with ali and sepsis [ ] . with only a total of eight papers in pubmed at that time, we next directly sequenced the pbef gene in subjects with ali, sepsis, and healthy controls and conducted a pbef snp-based association study in ali subjects of european and african-american descent [ ] . we identified snps in the pbef gene with two promoter snps, t- g and c- t, associated with ali and sepsis. genotyping of pbef c- t and t- g snps revealed significant associations of sepsis and ali, with the strongest association found with the - c/- g haplotype. univariate analysis found carriers of the g allele (t g) to have . -fold higher risk of developing ali as compared with controls (p = . ) [ ] . these results were subsequently confirmed in a comparable but distinct replicate ali population [ ] . interestingly, the - g/- c haplotype was also associated with increased icu parient mortality, whereas the - t/- t haplotype was associated with fewer ventilator days and decreased icu patient mortality [ ] . a key challenge in genomic explorations is the ability to confirm the contribution of a snp to a dysfunctional-geneinvolved disease process. additional reports have highlighted the capacity for the pbef gene to have an influence far beyond any b-cell regulatory function, with a key role in regulating vascular permeability [ ] as well as inhibiting neutrophil apoptosis [ ] . to further explore mechanistic participation of pbef in ali and vili, we focused on the contribution of pbef to endothelial function. our prior immunohistochemical staining of canine-injured lung tissues localized pbef expression to vascular endothelial cells, in addition to infiltrating neutrophils and type alveolar epithelial cells [ ] . our in vitro studies showed that expression of pbef in pulmonary artery endothelial cells increases thrombin-mediated vascular permeability [ ] , suggesting that enhanced pbef expression may mediate the early increase in vascular permeability that is characteristic of ali. neutrophils harvested from the circulation of septic and ali patients show marked inhibition of the apoptotic process in association with evidence of enhanced respiratory burst capacity [ , ] , with both activities largely restored with administration of pbef antisense oligonucleotides. our initial in vitro studies further demonstrated recombinant human pbef (rhpbef) as a direct rat neutrophil chemotactic factor, with in vivo studies demonstrating marked increases in bal fluid leukocytes (polymorphonuclear leukocytes, pmns) following intratracheal injection in c bl/ j mice [ ] . these changes were accompanied by increased bal fluid levels of pmn chemoattractants (kc and mip ) and modest increases in lung vascular and alveolar permeability. we also noted synergism between rhpbef challenge and a model of limited vili and observed dramatic increases in bal fluid pmns, bal protein, and cytokine levels (il- , tnfa, kc) compared with either challenge alone. gene expression profiling identified induction of ali-and vili-associated gene modules (nf-kb, leukocyte extravasation, apoptosis, toll-receptor pathways). heterozygous pbef +/− mice were significantly protected (reduced bal fluid protein levels, bal fluid il- levels, peak inspiratory pressures) when exposed to a model of severe vili ( h, ml/kg tidal volume) and exhibited significantly reduced gene expression of vili-associated modules. finally, strategies to reduce pbef availability (neutralizing antibody) resulted in significant protection from vili [ ] . pbef is now recognized as associated with modestly increased risk of type diabetes and elevated levels of acute-phase proteins [ ] and a c- g snp has been associated with an increased diastolic blood pressure in obese children [ ] . these studies implicate pbef, now associated with a number of inflammatory disorders such as inflammatory bowel disease, multiple sclerosis, cystic fibrosis, and asthma [ ] [ ] [ ] , as a key inflammatory mediator intimately involved in both the development and the severity of ventilator-induced ali. growth arrest dna damage inducible a (gadd a), a member of an evolutionarily conserved gene family, is implicated as a stress sensor that modulates the response of mammalian cells to genotoxic or physiological stress [ , ] . gadd a is a small -kda predominantly nuclear protein that interacts with other proteins implicated in stress responses, including proliferating cell nuclear antigen, p , cdc /cyclin b , mekk , and p kinase [ , ] . gadd induces cell cycle arrest and apoptosis in most of cells as well as promoting dna repair functions and survival [ ] . growth arrest and dna damage gene (gadd ) also maintains genomic stability in a p -responsive manner [ ] . despite the multiple known functions of gadd , its role ali, endothelial/epithelial barrier dysfunction, or repair of injured lung is unknown [ ] . gadd exhibited differential expression in orthologous global gene expression profiling, in multispecies ali models [ ] , in region-specific lung tissue expression profiling [ ] , and was markedly upregulated in response to the vili [ ] . we explored the mechanistic involvement of gadd a in endotoxin (lps)and ventilator-induced inflammatory lung injury (vili) by comparing multiple biochemical and genomic parameters of inflammatory lung injury in wild-type c bl/ and gadd a −/− knockout mice exposed to high tidal volume ventilation (vili) or intratracheally administered lps [ ] . gadd a −/− mice were modestly susceptible to lpsinduced injury but were profoundly susceptible to vili, demonstrating increased inflammation and increased microvascular permeability. vili-exposed gadd a −/− mice manifested striking neutrophilic alveolitis with increased bal fluid levels of protein, igg, and inflammatory cytokines. expression profiling of lung homogenates revealed strong dysregulation in the b cell receptor signaling pathway in gadd a −/− mice, suggesting the involvement of phosphatidylinositol -kinase/akt signaling components. western blots confirmed a threefold reduction in akt protein and phosphorylated akt levels observed in gadd a −/− lungs. electrical resistance measurements across human lung endothelial cell monolayers transfected with small interfering rnas to reduce gadd a or akt expression revealed significant potentiation of lps-induced endothelial barrier dysfunction which was attenuated by overexpression of a constitutively active akt transgene. whereas other lung injury studies failed to demonstrate a role for gadd in hyperoxic lung injury [ , ] , our studies validate gadd a as a novel inflammatory lung injury candidate gene and a significant participant in vascular barrier regulation via effects on akt-mediated endothelial signaling [ ] . thus, both akt and gadd are extremely attractive ali candidate genes. the human gadd a gene contains validated snps (national center for biotechnology information snp database) whose role in ali pathogenesis is completely unknown [ ] . we are currently pursuing further characterization of the role of gadd a and its association of genetic variants with sepsis and ali. the identification of novel pathways involved in the pathobiology of ali also opens doors for the exploration of new therapeutic targets for the disease. as such, the use of agents that attenuate the endothelial barrier dysfunction and the inflammatory response characteristic of ali have shown promise in preclinical studies which will hopefully lead to their use in trials of human ali (fig. ). s p, an important lipid mediator generated by the phosphorylation of sphingosine by sphingosine kinase, decreases endothelial permeability to both water and solute via cytoskeletal reorganization and adherens junction assembly [ , ] . s p-induced barrier protective effects could serve to attenuate the increased pulmonary vascular permeability essential factor in the development of ali. the s p analogue fty ( . mg/kg),when administered to c bl/ mice with endotoxin-induced lung injury, decreases lung edema formation, solute transport across the alveolar capillary endothelium, and inflammatory cell infiltration into lung parenchyma [ ] . similarly, the prophylactic administration of s p attenuates both alveolar and vascular barrier dysfunction while significantly reducing shunt formation associated with lung injury in rodent and canine models of ali induced by combined intrabronchial endotoxin administration and hightidal-volume mechanical ventilation [ ] . in a recent study of a canine model of ali, we demonstrated that when bacterial endotoxin was instilled intratracheally followed in h by intravenous administration of s p ( mg/kg) or vehicle and h of high-tidal-volume mechanical ventilation [ ] , s p treatment attenuated the severity of ali-induced increases in shunt fraction and the presence of both protein and neutrophils in bal fluid compared with vehicle controls. interestingly, bal fluid cytokine production was not altered fig. mechanism-based novel therapies for ali. the identification of novel pathways involved in the pathobiology of ali also facilitates the exploration of new therapeutic targets. sphingosine -phosphate (s p ) attenuates the endothelial barrier dysfunction associated with ali, whereas blocking of pbef attenuates vali significantly by intravenous administration of s p and s p potentiated the endotoxin-induced systemic production of the inflammatory cytokines tnfa, c-x-c chemokine ligand- , and il- , without resulting in end-organ dysfunction. these data suggest that s p may represent a viable therapy for the prevention and treatment of ali. as previously described in this chapter, pbef appears to play a central role in the promotion of several pathogenetic aspects of ali and vali. therefore, interventions aimed at attenuating the effects of pbef could have a potential therapeutic effect in these disorders. to begin to address the potential for pbef to serve as a therapeutic target in ameliorating vili, we assessed the effect of pbef neutralizing antibody on rhpbef-stimulated lung inflammation [ ] . simultaneous instillation of rhpbef and pbef neutralizing antibody produced dramatic reductions in rhpbef-induced neutrophil recruitment. further, the intratracheal delivery of pbef neutralizing antibody ( min before high-tidal-volume mechanical ventilation) abolished vili-induced increases in total bal fluid cell counts and significantly decreased neutrophil influx into the alveolar space as well as vili-mediated increases in the level of lung tissue albumin. ali is a major cause of morbidity and mortality in critically ill patients. given the unacceptably high mortality rate observed in ali and the paucity of novel therapies and biomarkers, it is essential to recognize molecular targets associated with ali to identify individuals at risk and to develop novel therapeutic targets and biomarkers. it is clear that derangements in endothelial cell barrier regulation play a major role in the pathobiology of ali and genetic variants regulate endothelial cell barrier function, thereby determining ali risk or subsequent severity of outcome. high-throughput gene sequencing and expression technologies, and complete genome sequencing of model organisms, have allowed for the performance of large-scale analyses of the genome in ali. in this chapter, we have highlighted how global gene expression profiling in multispecies ali models served to broaden our net knowledge of ali-implicated genes and provide a basis for hope that increased insights and therapies may be forthcoming. as genotyping becomes more rapid and easily accessed, combining advanced bioinformatics techniques with high-throughput methods will be the future practice of personalizing treatment strategies. continued challenges will be the gene-gene and gene-environment interactions, which add complexity to our understanding of the genome. these novel genetic approaches may prove exceptionally useful in ushering in the era of personalized medicine for critically ill individuals. the acute respiratory distress syndrome genomics of acute lung injury clinical predictors of the adult respiratory distress syndrome race and gender differences in acute respiratory distress syndrome deaths in the united states: an analysis of multiple-cause mortality data ( - ) ozone-induced acute pulmonary injury in inbred mouse strains making genomics functional: deciphering the genetics of acute lung injury wading into the genomic pool to unravel acute lung injury genetics susceptibility to neoplastic and non-neoplastic pulmonary diseases in mice: genetic similarities fate of angiotensin i in the circulation angiotensin ii induces apoptosis of human endothelial cells. protective effect of nitric oxide haemodynamic and endocrine effects of type angiotensin ii receptor blockade in patients with hypoxaemic cor pulmonale angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome angiotensin ii, via at and at receptors and nf-kb pathway, regulates the inflammatory response in unilateral ureteral obstruction inflammation and angiotensin ii angiotensin ii and the fibroproliferative response to acute lung injury angiotensin ii induces apoptosis in human and rat alveolar epithelial cells abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor angiotensin-converting enzyme protects from severe acute lung failure an insertion/deletion polymorphism in the angiotensin i-converting enzyme gene accounting for half the variance of serum enzyme levels evidence, from combined segregation and linkage analysis, that a variant of the angiotensin i-converting enzyme (ace) gene controls plasma ace levels alhenc-gelas f ( ) angiotensin i-converting enzyme in human circulating mononuclear cells: genetic polymorphism of expression in t-lymphocytes genetic polymorphisms associated with susceptibility and outcome in ards polymorphism of the angiotensin-converting enzyme gene affects the outcome of acute respiratory distress syndrome differences in frequency of the deletion polymorphism of the angiotensinconverting enzyme gene in different ethnic groups angiotensin-converting enzyme gene (ace) insertion/deletion polymorphism in mexican populations elevated plasma levels of soluble tnf receptors are associated with morbidity and mortality in patients with acute lung injury high berylliumstimulated tnf-a is associated with the - tnf-a promoter polymorphism and with clinical severity in chronic beryllium disease tumor necrosis factor gene polymorphism and septic shock in surgical infection ga and tnfb polymorphisms in acute respiratory distress syndrome influence of tnfa gene polymorphisms on tnfa production and disease single nucleotide polymorphism in the tumor necrosis factor-alpha gene affects inflammatory bowel diseases risk il gene-wide haplotype is associated with susceptibility to acute lung injury cytokine balance in the lungs of patients with acute respiratory distress syndrome a prospective study of inflammation markers in patients at risk of indirect acute lung injury persistent elevation of inflammatory cytokines predicts a poor outcome in ards. plasma il- beta and il- levels are consistent and efficient predictors of outcome over time pressure-time curve predicts minimally injurious ventilatory strategy in an isolated rat lung model science review: searching for gene candidates in acute lung injury orthologous gene-expression profiling in multispecies models: search for candidate genes use of consomic rats for genomic insights into ventilator-associated lung injury the association of interleukin haplotype clades with mortality in critically ill adults vascular endothelial growth factor and related molecules in acute lung injury lung overexpression of the vascular endothelial growth factor gene induces pulmonary edema vascular endothelial growth factor may contribute to increased vascular permeability in acute respiratory distress syndrome decreased vegf concentration in lung tissue and vascular injury during ards a common c/t mutation in the gene for vascular endothelial growth factor is associated with vascular endothelial growth factor plasma levels vascular endothelial growth factor gene polymorphism and acute respiratory distress syndrome genotypes and haplotypes of the vegf gene are associated with higher mortality and lower vegf plasma levels in patients with ards chemokine receptors as hiv- coreceptors: roles in viral entry, tropism, and disease involvement of chemokine receptors in breast cancer metastasis myosin iia is involved in the endocytosis of cxcr induced by sdf- a the a-chemokine, stromal cell-derived factor- a, binds to the transmembrane g-protein-coupled cxcr- receptor and activates multiple signal transduction pathways rab -family interacting protein and myosin vb are required for cxcr recycling and receptor-mediated chemotaxis role of tyrosine phosphorylation in ligand-independent sequestration of cxcr in human primary monocytes-macrophages a common stromal cell-derived factor- chemokine gene variant is associated with the early onset of type diabetes genetic restriction of aids pathogenesis by an sdf- chemokine gene variant. alive study, hemophilia growth and development study (hgds) high-resolution haplotype structure in the human genome complement factor h polymorphism in age-related macular degeneration a genome-wide association study identifies il r as an inflammatory bowel disease gene a genome-wide association study identifies novel risk loci for type diabetes genomewide association studies of stroke microarray analysis of regional cellular responses to local mechanical stress in acute lung injury novel interaction of cortactin with endothelial cell myosin light chain kinase cytoskeletal regulation of pulmonary vascular permeability activated protein c mediates novel lung endothelial barrier enhancement: role of sphingosine -phosphate receptor transactivation novel polymorphisms in the myosin light chain kinase gene confer risk for acute lung injury transactivation of sphingosine -phosphate receptors is essential for vascular barrier regulation. novel role for hyaluronan and cd receptor family regulation of endothelial cell gap formation and barrier dysfunction: role of myosin light chain phosphorylation regulation of endothelial cell gap formation and paracellular permeability protein kinase involved in lung injury susceptibility: evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment critical role of non-muscle mlck in ventilator-induced lung injury myosin light chain kinase in endothelium: molecular cloning and regulation adherent neutrophils activate endothelial myosin light chain kinase: role in transendothelial migration variation in the mylk gene is associated with development of acute lung injury after major trauma a variant of the myosin light chain kinase gene is associated with severe asthma in african americans macrophage migration inhibitory factor: a regulator of glucocorticoid activity with a critical role in inflammatory disease macrophage migration inhibitory factor role for macrophage migration inhibitory factor in acute respiratory distress syndrome mif as a glucocorticoid-induced modulator of cytokine production macrophage migration inhibitory factor governs endothelial cell sensitivity to lps-induced apoptosis intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (mif) in endothelium macrophage migration inhibitory factor in acute lung injury: expression, biomarker, and associations role of macrophage migration inhibitory factor (mif) in human and animal models of acute lung injury (ali) and sepsis: association of a promoter polymorphism and increased gene expression a novel dna vaccine-targeting macrophage migration inhibitory factor improves the survival of mice with sepsis role of macrophage migration inhibitory factor (mif) in allergic and endotoxininduced airway inflammation in mice aquaporin- : a candidate gene in sepsis and lung injury a novel ¢-flanking region polymorphism of macrophage migration inhibitory factor is associated with systemic-onset juvenile idiopathic arthritis sphingosine -phosphate promotes endothelial cell barrier integrity by edg-dependent cytoskeletal rearrangement regulation of sphingosine -phosphate-induced endothelial cytoskeletal rearrangement and barrier enhancement by s p receptor, pi kinase, tiam /rac , and alpha-actinin regulation of the micromechanical properties of pulmonary endothelium by s p and thrombin: role of cortactin protective effects of high-molecular weight polyethylene glycol (peg) in human lung endothelial cell barrier regulation: role of actin cytoskeletal rearrangement synthesis and biological evaluation of g-aminophosphonates as potent, subtypeselective sphingosine -phosphate receptor agonists and antagonists enhancement of capillary leakage and restoration of lymphocyte egress by a chiral s p antagonist in vivo protective effects of sphingosine -phosphate in murine endotoxin-induced inflammatory lung injury tipping the gatekeeper: s p regulation of endothelial barrier function effect of s p receptor agonists on murine lung airway function differential regulation of sphingosine- -phosphate-and vegf-induced endothelial cell chemotaxis. involvement of g ia -linked rho kinase activity endothelial cell barrier enhancement by atp is mediated by the small gtpase rac and cortactin sphingosine- -phosphate receptor variant increases promoter activity and decreases susceptibility to human asthma hepatocyte growth factor/scatter factor induces a variety of tissue-specific morphogenic programs in epithelial cells the biological role of hgf-met axis in tumor growth and development of metastasis ) c-met mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions involvement of hepatocyte growth factor in formation of bronchoalveolar structures in embryonic rat lung in primary culture hepatocyte growth factor may act as a pulmotrophic factor on lung regeneration after acute lung injury intratracheal administration of hepatocyte growth factor/scatter factor stimulates rat alveolar type ii cell proliferation in vivo met receptor dynamics and signalling c-met signalling: spatio-temporal decisions cd regulates hepatocyte growth factor-mediated vascular integrity. role of c-met, tiam /rac , dynamin , and cortactin keratinocyte growth factor and hepatocyte growth factor in bronchoalveolar lavage fluid in acute respiratory distress syndrome patients hepatocyte growth factor and keratinocyte growth factor in the pulmonary edema fluid of patients with acute lung injury. biologic and clinical significance pre-b-cell colonyenhancing factor as a potential novel biomarker in acute lung injury pre-b-cell colony-enhancing factor gene polymorphisms and risk of acute respiratory distress syndrome pre-b-cell-colonyenhancing factor is critically involved in thrombin-induced lung endothelial cell barrier dysregulation pre-b cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase- activity essential role of pre-b-cell colony enhancing factor in ventilator-induced lung injury a visfatin promoter polymorphism is associated with low-grade inflammation and type diabetes effects of genetic variation in the visfatin gene (pbef ) on obesity, glucose metabolism, and blood pressure in children genome-wide search for atopy susceptibility genes in dutch families with asthma two stage genome-wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes , and chromosome q - and multiple sclerosis: evidence for a genetic susceptibility effect in vicinity to the protachykinin- gene egad, more forms of gene regulation: the gadd a story gadd in the response of hematopoietic cells to genotoxic stress liebermann da genotoxic-stress-response genes and growth-arrest genes gadd, myd, and other genes induced by treatments eliciting growth arrest myeloid differentiation (myd)/growth arrest dna damage (gadd) genes in tumor suppression, immunity and inflammation genomic instability in gadd a −/− cells is coupled with s-phase checkpoint defects inhaled carbon monoxide confers antiinflammatory effects against ventilatorinduced lung injury gadd a is a novel candidate gene in inflammatory lung injury via influences on akt signaling -independent induction of gadd and gadd in mouse lungs exposed to hyperoxia loss of gadd a does not modify the pulmonary response to oxidative stress modulation of lipopolysaccharide-induced gene transcription and promotion of lung injury by mechanical ventilation sphingosine -phosphate reduces vascular leak in murine and canine models of acute lung injury sphingosine -phosphate rescues canine lps-induced acute lung injury and alters systemic inflammatory cytokine production in vivo key: cord- - j cdt b authors: chiang, eddie t.; wang, ting; garcia, joe g. n. title: acute lung injury: the injured lung endothelium, therapeutic strategies for barrier protection, and vascular biomarkers date: - - journal: textbook of pulmonary vascular disease doi: . / - - - - _ sha: doc_id: cord_uid: j cdt b the vascular endothelium can be considered as an organ/tissue which comprises a monolayer of endothelial cells which serve as a semipermeable cellular barrier separating the inner space of blood vessels from its surrounding tissue and to control the exchange of fluids and cells between the two compartments. since the pulmonary circulation receives the entire cardiac output, the large surface area of the lung microvasculature is well suited for sensing mechanical, chemical, and cellular injury by inhaled or circulating substances. this endothelial barrier is dynamically regulated through exposure to these various stimuli of physiological and pathological origin and serves to regulate multiple key biological processes (including lung fluid balance and solute transport between vascular compartments). for example, an increase in vascular permeability is a necessary feature of the body’s defense mechanism to provide injured tissues with access to leucocytes, resulting in tissue edema due to fluid extravasation. however, during conditions of intense lung inflammation such as observed in acute lung injury or its severer form of acute respiratory distress syndrome, the large surface area becomes a liability and provides the opportunity for profound vascular permeability resulting in massive fluid accumulation in the alveolar space and progressively leading to pulmonary failure. alterations in vascular permeability occur not only in acute inflammatory lung disorders primarily caused by sepsis, pneumonia, and trauma which result in high rates of patient morbidity and mortality, but are an attractive target for therapeutic intervention in subacute lung inflammatory disorders such as ischemia–reperfusion injury, radiation lung injury, and asthma. thus, understanding the mechanisms of endothelial barrier dysfunction is vital for the management and treatment of key and enigmatic pulmonary disorders. the vascular endothelium can be considered as an organ/ tissue which comprises a monolayer of endothelial cells (ecs) which serve as a semipermeable cellular barrier separating the inner space of blood vessels from its surrounding tissue and to control the exchange of fluids and cells between the two compartments. since the pulmonary circulation receives the entire cardiac output, the large surface area of the lung microvasculature is well suited for sensing mechanical, chemical, and cellular injury by inhaled or circulating substances. this endothelial barrier is dynamically regulated through exposure to these various stimuli of physiological and pathological origin and serves to regulate multiple key biological processes (including lung fluid balance and solute transport between vascular compartments). for example, an increase in vascular permeability is a necessary feature of the body's defense mechanism to provide injured tissues with access to leucocytes, resulting in tissue edema due to fluid extravasation. however, during conditions of intense lung inflammation such as observed in acute lung injury (ali) or its severer form of acute respiratory distress syndrome (ards), the large surface area becomes a liability and provides the opportunity for profound vascular permeability resulting in massive fluid accumulation in the alveolar space and progressively leading to pulmonary failure. alterations in vascular permeability occur not only in acute inflammatory lung disorders primarily caused by sepsis, pneumonia, and trauma which result in high rates of patient morbidity and mortality [ , ] , but are an attractive target for therapeutic intervention in subacute lung inflammatory disorders such as ischemia-reperfusion injury [ ] , radiation lung injury [ , ] , and asthma [ , ] . thus, understanding the mechanisms of endothelial barrier dysfunction is vital for the management and treatment of key and enigmatic pulmonary disorders. a key concept of the dynamically regulated lung ec barrier is the notion that two general pathways, transcellular and paracellular, that describe the movement and flow of fluid, macromolecules, and leukocytes into the interstitium (and subsequently the alveolar air spaces) produce clinically significant pulmonary edema during inflammatory lung processes (fig. ) . the transcellular pathway utilizes a tyrosine kinase dependent, gp -mediated transcytotic albumin route, an active process of albumin transport in which endocytic vessels fuse with the endothelium in response to surface glycoprotein (gp ) receptor ligation [ ] . however, there is general consensus that the primary mode of fluid and transendothelial leukocyte trafficking occurs by the paracellular pathway as shown by the elegant electron microscopy studies of majno and palade [ , ] , who demonstrated lung ec rounding and paracellular gap formation at sites of active inflammation within the lung vasculature. disruption of the integrity of the ec monolayer is now recognized as a cardinal feature of inflammation, ischemiareperfusion injury, and angiogenesis and occurs in response to a variety of mechanical stress factors, inflammatory mediators, and activated neutrophil products [reactive oxygen species (ros), proteases, cationic peptides]. the dramatic cell shape change which results in paracellular gap formation implicates the direct involvement of endothelial structural components composed of cytoskeletal proteins (microfilaments and microtubules). thus, although once perceived as a passive cellular barrier, ecs are now recognized as a highly dynamic tissue contributing to the multiple dimensions of ec function, including interactions with a number of barrier-regulatory effectors via the endothelial cytoskeleton. the duration and outcome of inflammatory disease processes depends upon the balance between the severity of endothelial injury caused by adhesive biophysical forces, mechanical shear stress (ss), or receptor ligation by specific inflammatory mediators and the efficiency of endogenous repair mechanisms to restore vascular integrity [ , ] . in this chapter, we will ( ) address the role of cytoskeletal rearrangement in mechanistic regulation of pulmonary vascular barrier function and permeability, ( ) define current strategies designed to enhance the integrity of the lung vascular endothelium, and ( ) identify vascular biomarkers and potential prognostic determinants of acute inflammation. it is now well accepted that dynamic cytoskeletal elements, actin, microtubules, and intermediate filaments (ifs) , are key elements of vascular barrier regulation. the vast majority of the studies contributing to this recognition have focused on agonist-mediated signaling to the actomyosin cytoskeleton with subsequent effects on lung vascular barrier-regulatory properties. historically viewed as separate and distinct cytoskeletal systems, microtubules and actin filaments are now known to interact functionally during dynamic cellular processes. the microtubule scaffolding complex [ , ] , with a central role of tubulin dynamics, actively contributes to cytoskeletal rearrangement and in transducing competing barrier-regulatory forces, often in close collaboration with microfilament elements. much less is known about ifs, an enigmatic component of the ec cytoskeleton consisting of dimer structured a-helical proteins which combine to form fibrils. if proteins are expressed in a specific manner, with vimentin the primary if protein found in ecs. the role of ifs in regulating ec barriers represents a fertile area for future investigations as only limited information is available [ , ] . nevertheless, cytoskeletal constituents together provide the capacity for dynamic regulation of cell shape and, as a consequence, of moment-to-moment adaptation to an ever-changing vascular environment. actin, a globular protein with a centrally located atp-binding site, is critical to many cellular processes, including cell motility, cell division, cell signaling, and as we and others have shown, ec permeability [ ] [ ] [ ] . g-actin reversibly assembles to form polymerized actin fibers called filamentous actin (f-actin) or actin microfilaments ( -nm diameter), conferring strength to structural elements regulating cell shape, particularly when accompanied by phosphorylated myosin. dynamic remodeling of actin filaments within peripherally distributed cortical bands is essential for maintenance of endothelial integrity and basal barrier function, with inhibition of actin polymerization (cytochalasin d) directly increasing ec permeability [ ] . edemagenic agents initiate dramatic cytoskeletal rearrangement characterized by the loss of peripheral actin filaments with a concomitant increase in organized actin cables that span the cell, known as "stress fibers." critically involved in regulating the spatial locale and level of actin cycling (polymerization-depolymerization) are numerous actin-binding proteins which serve as cross-linking/ bundling proteins, polymerization/depolymerization proteins, and capping/severing proteins. one key actin-binding protein and central regulator of the ec contractile apparatus is the ca + /calmodulin-dependent nonmuscle isoform of myosin light chain kinase (nmmlck). phosphorylation of the substrate myosin light chain (mlc) by nmmlck is central to paracellular gap formation and increased permeability by many edemagenic agents, including thrombin [ ] and vascular endothelial growth factor (vegf) [ ] , both in vitro and in preclinical models of inflammatory lung injury. studies with nmmlck knockout mice have revealed protection from sepsis-induced ali and our laboratory has shown that nmmlck knockout mice, as well as mice treated with an inhibitory peptide which reduces mlc kinase (mlck) activity, are protected against ventilator-induced lung injury (vili) [ ] . in addition, we have shown that genetic variants (single-nucleotide polymorphisms) in mylk, the gene on chromosome q encoding mlck, confer significant susceptibility to sepsis, and sepsisand trauma-induced ali [ ] , as well as contributing to risk of severe asthma in african americans, another inflammatory lung disorder [ ] . a key regulatory feature of nmmlck is the posttranslational modification (ptm) by increased levels of nmmlck tyrosine phosphorylation catalyzed by either p src kinase or c-abl kinase, or by inhibition of tyrosine phosphatases (vanadate). this ptm serves to increase kinase activity and modulates ec barrier responses [ , [ ] [ ] [ ] . diperoxovanadate, a potent tyrosine phosphatase inhibitor, also increased nmmlck activity, the number of stress fibers, and ec contraction via activation of p src kinase. the nmmlck isoform binds cortactin, another actin-binding protein and ec barrier regulator which localizes to numerous cortical structures within cells [ ] . the sh domain in cortactin binds the proline-rich areas in nmmlck [ , , ] , with this interaction enhancing cortical actin formation and tensile strength. the central region of cortactin binds and cross-links actin filaments, with its c-terminus site for p src kinase-mediated phosphorylation which reduces cross-linking activity. tyrosine phosphorylation of cortactin by p src potentiates and stabilizes actin polymerization, and strengthens cortactin-nmmlck interactions [ ] , and is a key step in a sequence of events that produce cytoskeletal changes, reassembly of adherens junctions (ajs), and barrier restoration during lung inflammation. microtubules are -nm polymers of a-tubulin and b-tubulin that form a lattice network of rigid hollow rods spanning the cell in a polarized fashion from the nucleus to the periphery while undergoing frequent assembly and disassembly [ , ] . important functions of microtubules include intracellular transport of vesicles and organelles, as well as signal transduction and cytoskeletal structure. in addition, microtubules act in concert with the actin cytoskeleton to promote ec barrier integrity. microtubules and actin filaments exhibit complex, but intimate functional interactions during dynamic cellular processes [ ] [ ] [ ] [ ] . microtubule disruption with an agent such as nocodazole or vinblastine induces rapid assembly of actin filaments and focal adhesions, isometric cellular contraction that correlates with the level of mlc phosphorylation, increased permeability across ec monolayers, and increased transendothelial leukocyte migration, events that can be reversed or attenuated by microtubule stabilization with paclitaxel [ , ]. the mechanisms involved in these effects are poorly understood but are likely to be mediated through interaction with actin filaments, suggesting significant microfilament-microtubule cross talk. disruption of microtubules causes actin cytoskeletal remodeling, cell contraction, and decreased transendothelial resistance through a rho kinase induced phosphorylation of mypt , a mlc phosphatase [ , ] . nocadozole causes formation of stress fibers and myofilament assembly accompanied by increases in mlc phosphorylation, remodeling of ajs [ , ] , and barrier disruption [ ] . microtubule stabilization with paclitaxel inhibits the formation of stress fibers and preserves cellular shape and intercellular contacts [ ] . although these effects are poorly understood, microfilament-microtubule cross talk represents an intriguing area of ec barrier regulation [ , ]. ifs, the third major element involved in ec cytoskeletal structure, were defined on the basis of their - -nm filament structure which distinguished them from -nm microfilament and -nm microtubules. despite greater diversity than the highly conserved components of either actin microfilaments or microtubules, if proteins share a common dimer structure containing two parallel a-helices which combine to form polar fibrils that associate with an array of if-binding proteins while connecting to the nuclear envelope, peripheral cell junctions, and other cytoskeletal components. if proteins are expressed in a highly cell specific manner, with vimentin being the primary if protein found in ecs and other cells of mesenchymal origin. although these data suggest potential roles for ifs in ec cytoskeletal structure and barrier function, these effects are likely to be subtle and subject to compensation by biological redundancy and the function of ifs in ec barrier regulation is much less understood [ ] . assembly of ifs is a complex process likely highly regulated by signaling cascades associated with cell motility. vimentin is a dynamic structure undergoing constant assembly/disassembly, as well as anterograde and retrograde movements. microtubulebased movement of ifs is likely critical for assembly and maintenance of the vimentin if network [ , ] . the physical and dynamic properties of the vimentin network in the vascular ec are likely important in regulation of cell shape and resistance to hemodynamic stress that accompanies blood flow and resistance to shear strain, physiological changes regulated by the if cytoskeleton, and if-associated proteins which serve as internal scaffolding for ecs, linked to the plasma membrane, and to junctional contacts. vimentin protein expression is higher in macrovascular ec lining vessels subjected to the highest hemodynamic strain, such as the aorta, compared with microvascular ec lining vessels under less ss. vimentin knockout mice develop normally without gross blood vessel abnormalities, but with reduced mesenteric artery vessel dilation in response to flow [ , ] . downstream responses to flow may be the result of intracellular mechanosignaling events triggered by deformation of the if cytoskeleton. changes in unidirectional laminar flow results in rapid adaptation of the ec vimentin network, with directional displacement within minutes of initial exposure. as noted with microfilaments and microtubules, over a period of hours, cytoskeletal filaments align themselves in the direction of flow, with significantly larger change in the vimentin distribution around the nucleus compared with displacement occurring in the cytosol closer to the substrate. these observed spatial changes may be a means of distribution of local shear force transmission throughout the cell and therefore convey cell signaling messages via a mechanosignaling pathway. thus, vimentin ifs are likely critical for maintaining the structural integrity of ecs under ss, and may also be a conduit for signaling cascades triggered by mechanical force, again an exciting area for future examination. dynamic equilibrium exists between ec contractile forces and the adhesive protein-cytoskeleton linkages with cellcell and cell-matrix interactions necessary for proper barrier function. a major contributor to the intact cellular barrier is the tight apposition of individual ecs with neighboring cells via intercellular junctions which collectively contribute to basal endothelial barrier function. the two primary types of intercellular contacts between ecs are ajs and tight junctions (tjs), both of which link the ec actin cytoskeletons of neighboring cells to each other while providing mechanical stability and mediating signal transduction [ ] (fig. ) . in addition to cell-cell junction stability, cell-matrix interaction also contributes to stability of the barrier function. specific components of the focal adhesion complex, i.e., the integrin-based linkage between the extracellular matrix (ecm) and the endothelial cytoskeleton, provide strong tethering of the endothelium to the vessel wall and thus enhanced barrier integrity. ajs are composed of cadherins bound together in a homotypic-and ca + -dependent fashion to link adjacent ecs [ ] . cadherins interact through their cytoplasmic tail with the catenin family of intracellular proteins (primarily b-catenin), which in turn provide anchorage to the actin cytoskeleton [ ] . the primary adhesive protein present in human endothelial ajs, vascular endothelial cadherin (ve-cadherin) [ ] , is critical to maintenance of ec barrier integrity as demonstrated by increased vascular permeability induced in mice after infusion of ve-cadherin blocking antibody [ ] . similarly, in cultured ecs, ve-cadherin blocking antibody enhanced neutrophil transendothelial migration while producing reorganization of the actin cytoskeleton [ ] . anchorage of ve-cadherin to the actin cytoskeleton is crucial to maintaining barrier integrity since a cytoplasmic-deleted ve-cadherin which cannot anchor to the actin cytoskeleton still forms cadherin-cadherin binding but results in increased vascular permeability [ ] . tjs, or zona occludens, are areas that surround the entire apical perimeter of adjacent cells and are formed by the fusion of the outer layers of the plasma membranes. these associations are sufficiently tight as to form a virtually impermeable barrier to fluid [ , ] and are composed of occludins, claudins, and junctional adhesion molecules coupled to cytoplasmic proteins [ ] (fig. ). similar to ve-cadherin at ajs, ecs express a cell-type-specific transmembrane adhesion protein, claudin- , at tjs. the cytoplasmic components of tjs are linked to the ec actin cytoskeleton by the zona occludens family (zo- ). tjs are particularly abundant and prominent in the brain microvasculature and epithelial cells, where strict control of permeability is needed. in contrast, most microvascular beds of ecs, particularly leaky lung microvasculature, have less defined tj structures and more prominent aj structures. therefore, ajs and not tjs have historically been considered the primary targets involved in junctional protein dissociation resulting in increased paracellular permeability, but there is growing evidence that tjs may play a larger role in the regulation of paracellular permeability in the lung than previously thought [ ] . finally, focal adhesions are intimately involved in lung ec barrier regulation via signaling between the cytoskeleton to the ecm. focal adhesions are attachments of ecs to the underlying ecm and are mediated by ecm proteins (i.e., collagen, fibronectin, laminin, etc.), integrins, and cytoplasmic adhesion plaques (containing vinculin, talin, and paxillin) [ , ] . integrins couple the ecm to the cytoskeleton and transmit signals from the surrounding environment and play a key role in the formation of cell adhesion complexes which attach to the actin cytoskeleton via the cytoskeletal proteins actin, vinculin, talin, and a-actinin. focal adhesions, primarily through integrins, form a bridge for bidirectional signal transduction between the actin cytoskeleton and the cell-matrix interface. disruptions of the integrin-ecm connection can increase ec permeability [ , ] and integrins modulate ec permeability to ss and inflammatory mediators [ ] . integrin-ecm binding stimulates tyrosine phosphorylation of proteins such as paxillin, cortactin, and focal adhesion kinase (fak), as well as calcium influx [ , ] . fak is the principal kinase which catalyzes the downstream reactions of integrin engagement and focal adhesion assembly [ ], with fak activity regulated by tyrosine phosphorylation mediated by the src family. activation of fak through tyrosine phosphorylation produces cell contraction and increased ec barrier permeability. we previously reported that integrin b expression is dramatically upregulated upon challenge with the barrier-protective agent simvastatin [ ] . furthermore, the upregulation of integrin b attenuates endotoxin-and ventilator-induced expression of inflammatory cytokines interleukin (il)- and il- [ ], suggesting a novel mechanism of modulating endothelial barrier function via integrin b and focal adhesion signaling. inflammatory mediators increase vascular permeability by disrupting endothelial junctions and focal adhesion complexes as well as inducing cellular contraction to open paracellular gaps [ , - ]. as tjs and ajs are ideally situated in a locale between cell-cell junctions, they logically are key participants in the control of vascular paracellular permeability and monolayer integrity. recent studies in brain ecs have focused on the importance of claudins in tj formation and maintenance [ , ] . mice with claudin- gene knocked out did not have a morphologically altered vascular network or tj structures, but the claudin- -deficient pups died within h of birth owing to size-selective loosening of the bloodbrain barrier against molecules of less than kda. it appears that moderate redundancy among the claudin isoforms may allow for the formation of the tj, but not for the complete function of the tj. claudin- appears to act in concert with claudin- to form the tightly organized strand network, but in claudin- mutants, claudin- can only maintain the barrier against larger molecules [ ], suggesting claudin- is a structural barrier, whereas claudin- is crucial for the dynamic regulation of tj permeability. gene inactivation of claudin- and occludin also has no effects on vascular morphology or barrier permeability, suggesting a minor role in tj function in endothelium as compared with claudin- and claudin- inactivation of these genes in mice does not cause any defect in the development of the vascular system in the embryo, but in adult mice these molecules play an important role in modulating leukocyte diapedesis through ecs. jam-cs however, is unique in that unlike other junctional proteins, it increases endothelial permeability when expressed at the ec surface, suggesting a role in promoting and/or organizing junction formation [ ] . this activity is mediated by ve-cadherin activity and actin organization, as well as by kinases and phosphatases that modulate tj protein phosphorylation and endothelial permeability. many of the studies on tj have been using brain ecs, where the adhesion molecules are prominent. nevertheless, recent studies on lung ecs have demonstrated that despite the less prominent formation of tjs as compared with ajs, tjs may play a critical role in the endothelial barrier dysfunction associated with exposure to particulate matter from air pollution, which has been shown to induce a gradual and prolonged barrier dysfunction in cultured lung endothelium [ , ] . ajs were found unexpectedly to be unaltered but the tjs, specifically zo- , were degraded through a calpain-dependent proteasome pathway, a novel mechanism of lung endothelial barrier regulation. in contrast to tjs, the regulation of lung vascular integrity involving ajs has been well characterized. although ve-cadherin is present in high concentration in all ecs, different types of vessels appear to modify ve-cadherin expression to complement the vascular barrier function of that particular vessel. four modes of aj protein regulation of permeability have been described, all involving ve-cadherin: phosphorylation, internalization, cleavage, and expression. simultaneous coordination of ve-cadherin phosphorylation and internalization appears to be crucial for a rapid response to an increase in permeability [ ], whereas ve-cadherin cleavage and expression are progressive alterations. edemagenic stimuli induce tyrosine phosphorylation of aj proteins (ve-cadherin, b-catenin, and p catenin), which parallels increases in permeability, with the tyrosine kinase src implicated in the phosphorylation of aj proteins as it directly associates with the ve-cadherin/catenin complex, and src gene inactivation or treatment with inhibitors blocks vegf-induced ve-cadherin phosphorylation [ ] . phosphorylation of ve-cadherin is dependent on kinase activation as well as inhibition of associated phosphatases such as the endothelial-specific phosphatase ve-ptp, which also associates with ve-cadherin, and inactivation of the ve-ptp gene leads to a phenotype comparable to that of ve-cadherin null embryos, suggesting that vessels cannot form correctly if ve-cadherin is constantly phosphorylated [ ] . permeability may also be regulated by ve-cadherin internalization. typically, p catenin binds to ve-cadherin and acts as a plasma membrane retention signal to prevent ve-cadherin internalization; however, upon challenge with barrier-disrupting stimuli, activated src phosphorylates vav , a guanine exchange factor (gef) for rac, which then phosphorylates ve-cadherin at ser , inducing b-arrestin recruitment and promoting clathrin-dependent ve-cadherin internalization [ ]. angiopoietin induces src trapping by mdia, reducing its activity at ajs, and thus reducing vascular permeability [ ] . the third pathway that may induce vascular permeability is ve-cadherin cleavage. ve-cadherin is particularly susceptible to enzymatic proteolysis, specifically elastase and adam- , which are released in high amounts by leukocytes, promoting ve-cadherin cleavage, cell extravasation, and vascular leakage. lastly, permeability control may also be achieved through ve-cadherin gene expression. the ve-cadherin promoter contains several binding sites for transcription factors, tal- , erg, and hypoxia-inducible factors. therefore, the interendothelial junction is a key site of regulating vascular permeability, with various stimuli targeting either the tj or the aj, or both. furthermore, there are various combinatory modes of regulating the aj that promote dissociation of adhesion proteins in the cell-cell junction. however, additional factors often accompany junctional dissociation, such as disbanding of cortical cytoskeleton and increase in cellular contraction, which augment the barrier dysfunction. the monolayer integrity is regulated by the dynamic equilibrium which exists between contractile forces and tethering forces [ , , ] . transcellular stress fiber formation and activation of actomyosin interaction, along with the cortical actin ring disassembly, results in contractile tension that induces cell rounding, which contributes to cell-cell gap formation ( fig. ) , with inhibition of this cytoskeletal reorganization attenuating barrier dysfunction [ , ] . contraction triggered in ecs is regulated by nmmlckcatalyzed mlc phosphorylation on thr and ser which increases actomyosin atpase activity and shifts the equilibrium between the folded and unfolded myosin forms [ ], thus providing the assembling and functioning of the contractile apparatus of the cells. the mylk gene on chromosome in humans encodes three proteins: the nmmlck isoform, the smooth muscle mlck isoform , and telokin [ ] [ ] [ ] [ ] . in smooth muscle, nmmlck is expressed at relatively low level, being present together with a shorter smooth muscle isoform, whereas only nmmlck can be detected in ecs [ ] and exists as a , amino acid high molecular weight ( -kda) protein. the nmmlck shares essentially identical catalytic and cam regulatory motifs with smooth muscle mlck, but contains a unique amino acid n-terminal domain comprising potential novel ptm sites [ ] . inflammatory agonists such as vegf and thrombin produce rapid increases in mlc phosphorylation, reflecting coordinated nmmlck activity and the small gtpase rho and its effector, rho kinase, result in phosphorylation and, thereby, inhibition of the mypt myosin phosphatase, resulting in stabilization and accumulation of phosphorylated mlc. the aggregated result is actomyosin interaction and ec permeability which is significantly attenuated by mlck or rho kinase inhibitors [ , [ ] [ ] [ ] . despite the clear contribution of mlck/rho kinase driven increases in mlc phosphorylation to tension development and increased vascular permeability, mlck-independent pathways are also involved in the regulation of cellular contraction. protein kinase c (pkc)-mediated pathways exert a prominent effect on barrier regulation in a time-and speciesspecific manner without significantly increasing mlc phosphorylation and without inducing formation of actin stress fibers, but with alterations in other components of the endothelial cytoskeleton [ , , ] . pkc-mediated increases in ec permeability involve phosphorylation of caldesmon, an actin-, myosin-, and calmodulin-binding protein present in smooth muscle actomyosin cross-bridges as a -kda protein and in ecs as a -kda protein [ ] . the phosphorylation of caldesmon alters smooth muscle crossbridge activity [ ] . caldesmon-mediated regulation of actomyosin atpase in smooth muscle is also modified by the actin cross-linking protein filamin and gelsolin [ ] . although filamin participates directly in barrier regulation via cam kinase ii activation [ ], its effects on actin cytoskeletal rearrangement are regulated through rho family gtpases [ , ], thereby providing another link with a known modulator of ec barrier function. the cytokine tumor necrosis factor a (tnf-a) induces slow-onset barrier disruption in cultured ecs independent of mlck activity [ ] . finally, p kinase activation also has been linked to contractile regulation in smooth muscle [ ] , ec migration [ , ], and lipopolysaccharide (lps)-induced ec permeability [ ] . the mechanism through which p exerts these effects is unclear but may involve the actin-binding protein hsp [ ], a known p mitogen-activated protein kinase (mapk) target whose actin-polymerization-inhibiting activity dramatically decreases after phosphorylation [ , ] in association with stress fiber development [ , ]. the pulsatile nature of blood pressure and flow exposes blood vessels to constant hemodynamic forces in the form of ss and cyclic stretch (cs). the flow of blood parallel to the vessel surface produces fluid ss from the friction of blood against the vessel wall. in contrast, cs is an important mechanical force generated in the lung circulation either by circulating blood, which results in the rhythmic, pulsatile distension of the arterial wall, or by tidal breathing. the endothelium converts these mechanical stimuli to intracellular signals that effector cellular functions including proliferation, migration, remodeling, apoptosis, and permeability, as well as gene expression. the cytoskeleton is the key structural framework for the ecs to transmit mechanical forces between its luminal, abluminal, and junctional surfaces to its interior, including the cytoplasm, nucleus, and focal adhesion sites. changes in mechanical stress among the organs in the body, the lung exists in a high-oxygen environment and is susceptible to injury by oxidative stress. cigarette smoking and inhalation of airborne pollutants/toxins/ oxidant gases and particulate matter result in direct lung damage as well as the activation of lung inflammatory responses [ - ]. long-term exposure of lungs to higher oxygen tension (hyperoxia), as observed with premature infants and critically ill patients on ventilators, causes oxidative stress and lung injury [ ]. thus, increased ros production has been directly linked to inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and ards. ros are essential for normal lung/endothelial function [ ] , but an imbalance of the redox equilibrium may contribute to pulmonary edema [ , ] . the imbalance of oxidants produced to oxidants detoxified, i.e., a change in the redox equilibrium appears important in the development of various inflammatory lung diseases, and increased ros production have been directly linked to oxidation of dna, proteins, lipids and sugars, remodeling of ecm, alteration of mitochondrial respiration, and apoptosis. furthermore, increased levels of ros have been implicated in initiating signaling cascades of activation of transcription factors (nf-kb and ap- ), chromatin remodeling, and gene expression of proinflammatory mediators [ , ] . also, ros generated by phagocytes that have been recruited to sites of inflammation and excess generation of ros by vascular cells are a major cause of edema and lung injury. generation of ros and ros signaling in lung endothelium alter vascular permeability in vivo [ , ] and in endothelial monolayers [ , , ] . despite several potential sources of ros [mitochondrial electron transport chain, cytochrome p- enzymes, xanthine oxidase, nitric oxide synthases, myeloperoxidase (mpo) system], the vascular nadph oxidase family of proteins has been shown to be a major contributor of endothelial ros in response to hyperoxia [ ] since nadph oxidase mediated superoxide production increases endothelial permeability [ , ] . a variety of agonists, cytokines, growth factors, and mechanical forces alter pulmonary vascular barrier properties and serve to increase vascular permeability [ , , , , , , , ] . the serine protease thrombin represents an ideal model for the examination of agonist-mediated lung endothelial activation and barrier dysfunction as thrombin evokes numerous ec responses that regulate hemostasis and thrombosis, and is recognized as an important mediator in the pathogenesis of ali [ ] . thrombin increases ec leakiness to macromolecules by ligating and proteolytically cleaving the extracellular n-terminal domain of the thrombin receptor, a member of the family of pars [ ] [ ] [ ] . the cleaved n-terminus, acting as a tethered ligand, activates the receptor and initiates a number of downstream effects, including cytoskeletal rearrangement (fig. ) . in vivo studies have detailed events which followed thrombin infusion into the pulmonary artery of the chronically instrumented lung lymph sheep model initiating a cascade of events that culminate in intravascular coagulation, inflammation, and vascular leak [ ] [ ] [ ] . naturally occurring agonists, such as the cytokines tnf-a and il- b, have a prominent effect early in ali, causing microthrombosis, and eliciting a cascade of inflammatory signals which result in capillary endothelial production of p-selectin, an adhesion molecule which enhances leukocyte-ec migration [ ] [ ] [ ] and actin reorganization, and paracellular gap formation [ ] . tnf-a also increases tyrosine phosphorylation of ve-cadherin, leading to increased paracellular gaps in human lung endothelium [ ] . much less is known about pre-b-cell colony-enhancing factor (pbef), a relatively unknown cytokine we identified via functional genomic approaches as a novel ali candidate gene [ , ] . pbef is also known as visfatin, following its identification as a visceral fat hormone [ ] , and nicotinamide phosphoribosyltransferase (nampt), as it serves as the rate-limiting component in the nad biosynthesis pathway that catalyzes the conversion of nicotinamide and phosphoribosylpyrophosphate into nicotinamide mononucleotide. we demonstrated pbef as a novel biomarker in sepsis and sepsis-induced ali with genetic variants conferring ali susceptibility [ , ] . furthermore, pbef is highly expressed in polymorphonuclear neutrophils (pmns) of sepsis subjects, with expression upregulated by mechanical force and inflammatory cytokines, and is involved in ec barrier regulation [ , , ] . we explored the mechanistic participation of pbef in ali and vili and demonstrated that recombinant human pbef is a direct neutrophil chemotactic factor and elicits marked increases in the levels of bronchoalveolar lavage (bal) pmns and pmn chemoattractants (kc and mip- ) after intratracheal injection in mice [ ] , changes accompanied by modest increases in lung vascular and alveolar permeability. dramatic increases in bal pmns, bal protein, and cytokine levels (il- , tnf-a, kc) were observed in recombinant human pbef-and vili-challenged mice [ ] , whereas heterozygous pbef +/− mice were significantly protected (reduced bal protein levels, bal il- levels, peak inspiratory pressures) when exposed to a model of severe vili and exhibited significantly reduced expression of vili-associated gene expression modules. the role of the renin-angiotensin system in pulmonary vascular regulation is now well recognized with angiotensin ii, a key component of the renin-angiotensin system, generated primarily by angiotensin-converting enzyme (ace) from angiotensin i and its effects are mediated through angiotensin type i (at- ) and angiotensin type ii (at- ) receptors which are expressed in the normal lung. the pulmonary endothelium represents a major site of ace expression and angiotensin ii production, with ace , a homologue of ace, expressed in the lung inactivating angiotensin ii, leading to the downstream generation of angiotensin - , which acts through at- receptors to induce vasodilatation. although components of the renin-angiotensin system have been implicated in a variety of lung diseases, including pulmonary hypertension and fibrotic lung diseases, the system has been strongly linked to the pathophysiology of pulmonary vascular leak syndromes. for example, ace serves as the receptor for the coronavirus, first identified in , responsible for severe acute respiratory syndrome [ , ] , with a mortality rate of more than % in the elderly. ace and at- serve a protective role in ards, whereas ace , angiotensin ii, and at- mediate lung edema and injury associated with ards. a role for ace via angiotensin ii and/ or bradykinin in ali was proposed [ ] . reductions in ace activity by captopril attenuated the inflammatory response and apoptosis, whereas blocking bradykinin receptors did not attenuate the anti-inflammatory and antiapoptotic effects of captopril [ ] . captopril did not attenuate ace activity or necrosis, indicating that inflammation and apoptosis in vili is due to ace-mediated ang angiotensin ii production [ ] . new blood vessel formation, or angiogenesis, is defined by the generation of new capillaries by ecs either by sprouting or by splitting from pre-existing vessels. sprouting angiogenesis involves ec detachment from the basement membrane, migration, and subsequent proliferation, tube formation, and, finally, functional maturation of the new vessel [ ] . vegf is key in vasculogenesis as mice lacking the vegf receptor flt- fail to develop fully functional blood vessels [ ] . inhibition of vegf as a promising therapeutic strategy in the management of patients with advanced malignancies [ ] . pulmonary hypertension is a devastating disease with many similarities to neoplastic processes and is characterized by aberrant angiogenesis, with vegf serving as a target in pulmonary hypertension [ , ] . vegf increases ec permeability and was originally named "vascular permeability factor" for its profound effects on vascular barrier function [ ] . vegf levels are highest in the lungs and plasma and vegf levels are increased in patients with ards compared with the other groups [ ] . vegf increases cytosolic calcium levels and levels of mlc phosphorylation at high doses and vegf inhibition decreases ec permeability [ , ] . additional angiogenic factors with barrier-regulatory properties include angiopoietin and angiopoietin , which are critical for normal vascular development. the angiopoietin family is compopsed of vascular growth factors which are ligands to the family of tyrosine kinases that are selectively expressed in the vascular endothelium. vegf induces ec differentiation and migration, whereas angiopoietin stabilizes vascular networks [ ] [ ] [ ] . angiopoietin and angiopoietin modulate ec permeability by altering the state of ajs and specifically inhibit vascular leakage in response to vegf or other barrier-disruptive agents, as well as promoting vessel maturation. angiopoietin antagonizes angiopoietin and promotes barrier dysregulation by blocking the ability of angiopoietin to activate its receptor [ ] . understanding the mechanisms of barrier dysfunction offers the advantages to design therapeutic strategies which target barrier-integrity preservation or reverse established barrier dysfunction by restoring vascular integrity. prior to the last decade, permeability-reducing strategies primarily consisted of cyclic amp (camp) augmentation, producing only modest barrier enhancement [ ] [ ] [ ] [ ] . more recently, a number of barrier-promoting agents have been identified which share common signal transduction mechanisms which are distinct from camp signals and target the endothelial actin cytoskeleton to facilitate barrier-restorative processes. the dynamic process of actin polymerization allows for the rapid reorganization of actin structures, with profound functional consequences for barrier regulation that are highly dependent on the exact spatial location of this actin rearrangement occurring as either barrier-disrupting cytosolic stress fibers or as a barrier-enhancing thickened cortical actin ring. we have demonstrated that the quiescent ec phenotype is characterized by a cortical actin ring and few stress fibers, a structure which favors cell-cell adhesion and cell-matrix tethering. we have conceptualized a paradigm whereby barrier recovery after edemagenic agonists involves development of a cortical actin ring to anchor cellular junctions and a carefully choreographed (but poorly understood) gap-closing process via formation of rac gtpase-dependent lamellipodial protrusions into the paracellular space between activated ecs (fig. ) within these lamellipodia, signals are transduced to actin-binding proteins (nmmlck and cortactin) and phosphorylated mlcs in spatial-specific cellular locations. lamellipodia also require formation of focal adhesions (regulated by the cytoskeleton) critical to the establishment of the linkage of the actin cytoskeleton to target effectors that restore cell-cell adhesion and cell-matrix adhesion. this process is essential to the restoration of endothelial barrier in response to exposure to agonists such as sphingosine -phosphate (s p), hepatocyte growth factor (hgf), simvastatin, activated protein c (apc), atp, oxidized phopholipids, and hyaluaron [ , , [ ] [ ] [ ] [ ] [ ] . central to these events is the activation of small gtpases, rac and cdc [ ] , which follows ligation of barrier-protective receptors and drives cortical actin remodeling and lamellipodia formation (fig. ). in addition to lamellipodia, there is increased actin polymerization at the cell periphery (i.e., the cortical actin ring) which occurs with increased force driven by the actin-binding proteins cortactin and nmmlck, which also translocate to this spatially defined region. like lamellipodia formation, rac gtpase-dependent increases in the level of cortical actin follow exposure to multiple barrier-enhancing levels of ss or to potent barrier-enhancing agonists [ , , ] , including s p [ , ] , hgf [ ], atp [ ] , simvastatin [ ] , apc [ ] , prostaglandin e [ ] , and oxidized phospholipid -palmitoyl- -arachidonoyl-sn-glycero- -phosphochlorine (oxpapc) [ ] (table ). these observations serve to highlight the importance of the cellular location of cytoskeletal proteins in maintaining or enhancing ec barrier function, with cortactin directly interacting with nmmlck, an association which is increased by p src tyrosine phosphorylation of either cortactin or nmmlck [ ] . rac activation is in conjunction with akt-mediated phosphorylation events known to be involved in ec proliferation and migration [ ] and ec barrier enhancement. akt-induced phosphorylation of the s p receptor is important in barrier enhancement produced by high molecular weight hyaluronan [ , ] . [ , , ] s p s p receptor s p induces rapid and potent endothelial barrier enhancement through reduction of the numbers of central actin stress fibers and enhancement of cortical actin formation to stabilize cell-cell junctions. s p attenuated endotoxin-induced pulmonary edema in mice and canine models of injury [ , , , , ] simvastatin patients on cholesterol-reducing statin regimens have exhibited improved vascular function. the hmg-coa reductase inhibitor mitigates vegf signaling through rhoa inhibition and rac activation. in vitro, simvastatin pretreatment protects ec from thrombin-induced stress fiber formation and barrier dysfunction [ , , , ] atp g i /g o protein, but not atp receptor atp induces endothelial barrier enhancement through a rac-dependent cytoskeletal rearrangement with reduction of the numbers of central actin stress fibers with increase cortical actin formation. in vivo, nonhyrolyzable atp protected mice from endotoxin-induced lung injury [ , , ] hgf cdc . oxpapc accentuates peripheral f-actin in a unique ziplike configuration with novel interaction between focal adhesion and aj complexes. in vivo, oxpapc protects rats from mechanical-stress-induced lung injury [ , [ ] [ ] [ ] ] mntx mop-r that inhibits s p receptor pretreatment with mop-r antagonist protects ecs from thrombin-and lps-induced barrier dysfunction through an mop-r-independent antagonism involving inhibition of rhoa-dependent s p receptor. the fda recently approved mntx for treatment of postoperative ileus, and it may rapidly translate into a treatment for pulmonary edema [ ] [ ] [ ] anti-pbef neutralizing antibody extracellular pbef pbef is significantly upregulated in the lung during injury. extracellular release of pbef promotes endothelial barrier dysfunction and neutrophil extravasation. anti-pbef neutralizing antibodies, which target extracellular pbef without altering beneficial intracellular pbef, protected lungs from ventilator-induced lung injury in mice [ , ] adherens junction, activated protein c, endothelial cell, endothelial protein c receptor, filamentous actin, hepatocyte growth factor, hydroxy- -methylglutaryl coenzyme a, lipopolysaccharide, mitogen-activated protein kinase, myosin light chain kinase, methylnaltrexone, mu opioid receptor, phosphatidylinositol -kinase, -palmitoyl- -arachidonoyl--glycero- -phosphochlorin, pre-bcell colony-enhancing factor, protein kinase c, sphingosine -phosphate, vascular endothelial growth factor historically, cyclic nucleotides have represented the sole strategy for retarding the edema phase observed in inflammatory lung syndromes, possibly via camp-dependent protein kinases that phosphorylate proteins such as mlck and inhibit f-actin reorganization [ , , , ] . we examined nmmlck as a molecular target involved in increase of lung epithelial and ec barrier permeability utilizing genetically engineered mice and complementary strategies to reduce nmmlck activity or expression. both mlck inhibition (membrane-permeant oligopeptide, pik) and silencing of nmmlck expression in the lung significantly attenuate lps-induced lung permeability and inflammation. we also targeted pulmonary vessels and utilized ace antibody-conjugated liposomes with nmmlck small interfering rna (sirna) as cargo in a murine vili model, again with significant attenuation of vili. furthermore, nmmlck −/− knockout mice were significantly protected when exposed to a model of severe vili. thus, the multidimensional cytoskeletal protein nmmlck represents an attractive target for reducing lung vascular permeability and lung inflammation in the critically ill [ , , ] . s p is a sphingolipid resulting from the phosphorylation of sphingosine, a product of sphingomyelinase catabolism of sphingomyelin, catalyzed by sphingosine kinase [ ] . s p ligates a family of receptors known as s p receptors (also termed endothelial differentiation gene or edg receptors) with prominent effects on the vasculature, promoting ec mitogenesis, chemotaxis, and angiogenesis. our earlier studies were the first to demonstrate that s p is the most potent ec chemoattractant in serum [ ] and to link s p and its receptor ligation to enhanced vascular barrier regulation and demonstrated that physiological doses of s p induce ec activation, marked cytoskeletal rearrangement, and stabilization of lung ec barrier function in vitro [ ] . this novel function for s p was of particular relevance to clinical medicine as thrombocytopenia is well known to be associated with increased vascular leak [ ] and although the mechanism of this effect was unknown, we demonstrated that activated platelets are an important source of s p and directly enhance barrier function via s p receptor ligation [ ] . platelets contain significant levels of sphingosine kinase but reduced levels of sphingosine lyase, thereby serving as enriched sources for the barrier-promoting s p [ ] . ligation by s p of the barrier-enhancing g i -protein-coupled s p receptor (also known as edg ) [ , , , ] increases rac gtpase activity [ ] , cytosolic calcium level [ ] , and aggregation of key barrier-regulatory signaling components into caveolin-rich lipid rafts, including the rac gtpase target p -associated ser/thr kinase (pak) and its downstream target cofilin, an actin-binding protein [ ] , nmmlck, cortactin, and c-abl. pak and cofilin allow polymerization-depolymerization cycling to occur and thus facilitate rearrangement of actin from primarily transcytoplasmic to primarily cortical in a spatially distinct organization as a cortical actin cellular ring, processes which are integral to ec barrier function [ ] . increases in mlc phosphorylation within a peripheral distribution within the cortical actin ring [ ] provide strength to this spatially directed scaffolding force and enhance cell-cell tethering as we described via atomic force microscopy [ ] . immunofluorescence studies demonstrated that overexpressed green fluorescent protein-nmmlck distributes along cytoplasmic actin fibers, but rapidly translocates to the cortical regions of the cell after s p treatment, rapidly catalyzing mlc phosphorylation. in addition, confocal microscopy studies showed ecs challenged with s p demonstrate colocalization of nmmlck with the key actin-binding and ec barrierregulatory protein cortactin [ ] . the interaction of cortactin and nmmlck decreases cortactin-stimulated actin polymerization [ , ] and is essential to s p barrier protection. the p src is not involved in this pathway, but other tyrosine kinases such as c-abl are likely involved [ ] . s p-induced cytoskeletal rearrangement produces increased linkage of actin to aj components, as well as s p-induced phosphorylation of focal-adhesion-related proteins paxillin and fak, with translocation of these proteins to the ec periphery, further implicating s pinduced cell-cell adhesive changes as part of the mechanism of s p-induced barrier enhancement [ , ] . the potential utility of s p in restoring lung water balance in patients with inflammatory injury was underscored in studies involving small-and large-animal models of ali in which s p provided dramatic attenuation of lps-mediated lung inflammation and permeability [ , ] . mice treated with s p had significantly less histological evidence of inflammatory changes/lung injury, with decreased neutrophil alveolitis on bal and decreased lung mpo activity [ ] . interestingly, mice treated with s p after intratracheal administration of lps also showed an attenuated renal inflammatory response compared with controls, measured by tissue mpo activity and evans blue dye extravasation as a measure of capillary leak. s p also protected against intrabronchial lps-induced ali and concomitant vili in a canine model, with decreased shunt fraction, decreased bal protein, decreased extravascular lung water, and improved oxygenation [ ] . use of a large-animal canine model allowed investigation of regional lung changes in ali and the effect of s p on these changes. computed tomography scans of animals subjected to lps/vili found that animals treated with s p had a dramatic improvement in alveolar air content (with decreased edema) in all lung regions [ ] . additional in vivo studies found that s p protects against vili in a murine model as assessed by evans blue dye extravasation [ ] . we have also evaluated a potential role for s p in ameliorating lung ischemia-reperfusion injury, a common sequela of lung transplantation, which is characterized by alveolar damage, edema, and inflammation in donor lungs and is a significant cause of transplant failure. utilizing a rat model of ischemia-reperfusion injury (pulmonary artery ligation and reperfusion), we determined that rats pretreated with s p exhibited reduced lung vascular permeability and inflammation compared with controls [ ] . lung mpo activity, an index of parenchymal leukocyte infiltration, and levels of il- , il- b, and il- were also attenuated in s ptreated animals exposed to ischemia-reperfusion injury [ ] . together, these findings suggest that s p may serve as an effective permeability-reducing agent in diverse conditions which share an element of lung inflammatory burden. despite the profound attractiveness of s p as a therapeutic agent which targets the endothelium in high-permeability states, s p has several attributes which limit its potential utility as a permeability-reducing strategy. with an affinity for ligation of the s p receptor, intratracheal s p has been implicated as a cause of pulmonary edema via endothelial/ epithelial barrier disruption [ ] . s p also causes bradycardia via ligation of cardiac s p receptor [ ] . these findings generated increased interest in fty , a derivative of the natural immunosuppressant myriocin [ ] , and a recently described immunosuppressive agent that causes peripheral lymphopenia by inhibiting cellular egress from lymphoid tissues. fty is structurally similar (but not identical) to s p and is phosphorylated by sphingosine kinase to fty -phosphate, which is an agonist at s p receptors [ ] . this characteristic prompted investigation of the effect of fty on ec barrier function. fty did not have superior efficacy compared with mycophenolate mofetil in preventing renal transplant rejection [ ] , but it is in phase iii clinical trials as an immunosuppressant in multiple sclerosis patients. the clinical availability of fty makes it attractive as a potential mediator of ec barrier function in patients with ali. our in vivo studies demonstrated that intraperitoneally administered fty protected against intratracheally administered lps in a murine model of ali, as measured by evans blue dye extravasation [ ] . the mechanism of fty -induced ec barrier enhancement diverges from the mechanism described for s p in several ways, including the delayed kinetics of the rise in total energy requirement (ter) compared with s p [ ] . decreased expression of the s p receptor prevented an s p-induced increase in ter but only partially altered fty -induced ter increases. unlike s p, fty did not result in threonine phosphorylation of the s p receptor, nor did inhibition of phosphatidylinositol -kinase (pi- -kinase) prevent fty -induced ec barrier enhancement [ ] . furthermore, fty did not cause the increased intracellular calcium level, the mlc phosphorylation, or the cytoskeletal rearrangement seen in response to s p [ ] . downregulation of rac or cortactin using sirnas attenuated the barrier-enhancing effect of s p, but not that of fty [ ] . although fty is an s p receptor agonist, its mechanism of barrier enhancement is distinct from that of s p and does not require the s p receptor. we are currently pursuing novel s p and fty analogues for use in inflammatory lung injury models [ ] [ ] [ ] . another class of prominent barrier-protective agonists under intense scrutiny is the statin family of compounds known as -hydroxy- -methylglutaryl coenzyme a reductase (hmg-coa reductase) inhibitors [ ] . these drugs inhibit cholesterol synthesis in the liver, are commonly used in clinical practice as lipid-lowering agents, and prevent acute coronary events. a plethora of reports have now demonstrated that the benefits of statin therapy cannot be entirely attributed to decreased serum cholesterol level. we have been interested in the effect of statins on endothelial function in ali as an ever-growing body of literature demonstrates improved outcomes in patients with sepsis who are treated with statins, with decreased mortality in bacteremic patients admitted to the hospital while on statin therapy [ ] . a retrospective study in human patients with multiple organ dysfunction syndrome found that those receiving statins had significantly lower -day mortality and hospital mortality compared with matched controls not receiving statin therapy [ ] . animal studies suggest dramatically improved survival in mice treated with simvastatin prior to initiation of sepsis by cecal ligation and puncture compared with mice which were not pretreated with simvastatin [ ] . we have pursued the mechanism of statin action on the endothelium and found that simvastatin attenuated thrombininduced stress fiber formation, paracellular gap formation, and barrier dysfunction [ ] . co-incubation with mevalonate (the product of hmg-coa reductase activity) eliminated the protective effect of simvastatin against thrombin-induced ec permeability, indicating this effect is due to hmg-coa reductase inhibition and did not involve either intracellular increased camp levels or increased levels of endothelial nitric oxide synthase. statins inhibit geranylgeranylation of small gtpases, essential for gtpase interaction with cell membranes [ ] , and translocation of the small gtpases rac and rho to the plasma membrane. ec pretreatment with simvastatin prevented thrombin-induced translocation of rho to the plasma membrane [ ] and simvastatin was found to confer greater protection against thrombin-induced barrier dysfunction than rho inhibition alone. rac inhibition may be protective via decreased activation of nadph oxidase and resultant superoxides that induce barrier dysfunction, and this was also found to be important in simvastatin-induced ec barrier protection [ ] . simvastatin pretreatment resulted in reduced diphosphorylated mlc levels, reduced numbers of stress fibers, increased rac gtpase activation [ ] , cortactin translocation to the ec periphery [ ] , and increased cortical actin and decreased paracellular gap formation after thrombin treatment. unlike s p, simvastatin does not cause an increased baseline ter [ ] . simvastatin elicits changes in ec gene expression with downregulation of caldesmon and the thrombin receptor par- , as well as upregulation of integrin b (known to function in cell-cell adhesion), rac , and gefs, which may regulate rho gtpase activity [ ] . the importance of new protein synthesis to the barrier protective effect of simvastatin was established by the elimination of the protective effect by coincubation of ecs with simvastatin and the protein synthesis inhibitor cycloheximide [ ] . in vivo data from an intratracheal-lps murine model of ali support the in vitro finding that simvastatin is protective of ec barrier function and against markers of inflammatory lung injury compared with controls, with decreased bal neutrophil count and mpo activity, decreased vascular permeability, and a marked reduction of inflammatory histological changes [ ] . investigation of gene expression in lung tissue of mice pretreated with simvastatin in this lps-induced model of ali found that simvastatin caused differential regulation of several families of genes, including inflammatory and immune response genes, as well as nfkb regulation and cell adhesion genes [ ] . simvastatin may prove to be clinically relevant in treating ali, as ali typically has a prolonged course, and treatment with simvastatin along the trajectory of the illness may be beneficial. to this end, a blinded, randomized controlled clinical trial of simvastatin in ali is currently under way. atp is found in abundance in the ec microenvironment and participates in ec barrier regulation, with constitutive release of atp across the ec apical membrane in basal conditions [ , ] . atp reduced ec albumin permeability in a concentration-dependent manner in ecs from a variety of origins, including porcine aorta and pulmonary artery, bovine aorta, and human umbilical vascular endothelial cells [ ] . the mechanism of atp-induced ec barrier enhancement involves g i /g o proteins [ ] but does not involve adenosine receptors [ ] , increased pkc activity, or increases in cyclic gmp levels [ ] . however, atp-induced decreases in ec permeability were found to involve the phospholipase c signaling pathway [ ] , as well as alterations in ec mlc phosphorylation [ , ] . we demonstrated that atp produces ca + -and p / mapk-independent increases in cell-cell interfaces (ve-cadherin staining) and increased thickness and continuity of zona occludens (zo- ) in tjs [ ] , mediated in part via camp-independent activation of protein kinase a (pka). we also noted that atp produced a biphasic effect on mlc phosphorylation, with an initial increase followed by a decrease in levels of phosphorylated mlc. however, the delayed decrease in the levels of phosphorylated mlc was prevented by phosphatase inhibitors, emphasizing the importance of g-protein-mediated phosphatase activity in the atp-induced decrease in mlc phosphorylation and atp-induced barrier enhancement [ ] . similar to s p (as well as hgf, apc, etc.), atp-mediated barrier enhancement required rac-dependent cytoskeletal rearrangement with decreased numbers of central actin stress fibers, increased cortical distribution of actin, peripheral mlc phosphorylation, and cortactin translocation to the cortical actin ring [ ] . in addition, a rapid, transient increase in mlc diphosphorylation was observed after atp stimulation, with phosphorylated mlc localized at the cell periphery, a stark contrast to the central, stress-fiber-associated phosphorylated mlc seen in ecs treated with thrombin [ ] . as an extension of these in vitro studies, the effect of purinergic stimulation was assessed in a murine model of ali with intratracheally administered lps. as atp is rapidly degraded intravascularly, the nonhydrolyzable analogue atpgs was used for in vivo studies. mice given atpgs intravenously concomitant with intratracheal administration of lps were protected from lps-induced ali compared with controls as assessed by neutrophil infiltration and mpo activity [ ] . atpgs also attenuated the lung microvascular permeability elicited by lps, with decreased bal protein and decreased evans blue-albumin extravasation in mice treated with atpgs compared with controls [ ] . atpgstreated animals were also protected from the lps-induced decrease in body weight that was seen in control mice [ ] . in addition, in vitro studies found that atpgs alone produced an increased ter in ecs and also showed delayed protection against the reduction in ter caused by lps [ ] . alterations in vascular permeability are requisite steps in the angiogenic process [ , ] . we were the first to report that hgf, a well-known angiogenic factor, like s p, is a potent ec barrier-protective agonist [ ] and acts via stabilization of the ec actin cytoskeleton. hgf-mediated ec protection from the barrier-disrupting effect of thrombin [ ] evolves via increased rac activation involving the racspecific gef tiam as well as decreased rho activation with increased pak phosphorylation [ ] . hgf signals via a tyrosine kinase receptor, c-met, and serves to recruit cd v , a key transactivated receptor for cd , into caveolin-enriched microdomains (cems) or lipid rafts [ ] . in experiments using sirna, both c-met and cd were found to be important in hgf-induced increases in ec ter [ ] . furthermore, pretreatment of ecs with the ceminterfering compound methyl-b-cyclodextran also prevented hgf-induced increases in ter [ ] . in addition, rac activation by hgf was found to require cem formation, c-met, cd , tiam , and dynamin- [ ] . in a mouse model of lps-induced ali, hgf was protective against markers of lung inflammation, an effect not noted in cd knockout mice [ ] . the signaling mechanism involved in hgfinduced ec barrier enhancement is complex, with important roles for c-met, cd , and cem formation. hgf produced rac-dependent increases in the levels of cortical actin, cortactin translocation, and cortical levels of phosphorylated mlc [ ] . further mechanistic studies found that hgfinduced ec barrier enhancement critically involves pi- -kinase activity, distinguishing the mechanism of hgfinduced barrier enhancement from that of s p [ ], with important roles for mapks (erk and p ) and pkc in hgf-induced ec barrier enhancement [ ] . attention to the role of improved cell-cell or cell-matrix adhesion elicited by hgf found that hgf produced increased b-catenin localization to the ec periphery alongside cortical actin and increased association of b-catenin with ve-cadherin [ ] . the cell signaling effectors of hgf (pi- -kinase, erk, p , pkc) were found to converge at phosphorylation of glycogen synthase kinase- b, which regulates the association of b-catenin and cadherin, thereby controlling cell-cell adhesion [ ]. apc is a serine protease that modulates coagulation and inflammation. in , the food and drug administration approved xigris ® , or recombinant human apc (rhapc), also known as drotrecogin alfa (activated), for treatment of severe sepsis in adults after a randomized trial found a -day survival benefit in treated patients [ ] . because severe sepsis involves ali and systemic increased vascular permeability, the effect of apc on pulmonary ec permeability is intriguing. interest in the effect of the anticoagulant apc on ec permeability is also related to the well-described role of the procoagulant thrombin in ec barrier disruption. furthermore, the mechanism of the survival benefit imparted by treatment with rhapc is unclear, as apc given to human subjects in the setting of endotoxin infusion improved hemodynamics but did not have an anti-inflammatory or antithrombotic effect [ ] , suggesting that a different mechanism may be involved. we demonstrated that apc prevented and was able to reverse thrombin-induced increased permeability [ ] . apc also increased mlc phosphorylation and the level of actin at the ec periphery and decreased the number f central stress fibers. the barrier-enhancing effect of apc was found to be mediated by rac activation, similar to the barrier-enhancing effect of s p, simvastatin, and hgf [ ] . the endothelial protein c receptor (epcr) is critical to apc-induced barrier enhancement and mlc phosphorylation. furthermore, epcrmediated transactivation of the s p receptor via pi- -kinase is essential and involves direct interaction between epcr and s p receptor [ ] . this novel pathway for apc-induced ec barrier enhancement may contribute significantly to the survival benefit offered by rhapc in patients with severe sepsis. more recent work has focused on apc in animal models of ali. using a rat model of intestinal ischemia-reperfusion injuryinduced ali, investigators found that apc treatment just prior to reperfusion attenuated subsequent pulmonary edema, which was accompanied by fewer neutrophils on histological examination and a marked improvement in the histological appearance compared with animals that did not receive apc [ ] . in addition, rats treated with apc prior to intestinal reperfusion had lower serum levels of tnf-a, il- , and d-dimer compared with controls [ ] . investigation of apc in a mouse model of vili found that apc pretreatment was protective against vili caused by high tidal volume ventilation, with mice pretreated with apc exhibiting significant reductions in bal protein and evans blue dye extravasation compared with controls [ ] . oxidized phospholipids are derived from oxidized low-density lipoproteins and have been the focus of much investigation in the areas of vascular injury and inflammation [ ], with increased levels noted in ali [ ] . oxidized phospholipids resulting from the oxidation of oxpapc activate mapks erk, and c-jun n-terminal kinase, but not p or its downstream target, hsp [ ], and increased the activity of both pkc and pka [ ] and src kinases, processes involved in oxpapc-mediated ec barrier enhancement, whereas rho, rho kinase, erk, p , and pi- -kinase were not involved [ ] . furthermore, oxpapc resulted in phosphorylation of the actin-binding protein cofilin as well as phosphorylation of the focal adhesion proteins fak and paxillin, indicating that oxpapc may affect the ec actin cytoskeleton and cellcell adhesions [ ] . oxpapc protects against ec barrier dysfunction in vitro [ , ] after thrombin and lps stimu lation [ ] . oxpapc accentuates peripheral f-actin in a unique, ziplike configuration [ , , ] and results in continuous focal adhesions with accumulation of b-catenin [ ] . the signaling pathways involved in oxpapc-mediated endothelial barrier protection involve rac and cdc [ ] , the rac effector pak [ ] , the upstream rac/cdc specific gefs tiam and bpix [ ] , and the actin-binding proteins cortactin and arp [ ] . oxpapc was found to cause a novel interaction between focal adhesion and aj complexes, a process mediated by association of paxillin and b-catenin and dependent upon rac and cdc [ ] . in vivo studies have shown that intravenous oxpapc delivery results in significant attenuation of lps-induced inflammation in a rat model [ ] and vili [ ] . oxpapc protects ecs from mechanical-stress-induced injury via cytoskeletal rearrangements and changes in rho and rac activation and remains a potential therapy for the profound pulmonary edema associated with inflammatory states. methylnaltrexone (mntx) is a peripherally restricted mu opioid receptor (mop-r) antagonist recently approved by the food and drug administration for the treatment of postoperative ileus and also recently found to work synergistically with -fluorouracil and bevacizumab to inhibit vegf-induced pulmonary ec proliferation and migration [ ] . antagonists of mop-r are of interest as potential ec barrier-enhancing agents because of the barrier-disruptive properties of the mop-r agonist morphine [ ] . pretreatment of human pulmonary microvascular ecs with . mm mntx was found to protect against the decrease in ter caused by the mop-r agonists morphine and damgo and also protected against the barrier-disruptive effects of thrombin and lps, which act independently of mop-r [ ] . mntx augments the barrier-enhancing effect of s p [ ] . ec pretreatment with naloxone, a charged mop-r antagonist, protected against morphine and damgo-induced barrier disruption, but was not protective against barrier disruption caused by thrombin or lps. these data, together with the observation that sirna targeting mop-r had a minimal effect on mntxinduced protection against thrombin and lps, suggest that the protective effect of mntx cannot be attributed to mop-r antagonism alone [ ] . further experiments found that mntx confers its barrier-protective effect by inhibiting the association of the rhoa-activating gef p rhogef with the s p receptor and resultant rhoa activation that is caused by barrier-disrupting agents [ ] . complementary in vivo experiments found that intravenous administration of mntx after ali had been established via intratracheal administration of lps was protective against ali at h, as assessed by histological examination and bal protein and tnf-a levels [ ] . as noted already, pbef is a biomarker in sepsis and sepsisinduced ali and intratracheal injection of recombinant pbef into mice results in increased lung inflammation and vascular permeability [ , ] , indicating that extracellular pbef promotes endothelial barrier dysfunction. intracellular pbef may have a contrasting beneficial response in ali function via effects on cell apoptosis. neutrophils in sepsis patients increase expression of pbef, which promotes cell survival through the enzymatic process of nad biosynthesis via nicotinamide phosphoribosyltransferase (nampt) activity, a feature cancer cells have utilized to prevent cell death. the nampt inhibitor fk- is currently in trials as a cancer drug to promote apoptosis. thus, pbef therapies are complicated, with intracellular pbef appearing to have beneficial effects in cells by promoting cell survival, whereas extracellular pbef appears to induce inflammatory response. to specifically target extracellular pbef that may induce deleterious cellular response, we generated neutralizing antibodies against pbef to act as a molecular sponge for extracellular pbef without altering intracellular pbef function, which may be beneficial for the cell. using a mouse model of lung injury, we demonstrated that the anti-pbef neutralizing antibodies significantly protected lungs from vili by reducing the availability of extracellular pbef from sensitizing the lung endothelium [ ] . the study implicates pbef as a key inflammatory mediator intimately involved in both the development and the severity of ventilator-induced ali and demonstrated that anti-pbef neutralizing antibody has potential clinical utility. various molecules participating in the activation of inflammation in ali serve as indicators for the progression of normal to pathological biological processes, providing important tools to detect disease and support diagnostic and therapeutic decisions. ideally, vascular biomarkers have strong correlation between the presence/absence of a disease state and clinical outcome and provide predictive points of intervention to slow or reverse the disease. furthermore, the indication of a specific biomarker may allow for customized therapies that are more effective in different phases of the disease. new research and novel understanding of the molecular mechanisms of ali have revealed an abundance of exciting new biomarkers with high potential value as prognostic tools ( table ) . cofactor in the thrombin-induced activation of protein c in the anticoagulant pathway reduced plasma thrombomodulin level is predictive of higher mortality and worse system dysfunction [ , ] pai- inhibitor of plasminogen activator in plasma pai- level increase is predictive of death [ , ] sicam- marker of ec activation; adhesion molecules icam- level increase is predictive of death [ , ] il- cytokine; inflammation il- level increase is predictive of death [ , ] cytokine; inflammation secretion of extracellular pbef upon mechanical stress induces pulmonary edema and neutrophil extravasation in mice [ , ] icam- intercellular adhesion molecule- , interleukin, pai- plasminogen activator inhibitor- , sicam- soluble intercellular adhesion molecule- the importance of sphingolipids to maintain physiological vascular integrity has been well established and thrombocytopenia, a clinical condition in which there is a deficient number of circulating platelets, is associated with increased vascular leak [ ] via an unknown mechanism. activated platelets are an important source of s p and contain significant levels of sphingosine kinase but reduced levels of sphingosine lyase, thereby serving as enriched sources for the barrier-promoting s p [ ] which directly enhance barrier function via s p ligation [ ] . although the role of s p at physiological concentration is critical to maintaining normal endothelial barrier function, the differential ligation to s p receptors has differential responses. in contrast to the ligation to s p , the ligation of s p induces endothelial barrier dysfunction via activation of rho-dependent actin stress fiber and cell-cell gap formation. recently, we discovered that culture of ecs challenged with barrier-disrupting agents induces tyrosine nitration of s p receptors, which are released into media in microparticles or exosomes [ , ] . the occurrence of protein tyrosine nitration under disease conditions is now firmly established and represents a shift from the physiological signaling actions of •no to oxidative and potentially pathogenic pathways. protein tyrosine nitration is an irreversible ptm mediated by reactive nitrogen species, a process that suggests the regulatory function of proteins that undergo phosphorylation in signal transduction cascades might be seriously compromised by peroxynitrite-promoted nitration. we explored s p as a potential biomarker and observed from immunoblot analysis of serum from mice exposed to various models of vascular injury that they had significant tyrosine-nitrated s p expression [ , ] . in addition, we examined serum from patients with sepsis and ali and discovered tyrosine-nitrated s p receptor was correlated with disease progression [ , ] . therefore, our data indicate tyrosine-nitrated s p receptor is released from challenged ecs in microparticles and serves as a novel biomarker for vascular injury in various disease models. the family of pmn chemotactic cytokines, known as the cxc chemokines have been described and characterized and include il- , gro-a, gro-b, gro-g, ena- , and granulocyte chemotactic peptide (gcp)- . these chemokines are all produced by human alveolar macrophages and contain a glutamylleucyl-arginine (elr) motif that is critical to their neutrophil binding and chemotactic functions [ ] . il- is present in biologically significant concentrations in bal fluid from patients with ards, tracking pmn concentrations [ ] . although gro-a and ena- concentrations are higher than il- concentrations, il- is the predominant chemoattractant in ards bal fluid via its high-affinity binding to cxc chemokine receptors, cxcr and cxcr , on human pmns. unlike ena- , gro-a, gro-b, and gro-g with a high-affinity binding only to cxcr , il- and gcp- can bind to either receptor with high affinity [ ] . in the presence of a systemic inflammatory process such as severe sepsis, cxcr is tonically downregulated and the function of only cxcr receptor predominates [ ] . thus, of the multiple neutrophil chemotactic factors produced in humans, there appears to be a small group that is particularly relevant to patients with ards, with il- and its cognate receptor cxcr being the dominant receptor-ligand pair. il- also binds to its circulating high-affinity polyclonal igg and igg autoantibodies naturally [ ] , therefore preventing binding to cxc chemokine receptors on pmns [ ] . these autoantibodies are present in lung fluids from patients who are at-risk for ards as well as in patients after the onset of ards. the ratio of il- autoantibody:cytokine complex was significantly higher at the onset of ards than in patients at risk for ards. in addition, patients with ards with an elevated anti-il- -autoantibody:il- complex ratio are more likely to die than patients with lower concentrations of anti-il- -autoantibody:il- complex [ ] . thus, the anti-il- -autoantibody:il- complex ratio in lung fluid samples was more revealing than lung fluid protein concentrations to predict the development of ards in patients who were at-risk, and also for predicting mortality in patients with ards [ ] . the protein c pathway is one of the most important regulators of blood coagulation and serves as a critical link between coagulation and inflammation in sepsis and ali [ ] [ ] [ ] . protein c is a vitamin-k-dependent plasma glycoprotein that is synthesized by the liver and circulates as a two-chain biologically inactive zymogen. it is transformed to its active form, apc, by the thrombomodulin-thrombin complex on the cell surface. apc suppresses further thrombin formation by proteolytically inactivating coagulation factors va and viiia [ ] . the membrane-bound epcr potentiates this activation about -fold [ ] . recent evidence suggests that, in addition to its anticoagulant effects, apc also has anti-inflammatory properties. thus, the protein c pathway is important for the control and modulation of both coagulation and inflammation [ ] . apc inhibits the production of tnf-a via nfkb activation in monocytes and ecs [ ] , and inhibits neutrophil activation and chemotaxis through interaction with a cell-surface receptor similar to the epcr [ ] . decreased protein c activation on the pulmonary vascular endothelium surface may contribute to the widespread microvascular thrombosis that occurs in the acutely injured lung and may also be proinflammatory and proapoptotic. administration of apc attenuates experimental sepsis-induced lung injury. in human studies, an infusion of apc h prior to and h after administration of an intravenous injection of lps prevented lps-induced increase in tissue factor expression and thrombin formation in plasma after lps injection, as well as circulating levels of il- or tnf-a, markers of inflammation [ ] . loss of thrombomodulin and epcr from the cell surface results in a decreased ability to activate protein c, a phenomenon that has been implicated in the pathogenesis of sepsis and lung injury. release of the protein c pathway components throm-bomodulin and epcr into the plasma has been reported in experimental sepsis models [ ] . in clinical studies, plasma protein c levels were reduced in patients with severe sepsis, with % of patients meeting the criteria for acquired protein c deficiency. low levels of protein c were associated with ventilator dependency and a higher prevalence of ards and correlated with higher mortality [ ] . another study demonstrated that patients with severe sepsis varied markedly in their ability to generate apc [ ] . modulation of coagulation and inflammation through the activation of protein c is a critical mechanism in the pathogenesis of sepsis and ali [ ] . protein c levels and thrombomodulin levels are lower early in the course of ali and reduced plasma protein c and thrombomodulin levels are associated with higher mortality and more nonpulmonary organ system dysfunction, with the combination of low levels of protein c and other predictors such as high levels of plasminogen activator inhibitor- (pai- ) conferring an even higher risk of mortality. the prognostic value of protein c and thrombomodulin was not altered by exclusion of patients with coexisting sepsis [ ] . the balance between activation of coagulation and activation of fibrinolysis is likely an important determinant of the amount and duration of fibrin deposition in the injured lung, and the fibrinolytic system is profoundly altered in patients with ali/ards, both systemically and in the alveolar compartment. plasminogen activator (pa) and pai- regulate fibrinolysis, the dissolution of fibrin clots, through modulation of the conversion of plasminogen to plasmin, a major fibrinolytic enzyme [ ] . upregulation of pai- , the major inhibitor of fibrinolysis, appears to play a primary role in the shift from profibrinolytic to antifibrinolytic phenotypes in a variety of cell types, including ecs, indicating a risk factor for ali and sepsis. there are two forms of pa, urokinase-type pa (upa) and tissue-type pa (tpa). upa is a cell-surface protein that is responsible for activating fibrinolysis at the tissue level, whereas tpa is a soluble protein that activates intravascular fibrinolysis [ ] . two major endogenous pa inhibitors have been identified, pai- and pai- , which are produced by platelets, endothelial, mesothelial, and epithelial cells, including those of the lung [ ] . pai- is the major pa inhibitor in plasma and extravascular fluids and has been implicated in the fibrinolytic defect associated with ali [ ] . human lung ecs isolated from patients with ards constitutively express greater levels of pai- than controls with lower fibrinolytic potential as measured by the pa to pai- ratio. in limited ali/ards clinical studies, reduced fibrinolytic capacity and an increase in upa and in pai- activity was noted in ards patents, with levels of pai- higher in both pulmonary edema fluid and plasma of ali/ards patients, and correlated with mortality in patients with ali/ ards [ , ] . a variety of strategies are being explored to develop inhibitors of pai- that might be of therapeutic use in ali/ards or other diseases associated with high levels of pai- such as cardiovascular disease [ ] . intercellular adhesion molecule- (icam- ; cd ) is an adhesion molecule constitutively expressed in the normal lung and is a critical participant in pulmonary innate immunity [ ] . soluble icam- (sicam- ) represents a circulating form of icam- that is constitutively expressed or is inducible on the cell surface of different cell lines [ ] . structurally, icam- belongs to the immunoglobulin superfamily, serving as a counterreceptor for the leukocyte integrin lfa- . interaction between icam- , present on ecs, and lfa- facilitates leukocyte adhesion and migration across the endothelium; however, sicam- binding to lfa inhibits lymphocyte attachment to ecs [ ] . sicam- is found in bal fluid and the release of sicam- is induced by several cytokines and various factors, including il- , il- , tnf-a, interferon-g, and angiotensin ii via proteolytic cleavage of icam- or direct transcription from its messenger rna [ ] . studies correlating sicam- levels to disease have led to the identification of sicam- as a marker for diseases such as viral infections, autoimmune disease, atherosclerosis, coronary heart disease, cancers, and neurological disorders [ ] . increased bal sicam- has been described in adults with granulomatous lung diseases such as sarcoidosis, tuberculosis [ ] , hypersensitivity pneumonitis, and radiation pneumonitis [ ] , and in children exposed to second-hand smoke [ ] . importantly, the level of sicam- is increased in pediatric ards during high-frequency oscillatory ventilation [ ] and in ali patients [ ] . il- , a well-recognized ali candidate gene and ali biomarker [ , ] , and is produced by a wide range of cells, including ecs, in response to stimulation by endotoxin, il- b, and tnf-a [ ] . il- in the acute-phase response stimulates synthesis of c-reactive protein from hepatocytes in vitro and in vivo [ ] . elevated levels have been described in acute conditions such as burns, major surgery, and sepsis and may predict development of multiple organ failure and the severity of ards of different orgins, such as sepsis and acute pancreatitis [ ] . the elevation of the level of and persistence of circulating il- has been associated with increased mortality in critically ill patients with ards, sepsis, and trauma, and il- concentrations have been shown to be elevated in the bal fluid from patients with established severe ali [ ] . functional polymorphisms in the promoter region of the il- gene exist (g c), with the c allele associated with reduced gene promoter activity, lower circulating il- concentrations, and a lower mortality rate in patients with acute respiratory failure admitted to the icu [ ] . in the multispecies ali studies performed, significant il- gene expression across all species as well as differential region-specific expression in the canine ali model has been noted. all of these facts suggest that the role of il- in ali is complex and il- may have a dual role in the temporal response to sepsis and mechanical stress. the pbef gene is one of a handful of genes with extremely high level of expression across the range of ali models used and in human ali samples. whereas we were the first to report that pbef is significantly upregulated in the lung as well as in models of lung injury [ ] , the published literature on pbef is quite sparse [ , ] . this gene encodes for a proinflammatory cytokine, originally described for its role in the maturation of b-cell precursors, with gene expression upregulated in amniotic membranes from patients undergoing premature labor, especially with amniotic infections. pbef protein levels were significantly increased in both bal fluid and serum of human, murine, and canine ali models as well as in cytokine-or cs-activated lung microvascular endothelium [ , ] . triple immunohistochemical staining of canine lungs revealed colocalization of increased pbef expression in lung endothelium, type ii alveolar epithelial cells, and infiltrating neutrophils, as well as upregulation of pbef expression in inflammatory cytokine-stimulated human pulmonary microvascular ecs in vitro [ ] . these results support pbef as a potential biomarker in ali and potentially involved in inflammatory lung processes, a notion supported by recent studies in patients with sepsis which convincingly demonstrate that pbef inhibits neutrophil apoptosis [ , ] . common variants in the human pbef gene are also confirmed to be associated with susceptibility to sepsis-associated ali [ ] . the t allele in the c- t single-nucleotide polymorphism in the pbef promoter region was associated with a nearly twofold decrease in the reporter gene expression. this result is consistent with our observations from animal models of ali, human patients with ali, and in vitro cell culture experiments, and suggests that higher expression of pbef is implicated in the pathogenesis of ali. these results further suggest that genetically determined increased pbef expression contributes to susceptibility to ali. despite decades of frustration in the pursuit of potent barrierregulatory therapies, progress has now been made for alleviation of the human suffering associated with uncontrolled lung vascular leakage and alveolar flooding. novel biologically compatible agents have now been identified which can preserve or restore vascular integrity, leveraging new insights into the mechanisms which govern the integrity of the vascular endothelium, particularly the role of cytoskeletal linkages to junctional proteins. in addition, several endothelial target proteins or protein pathway participants also serve as potentially novel biomarkers in the management of these patients. the newly revised scientific armamentarium offers promise for the future management of pulmonary edema associated with increased vascular leak in the critically ill as well as other lung conditions which exhibit strongly dysregulated barrier function such as radiation pneumonitis, acute chest syndrome in sickle cell patients, and in subacute inflammatory disorders such as asthma. nearly each barrier-regulatory agent discussed herein has been successfully evaluated in preclinical models of ali and one agent, fty , is in phase iii trials, whereas three agents, statins, apc and mntx, are currently approved by the food and drug administration for other medical conditions. thus, the prospects for the rapid translation of these lung vascular barrier-protective strategies to clinical practice are high. additional translational bench-to-bedside genomic and genetic strategy approaches combined with dissection of the basic mechanisms of endothelial structure/function during inflammation will lead to greater specificity in advancing clinical trials of agents for the treatment of inflammatory lung injury in a manner which represents personalized medicine for critically ill individuals. 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effects of oxidized phospholipids on endotoxininduced lung injury synergistic effects of methylnaltrexone with -fluorouracil and bevacizumab on inhibition of vascular endothelial growth factor-induced angiogenesis dual effects of morphine on permeability and apoptosis of vascular endothelial cells: morphine potentiates lipopolysaccharide-induced permeability and apoptosis of vascular endothelial cells attenuation of vascular permeability by methylnaltrexone: role of mop-r and s p transactivation critical role of pbef expression in pulmonary cell inflammation and permeability sphingosine- -phosphate receptor (sip ) is a novel biomarker for acute lung injury and idiopathic pulmonary fibrosis sphingosine- -phosphate receptor (s p ) is a novel biomarker for acute lung injury cytokinemediated inflammation in acute lung injury interleukin (il- ) in the bronchoalveolar lavage fluid from patients with the adult respiratory distress syndrome (ards) and patients at risk for ards granulocyte 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soluble intercellular adhesion molecule- in children with colds plasma soluble intercellular adhesion molecule- (sicam in pediatric ards during high frequency oscillatory ventilation: a predictor of mortality elevated pulmonary edema fluid concentrations of soluble intercellular adhesion molecule- in patients with acute lung injury: biological and clinical significance functional genomic insights into acute lung injury: role of ventilators and mechanical stress il gene-wide haplotype is associated with susceptibility to acute lung injury inflammatory mediators in chronic obstructive pulmonary disease c-reactive protein, inflammation, and innate immunity cytokine balance in the lungs of patients with acute respiratory distress syndrome genetic polymorphisms associated with susceptibility and outcome in ards cloning and characterization of the cdna encoding a novel human pre-b-cell colony-enhancing factor genomic organization of the gene coding for human pre-b-cell colony enhancing factor and expression in human fetal membranes making genomics functional: deciphering the genetics of acute lung injury biomarkers of inflammation, coagulation and fibrinolysis predict mortality in acute lung injury protein c and thrombomodulin in human acute lung injury key: cord- -ai el j authors: li, yaqi; tang, peiyuan; cai, sanjun; peng, junjie; hua, guoqiang title: organoid based personalized medicine: from bench to bedside date: - - journal: cell regen doi: . /s - - -z sha: doc_id: cord_uid: ai el j three-dimensional cultured organoids have become a powerful in vitro research tool that preserves genetic, phenotypic and behavioral trait of in vivo organs, which can be established from both pluripotent stem cells and adult stem cells. organoids derived from adult stem cells can be established directly from diseased epithelium and matched normal tissues, and organoids can also be genetically manipulated by crispr-cas technology. applications of organoids in basic research involve the modeling of human development and diseases, including genetic, infectious and malignant diseases. importantly, accumulating evidence suggests that biobanks of patient-derived organoids for many cancers and cystic fibrosis have great value for drug development and personalized medicine. in addition, organoids hold promise for regenerative medicine. in the present review, we discuss the applications of organoids in the basic and translational research. two-dimensional ( d) cultured cell lines have been the main in vitro research tool for the past decades. cell lines are relatively cheap, easy to handle and can be applied to multiple experimental techniques. however, the establishment of a cell line is time-consuming and involves extensive genetic and phenotypic adaption to culture conditions. thus, most cell lines are derived from tumors or have acquired oncogenic potential in vitro, while matching normal cells are usually lacking. the main problem of cell lines is the homogeneity of the cells, short of differentiated cell types in the original tissue. these problems limit the use of cell lines in personalized medicine and make them less suited to tissue physiology research requiring differentiated cell types. in cancer research, the preclinical research model that can phenocopy tumor heterogeneity is highly needed for research on the mechanisms of cancer progression and acquired drug resistance. in , the first patient-derived xenograft (pdx) models were successfully established (toolan ). in this model, primary tumor tissue is transplanted into immune-deficient mice, while tumor structure and the relative proportion of tumor cells and stromal cells are largely preserved (byrne et al. ). thus, pdxs better retain the complexity and heterogeneity of the parental tumor than do cell lines, but establishment is still inefficient and early tumors are hard to establish (john et al. ) . besides, genetic manipulations cannot be carried out, and high-throughput analyses are expensive and hampered by complex logistics. last decade has witnessed a booming development of three-dimensional ( d) cell culture technologies. the advent of organoids avoids many of the disadvantages associated with cell lines and pdx. an organoid is characterized as a d structure, grown from stem and progenitor cells and consisting of variant organ-specific cell types, that self-organize via cell differentiation and spatially restricted lineage commitment (clevers ) . organoids can be grown from two types of cells: (i) pluripotent stem cells (pscs), such as embryonic stem cells (escs) and induced pluripotent stem cells (ipscs), or (ii) adult stem cells (ascs) (clevers ; rookmaaker et al. ) . organoids are proved amenable to all standard laboratory techniques, as well as to genetic modification drost et al. ; schwank et al. ) . organoids can be fast expanded, cryopreserved and applied to high-throughput analyses. though organoid cultures cannot mimic interactions with vasculature and stroma, organoids are a promising research model bridging the gap between cell lines and pdxs ( fig. ) (drost ; sachs and clevers ) . since cell lines of escs and ipscs were established, researchers began to apply insights to induce these stem cells to generate differentiated cell types (chen et al. ; cherry ) . yoshiki sasai and colleagues firstly dug deeper by questioning whether such an in vitro model could mimic in vivo development and thus developed methods to culture brain, retina and pituitary structures 'in a dish' eiraku et al. ) . later, ipscs-derived organoids from optic cup, intestine, stomach, liver, lung, thyroid and kidney, were followed (chen et al. ; kurmann et al. ; mccracken et al. ; mccracken et al. ; nakano et al. ; takasato et al. ; takebe et al. ) . of note, each germ layer (endoderm, mesoderm, and ectoderm) is represented among this set of organs. typically, ipscs are expanded and subsequently differentiated through a multi-step protocol that moves towards a fully differentiated structure, and specific cocktails of growth factors are required for each step (fig. ) . the differentiation process usually takes about - months, which depends on the specific type of organ (mccracken et al. ) . the structure of ipscs-derived organoids is complex and may contain mesenchymal, as well as epithelial and endothelial components. because differentiation protocols recapitulate development in vitro, ipscs-derived organoids are excellent models for studying development (takasato et al. ) , genetic diseases (freedman et al. ) , and infectious disease (garcez et al. ) . another air-liquid interface (ali) method was introduced allowing for the preservation of both epithelium and matched in vitro stromal microenvironment (neal et al. ). the ali method employs a boyden chamber-like structure where primary tissue is seeded in ecm (extracellular matrix) gel in an inner transwell tm fig. comparison of cell lines, patient-derived xenografts and organoids. cell lines have low cost, are easy to handle and can be applied to multiple experimental techniques. pdxs preserve tumor heterogeneity and tumor-stromal interactions. pdos can be derived from both epithelial cancer cells and normal epithelium and cultured in an extracellular matrix (ecm) -providing basement membrane extract dish which is exposed to air to enhance oxygenation (dimarco et al. ; li et al. ; ootani et al. ). culture medium is added to the outer dish and can diffuse through the permeable transwell tm into the inner dish (fig. ) . ali method has been applied in pscsderived organoid culture lately. koike and colleagues (koike et al. ) reported the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from ali culture of anterior and posterior gut spheroids differentiated from human psc. adapted ali culture of human cerebral organoids (giandomenico et al. ) and neocortical organoid (qian et al. ) derived from pscs were also developed. complementary to pscs-derived organoids that recapitulate development in vitro, ascs-derived organoids model adult tissue repair (clevers ) and can be established only from regenerative tissue compartments. in , researches began to explore d-culture by culturing primary cells on a reconstituted basement membrane from engelbreth-holm-swarm (ehs) tumor (li et al. ; shannon et al. ). li and colleagues found mammary epithelial cells cultured on ehs matrix could form ducts, ductules, and lumina and resemble secretory alveoli. shannon and colleagues cultured adult rat type ii cells on ehs matrix with feeder layer cells (mainly fibroblasts) and revealed that cell-matrix interactions help type ii cells preserve their original cubical shape and morphological characteristics of variable differentiated cells. exploration based on ascs-derived d culture has been led to a new stage. two decades later, ascs-derived organoids were successfully developed from lgr -positive intestinal stem cells in culture conditions modeling the stem cell niche of intestine sato et al. ). by providing the wnt agonist r-spondin, epidermal growth factor (egf), and the bone morphogenetic protein (bmp) inhibitor noggin, and embedding the cells in an extracellular matrix (ecm) -providing basement membrane extract (wenr method, culture strategy and applications of pluripotent stem cell (psc)-derived organoids. pscs-derived organoids can be differentiated toward each of the three germ layers (endoderm, mesoderm, and ectoderm) under specific differentiation signals. pscs-derived organoids can be applied to studying development, genetic diseases, and infectious disease spondin- ), lgr -positive stem cells are able to selforganize, proliferate and form differentiated crypt-villuslike organoids (fig. a, b ). since then, by modifying cocktails of growth factors and cell isolation procedures, cultures of patient-derived organoids (pdos) have been successfully established for various human tissues by biopsy or resection, including the esophagus , stomach (bartfeld et al. ) , colon , liver (broutier et al. ; hu et al. ; huch et al. ) , pancreas (boj et al. ) , salivary gland (nanduri et al. ) , fallopian tube (kessler et al. ) , ovary (hill et al. ; kopper et al. ) , prostate karthaus et al. ) , breast , airway (sachs et al. ) , taste buds (ren et al. ) , endometrium (turco et al. ) , kidney (schutgens et al. ) , bladder (lee et al. ) , thyroid (saito et al. ) , biliary tract (saito et al. ) , oral mucosa (driehuis et al. a ) and glioblastoma (fig. , table ) . a counterintuitive phenomenon is found that normal epithelium organoids often outgrow tumor organoids, which, in some instances, can be prevented by using cancer-specific selection methods. for example, tumor organoids from colorectal cancer (crc) can be selectively expanded upon withdrawal of wnt a and r-spondin . nearly all crcs harbor activating mutations in the wnt pathway or fusion of rspo(r-spondin- ) genes, allowing for the expansion of cancer cells without wnts and r-spondins, while normal epithelial cells arrest (nusse ; sato et al. ; seshagiri et al. ; van de wetering et al. ) . another approach to culture tumor cells selectively is to stabilize wild-type p by adding the mdm inhibitor nutlin- (drost et al. ) . tumor cells are not affected by nutlin- due to a loss of tp (olivier et al. ) , while normal cells in culture present cell cycle arrest and death, allowing for the selection of tumor cells. in general, pdos using wenr method can be derived from any epithelium of normal tissues as well as malignant or otherwise diseased tissues within approximately days after embedding the cells into ecm matrix ( fig. c; fig. ). pdos can be expanded long term and cryopreserved while remaining genetically stable, making organoids an ideal tool for disease modeling. in addition, this type of organoid culture allows the direct parallel expansion of diseased cells and matched normal cells from individual patients, which allows for the generation of living tumor organoid biobank and facilitates its potential application in personalized therapy (fig. ) . however, to date, nearly all pdos types represent only the epithelial parts of organs, and there is an absence of stroma, nerves, and vasculature. adopting ali method, researchers can generate ascs-derived organoids from various murine tissues including small intestine, colon, stomach, and pancreas (li et al. ; ootani et al. ), then extending to culture clinical tumor samples (neal and kuo ; neal et al. ) , accurately recapitulating stem cell populations and their multi-lineage differentiation. the ali model preserves tumor microenvironment with tumor parenchyma and stroma, including functional tumor infiltrating lymphocytes (tils), providing a promising model for immunotherapy research for patients with cancer (neal et al. ) . in the remainder of this review, we will discuss how pscs-derived organoids and ascs-derived organoids are applied in basic and translational research. comparison of different culture methods for adult stem cells-derived organoids. in the wenr method, epithelial organoids are derived from tumor biopsies directly in matrigel with cocktail growth factors, with long-term expansion but no tumor micro environment. in the air-liquid interphase (ali) method, tumor biopsies are cultured in ali in the entire tumor microenvironment as a cell suspension of all cell types, including immune cells and other non-epithelial cell types, but with limited expansion tissue physiology organoids as a research tool for stem cell biology organoids is an ideal in vitro tool for the identification of novel stem cell markers, and the study of physiological phenomena requiring the coculture of multiple cell types. lgr + cells located at the crypt base was verified as the real intestinal stem cells (barker et al. ) . enlightened by the finding that lgr + intestinal stem cells can undergo thousands of cell divisions in vivo, sato and colleagues sato et al. ) successfully established the epithelial organoids from a single lgr + stem cell, which are also known as "miniguts". wnt signals, notch signals, egf signals and bmp signals together contribute the stem cell niche homeostasis (clevers ) . other stem cell biomarkers have been explored to initiate intestinal organoid cultures, including cd (von furstenberg et al. ) , ephb (jung et al. ) , and cd +/grp (wang et al. ) . mini-guts contain multiple differentiated cell types. rapidly dividing, transit-amplifying (ta) daughter cells derived from lgr + cells can differentiate to enterocytes, fig. morphology of several types of human adult stem-cell organoids. a a schematic diagram showing the growth pattern of organoids. typically, isolated cells or functional aggregates are embedded in extracellular matrix domes and cultured in media with essential niche factors. they gradually build tissue-like d structures within - weeks. b bright-field image of a typical murine small intestinal organoid culture. c brightfield image and he staining image of typical human normal colon epithelium, adenoma and adenocarcinoma organoids paneth cells, goblet cells, enteroendocrine cells, tuft cells, and the m cells that cover peyer's patches (clevers ) ,contributing to the study of physiology of cryptvillus axis. lendeboom and colleagues (lindeboom et al. ) applied a multi-omics framework on stem cellenriched and enterocytes-enriched mouse intestinal organoids to reveal multiple layers of gene expression regulation contributing to lineage specification and plasticity of intestine and found that hnf g as a major driver of enterocyte differentiation. as another example, beumer and colleagues (basak et al. ; beumer et al. ) used organoids to study the effect of growth factors on hormone expression in enteroendocrine cells after establishing a protocol to obtain enteroendocrine cells in organoids. in organoids, hormones in enteroendocrine cells were differentially expressed based on the presence or absence of bmp . this finding was then studied in a mouse model, and it was found that the bmp gradient along the crypt-villus axis in vivo dictates a switch in expressed hormones in enteroendocrine cells that migrate up this bmp gradient. beumer and his colleagues ) further constructed an organoid-based platform for functional studies of human enteroendocrine cells, which can be induced by transient expression of neurog . by using single-cell mrna sequencing and mass-spectrometry, they revealed differences of human enteroendocrine cells with mice, and several secreted products were identified and validated by functional experiments. the mini-gut culture approach has been applied to the generation of organoids derived from the epithelial compartments of a variety of murine and human tissues of ecto-, meso-and endodermal origin, and promotes the study of stem cell biology of other tissues except for intestine. for example, long-term expanding organoids modeling mature pyloric epithelium can be efficiently generated from single lgr + stem cells located at the base of pyloric glands (barker et al. ) . later, strange and colleagues (stange et al. ) discovered that troy + chief cells can spontaneously dedifferentiate to act as multipotent epithelial stem cells in vivo, particularly upon damage. importantly, single troy + chief cells can initiate long-term expanding gastric organoids, containing various cell types of corpus glands. the finding further confirms troy + chief cells' role as "reserve" stem cells upon challenge of tissue homeostasis. organoid culture allows for the generation of specific cell types that were previously impossible in d cultures. for example, hepatocytes can be successfully established and expanded in organoid culture peng et al. ) . based on adult bile duct-derived bipotent progenitor organoids , culture table comparison of the conditioned media requirements for the patient-derived organoids culture of respective cancer types conditions were developed that supported the growth of human hepatocyte organoids. the organoids proliferate greatly after transplanting into mice ). the resulting hepatocytes maintained its original physiological functions, including secreting cytoplasmic glycogen particles, forming bile canaliculi, and expressing albumin and cytochrome p enzymes. based on organoid culture system of hepatocytes, peng and colleagues (peng et al. ) described a unique effect of tumor necrosis factor-α, a cytokine essential for liver regeneration and found that the addition of regeneration-enhancing cytokines in facilitating the in vitro expansion of cell types that are otherwise difficult to culture. as another example, yin and colleagues (yin et al. )showed modulation of wnt and notch signaling in intestinal organoids to direct lineage differentiation into mature enterocytes, goblet cells and paneth cells. specifically, the combination of iwp- (inhibitor of wnt production ; wnt pathway inhibitor) and vpa (valproic acid; notch activator) specifically induced enterocyte differentiation, presumably by combining the effects of both inhibitors, in which iwp- induced enterocyte differentiation while vpa suppressed the differentiation of lgr + stem cells toward secretory cell types. the combination of dapt (notch inhibitor) and chir (chicken immunoglobulin-like receptor; gsk β inhibitor) mainly induced paneth cell differentiation, and the combination of iwp- and dapt primarily induced goblet cell differentiation. these methods provide new tools for the study and application of multiple intestinal epithelial cell types. organoids can be established from a single cell, which makes it possible to study the mutational status of single stem cells. the gradual accumulation of genetic mutations in stem cells throughout life is related to a variety of age-related diseases, including cancer. in this way, blokzijl and colleagues (blokzijl et al. ) were able to unveil mutation rates and patterns in normal stem cells throughout life by whole-genome sequencing (wgs) analysis (with peripheral blood as a reference for germline mutations). interestingly, the mutation rate, with around novel mutations per year per stem cell, was applications of adult stem cells-derived organoids. a organoids derived from normal tissue are useful for studying physiology. for disease modeling, organoids can be genetically engineered to model genetic and malignant diseases by using crispr-cas . normal organoids can also be infected with different types of pathogens to model infectious disease. normal organoids can be transplanted to wounds for tissue repair. b tumor-derived organoids can be used for basic research by genetic modification and modeling rare cancer. for translational research, tumorderived organoids can be used for biobanking, genetic repair and drug screening studies, both for personalized medicine (to choose the most effective treatment for a specific patient) and drug development (to test a compound library on a specific set of tumor organoids), as well as immunotherapy research similar in liver, small intestine, and colon stem cells, regardless of the large variation in cancer incidence of these organs. however, the types of mutations detected and the resulting mutational signatures in colon and small intestine cells were different from those in liver cells. to be pointed out, the inter-individual variation in mutation rate and spectra are low, indicating organspecific activity of common mutational processes throughout life. disease modeling crispr-cas technology as a useful tool for disease modeling of organoids the clustered regularly interspaced short palindromic repeats (crispr) associated protein (cas )/crispr system has become a major technology for mammalian genome editing. the system consists of cas nuclease derived from streptococcus pyogenes and guide rna which can recognize and target a specified dna sequence preceding the motif sequence adjacent to the. crispr-cas can generate dna double-strand breaks at specific genomic sites. mammalian double-strand dna breaks can be repaired by two ways, nonhomologous end joining (nhej) and homology-directed repair (hdr). nhej inserts indels randomly in the process of repair, and biallelic introduction of indels leads to gene knock-out (komor and badran ) . on the other hand, hdr can replace the damaged allele by existing intact genome, thus when tailored dna templates are co-delivered with crispr-cas , hdr can be used for gene knock-in (komor and badran ) . although the crispr-cas technology has broadened its applications to a series of purposes, including dna base editing, rna targeting, epigenome editing and gene expression manipulation (adli ; komor and badran ) , the use of crispr-cas on organoids still basically harness nhej and hdr to engineer genes of interest. indeed, organoids are ideal model for investigating gene function by genome editing, as the organoid system allows for fast expansion with stable genetics and phenotype. previous studies have successfully achieved genome editing by installing crispr-cas into organoids using various approaches, including liposomal transfection, electroporation and viral infection (fig. ) . however, variable experimental conditions limit the efficiency of genome editing in organoids, including the recovery after single-cell isolation, approaches for crispr-cas delivery, and the cleavage efficiency of the guide rna. selection and enrichment of positive organoids are necessary after crispr-cas -mediated genome editing, or otherwise, labor-intensive organoid cloning, followed by sequencing of expanded organoid clones is needed. recently, ringel and his colleagues (ringel et al. ) developed a genome-wide pooled-library crispr screen approach by capturing sgrna (single-guide rna) integrations in single human intestinal organoids to dissect oncogenic signaling pathways. their screening method would be broadly applicable to various organoid models and selection assays, which may contribute to dissecting human disease mechanisms and facilitating biological discovery in primary d tissue models. currently, organoids have become a useful tool to model genetic diseases. generally, two types of methods have been adopted: (i) organoids established from patientderived biopsies; (ii) specific genetic mutations introduced to wild-type organoids using crispr-cas technology. cystic fibrosis (cf) is the best example for pdo modeling human genetic disease. cf is a monogenic channelopathy caused by inactivating mutations in the cf transmembrane conductance regulator (cftr) gene. the disease involves multiple organs, including the intestine, lung, pancreas, liver, kidney and sweat gland. in gastrointestinal organs and lungs, decreased cftr function results in reduced chloride transport through cftr toward the extracellular space, leading to a reduced water flow by osmosis and, hence, increased density of mucus. in the sweat glands, loss of cftr function leads to a high saline concentration in sweat. organoid model was firstly derived from rectum of cf patients. early work with organoids derived from the rectum of cf patients revealed cftr function: wild-type organoids rapidly swell upon opening the cftr channel in a cyclic adenosine monophosphate (camp)dependent manner through the addition of forskolin (fsk) ). rectal organoids from cf patients do not respond to fsk, but it is restored upon pre-incubation with cftr-restoring compounds or upon correction of the cftr mutation by crispr-cas (schwank et al. ). based on this finding, this organoid-based fsk-induced swelling assay began to be used to test drug response on organoids isolated from patients harboring different cftr mutations, including rare variants . lung is another organ that can be severely harmed by cf. due to cftr mutation, a thick sticky mucus forms in the lungs, which impairs breathing and provides a fertile environment for pathogens reproduction, leading to premature respiratory failure. cf airway organoids can be established from patient-derived ipscs (firth et al. ) , bronchial epithelial cells based on both ali (fulcher et al. ) and wenr cultures (sachs et al. ) or bronchoalveolar lavage fluids (no biopsy needed) (sachs et al. ; sondo et al. ). airway organoids from cf patients had an increased mucus layer, recapitulating the disease phenotype. fsk-induced swelling in airway organoids was reduced compared with organoids from normal controls and could be restored with cftrrestoring compounds. however, in contrast to rectal organoids, fsk-induced swelling in lung organoids did not depend on cftr alone, it was also influenced by the chloride transporter tmem a, which is set as an alternative therapeutic target for cf patients. therefore, airway organoids may function as an additional platform for assessing drug response to cf, particularly for drugs acting on tmem a. besides, liver, pancreatic and kidney organoids derived from cf patients can also be established. sampaziotis and colleagues (sampaziotis et al. ) generated ipscs from skin fibroblasts of a cf patient with homozygous f mutation and differentiated them into cholangiocyte-like cells (clc). cf-clcs expressed displayed defective expression of cftr protein. cf-clc organoids treated with experimental cf drug vx increases cftr function and improves intraluminal fluid secretion. cf pancreatic organoids can be established from pscs of cf patients, including both human escs and an ipsc line, to generate differentiated pancreatic ductal epithelial cells (pdecs) (simsek et al. ). pdecs derived from cf-ipscs showed decreased expression of cftr protein and damaged chloride ion channel activity, reappearing functional defects of patients with cf at the cellular level. in addition, a tubuloid line from urine of a cf patient was established (schutgens et al. ) . at the morphological level, the kidney tubuloids maintained folded over long-term culture, instead of the typical cystic phenotype, which was probably due to the lack of cftr function caused by cftr mutations f del/s n. in tubuloids derived from urine of cf patients, fsk caused slight swelling in a concentration-dependent manner, suggesting residual cftr function, while after pre-incubation with the fig. genome editing in organoids by crispr-cas technology. workflow of genetic engineering in organoids using crispr-cas . either nhej or hr can be exploited for gene knock-out or knock-in, respectively. in most cases, expansion of single organoid clones after the selection procedure is necessary to obtain isogenic organoid populations cftr-potentiator drug vx- (ivacaftor, kalydeco), swelling increased significantly. all the above cf organoid models of different related organs allow in vitro assessment of treatment response and development of novel drugs. for a discussion of the use of cf-pdo for personalized medicine, see section . . additionally, intestinal organoids harboring inactivation mutation of ttc a have been successfully derived from patients with intestinal atresia, which recapitulates how ttca deficiency results in the loss of apical-basal cell polarity in the intestinal epithelium that can be rescued by adding rho kinase inhibitors (bigorgne et al. ) . besides, intestinal organoids were derived from patients with microvillus inclusion disease (mvix) caused by homozygous truncating mutations of syntaxin- (stx ) gene. the model revealed that partial loss of brush border microvilli and subapical accumulation of vesicles are typical histological phenomena of the disease (wiegerinck et al. ). liver organoids have been generated from patients with α -antitrypsin (a at) deficiency. accumulation of mutant a at in the endoplasmic reticulum in the liver leads to fibrosis or cirrhosis. liver organoids derived from the patients indeed contained a at aggregates and presented increased apoptosis, which might contribute to fibrosis and cirrhosis . alagille syndrome is caused by loss-of-function mutations in jag or notch and leads to partial or complete biliary atresia. accordingly, organoids generated from a patient with alagille syndrome can not differentiate toward the biliary fate, whereas in proliferate conditions, no differences were observed compared with healthy controls (andersson et al. ; sachs et al. ) . ipscs-derived organoids can also be manipulated by crispr-cas technology to model diseases in different tissues. in human ipscs-derived kidney organoids, knock-out of podocalyxin and pkd genes freedman et al. ) recapitulated defects that mimic nephrotic syndrome and polycystic kidney disease respectively, as well as contributed to understanding the functions of the genes in the pathogenesis context. engineered ipscs-derived liver organoids helped in illustrating the various functions that different mutations of jag gene can have in the development of bile ducts and genesis the alagille syndrome: the c x mutation of jag can causes significant alterations, while the g d mutation does not affect organoid properties (guan et al. ) . in brain tissue, patient-specific ipscs-derived brain organoids can be used to model lissencephaly (bershteyn et al. ) , down syndrome , and neuronal heterotopia (klaus et al. ) . engineered ipscs-derived brain organoids were established to model microcephaly by rna interference of reprogramming factors (lancaster et al. ) , autism by overexpression of the transcription factor foxg (forkhead box g ) (mariani et al. ) , macrocephaly by deletion of pten (li et al. ) , timothy syndrome by introducing mutations in the cav . calcium channel-interneurons (birey et al. ) and aicardi-goutières syndrome by introducing inactivation mutation of trex- ( thomas et al. ) . fused organoids culture was recently established to understand more complex biology, which was more applied in brain study. bagley and colleagues (bagley et al. ) firstly showed a co-culture method combining brain regions of choice within one organoid tissue and they generate a dorsal-ventral axis by fusing organoids of dorsal and ventral forebrain identities. combined with reprogramming technology, their novel fusions of organoids culture should offer researchers the possibility to analyze complex neurodevelopmental defects using cells from neurological disease patients and to test potential therapeutic compounds. xiang and his colleagues (xiang et al. ) successfully established and fused medial ganglionic eminence (mge) and cortex-specific organoids from human pluripotent stem cells followed by live imaging, to investigate mge development and human interneuron migration and integration, which offers deeper insight into molecular dynamics during human brain development . the same research team developed a new d system to create the reciprocal projections between thalamus and cortex by fusing the two distinct region-specific organoids, providing a platform for understanding human thalamic development and modeling circuit organizations and related disorders in the brain (xiang et al. ) .generally, engineered organoids can faithfully recapitulate genetic diseases and thus provide a valid resource for basic research and for development of novel therapeutics. organoids are closed d structures that exhibit the apical side of the epithelium towards the lumen and the basal membrane towards the outside. the apical membrane within the lumen is initially exposed to pathogens in vivo. three different methods have been established to reproduce the interaction between microbes and the host in the organoids culture (fig. ). in the first method, organoids are mechanically sheared or digested into single-cell suspensions or large particles and then they are incubated with pathogens, which leads to infection of the cells. after embedded in the d matrix, infected cells can reform organoids that can be used to model infectious disease (dang et al. ; forbester et al. ; nigro et al. ; zhang et al. ). this method is easy to handle and do not require special equipment. but, the efficiency of infection varies among different pathogens and it can not reflect the initial interaction between microbes and the host. besides, during the process not only the apical side but also the basal side of cells are exposed, thus nonspecific responses may be introduced due to the interaction of pathogens with the basal side of the cells. in the second method, pathogens are directly injected in to the lumen of the organoids by a microinjection, thus the initial interaction of pathogens and the early response of the host cells can be captured and either apical or basal interaction can be investigated separately (bartfeld et al. ; leslie et al. ; mccracken et al. ). this method is now the mainstream method to build infection model. however, this method needs special such as a microinjector and it is hard to perform quantitatively due to the different sizes of organoids. in the third method, when single cells digested from organoids are seeded onto a d matrix-coated dish, they grow as d monolayer with the apical side exposed. adding pathogens directly into the culture media allows interaction between microbes and the host cells (ettayebi et al. ) . the d culture contains various differentiated cells and allows quantitative experiments, while it does not resemble the in vivo d structure of host tissues. based on the above approaches, organoids have been adopted to model viral, bacterial, and parasitic infectious diseases of different tissues, including diseases caused by pathogens that previously could not be studied in vitro (table ) . these models recapitulate features of in vivo infection and could help identify therapeutic targets and develop novel drugs and vaccine. pscs-derived organoids were firstly used to model viral disease. in neuroscience, the development of pscsderived brain organoids help deciphered the sequence of disease progression in zika virus (garcez et al. ; qian et al. ) . zika virus (zikv) mainly spreads by the aedes aegypti mosquito and its infection can lead to microcephaly, which was set as a public health emergency by fig. approaches of studying infectious diseases using organoids. organoids can be micro-injected with the microbe, thus the microbe is in direct contact with the apical side of epithelial cells, which became the mainstream method to build infection model. organoids can also be sheared into smaller aggregates, incubated with pathogens and re-seed into matrigel. alternatively, d organoids can be digested by enzyme into single cells and grown as d monolayer cultures, and then microbes are added into the culture media world health organization (who) in . however, the pathogenesis of the zikv infection was not fully understood until brain organoids emerged. multiple research demonstrated that zikv infection can cause disorder of the cortical layers of cerebral organoid, abrogating growth and thus halting neurogenesis. researchers found that zikv infection leads to the activation of the toll-like receptor (tlr ), contributing to deregulated neurogenesis and decreased functional neurons (cugola et al. ). gabriel and colleagues (garcez et al. ) further illustrated that two new isolated zikvs have different patterns of pathogenicity. unlike the highly passed mr strain of zikv, the new strains, infected apical proliferating progenitors, interfering with centrosomal protein assembly, which in turn led to their premature differentiation and apoptosis, resulting in microcephaly (wells et al. ) . the organoids model of zikv infection also promotes the development of treatment. in a high-throughput drug screen of compounds, caspase- activity inhibitors, emricasan and niclosamide, were found to be effective in limiting zikv induced death of neural cortical progenitor and zikv replication (xu et al. ) . except for zikv, japanese encephalitis virus (jev) infection, leading to japanese encephalitis (je), was modeled in generated telencephalon organoid . researchers found that jev infection caused decline of cell proliferation and increase of cell death, and infected astrocytes and neural progenitors. in addition, they revealed variable antiviral immunity in brain organoids of different stages of culture, which also provide clues to develop effective therapeutics of such diseases. another example of pscs-derived organoids application in human disease is with hepatitis b virus (hbv). in recent decades, treatments against hbv infection have improved; however, the development of personalized treatments has been hindered by the absence of personalized infection models. nie and colleagues (nie et al. ) generated pscs-derived liver organoids that recapitulated the genetic background of the donor, and found hbv infection in pscs-derived liver organoids could reproduce the life cycle of hbv and hbv-induced hepatic dysfunction, indicating that pscs-derived liver organoids may provide a promising personalized infection model for the development of personalized treatment for hepatitis. in recent years, ascs-derived organoids have become the main force in infectious diseases modeling. ascsderived organoids can be adopted to model the viral infection of intestinal organoids. gastric diarrhea in humans is mostly caused by human norovirus (hunov) and rotavirus infection (zheng et al. ) . although both of these viruses are rampant, no proper vaccine has been developed owing to the lack of in vitro culture method supporting their replication. intestinal organoids that were cultured as monolayers allowed for extensive replication of multiple strains of noroviruses. for some strains, the addition of bile to the culture medium was required for replication (ramani and atmar ) , indicating that not only are in vivo-like host cells required for productive infection but also an in vivo-like environment is relevant as well. ettayebi and colleagues (ettayebi et al. ) reproduced hunov infection in an organoid-virus co-culture system, with only a specific gii. hunov strain requiring the presence of bile. furthermore, lack of histo-blood group antigen (hbga) expression in intestinal organoids limits hunov replication, suggesting that this culture system allows the evaluation of potential treatments and preventions. similarly, researchers have shown that rotavirus strain (simian sa ) from clinical samples can proliferate in pscs-derived intestinal organoids (finkbeiner et al. ; yin et al. ). in urinary system, bk virus, which is a tubule-specific circular dna virus, infects - % of transplanted kidneys, leading in - % of these infected kidneys to the loss of the donor organ and no curative treatment exists (hirsch et al. ) . infection of kidney tubuloids (kidney-derived organoids in which only the tubular epithelium of the kidney is represented and glomeruli are lacking) with bk virus yielded a patchy infection with enlarged nuclei (due to intranuclear basophilic viral inclusions), similar to what is observed in kidney biopsies from patients with bk virus nephropathy (bohl and brennan ; schutgens et al. ) . respiratory infections pose a major global disease burdens (ferkol ) . respiratory syncytial virus (rsv) alone causes hundreds of thousands deaths annually among children, mostly in developing countries (nair et al. ) . ipscs-derived organoids of human airway epithelium can be infected by rsv virus, which can reproduce the morphological features of rsv infection in the distal lung (chen et al. ). persson and colleagues (persson et al. ) established a ali culture system for infection of human airway epithelium with rsv virus and they found that rsv has the potential to influence the cellular composition of the airway epithelium. besides, ascs-derived airway organoids using wenr methods can also be infected with rsv, recapitulating syncytia formation, cytoskeletal changes, and shedding of epithelial cells (mueller et al. ) . rsv-infected organoids attracted neutrophils more than did mockinfected control organoids, making this the first organoid model suitable for studying neutrophil-epithelium interactions (sachs et al. ) . intriguingly, rsv infection strongly increased organoid motility and ultimately resulted in organoid fusion. influenza viruses also pose a major public health problem worldwide, and novel emerging viruses may be lethal, as evidenced by the poultry-derived h n virus infection that has had a % mortality rate since . the infection of differentiated airway organoids with distinct strains of influenza virus can discriminate between poorly infective and highly infective strains (zhou et al. ) . importantly, hui and colleagues (hui et al. ) compared human and avian strains of influenza a virus in in vitro human bronchus and airway organoids, and found that the infection of airway organoids yielded similar results regarding virus replication and cytokine response. in addition, infection of airway organoids with enterovirus (ev ) showed that ev replication kinetics are strain-dependent and the model help identify new infectivity makers for ev (van der sanden et al. ) . the year of witnessed the outbreak of coronavirus disease- (covid- ) caused by the virus severe acute respiratory syndrome-coronavirus (sars-cov- ) which presents influenza-like symptoms ranging from mild disease to severe lung injury and multi-organ failure, eventually leading to death, especially in older patients with other co-morbidities. the who has declared that covid- is a public health emergency of pandemic proportions. organoids have been used as a great platform to research how covid- affects human and causes damage and for identifying possible drug targets for covid- . lamers and colleagues (lamers and beumer ) infected enterocytes in human small intestinal organoids with sars-cov and sars-cov- and found that the intestinal epithelium supports sars-cov- replication, and organoids can be served as an experimental model for coronavirus infection and biology. zhou and colleagues (zhou et al. ) established the first expandable organoid culture system of bat intestinal epithelium which were fully susceptible to sars-cov- infection and sustained robust viral replication. they also found active replication of sars-cov- in human intestinal organoids indicating that the human intestinal tract might be a transmission route of sars-cov- . in addition, monteil and colleagues (monteil et al. ) found that sars-cov- can directly infect engineered human blood vessel organoids and human kidney organoids, which can be inhibited by human recombinant soluble ace (hrsace ), demonstrating that hrsace can be a possible drug for early stages of covid- . salmonella typhi (s. typhi) and clostridium difficile (c. difficile) are the two major bacterial intestinal pathogens that can cause diarrhea and gastrointestinal failures in humans. these pathogens infection has been successfully modeled using pscs-derived intestinal organoids. forbester and colleagues (forbester et al. ) microinjected live s. typhi into the lumen of the ipscs-derived intestinal organoids and revealed that upon injection, nf-κb signaling was activated and inflammatory factors were secreted, which was consistent with previous findings in animal models. likewise, the spence lab (leslie et al. ) microinjected c. difficile toxin a (tcda) and toxin b (tcdb) into the lumen of ipscs-derived intestinal organoids to model anaerobe c. difficile infection (cdi). the injection of tcda recapitulates the impair of the epithelial barrier function and structure observed in organoids colonized with viable c. difficile. in another study, the worrell lab (engevik et al. ) observed a decreased expression of nhe (sodium/hydrogen exchanger ) and muc (mucoprotein ) protein in c. difficile infected organoids compared to normal organoids, which may help creating a favorable environment for its colonization. bacterium helicobacter pylori (h. pylori) infection is a major risk factor for peptic ulcers, gastric adenocarcinoma and gastritis (salama and hartung ) . both gastric organoids derived from ipscs and ascs can be used to model h. pylori infection, by microinjecting h. pylori strain into the lumen of organoids (mccracken et al. ) , which can ensure the apical side exposed to h. pylori. luminal injection of h. pylori induces a potent nf-κb-mediated inflammatory response (bartfeld et al. ) , connecting excessive microbial colonization of h. pylori with the occurrence of gastric cancer. in a followup study, researchers adopted gastric organoids to find out how h. pylori finds its gastric niche: a potent chemoattractant, urea, which produced by gastric epithelium is essential for the colonization of h. pylori in the gastric mucosa (huang et al. a) . chronic salmonella infection of the gall bladder is associated with gallbladder carcinoma (shukla et al. ) . scanu and colleagues (scanu et al. ) showed that after salmonella infection, mouse gallbladder organoids exhibited characteristics of loss of polarity, familiar with those showed in the mouse model of gallbladder cancer. another study found that gallbladder organoids preexposed to salmonella that lack functional tp showed neoplastic transformations by activating akt (protein kinase b) and mapk (mitogen-activated protein kinase) pathway and could grow in culture media free of growth factors (scanu et al. ) . the protozoan parasite cryptosporidium causes lifethreatening diarrhea in immunocompromised individuals (e.g. people living with hiv and malnourished children), and infection may spread to the lungs (checkley et al. ) . drug development requires detailed pathophysiology information of cryptosporidium, but the lack of an optimal in vitro culture system hinders the experimental approaches. heo and colleague (heo et al. ) infected epithelial organoids derived from human small intestine and lung with cryptosporidium and found that the parasite can reproduce within the organoids and complete its complex asexual and sexual life cycles for multiple rounds. plasmodium parasites can cause malaria, which poses a significant global health burden, with over million cases every year. plasmodium parasites are maintained between anopheles mosquitoes and mammalian hosts in a complex life cycle, and models to study them are challenging to establish, particularly for plasmodium species that infect humans (mellin and boddey ) . recently, several studies reported the application of ipscs-derived hepatocyte-like cells to model in vitro liver stage infections with p. berghei, p. yoelii, p. falciparum, and p. vivax (ng et al. ) . it was found that p. yoelii and p. falciparum infections of organoids recapitulated the primaquine sensitivity found in vivo. chua and colleagues (chua et al. ) infected organoids derived from simian and human hepatocytes, with p. cynomolgi and p. vivax and found that organoids could support the complete liver stage of both simian and human parasites, from initial infection with sporozoites, to the release of merozoites capable of erythrocyte infection. this study also illustrated the use of infected organoids to evaluate the response to an anti-relapse drug, highlighting the potential for organoids as a parasite drug screening platform, particularly in parasites with life-cycles longer than their host cells. though organoids derived from tumor and matched normal epithelial tissues provide valuable research tools for cancer biology, one of the most remarkable improvements in organoid research is the capacity to manipulate the genomes, transcriptomes and epigenomes of normal epithelial organoids to study the role of specific alterations in the process of tumorigenesis. murine organoid cultures were firstly used to study the early stages of tumorigenesis. li and colleagues (li et al. ) adopted the ali culture approach combined with genetically engineered mouse model and the retrovirus-mediated delivery of shrna constructs, to model multi-step tumorigenesis in organoids derived from digestive tract, including the colon, stomach, and pancreas. pancreatic and gastric organoids exhibited dysplasia as a result of expression of kras g d , p loss or both. while colon organoids needed assembled apc, p , kras g d and smad mutations for malignant transformation to invasive adenocarcinoma-like morphology. all engineered organoids presented histologic characteristics of adenocarcinoma after subcutaneous implantation was performed to immunocompromised mice. following research tried to model multi-step tumorigenesis of conventional crc, which is characterized by chromosomal instability (cin) (fig. ). drost and colleagues (drost et al. ) adopted crispr-mediated knock-out of the tumor suppressors apc, tp , and smad , married with crispr-mediated knock-in of the oncogenes kras g d to model multi-step tumorigenesis. after being selected by niche factors in the culture media, cultures of organs were successfully built with complex oncogenic multi-gene modules that contain up to four simultaneous changes. the -hit akst (apc, kras g d , smad , and tp ) organoids could grow without stem cell niche factors such as wnt- , rspondin- and egf. akst organoids were able to generate tumors with characteristics of invasive carcinoma upon subcutaneous implantation into immunocompromised mice. matano and colleagues (matano et al. ) applied a similar method to model tumorigenesis by inserting an additional crispr-mediated knock-in of the oncogene pik ca e , in addition to akst. both studies showed that organoids with apc and tp mutations showed extensive aneuploidy, which is the hallmark of the cin pathway. xenotransplantation of engineered colorectal tumor organoids makes it possible to study cancer stem cells in vivo (de sousa e melo et al. ; shimokawa et al. ) and leads to metastatic diseases, making organoids a useful research tool to study metastasis mechanisms (fumagalli et al. ; roper et al. ) . de sousa e melo and colleagues (de sousa e melo et al. ) combined crc mouse with the lgr dtr/egfp allele. the resulting animals carry two of the most frequently mutated genes, apc and kras g d , and in addition, express a diphtheria toxin receptor fused to an egfp under the endogenous regulatory region of lgr , allowing specific elimination and visualization of lgr -positive stem cells. using this model, it was found that in the absence of cancer stem cells, liver metastases did not occur, whereas primary tumors did not regress, indicating that lgr -positive cancer stem cells are required for metastasis. in another study, fumagalli and colleagues (fumagalli et al. ) orthotopically transplanted cris pr-mediated kras, apc, tp , and smad comutated human colon organoids into mice and showed that metastases to the liver and lungs occurred in % of the mice. almost no metastasis occurred when organoids carrying mutations in only three of these four genes were transplanted to mice; however, the lack of the fourth mutation could be overcome by providing the niche factor upstream of the absent mutation. for example, organoids with triple mutants lacking smad inactivation metastasized when noggin was added to the cells. these findings indicate that metastatic potential is directly related to the loss of niche factor dependency. crc that arises from the serrated neoplasia pathway is different from crc that arises from the conventional cin pathway. the activation of oncogene braf initiated the serrated pathway, followed by the extensive hypermethylation of cpg island methylator phenotype, and subsequent inactivation of tumor suppressor genes (bae and kim ). fessler and colleagues (fessler et al. ) firstly built the organoids to model the serrated pathway of crc by introducing the braf v e mutation into normal human colon epithelial organoids via homologous recombination. they revealed that induction of a mesenchymal phenotype upon tgfβ treatment prevails in the braf v e mutated organoids generating sessile serrated adenomas (ssas). in a recent study, by fig. assessing different tumorigenicity and metastases mechanisms of combinative mutations in colon cancer based on normal colon organoids by using crispr-cas technology analyzing the genomic data from tcga, crcassociated gene alterations including braf v e , cdkn a, tgfbr , znrf and rnf , were selected to be introduced into murine colon organoids using crispr-cas technology to model the serrated pathway (lannagan et al. ) . upon subcutaneous implantation into immunocompromised mice, these engineered organoids could generate tumors with characteristics of serrated crc, including desmoplastic stromal responses, infiltrative growth, mucinous differentiation, tumor budding, and the formation of colon tumors spontaneously metastasizing to the liver. to be pointed out, transplantation of braf-mutated organoids failed to generate tumors, while injection of organoids with both mutations of braf and tgfbr led to invasive adenocarcinoma. implantation of organoids with those and additional mutations (cdkn a, znrf and rnf ) resulted in increased tumor initiation and decreased survival time. moreover, to study the development of the r-spondin-driven serrated pathway, another study also introduced crispr-mediated gene fusions on braf v e and/or tp accompanied with the r-spondin fusion eif e (eukaryotic translation initiation factor subunit e )-rspo to normal human colon epithelial organoids (kawasaki et al. ). the resulting braf v e organoids showed aberrant crypt formation ability with poor engraftment capacity, while organoids with mutations of braf v e and tp with eif e-rspo fusion generated flatly elevated lesions and hyperplastic crypt structures with 'v'-shaped serration and basal dilation, together with enhanced engraftment capacity compared with only braf v e mutated organoids. the loss of dna mismatch repair enzymes, such as mutl homolog (mlh ), is common in crcs, resulting in tumors with high mutational load. these tumors are characterized by microsatellite instability (msi), as repetitive short sequences in the genome, which leads to changes in copy number following the loss of mismatch repair enzymes. drost and colleagues used crispr-mediated knock-out of mlh and cultured organoids for months to allow accumulation of mutations. subsequent dna analyses of cultured organoids revealed an increase in mutational load compared with controls, which was similar to that of msi colorectal tumors. this study shows that organoids faithfully recapitulate in vivo mutagenesis and allow for the identification of mechanisms of tumor development. in addition, rare type of colorectal cancer can also be modeled based on organoid culture. li and colleagues ) have established a novel organoid line of colon signet-ring cell carcinoma (srcc), which accounts for only % of colorectal cancers. the novel organoid line resembles the primary tumor histologically and molecularly, and can efficiently generate tumors on xenografts. targeted dna sequencing with drug screening of compound identified that jak (janus kinase ) might be a potential treatment target. an in vitro drug screening experiment exhibited that srcc organoids can be sensitively treated by at and pacritinib, two jak inhibitors, which was consistent with the in vivo xenograft response. the study provides a novel in vitro research tool for colorectal srcc and sets an example for personalized medicine based on organoids for rare cancer. pancreatic ductal adenocarcinoma (pdac) accounts for about % of all pancreatic malignancies and over % pdac patients harbor activation of the oncogene kras. kras can consequently induce inactivation of various tumor suppressor genes including tp , cdkn a, smad and brca to accelerate pdac development and progression (kanda et al. ; morris and wang ) . establishment of murine pancreatic organoids bearing a lox-stop-lox (lsl) krasg d allele provided valuable insights for the development of pdac. in one study (li et al. ) , pancreatic organoids derived from lsl-kras g d (k), tp flox/flox (p), and lsl-kras g d ; tp flox/flox (kp) mice were successfully built using ali method. k, p, and kp organoids exhibited in vitro dysplasia and increased invasive behavior, and could in vivo generate well differentiated to poorly differentiated adenocarcinoma upon implantation into immunocompromised nog mice. kp organoids had most poorly differentiated morphology, with significant loss of ecadherin, indicating increased epithelial to mesenchymal transition. researchers of another study generated lowgrade preinvasive pancreatic intraepithelial neoplasias (panins) in vivo by crossing the murine lsl-kras g d allele to a pancreas-specific pdx -cre driver and organoids derived from panin lesions could be long-term expanded a produce panin-like lesions that persisted for up to months upon transplantation (boj et al. ) . two studies have adopted crispr-cas technology to manipulate pdac driver genes including kras g v , cdkn a, tp and smad (kcts) in normal human pancreatic ductal organoids seino et al. ) . upon orthotopically transplantation to immunocompromising mice, different combinations of mutants in organoids exhibited distinct panins features, but only kcts organoids displayed pdac histopathological transformation . notably, kc and kt organoids died after wnt removal for - weeks, while kct and kcts organoids survived and expanded for at least months, suggesting that cdkn a and tp mutations are essential for organoids to grow independently of stem cell niche factors . in all, these studies indicate that organoids combined with crispr-mediated sequential mutations can recapitulate tumorigenesis and progression from panin to pdac. moreover, organoids could also be used to explore the roles of various factors such as the redox regulator nrf and the transcriptional enhancer foxa in pdac progression (chio et al. ; roe et al. ) . further researches are need to apply the improved knowledge of molecular mechanisms to clinics. gastric cancer are classified into four molecular subtypes based on deep sequencing: epstein-barr virus positive, msi, cin and genomically stable (cancer genome atlas research ). nanki and colleagues adopted organoids to illustrate the genotype-phenotype associations of gastric cancer. phenotype analyses of organoids derived from gastric cancer patients indicated multiple genetic and epigenetic ways to confer wnt and r-spondin niche independency. they found that induction of rnf and znrf mutations was sufficient for gastric cancer organoids to gain r-spondin independency. the phenomenon was then validated in crispr-cas engineered gastric organoids. in a similar study, seidlitz and colleagues (seidlitz et al. ) generated human and murine gastric cancer organoids which recapitulated the typical features and altered pathways of each of four molecular subtypes of gastric cancer. the combination of organoids and crispr-cas technologies promotes research on the molecular mechanisms of gastric cancer tumorigenesis and progression, thereby accelerating the development of preclinical gastric cancer models for novel drug development and personalized medicine. recently, dekkers and colleagues attempted to model multi-step carcinogenesis in breast epithelial organoids derived from human reduction mammoplasties using crispr-cas technology. they introduced crispr-mediated knock-out of four breast cancer-associated tumor suppressor genes, including p , pten, rb and nf (ptrn) to mammary progenitor cells. mutated organoids could long-term expanded and generated er+ luminal tumors upon transplantation into mice for out of ptr-mutated and out of ptrn-mutated organoid lines. these organoids had various response to endocrine therapy or chemotherapy, indicating the potential application of this model to facilitate our understanding of the molecular mechanisms in specific subtypes of breast cancer. in prostate cancers, - % of tumors harbor a gene fusion between the androgen receptor (ar)-responsive transmembrane protease serine (tmprss ) gene and e transformation-specific (ets) family transcription factor, most often the oncogene ets-related gene (erg) (tomlins et al. ) . tmprss and erg, both located on chromosome , are separated by about million base pairs. using crispr-cas , a tmprss -erg fusion was successfully introduced into mouse prostate organoids using a template that brought these two dna regions together. this genetic alteration resulted in armediated overexpression of erg, an effect that can be prevented by androgen receptor antagonist, consistent with those seen in vivo (driehuis ) . living organoid biobanks as a tool for personalized treatment and drug development as mentioned above, organoids can be efficiently established from patient-derived normal and tumor tissue samples, which can be cryopreserved and stored in living organoid biobanks. pdos resemble the tumor epithelium they were derived from both phenotypically and genetically. however, molecular profile alone is not sufficient to adequately predict drug sensitivity. even in patients with the same genotype, drug response varies. besides, some mutations are rare, thus clinical trials are impossible and efficacy testing is required for drugs to be conducted on an individual basis. thus, combined molecular and therapeutic profiling of pdos may help predict treatment response and contribute to personalized cancer treatment and drug development. a number of organoid biobanks have been reported since (table ) . a colon cancer derived biobank of lines was established in , setting an example for a standard biobank . all samples performed rna sequencing and whole genome sequencing analysis. the molecular characteristics of pdos cover all five consensus molecular subtypes of crc. the mutations in the organoids were largely concordant with the original tumors, which was validated in a set of organoids established of colorectal metastases (weeber et al. ) . high-throughput screening of a panel of compounds found that differences in drug sensitivity among the organoid lines that in some cases correlated with specific mutation. for example, rnf mutant organoids were sensitive to wnt secretion inhibitors, and kras-mutant organoids were resistant to the egfr (epidermal growth factor receptor) inhibitors, including cetuximab and afatinib. later, schutte and colleagues (schütte et al. ) reported a biobank of organoid lines from crc. they found that organoid models reproduce most the genetic and transcriptomic characteristics of the donors, but (vlachogiannis et al. ) determined less complex molecular subtypes for the absence of stroma. drug screening with therapeutic compounds representing the standard of care for crc, combined with molecular profiles, helped identify a signature outperforming ras/raf mutation which has predictive value for sensitivity to the egfr inhibitor cetuximab. drug response in organoids and clinical response was also observed to prove that the in vitro organoid response correlates with the in vivo response. a clinical study of pdos derived from metastatic gastroesophageal and crc showed a strong correlation ( % sensitivity, % specificity, % positive predictive value, and % negative predictive value) between the in vitro organoid response to a set of targeted therapies and chemotherapies and the response of the tumor in patients (vlachogiannis et al. ) . another study adopted organoids for colon cancer chemoprediction showing that pdos test predicted more than % of patients' response treated with irinotecan-based therapies (ooft et al. ) . together, these studies indicate the potential of tumor-derived organoids to predict patients' responses. recently, two studies showed the applications of pdos derived from rectal cancer to predicting patient responses to neoadjuvant chemoradiation therapy. yao and colleagues (yao et al. ) generated a rectal cancer derived biobank (n= ) and tested pdos' sensitivity to -fu, irinotecan, or radiation. they incorporated a correlation between in vitro responses in organoids and the histopathologically determined tumor regression scores (trgs) after surgical resection to define prognostic cut-offs. using these parameters, the in vitro responses could predict clinical responses with an impressive area under the curve (auc) of . and an accuracy of %. in the other study, ganesh and colleagues (ganesh et al. ) established pdo lines from rectal cancer to test responses to neoadjuvant chemoradiation therapy, including the standard folfox chemotherapy and radiation. the pdo responses significantly reflected the patients' progression-free survival. moreover, pdos generated invasive rectal cancer followed by metastasis upon transplantation into murine rectal mucosa, exhibiting the same in vivo metastatic route as in the corresponding patients. for pancreatic cancer, boj and colleagues (boj et al. ) were the first to successfully developed organoids from patient-derived pdacs. subsequently, seino and colleagues ) generate an extensive organoids biobank of pdacs (n= ) covering both classical and basal subtypes according to gene expression signatures. they found basal-type pdacs derived organoids are more independent of wnt signaling, which are more invasive and aggressive clinically, indicating that progression of pdacs are accompanied by loss of stem cell niche dependence. recently, tiriac and colleagues (tiriac et al. ) generated a much larger pdac biobank (n= ) and exposed a subset of these organoid lines to the standard-of-care chemotherapies. their sensitivities paralleled clinical responses in patients. besides, gene expression signatures of chemosensitivity based on organoids were developed to help predict responses to chemotherapy in both the adjuvant and advanced disease settings. by high throughput drug screening, they nominated alternative treatment strategies for chemorefractory pdo. another study also used pdos (n= ) to identify novel therapeutics to target pancreatic tumor cells in a biobank covering different histological subtypes, including pdacs, acinar cell carcinoma, cholangiocarcinoma, adenosquamous-pdacs, intraductal papillary mucinous neoplasm (ipmn)-derived pdacs and papilla of vater adenocarcinomas (driehuis et al. c) . pdos were exposed to therapeutic agents currently not exploited in the clinic. the prmt (protein arginine methyltransferase ) inhibitor, ezp , was shown to target mtap (methylthioadenosine phosphorylase)-negative tumors, but also appeared to constitute an effective therapy for a subset of mtap-positive tumors, indicating the importance of personalized approaches for cancer treatment. huch and colleagues (broutier et al. ) described a liver tumor biobank (n= ) containing hepatocellular carcinoma and cholangiocarcinoma, as well as the rarer lymphoepithelioma-like cholangiocarcinoma. in drug screening experiments with compounds, the erk (extracellular regulated protein kinases) inhibitor sch was found to effectively inhibit the growth of tumor organoids, which was validated in vivo using xenotransplanted organoid lines in mice, histological types were not comprehensively reported highlighting sch as a possible therapeutic agent. biliary tract carcinomas-derived organoids biobank was also established, covering intrahepatic cholangiocarcinoma, gallbladder cancer, and neuroendocrine carcinoma of the ampulla of vater (saito et al. ) . gene expression profiling of the organoids indicated that sox , klk and cpb could be potential prognostic biomarkers. drug screening using a compound library of drugs showed that the antifungal drugs, amorolfine and fenticonazole, significantly suppressed the growth of biliary tract carcinomas organoids with little toxicity to normal biliary epithelial cells. the organoids biobank of metastatic prostate cancer covering both ar (androgen receptor)-positive and -negative subtypes was the first reported biobank established by gao and colleagues ). the biobank captured the most common genetic aberrations in prostate cancer, including tmprss -erg fusion, homozygous deletions of pten and chd , as well as typical copy number variations. to be pointed out, organoids derived from circulating tumor cells were also successfully established in this biobank, showing that at least in some cases, organoids can be established from less invasive blood samples. a bladder cancer derived organoids biobank (n= ) was established containing urothelial carcinomas and one squamous cell carcinoma (lee et al. ) . organoid lines were interconvertible with orthotopic xenografts and recapitulated the mutational spectrum of the corresponding tumor type, including activation of fgfr and mutations in epigenetic regulators such as arid a. drug screening of compounds based bladder tumor organoids showed partial correlations with mutational profiles as well as treatment resistance, and the drug responses can be validated using xenografts in vivo. a biobank of breast cancer organoids (n= ) has been described covering major histological subtypes (invasive ductal carcinoma and invasive lobular carcinoma) and all molecular subtypes based on gene expression . organoid morphologies matched the histopathology of the original tumors, and hormone receptor [estrogen receptor (er), progesterone receptor (pr)], her status and copy number variations were retained. er and pr status have predictive value for the outcome of endocrine therapy (e.g. tamoxifen), while her is a target for targeted therapy (e.g. trastuzumab) and also has predictive value for chemotherapy outcome. the response of breast cancer-derived organoids to her inhibitor afatinib and to endocrine therapy tamoxifen were consistent with in vivo xeno-transplantations and patient response in clinic. an organoid biobank of high-grade serous ovarian cancer (hgsc) (n= ) was established by hill and colleagues (hill et al. ) . up to % of all patients with hgsc have dna repair defects, typically mutation of brca or brca . these patients were thought to benefit from treatment with poly (adp-ribose) polymerase (parp) inhibitors. in the clinical setting, mutation analysis alone is not sufficient to adequately predict drug sensitivity. the study showed that functional assays in organoids are a better predictor than the genomic analysis in clinic, implying that functional assays in organoids may improve the prediction of drug sensitivity beyond what can be achieved with genomic analysis alone. kopper and colleagues (kopper et al. ) established a second ovarian cancer biobank (n= ) that captured all of the main histological subtypes, including borderline tumors, endometroid carcinomas, mucinous carcinomas, lgsc and hgsc. notably, a novel single-cell dna sequencing method was used to demonstrate intra-patient heterogeneity was preserved in organoids when compared with the original tumor. pdos can be used for drug-screening analyses and capture distinct responses of different histological subtypes to platinum-based chemotherapy, including acquisition of chemoresistance in recurrent disease. besides, pdos can also be xenografted, enabling in vivo drug sensitivity analyses. taken together, pdos of ovarian cancer have potential application for translational research and precision medicine. driehuis and colleagues (driehuis et al. a ) established an organoid biobank (n= ) derived from head and neck squamous cell carcinoma (hnscc). pdos recapitulates genetic and molecular characteristics of original hnsccs and can generate tumors upon transplantation to immunocompromised mice. the authors observe different responses to commonly used drugs in clinic including cisplatin, carboplatin, cetuximab, and radiotherapy in vitro. besides, drug screens exhibit selective sensitivity to targeted drugs that are not normally used in clinic for patients with hnscc. these findings may inspire the personalized treatment of hnscc and expand the list of hnscc drugs. in another study, the authors reported pdos derived from hnscc can also be used to evaluate their response to targeted photodynamic therapy and to ensure the safety of the treatment at the same time by testing it on organoids derived from matched normal tissues (driehuis et al. b ). all organoids discussed above were derived from tumors of epithelial origin, known as carcinomas. recent advances show organoids derived from primary glioblastoma tissue, setting the stage for growing organoids from non-epithelial tumors (hubert et al. ) . glioblastoma presents great heterogeneity, and thus it's difficult to generate an ideal in vitro model which can recapitulate the in vivo situation of the donor. by modifying the method to develop cerebral organoids, the pdos can be successfully derived both from primary lesion of glioblastoma and from brain metastases. once formed, pdos presented hypoxia gradients and mimicked cancer stem cell heterogeneity with rapidly dividing outer cells surrounding the hypoxic core of differentiated cells and diffuse, quiescent cancer stem cells. drug testing based on organoids showed that nonstem cells were sensitive to radiation therapy, whereas adjacent cancer stem cells were radioresistant. orthotopic transplantation of pdos resulted in tumors recapitulating histological features of the parental tumor. based on this method, jacob et al. ( ) established a larger organoid biobank derived from glioblastoma (n= ), recapitulating the histological characteristics, cellular diversity, gene expression, and mutational profiles of their donors. the organoids generated rapid, aggressively infiltrated tumors upon transplantation into adult rodent brains. the authors observe different responses to exposure of radiation with concurrent temozolomide, which was consistent with the patients' response and survival in clinic. notably, the authors further demonstrate the utility of organoids in modeling immunotherapy by co-culture of chimeric antigen receptor t (car-t) cells with organoids. they observed specific tumor cells were targeted and killed by car-t cells. the study expands the application of pdos in personalized treatment to include immunotherapy. several biobanks containing organoids derived from mixed cancers were established for pan-cancer research. pauli et al. ( a pauli et al. ( , b ) developed a robust precision cancer platform, by integrating whole exome sequencing with a living biobank which allows high throughput drug screens on pdos. the biobank included tumors derived from prostate, breast, colorectum, esophagus, brain, pancreas, lung, small intestine, ovary, uterus, soft tissue, bladder, ureter and kidney. in another study, to model tumor immune microenvironment, kuo and colleagues (neal et al. ) established pdos based on ali method from > individual patient tumors of distinct organs and histological subtypes. these pdos included common cancers such as colon, pancreas, and lung, and rarer histologies such as bile duct ampullary adenocarcinoma, brain schwannoma, and salivary gland pleomorphic adenoma. pdos in this biobank retain immune cells and should enable immuno-oncology investigations and facilitate personalized immunotherapy testing. as mentioned above, cf is a lethal genetic disease caused by cftr mutations that impairs the function of many organs including the intestine, lung, pancreas, sweat gland, liver, and kidney. the disease is characterized by the buildup of viscous, sticky mucus which clogs airways, causes chronic digestive system problems and leads to cf-related diabetes. approximately , cfcausing mutations of cftr have been described, and drug efficacy varies among the different genotypes (cutting ) . thus, there is a need for a personalized medicine approach to predict treatment response. beekman and colleagues (dekkers et al. ) first established an organoid biobank derived from rectum of cf patients with different cftr genotypes. based on the biobanking, they developed a personalized medicine approach by using fsk-induced swelling assay to select clinical responders to cf modulators. two patients with the rare and uncharacterized f del/ g r genotype responded in vitro to a specific cf modulator, ivacaftor (kalydeco, vertex pharmaceuticals). the responses were consistent with their in vivo clinical response to the treatment, reflected by improved pulmonary function and sweat chloride tests. in a prospective follow-up study involving participants (berkers et al. ) , the predictive power of the fsk assay was further substantiated, as the in vitro assay correlated with changes in pulmonary function and sweat chloride tests conducted in vivo. besides cf-rectal organoids, pancreatic organoids and cholangiocyte-like organoids derived of cf-ipscs have also shown the potential for drug screening (sampaziotis et al. ; simsek et al. ). in the netherlands, the licensing of orkambi (lumacaftor/ivacaftor, vertex pharmaceuticals) allows treatment of cf patients solely based on a positive organoid swelling response, demonstrating the potential of organoid-based assays for delivering personalized medicine (fig. ) . in summary, organoid biobanks have been established for multiple tumor types (table ) , including nonepithelial glioblastoma, and several principals can be concluded. first, pdos can be established from small biological samples, such as biopsies and even circulating tumor cells, and these organoids can generate tumors upon xenotransplantation. second, the pdos in biobanks recapitulate histological and genetic aspects of the original tumors, which holds not only for localized primary tumors but also for metastatic tumors. third, high-throughput drug screening experiments in organoids correlate with the response in patients and lead the identification of new therapeutic targets. combining genetic sequencing with functional assays in pdos will facilitate personalized treatment, even including immunotherapy, which will be discussed in detail in section . . schwank and colleagues (schwank et al. ) firstly demonstrated that it is feasible to repair genetic defects in organoids. intestinal organoids derived from two cf patients with a homozygous cftr f deletion were repaired using crispr-cas technology. after repair, fsk-induced swelling was restored, functionally demonstrating cftr activity. later, firth and colleagues (firth et al. ) generated ipscs from cf patients with a homozygous deletion of cftr f and corrected this mutation using crispr technology to target corrective sequences to endogenous cftr genomic locus, combining with selection system. the corrected ipscsorganoids were able to differentiate mature airway epithelial cells with normal cftr expression and function. however, cftr gene repair in organoids and subsequent transplantation into patients is hard to be applied in the clinic. first, the loss of cftr functions results in disease in multiple organ systems, which would require the transplantation of organoids into multiple tissue sites. second, a high percentage of repaired cells per organ would be required for functional restoration. third, ethics problems in organoids transplantation are controversial and needs to reach a consensus in the research field. human and murine organoids have been orthotopically transplanted into mice to model disease or to show tumorigenic potential. here, we discuss studies that used organoid transplantation as therapy. yui and colleagues (yui et al. ) firstly exploited the feasibility to apply organoids to repair damaged epithelium. gfp + murine colon organoids derived from lgr + stem cells were reintroduced into mice with dss (dextran sulfate sodium salt) -induced acute colitis. transplanted cells readily integrated into the mouse colon and covered superficially damaged tissue. weeks after transplantation, the donor cells constituted a single-layered epithelium, which formed functionally and histologically normal crypts that were able to self-renew. further, engrafted mice had higher body weights than ungrafted ones, indicating that donor cells contributed to the recovery of dss-induced acute colitis. although further optimization is still needed, the current study indicates that in vitro expansion and transplantation of organoids may be a promising treatment choice for patients with severe gastrointestinal epithelial injuries. orthotopic transplantation of liver organoids has also shown promising results. in a mouse model of toxicityinduced acute liver failure, transplantation of mouse differentiated biliary duct organoids derived from lgr + stem cells generated detectable organoid-derived nodules in - % of cases . although engraftment rate was low (approximately %), a significant increase in survival of the grafted group was observed compared to the ungrafted group, indicating that the transplanted cells contributed to liver function repair ). in follow-up studies using the same injury model, the transplantation of mouse (peng et al. ) and human fetal ) hepatocyte organoids, generated much more extensive engraftment indicating that the efficiency of engraftment may be enhanced by transplantation of the most physiologically relevant cell type. recently, xiao and colleagues (xiao and deng ) generated induced sensory ganglion organoids exhibiting fig. organoids for modeling cystic fibrosis (cf) and its multiple applications. organ-specific pathologies of cf can be studied separately using organoids derived from distinct tissues and applied to personalized drug screening molecular features, subtype diversity, electrophysiological and calcium response properties, and innervation patterns characteristic of peripheral sensory neurons, which may serve as a source for cell replacement therapy. yoshihara and colleagues (yoshihara and o'connor ) generated human islet-like organoids (hilos) from ipsc, which provides a promising alternative to cadaveric and device-dependent therapies in the treatment of diabetes. hilos contain endocrine-like cell types that, upon transplantation, rapidly re-establish glucose homeostasis in diabetic nod/scid mice. overexpression of the pd-l protected hilo xenografts such that they were able to restore glucose homeostasis in immune-competent diabetic mice. organoids combined with d printing were recently introduced to build the d architecture of tissue, which may find widespread applications in regenerative medicine. firstly, zhang and his colleagues (zhang et al. ) established the technique that can be transformed into human cardiomyocytes derived from ipscs to construct endothelialized human myocardium. then, creff and colleagues (creff et al. ) provided the possibility of creating artificial d scaffolds that match the size and structure of mouse intestinal crypt and villi. moreover, homan and colleagues (homan et al. ) built a model that had the ability to induce substantial vascularization and morphogenesis of renal organs in vitro under flow conditions opening up a new way for the study of renal development, disease and regeneration. though above studies indicate the potential application of organoids in regenerative medicine, many problems need to be solved before organoids are put into clinical use. for example, integration upon transplantation requires optimization, and animal-based d ecm matrix used for organoid culture need to be replaced with a synthetic matrix. the tumor microenvironment consists of various nonepithelial cell types, including immune cells and stromal cells, which greatly affects therapeutic responses. however, it is a major challenge to model tumor microenvironment. much of our knowledge regarding the tumor microenvironment was studied on cell lines and pdx models. however, cell lines are insufficient to recapitulate the heterogeneity of tumor cells, while the microenvironment of pdx models mainly depend on the mouse immune system, which cannot adequately recapitulate the human immune system. cancer immunotherapy has emerged as a promising therapeutic developments that take advantage of a patient's own immune system to eradicate tumor cells (mellman and coukos ) , and several organoid-based models have been established to study immunotherapy response (fig. ) . recently, a holistic approach based on the ali method (mentioned in section . ), which preserved the tumor epithelium and its stromal microenvironment in vitro, was described using pdos of various cancer types, including colorectal and lung cancers (neal et al. ). in addition to stromal fibroblasts, cellular immune components such as tumor-associated macrophages, ctls (cytotoxic t lymphocyte), t h cells (t helper cells), b cells, natural killer (nk) cells and natural killer t (nkt) cells were also readily maintained for up to days in the organoid cultures. the organoid cultures also preserved the t cell receptor (tcr) heterogeneity of the t cells found in the parental tumor. the authors used these organoids to model immune checkpoint blockade, leading to the expansion and activation of tumor antigen-specific t cells and subsequent killing of tumor cells. adoptive cell therapy is a promising immunotherapy. in this method, autologous immune cells are expanded in vitro followed by the subsequent transplantation of these cells back into the patient, to enhance the immune response against a tumor. using this strategy, durable regression of melanoma was achieved by in vitro expansion of autologous tumor-infiltrating lymphocytes (tils) (rosenberg ) . however, this approach requires resected specimens from which tils can be obtained. a strategy to avoid resection is to isolate peripheral blood lymphocytes, activate these cells in vitro by co-culture with tumor cells. for this strategy, tumor-derived organoids are a highly useful source of tumor cells for coculture: tumor organoid cultures can be efficiently established from a small tissue sample through biopsy, and tumor-derived organoids are heterogeneous and recapitulate the genetic and histological characteristics of the parental tumors. dijkstra and colleagues (dijkstra et al. ) were thus able to obtain tumor-reactive t lymphocytes from peripheral blood lymphocytes after weeks of co-culture with tumor organoids derived from non-small cell lung cancer and msi-h crc. before coculture, organoids were stimulated with ifn-γ to enhance antigen presentation. pd- blocking antibody, il- , and anti-cd were added to enhance t cell activation. after co-culture, t lymphocytes were activated, as demonstrated by expression of ifn-γ and cd a. accordingly, after an additional days of co-culture of activated t lymphocytes with tumor organoids, the survival of the tumor organoids was reduced, while matched normal organoids were unaffected. cancers with low mutational burden and stable tumor antigen presentation may be suitable targets for chimeric antigen receptor (car)-engineered t cells. studies on b cell malignancies showed promising results (june ) . in solid tumors, the therapeutic application of car t cells has been hindered by side effects that arise from targeting overexpressed native antigens that are, not exclusively expressed by tumors. thus, for solid tumors, preclinical models are needed to allow for carmediated cytotoxicity testing. recently, a luciferasebased quantification assay has been developed to test car t cell-mediated cytotoxicity against pdos (schnalzger et al. ) . instead of using carengineered t cells, the researchers adopted an nk cell line with non-mhc restricted cytolytic activity to target research. the efficiency of the system was confirmed by using car-engineered nk- cells directed toward epcam, an epithelial marker overexpressed by cancers. car-mediated cytotoxicity was observed against organoids derived from both normal colon tissue and crc tissue presenting either peptide (schnalzger et al. ). subsequently, the authors engineered cars targeting egfrviii, a neoantigen that is widely expressed by solid cancers. in a competitive co-culture assay, egfrviiispecific car nk cells killed egfrviii-expressing organoids derived from tumors efficiently but not organoids derived from normal tissue. finally, the team generated cars targeting antigens specific to subgroup of crc that overexpress the wnt ligand receptor fzd upon loss of expression of its antagonists rnf /znrf . to test possible side effects of fzd-specific cars, the authors evaluated cytotoxicity against normal colon fig. co-culture systems of organoid and immune cell in immuno-oncology research. two main methods are currently being adopted: a holistic approach (up). tumor biopsies are cultured in ali in the entire tumor microenvironment as a cell suspension of all cell types, including immune cells and other non-epithelial cell types. b reductionist approach (down), epithelial organoids are derived from tumor biopsies and are then co-cultured with autologous immune cells derived from the peripheral blood of the same patient. crc, colorectal cancer; ali, air-liquid interphase; ecm, extracellular matrix; nk cell, natural killer cell organoids as well as different gene-edited organoid lines deficient for both rnf and znrf or for apc. these co-culture assays illustrated that the cytolytic activity of the fzd-specific car nk cells was not specific to the mutated organoid lines, suggesting that such approaches may have marked side effects if used therapeutically. though effective target has not been found, the platform can be widely used to evaluate car efficacy and tumor specificity in a personalized manner. in summary, co-cultures of cancer organoids and immune cells have become a highly promising strategy for personalized immunotherapy for cancer patients. organoids are robust research tools for the study of human development and disease. however, there are hurdles and limitations associated with using organoids (fig. ) . first, culture approaches are not standardized. ascsderived organoids are established under distinct culture conditions in each laboratory. to reduce the cost, costefficient small molecule compounds were used to replace growth factors yin et al. ) . besides, homemade niche factors produced by various cell lines has been used commonly to culture organoids. however, this trend will result in experimental variation into organoid studies across different research labs. second, organoid culture requires the use of matrigel or other animal-based matrix extract to enable cells to aggregate into d structures. these extracts suffer from batch-to-batch variability in their composition, which may affect the reproducibility of experiments. in addition, they may carry unknown pathogens and are potentially immunogenic when transplanted to humans, limiting the application of organoids in a clinical transplantation setting. this may be solved by culturing with clinical grade collagen, which has been successfully used for colon organoids culture and expansion (yui et al. ) . steps toward fully defined culture conditions have been made with the development of a synthetic polyethylene glycol-based gel that sustains the short-term growth of mouse asc-derived intestinal organoids (gjorevski et al. ). however, this matrix remains to be optimized for the long-term expansion of intestinal organoids and f non-intestinal organoids. third, obtaining pure tumor organoids is another critical problem for researchers. many studies have reported fig. schematic summarize of current limitations in organoids culture development (yellow) and methods to overcome these issues (brown) that tumor organoids can be overgrown and contaminated by matched normal organoids. one of the most widely used methods to acquire pure tumor organoids is to select tumor cells that harbor the most common mutations in its cancer type. for example, tumor organoids derived from crc can be selectively upon withdrawal of wnt a and r-spondin . however, this method is not applicable to all cancers, and some specific cancer subtypes. more importantly, intra-tumoral heterogeneity would inevitably be lost upon selection. some labs have suggested obtaining pure tumor organoids based on growth phenotypes. however, not all tumor organoids have clear morphological differences to normal organoids. more researches are needed to establish appropriate approaches for obtaining pure tumor organoids. fourth, for ascs-derived organoids, only the epithelial compartment of organs is represented, while blood vessels, immune cells, stroma, and nerves are lacking. as mentioned above, many groups have focused coculturing organoids with various types of cells, analogous to what has already been done with immune cells (dijkstra et al. ) , or adopted unconventional organoid culture methods, such as the ali method (neal et al. ; ootani et al. ). besides, several psc-based organoids are able to undergo mesenchymal differentiation to generate subepithelial myofibroblasts and smooth muscle . last, the development of a model based on living human tissues that can be stored and expanded in biobanks-potentially forever-has raised a set of ethical issues regarding informed consent and ownership (bredenoord et al. ) . for organoid biobanks, patient consent is required. the most common type of patient consent restricts the use of a patient's material to only a specific research aim. however, biobanks are useful for researchers in multiple fields, and the use of biobanks in a combination of fields may provide potentially synergistic data. to solve this problem, bredenoord and colleagues (boers and van delden ; bredenoord et al. ) have suggested that a broad consent be used for governance. this broad consent would allow donors to make informed decisions about how their samples are used after they have been provided with relevant information about the establishment and regulation of the biobank. another issue that has arisen with the development of living biobanks is ownership. organoids are increasingly used by commercial parties as tools for drug development or in validation studies. such uses will inevitably result in patentable compounds. it may be helpful to include regulations covering the distribution of any financial gains from intellectual property among stakeholders in the governance of the biobanks. furthermore, in terms of the culture conditions of tumor organoids, intra-tumoral heterogeneity could be lost during passages for that the culture media might not favor the growth of all tumor subclones equally effectively. additionally, novel mutations would be acquired during long-term expansion. collectively, these drawbacks deserve further attention, and more work is needed to improve organoids culture technologies. despite these limitations, organoid technology holds great promise as a robust tool for basic, translational and clinical research for human development and disease modeling. in this review, we have demonstrated the potential of organoid technology for modeling genetic, infectious and malignant disease, as well as for drug development and personalized medicine. the basic and translational applications of organoids are expected to expand in the future. regarding epithelial genetics, organoids have potential especially for rare diseases due to their high expansion ability and genetic stability. in infectious diseases, the mechanisms of virus-induced malignant transformation, for example, by epsteini-barr virus in gastric and nasopharynx cancers, may be studied in long-term cocultures of the virus with normal epithelium from the respective organ. in the studies of malignancies, culture conditions need to be developed for many types of carcinomas and for sarcomas and melanomas. the optimization of drug screening will be a pivotal use of organoids in personalized medicine by adopting biobanks of different cancers. this enables many compounds to be screened for a specific disease or a specific compound to be screened for many forms of a given disease. moreover, multiple organoids can be derived from the same cancer patient over time to assess drug response and developing resistance to targeted drugs and to predict patient outcome. in the area of regenerative medicine, there is still a long way to go for the transplantation of organoids as therapy. several hurdles, including the development of a non-animal-based alternative for matrigel and efficient delivery procedures, remain to be overcome. in conclusion, the worldwide application of organoids has contributed to unprecedented advances in research on human development and diseases. even though current organoid systems show some limitations and require further optimization for use in disease modeling and personalized medicine, they will continue to be valuable tools in basic and translational research. ecm: extracellular matrix; pdos: eatient-derived organoids; wenr method: wnt a+egf+noggin+r-spondin- nico: nicotinamide; rspo: r-spondin- ; pge : prostaglandin e dht: dihydrotestosterone; crc: colorectal cancer; ali: air-liquid interface tumor infiltrating lymphocytes; ta: transit-amplifying; wgs: wholegenome sequencing; crispr: clustered regularly interspaced short palindromic repeats; cas : crispr associated protein ; nhej: non-homologous end joining; hdr: homology-directed repair cftr: cf transmembrane conductance regulator; camp: cyclic adenosine monophosphate; fsk: forskolin; clc: cholangiocyte-like cells pdecs: pancreatic ductal epithelial cells; mvix: microvillus inclusion disease syntaxin- ; a at: α -antitrypsin; ags: aicardi-goutières syndrome zikv: zika virus; who: world health organization; tlr : toll-like receptor japanese encephalitis virus; je: japanese encephalitis; hbv: hepatitis b virus; hunov: human norovirus; hbga: histo-blood group antigen rsv: respiratory syncytial virus salmonella typhi clostridium difficile; cdi: c. difficile infection helicobacter pylori; cin: chromosomal instability hiv: human immunodeficiency virus; shrna: short hairpin rna; ssas: sessile serrated adenomas; mlh : mutl homolog ; msi: microsatellite instability pdac: pancreatic ductal adenocarcinoma; srcc: signet-ring cell carcinoma androgen receptor; tmprss : transmembrane protease serine ; ets: e transformation-specific; erg: ets-related gene auc: area under the curve; ipmn: intraductal papillary mucinous neoplasm hgsc: high-grade serous ovarian cancer; lgsc: low-grade serous ovarian cancer; parp: poly (adpribose) polymerase; hnscc: head and neck squamous cell carcinoma; car-t: chimeric antigen receptor t; nk cell: natural killer cell; nkt cell: natural killer t cell; tcr: t cell receptor chimeric antigen receptor; dss: dextran sulfate sodium; ifnγ: interferon-gamma; mhc: major histocompatibility complex nhe : sodium/hydrogen exchanger ; muc : mucoprotein ; akt: protein kinase b; mapk: mitogen-activated protein kinase; eif e: eukaryotic translation initiation factor subunit e egfr: epidermal growth factor receptor; prmt : protein arginine methyltransferase ; mtap: methylthioadenosine phosphorylase erk: extracellular regulated protein kinases; ar: androgen receptor severe acute respiratory syndrome-coronavirus ; mge: medial ganglionic eminence; hrsace : human recombinant soluble ace ; iwp- : inhibitor of wnt production ; vpa: valproic acid dapt: dual 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authors: wang, renxi; xiao, he; guo, renfeng; li, yan; shen, beifen title: the role of c a in acute lung injury induced by highly pathogenic viral infections date: - - journal: emerg microbes infect doi: . /emi. . sha: doc_id: cord_uid: p i m the complement system, an important part of innate immunity, plays a critical role in pathogen clearance. unregulated complement activation is likely to play a crucial role in the pathogenesis of acute lung injury (ali) induced by highly pathogenic virus including influenza a viruses h n , h n , and severe acute respiratory syndrome (sars) coronavirus. in highly pathogenic virus-induced acute lung diseases, high levels of chemotactic and anaphylatoxic c a were produced as a result of excessive complement activaiton. overproduced c a displays powerful biological activities in activation of phagocytic cells, generation of oxidants, and inflammatory sequelae named “cytokine storm”, and so on. blockade of c a signaling have been implicated in the treatment of ali induced by highly pathogenic virus. herein, we review the literature that links c a and ali, and review our understanding of the mechanisms by which c a affects ali during highly pathogenic viral infection. in particular, we discuss the potential of the blockade of c a signaling to treat ali induced by highly pathogenic viruses. the epithelium of the lung is vulnerable to damage caused by inhaled microorganisms and other noxious particles. many studies suggested the presence of complement components at the alveolar epithelium, where inhaled airborne particles and microorganisms are deposited. [ ] [ ] [ ] in addition, the complement system has been implicated in the development of acute lung diseases induced by highly pathogenic viruses including influenza a virus h n , h n , h n , severe acute respiratory syndrome coronavirus (sars-cov), middle east respiratory syndrome coronavirus (mers-cov). however, the specific contributions of complement to lung diseases based on innate and adaptive immunity are just beginning to emerge. elucidating the role of complement-mediated immune regulation in these diseases will help identify new targets for therapeutic interventions. complement activation leads to the formation of bioactive molecules, including the anaphylatoxins, c a and c a, and the lytic membrane attack complex (c b- ). the complement-activated product c a is a strong chemoattractant and is involved in the recruitment of inflammatory cells such as neutrophils, eosinophils, monocytes, and t lymphocytes, in activation of phagocytic cells and release of granulebased enzymes and generation of oxidants. c a also displays other powerful biological activities including inducing ''cytokine storm.'' on the other hand, blockade of c a signaling has demonstrated potential benefits in the treatment of acute lung injury (ali) induced by highly pathogenic viruses. in this article, we summarize recent developments in our understanding of the role of c a in mediating aute lung injury induced by highly pathogenic viruses. highly pathogenic virus due to high mutation rates of viruses, every several years to decades a highly pathogenic virus emerges. especially in the recent decades, there were more than five highly pathogenic viruses such as sars coronavirus in , avian influenza a/h n virus in , h n virus in , h n virus in , and mers coronavirus in . as exemplified by coronaviruses and influenza viruses, bats and birds are natural reservoirs for providing viral genes during evolution of new virus species and viruses for interspecies transmission. , this is the primary cause of an outbreak by jumping directly from bird to human. in two months, laboratory-confirmed cases and deaths have been reported globally. there is an h n vaccine for human use, but there is currently no h n , sars or mers vaccine available. current vaccination strategies are still inadequate at providing protection against epidemic outbreaks. thus, it is urgent to explore the mechanism by which highly pathogenic viruses induce diseases. acute lung injury induced by highly pathogenic viral infections although highly pathogenic virus infections have the different epidemiology, there is a similar rapid progression to acute respiratory distress syndrome (ards). for example, histopathological changes in the lung from patients infected with h n are highly similar to those of patients with sars. except for influenza a h n virus, avian influenza a h n virus in also caused severe pneumonia. postmortem biopsy of patients infected with h n in showed acute diffuse alveolar damage: patient , who died days after symptom onset, had intra-alveolar hemorrhage, whereas patients and , who died days after symptom onset, had pulmonary fibro proliferative changes. patients infected with h n develop rapidly progressive pneumonia, further resulting in ali or ards. , ali may be a critical cause of death in patients with h n infection. , like h n infection, h n also causes serious lung pathology. in addition, sars-cov infection caused ali that may progress to life-threatening ards. mers-cov infection resulted in a more severe pneumonia than sars-cov infection. respiratory distress is the most common cause of death in patients infected with highly pathogenic virus. in terms of therapy, lung protective ventilation is the cornerstone of supportive care. extracorporeal membrane oxygenation is routinely used in many centers for the treatment of severe respiratory tract infections. however, due to few effective treatment options, ali is often fatal for patients infected with highly pathogenic viruses. this suggests that serious lung pathology should be of particular concern. after a microorganism infection begins, the host quickly activates the complement system to clear infected pathogens. during the complement activation, the high levels of products such as c a are commonly involved in exacerbated inflammatory reactions that can cause direct harm to the host following infections. [ ] [ ] [ ] iav belongs to the orthomyxoviridae family with single-stranded negative-sense rna virus, and has the capacity to activate the complement system. in addition, the avian influenza hemagglutinins typically bind alpha - sialic acid receptors, whereas human influenza hemagglutinins bind alpha - sialic acid receptors. thus, h n replicates in the lower respiratory tract, then causes complement activation. this suggests that upon influenza infection, the high levels of c and c including fragments c a and c a are produced. complement activation possibly contributes to the observed tissue damage in severe viral infection. studies demonstrated that ali in h n -infected mice was caused by excessive complement activation such as release of c a. thus, complement activation plays a critical role in the pathogenesis of virus-induced acute lung injury. among the complement activation products, the anaphylatoxin c a is one of the most potent inflammatory peptides. increased levels of c a were found in bronchoalveolar lavage fluid (balf) and serum from patients infected with fatally h n pandemic virus. , c a had also been found to increase in balf of mice infected with highly pathogenic avian influenza h n but not following seasonal iav infection. on the other hand, balf from recovered patients with ards demonstrated significantly reduced c a-dependent chemotactic activity. thus, c a might play a critical role in the pathogenesis of virus-induced acute lung injury. c a-mediated inflammatory cells migrate into lung tissue compared to normal controls, sars patients had increased cellularity of balf with increased alveolar macrophages. thus, mononuclear cell infiltration might have an important role in the pathogenesis of ali induced by highly pathogenic viruses like sars. anaphylatoxin c a has been implicated in the pathogenesis of ards by mediating neutrophil attraction, aggregation, activation, and subsequent pulmonary endothelial damage. [ ] [ ] [ ] [ ] reversely, c adependent chemotactic activity is significantly decreased in recovered patients with ards. these suggest that c a-mediated mobilization and activation of immune cells might be the central events to tissue injury caused by highly pathogenic viral infections. two chemoattractants c a and interleukin (il- ) can be synthesized by cells in the lung (e.g., macrophages, epithelial cells, endothelial cells, smooth muscle cells and neutrophils). il- levels have also been found to correlate with neutrophil numbers and the degree of lung dysfunction. c a could strongly amplify il- expression from human whole blood cells induced by lipopolysaccharides and other types of toll-like receptors agonists via extracellular-signal-regulated kinases / and p , but not c-jun n-terminal kinase. the data suggest that c a might be a critical effector molecule to mediate lymphocyte attraction by itself or indirectly by enhancing the production of il- . altogether, c a-mediated lymphocyte attraction plays a critical role in the pathogenesis of ali induced by highly pathogenic viruses. neutrophil extracellular traps (nets) are primarily composed of dna from neutrophils, which bind pathogens with antimicrobial proteins. nets are beneficial in antimicrobial defense and can help fight against invading pathogens. however, an excess of nets contributes to the pathology of a number of diseases including those of the lung. nets are found in infection-related ali models of influenza virus. , in vitro studies demonstrated that c a, in association with granulocyte-macrophage colony-stimulating factor, is able to induce the release of nets. c a is also able to activate macrophages and endothelial cells and to promote vascular leakage and the release of nets. thus, nets are induced by c a during iav infection and are associated with alveolar damage in iav-induced pneumonitis. the excess of net components are potent factors in lung injury. net increases the permeability of the alveolar-capillary barrier by cleaving endothelial actin cytoskeleton, e-cadherin and vecadherin. the antimicrobial peptide ll- in net structures presents cytotoxic and proapoptotic properties towards endothelial and epithelial cells. net also induces the release of proinflammatory cytokines. the data suggest that c a-mediated neutrophil extracellular traps aggravate ali in patients infected with highly pathogenic virus. c a-mediated release of reactive oxygen species c a is a strong chemoattractant for neutrophils and monocytes; it then activates these cells to generate oxidative burst with release of a study demonstrated that ros are primary pathogenic molecules in pneumonia from mice infected with influenza virus. the amount and duration of exposure of generated ros, released from respiratory, immune, and inflammatory cells, determined the extent of lung damage. in lung fibroses, inflammatory cells produce a significantly greater amount of ros. critically, antioxidant treatment significantly reduces lung damage and mortality in influenza-infected mice. these studies demonstrated a critical role of reactive oxygen intermediates (rois) in virus-induced epithelial damage. c a-c ar interaction plays a critical role in oxidative burst. interception of c a/c ar signaling with a c ar antagonist significantly inhibited oxidative burst in neutrophils induced with e. coli. similarly, anti-c a blocked the oxidative burst in whole blood induced with neisseria meningitides. phosphorylation of p phox is essential for assembly of nadph oxidase and the subsequent production of o and h o . c a is a strong activator of mitogen-activated protein kinase (including p /p ), which is an important kinase for p phox phosphorylation. except for directly affecting tissue damage, oxidant production might also be involved in signal transduction pathways. il- expression is enhanced by the oxidant sensitive transcription factor nuclear factor-kb activated in the lungs of influenza-infected mice. this means that oxygen-derived free radicals might exert much greater effects on the pathogenesis of the disease by indirectly inducing other proinflammatory mediators. thus, c a-mediated oxygen-derived free radicals are thought to be important events in the pathogenesis of the disease. c a-mediated release of histones histones are essential regulators of genome function in eukaryotic cells. the ns protein of influenza a h n subtype possesses a histone-like sequence (histone mimic), and could target the human rna polymerase-associated factor transcription elongation complex which has a crucial role in the antiviral response. thus, the virus used ns histone mimic to suppress human rna polymerase-associated factor transcription elongation complex-mediated antiviral response. diversely modified histone regulates gene replication, repair and transcription. after activation with influenza, h k me reduced association of interferon i (ifn-i) and ifn-iii promoters in dendritic cells (dcs) to suppress antiviral gene expression. in contrast to ifns, the association of tumor necrosis factor-a (tnf-a) promoter was not disturbed. histone can be excreted into cells to reduce intracellular histone to suppress antiviral gene expression. in the setting of ali both in humans and in mice, histone presence has been found in balf. in addition, when polymorphonuclear leukocytes are incubated in vitro or in vivo with c a, neutrophil extracellular histones-contained extracellular traps (nets) develop. these results suggest that engagement of c a with its receptors led to the appearance of extracellular histones in balf. extracellular histones significantly enhance inflammatory response by inducing nucleotide-binding domain and leucine-rich repeat containing family, pyrin domain containing (nlrp ) inflammasome. furthermore, airway instillation of histones resulted in intense lung injury and inflammation, together with fibrin clots in pulmonary veins. c a-mediated release of histones has an important contribution to the pathogenesis of ali. the process of leukocyte adhesion to endothelial cells is the first critical step in neutrophil migration into an area of inflammation. adhesion molecules on the surface of endothelial cells have an important role in inflammatory cell migration. in fact, c a can regulate the expression of adhesion molecules. c a directly activates endothelial cells to upregulate adhesion molecules such as p-selectin. in addition, c a and tnf-a cooperate to enhance upregulation of intercellular adhesion molecule and e-selectin. thus, c a is an effective mediator in the first step in inflammatory cell migration into the lung. adhesion molecules on the surface of inflammatory cells also have an important role in inflammatory cell migration. in vitro studies demonstrated upregulation of cd lb/cd expression on neutrophils induced by c a. in addition, c a also induced the expression of b and b integrin on blood neutrophils. , thus, enhanced adhesive interactions of neutrophils to endothelial cells promote inflammatory cell migration into inflammatory sites. the adhesion molecules effectively enhanced pro-inflammatory cytokines such as tnf-a production by pulmonary macrophages, which, in turn, promotes the inflammatory response. blockade of cdllb, cd , intercellular adhesion molecule , or p-selectin significantly reduced ali damage by neutrophil content of the lungs. anti-c a might protect tissue injury in various organs by limiting neutrophil sequestration through downregulating the expression of adhesion molecules. these studies suggest that c a-mediated upregulation of adhesion molecules promotes the inflammatory response. c a-mediated adaptive immune response c a induces innate immune cells including mast cells, neutrophils, and macrophages to release cytokines such as il- , tnf-a and macrophage inflammatory proteins- a. il- is a strong activator of cd t cells, whereas tnf-a promotes transendothelial migration of t cells by up-regulating vascular adhesion molecules and induces ifn-c expression in t cells. these data demonstrate that c a indirectly induces adaptive immune response by activating innate immune cells. apart from innate immune cells, human dcs , and t cells also express the c a receptor (c ar, cd ). thus, c a is also a potent chemoattractant for human t cells, , b cells, and dcs. , , , in addition, during the early inflammatory stage of a pathogen infection, dcs used c a as a homing signal to take up ag, and then were primed for helping t-cell function. thus, c a induces adaptive immune response by recruiting for dcs. cd and cd l on t cells are important signaling for t-cell proliferation and differentiation induced by interaction of locallyproduced c a with c ar on antigen-presenting cells (apcs). accordingly, c a could not activate cd / cd / and cd / apcs to induce t cell activation. the data suggest that the local interaction of c a and c ar on apcs is critical to cd t cell proliferation and differentiation. the binding of the c a to the c ar also plays an important role in cd t cell responses. cd t cell activation during influenza infection requires c a, which acts as a chemoattractant for t lymphocytes. , thus, it is conceivable that c a might elicit cd t cell response upon the input stimuli. accordingly, c ar antagonist reduced the frequency and absolute numbers of flu-specific cd t cells. in patients infected with influenza a virus like h n , inflammatory cytokines such as il- b, il- , and il- play a major role in mediating and amplifying ali and ards by stimulating by chemotaxis c a. c a induces innate immune cells including mast cells, neutrophils, and monocytes/macrophages to release proinflammatory cytokines such as il- , tnf-a and macrophage inflammatory proteins- a. in addition, c a also stimulates adaptive immune cells such as t and b cells to release cytokines such as tnf-a, il- b, il- , and il- . , many cytokines, triggered by highly pathogenic viruses like h n , has been called a ''cytokine storm''. cytokines were rapidly induced at h post infection with h n . the pro-inflammatory cytokines including il- b and tnf-a might contribute to the severity of disease by promoting maximal lung inflammation caused by h n viral infection. compared to healthy volunteers, h n -infected patients have significantly higher levels of cytokines such as il- , ifn-c-inducible protein , il- , ifn-c, and tnf-a. a dangerous cytokine storm also occurs in sars. the representative sars-cov ssrnas had powerful immunostimulatory activities in inducing pro-inflammatory cytokines tnf-a, il- and il- . elevated levels of some proinflammatory cytokines including moncyte chemoattractant protein- , transforming growth factor-beta , tnf-a, il- , and il- , produced by cells infected by sars-cov, might cause ali. in addition, a cytokine could induce other cytokines to further enhance the proinflammatory response. take for example, elevated levels of tnf-a induced other cytokines like il- . thus, cytokine storm plays an important role in ali. anti-tnf-a (etanercept) significantly reduced the damage of ali. the inhibition of macrophage migration inhibitory factor alleviated h n influenza virus pneumonia in murine model by causing a significant reduction in pulmonary inflammatory cytokines il- b, il- and tnf-a and ifn-c-inducible protein a widely used antiviral agent arbidol hydrochloride efficiently inhibits both h n strains and diminishes both viral replication and acute inflammation through suppression of inflammatory cytokines such as il- b, il- , il- , and tnf-a. these studies indicate that blockade of cytokine storm is effective in treatment of infections with highly pathogenic virus. the severe h n patients were in a state of immune paralysis with general leukopenia, low antigen-presenting capacity and impaired t cell response. those suffering fatal infections with h n have particularly low proportions of peripheral blood t lymphocyte subgroups. previous studies have demonstrated that c a induces thymocyte apoptosis, which in turn results in decreased number of t cells in circulation and attendant immunosuppression. , this suggests that in a striking contrast to neutrophils, thymocytes apparently receive pro-apoptotic signals from c a. during sars-cov infection, il- and il- induced by c a inhibits the t-cell-priming ability of dcs. compared to significant up-regulation of inflammatory chemokines, the sars-cov-infected dcs showed low expression of antiviral cytokines (ifn-a, ifn-b, ifn-c, and il- p ) . these studies are in accordance with the conclusion that the n-protein of sars-cov induced ali by resulting in imbalance of pro-inflammatory and anti-inflammatory cytokines. many inflammatory and anti-viral genes were differentially expressed in sars patients. plenty of pro-inflammatory cytokines such as il- , tnf-a, and il- significantly increased, whereas a number of ifnstimulated genes like double-stranded rna-dependent protein kinase, interferon-induced guanylate-binding protein- and , c-x-c motif chemokine decreased in the acute severe case. like sars-cov, mers-cov viruses were unable to significantly stimulate the expression of antiviral cytokines (ifn-a and ifn-b) but induced comparable levels of tnf-a and il- . c a-c ar interaction might potentiate the mitochondrial apoptotic pathway and/or enhance the expression of proapoptotic factors, such as tnf-a, which has been linked to thymocyte apoptosis, in turn reducing the expression of antiviral cytokines. this suggests that c a-mediated immune paralysis plays a critical role in mediating pathogenic damage in severe patients infected with highly pathogenic virus like h n . to evaluate the effect of c a blockade, omci, a potent arthropodderived inhibitor of c activation that binds to c and prevents release of c a by complement activation, was used to treat mice infected with h n pandemic virus. omci significantly inhibited neutrophil and macrophage infiltration in the airways, nets formation, death of leukocytes, lung epithelial injury and overall lung damage. the study suggests that targeting c a could be a promising approach to reduce excessive inflammatory reactions associated with the severe forms of iav infections. c ar was found to be expressed on upper (bronchial) and lower (alveolar) airway epithelial cells. an adenovirus construct (sirna) was used to silence mrna for c ar in the lung and resulted in buildup of polymorphonuclear leukocytes, and lower levels of proinflammatory mediators in bronchoalveolar lavage fluid. antagonism of c a receptors also significantly inhibited the development of ards induced by intravenous infusion of cobra venom factor, including neutrophil migration and bronchoalveolar vascular leakage, blood pressure alterations, pro-inflammatory cytokines including tnf-a levels in bronchoalveolar lavage fluid. the study indicates that c a signaling greatly contributes to inflammation and injury in the lung and was targeted to treat highly pathogenic virus infection. in addition, interception of c a signaling has recently shown promising beneficial effects in small animal models of ali/ards by reducing pro-inflammatory cytokines. polyclonal anti-c a antibody led to significantly reduced inflammation in lungs, alleviating ali in h n -infected mice. the study indicates that inhibition of c a might be an effective clinical intervention for h n -induced ali. however, studies in knockout mice demonstrated that c was required for protection from influenza infection, proper viral clearance, and associated with changes in cellular infiltration. the data are in accordance with the fact that complement c a is the leading mediator of the over-inflammatory response which induced ali, whereas the lytic membrane attack complex (c b- ) provide a protective role in controlling viral infection. thus, we developed a neutralized humanized anti-human c a antibody which only blocked c a effects but did not affect the formation of c b- membrane attack complex. in vitro experiments demonstrated that a novel, neutralizing, humanized anti-human c a antibody blocked the ability of c a to induce granulocytes to express cd b while not affecting the ability of c b to form the membrane attack complex. african green monkeys were inoculated with h n virus and then treated intravenously with anti-human c a antibody. anti-c a treatment in h n -infected monkeys substantially attenuated ali by reducing the lung infiltration of macrophages and neutrophils, and the levels of inflammatory mediators. the data suggest that humanized anti-human c a antibody might provide a potential therapeutic reagent for h n -infected patients. the role of c a in the different viral infections and the effect of c a blockade on acute lung injury were described in table that the neutralized humanized anti-human c a antibody would be a potential therapeutic option for h n -infected patients. the complement system, a part of innate immunity, plays a critical role in host defense against pathogens. unregulated complement activation is likely to play a crucial role in the pathogenesis of lung diseases. the complement-activated product c a displays powerful biological activities in the activation of phagocytic cells, generation of oxidants, release of histones and cytokine storm, and so on. in particular, cytokine storm is believed to be responsible for many of the deaths during the influenza pandemic, during the sars epidemic in , mers-cov in , and the human deaths from h n , h n and h n . there is growing awareness that there are key similarities in the contribution to the cytokine storm and the manifestation of lung pathology among the chronic respiratory diseases, and the cause of death such as bleeding from ebola virus. c a, as a key trigger to induce cytokine storm, could be an ideal target for many lung inflammatory diseases, and it would be important to assess the therapeutic potentials of c a blockade in human clinical trials. we have evidence that humanized anti-c a antibody greatly reduced lung histopathologic injury, as well as decreased lung infiltration of macrophages and neutrophils and the levels of pro-inflammatory cytokines including tnf-a in a monkey model of ali induced by h n and herbicide, paraquat (shihui sun et al, unpublished data). thus, it is reasonable to speculate that blockade of c a with a humanized anti-human c a antibody would be a potential therapeutic target for highly pathogenic viral infection-induced acute lung injury. anti-c a ab treatment also reduced lung injury and neutrophil infiltration especially on day after h n virus infection. also, anti-c a ab treatment increased survival rate, with % mortality in the c a ab group compared with % mortality in the control group on day after h n virus challenge. hpai h n virus infected murine model h n influenza virus infected mice had increased levels of c a activation byproducts as compared to mice infected with either seasonal or pandemic h n influenza viruses. h n -infected monkey model anti-c a treatment in h n -infected monkeys substantially attenuated ali: it markedly reduced the lung histopathological injury and decreased the lung infiltration of macrophages and neutrophils. moreover, the treatment decreased the intensity of sirs; the body temperature changes were minimal and the plasma levels of inflammatory mediators were markedly reduced. the treatments also significantly decreased the virus titers in the infected lungs. ab, antibody; hpai, highly pathogenic avian influenza; sirs, systemic inflammatory response syndrome c a in acute lung injury rx wang et al. production of c by human alveolar macrophages complement biosynthesis by human bronchoalveolar macrophages pulmonary alveolar type ii epithelial cells synthesize and secrete proteins of the classical and alternative complement pathways complement c activation during 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state of the art complement (c a)-induced granulocyte aggregation in vitro: a possible mechanism of complement-induced leukostasis and leukopenia oxygen radicals mediate endothelial cell damage by complement-stimulated granulocytes. an in vitro model of immune vascular damage neutrophils in chronic obstructive pulmonary disease regulation of il- production by complement activated product, c a, in vitro and in vivo during sepsis net balancing: a problem in inflammatory lung diseases excessive neutrophils and neutrophil extracellular traps contribute to acute lung injury of influenza pneumonitis doxycycline treatment attenuates acute lung injury in mice infected with virulent influenza h n virus: involvement of matrix metalloproteinases viable neutrophils release mitochondrial dna to form neutrophil extracellular traps neutrophil extracellular traps directly induce epithelial and endothelial cell death: a predominant role of histones mechanisms of cell death induced by the neutrophil 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infection new strategies for treatment of humans with acute lung injury/acute respiratory distress syndrome severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells chemokine up-regulation in sarscoronavirus-infected, monocyte-derived human dendritic cells a study of pulmonary inflammatory reaction induced by nprotein of sars-cov in rat models and effects of glucocorticoids on it gene expression profiles in peripheral blood mononuclear cells of sars patients attenuation of igg immune complex-induced acute lung injury by silencing c ar inlung epithelial cells complement inhibitors selectively attenuate injury following administration of cobra venom factor to rats protein-based therapies for acute lung injury: targeting neutrophil extracellular traps role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome preparing for the next pandemic confronting potential influenza a (h n ) pandemic with better vaccines the cytokine network in asthma and chronic obstructive pulmonary disease clinical features and pathobiology of ebolavirus infection this study was supported by national basic research program grants ( cb ), national nature and science fund ( , and ) and beijing natural science foundation ( , and ). key: cord- -qnxyhmw authors: chen, xuxin; tang, lu; feng, jian; wang, yi; han, zhihai; meng, jiguang title: downregulation of paralemmin- ameliorates lipopolysaccharide-induced acute lung injury in rats by regulating inflammatory response and inhibiting formation of tlr /myd and tlr /trif complexes date: - - journal: inflammation doi: . /s - - - sha: doc_id: cord_uid: qnxyhmw previous studies have demonstrated paralemmin- (palm ) participates in toll-like receptor (tlr) signaling. this study investigated the effect of palm knockdown on lipopolysaccharide (lps)-induced acute lung injury (ali) and its underlying mechanisms. we constructed a recombinant adenoviral vector containing short hairpin rna for palm to knockdown palm expression. a transgene-free adenoviral vector was used as a negative control. the ali rat model was established by lps peritoneal injection at -h post-transfection. results showed that downregulation of palm improved the survival rate, attenuated lung pathological changes, alleviated pulmonary edema, lung vascular leakage and neutrophil infiltration, inhibited the production of proinflammatory cytokines and activation of nuclear factor κb and interferon β regulatory factor , and promoted the secretion of anti-inflammatory cytokine interleukin- and expression of suppressor of cytokine signaling- in the ali rat model. however, palm knockdown had no effect on tlr , myeloid differentiation factor (myd ), and toll-interleukin- receptor domain-containing adaptor inducing interferon β (trif) expression. moreover, palm knockdown reduced the interaction of tlr with myd or trif induced by lps in rat lungs. therefore, the downregulation of palm protected rats from lps-induced ali and its mechanisms were partially associated with the modulation of inflammatory responses and inhibition of tlr /myd and tlr /trif complex formation. acute lung injury (ali) and its advanced stage, acute respiratory distress syndrome (ards), are clinical syndromes characterized by acute hypoxemic respiratory failure, decreased pulmonary compliance, and severe imbalance of ventilation and blood flow ratio resulting from excessive pulmonary inflammation, non-cardiogenic pulmonary edema, and diffuse alveolar damage [ , ] . because of the lack of specific and effective treatments for ali/ ards, the mortality rate among these patients remains as high as - % [ ] . the pathogenesis of ali/ards is complex, and one of the most crucial factors is an uncontrolled inflammatory response [ , ] . controlling the excessive inflammatory response during acute respiratory failure is an important therapeutic target for treating ali/ards. the toll-like receptor (tlr) signaling pathway plays a key role in host defense, innate immunity, and inflammation [ , , ] . prototypic signaling in inflammation occurs through the lipopolysaccharide (lps)-tlr pathway. lps induces the dimerization of tlr that initiates signal transduction involving multiple adaptors, leading to the activation of inflammatory transcription factors including nuclear factor kappa beta (nf-κb) and interferon β regulatory factor (irf ), which mediates the upregulation of inflammatory cytokine gene expression leading to an inflammatory response [ , ] . however, unchecked, excessive or inappropriate tlr signaling activation can lead to severe inflammation and immunity-related tissue damage [ , ] . single immunoglobulin interleukin (ig il)- receptor-related molecule (sigirr) is an important negative regulator of tlr signaling [ ] . through the negative regulation of tlr signaling, sigirr can depress the inflammatory response to lps, block the transduction cascade, inhibit nf-κb activation, decrease the production of inflammatory mediators, and ultimately attenuate organ injury [ ] . trapping key components of the tlr signaling pathway is the mechanism by which sigirr inhibits tlr signaling transduction [ , ] . the toll-interleukin- receptor (tir)-domain of sigirr is necessary for sigirr to interact with tir-domain containing adaptors [ ] . paralemmin (palm)- belongs to the palm protein family and was first described in xenopus laevis as xlgv / xlcaax- by cornish et al. [ ] . in our previous study, we used the c-terminal part of sigirr containing the tir domain as bait to screen sigirr-binding proteins in a human lung cdna library and found that palm- is a novel interactive partner of sigirr [ ] . we also showed that silencing palm expression inhibited the release of inflammatory cytokines induced by lps in vitro [ ] . moreover, previous research demonstrated that the downregulation of palm was protective against lps-induced ali in mice [ ] . although the downregulation of palm has been shown to benefit lps-induced ali in mice, the explicit underlying mechanisms against ali, including whether the downregulation of palm has a protective effect against lps-induced ali in other animal models, a negative effect on the expression of adaptors in the lps-tlr pathway, blocks lps-tlr signal transduction, and regulates the inflammatory transcription factors nf-κb and irf , remain unclear. indeed, species diversity usually leads to different research results, even under the same experimental conditions. for example, comprehensive physiologic studies of lung injury are better in rats than in mice [ ] . therefore, the present study investigated the effect of the downregulated expression of palm on lps-induced ali using a rat model. to elucidate the potential mechanisms involved, the production of pro-and anti-inflammatory cytokines in bronchoalveolar lavage fluid (balf) and serum, and the expression of tlr , myeloid differentiation factor (myd ), tir domain-containing adaptor inducing interferon β (trif), and nf-κb and irf activities in the lungs were examined. additionally, the interaction of tlr with myd or trif in rat lung was also detected by coimmunoprecipitation. cells in the exponential growth phase were used in the experiments described below. hek cells (microbix, toronto, canada) were grown in dulbecco's modified eagle's medium (gibco/brl, grand island, ny), supplemented with % fetal bovine serum (gibco/ brl), u/ml penicillin, u/ml streptomycin, and mmol/l l-glutamine (gibco/brl) at °c in a humidified chamber with % co . hek cells were seeded in m flasks or -well plates ( × /well) prior to transfection. in accordance with the principles of rna interference sequence design, the sequence ′-agatcttgatggag ggttt- ′ was chosen as the target sequence. the primers for this target sequence were ′-ccggggagatcttg atggagggtttctcgagaaaccctccatca agatctcctttttg- ′ (sense) and ′ -a att c a a a a a g g a g at c t t g at g g a g g g t t t c tcgagaaaccctccatcaagatctcc- ′ (antisense) (genechem co. ltd., shanghai, china). the short hairpin oligonucleotide and complementary strand targeting the consensus sequences of palm were designed and inserted into the shuttle plasmid pgv (genechem) to generate the plasmid pgv -palm -shrna. the pgv -palm -shrna plasmid was identified by pcr and dna sequencing. recombinant viruses were generated using packaging cells cotransfected with the shuttle plasmid and the framing plasmid (pbhglox) via the admax™ system (microbix). viral stocks were purified using cscl gradients. the recombinant virus was named ad.palm -shrna. a control vector containing no transgene (ad.v) was constructed using the same method. titers of the recombinant adenoviruses were determined by means of % tissue culture infectious dose, and for ad.palm -shrna and ad.v, this was × plaqueforming units (pfu)/ml and × pfu/ml, respectively. adult wistar rats ( weeks old) were purchased from the laboratory animal center of the academy of military medical sciences (certificate number scxk-army- - , beijing, china). all rats used in this research were raised and used in accordance with the arrive (animal research: reporting of in vivo experiments) guidelines on the use of laboratory animals [ ] and the care and housing of rats were approved by the animal care and use committee of navy general hospital (approval number: scxk-army- - ). animals were housed in specific pathogen-free conditions at a regular temperature ( ± °c) and humidity ( ± °c) with a standard diet and clean water provided ad libitum. after acclimatization for week in the animal housing facility, rats were anesthetized by ether (fuyu chemical co., ltd., tianjin, china) inhalation as follows: cotton wool containing ether was placed in an animal anesthesia bottle and the rats were placed into this bottle for min. after the rats were lightly anesthetized, they were removed and inoculated intranasally with a total of × pfu (in μl) of ad.palm -shrna (ad.palm -shrna group) or ad.v (ad.v group) using a -ml blunt head syringe as previously described [ , ] . forty-eight hours later, lps (escherichia coli : b , sigma-aldrich, st. louis, mo, usa) at mg/kg (dissolved in saline) was injected into the peritoneal cavity of conscious rats to establish the a l i r a t m o d e l . r a t s w e r e s a c r i f i c e d w i t h a supraphysiological dose of pentobarbital sodium ( mg/kg) at , , , , , or h after lps challenge (n = per time point in each group). after each rat was sacrificed, an -gauge sterile catheter (carelife co. ltd., shanghai, china) was inserted into the left bronchus to collect balf as previously described [ ] . the right lungs were rapidly removed from all rats and washed in rnasefree ice-cold saline. the upper lobe of the right lung was frozen in ml trizol reagent (invitrogen, shanghai, china) and stored in liquid nitrogen for rna extraction. the middle lobe of the right lung was frozen in ml cell lysis buffer (beyotime chemical co., jiangsu, china) and stored in liquid nitrogen for protein isolation and tissue homogenization. the lower lobe of the right lung was immediately fixed with % paraformaldehyde phosphate buffer solution (bosterbio, wuhan, china) for histological evaluation. blood samples were obtained from the right side of the heart ( - ml per rat), allowed to clot for . h on ice, and then centrifuged at ×g for min ( °c). the sera were stored in small aliquots at − °c until the cytokine levels were measured. total rna was extracted from the lung tissue using trizol reagent (invitrogen) according to the manufacturer's instructions. the rna was further purified by an rna cleanup kit (cwbio co., beijing, china). reverse transcription (rt) of the lung samples was performed using the takara primescript rt reagent kit (takara, dalian, china). then, quantitative real-time polymerase chain reaction (qrt-pcr) was performed using itaq universal sybr green supermix (takara) according to the manufacturer's instructions. the primers for rat palm were ′-gaggcagggatcttgatgtc- ′ (sense) and ′-gcccaacaccctcaagacta- ′ (antisense) (takara). the primers for rat β-actin were ′-ggag attactgccctggctccta- ′ (sense) and ′-gact catcgtactcctgcttgctg- ′ (antisense) (takara). β-actin was selected as an internal standard. the pcr parameters were set as °c for s, followed by cycles of °c for s and °c for s. all reactions were performed in triplicate, and reports were generated by rotor-gene real-time analysis software . (corbett research, australia). the relative expression of the target genes (palm and β-actin) was calculated by the −△△ct method. protein was extracted from the lung tissue as previously described [ , ] . protein concentrations were measured by the bicinchoninic acid (bca) method (sigma-aldrich) at nm with a μquant microplate spectrophotometer (biotek inc., winooski, vt, usa). protein samples were analyzed by western blotting. equal amounts of protein were separated by % sodium dodecyl sulfate (sds)/polyacrylamide gel electrophoresis (page) and transferred onto a polyvinylidene fluoride filter membrane in a semidry blotting apparatus (bio-rad co. ltd., shanghai, china). to reduce non-specific binding, the membrane was blocked in a % (w/v) non-fat milk (beyotime) solution in tris-buffered saline-tween (tbst: mm nacl, mm tris/hcl, ph . and . % tween ) (beyotime) at °c for h. the membranes were then incubated with anti-palm- (cat. no. sc- , ; polyclonal goat anti-rat; santa cruz) ( : in tbst) and anti-β-actin antibodies (cat. no. af ; monoclonal mouse anti-rat; beyotime) ( : in tbst) overnight at °c. after extensive washing with tbst, the membranes were incubated with horseradish peroxidase (hrp)-conjugated secondary antibody ( : in tbst) (beijing golden bridge biotech, beijing, china) at room temperature for h. the blots were washed twice in tbst buffer and the signals were detected by enhanced chemiluminescence following the manufacturer's instructions (beyotime). the membrane was photographed using the gel documentation and analysis system (gbox-hr, syngene, usa), and band intensities were measured with adobe photoshop version . . software (adobe systems, usa) and normalized to the expression of β-actin for quantitative analysis. a second cohort of rats (n = ) was used to evaluate the effect of ad.palm -shrna on the outcome of lpsinduced ali. rats were divided into two groups (n = rats per group) and given the same treatment as previously described. seven-day survival rates were recorded and analyzed. lung tissues were treated with a % paraformaldehydephosphate buffer solution (bosterbio) (ph = . ) overnight at °c, dehydrated, embedded, and sectioned to . -μm thickness. tissue slides were then stained with hematoxylin and eosin (he) (bosterbio) and analyzed under identical light microscope conditions (olympus co., ltd., tokyo, japan). lung injury was scored by a blinded observer according to the following three criteria: ( ) alveolar and interstitial edema, ( ) alveolar hemorrhage, and ( ) infiltration or aggregation of neutrophils. each criterion was graded according to a four-point scale: = normal, = mild change, = moderate change, and = severe change. the scores for criteria to were summed to represent the lung damage score (total score: - ) [ , ] . the left lungs were lavaged five times with pbs ( ml each time) and approximately % of the instilled volume was retrieved [ , ] . balfs were then centrifuged at ×g for min ( °c), and the clear supernatant was transferred to sterile eppendorf tubes and stored at − °c for the analysis of total protein concentration [ , ] by the bca method (sigma-aldrich). adult wistar rats (n = per group) were treated as described in the bmaterials and methods^section bestablishment of ali rat model and experimental design.^at h after lps challenge, the right lungs were harvested and stored in liquid nitrogen for the analysis of nf-κb phospho-p , phospho-irf , tlr , myd , trif, suppressor of cytokine signaling (socs ) protein levels and coimmunoprecipitation assay, and the left lungs were harvested for the analysis of lung wet/dry weight ratio. the lung tissues were weighed immediately then subjected to desiccation in a °c oven until a stable dry weight was achieved after h. the scales and oven were purchased from whaisp scientific instrument co., ltd. (wuhan, china). the wet/dry weight ratio was calculated by dividing the wet weight with the dry weight to quantify the magnitude of pulmonary edema [ ] . balf was prepared as described above, and balf pellets were resuspended in cold pbs ( °c). balf ( μl) was mounted on a glass slide by centrifugation and the glass slide was stained with he (bosterbio). for analysis of the neutrophil cell count, we counted cells per slide in randomly selected high-powered fields (× ) under identical light microscope conditions (olympus). balf neutrophil cell counts were expressed as cell count/ml of balf [ ] . to measure tissue myeloperoxidase (mpo) activity, frozen lungs were thawed and mpo was extracted following the homogenization and sonication procedure described previously [ ] . mpo activity in the supernatant was measured at an absorbance of nm, and changes in mpo activity were determined following the decomposition of h o in the presence of o-dianisidine. the specific activity of mpo in the lung was expressed as u/g of the wet lung tissue. serum and balf were prepared as described above. levels of tumor necrosis factor-α (tnf-α), interleukin- β (il- β), il- (r&d systems, inc., minneapolis, mn), and interferon β (ifn-β) (cusabio, wuhan, china) in balf and serum were measured by commercially available enzyme-linked immunosorbent assay (elisa) kits for rat, following the manufacturer's instructions. in brief, diluted standards or samples were added to -well plates precoated with affinity purified polyclonal antibodies specific for tnf-α, il- β, il- , and ifn-β, respectively. enzyme-linked polyclonal antibodies were then added to the wells prior to incubation at °c for min, followed by five final washes. the intensities detected at nm were measured after the addition of substrate solutions for min. each sample was assayed in triplicate. levels of tnf-α, il- β, il- , and ifn-β were calculated according to standard curves. nuclear extracts were prepared using the ne-per nuclear-cytoplasmic extraction reagents kit (thermo fisher scientific, usa) according to the manufacturer's protocol. the extracts were stored at − °c, and the protein concentration of the nuclear extracts was quantified by the bca method (sigma-aldrich). nf-κb p dna binding activity was assessed on isolated nuclear extracts by elisa using the transam™ nf-κb transcription factor assay kit according to the manufacturer's protocol (active motif, carlsbad, ca) [ ] . each sample was assayed in triplicate. equal amounts of raji nuclear extract were used as positive controls. western blot analysis of nf-κb phosho-p , phospho-irf , tlr , myd , trif, and socs lung tissues samples were obtained as described in the bmaterials and methods^section blung wet/dry ratios.^the nuclear extract and total protein were prepared, and the procedure of western blot analysis was performed as described above. expressions of nf-κb phospho-p and phospho-irf in the nuclear extracts were analyzed by immunoblotting with phospho-specific anti-nf-κb p (cat. no. an ; monoclonal rabbit antirat; beyotime) ( : in tbst) antibody or phosphospecific anti-irf (cat. no. ab ; polyclonal rabbit anti-rat; abcam, cambridge, uk) ( : in tbst) antibody, and histone h (cat. no. sc- , ; monoclonal mouse anti-rat; santa cruz) ( : in tbst) was used as a lysate control. expressions of tlr , myd , trif, and socs in total protein were analyzed by immunoblotting with anti-tlr (cat. no. sc- , ; monoclonal mouse anti-rat; santa cruz) ( : in tbst), anti-myd (cat. no. ; monoclonal rabbit anti-rat; cell signaling technology) ( : in tbst), rabbit polyclonal anti-trif (cat. no. ab ; polyclonal rabbit anti-rat; abcam, cambridge, uk) ( : in tbst), or anti-socs (cat. no. sc- , ; monoclonal mouse anti-rat; santa cruz) ( : in tbst). β-actin (beyotime) ( : in tbst) was used as a lysate control. corresponding hrpconjugated secondary antibodies (beijing golden bridge) were used to display the protein signal. to examine endogenous protein-protein interactions, coimmunoprecipitation assays were performed. lung tissue samples were obtained as described in the bmaterials and methods^section blung wet/dry ratios.^protein was extracted from the lung tissue as previously described [ ] . the rat lung tissue was homogenized and lysed in tne buffer ( mm tris, ph . , mm nacl, mm edta, % np- , % glycerol with protease inhibitor cocktail [sigma-aldrich]) ( ml of tne/g lung lysate) and clarified by centrifugation at , ×g for min at °c. after centrifugation, proteins were quantified using the bca method (sigma-aldrich). approximately mg samples of rat lung lysates were incubated with a primary rat-specific anti-tlr ( ) antibody ( : in tbst) (cat. no. sc- , ; monoclonal mouse anti-rat; santa cruz), and the same amount of lysate was incubated with normal rat igg (santa cruz) as a negative control at °c for h and then with μl of protein a/g-agarose (beyotime) at °c with rocking overnight. the pellets obtained after centrifugation were washed five times with washing buffer ( mm tris (ph . ), mm mgcl , mm edta, and mm pmsf). the pellets were resolved by sds-page and boiled for min. after centrifugation, the supernatants were obtained as immunoprecipitates for western blotting analysis by using anti-tlr antibody (santa cruz), anti-myd antibody (cell signaling technology), or anti-trif antibody (abcam). data are expressed as the means ± standard error of the mean (sem). statistical significance was estimated by one-way analysis of variance (anova) followed by the student-newman-keuls test. kaplan-meier curves and the log-rank test were used to assess survival data. statistical significance was determined for p values less than . . lung tissues were obtained at , , , , , and h after lps injection to detect palm expression using quantitative real-time polymerase chain reaction (qrt-pcr) and western blot analysis. in the ad.v group, palm gene and protein expression in the lung tissues increased significantly, peaking at h after lps challenge (p < . vs. -h time point; p > . -h vs -h time point) (fig. ) , indicating that lps enhanced palm expression in a time-dependent manner. this result was consistent with our previous report in vitro [ ] . the administration of ad.v did not influence palm expression before lps stimulation (p > . vs. normal rats) (fig. ) . compared with the ad.v group and normal rats, palm mrna and protein expression in the ad.palm -shrna group were significantly decreased, although increases were noted after lps stimulation at h (p < . vs. ad.v group at each time point) (fig. ) . moreover, the palm expression levels in the ad. palm group decreased by about % compared with those in the ad.v group at each time point. to determine the effect of the downregulation of palm expression on the outcome of lps-induced ali, survival rates of the ad.v and ad.palm -shrna groups were compared using the log-rank test. compared with the ad.v group, the survival rate of the ad.palm -shrna group was significantly increased. rats receiving the ad.palm -shrna and control adenoviral vectors had an overall survival rate of and %, respectively, over days (p < . ) (fig. ) . histopathological staining showed that lung sections from the ad.v group displayed typical histological features of ali after lps challenge, including the infiltration of numerous neutrophils, alveolar congestion and hemorrhage, and marked swelling of the alveolar walls. moreover, the magnitude of lung injury was increased over time ( fig. a (a-e) ). ad.palm -shrna pretreatment improved these lps-induced histological changes of lung tissue compared with the ad.v group (fig. a (a-j) ). in addition, we used a pathological scoring system to quantify the severity of lung tissue damage in both groups. as shown in fig. b , the lung injury scores of both groups were very low before lps stimulation, demonstrating that the adenovirus vector had no impact on lung injury (fig. b) . furthermore, the lung injury scores in the ad.palm -shrna group were significantly decreased at each time point after lps challenge compared with those in the ad.v group (p < . vs. ad.v group at each time point) (fig. b) . because protein concentrations in the balf and lung water content are usually used to quantify the increase of vascular permeability in ali, we measured total protein levels in balf and lung wet/dry ratios in both groups. the concentration of total protein in the balf of both groups increased after lps-stimulation (p < . vs. -h time point) (fig. a) . however, total protein levels in the balf of the ad.palm -shrna group were significantly lower than those in the ad.v group after lps stimulation (p < . vs. ad.v group at each time point) (fig. a) . this result also demonstrated that the total protein level in the balf of both groups reached a peak at h after lps stimulation (fig. a) , indicating that lung vascular leakage was most severe at the -h time point. consequently, the lung wet/dry ratios at this time point were measured to confirm changes of lung vascular permeability. we found that lps led to increased lung wet/dry ratios in both ad.v and ad.palm -shrna groups (p < . vs. normal rats) (fig. b) , and ad.palm -shrna pretreatment markedly attenuated lps-induced pulmonary edema (p < . vs. lps-treated and lps + ad.v-treated rats) (fig. b) . balf neutrophil counts commonly indicate the severity of inflammation in ali. as shown in fig. a , the balf neutrophil count was significantly increased in the ad.v group after lps injection (p < . vs. -h time point) (fig. a) . however, in the ad.palm -shrna group, the balf neutrophil count was significantly decreased at each time point after lps exposure (p < . vs. group ad.v at each time point) (fig. a) . myeloperoxidase (mpo) activity is an index of neutrophil sequestration. thus, the mpo activity in lung tissue was detected to determine the extent of neutrophil infiltration. mpo activity was normalized by the weight of wet lung tissue. in unstimulated rats, mpo levels in the lung tissues were very low ( . ± . u/g wet lung tissue in the ad.v group and . ± . u/g wet lung tissue in the ad.palm -shrna group) (fig. b) . mpo activities in lung tissues increased sharply after lps injection in both groups (p < . vs. -h time point) (fig. b) . nevertheless, increases of mpo activity in the lung were significantly decreased in the ad.palm -shrna group at each time point after lps exposure compared with that in the ad.v group (p < . vs. group ad.v at each time point) (fig. b) . increased levels of proinflammatory cytokines are an indication of local pulmonary injury/systemic inflammation in ali, as reported previously [ , , ] . thus, we evaluated levels of the proinflammatory cytokines tnf-α, il- β, and ifn-β in the balf and serum. when ad.palm -shrna and ad.v were administered without lps treatment, there were no significant differences in cytokine levels in balf and serum between the two groups (p > . ) (fig. ) . after lps stimulation, the levels of tnf-α (fig. a, b) , il- β (fig. c, d) , and ifn-β (fig. e, f) in balf and serum were significantly increased in the ad.v group. in contrast, ad.palm -shrna pretreatment reduced the elevated levels of tnf-α (fig. a, b) , il- β (fig. c, d) , and ifn-β (fig. e, f) in balf and serum (p < . vs. ad.v group at each time point) (fig. ) . il- is an anti-inflammatory cytokine with negative immune regulatory effects in the tlr inflammatory signal transduction pathway [ ] . thus, we evaluated the levels of anti-inflammatory cytokine il- in the balf and serum. lps exposure increased the levels of il- in the balf (fig. a) and serum (fig. b) . in the ad.palm -shrnatreated group, the lps-induced increase of il- was further enhanced compared with the ad.v-treated group (p < . vs. ad.v group at each time point) (fig. ) . socs is an important negative regulator of inflammation and plays a protective role in lps-induced lung inflammatory responses [ , ] . previous studies have shown that il- induced socs expression [ , ] . thus, we evaluated the protein levels of socs in lung tissues using western blot analysis. the results showed that lps induced the expression of socs in all lps-treated rats (p < . vs. normal rat) (fig. c) . however, ad.rpalm pretreatment further increased the elevated socs protein levels in lung tissues (p < . vs. lps-treated and lps + adv-treated rats) (fig. c) . nf-κb is considered a key transcription factor in lps-tlr signaling that mediates lps-induced ali [ ] . thus, the nf-κb-dna-binding activity and nf-κb phospho-p protein level in rat lungs was determined as described above. the elisa result showed that all lps-injected rats exhibited an increase in nf-κb-dna-binding in comparison with lps-free rats (p < . vs. -h time point) (fig. a) . ad.palm -shrna pretreatment significantly attenuated the lps-induced increase in nf-κb activation compared with the ad.v group (p < . vs. ad.v group at each time point) (fig. a) . elisa demonstrated that the activity of nf-κb reached a peak at h after lps stimulation (fig. a) . therefore, we also examined the nf-κb phospho-p protein level in nuclear extracts at this time point using western blot analysis to confirm the inhibitory action of ad.palm -shrna on lps-induced nf-κb activation. as shown in fig. b , at h after lps stimulation, the nf-κb and phospho-p protein levels in the ad.palm -shrna group were reduced by about % compared with those in lps-treated and lps + ad.v-treated rats (p < . ) (fig. b) . irf is also an important transcription factor in cells and activates the ifn-β gene to regulate the release of proinflammatory cytokines induced by lps [ ] . we therefore examined the phospho-irf protein levels in nuclear extracts using western blot analysis to detect the influence of palm on lps-induced irf activation. at h after lps stimulation, phospho-irf protein levels were significantly increased in all rats. however, ad.rpalm pretreatment reduced the elevated phospho-irf protein levels (p < . vs. lps-treated and lps + ad.vtreated rats) (fig. c) . effects of downregulation of palm on lps-induced tlr , myd , and trif protein expression tlr , myd , and trif are important adaptors in the tlr signaling pathway [ ] . thus, we examined tlr , myd , and trif expression levels in lung tissues using western blot analysis to detect the influence of palm on their expression in the ali rat model. as shown in fig. , lps significantly induced the expression of tlr , myd , and trif in lung tissues (p < . vs. normal rats). however, palm shrna and control adenovirus did not have significant effects on tlr , myd , and trif protein expression in lps-induced ali rats (p > . vs. lps-treated and lps + ad.v-treated rats) (fig. ) . myd and trif are recruited to the tlr intracellular domain after stimulation by lps, which triggers the formation of tlr /myd and tlr / trif complexes [ ] . as shown in fig. , the formation of tlr /myd and tlr /trif complexes was significantly increased in lung tissues after lps stimulation (p < . vs. normal rats). pre-treatment with ad.palm -shrna reduced the i n t e n s i t y o f t h e m y d a n d t r i f b a n d coimmunoprecipitated using anti-tlr antibody (p < . vs. lps-treated and lps + ad.v-treated rats). however, the control adenovirus (ad.v) pre-treatment did not have significant effects on lpsinduced tlr /myd and tlr /trif complex formation in comparison with lps-treated rats (p > . vs. lps-treated rats) (fig. ) . fig. . the downregulation of palm has no effect on lps-induced tlr , myd , and trif protein expression. rats were instilled with μl of ad.palm -shrna or ad.v intranasally. at h, lung injury was induced by a peritoneal injection of lps ( mg/kg). normal and ali rats were used as control groups, and lung tissues were collected at h after lps challenge. a representative immunoblots of tlr , myd , and trif. β-actin served as an internal control. b quantification of densitometric measurement as a ratio of tlr , myd , and trif relative to β-actin. data are expressed as means ± sem (n = ). *p < . vs. normal rats. fig. . effects of downregulation of palm on lps-induced tlr /myd and tlr /trif complex formation in lungs. rats were instilled with μl of ad.palm -shrna or ad.v intranasally. at h, lung injury was induced by a peritoneal injection of lps ( mg/kg). normal and ali rats were used as control groups, and lung tissues were collected at h after lps challenge. a representative coimmunoprecipitated bands of myd and trif by anti-tlr antibody. β-actin served as an internal control. b quantification of densitometric measurement of palm knockdown on interactions of tlr with myd and trif. data are expressed as means ± sem (n = ). *p < . vs. normal rats and #p < . vs. lps + ad.palm -shrna-treated rats. the present study revealed that palm expression in rat lung was upregulated in a time-dependent manner in response to lps. furthermore, the downregulation of palm improved the survival rate of rats, ameliorated the severity of lung injury, alleviated pulmonary edema, lung vascular leakage and neutrophil infiltration, inhibited the production of proinflammatory cytokines (tnf-α, il- β, and ifn-β) and activation of nf-κb and irf , and promoted the secretion of the antiinflammatory cytokine il- and the expression of socs in the lps-induced ali rat model. however, the downregulation of palm did impact the upregulation of tlr , myd , and trif protein expression induced by lps in rat lung tissues. moreover, the downregulation of palm impeded the interaction of tlr with myd or trif induced by lps in rat lungs. these results suggest that the downregulation of palm exerts a potential protective effect against lpsinduced ali in rats and may be a promising potential treatment for ali. its protective effects were partially associated with the downregulation of proinflammatory cytokines, upregulation of the anti-inflammatory cytokine (il- ) and negative regulator of inflammation (socs ), and the inhibition of nf-κb and irf activities. its mechanisms may be attributed to the modulation of inflammatory responses and inhibition of tlr / myd and tlr /trif complex formation. palms are a small protein family that includes palm , palm , palm , and palmdelphin (palmd) [ ] , which are highly expressed in the brain, kidney, adrenal gland, mammary gland, and breast cancers [ ] [ ] [ ] . previous studies have verified that palm is implicated in controlling cell shape, plasma membrane dynamics, cell motility, invasiveness and metastatic potential of cancer cells, modulation of cell migration and maturation, and tumor lymphangiogenesis [ , , ] . however, little is known about the biological functions of the other palm isoforms. hultqvist and colleagues [ ] considered that there is functional diversification and specific biological roles among palm isoforms. palm was first reported in by cornish et al. [ ] and was speculated to act as an adaptor to link intrinsic membrane proteins to each other, to the cytoskeleton, or to motor proteins [ , ] . we reported for the first time in that palm is a novel interactive partner of sigirr, and we also found that lps upregulated the expression of palm [ ] . our present study confirms the findings of our previous studies, which demonstrated the modulation of palm expression in response to lps [ , ] . our results revealed that palm expression was detected in the lung tissue of normal rats, and that the control adenovirus ad.v had no effect on palm expression. after lps stimulation, palm protein and mrna expression levels in the lungs of the ad.v group were significantly upregulated in a time-dependent manner, which was in line with our previous result in vitro [ ] . moreover, the expression pattern of palm was similar with those of the adaptors in tlr signaling, including myd and trif [ ] [ ] [ ] [ ] . the pattern of palm upregulation might be related to its functional involvement in lps-tlr signal transduction, similar to the adaptors [ ] . in our present study, palm gene and protein expression in lung tissues was downregulated by administering a recombinant adenovirus expressing shrna for rat palm . although palm expression in the ad.palm -shrna group increased after lps stimulation for h, this expression level remained lower than that of the normal and ad.v group rats during the experiment. this result demonstrated that the palm gene transcript in lung tissue was silenced successfully using the interference adenoviral vector-mediated rna through intranasal instillation. increased palm expression levels in the lungs of the ad.palm -shrna group may also be caused by enhanced palm gene transcription during the process of inflammatory signal transduction. an uncontrolled inflammatory response is one of the most crucial factors in the pathogenesis of ali [ , ] . during the early stage of ali, inflammatory and immune responses involve a vast of array of mediators; therefore, inhibition of the inflammatory cascade response is critical for the treatment of this disorder [ ] . downregulation of palm gene transcription to impede tlr signal transduction at the early stage may inhibit the excessive inflammatory responses and improve the outcome of inflammatory diseases, including ali/ ards. thus, we determined the survival rate, lung histological changes, pulmonary edema, lung vascular leakage, neutrophil infiltration, and mpo activity in the lung to assess the effect of downregulation of palm on lps-induced ali in rats. our findings showed that the downregulation of palm expression significantly improved the aforementioned assessment indexes, similar to previous studies [ , ] . however, in these previous studies, the underlying mechanisms have not been explored. lps induces the dimerization of tlr , resulting in conformational changes of the tlr homodimer that induces the recruitment of adaptor proteins containing tir domains. tir domains of tlr recruit tir domaincontaining adaptor proteins, myd -adaptor-like (mal) and myd (myd -dependent pathway), or trif and toll-il- receptor-containing adapter molecule (ticam- ) (myd -independent pathway) [ ] . activation of the myd -dependent pathway induces the activation and translocation of transcription factors such as nf-κb and activator protein- (ap- ) in the nucleus and induces the production of proinflammatory cytokines [ ] . activation of the myd independent pathway leads to the activation and translocation of the transcription factor irf in the nucleus to induce the production of type i interferon [ ] . the myd -independent pathway also plays a key role in the late-phase activation of nf-κb [ ] . in the present study, we found that the downregulation of palm suppressed nf-κb-dna-binding activity and decreased nf-κb phospho-p and phospho-irf protein levels in the nucleus of ali rat lung tissues. these findings suggested that the downregulation of palm inhibited the activation of nf-κb and irf in the ali rat model. the increase of proinflammatory cytokines in the balf are thought to be indices of local pulmonary inflammation in ali [ , , ] , and the levels of inflammatory mediators in serum generally reflect the extent of systemic inflammatory response and the prognosis of inflammatory diseases [ , , , ] . in our present study, a significantly increased survival rate was observed in the ad.palm -shrna group, accompanied with a decrease in inflammatory mediator levels in the balf and serum. these results suggest that this therapy directed to the lung through intranasal instillation inhibited local inflammation in the lungs and suppressed systemic inflammation to improve the outcome of ali. mitigation of pulmonary inflammation and reduction of alveolar capillary permeability decreased the translocation of inflammatory mediators from lung tissues into the systemic circulation ameliorating severity of the systemic inflammatory response [ , ] . the negative effect of the downregulated expression of palm on the production of proinflammatory cytokines and the infiltration of neutrophils might be ascribed to its negative regulatory function on transcription factors nf-κb and irf . alleviation of inflammatory responses can finally lead to the amelioration of lung tissue damage. our results also revealed that the downregulation of palm did not impact the upregulation of tlr , myd , and trif protein expression after ali in lung tissues. previous studies have shown that palms are associated with lipid rafts and have been proposed to function as adaptors between membrane proteins or with the cortical cytoskeleton [ , ] . with this in mind, we speculated that the inhibition of palm expression to impair its function with adaptors in the lps-tlr signaling pathway may be a potential mechanism for the downregulation of palm protection against lpsinduced inflammatory responses and lung injury. downregulating the palm suppression of transcription factors nf-κb and irf might not be mediated by inhibiting the expression of the adaptors tlr , myd , and trif in lps-induced inflammation. therefore, we further investigated the effect of the downregulation of palm on the interaction of tlr with myd or trif in lung tissues of ali rats. the result of coimmunoprecipitation analysis showed that palm knockdown inhibited the interaction of tlr with myd or trif. our previous study demonstrated that palm interacted with the tir domain of sigirr [ ] . moreover, our in vitro study reveals that palm interacts with myd , irak- , traf- , and ticam- , important adaptors in the tlr signaling pathway, in a ligand-dependent manner (accepted but unpublished data). therefore, we presumed that palm might act as a bbridge^to link tlr to the adaptor proteins (myd , irak- , traf- , and tiacm- ) in lps-tlr signal transduction. downregulation of palm expression might diminish the bridging efficacy of palm during lps-tlr signal transduction, which might reduce further the interaction of tlr with myd or trif. inhibition of tlr /myd and tlr /trif complex formation may be a potential mechanism of the suppression of nf-κb and irf activation in lps-induced inflammation. further investigation is still needed to illuminate the detailed molecular mechanisms of the regulatory effect of palm on lps-tlr signaling. il- is a negative feedback regulatory molecule in the tlr signaling pathway that degrades the nf-κb p subunit and inactivates mitogen-activated protein kinase (mapk) signaling [ ] . in this study, we also found that lps-induced il- expression in the balf and serum was significantly increased after palm gene silencing. this finding suggests that palm inhibits anti-inflammatory cytokine il- secretion in lps-induced ali. however, the downregulation of palm decreased the inhibitory effect of palm on il- secretion. promotion of anti-inflammatory cytokine il- secretion may also be a possible mechanism of downregulation of palm protection against lps-induced lung injury. the detailed mechanisms of palm inhibition against lpsinduced il- secretion warrant further research. previous studies have shown that socs proteins have pivotal roles in attenuating cytokine and tlr signaling in myeloid cells [ ] . in mice deficient for socs in hematopoietic and endothelial cells, injection with granulocytecolony stimulating factor (g-csf) induced a pronounced inflammatory response involving enhanced neutrophilia and neutrophil infiltration of multiple tissues [ ] . furthermore, the exogenous delivery of socs significantly enhanced the survival of mice challenged with lps-induced endotoxic shock [ ] . in the present study, we demonstrated that the downregulation of palm promoted the expression of socs in the lung tissues of ali rats. it was reported that il- induced socs expression to inhibit the expression of inflammatory cytokines in lpsstimulated macrophages [ ] . thus, downregulating the palm promotion of socs expression might be indirectly mediated by enhancing the expression of il- in lps-induced inflammation. the detailed molecular mechanisms of the regulatory effect of palm on socs expression require further investigation. the lps model of lung injury displays the key features of clinical ards, including inflammation, pulmonary edema, and mortality, and is still widely used to investigate the pathogenesis and treatment of ali and ards [ , , , ] . therefore, in the present study, we used the lps-induced ali rat model to investigate the effect of the downregulation of palm on ali. the rat ali model may be superior to the mouse ali model based on the following reasons. first, the availability of the rat genome will be extremely useful, as opportunities for comprehensive physiologic studies of lung injury are better in rats than in mice [ , ] . second, rats have a larger blood volume in contrast to mice, which benefits the analysis of cytokine levels in serum, to further illustrate the relationship between the local and systemic inflammation and the mechanisms of ali. the palm gene family is an early gene family that has been maintained throughout vertebrate evolution and may be important for the development and plasticity of complex nervous systems [ ] . at present, there are no relevant literature reports on palm knockouts; therefore, whether palm knockout possesses potentially fatal consequences remains unclear. the effect of palm knockout on the entire animal and the pathophysiological changes of ali in palm knockout animals will be investigated in future studies by constructing palm gene knockout rat models. with respect to ali/ards, transgene expression does not need to last a lifetime and a single administration of adenoviral vector could cover the critical period of ali/ ards [ ] . we therefore chose an adenoviral vector as a transient gene transfer tool in our study. our results showed that although palm expression increased in the palm knockdown group after lps stimulation for h, this expression level did not exceed that of normal rats at all time points. this finding indicates that the rna interference activity of ad.palm -shrna lasted for the duration of the experiment. moreover, in this study, we did not determine the effect of palm on inflammatory cells and immune cells, such as dendritic cells and macrophages, as it was not clear whether palm was expressed in these cells; however, this was not the main emphasis of the present study. future research will focus on this emerging issue using in vitro experiments. in summary, our results demonstrated that palm expression was induced by lps in a time-dependent manner, and that downregulation of palm in the lung, via interfering adenoviral vector-mediated rna, improved the survival rate in an lps-induced rat model. this protective effect of palm -knockdown on ali might be mediated by attenuating pulmonary pathological injury, alleviating alveolar capillary permeability, and decreasing neutrophil infiltration. downregulation of proinflammatory cytokine secretion, upregulation of anti-inflammatory cytokine secretion and socs expression, and inhibition of nf-κb and irf activities likely represent the mechanisms for the downregulation of palm protection against lpsinduced inflammatory responses and lung tissue injury. downregulating palm suppression of lps-tlr signaling might not be mediated by modulating the expression of tlr , myd , and trif, but rather through inhibiting the interaction of tlr with myd or trif. these results suggest that modulating palm expression may be a promising potential treatment for ali/ards. future research will focus on the emerging issues from this study and on the detailed molecular mechanisms of the palm regulation of lps-tlr signaling. programmed cell death receptor ligand modulates the regulatory t cells' capacity to repress shock/sepsis-induced indirect acute lung injury by recruiting phosphatase src 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injury and mortality in mice intratracheal instillation of keratinocyte growth factor decreases hyperoxia-induced mortality in rats socs is a critical physiological negative regulator of g-csf signaling and emergency granulopoiesis gene delivery of socs protects mice from lethal endotoxic shock rat genetics: attaching physiology and pharmacology to the genome the authors thank the national natural science foundation of china (chen, x.x., no. ; feng, j., no. ) and the innovative cultivation foundation of navy general hospital of the pla (chen, x.x., no. cxpy ) for their great support in financing these researches. conflict of interest. the authors declare that they have no conflict of interests.funding. this study was funded by the national natural science foundation of china (chen, x.x., no. ; feng, j., no. ) and the innovative cultivation foundation of navy general hospital of the pla (chen, x.x., no. cxpy ). key: cord- - o z authors: martin, thomas r.; wurfel, mark m. title: a triffic perspective on acute lung injury date: - - journal: cell doi: . /j.cell. . . sha: doc_id: cord_uid: o z acute lung injury (ali) is a leading cause of death in people infected with h n avian influenza virus or the sars-coronavirus. imai et al. ( ) now report that ali is triggered by the signaling of oxidized phospholipids through toll-like receptor (tlr ) and the adaptor protein trif. these findings provide insight into the molecular pathogenesis of ali, a condition for which treatment options are currently very limited. is thicker and is not vascularized, thus resulting in a diffusion distance between air and dermal capillaries that is too great to serve as an efficient means of o uptake. finally, does dysregulation of cutaneous blood flow have any effect on body temperature homeostasis? remarkably, mice lacking vhl in their keratinocytes die from hypothermia when subjected to cold stress due to a failure of cutaneous vasoconstriction (r. johnson et al., personal communication) . considering the complex homeostatic mechanisms that are subserved by the cutaneous vascu-lature, the study by boutin et al. elegantly demonstrates that beauty is not the only characteristic that is skin deep! acute lung injury (ali) affects more than , people in the us each year, with approximately , deaths, making it an important cause of morbidity, mortality, and health care expenditure (rubenfeld et al., ) . bacterial and viral infections are important risk factors for ali, but aspiration of gastric contents, major trauma, and repeated transfusions are additional risks. ali is also a leading cause of death in people infected with h n avian influenza virus or the coronavirus that causes sars (severe acute respiratory syndrome). in this issue, imai et al. ( ) report surprising insights from murine studies that provide a new perspective on the mechanisms contributing to ali in humans. the alveolar membrane of the lungs is the largest surface area of the body that is in continuous contact with the out-side environment, and a complex set of defenses have evolved to protect it against inhaled particulates and microbes. the alveolar wall is a delicate structure, consisting of a thin alveolar epithelial layer, a basement membrane composed of collagens, glycoproteins, and glycosaminoglycans, and a thin endothelial cell layer. surfactant phospholipids and associated proteins lining the alveolar surface are critical in reducing surface tension in alveolar fluid, so that alveoli do not collapse at low lung volumes. cells called type ii pneumocytes in the alveolar walls produce surfactant and actively transport sodium ions from the lumen to the interstitium, facilitating passive water movement from the alveoli to the interstitium and lymphatics in order to keep the airspaces dry. acute damage to epithelial or endothelial cells in the alveolar membrane causes the clinical syndrome of ali, in which the alveolar spaces fill with proteinaceous exudates, producing severe alterations in gas exchange, critical hypoxemia, and death in the absence of aggressive medical care. the hallmark findings of ali include acute neutrophilic inflammation and an array of proinflammatory cytokines in the lungs, suggesting that activation of innate immunity is an initial event, whether or not overt infection is present. activation of innate immune pathways combined with the physical stresses created by mechanical ventilation cause a synergistic increase in lung injury, but the mechanisms underlying ali are not clear (dos santos and slutsky, ) . in order to identify susceptibility factors for lung injury, imai and colleagues screened several strains of mice using a a triffic perspective on acute lung injury thomas r. martin , * and mark m. wurfel simple model of lung injury, intratracheal instillation of . n hydrochloric acid (hcl), which approximates severe gastric acid aspiration. surprisingly, mice with an inactivating mutation in toll-like receptor (tlr ) were protected from lung injury in this noninfectious model. tlr is the primary receptor for gramnegative bacterial lipopolysaccharide (lps) and also recognizes endogenous stimuli termed "alarmins" at sites of inflammation (oppenheim et al., ) . in macrophages, tlr signals via two different intracellular adaptor proteins, myd and trif (tir-domain-containing adaptor-inducing interferon-β), leading to two distinct intracellular signaling programs (beutler, ) . the myd pathway causes rapid nf-κb activation and cytokine production. the trif pathway leads to the production of type i interferons via interferon regulatory factor (irf- ) and also causes delayed nf-κb activation via activation of tnf receptorassociated factor (traf ) (hoebe et al., ; sato et al., ) . surprisingly, imai and colleagues found that trif-deficient mice and mice lacking traf in myeloid cells were protected from hclinduced injury, whereas myd knockout mice were not, suggesting that the trif pathway, acting through traf , is the major effector pathway in this noninfectious model. they also showed that trif-dependent lung injury is likely to be mediated by production of interleukin (il- ), as il- -deficient mice were also protected from injury. the finding that the tlr -trif pathway mediated injury in the absence of an infectious agent raised questions about the identity of the stimulus for tlr , and the mechanism responsible for preferential activation of the trif pathway. the lung lavage fluid of hcl-treated mice contained oxidized phospholipids (oxpls) detected by immunocytochemistry. an anti-oxpl antibody significantly reduced the proinflammatory activity of lung lavage fluid on lung macrophages in vitro. intratracheal instillation of synthetically oxidized phospholipids caused lung inflammation in normal and surfactantdepleted mice, whereas mice lacking tlr were protected. the monoclonal antibody used to detect oxpl provided a clue to the specific oxpl responsible because it recognizes phospholipids containing oxidized phosphatidylcholine (e.g., -palmitoyl- -arachidonoyl-phosphatidylcholine, oxpapc). oxpapc was shown to stimulate il- production from lung macrophages via the tlr -trif-traf pathway in vitro, independently of myd . these findings contrast sharply with signaling initiated by lps, which occurs predominantly through tlr -myd . in the complex inflammatory response initiated by hcl in the lungs, one might expect that tlr would be activated by several different endogenous stimuli; however, mice lacking tlr , trif, or traf all resisted hclas well as oxpapc-induced inflammation, supporting a role for oxpapc as an important stimulus of tlr activation in this model. because patients infected with the influenza virus or sars-coronavirus often develop severe lung injury, the authors looked for oxpl in the lungs of mice infected with an inactivated h n avian influenza virus. as in the hcl injury model, immunohistochemical analysis identified oxpapc in the lungs, but mice lacking tlr or trif had lung inflammation that was much less severe. mice lacking the ncf protein, which lack an active nadph oxidase complex, were protected from viral lung inflammation and did not form oxpapc in the airspaces, further supporting a key role for oxidation of phospholipids in the pathogenic pathway. high levels of oxpapc were also detectable in the lungs of animals with experimental pulmonary infections due to bacillus anthracis or yersinia pestis, suggesting that oxpl-mediated lung injury is of more general significance. the relevance of this new mechanism for human lung injury was demonstrated by the observation that significant amounts of oxpapc were present in lung tissue samples from two patients with lethal h n avian influenza infection and nine patients with ali following sars-coronavirus infection. these experimental results have surprising implications for understanding the pathogenesis of ali. a central role of tlr in lung injury has been suspected because lps is present in the lungs of many patients with ali, whether or not overt bacterial infection is present (martin et al., ) . in addition, tlr is triggered by a number of different endogenous alarmins likely to be present in injured lungs (oppenheim et al., ) . similarly, the formation of oxidized phospholipids in the lungs is not surprising given the intensely oxidative, neutrophil-rich environment in the lungs of patients with ali (sittipunt et al., ) . however, the central role of tlr in acid-induced injury, the principal role of oxpl in triggering tlr signaling, the predominant role of the trif-traf signaling pathway (figure ) , and the general applicability of the findings to important respiratory viral infections are all unexpected. paradoxically, the intravenous administration of oxpapc protects mice from lps-induced lung injury by protecting the work of imai et al. ( ) provides evidence that acute lung injury involves oxidized phospholipids acting through toll-like receptor (tlr ). in this model, injury to the lungs through acid aspiration or viral infection leads to activation of nadph oxidase (nadph ox) and production of reactive oxygen species (ros), which oxidize -palmitoyl- -arachidonoyl-phosphatidylcholine (papc, ox-papc). oxpapc activates tlr expressed by myeloid cells (an alveolar macrophage is shown), and the intracellular signal is transduced by the adaptor proteins trif and traf , leading to interleukin (il- ) production, inflammation, and alveolar damage. pmn, polymorphonuclear leukocyte. the endothelial barrier, suggesting that oxpapc might have different effects in the alveolar and intravascular compartments (nonas et al., ) . these seemingly discrepant findings could be reconciled in part if systemic challenge with oxpapc directly (via tlr ) or indirectly desensitizes the activation of circulating leukocytes. given that the myd pathway is critical to the host response to bacterial infections (skerrett et al., ) , the results of imai and colleagues suggest that new strategies to modulate the trif-traf pathway, while leaving the myd pathway largely intact, might be beneficial in some forms of ali. although the proximal event that creates the initial oxidative environment in the lungs remains unclear, neutrophil recruitment and activation are likely to be important because of the neutrophil's potent respiratory burst and because of the protection noted in ncf -deficient mice. likewise, the key molecular "switch" that controls whether trif or myd is activated by tlr remains a key unanswered question. almost years after the clinical description of ali, we have only one treatment that definitely improves survival, and this involves reducing the volume of air applied to the lungs during mechanical ventilation (acute respiratory distress syndrome network, ). the work of imai and colleagues points to potential molecular approaches that could further improve outcomes for this clinically important syndrome. courtship in the fruit fly drosophila melanogaster is largely the domain of the male and consists of a series of intricate behaviors designed to achieve successful copulation. these behaviors include following, tapping and licking the female, and the extension of the male wing that is closest to the female and its vibration to generate male courtship song (reviewed by billeter et al., a) . these behaviors depend on complex sensory and motor neural circuitry acting on specific effector tissues such as the limbs, wings, proboscis, and abdominal muscles of the male. the action of the neurons involved in these circuits can be related directly to the behavior they modulate such as courtship song production, which is crit-ical for copulatory success. this behavioral output is robust and easily quantified, and so lends itself to structure/ function analyses. the ability to perform these sexspecific behaviors is dependent on the existence of a sexually dimorphic nervous system. differences, both in neuronal numbers and projection patterns, acute respiratory distress syndrome network key: cord- - uze rp authors: dixon, barry; santamaria, john d; campbell, duncan j title: a phase trial of nebulised heparin in acute lung injury date: - - journal: crit care doi: . /cc sha: doc_id: cord_uid: uze rp introduction: animal studies of acute lung injury (ali) suggest nebulised heparin may limit damage from fibrin deposition in the alveolar space and microcirculation. no human studies have been undertaken to date. we assessed the feasibility, safety and potential anticoagulant effects of administration of nebulised heparin to patients with ali. methods: an open label phase trial of four escalating doses of nebulised heparin was performed. a total of ventilated patients with ali were studied. the first group was administered a total of , u/day, the second group , u/day, the third group , u/day and the fourth group , u/day. assessments of lung function included the pao( )/fio( )ratio, lung compliance and the alveolar dead space fraction. monitoring of anticoagulation included the activated partial thromboplastin time (aptt) and the thrombin clotting time. bronchoalveolar lavage fluid was collected and the prothrombin fragment and tissue plasminogen activator levels were assessed. analysis of variance was used to compare the effects of dose. results: no serious adverse events occurred for any dose. the changes over time for the pao( )/fio( )ratio, lung compliance and the alveolar dead space fraction levels were similar for all doses. a trend to increased aptt and thrombin clotting time levels was present with higher doses (p = . and p = . , respectively). for the highest dose, the aptt reached seconds; following cessation of nebulised heparin, the aptt fell to seconds (p = . ). in bronchoalveolar lavage samples a trend to reduced prothrombin fragment levels was present with higher doses (p = . ), while tissue plasminogen activator levels were similar for all doses. conclusion: administration of nebulised heparin to mechanically ventilated patients with ali is feasible. nebulised heparin was not associated with any serious adverse events, and at higher doses it increased aptt levels. larger trials are required to further investigate the safety and efficacy of nebulised heparin. in these trials due consideration must be given to systemic anticoagulant effects. trial registration: australian clinical trials registry actrn . acute lung injury (ali) is a serious clinical problem. estimates are that , cases of ali develop in the united states each year, which are associated with , deaths and . million hospital days [ ] . the -day mortality for ali is % [ ] . there is currently no method to prevent or treat ali ali is characterised by the rapid onset of respiratory distress in the setting of an inflammatory insult to the lungs [ , ] . inflammatory insults include sepsis, trauma, hypotension, cardiopulmonary bypass, pancreatitis, aspiration and multiple transfusions. septic insults are by the commonest cause of ali. pneumonia triggers % of cases, and sepsis elsewhere in the body causes % of cases [ ] one mechanism by which inflammation causes ali is deposition of fibrin in the alveolar space and microcirculation. fibrin deposition in the alveolar sacs gives rise to a hyaline membrane, and deposition in the microvasculature results in thrombosis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . nebulised heparin may limit fibrin deposition in the alveolar space and microcirculation through heparin's anticoagulant and fibrinolytic actions [ ] [ ] [ ] [ ] . studies in animal models of ali have demonstrated that nebulised heparin improved the pao /fio ratio and reduced histological ali = acute lung injury; aptt = activated partial thromboplastin time; bal = bronchoalveolar lavage; elisa = enzyme-linked immunosorbent assay; pao /fio = arterial oxygen partial pressure to inspired oxygen fraction ratio; ptf = prothrombin fragments; tct = thrombin clotting time; t-pa = tissue plasminogen activator. (page number not for citation purposes) damage [ , ] . in addition, in the setting of lung injury triggered by cardiac surgery, a preoperative heparin infusion reduced evidence of pulmonary microvascular thrombosis [ ] . we are unaware of previous trials of nebulised heparin in patients with ali. we therefore undertook the present trial to assess the feasibility, safety and potential anticoagulant effects of nebulised heparin in mechanically ventilated patients with ali. in addition, we assessed the effects on intrapulmonary coagulation activation and fibrinolysis. the study was approved by the st vincent's hospital human research ethics committee. consent was obtained from the patient or next of kin before participation in the study. the present study was an open-label, escalating-dosage phase trial of nebulised heparin (heparin sodium, , u/ ml; cp pharmaceuticals ltd, wrexham, uk) in mechanically ventilated patients with ali. four doses were studied. each dose was assessed in four patients over days. the first group was administered , u/day, as , u hourly (four nebulisations); the second group received , u/ day, as , u hourly (four nebulisations); the third group received , u/day, as , u hourly (four nebulisations); and the fourth group was administered , u/day, as , u hourly (eight nebulisations). the final nebulisation of heparin was administered at hours from baseline in the , u/day, , u/day and , u/day groups, and at hours in the , u/day group. we studied patients admitted to the intensive care unit that met the following inclusion and exclusion criteria. the inclusion criterion was the initiation of mechanical ventilation for acute respiratory dysfunction characterised by a pao / fio ratio < mmhg, where the acute respiratory dysfunction was primarily due to a direct or indirect inflammatory insult to the lung. the exclusion criteria were > hours since the inclusion criterion was met; hypoxemia predominantly due to a cause other than ali, such as congestive heart failure, pulmonary embolism, chronic obstructive airways disease or asthma; systemic anticoagulation (including activated protein c), potential need for haemofiltration and therefore anticoagulation; pulmonary haemorrhage in the previous months, uncontrolled bleeding, significant bleeding disorder; allergy to heparin, including heparin-induced thrombocytopenia; age < years or > years; patient unlikely to survive hours; bronchoscopy not possible due to severe hypoxia; previous intubation and venti-lation during current admission; noninvasive ventilation for more than hours prior to intubation; or pregnancy. heparin was nebulised with an aeroneb pro nebulizer (aerogen ltd, galway, ireland) over minutes. the nebuliser was placed in the inspiratory line cm from the y of the circuit. the heat and moisture exchanger was removed during nebulisation. patients were ventilated in a pressure-support mode of ventilation and upper pressure levels were maintained at or below cmh o. the p a o /f i o ratio, lung compliance and the alveolar dead space fraction were measured at baseline and at , , , , , , , , , , and hours. we measured the alveolar dead space fraction because previous studies have suggested this variable may reflect the extent of microvascular thrombosis in ali [ , ] . evidence of blood staining of respiratory secretions was assessed by the bedside nurse following routine pulmonary suctioning. the activated partial thromboplastin time (aptt) and the thrombin clotting time (tct) were assessed at the same time points as those of lung function, and at , and hours in the , u/day group. prothrombin fragments (ptf) and tissue plasminogen activator (t-pa) levels in bronchoalveolar lavage (bal) fluid were assessed at baseline and following the final nebulisation (bal was undertaken on average . ± hours following the final nebulisation). standard formulae were used to calculate lung compliance. the alveolar dead space fraction was measured with the cosmo plus respironics monitor (novametrix medical systems, wallingford, ct, usa) [ ] . the bronchoscope was wedged in the distal airway. the initial ml of warm saline injected was discarded. five further ml aliquots were instilled and aspirated. a portion of the aspirated fluid was spun at , × g for minutes at °c. the supernatant was stored at - °c. samples were assayed by elisas for ptf levels (enzygnost f + monoclonal assays; behring, marburg, germany) and for t-pa antigen levels (tin-telize tpa, biopool international, ventura, ca, usa). based on previous studies we determined that four patients in each group would be adequate to detect a major anticoagulant effect [ , ] . analysis of variance was used to compare the effect of heparin dose on the p a o /f i o ratio, lung compliance, the alveolar dead space fraction, the aptt, the tct and intrapulmonary ptf and t-pa levels. fisher's exact test compared categorical variables. student's t test compared normally distributed variables. data are reported as the mean ± standard deviation. statistical analysis was performed with the jmp program (sas institute, inc., cary, nc, usa). sixteen patients were enrolled. the mean patient age was ± years, and the acute physiology and chronic health evaluation score ii was ± . the baseline p a o /f i o ratio was ± mmhg, lung compliance was ± ml/ cmh o and the alveolar dead space fraction was . ± . . prophylactic subcutaneous heparin was administered to of the patients studied. the commonest aetiological factor for ali was pneumonia ( table ) . the time from intubation to initial heparin nebulisation was ± hours. the mean mechanical ventilation time was ± days, the intensive care length of stay was ± days and the hospital length of stay was ± days. the tracheostomy rate was % and the hospital mortality was %. the changes over time in the p a o /f i o ratio, lung compliance and the alveolar dead space fraction were similar for all doses studied. there were no statistically significant differences found for the dosage or for the interaction between dosage and time (figures to ). one patient in the , u/day group developed bloodstained respiratory secretions after the seventh dose. this was not associated with any deterioration in lung function. the blood staining resolved following withdrawal of nebulised heparin. the mean aptt for each group following the final nebulisation, in order of increasing dose, was seconds (normal range < seconds), seconds, seconds and seconds (p = . , analysis of variance, comparison by dose) (figure ). the mean tct for each group following the final nebulisation, in order of increasing dose, was seconds (normal range < seconds), seconds, seconds and seconds (p = . , analysis of variance, comparison by dose) ( figure ). for the higher dose groups, both the aptt and tct fell following cessation of nebulised heparin. for the highest dose, the aptt fell from seconds to seconds (p = . ) (figure ). the ptf levels in bal fluid in the , u/day group were higher following the final nebulisation, while in the , u/ day, , u/day and , u/day groups the ptf levels remained similar to baseline levels following the final nebulisation (p = . , analysis of variance, comparison by dose) (figure ). the t-pa levels were similar to baseline levels for all doses following the final nebulisation ( figure ). changes in arterial to inspired oxygen ratio with nebulised heparin dosage changes in arterial to inspired oxygen ratio with nebulised heparin dosage. percentage change from baseline in the arterial to inspired oxygen ratio (p a o /f i o ) (mean ± standard deviation). changes in lung compliance with nebulised heparin dosage changes in lung compliance with nebulised heparin dosage. percentage change from baseline in the lung compliance over time for each dose (mean ± standard deviation). (page number not for citation purposes) changes in alveolar dead space fraction with nebulised heparin dosage changes in alveolar dead space fraction with nebulised heparin dosage. percentage change from baseline in the alveolar dead space fraction (adsf) (mean ± standard deviation). we assessed the feasibility, safety and potential anticoagulant effects of nebulised heparin in mechanically ventilated patients with ali. we found administration of nebulised heparin to mechanically ventilated patients with ali was feasible, was not associated with serious adverse events, and increased aptt levels at higher doses. the changes in the pao /fio ratio, lung compliance and the alveolar dead space fraction were similar for all doses. in one patient in the , u/day group, blood staining of the respiratory secretions was present after the seventh dose. this staining resolved following withdrawal of heparin. we found evidence of dose-dependent effects on aptt and tct levels. for the , u/day group, the levels remained within the normal range; however, for the , u/day, , u/day and , u/day groups, the aptt and tct levels were raised on the second day. peak levels were reached following the final nebulisation, and thereafter levels fell. for the , u/day group, the aptt reached the therapeutic range ( seconds) and fell acutely to seconds following cessation of nebulised heparin. previous clinical studies have investigated the potential of systemic anticoagulation using nebulised heparin -to date, without success [ , ] . unlike these studies we used repeated doses of nebulised heparin. our finding that the aptt and tct levels increased only after repeated doses suggests that pulmonary processes, such as storage of heparin in endothelial cells and metabolism by heparinases, may initially limit heparin reaching the systemic circulation. these processes may, however, become saturated following repeated heparin doses [ ] . in future trials of nebulised heparin, due consideration must be given to this systemic anticoagulant effect. we also examined whether nebulised heparin limited coagulation and increased fibrinolysis in the lungs. for the , u/ day group, the ptf levels in bal fluid doubled from baseline levels following the final nebulisation. for the , u/day, , u/day and , u/day groups, however, the ptf levels did not increase. previous trials in patients with ventilated-associated pneumonia also found a doubling of coagulation levels in bal fluid over the first few days [ , ] . while inconclusive, our findings raise the possibility that nebulised heparin, at higher doses, limited coagulation activation in the lungs. nebulised heparin did not increase t-pa levels in bal fluid for any of the doses studied. one of the strengths of the present study was the nebulisation system used. the evidence of a dose-related effect on systemic aptt and tct levels suggested significant amounts of heparin reached the alveolar spaces. this finding is consistent with previous studies [ ] . another strength of the study was the inclusion of genuinely high-risk patients with ali. the aver-age p a o /f i o ratio at baseline was mmhg, the tracheostomy rate was % and the hospital mortality was %. limitations of the present study included the absence of a control group, the small number of patients enrolled and the relatively short time ( days) over which heparin was nebulised. the size of the study reflected the need for caution, as nebulised heparin had not previously been administered to patients with ali. furthermore, we determined that four patients in each group would provide adequate power to detect a major anticoagulant effect. our study was consequently too small to draw conclusions regarding efficacy or potential infrequent deleterious effects. previous studies in animal models of ali have demonstrated significant improvements in pulmonary function with inhaled heparin and other glycosaminoglycans [ , , ] . heparin has a range of anticoagulant actions and also promotes fibrinolysis through increased t-pa levels [ ] [ ] [ ] [ ] . compared with the intravenous route, nebulisation delivers high concentrations of heparin to the alveolar space with a reduced risk of adversely effecting systemic coagulation. administration of nebulised heparin to mechanically ventilated patients with ali is feasible. the heparin administration was not associated with any serious adverse events, and increased aptt levels at higher doses. larger trials are required to further investigate the safety and efficacy of nebulised heparin in ali. in these trials, due consideration must be given to systemic anticoagulant effects. (page number not for citation purposes) ies had no role in the study design, data collection, analysis and interpretation of the data or in the writing and publication of the manuscript. incidence and outcomes of acute lung injury incidence and mortality of acute lung injury and the acute respiratory distress syndrome in three australian states the acute respiratory distress syndrome definitions, mechanisms, relevant outcomes, and clinical trial coordination pathophysiology of the respiratory distress syndrome lung pathology of fatal severe acute respiratory syndrome case records of the massachusetts general hospital. weekly clinicopathological exercises. case - disseminated intravascular coagulation in newborn infants. prevalence in autopsies and significance as a cause of death the pulmonary vascular lesions of the adult respiratory distress syndrome intravascular coagulation associated with the adult respiratory distress syndrome pulmonary pathology in acute respiratory insufficiency: lung biopsy as a diagnostic tool the microembolism syndrome pathological manifestations of septic shock infection and disseminated intravascular coagulation heparin reverses the procoagulant properties of stimulated endothelial cells neri serneri gg: tissue factor reduction and tissue factor pathway inhibitor release after heparin administration tissue factor and plasminogen activator inhibitor type expression in human stimulated monocytes is inhibited by heparin the effect of heparin and other glycosaminoglycans on levels of tissue plasminogen activator and plasminogen activator inhibitor in cultured human umbilical vein endothelial cells heparin nebulization attenuates acute lung injury in sepsis following smoke inhalation in sheep prevention of bleomycin-induced lung fibrosis by aerosolization of heparin or urokinase in rabbits elevated pulmonary dead space and coagulation abnormalities suggest lung microvascular thrombosis in patients undergoing cardiac surgery pulmonary dead-space fraction as a risk factor for death in the acute respiratory distress syndrome effect of inhaled heparin on lung function and coagulation in healthy volunteers anticoagulant effects and tissue factor pathway inhibitor after intrapulmonary low-molecular-weight heparin endothelial sequestration of heparin administered by the intrapulmonary route clinical and hemostatic responses to treatment in ventilator-associated pneumonia: role of bacterial pathogens poll t van der: local activation of coagulation and inhibition of fibrinolysis in the lung during ventilator associated pneumonia lung deposition and clearance of inhaled ( m)tc-heparin in healthy volunteers the effects of aerosolized dextran in a mouse model of pseudomonas aeruginosa pulmonary infection the present study was supported by the st vincent's hospital research endowment fund and the intensive care foundation. the funding bod- the authors declare that they have no competing interests. bd designed the study, collected the data, performed the statistical analysis and drafted the manuscript. jds and djc participated in its design, and coordinated and helped to draft the manuscript. all authors read and approved the final manuscript. key: cord- - gbjdr u authors: fu, lin; fei, jun; xu, shen; xiang, hui-xian; xiang, ying; tan, zhu-xia; li, meng-die; liu, fang-fang; li, ying; han, ming-feng; li, xiu-yong; zhao, hui; xu, de-xiang title: acute liver injury and its association with death risk of patients with covid- : a hospital-based prospective case-cohort study date: - - journal: nan doi: . / . . . sha: doc_id: cord_uid: gbjdr u background: coronavirus disease (covid- ) is a newly respiratory infectious disease caused by severe acute respiratory syndrome coronavirus- (sars-cov- ) with multiple organ injuries. the aim of this study was to analyze sars-cov- -induced acute liver injury (ali), its association with death risk and prognosis after discharge. methods: three-hundred and fifty-five covid- patients were recruited. clinical data were collected from electronic medical records. ali was evaluated and its prognosis was tracked. the association between ali and death risk was analyzed. results: of covid- patients, were common, severe, and critical ill cases, respectively. on admission, ( . %) patients were with hypoproteinemia, ( . %) with cholestasis, and ( . %) with hepatocellular injury. as expected, ali was more common in critical ill patients. by multivariate logistic regression, male, older age and lymphocyte reduction were three important independent risk factors predicting ali among covid- patients. death risk analysis shows that fatality rate was higher among patients with hypoproteinemia than those without hypoproteinemia (rr= . , p< . ). moreover, fatality rate was higher among patients with cholestasis than those without cholestasis (rr= . , p< . ). follow-up observation found that more than one hepatic functional indexes of two-third patients remained abnormal days after discharge. conclusions: ali at early stage elevates death risk of covid- patients. sars-cov- -induced ali has not recovered completely days after discharge. onset, admission and death were recorded. the onset time was defined as the date when patients' any symptom and sign were found. patient's pharyngeal swab specimens were collected for extraction of sars-cov- rna. real-time rt-pcr was used to detect viral nucleic acid using a covid- nucleic acid detection kit following experimental instructions (shanghai the clinical characteristics of covid- patients were analyzed. as shown in table , common case, defined as oxygenation index higher than , was . %. for severe case, whose oxygenation index was from to , was . %. for critically ill case, whose oxygenation index was lower than , was . % ( table ). the demographic characteristics of covid- patients were then analyzed. of covid- patients, males accounted for . % and females accounted for . % (table ). there were patients younger than , cases aged between and , and patients older than ( table ). as shown in table , patients were with diabetes, with hypertension and with hepatic diseases. finally, blood lymphocytes were analyzed among covid- patients. as shown in table , . % ( / ) patients were with lymphopenia. the association between the severity and ali was analyzed among covid- patients. as shown in table , all hepatocellular injury markers, including total bilirubin, direct bilirubin, indirect bilirubin, alt and ast, were higher in critically ill patients than those of common cases. by contrast, total protein, albumin and albumin/globulin ratio, three markers of hypoproteinemia were lower in critically ill patients than those of common cases. despite of no difference on serum tba, alkaline phosphatase and glutamyl transferase, two markers of cholestasis, were higher in critically ill patients than those of common cases. the association between oxygenation index and hepatic functional indexes was analyzed. as shown in figure , there was a weak negative correlation between oxygenation index and alt, ast, ast/alt, and glutamyl transferase. by contrast, there was a weak positive correlation between oxygenation index and albumin among covid- patients ( figure ). . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint the effects of demographic characteristics on hepatic functional indexes were analyzed. as shown in table , the levels of direct bilirubin, indirect bilirubin, alt, alkaline phosphatase and glutamyl transferase were higher in males than in females. by contrast, the level of albumin was lower in males than in females. further analysis showed that the levels of total bilirubin, alt, alkaline phosphatase and glutamyl transferase were higher in patients older than than those of younger patients. by contrast, the level of albumin was lower patients older than than those of younger patients ( table ). the effects of comorbidity on hepatic functional indexes were then analyzed. as shown in table , alkaline phosphatase was slightly increased in covid- patients with hypertension as compared with those without hypertension. further analysis showed that the levels of alkaline phosphatase and glutamyl transferase were slightly increased in covid- patients with diabetes as compared with those without diabetes. by contrast, the level of albumin was slightly decreased in covid- patients with diabetes as compared with those without diabetes. of interest, there was no significant association between hepatic functional indexes and comorbidity with liver disease (table ) . finally, the association between blood lymphocytes and ali was analyzed among covid- patients. as shown in table , the levels of alt and glutamyl transferase were higher in covid- patients with lymphopenia than those without lymphopenia. by contrast, the levels of albumin and globulin were lower in covid- patients with lymphopenia than those without lymphopenia ( cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . hypoproteinemia, and . ( % cl: . , . ; p= . ) for hepatocellular injury, respectively (supplemental table ). the association between comorbidities and ali was analyzed among covid- patients. the ors of diabetes were . ( % cl: . , . ; p= . ) for cholestasis, . ( % cl: . , . ; p= . ) for hypoproteinemia, respectively. no significant relationship was observed between ali and comorbidity with hypertension and hepatic disease among covid- patients (supplemental table ). the effects of ali at the early stage on death risk are presented in table . among covid- patients with cholestasis, . % were died. the fatality rate was higher among covid- patients with cholestasis than those without cholestasis ( . % vs . %; rr= . , % cl: . , . ; p< . ). as shown in table , there was no significant association between hepatocellular injury and death risk among covid- patients. the prognosis of sars-cov- -induced ali was tracked days after discharge. as shown in table , no significant change was observed between hepatic functional indexes on days after discharge and those on admission. although the percentage of patients with hypoproteinemia was lower on days after discharge than on admission ( . % vs . %, p< . ) (supplemental table ), . % albumin, . % pre albumin and . % albumin/globulin ratio remained below normal range ( table ). table , the percentage of patients with serum alt elevation was higher on days after discharge than on admission ( . % vs . %, p< . ). in addition, patients with serum ibil elevation was higher on days after discharge than on admission ( . % vs . %, p< . ). no significant difference on the percentages of . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . patients with abnormal value for direct bilirubin, tba, alkaline phosphatase, glutamyl transferase and ast was observed when admission and days after discharge (table ). further analysis showed that no significant difference on the percentages of patients with cholestasis and hepatocellular injury was observed when admission and discharge, while the percentages of patients with hypoproteinemia was significantly reduced on days after discharge as compared with on admission (supplemental table ). the present study aimed to analyze sars-cov- -induced ali, its association with death risk and the prognosis after discharge. the major findings of this study include: ( ) ali is more common in the critically ill covid- patients; ( ) accumulating data demonstrated that sars-cov- infection caused multiple organ injuries, including myocardial dysfunction, lymphopenia and even acute renal these results provide evidence that ali on admission is associated with the severity of covid- patients. several studies found that elderly covid- patients had more severe symptoms and signs than younger cases [ , ] . the present study analyzed the influence of gender and age on sars-cov- -induced ali. we showed that the levels of serum total bilirubin, alt, alkaline phosphatase and glutamyl transferase were higher in males than in females. by contrast, serum albumin level was lower in males than in females. further analysis showed that total bilirubin, alt, alkaline phosphatase and glutamyl transferase were higher in older patients than younger ones. by contrast, albumin and globulin were lower in older patients than younger ones. according to several clinical reports, covid- patients with comorbidities had worse prognosis [ , ] . indeed, this study found that . % covid- patients were with diabetes, . % with hypertension, and . % with liver disease. to explore the influence of comorbidities on ali, the present study analyzed hepatic functional indexes among different groups. our results showed that alkaline phosphatase was slightly higher in covid- patients with hypertension than those without hypertension. in addition, alkaline phosphatase and glutamyl transferase were slightly higher in patients with diabetes than those without diabetes. by contrast, albumin was slightly lower in patients with either diabetes or hypertension than those neither diabetes nor hypertension. unexpectedly, comorbidity with liver disease did not influence hepatic functional indexes of covid- patients. lymphocytopenia is one of the important early manifestations during the pathogenesis of covid- [ , ] . this study analyzed the influence of lymphocytopenia on sars-cov- -induced ali. we found that almost all hepatic functional indexes were worse among patients with lymphocytopenia than without lymphocytopenia. to exclude potential confounding factors, multivariable logistic regression was used to further analyze the impact of gender, age and comorbidities on sars-cov- -induced ali. we found that male, older age, comorbidity with diabetes and lymphocytopenia . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . were independent risk factors of cholestasis. in addition, older age, lymphocytopenia and comorbidity with diabetes were independent risk factors of hypoproteinemia. moreover, male and lymphocytopenia were independent risk factors of hepatocellular injury. taken together, the present study provide evidence that male, older age, comorbidity with diabetes and lymphocytopenia are major risk factor of ali among covid- patients. the influence of ali on the prognosis of covid- is unclear. the present study analyzed the impact of ali on death risk of covid- patients. our results showed that the fatality rate was higher in covid- patients with hypoproteinemia than those without hypoproteinemia. moreover, the fatality rate was higher in covid- patients with cholestasis than without cholestasis. our results suggest that hypoproteinemia and cholestasis at the early stage elevate death risk of covid- patients. it is especially interesting whether sars-cov- -induced ali recovers in a short time after discharge. in the present study, covid- patients were followed up and measured hepatic functions days after discharge. unexpectedly, no significant difference on the values of serum direct bilirubin, indirect bilirubin, tba, alkaline phosphatase, glutamyl transferase and ast was observed when admission and days after discharge. although serum albumin level was rebounded on days after discharge, % covid- patients remained below normal range. our results indicate that hepatic functional indexes of two-third covid- patients remain abnormal days after discharge. therefore, further follow-up is required to further evaluate whether sars-cov- infection causes permanent liver injury. the mechanism through which sars-cov- evokes ali remains unclear. accumulating data indicate that sars-cov- infection causes multiple organ injuries, such as lymphopenia, myocardial dysfunction and acute renal failure [ , [ ] [ ] [ ] ] . in the present study, we showed that oxygenation index, an index of respiratory function, was positively correlated with serum albumin. by contrast, there was a weak negative correlation between oxygenation index and alt, ast, ast/alt, and glutamyl . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . transferase among covid- patients. these results suggest that respiratory failure may contribute, at least partially, to sars-cov- -induced ali. several studies demonstrated that ace , as a receptor for sars-cov- , was expressed in cholangiocytes and hepatocytes [ , ] . therefore, this study does not exclude that sars-cov- evokes ali partially through infecting liver. it is required to further explore whether human liver is another target of sars-cov- injection. in summary, this study aimed to investigate sars-cov- -induced ali among covid- patients from two hospitals. our results revealed that sars-cov- -induced ali was more common in critically ill patients. in addition, male elderly covid- patients with diabetes mellitus and lymphopenia were more susceptible to ali. we provide evidence that ali at the early stage elevates death risk of covid- patients. importantly, sars-cov- -induced ali has not recovered completely days after discharge. therefore, it is necessary to further evaluate whether sars-cov- infection causes permanent liver injury. we thank all members of respiratory and critical care medicine in the second . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . https://doi.org/ . / . . . doi: medrxiv preprint . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. . cc-by-nc-nd . international license it is made available under a author/funder, who has granted medrxiv a license to display the preprint in perpetuity. is the (which was not peer-reviewed) the copyright holder for this preprint . table . the association between the severity and hepatic function markers among covid- patients. table . hepatic function markers on admission and after discharge among covid- patients. the continuing -ncov epidemic threat of novel coronaviruses to global health -the latest novel coronavirus outbreak in wuhan, china severe acute respiratory syndrome coronavirus (sars-cov- ) and coronavirus disease- (covid- ): the epidemic and the challenges clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically-proven protease inhibitor structure, function, and antigenicity of the sars-cov- spike glycoprotein structural basis for the recognition of the sars-cov- by full-length human ace epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study cardiovascular implications of fatal outcomes of patients with coronavirus disease (covid- ) clinical and immunologic features in severe and moderate coronavirus disease clinical characteristics of deceased patients with coronavirus disease : retrospective study clinical characteristics of imported cases of covid- in jiangsu province: a multicenter descriptive study hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china multiple organ infection and the pathogenesis of sars liver-targeted angiotensin converting enzyme therapy inhibits chronic biliary fibrosis in multiple drug chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme cases in age, compared with "＜ " group, *p< . , **p< . ; compared with " - " group, ¶ p< . , ¶ ¶ p< . . cases in hypertension, compared with "yes" group, *p< . , **p< . . cases in diabetes key: cord- - hx kc authors: movia, dania; prina-mello, adriele title: preclinical development of orally inhaled drugs (oids)—are animal models predictive or shall we move towards in vitro non-animal models? date: - - journal: animals (basel) doi: . /ani sha: doc_id: cord_uid: hx kc simple summary: this commentary focuses on the methods currently available to test the efficacy and safety of new orally inhaled drugs for the treatment of uncurable respiratory diseases, such as chronic obstructive pulmonary disease (copd), cystic fibrosis or lung cancer, prior to entering human experimentation. the key question that the authors try to address in this manuscript is whether there is value in using and refining current animal models for this pre-clinical testing, or whether these should be relinquished in favor of new, more human-relevant non-animal methods. abstract: respiratory diseases constitute a huge burden in our society, and the global respiratory drug market currently grows at an annual rate between % and %. inhalation is the preferred administration method for treating respiratory diseases, as it: (i) delivers the drug directly at the site of action, resulting in a rapid onset; (ii) is painless, thus improving patients’ compliance; and (iii) avoids first-pass metabolism reducing systemic side effects. inhalation occurs through the mouth, with the drug generally exerting its therapeutic action in the lungs. in the most recent years, orally inhaled drugs (oids) have found application also in the treatment of systemic diseases. oids development, however, currently suffers of an overall attrition rate of around %, meaning that seven out of new drug candidates fail to reach the clinic. our commentary focuses on the reasons behind the poor oids translation into clinical products for the treatment of respiratory and systemic diseases, with particular emphasis on the parameters affecting the predictive value of animal preclinical tests. we then review the current advances in overcoming the limitation of animal animal-based studies through the development and adoption of in vitro, cell-based new approach methodologies (nams). respiratory diseases constitute a huge burden in our society. it has been calculated that, worldwide, around million people are living with asthma [ ], million with chronic obstructive pulmonary disease (copd) [ ] , and more than , people with cystic fibrosis [ ] . furthermore, million people are affected by idiopathic pulmonary fibrosis (ipf) [ ] , and million people contract tuberculosis (tb) annually [ ] . in addition to this, lung cancer continues to be the leading cause of cancer death worldwide, accounting for . million deaths in [ ] ; whereas, pneumonia still constitutes the inhalation is the preferred administration method for treating respiratory diseases [ ] , as: (i) it delivers the drug directly at the site of action, resulting in a rapid therapeutic onset with considerably lower drug doses, (ii) it is painless and minimally invasive thus improving patients' compliance, and (iii) it avoids first-pass metabolism, providing optimal pharmacokinetic conditions for drug absorption and reducing systemic side effects [ ] [ ] [ ] . it should be noted here, inhalation differs from intranasal administration for the drug portal-of-entry (poe) and targeted site of action. intranasal drugs are sprayed into the nostrils, producing a local effect in the nasal mucosa; whereas, inhalation occurs through the mouth, with the oids, also referred to as orally inhaled drug products (oips), having their efficacy in the lungs. notably, attempts have been made to develop oids that exert their therapeutic action outside the lung, for the treatment of systemic diseases [ ] . the latter include, for example, migraine headaches, treated with aerosols of ergotamine or hydroxyergotamine, and type /type diabetes, for which inhaled insulin products have been developed (e.g., exubera-withdrawn in due to poor revenue-and afrezza-the uptake of which has also been impacted by socio-economic issues). oid therapeutic categories currently approved for the clinical treatment of respiratory diseases include drugs for the treatment of asthma and copd, such as β adrenergic agonists (e.g., albuterol, formoterol) and muscarinic antagonists (e.g., ipratropium, tiotropium) inducing bronchodilation, or glucocorticosteroids (e.g., fluticasone and budesonide) reducing inflammation. oids for the treatment of cystic fibrosis are also available for clinical use, with most of them falling into the therapeutic categories of mucolytics (e.g., saline and acetyl choline), aiming at thinning the mucus for facilitating its clearance from the patient's lungs. alternatively, leukocyte dnase, reducing inflammation, and antimicrobial agents (e.g., tobramycin), treating the bacterial infection characteristic of this disease, are also administered as oids. various devices can be used to administer oids to patients, including dry-powder inhalers (dpis), pressurized metered-dose inhalers (pmdis) and nebulizers. these devices have been extensively discussed in several recent works [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . briefly, dpis deliver powder particles carrying the drug; pmdis and nebulizers generate liquid droplets containing the drug. to be effective, an inhalation device must be easy to use and forgiving of poor patient's compliance, while providing reproducible effective dosing. thus, a through characterization of the performance of the inhalation device is required at regulatory level, when developing an oid. such characterization is based on in vitro, ex vivo and in vivo (on human volunteers) tests, as extensively described in the scientific literature [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] animal models are not used in the characterization of the efficiency and reproducibility of inhalation delivery devices. this is due to the fact that, dpis and pmdis are breath-actuated and therefore not compatible with animal exposure; whereas for nebulizers modifications are needed in line with the animal model adopted. thus, our manuscript, which focuses on the potential reduction and replacement of animals studies in oid development, does not discuss the impact of inhalers' performance on the effectiveness of inhalation therapies [ ] , a current challenge discussed in detail elsewhere [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . despite the major advantages over i.v. administration of drugs, inhalation therapy encounters several obstacles in achieving an effective therapeutic dose for the successful treatment of respiratory and/or systemic diseases. below, we describe the journey of an oid once administered and the human-specific features that, in the authors' opinion, strongly impact on the current low translation rate of oids, as these are poorly replicated in the current preclinical models. when an oid is administered to a patient, its liquid or powder aerosol enters the human respiratory system via the oropharynx. oid deposition in the oropharynx is invariably wasteful, reducing the oid dose reaching the lungs. this indeed constitutes the first feature to keep into account for developing an effective inhalation therapy [ ] . rodent models cannot reproduce this feature, as they are obliged nose-breathers. however, other animal models (e.g., dogs) can be used to overcome the limitations posed by rodents. also, oid deposition in the oropharynx must be minimized in clinics to avoid severe side-effects in the patients. side-effects can be due to both local and systemic toxicity, as oids accumulating in the mouth and throat enter the body through swallowing. achieving an optimal oid deposition pattern in the patients' lung is the second feature to keep into account for an effective inhalation therapy [ ] . to reach its site of action and/or absorption, the oid needs to pass through the so-called extrathoracic (or et) region of the larynx, enter the tracheobronchial region and reach the small and/or peripheral (alveoli) airways. drug absorption and translocation into the blood flow can in fact occur from all parts of the lung, but it occurs more readily in the alveoli [ ] , where there is a large surface area and a relatively thin layer of epithelial and endothelial cells separating the inhaled drug from the blood flow. the oid journey within the complex, branched structure of the human lung is influenced by two main parameters of the particles/droplets carrying the drug [ ] : (i) velocity [ ] ; and (ii) aerodynamic size distribution (the so-called apsd) [ ] . both parameters strongly impact on the drug deposition pattern and, subsequently, on the effectiveness of the inhalation therapy. velocity is defined by the delivery system employed in the oid administration. generally, high velocity results in increased deposition in the oropharynx and tracheobronchial regions; whereas, low velocity generates a peripheral deposition pattern [ ] . it goes without saying that oids cannot reach those part of the respiratory tract where velocity is null, i.e., those parts of the lung that are not ventilated. this is particularly relevant to consider when developing oids against respiratory diseases [ ] , which are characterized by the partial or full obstruction of the respiratory tract (e.g., asthma, copd, cystic fibrosis and lung cancer). combination of drugs where bronchodilators or mucolytics are used in a synergistic manner with other drug therapies, can be used to modulate oid velocity and increase the efficacy of the inhalation therapy. in parallel, the deposition mechanism of the aerosol particle/droplets in the bronchial tree changes depending on their apsd [ ] . droplets/particles with large aerodynamic size deposit by impaction or interception mechanisms in the oropharynx or just beyond the trachea bifurcation. the smaller droplets/particles deposit in the smaller airways by sedimentation, subject to gravity. among those, the droplets/particles with aerodynamic size below µm further move to the alveoli by diffusion or brownian motion. it should be noted here, droplets/particles deposition follows stokes' law [ ] . the consequence is that, since most of the droplets/particles are near spherical, their aerodynamic size can be small despite being geometrically large. this happens when particles/droplets have low density, which is determined by the composition of the oid formulation. oid deposition pattern is currently evaluated in in vitro, cell-free experiments, achieving good predictive value [ ] . once the oid deposits on the airways, removal mechanisms, such as mucociliary clearance in the conducting airways and macrophage clearance in the alveolar space, can be responsible for the drug elimination and/or degradation [ ] , thus hindering the local efficacy and/or the systemic absorption of the oid. mucociliary clearance is the upward movement of mucus driven by beating cilia towards the pharynx, where mucus is subsequently swallowed and pass into the gastrointestinal tract [ ] . in macrophage clearance, the oid is phagocytosed by alveolar macrophages and cleared by transport to the lung-draining lymph nodes [ , ] . compared with mucociliary clearance, macrophage clearance is far slower [ ] and, therefore, its action is typically assumed to be negligible for oids, unless the drug is known to be degraded by alveolar macrophages [ ] . absorptive drug clearance is yet another clearance mechanism by which an oid is cleared from the lung through the blood circulation, a mechanism that is heavily dependent on perfusion. perfusion levels, however, vary between the different lung regions. in the alveoli, perfusion levels are the highest and drugs have a very short half-life; by contrast, in the tracheobronchial region, the perfusion rate is lower, thus offering a longer drug bioavailability [ ] . removal mechanisms constitute the third feature to keep into account for developing an effective inhalation therapy. as described in detail in section . . , this feature is species-specific [ ] and, therefore, human-specific removal mechanisms are not replicated by animal models. notably, human-specific removal mechanisms can be reproduced by in vitro, cell-based nams [ ] [ ] [ ] [ ] , as discussed in detail in section . . to exert local or systemic efficacy, oid dissolution and absorption are indeed necessary [ ] . the thickness and constitution of the pulmonary lining fluid, which can be modified by lung diseased states [ ] , influence oid dissolution and, subsequently, absorption [ ] , constituting the fourth feature to keep into account for developing an effective inhalation therapy. while the mucus layer (produced by goblet cells in the bronchial region) acts as a physical barrier, surfactants produced by alveolar cells in the peripheral airways reduce surface tension and facilitate drug dissolution [ ] . noteworthy, oid dissolution rates strongly depend on disease-specific airway characteristics (e.g., copd is characterized by a thick mucus, hindering oid efficacy), which are not replicated by conventional preclinical models. noteworthy, in vitro, cell-based nams have the potential to reproduce the disease-specific composition of pulmonary lining fluid [ ] . finally, the multicellular composition of the lung is the fifth feature to keep into account for developing an effective inhalation therapy, by playing an important role in defining oid delivery efficiency. for example, mast cells have protective functions against inhaled drugs; dendritic cells, together with macrophages, are the first line of defense of the lung immune system, sampling for and removing constantly any exogenous material such as drugs. clara cells are involved in oid metabolism. interestingly, the human lung has relatively low metabolic activity as compared to the gastro-intestinal tract or the liver [ , ] . this constitutes a distinct advantage for inhalation therapy over oral drug administration. however, protease activity is generally increased in lung diseases as a result of chronic inflammation (e.g., enhanced activity of cytochrome p in patients affected by lung cancer [ , ] or copd [ ] ); this can indeed reduce the biopersistence and bioavailability of some oids (e.g., insulin [ ] ). protection against metabolic activity has been achieved in inhalation therapy by drug encapsulation into carriers (e.g., liposomes [ ] [ ] [ ] [ ] [ ] ). animal models and humans differ in the metabolism and distribution/types of cell populations lining the airways. for example, it has been shown that the average number of cells per alveolus for rats versus humans is: vs. , for endothelial cells, vs. for interstitial cells, vs. for epithelial type ii cells, vs. for epithelial type i cells, and . vs. for alveolar macrophages [ ] . this has important clinical implications during the oid development. notably, the human-specific composition and metabolism of the lung can indeed be replicated more closely by adopting in vitro, cell-based nams, as described in the following sections. based on the multiple mechanisms and processes described above, it is evident that oid development is not an easy task. overall, a sound understanding of the features involved in the oid journey is necessary to use the most predictive preclinical models to overcome the complex, intrinsic challenges associated with inhalation therapy. interestingly, such challenges have certainly not hindered the interest of the pharmaceutical industry in inhalation therapy. based on a search carried out by the authors in july , inhalation clinical trials for new, combination, and existing products, encompassing drug interventions, different conditions and rare diseases, have been logged on clinicaltrials.gov in the last four years (search terms: interventional studies; inhalation; start date from / / to / / ). to put this into context, a total of , interventional studies, comprising drug interventions, have been registered on clinicaltrials.gov in the same time period. consequently, inhalation clinical trials make for the . % of the total number of interventional studies registered in the time period under consideration ( - ), and . % of the total drug interventions examined. it is important to observe that more than half of these inhalation studies are for systemic conditions, thus demonstrating an interest that expands beyond the domain of respiratory diseases. preclinical studies of new oid candidates generally start from compound profiling in high-throughput in vitro studies [ ] . compounds with promising efficacy results progress to in vivo studies. three preclinical animal-based studies are currently required by regulatory authorities before approving the request of clinical study for a novel oid. these are: (i) the range finding study, (ii) the repeat dose study, and (iii) the carcinogenicity study. other specialized studies can be necessary, such as safety pharmacology studies, reproductive studies, and neonatal/juvenile studies for pediatric oids. animal-based inhalation studies are carried out mainly in rats, mice or rabbits by exposure in restraint tubes [ ] . dogs and primates can also be used for testing oids in more realistic settings, via facemasks or helmets [ ] . although high-throughput cell-based assays can provide insightful information at the early stages of preclinical development, the cell models used fall short in recapitulating the complex interactions between different cell types and tissues/organs occurring in human. conventional in vitro models are in fact formed by one cell type grown as a flat, two-dimensional culture; thus, they are a simplistic representation of the human lung tissue [ ] . furthermore, many in vitro assays use transformed cell lines that exhibit gene and protein expression that strongly differ from their primary counterpart [ ] . on the other hand, various uncertainties characterize the animal-based preclinical studies currently required for regulatory purposes. the first level of uncertainty is associated with the type of devices used to administer the oid to the animal. while clinical nebulizers can be used in the preclinical environment (upon small modifications), dpis and pmdis cannot be employed to expose animal models at the preclinical screening level, as these devices are breath actuated. to overcome this issue, specialized equipment is used to expose the animal to an aerosol in a restrained environment. aerosol of powders is achieved via, for example, rotating brush generators or wright dust feed. an algorithm-based extrapolation [ ] is then applied to define dose ranges to be used in clinical trials. the delivered dose is calculated as the amount of oid per unit of body weight that is presented to the animal. due to the two parameters (velocity and aerodynamic size distribution) affecting oid deposition patterns in the lungs, as discussed in the section above, and to the species of the animal model used, the deposited dose is only a fraction of the delivered dose. the fda assumes % deposition in humans, % in rats and % in dogs or non-human primates, irrespective of any information that has been produced by the submitting company [ ] . this indeed generates uncertainties when calculating clinical overages. the second level of uncertainty in in vivo studies is posed by the animal model itself [ ] . for example, rodents are obligate nose breathers; this strongly influences how inhaled compounds deposit in the respiratory tract. this and other interspecies differences have been extensively discussed by the authors in a recent perspective [ ] . preclinical studies during oid development requires a clear understanding of such interspecies differences and their impact on the screening outcomes in terms of oid efficacy, toxicity and recovery from adverse effects. although not required at regulatory level, disease animal models are also used in preclinical research, particularly in the oncological field, as proof of concept for demonstrating oid efficacy. the authors have performed a literature search on pubmed using the searching terms "(inhaled drug) and (in vivo) and (efficacy)". the search results showed that, in the last five years, articles used disease animal models to test the efficacy of oids. however, animal use as disease models needs to be viewed cautiously. in animal models, disease features are reproduced by applying exogeneous stimuli (e.g., allergens, irritant gas exposures, cigarette smoke, etc.) [ ] . this modelling process is however incomplete, as the use of single stimuli does not mimic the disease etiology and chronicity observed in patients. the next section of this commentary focuses on this specific aspect, complementing the authors' previous publication [ ] and further discussing if and how new approach methodologies (nams) could become useful in the attempt to overcome the limitations of current animal models and increase oid translation rate. for completeness, it should be mentioned here that the abbreviation "nams" is often used in toxicology to refer broadly to any non-animal technology, methodology, approach, or combination thereof that can be used to provide information on chemical hazard and risk assessment. examples of nams include non-mammalian model systems, (e.g., caenorhabditis elegans [ ] [ ] [ ] , drosophila melanogaster [ ] [ ] [ ] , zebrafish [ ] [ ] [ ] and dictyostelium [ ] ) and computational (in silico) approaches [ ] , which indeed offer opportunities for mimicking human respiratory diseases in a predictive manner. however, the scope of the nams considered in our commentary includes only in vitro, non-animal cell models for the testing of oids. based on the most recent advances in tissue-engineering technologies, in vitro cell-based nams for screening the efficacy of oids can be classified in three main categories [ ] : (i) tissue-mimetic lung cultures grown at the air-liquid interface (ali); (ii) lung organoids; and (iii) lung-on-chip. ali cultures mimic one of the main properties of the lung epithelium, i.e., the direct contact with the gas phase (air). this provides a tissue-mimetic environment that makes it possible for airway epithelial cells to proliferate and differentiate in vitro into a pseudostratified, ciliated epithelium that produces mucus. thus, ali cultures provide an excellent method for testing oid dissolution and absorption, while enabling testing of the drug in its aerosol form. whitcutt et al., were among the first research groups to report mucociliary differentiation in ali cultures [ ] . today, ali cultures are known to be particularly useful in understanding the mechanisms of respiratory diseases, including the cell-cell and cell-extracellular matrix interactions during airways remodeling [ ] [ ] [ ] . also, they can replicate some of the key features that need to be kept into account when developing an inhalation therapy, namely (i) the constitution and thickness of the pulmonary lining fluid [ ] and (ii) mucociliary clearance [ ] [ ] [ ] . for example, ali cultures have been used to model the effects of smoke exposure on epithelial cells [ ] and the authors have created a complex, diseased ali culture model capable of reproducing the chemoresistance mechanisms observed in patients affected by non-small-cell lung cancer [ , ] . also, culturing human airway epithelial cells isolated from patients, makes it possible to conduct patient-specific research and drug-screening, for example in cystic fibrosis, asthma and copd [ ] [ ] [ ] [ ] . with the aim of further increasing the predictive value of this in vitro nam, ali co-cultures have also been developed. in ali co-cultures, the lung cell populations are mixed or partially separated, depending on the experimental set-up. in general, the immune cells are cultured in direct contact with the epithelial cells; whereas, fibroblasts and endothelial cells are separated from the epithelial cells by the transwell permeable membrane. cell separation is due to the relative difference in the culturing conditions of the various cell types and the consequent need to separate them. this constitutes one of the main limitations of ali models, as separated cells cannot establish physical (cell-to-cell) interactions as per in vivo conditions. this indeed affects the detected responses during oid preclinical testing. the second type of in vitro, cell-based nams currently available for oid testing are lung organoids. these are grown from human induced pluripotent stem cells (ipscs) cultured within a natural or synthetic extracellular matrix to form three-dimensional ( d), hollow cell spheroids of basal, ciliated and secretory cells [ ] . through differentiation and self-organization of the ipscs, an in vitro culture with lung tissue-specific morphogenetic and histological properties is formed [ ] . to date, several organoids representative of the various human lung regions [ ] and assessing a variety of pulmonary diseases [ , , ] have been developed. in the context of oid preclinical testing, lung organoids can be used for modeling respiratory diseases and, therefore, as a platform for screening the efficacy of inhalation therapies [ , ] . indeed, technical limitations are inherent with the use of lung organoids. lungs are in fact subjected to mechanical deformation during breathing cycles, a deformation that is currently hard to model in organoids. furthermore, there is still a lack of established in vitro lung organoids with a functional representation of the vasculature network. most importantly, lung organoids lack an important feature for oid testing, i.e., the direct contact of epithelial cells with the air. as mentioned above, lung organoids are spherical cultures. they present an interiorized lumen, with epithelial cells facing inwards rather than outwards; this makes drug administration extremely difficult and reduces the application of organoids in the screening of oid absorption. microfluidic technologies allow to add further complexity and functionality to the in vitro ali models described above. the so-called "lung-on-chip" is a microfluidic-based in vitro system in which lung epithelial cells are grown on one side of a membrane, and stromal cells on the other surface. liquid and air are circulated through the system to mimic air and blood flow in the lung. the applications of lung-on-chip range from basic research to drug discovery [ ] , where the oid can be introduced in the air flow as per in vivo conditions. probably the most famous example of this in vitro, cell-based nam is the breathing lung-on-chip developed by huh and co-workers at the wyss institute of harvard university (usa), capable of reproducing both the physiological and pathological responses of the human lung, a rudimentary circulatory system and the mechanical stress associated with breathing [ ] [ ] [ ] . the immediate application of lung-on-chip has been for toxicity testing [ , ] ; more recently, this model has been exploited for improving understanding of the complex lung disease processes and their responses to therapeutics [ ] [ ] [ ] , with applications extending even to the most recent need of a fast drug discovery for covid- treatment [ ] . lung-on-chip systems allow, in fact, the in vitro creation of highly tissue-mimetic lung disease models [ , ] , thus allowing, for example, to model the human response and the effects of existing and novel therapeutics when the lung is infected by the influenza virus or by viral pseudoparticles expressing spike protein of sars-cov- , the virus responsible for covid- development [ ] . the clear advantage of lung-on-chip systems over ali cultures or lung organoids is the possibility of mimicking the pulmonary mechanical stretch during in-and exhalation, while replicating the air-blood barrier for studying oid absorption. furthermore, lung-on-chip models allow evaluating the impact of the mucociliary clearance mechanism overcoming the lack of directionality in cilia beating function characteristic of fully-differentiated in vitro ali models [ ] . nevertheless, the lung-on-chip models share some of the limitations of ali cultures, i.e., the impairment of physical crosstalk among different cell types. in fact, even in the most recent and advanced developments in "tumor-on-a-chip" cell culture technology, successfully used to create in vitro human orthotopic models of non-small-cell lung cancer [ ] , the lung cancer cells (cultured under ali conditions) are physically separated from the lung endothelial cells by a porous, permeable membrane [ ] . it is noteworthy to mention that, in the respiratory disease field, two additional categories of in vitro, cell-based nams exists, although these have not been used for oid testing to date. the first category is constituted by explant or ex vivo cultures, namely isolated perfused lungs and precision cut lung slices. these are better representations of the in vivo situation than any of the previous three nam types mentioned above. the use of ex vivo cultures in oid testing is however hindered by the hurdles associated with their manipulation, and by donor-specific differences that make the oid screening outcomes often not significant or difficult to interpret [ ] . the second category includes the engineered, reconstructed lung organs [ ] . these are formed from several cell types co-cultured within scaffolds that aim at replicating the composition and architecture of the human lung acellular stroma [ ] . mechanical or biochemical stimuli can be added to tailor the properties of the scaffold and increase the similarity to the lung stroma in vivo. the first engineered lung organ was built from a decellularized lung matrix used as scaffold [ ] . more recently, d bioprinting techniques have been used to produce the lung organs in vitro. for d bioprinting, cells are combined with bioactive hydrogels composed of synthetic (e.g., polyethylene glycol, pluronic) or natural (collagen, chitosan, fibrin, gelatin, matrigel, alginate) polymers [ ] . the use of reconstructed lung organs in oid preclinical screening is currently hampered by the low throughput of these methods. to summarize, in this commentary we have presented an overview of the in vitro, cell-based nam systems that, to date, have been successfully employed to fill the technological gap that is believed to hindering the effective oid translation from the lab bench to the clinic. in the past, oid failure at clinical trial stage was mainly due to poor pharmacokinetics and bioavailability. today, these are rarely a cause of failure, as the pharmaceutical industry greatly invested in the development and application of much more accurate prediction and modelling approaches. lack of efficacy is now the most common cause of oid attrition [ ] ; this appears to be associated to the fact that preclinical animal models are poorly representative of human respiratory diseases [ ] . improved in vitro non-animal methods could provide a more human-relevant predictive value so that compounds would fail earlier in their course of development [ ] . furthermore, we have provided a brief overview of those in vitro, cell-based nams that, in the future, we believe they could be adapted towards oid testing. although in vitro, cell-based nams still have limitations, the advantages associated with their use is evident and future efforts should aim at validating these systems for regulatory acceptance [ ] . in the development of oids, we should therefore invest in moving away from animal studies. in the last decades, significant funding and precious time have been spent on developing animal models, despite the known species differences that make the results obtained from such models often unreliable when translated to humans. as dr. francois busquet and colleagues from the center for alternatives to animal testing-europe state for covid- , human-relevant approaches offer crucial advantages of speed and "much more robust and exacting data than any animal experiment could deliver" [ ] . in this instance, we believe it is important to highlight that directive / /eu on the protection of animals used for scientific purposes aims non only at reducing but at the "full replacement of procedures on live animals for scientific and educational purposes, as soon as it is scientifically possible to do so" [ ] . consistently with this aim, in the netherlands has been the first eu member state to present a roadmap for phasing out animal testing in the safety research on chemical substances, food ingredients, pesticides and medicines (including veterinary medicines) [ ] . the recent advances in tissue engineering, microfluidic and organ-on-chip technologies are providing researchers with tools for the development of human-relevant, in vitro nams. thus, it is essential now that the 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tissue-informed engineering strategies for modeling human pulmonary diseases three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration d in vitro/ex vivo systems animal models of asthma: value, limitations and opportunities for alternative approaches human tissue models for a human disease: what are the barriers? thorax harnessing the power of novel animal-free test methods for the development of covid- drugs and vaccines on the protection of animals used for scientific purposes this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank moreno carrer for the technical assistance. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord- -l ufs k authors: tomita, kengo; saito, yuna; suzuki, tokiko; imbaby, samar; hattori, kohshi; matsuda, naoyuki; hattori, yuichi title: vascular endothelial growth factor contributes to lung vascular hyperpermeability in sepsis-associated acute lung injury date: - - journal: naunyn schmiedebergs arch pharmacol doi: . /s - - - sha: doc_id: cord_uid: l ufs k vascular endothelial growth factor (vegf) is a prime regulator of vascular permeability. acute lung injury (ali) is characterized by high-permeability pulmonary edema in addition to refractory hypoxemia and diffuse pulmonary infiltrates. in this study, we examined whether vegf can be implicated as a pulmonary vascular permeability factor in sepsis-associated ali. we found that a great increase in lung vascular leak occurred in mice instilled intranasally with lipopolysaccharide (lps), as assessed by igm levels in bronchoalveolar lavage fluid. treatment with the vegf-neutralizing monoclonal antibody bevacizumab significantly reduced this hyperpermeability response, suggesting active participation of vegf in non-cardiogenic lung edema associated with lps-induced ali. however, this was not solely attributable to excessive levels of intrapulmonary vegf. expression levels of vegf were significantly reduced in lung tissues from mice with both intranasal lps administration and cecal ligation and puncture (clp)-induced sepsis, which may stem from decreases in non-endothelial cells-dependent vegf production in the lungs. in support of this assumption, stimulation with lps and interferon-γ (ifn-γ) significantly increased vegf in human pulmonary microvascular endothelial cells (hpmecs) at mrna and protein levels. furthermore, a significant rise in plasma vegf levels was observed in clp-induced septic mice. the increase in vegf released from hpmecs after lps/ifn-γ challenge was completely blocked by either specific inhibitor of mitogen-activated protein kinase (mapk) subgroups. taken together, our results indicate that vegf can contribute to the development of non-cardiogenic lung edema in sepsis-associated ali due to increased vegf secretion from pulmonary vascular endothelial cells through multiple mapk-dependent pathways. sepsis is a potentially life-threatening medical emergency that is caused by the body's extreme response to an infection. the advent of the new definition of sepsis, which has been published recently, prompts a reappraisal of organ dysfunction as the hallmark of sepsis (singer et al. ). sepsis affects every major organ, including the lung, liver, and kidney, within the body, ultimately leading to the failure of one or more organs. the respiratory system is the most affected organ of the body, and lung dysfunction is the first step in the development of multiple organ failure in septic patients. acute lung injury (ali) and its most extreme form, acute respiratory distress syndrome (ards), are the manifestations of lethal and complex respiratory dysfunction and are characterized by explosive and diffuse pulmonary infiltrates, leading to noncardiogenic alveolar edema and, ultimately, refractory hypoxemia (tsushima et al. ; matuschak and lechner ) . vascular endothelial growth factor (vegf), also known as vegf-a, is a glycoprotein originally isolated as a tumor cellsecreted vascular permeability factor (plouet et al. ) and, since then, it has long been documented that vegf serves as the prime regulator of vascular permeability (bates ) . while vegf has been subsequently shown to have additional potent mitogenic and angiogenic properties (ferrara ; sharma et al., ) , significant amounts of vegf are known to exist in the normal human lung without significant mitogenesis or angiogenesis (barratt et al. ). in the normal lung, vegf may function as a survival factor for epithelial cells and endothelial cells in a paracrine manner (gerber et al. ; mura et al. ; roberts et al. ). in the meanwhile, vegf may contribute to the development of non-cardiogenic pulmonary edema associated with ali/ards. lung-targeted overexpression of human vegf , using an adenoviral gene vector, has been shown to result in pulmonary edema and increased vascular permeability in mice (kaner et al. ) . in addition, it has been revealed that the high ventilation-induced increase in pulmonary microvascular permeability in mice can be attenuated by knockdown of vegf by short-interfering rnas (li et al. ) . moreover, pretreatment with adenovirus-encoding soluble vegf receptor (vegfr) has been found to prevent ischemia-reperfusion-induced lung injury in rats (godzich et al. ). however, a role of vegf in pulmonary vascular hyperpermeability accompanied by sepsis-associated ali is not fully understood. in the present study, by conducting in vivo and in vitro experiments using rodent models of sepsis-associated ali and human pulmonary microvascular endothelial cell line, respectively, we attempted to test the hypothesis that vegf may contribute to non-cardiogenic high vascular permeability pulmonary edema in ali associated with sepsis. all animal experimental procedures were conducted in accordance with the national institute of health guidelines on the use of laboratory animal and with approval of the care and use committee of the university of toyama. in the first series of experiments, we used the cecal ligation and puncture (clp)-induced sepsis mouse model. clp-induced sepsis is regarded as a highly clinically relevant model of polymicrobial sepsis, because it reproduces many hallmarks of sepsis occurring in human patients (hubbard et al. ) . the surgical procedure to generate clp-induced sepsis was conducted according to our previous reports (tomita et al. ; kawakami et al. ; yamashita et al. ) . in brief, male balb/c mice (sankyo lab service, tokyo japan), - weeks old, were anesthetized with - % sevoflurane by inhalation, and a middle abdominal incision was made. the cecum was mobilized, tightly ligated ( cm from the cecum tip), punctured twice with a -gauge needle, and gently squeezed to expel small amounts of feces. then, the bowel was repositioned to the peritoneal cavity, and the laparotomy site was closed with sterile suture (the skin and muscle were sutured separately). sham-operated control underwent the same procedure except for ligation and puncture of the cecum. both groups of animals were fed the same diet and water ad libitum, and housed in an environment with controlled temperature, constant humidity, and a daily -h light-dark cycle ). all animals after clp surgery were lethargic, showed lack of interest in their environment, displayed piloerection, and had crusty exudates around their eyes, as opposed to sham-operated animals that were healthy, moving freely and eating (matsuda et al. ). all mice received subcutaneous injection of . ml sterile normal saline immediately after surgery. at h after surgery, blood samples were collected and inflation-fixed lungs were harvested from mice treated with - mg/kg ketamine and mg/kg xylazine hydrochloride. in the second series of experiments, mice, under light anesthesia, were instilled intranasally with μg of lipopolysaccharide (lps) (escherichia coli :b ; list biological laboratories, campbell, ca, usa) in μl of sterile . % nacl solution. control animals received equivalent volume of vehicle saline solution. when bevacizumab, which neutralizes vegf and blocks its signal transduction through vegf receptors (papadopoulos et al. ) , was used, it was intravenously given to mice at a dose of μg at min before administration of lps. animals were euthanized at h after lps challenge. all experimental data were analyzed in a blinded fashion. the immortalized human pulmonary microvascular endothelial cell line (hpmec-st . r), which was developed by means of co-transfection of a plasmid encoding the catalytic component of telomerase and a plasmid encoding the simian virus large t antigen (krump-konvalinkova et al. ; unger et al. ) , was kindly provided by drs. c. james kirkpatrick and ronald e. unger at johannes gutenberg university (mainz, germany). hpmec-st . r cells were routinely maintained on tissue culture plastic ware in m medium (sigma-aldrich, st. louis, mo, usa) supplemented with % (v/v) heat-inactivated fetal bovine serum, μg/ml endothelial cell growth supplements (sigma-aldrich), μg/ml sodium heparin (sigma-aldrich), % (v/v) penicillin/streptomycin (nacalai tesque, kyoto, japan), and μg/ml g (nacalai tesque). cells were cultured at °c under a humidified atmosphere containing % co and % air. in hpmec-st . r cells, the presence of interferon (ifn)-γ can highly amplify the inflammatory responses to lps ). thus, cells were stimulated with μg/ml lps and ng/ml ifn-γ (r&d systems, minneapolis, mn, usa). when mitogen-activated protein kinase (mapk) inhibitors, pd ( μm; cayman chemical, ann arbor, mi, usa), sb ( μm; adipogen life sciences, epalinges, switzerland), sp ( μm; cayman chemical), and jnk-in- ( μm; merck, darmstadt, germany) were used, they were added to the medium for min before challenge with lps and ifn-γ. lps/ ifn-γ stimulation was stopped at the indicated time points by aspirating off the culture medium and then adding ice-cold pss before harvesting. blood levels of tumor necrosis factor (tnf)-α, interleukin (il)- β, il- , and monocyte chemoattractant protein- (mcp- ) were measured by the use of commercially available enzyme-linked immunosorbent assay (elisa) kit (r&d systems, minneapolis, mn) according to the manufacturer's instructions. to measure the concentrations of vegf in serum and culture medium samples, mouse vegf quantikine elisa kit (r&d system) and human vegf duoset elisa (r&d system) were used, respectively. the plate was read on a microplate reader (nippon-intermed, tokyo, japan). assays were performed in duplicate. to estimate pulmonary microvascular permeability, igm was selected as a marker of permeability and its concentration in bronchoalveolar lavage (bal) fluid was determined immunologically by igm mouse uncoated elisa kits with plates (thermo fisher scientific, rockford, il, usa). a polyethylene catheter was inserted into the trachea, bal was performed by repeatedly infusing and removing ml of pbs, and the third drainage of effluent was kept as bal fluid. total rna was isolated from hpmec-st . r cells and lung tissues with the use of sepazol®-rna i super g (nacalai tesque) according to the manufacturer's manual. revertra ace qpcr rt master mix (toyobo, osaka, japan) was used for the reverse transcription reaction, and real-time pcr analyses were performed using powerup™ sybr® green master mix (thermo fisher scientific), as described in the manufacturers' instructions. values were normalized to the housekeeping gene gapdh according to the manufacturer's protocol (mx p real-time pcr system; agilent technologies inc., santa clara, ca, usa). additional details are described by our laboratory (kawakami et al. ; yamashita et al. ; suzuki et al. ). the pcr primers were designed as follows: forward '-tgcagattatgcggatcaaacc- ′ and reverse ′-tgcattcacatttgttgtgctgtag- ′ for vegf, forward ′-caggcccagtttctgccatt- ′ and reverse ′-ttccagctcagcgtggtcgta- ′ for vegfr , forward ′-ccagcaaaagcagggagtct gt- ′ and reverse ′-tgtctgtgtcatcggagtga tatcc- ′ for vegfr , and forward ′-tgtg tccgtcgtggatctga- ′ and reverse ′-ttgc tgttgaagtcgcaggag- ′ for glyceraldehyde- phosphate dehydrogenase (gapdh). hpmec-st . r cells were grown in -mm dish, harvested, and lysed in μl of ripa buffer ( mm tris-hcl, mm nacl, % np- , % sodium deoxycholate, . % sds, ph . ) containing protease inhibitor cocktail on ice. the lysates were centrifuged at , ×g for min at °c and the resulting supernatants were collected. the protein concentration in the remaining supernatant was measured using bca protein assay kit (nacalai tesque). blotting procedure, chemiluminescent detection, and densitometric analysis were carried out as described in our previous reports (kawakami et al. ; suzuki et al. ) . samples ( - μg of protein) were separated with % sds-page gel electrophoresis and then transferred to polyvinylidene difluoride filter membrane. the membrane was probed with the following primary antibodies: anti-human extracellular signal-regulated protein kinase (erk) / mouse monoclonal antibody ( : ; cell signaling, danvers, ma, usa), anti-human phospho-erk / (thr- /tyr- ) rabbit monoclonal antibody ( : ; cell signaling), anti-human c-jun nterminal kinase (jnk) rabbit monoclonal antibody ( : ; cell signaling), anti-human p rabbit monoclonal antibody ( : ; cell signaling), anti-human phospho-p (thr- /tyr- ) mouse monoclonal antibody ( : ; cell signaling), anti-human jnk (thr- /tyr- ) mouse monoclonal antibody ( : ; cell signaling), and anti-human gapdh chicken polyclonal antibody ( : ; emd millipore, billerica, ma, usa). irdye®-labeled secondary antibodies were purchased from li-cor bioscience (lincoln, ne, usa) and odyssey clx infrared imaging system (li-cor bioscience) was employed for primary antibody detection. gapdh was used as the loading control. results are presented as mean ± standard error. data were analyzed by the use of prism software (version ; graphpad software, san diego, ca, usa). statistical analysis was performed by student's t test or one-way analysis of variance (anova) followed by tukey's multiple-comparison test. differences were considered to be statistically significant when a p value was < . . the clp rodent model, which causes peritonitis and leads to a polymicrobial sepsis, represents an indirect insult similar to the pathogenesis of ali/ards (villar et al. ) . indeed, we have clearly demonstrated that mice - h after clp surgery display marked hypoxemia, increased lung vascular permeability, and histological damage in lungs, including wall thickening, inflammatory infiltrate, and hemorrhage (takano et al. ; oishi et al. ; imaizumi et al. ) . when blood levels of pro-inflammatory cytokines were measured using an elisa, the sham-operated control animals had extremely low levels of the cytokines examined here. the mice h after clp-induced sepsis exhibited marked elevations in blood levels of tnf-α, il- β, il- , and mcp- (c) the mrna levels of vegf, vegfr , and vegfr in lung tissues were quantified by real-time pcr. lung tissues were harvested h after surgery. values are normalized to gapdh (n = ). * p < . , ** p < . , and *** p < . vs. the sham-operated control group (fig. a) . sepsis induction by clp also resulted in a significant elevation in plasma vegf protein levels in mice (fig. b) . plasma vegf in clp mice was increased . -fold in comparison with sham-operated control animals. however, the vegf mrna level was significantly downregulated in lung tissues from mice with clp-induced sepsis (fig. c) . we further examined vegfr mrna levels in clp mouse lungs. vegf regulates vascular permeability by activating receptors, vegfr (flt- ) and vegfr (kdr/flk ) (shibuya ) . as shown in fig. c , vegfr mrna was increased and vegfr mrna was decreased in lung tissues of clp mice compared with sham-operated controls. to assess changes in pulmonary vascular permeability, bal supernatant was analyzed for igm using an elisa. intranasal challenge with lps resulted in a highly significant . -fold increase in pulmonary vascular permeability (fig. a) . treatment with bevacizumab, which neutralizes vegf and blocks vegfr signaling (papadopoulos et al. ) , significantly but partially attenuated lung vascular permeability as compared with that shown in mice challenged with lps alone. we also examined vegf mrna levels in lung tissues of mice intranasally instilled with lps. as seen in clp-induced septic mice, pulmonary mrna expression was significantly downregulated in mice challenged with lps (fig. b ). in the immortalized human pulmonary microvascular endothelial cell line hpmec-st . r, the time course of changes in gene expression of vegf and its receptors after coadministration of lps and ifn-γ were investigated. the mrna level of vegf was gradually increased, reached a maximum at - h, and then showed a return toward the baseline at h (fig. a) . the mrna level of vegfr showed a trend toward increasing throughout - h observation period (fig. b) . vegfr mrna remained virtually unchanged in a wide range of time after stimulation with lps and ifn-γ (fig. c) . when the amounts of vegf in culture media were measured by an elisa, lps/ifn-γ challenge resulted in a significant increase in the protein level of vegf (fig. b) . the mapk signaling cascades are generally thought to be important in the pathogenesis of ali/ards (newton and holden ; qian et al. ) . when activation of the three major subgroups of mapk family, erk / , p , and jnk, was assessed by increases in their phosphorylation levels, coadministration of lps and ifn-γ resulted in significant activation of all three families of mapks in hpmec-st . r cells (fig. a) . we thus examined whether expression of vegf in human pulmonary microvascular endothelial cells is regulated by mapks. when hpmec-st . r cells were treated with pd , an inhibitor of mapk kinase which is an erk / upstream activator, or sb , which is widely used as a specific inhibitor of p mapk, the lps/ifn-γinduced increase in vegf protein levels was strongly blocked (fig. b) . treatment with sp , an anthrapyrazolone inhibitor of jnk, also abrogated the vegf protein increase in hpmec-st . r cells stimulated with lps/ifn-γ (fig. b) . the striking inhibition of the lps/ifn-γ-induced vegf upregulation was similarly observed by treatment with another fig. involvement of vegf in lung vascular hyperpermeability in mice after lps administration. lps ( μg) was instilled intranasally and animals were euthanized at h after lps challenge. (a) lung vascular permeability was assessed by igm levels in bal fluid from mouse lungs (n = - ). bevacizumab ( μg) was intravenously injected to mice min before lps challenge. (b) the mrna levels of vegf in lung tissues were quantified by real-time pcr. values are normalized to gapdh (n = ). *** p < . vs. control. ## p < . vs. lps alone jnk inhibitor jnk-in- , which is far more selective for jnk than sp (brain et al. ) (fig. b ). we demonstrated in this study for the first time that vegf contributed to pulmonary vascular hyperpermeability when lps was intranasally administrated in mice. intranasal administration of lps has long been widely used as an appropriate model in which ali can be directly induced (gharib et al. ; bosmann et al. ; juschten et al. ) , and increased pulmonary vascular permeability is a critical and non-redundant pathological process involved in the ali development (herold et al., ) . we found that treatment with the vegf-neutralizing monoclonal antibody bevacizumab resulted in a significant inhibition of the increase in pulmonary vascular permeability caused by intranasal lps challenge, as evidenced by changes in igm levels in bal fluid from mouse lungs. previous reports have well established that the amounts of igm in bal fluid are related with alterations in alveolarcapillary barrier and lung vascular permeability (kantrow et al. ; matute-bello et al. ; johnston et al. ) . intriguingly, while bevacizumab is clinically used for treatment of advanced cancers of the lung, colon, brain, kidney, and others by counteracting the angiogenic effect of vegf, its ability to reduce the increase in vascular permeability associated with vegf expression may help relieve patients with the potentially serious morbidity and symptoms that accompany peritumoral edema (gil-gil et al. ) . our findings imply the active participation of vegf in non-cardiogenic high vascular permeability pulmonary edema associated with lpsinduced ali (fig. ) . however, the preventive effect of bevacizumab on lps-induced pulmonary vascular hyperpermeability was partial. our previous study has shown that treatment with n g -nitro-l-arginine, an inducible nitric oxide (no) synthase (inos) inhibitor, or diphenhydramine, a histamine h -receptor antagonist, significantly but incompletely inhibited lps-induced lung vascular permeability in mice (matsuda et al. ) , which suggests that no and histamine may also be partly responsible for mediating increased lung vascular leak following lps challenge. we thus assume that several vascular permeability molecules, including vegf, no, and histamine, can be excessively produced and thereby actually contribute to the development of pulmonary edema in sepsis-associated ali. indeed, great increases in gene and protein expression of inos and histidine decarboxylase, an enzyme that only forms histamine in mammals, have been observed in the lungs of mice after induction of sepsis with lps (matsuda et al. ) . unexpectedly, expression of vegf in lung tissues was significantly downregulated rather than upregulated in two murine models of ali, intranasal lps administration and clp-induced sepsis. this observation is consistent with a string of human studies showing a reduction in intrapulmonary vegf in the early stages of ali/ards (maitre et al. ; thickett et al. ; abadie et al. ) , although a conflicting result was reported in mice exposed to lps in a nebulization chamber (karmpaliotis et al. ) . the reduced levels of intrapulmonary vegf in ali/ards may be explained by direct injury to and clearance of epithelial type cells (medford and millar ) which are considered to be the main source of vegf in lungs (kaner and crystal ) . in this regard, caution would be required in the interpretation of the observed decrease in vegf levels in lung tissues, since the total amount of intrapulmonary vegf may be determined by the deduction of vegf released from different intrapulmonary components, such as epithelial cells and vascular endothelial cells, which can be variably affected by endotoxin. as reported in human studies (maitre et al. ; thickett et al. ; azamfirei et al. ), we found a significant rise in plasma levels of vegf in mice with clp- fig. involvement of mapk activation in vegf released from human pulmonary microvascular endothelial cells after challenge with lps and ifn-γ. hpmec-st . r cells were stimulated with μg/ml lps and ng/ml ifn-γ. (a) activation of mapks in hpmec-st . r after challenge with lps and ifn-γ. levels of phosphorylation and total expression of erk / , p , and jnk before and min after lps/ifn-γ challenge were determined by western blotting. in the top trace of each panel, typical western blots are shown. in the bottom trace, the summary of quantification of densitometric measurements as ratio of phospho-mapk relative to mapk is presented (n = - ). * p < . and ** p < . vs. unstimulated value. (b) effects of mapk inhibitors on vegf levels in hpmec-st . r cells stimulated with lps/ifn-γ for h. pd ( μm), sb ( μm), sp ( μm), or jnk-in- ( μm) was added h before lps/ifn-γ challenge. the vegf levels released from cells into the cell culture medium were measured by an elisa (n = ). ** p < . vs. control. ### p < . vs. lps/ ifn-γ alone induced sepsis. this rise in plasma vegf levels seems likely to be attributed to the release from vascular endothelial cells. we showed that stimulation with lps/ifn-γ resulted in a significant upregulation of vegf expression in human pulmonary microvascular endothelial cells at mrna and protein levels, implying that endotoxin positively regulates endothelial cell-derived vegf in a transcription manner. it should be added that neutrophils may also contribute to the increased vegf plasma levels in clp-induced septic mice, because these hemocytes have been shown to produce various vascular permeability molecules, including vegf, in certain circumstances such as inflammation (taichman et al. ; distasi and ley ) . in line with our recent report , stimulation with lps/ifn-γ significantly activated three major subgroups of mapk family, erk / , p , and jnk, in human pulmonary microvascular endothelial cells, as assessed by their phosphorylation levels. each specific inhibitor of these mapk subgroups completely blocked the increase in vegf protein levels caused by lps/ifn-γ challenge. this suggests that endotoxin upregulates vegf expression in pulmonary microvascular endothelial cells through multiple mapkdependent pathways. it is noteworthy that mapk signaling is linked to increased expression of growth factors, including vegf, in different cell types (li et al. ; pagès et al. ; rak et al. ; schrma et al. ) . vegf displays broad vascular functions, including vascular permeability, via binding to and activating its specific receptors, vegfr and vegfr (shibuya ) . in this study, in lung tissues from clp-induced septic mice, vegfr mrna was upregulated and vegfr mrna was downregulated compared with sham-operated controls. furthermore, lps/ifn-γ application showed a trend to increase vegfr mrna in human pulmonary microvascular fig. schematic diagram of the participation of vegf released from pulmonary vascular endothelial cells in noncardiogenic high vascular permeability pulmonary edema associated with lps-induced ali. see text for details endothelial cells. however, it is not clear at this time whether altered expression of vegfr can be involved as regulatory mechanisms to transduce vegf-mediated vascular hyperpermeability signals. several lines of evidence provide that vegfr may function as a decoy receptor and negatively regulate vegf functions (sato et al. ; cao ) . accordingly, what role, if any, is played by altered vegfr expression observed in this study in the pathophysiology of sepsis-associated ali awaits further study. in conclusion, the present results indicate that vegf can contribute to the development of non-cardiogenic pulmonary edema in sepsis-associated ali as a result of increased vegf secretion from pulmonary vascular endothelial cells through multiple mapk-dependent pathways (fig. ). as such, anti-vegf therapy may be of value in ali/ards. however, it is proposed that vegf also displays a protective effect on the alveolar epithelium following injury (medford and millar ) . therefore, a deeper understanding of the role of vegf in the lung will be required before treatment modulating vegf may be used in ali/ards. thus, it remains the subject of our ongoing study to precisely grasp vegf biology in the body, including the lungs, under sepsis. decreased vegf concentration in lung tissue and vascular injury during ards vascular endothelial growth factor: a possible mediator of endothelial activation in acute respiratory distress syndrome vascular endothelial growth factor in acute lung injury and acute respiratory distress syndrome vascular endothelial growth factors and vascular 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sepsis-induced acute lung injury model cardioprotective and functional effects of levosimendan and milrinone in mice with cecal ligation and puncture-induced sepsis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations acknowledgments we thank drs. takahiro imaizumi and takuya sakamoto for their expert help in the commencing time of this study.author contributions k.t., n.m., and y.h. conceived and designed the experiments. k.t.., y.s., t.s., and s.i. performed the experiments. k.t., y.s., and t.s. analyzed data. k.h. and y.h. wrote the article. all authors read and approved the manuscript. the authors declare that all data were generated in-house and that no paper mill was used.funding information this study was supported by grant-in-aid for young scientists ( k , k ) and for scientific research ( k , h ) from japan society for promotion of science. all animal studies were approved by the animal care and use committee of the university of toyama, which are based on the national institute of health guide for the care and use of laboratory animals and the arrive guidelines. the authors declare that they have no conflict of interest. key: cord- -god qzw authors: mao, kaimin; geng, wei; liao, yuhan; luo, ping; zhong, hua; ma, pei; xu, juanjuan; zhang, shuai; tan, qi; jin, yang title: identification of robust genetic signatures associated with lipopolysaccharide-induced acute lung injury onset and astaxanthin therapeutic effects by integrative analysis of rna sequencing data and geo datasets date: - - journal: aging (albany ny) doi: . /aging. sha: doc_id: cord_uid: god qzw acute lung injury (ali) and acute respiratory distress syndrome (ards) are life-threatening clinical conditions predominantly arising from uncontrolled inflammatory reactions. it has been found that the administration of astaxanthin (ast) can exert protective effects against lipopolysaccharide (lps)-induced ali; however, the robust genetic signatures underlying lps induction and ast treatment remain obscure. here we performed a statistical meta-analysis of five publicly available gene expression datasets from lps-induced ali mouse models, conducted rna-sequencing (rna-seq) to screen differentially expressed genes (degs) in response to lps administration and ast treatment, and integrative analysis to determine robust genetic signatures associated with lps-induced ali onset and ast administration. both the meta-analyses and our experimental data identified a total of degs in response to lps administration, and core degs (timp , ly i, cxcl , irf , cxcl , ccl , isg , saa , saa , tgtp , and gbp ) were identified to be associated with ast therapeutic effects. further, the core degs were verified by quantitative real-time pcr (qrt-pcr) and immunohistochemistry (ihc), and functional enrichment analysis revealed that these genes are primarily associated with neutrophils and chemokines. collectively, these findings unearthed the robust genetic signatures underlying lps administration and the molecular targets of ast for ameliorating ali/ards which provide directions for further research. acute respiratory distress syndrome (ards) is an acute inflammatory lung injury, associated with increased pulmonary vascular permeability, increased lung weight, and loss of aerated lung tissue [ ] . its less severe form is acute lung injury (ali). most patients need mechanical ventilation for support. the initial acute or exudative phase of ali/ards is characterized by the rapid onset of dyspnea, hypoxemia, respiratory failure, and bilateral infiltrates on chest radiographs that are consistent with pulmonary edema [ ] . ali/ards is common and has been associated with several clinical disorders, such as sepsis; pneumonia; aspiration of gastric contents, aging saltwater, or freshwater; major trauma; transfusion of blood products; acute pancreatitis; and drug reactions (for example, reactions to lipopolysaccharide) [ ] . in the past years, considerable progress has been made in understanding the epidemiology, pathogenesis, and pathophysiology of ards. however, ards is being increasingly recognized as a heterogeneous syndrome, generating momentum for the identification of clinical and biological features for classifying patients into subphenotypes that might be more responsive to specific therapies. lipid a (endotoxin), the hydrophobic anchor of lipopolysaccharide (lps), is a glucosamine-based phospholipid that makes up the outer monolayer of the outer membranes of most gram-negative bacteria [ ] . in recent years, lps, which has been most widely used in drug-associated ali models, can effectively induce a neutrophilic inflammatory response accompanied by an increase in intrapulmonary cytokines. many studies have shown that oxidative stress plays a major role in the pathogenesis of lung injury in a murine model of ali induced by lipopolysaccharide (lps) [ ] [ ] [ ] . in response to the increased formation of reactive oxygen species (ros), thioredoxin interacting protein (txnip) detaches from thioredoxin (trx), binds to the nucleotide-binding domain-like receptor protein (nlrp ), and then activates nlrp inflammasome [ ] . the activation of the nlrp inflammasome results in the maturation and release of pro-inflammatory cytokines, such as interleukin- β (il- β), which further aggravates the production of inflammatory cytokines (tumor necrosis factor-α (tnf-α), il- , inducible nitric oxide synthase (inos), and cyclooxygenase- (cox )) and induces oxidative stress [ ] [ ] [ ] . astaxanthin (ast) is a lipid-soluble, red-orange-colored xanthophyll carotenoid synthesized by many microorganisms and various types of marine life. the main producers of natural ast are microalgae and fungi. aquatic animals such as salmon, red seabream, shrimp, lobster and crayfish, which feed on ast-producing organisms, are significant dietary sources of ast for humans [ ] [ ] [ ] . it has been revealed that ast can prevent inflammatory processes by blocking the expression of pro-inflammatory genes as a consequence of suppressing nuclear factor kappab (nf-κb) activation [ ] . some studies also suggested that ast has a dosedependent ocular anti-inflammatory effect, through the suppression of no, pge , and tnf-alpha production, which is achieved by directly blocking nos enzyme activity [ ] . furthermore, ast has great therapeutic value for lung disease, such as an antifibrotic effect against the promotion of myofibroblast apoptosis based on dynamin-related protein- (drp )-mediated mitochondrial fission in vivo and in vitro [ ] and anti-inflammatory effect against lps-induced ali, as mentioned above [ , ] . however, at the transcriptional level, the mechanism of action of ast in the treatment of ali-/ ards-remains unclear. therefore, we hope to explore the molecular targets of ast against ali-/ ards-through further research, with the purpose of providing a new alternative for the clinical treatment of this acute lung disease. to determine the common molecular signatures underlying lps-induced mouse ali initiation, five microarray datasets were obtained from corresponding independent studies. the characteristics of the studies composing the five gene expression compendiums are listed in table and supplementary table . we extracted and annotated the five microarrays, which yielded a collection of unique genes from samples, including control and lps-induced ali mice. before the meta-analysis study, we comprehensively analyzed the five datasets by identifying the differentially expressed genes in each data set and evaluated overlapping significant genes. the overlapping results were used to generate a venn diagram (figure ), and three genes (ccl , zbp , and cxcl ) were identified in the common region, suggesting that these three genes were significantly correlated with lps management in mice in the five datasets. then, we performed a meta-analysis using networkanalyst (http://www.networkanalyst.ca), which is a comprehensive web-based tool designed to perform meta-analyses of gene expression data [ ] . an overview outlining the procedure of the analysis is presented in figure a . using three meta-analysis approaches, namely fisher's method, fixed effect model and voting count, , and differentially expressed genes, respectively, were identified. among these genes, were identified by all three methods ( figure b ), with ( . %) genes being upregulated and ( . %) being downregulated in the lps group compared with the control group. a full list of the common genes identified by the three meta-analysis methods is presented in supplementary table . a heat map of the top common degs across the five datasets is displayed in figure c . of note, the top upregulated genes (p< . ) were junb, vcam , ehd , ifrd, adm, cd , nadk, litaf, tubb , and ctps. the most significantly downregulated genes (p< . ) among the top common degs were acss and abcd . the merged data from this meta-analysis are listed in supplementary data . to further identify the robust expression signatures in lps-induced ali and investigate the transcriptional changes resulting from treatment of ali with ast, we divided mice into three groups, including the control group, lps group, and ast group. rna-sequencing (rna-seq) was performed to profile differentially expressed genes (degs) associated with lps-induced ali initiation and ast treatment. a total of degs were identified in the lps-induced ali group compared with the control group. among these genes, were table . then, we compared these genes with the degs obtained from the above meta-analysis, and generated two heat-maps of the common degs across the meta-analysis results and our experimental aging data, which are displayed in figure c and supplementary figure . in total, degs were detected in both published data and our experimental data, including upregulated and downregulated degs. to explore the therapeutic effect of ast against ali at the genetic level, we also compared the gene expression profile of the lps-induced ali group with that of the ast treatment group. in total, degs were identified after ast treatment ( figure b table ). we subsequently integrated the rna-seq and microarray meta-analysis data, and core degs (timp , ly i, cxcl , irf , cxcl , ccl , isg , saa , saa , tgtp , and gbp ) that were upregulated in ali models and downregulated significantly after ast treatment were identified ( table ) . to understand the function of the core degs, go enrichment analysis including molecular function (mf), biological process (bp) and cellular component (cc) categories (supplementary table ) was performed using the 'clusterprofile' package in r [ ] . in bp terms, the upregulated genes were associated with "cell chemotaxis," the "chemokine−mediated signaling pathway," and "neutrophil migration" ( figure a ). several studies have shown that neutrophil migration and related chemokine network regulation in the lung play roles in the pathogenesis and development of ali/ards [ ] [ ] [ ] . in the mf category, the core degs were associated with "glycosaminoglycan binding," "chemokine activity," and "receptor-ligand activity" ( figure b ). since glycosaminoglycan-cytokine interactions have been reported to support cellular mechanisms that cause acute inflammation [ ] , ast may affect these interactions by downregulating the genes involved to exert an anti-inflammatory effect. moreover, degs were enriched in the cc category involved in "high−density lipoprotein particles," "symbiont−containing vacuole membranes," and "plasma lipoprotein particles" ( figure c ). to further confirm the differences in the expression of the core degs (timp , ly i, cxcl , irf , cxcl , ccl , isg , saa , saa , tgtp , and gbp ) among the control group, lps group, and ast group, we divided mice into three groups and conducted qrt-pcr and ihc verification ( figure a - k, supplementary figure ). the results demonstrate that the relative expression levels of all genes were significantly upregulated in the lps group compared to the control group. more importantly, the expression levels of the above degs, as analyzed by qrt-pcr, were significantly inhibited after the application of ast. of the core genes, were tested by ihc, and the results were consistent with the qrt-pcr results, which further verifying the data (supplementary figure ) . overall, the rt-qpcr and ihc results were consistent with our integrative rna-seq analysis and metaanalysis, suggesting the critical role that the core dges might play in the mechanism by which ast alleviates ali/ards. as a life-threatening condition, ali/ards is an underrecognized condition, and its treatment is an unmet medical need. it is thought that inflammatory storm is the key factor in the occurrence of ali [ ] , and anti-inflammatory and antioxidant therapy should be the primary objective in ali/ards [ ] . to find the conserved genes responsible for lps-induced ali initiation and the effects of ast treatment, we identified robust changes in gene expression related to ali by meta-analysis and rna-seq using the gene expression omnibus (geo) database and mice, respectively. moreover, we performed functional enrichment analysis of core genes using the gene ontology (go) database to explore the possible molecular mechanisms that mediate the therapeutic effect of ast. before the meta-analysis of the five microarray datasets, we compared the differentially expressed genes in each dataset, and common differentially expressed genes (degs) were found in all five datasets: cxcl , zbp , and ccl . cxcl is abnormally expressed in the lung tissues of patients with idiopathic pulmonary fibrosis (ipf), and its circulating concentration is also highly correlated with the clinical manifestations and disease progression of individual patients. in the lung tissues of patients with ipf, cxcl may promote focal infiltration of nonproliferating b cells through the cxcl -cxcr axis [ ] . zbp is a host protein that was shown to be an innate sensor of viral infection, regulating cell death, inflammasome activation, and proinflammatory responses in a variety of situations, including infection and embryonic development [ ] . a previous study indicated that zbp is abnormally expressed in h n induced pneumonia associated with acute respiratory distress syndrome in mice [ ] . ccl (mcp ), which is elevated in pulmonary fibrosis, has been reported to mediate fibroblast survival through il- [ ] . since fibroproliferation is initiated early in lung injury, it has been observed that ccl is highly expressed in ards statistical analysis of significant differences between groups was achieved with one-way anova using prism software. ****p < . , ***p < . , **p < . , and *p < . were considered statistically significant. aging induced by severe sepsis [ ] . to reduce the study bias and increase the statistical power of individual microarray data, we performed a meta-analysis of five microarray gene expression profiles to assess the differentially expressed genes between lps-induced and control groups. consequently, differentially expressed genes (degs) were identified using three meta-analysis approaches. to further identify the robust expression signature related to lps-induced ali and investigate the transcriptional changes in response to the treatment of ali by ast, we performed rna-seq on three groups of mice and integrated the data with the results of the above mentioned meta-analysis. ultimately, we identified core degs that were significantly associated with ast treatment. saa , ly i, saa , irf , cxcl , ccl , timp , isg , gbp , tgtp , and cxcl were found to be overexpressed in the lps group compared with the control group but relatively downregulated in the ast group. our qrt-pcr and ihc verification of the core genes in the mice suggested that these genes might be the key mediators of the therapeutic effect of ast in ali/ards. among the core genes that were differentially expressed in response to ast mediation, two genes are members of the serum amyloid a (saa) family. saa is a critical acute-phase protein that is often increased by infection, trauma, cancer, or other causes of inflammation and plays an important role in the regulation of inflammatory responses [ ] . recent studies have indicated that an increased level of saa is positively correlated with the disease progression of covid , and can thus be a sensitive indicator for assessing the severity and prognosis of covid- [ ] . in our study, saa was the most significantly inhibited gene by ast application in lps-induced ali mice, and its downregulation was further confirmed by qrt-pcr and ihc. saa , the one of three isoforms of saa expressed in mice, is stimulated intensely in lps-induced acute systemic inflammation, which is consistent with our findings [ ] . high expression of saa in response to acute inflammation may be repressed by an interaction with noncoding rnas. it has been confirmed that mir- b- p may target saa to protect against lps-induced ali [ ] . additionally, lncrna malat can also target saa directly or indirectly to cause many diseases such as inflammation, diabetes and septic cardiomyocytes [ , ] . saa , another member of the saa gene family, is believed to have a pro-inflammatory effect, and its expression may aggravate tissue inflammation and damage [ ] . removing the n-and c-terminal sequences of saa can switch the protein to an anti-inflammatory role [ ] . however, other research has suggested that mice induced to express genetically modified human saa have a partial protective effect against the inflammatory response and lung injury caused by lps [ ] . moreover, saa is the direct target of mir- , which can protect nucleus pulposus cells from tnfainduced apoptosis in intervertebral disc degeneration [ ] . considering that saa might act as a biomarker of inflammatory disease, it is possible, that its downregulation induced by ast may partly indicate the antiinflammatory effect of ast. the deeper molecular mechanism underlying saa action in response to ast application deserves further exploration. interferon regulatory factor (irf ) is considered the master regulator of ifn-α against pathogenic infections [ ] . the excessive activation of irf promotes the development of acute lung injury (ali) caused by influenza a virus (iav), and attenuating irf activity can significantly prevent the progression of iavinduced ali in model mice [ ] . thus, the present finding that irf was upregulated by lps and downregulated in response to ast treatment may suggest that of ast protects against ali. regarding how irf regulates ifn production, mirna may act as an important mediator. previous research has shown that mir- c can downregulate irf and irf expression to mediate influenza a virus-induced ifnβ expression [ ] . additionally, mir- was shown to reduces the antiviral response by attenuating the traf -irf pathway to alter the cellular antiviral transcriptional landscape [ ] . however, whether mirna-irf interactions are involved in the pharmacological mechanism of ast remains to be further investigated. tissue inhibitor of metalloproteinase- (timp ), a member of timp family, is primarily recognized to regulate the degradation of the extracellular matrix by inhibiting the activity of matrix metalloproteinases (mmps) [ ] . it has been reported that an imbalance between mmp and timp plays a pivotal role in the pathogenesis of ards mainly through participating in airway remodeling, thus indicating the function of the mmp /timp ratio in the evolution of pulmonary fibrosis in ards [ ] . indeed, increased systemic levels of timp were proven to be associated with increased -day mortality in ards patients according to a large, prospective, multicenter study [ ] . additionally, other research has demonstrated that increased timp expression promotes an immune response, has a pro-inflammatory effect in the lungs after influenza infection and facilitates an injurious phenotype [ ] . the above observations not only support our present results regarding timp but also provide a considerable explanation for the increase in timp expression after lps application. intriguingly, given that timps are highly expressed in liver fibrosis and that the imbalance of mmps/timps promotes the progression of fibrosis, shen et al. found that astaxanthin is able to repress the activation of hepatic stellate cells (hscs) to ameliorate liver fibrosis through downregulating the expression of nf-κb and tgf-β and preserving the balance between mmp and timp [ ] . hence, it is reasonable to further investigate whether there is a similar mechanism by which ast downregulates the expression of timp to mitigate lps-induced ali. interferon-stimulated gene (isg ), which encodes the ubiquitin-like protein isg , which is primarily induced by type i interferons, is an essential player in regulating host signaling pathways such as damage repair responses and immune responses. isg can be induced by various pathogenic stimuli such as viral and bacterial infections, lipopolysaccharide (lps), retinoic acid, or certain genotoxic stressors [ ] . in accordance with our findings, previous studies have observed increased levels of isg conjugates in macrophages in response to lps treatment [ ] . moreover, research has found that systemic isg (mx , isg , ifit , and ifit ) expression within the first days of ards onset is associated with disease severity and prognosis. this response should be considered along with other identified genetic, environmental, and complex demographic factors as the cause of heterogeneity in ards prognosis [ ] . nevertheless, no data has been reported on the association between isg and ast in the literature. since the excessive recruitment of leukocytes appears to be a central contributor to the pathogenesis of ali, the elevation of proinflammatory cytokines and chemokines is considered the most important factor [ ] . similarly, we found that the expression of chemokines such as ccl , cxcl , and cxcl were increased after lps instillation but decreased after ast treatment. previous reports have documented an increased level of ccl in a mouse model of acute lps-induced lung inflammation [ ] . moreover, the expression of cxcl is also rapidly induced in ali murine models after lps administration [ ] . however, no data has on cxcl expression in ali models has been reported. therefore, we report for the first time the induction of cxcl after lps administration, which provides insights into the role of cxcl in the pathogenesis of ali. furthermore, the observation of decreased expression of ccl , cxcl , and cxcl may hint at the antiinflammatory properties of ast. although the roles of other degs (ly i, gbp , and tgtp ) have been described in many other diseases in detail, their regulatory mechanisms in ali-/ ards-are not fully understood. our results show that these degs are overexpressed to varying degrees in the lps group and that ast can effectively prevent this overexpression. further studies on the roles of these three genes in ali initiation and progression are need. to determine the functional mechanisms of these degs, go enrichment analyses were further conducted. according to the results, terms in biological process category, in cellular component category and in the molecular function category were enriched. the most significantly enriched terms in the bp category were associated with chemokines and neutrophils, indicating the dominant role of neutrophils and related chemokines in the pathogenesis and progression of lps-induced ali. in ali, the excessive recruitment of inflammatory cells and their mediators results in injury to endothelial and epithelial barriers [ ] . thus, agents such as ast, which can exert robust anti-inflammatory effects, may provide potential treatment prospects. despite this, several limitations of the current study need to be addressed. first, our research did not use the ali mouse models induced by other agents; thus, it did not address heterogeneity of ali initiation. second, given that findings in animal models of lps-induced lung injury may depend on the time point at which samples are obtained and physiological data are captured, the dynamic changes in lps-induced ali models may have been ignored to a certain extent [ ] . finally, in-depth research into the underlying mechanisms using knockout-gene mice for each differentially expressed gene will help further our understanding of the role of ast in ameliorating ali/ards. in conclusion, many genes were dysregulated in ali/ards. we not only identified genes that consistently differed in expression in the lps group compared to the control group but also revealed that ast can alleviate the abnormal expression of these genes and thus confer a certain therapeutic effect against ali/ards, suggesting the potential for ast to become a novel treatment for ali/ards. to identify the genes related to lps-induced acute lung injury in mice, five datasets (gse , gse , gse , gse , and gse ) were obtained from geo (gene expression omnibus, http://www.ncbi.nlm.nih.gov/geo) [ ] [ ] [ ] [ ] [ ] . lps and control treatments were used in this study. the detailed information (experimental design, transcriptome analysis, array information, data processing, and platform id) for these datasets can be obtained from the geo repository, and this information is partly aging summarized in table and is described in more detail in supplementary table . then, we conducted a microarray meta-analysis using networkanalyst . (https://www.networkanalyst.ca) [ ] . networkanalyst is a visual analytics platform for comprehensive gene expression profiling and metaanalysis. all gene probes were converted to a common entrez id using the gene/probe conversion tool in networkanalyst. following quantile normalization, all datasets were preprocessed through a log transformation and variance stabilizing normalization (vsn). each dataset was visualized in box plots to ensure an identical distribution among the samples. differential expression analysis was performed independently for each dataset using networkanalyst, with an fdr of . and a significance of p < . . the moderated t-test was based on the limma algorithm. for the meta-analysis, we used fisher's method, the fixed-effect model, and vote counting (combined p < . or vote counts ≥ were considered significant) to identify the differentially expressed genes (degs) and we selected the common degs identified by these three methods as the final degs. male c bl/ j mice ( - g, ~ -weeks-old) were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china). the mice were housed per cage under a h light/dark cycle in a laboratory at ± °c and % humidity. all experiment protocols conformed to the guidelines of the china council on animal care and use. these animal studies were approved by the institutional animal research committee of union hospital. the mice were randomly allocated into three groups: ( ) the control group (n= ), which was exposed to pbs alone and received an intraperitoneal injection of sterile saline; ( ) the lps group (n= ), which was exposed to pbs containing . mg/ml lps; and ( ) the ast group (n= ), which was intraperitoneally injected with ast ( mg/ml, dissolved in pbs) at a dosage of mg/kg body weight every day before one week of exposure to lps to evaluate its preventive and protective effects [ , ] , and intraperitoneally injected with mg/kg ast ( mg/ml, dissolved in pbs) hours after lps exposure in order to confirm the therapeutic effect of ast [ ] . ast was obtained from sigma-aldrich (st louis, mo, usa). for acute lps exposure, mice were exposed to an aerosol of phosphate buffer saline (pbs) alone or pbs containing . mg/ml lps for h, in a custom-built cuboidal chamber. the lps solution was aerosolized with a constant output ultrasonic nebulizer (model: b, yuwell, china) at a flow rate of ml/h. lps was purchased from sigma-aldrich (extracted from escherichia coli o : b , l ). the chamber was cm long, cm wide and cm high. total rna was extracted from mouse lung tissue samples with trizol® reagent (invitrogen, ca) following the manufacturer's protocol. the concentration and purity of the rna were measured by a nanodrop spectrophotometer (nanodrop technologies, technologies, wilmington, de, usa), the rna integrity was detected by agarose gel electrophoresis, and the rin was determined using an agilent bioanalyzer (agilent technologies, santa clara, ca, usa). the construction of a single library required a total of μg rna with a concentration of ≥ ng/μl and an od / ratio between . and . . then, oligo (dt) magnetic beads were subjected to capture mrnas that contained poly-a tails from the total rna. the resulting mrnas were subsequently randomly broken into small fragments of approximately bp by adding fragmentation buffer. the mrna fragments functioned as the templates for double-stranded cdna (dscdna) synthesis using the superscript double-stranded cdna synthesis kit (invitrogen, ca, usa). under the action of reverse transcriptase, a strand of cdna was synthesized by using random primers with mrna as a template, which was followed by two-strand synthesis to form a stable double-stranded structure. since there was a cohesive terminus in the double-stranded cdna structure, end repair mix was added to patch it into a blunt end, and an a base was added at the 'end to connect the yshaped adaptor. to purify and enrich the dscdna, cycles of pcr were performed, and clean dna beads were used to screen - bp bands. after quantification by tbs (picogreen, invitrogen, ca usa), high-throughput sequencing of the resulting libraries was performed on the illumina hiseq xten/novaseq sequencing platform (san diego, ca, usa), and the sequencing read length was pairedend (pe) . to ensure the accuracy of the subsequent biological information analysis, the raw sequencing data generated from rna-seq was firstly filtered to obtain high-quality sequencing data (clean data) to ensure the smooth progress of the subsequent analysis. quality control of the raw reads was performed using seqprep (https://github.com/jstjohn/seqprep) and sickle (https://github.com/najoshi/sickle). the processes were as follows. the first step was to remove the adapter aging from the reads and the reads that did not insert the fragment due to the self-connection of the adapter. second, bases with a low quality (quality value less than ) at the end of the sequence ( ' end) were trimmed. if there was still a quality value of less than for the remaining sequence, the whole sequence was discarded; otherwise, it was retained. third, reads with n ratios over % and sequences with lengths less than bp after quality trimming were also removed. finally, the error rate (%), q and q values, gc-content (%), and sequence duplication levels of the generated clean reads were assessed [ ] . after filtering the raw data, the clean data were aligned to the mouse reference genome grcm by 'bowtie ' software [ ] . then, read summarization was calculated by the 'feature count' tool. differently expressed genes (degs) between the lps samples and control samples were identified by t-test using the 'deseq ' r package, as were degs between the ast samples and lps samples [ ] . the raw p-value was adjusted to the false discovery rate by the benjamini method, and a false discovery rate (fdr) ≤ . and |log fc|≥ was chosen as the threshold. based on the hypergeometric distribution algorithm, go (gene ontology, http://www.geneontology.org/) biological process (bp), molecular function (mf) and cell component (cc) pathway enrichment analyses were performed using the 'clusterprofler' r package [ ] . a p-value ≤ . was set as the cutoff criterion. to validate the combined findings from rna-seq and microarray meta-analysis, the expression of core degs in the three groups was confirmed. rnaiso plus reagent (takara, tokyo, japan) was employed to extract total rna from mouse lung tissues from each group, and reverse transcription was performed to obtain cdna using primescript™rt master mix (takara, tokyo, japan) along with the gdna eraser kit (takara, tokyo, japan). relative mrna expression levels were determined using rt-pcr performed on bio-rad cfx maestro (bio-rad, usa) with tb green® premix ex taq™ ii (takara, tokyo, japan). all the above experimental steps were performed according to the manufacturer's instructions for the corresponding kit. glyceraldehyde- -phosphate dehydrogenase (gapdh) was selected as the reference, and the primer sequences are presented in supplementary table . qrt-pcr was performed under the following conditions: °c for min, followed by cycles at °c for s, °c for s, and °c for s. each analysis was implemented in triplicate, and the relative expression levels of the target genes were calculated by employing the -ΔΔct method [ ] . inmex and networkanalyst were applied for the network-based microarray meta-analysis. for qpcr data, statistical analysis of differences between groups was achieved by one-way anova using prism software (graphpad software inc., san diego, ca, usa). a twotailed test was used for all data, and differences with a p-value < . were considered significant. first of all, we used the search formula of lps[all fields] and ("lung"[mesh terms] or lung[all fields]) and ("mus musculus"[organism] and "expression profiling by array" [filter] ) to obtain results in geo datasets. by eliminating datasets of mirna sequencings, datasets not related to acute lung injury, and datasets that only researching on rna sequencing of specific cells such as macrophages and type ii alveolar epithelial cells, there were articles remained (gse , gse , gse , gse , gse , gse , gse and gse ). continuing to check the specific description of the sample in articles, we found some datasets were grouped with sample of n< and some mainly studied ali or ards induced by excessive ventilation or non-lps chemicals. in the end, there were datasets (gse , gse , gse , gse and gse ) that met the requirements of integrated analysis. we conducted a microarray meta-analysis using networkanalyst . combined three well-established meta-analysis approaches --fisher's method, fixed effect model, and vote counting. the features and main characteristics are given below (https://www.networkanalyst.ca). ( ) fisher's method (- *∑log(p)) is known as a 'weight-free' method and combines p values from multiple studies for information integration. ( ) effect size is the difference between two group means divided by standard deviation, which are considered combinable and comparable across different studies. in the fixed effects models (fem), the estimated effect size in each study is assumed to come from an underlying true effect size plus measurement error. ( ) vote counting is the simplest method in metaanalysis. differentially expressed gene is first selected based on a threshold to obtain a list of de genes for each study. the vote for each gene can then be calculated as the total number of times it occurred in all de lists. the final de genes can be selected based on the minimal number of votes set by the user. after the mice were sacrificed, the lung tissues were collected. immediately, the tissue was fixed in % paraformaldehyde for hours and embedded in paraffin. the embedded tissue was sliced into µm sections for staining. after the tissue sections were deparaffinized and rehydrated, they were heated in citrate buffer at °c for minutes to restore antigen activity. the sections were then incubated with . % hydrogen peroxide in methanol for minutes to inhibit endogenous peroxidase activity. after blocking nonspecific reactions with % normal bovine serum, the sections were incubated with rabbit polyclonal antibodies specific for ccl ( : , abcam), saa ( : , ab , abcam), ly i ( : , abcam), saa ( : - : , thermo), lrf ( : , thermo), timp ( : , thermo), isg ( : , thermo) and cxcl ( : , abcam). the treated samples were placed at °c for hours. the sections were then washed with pbs and incubated with horseradish peroxidase-conjugated secondary antibodies at °c for hours. the stained sections were imaged under an inverted phase contrast microscope. acute respiratory distress syndrome acute respiratory distress in adults. the lancet, saturday the acute respiratory distress syndrome lipopolysaccharide endotoxins cordycepin inhibits lps-induced acute lung injury by inhibiting inflammation and oxidative stress xanthohumol ameliorates lipopolysaccharide (lps)-induced acute lung injury via induction of ampk/gsk β-nrf signal axis linarin prevents lps-induced acute lung injury by suppressing oxidative stress and inflammation via inhibition of txnip/nlrp and nf-κb pathways curcumin and allopurinol ameliorate fructose-induced hepatic inflammation in rats via mir- a-mediated txnip/nlrp inflammasome inhibition troxerutin protects kidney tissue against bde- -induced inflammatory damage through cxcr -txnip/nlrp signaling astaxanthin: a review of its chemistry and applications astaxanthin, a carotenoid with potential in human health and nutrition biorefinery approach and environmentfriendly extraction for sustainable production of astaxanthin from marine wastes astaxanthin inhibits nitric oxide production and inflammatory gene expression by suppressing i(kappa)b kinase-dependent nf-kappab activation effects of astaxanthin on lipopolysaccharide-induced inflammation in vitro and in vivo astaxanthin prevents pulmonary fibrosis by promoting myofibroblast apoptosis dependent on drp -mediated mitochondrial fission astaxanthin alleviated acute lung injury by inhibiting oxidative/nitrative stress and the inflammatory response in mice astaxanthin prevents against lipopolysaccharideinduced acute lung injury and sepsis via inhibiting activation of mapk/nf-κb networkanalyst . : a visual analytics platform for comprehensive gene expression profiling and metaanalysis clusterprofiler: an r package for comparing biological themes among gene clusters inflammatory cytokines in patients with persistence of the acute respiratory distress syndrome neutrophils in the initiation and resolution of acute pulmonary inflammation: understanding biological function and therapeutic potential evidence for chemokine synergy during neutrophil migration in ards fernández-botrán r. modulation of acute inflammation by targeting glycosaminoglycan-cytokine interactions contribution of neutrophils to acute lung injury antiinflammatory activity of a novel family of aryl ureas compounds in an endotoxin-induced airway epithelial cell injury model c-x-c motif chemokine (cxcl ) is a prognostic biomarker of idiopathic pulmonary fibrosis zbp : innate sensor regulating cell death and inflammation dynamic gene expression analysis in a h n influenza virus mouse pneumonia model the cc chemokine ligand (ccl ) mediates fibroblast survival through il- complement inhibition decreases early fibrogenic events in the lung of septic baboons the cytokine-serum amyloid achemokine network serum amyloid a is a biomarker of severe coronavirus disease and poor prognosis serum amyloid a is a high density lipoprotein-associated acute-phase protein exosomes derived from microrna- b- p-overexpressing mesenchymal stem cells protect against lipopolysaccharide-induced acute lung injury by inhibiting saa long non-coding rna malat regulates hyperglycaemia induced inflammatory process in the endothelial cells il- induced lncrna malat enhances tnf-α expression in lps-induced septic cardiomyocytes via activation of saa emerging functions of serum amyloid a in inflammation suppression of lipopolysaccharide-induced inflammatory response by fragments from serum amyloid a serum amyloid a promotes lps clearance and suppresses lps-induced inflammation and tissue injury knockdown of mir- protects nucleus pulposus cells from tnf-a-induced apoptosis by targeting serum amyloid a irf : activation, regulation, modification and function attenuation of interferon regulatory factor activity in local infectious sites of trachea and lung for preventing the development of acute lung injury caused by influenza a virus mir- c mediates influenza a virus-induced ifnβ expression by targeting nf-κb inducing kinase mir- attenuates the host response to influenza virus by targeting the traf -irf signaling axis cytokine functions of timp- imbalance between matrix metalloproteinases (mmp- and mmp- ) and tissue inhibitors of metalloproteinases (timp- and timp- ) in acute respiratory distress syndrome patients serum mmp- and timp- in critically ill patients with acute respiratory failure: timp- is associated with increased -day mortality timp- promotes the immune response in influenza-induced acute lung injury isg in antiviral immunity and beyond lipopolysaccharide activates the expression of isg -specific protease ubp via interferon regulatory factor extremes of interferon-stimulated gene expression associate with worse outcomes in the acute respiratory distress syndrome role of chemokines in the pathogenesis of acute lung injury proteinaseactivated receptor- , ccl , and ccl regulate acute neutrophilic lung inflammation extracellular atp mediates the late phase of neutrophil recruitment to the lung in murine models of acute lung injury molecular dynamics of lipopolysaccharide-induced lung injury in rodents modulation of lipopolysaccharideinduced gene transcription and promotion of lung injury by mechanical ventilation bpifa regulates lung neutrophil recruitment and interferon signaling during acute inflammation altered gene expression profiles in the lungs of benzo[a]pyreneexposed mice in the presence of lipopolysaccharideinduced pulmonary inflammation effects of age on the synergistic interactions between lipopolysaccharide and mechanical ventilation in mice clara cells attenuate the inflammatory response through regulation of macrophage behavior astaxanthin suppresses cigarette smokeinduced emphysema through nrf activation in mice fastp: an ultra-fast all-inone fastq preprocessor fast gapped-read alignment with bowtie moderated estimation of fold change and dispersion for rna-seq data with deseq analysis of relative gene expression data using real-time quantitative pcr and the (-delta delta c(t)) method the authors declare that they have no conflicts interest. please browse full text version to see the data of supplementary tables to key: cord- -sut nf a authors: wang, shuang; zhao, jijun; wang, hongyue; liang, yingjie; yang, niansheng; huang, yuefang title: blockage of p x attenuates acute lung injury in mice by inhibiting nlrp inflammasome date: - - journal: int immunopharmacol doi: . /j.intimp. . . sha: doc_id: cord_uid: sut nf a nlrp inflammasome is engaged in the inflammatory response during acute lung injury (ali). purinergic receptor p x has been reported to be upstream of nlrp activation. however, the therapeutic implication of p x in ali remains to be explored. the present study used lipopolysaccharide (lps)-induced mouse model to investigate the therapeutic potential of p x blockage in ali. our results showed that p x /nlrp inflammasome pathway was significantly upregulated in the lungs of ali mice as compared with control mice. p x antagonist a suppressed nlrp /asc/caspase activation, production of il- β, il- a and ifn-γ and neutrophil infiltration but not the production of il- , resulting in a significant amelioration of lung injury. moreover, blockage of p x significantly inhibited nlrp inflammasome activation and il- β production in bone marrow derived macrophages. similar results were obtained using another p x inhibitor brilliant blue g (bbg) in vivo. thus, pharmacological blockage of p x /nlrp pathway can be considered as a potential therapeutic strategy in patients with ali. acute lung injury (ali) or a more severe ali defined as acute respiratory distress syndrome (ards) is the major cause of hypoxemic respiratory failure in adults during hospitalization with high morbidity and mortality [ ] . many conditions, such as trauma, infection and sepsis could lead to ali [ ] . despite the diversity in etiology, inflammation takes an indispensable part in the process of ali. although many medical therapies are used to treat ali/ards, there is still an in-hospital mortality rate over % [ ] . thus, development of novel therapeutic strategies especially targeting the overwhelming inflammation in ali remains urgent. nlrp is the best characterized family member of nod-like receptors (nlrs) that are involved in innate and adaptive immune responses by composing inflammasomes with other intracellular molecules such as inflammatory caspases and apoptosis-associated speck-like protein containing a card (asc) [ , ] . briefly, upon activation, nlrp interacts with asc which bridges nrlp to procaspase and allows for activation of caspase . once activated, caspase can cleave the proforms of il- β and il- into their mature and active forms [ ] . in the lungs, nlrp inflammasome activation and il- β production are augmented with lps challenge in the ali mice [ ] . p x is a membrane protein which can be activated by extracellular atp and act as upstream of nlrp inflammasome to mediate the production of il- β [ , ] . lps administration increased p x expression in the lung parenchyma and p x −/− mice showed decreased polymorphonuclear cell infiltration, less inflammatory cytokine production and reduced collagen deposition [ ] . however, it is still unknown how p x contributes to the pathogenesis of ali. in this study, we demonstrated that pharmacological blockade of p x by using selective antagonists effectively ameliorated ali in mice via inhibiting nlrp inflammasome pathway. male c bl/ mice were purchased and housed under specific pathogen-free conditions in the experimental animal center at sun yat-sen university, guangzhou, china. experiments were approved by the ethics committee of sun yat-sen university. mice were kept in these pathogen-free facilities and the experiments were performed in accordance with the national institutes of health guide for care and use of animals. lps-induced ali model was created as described previously [ ] . male c bl/ mice ( - weeks of age) were anesthetized with intraperitoneal international immunopharmacology ( ) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ketamine ( mg/kg) and xylazine ( mg/kg). mg/kg of lps (sigma-aldrich, st. louis, mo, usa) was delivered in μl of phosphatebuffered saline (pbs) to the lungs via a -gauge angiocath catheter used as an endotracheal tube. the animals were rolled from side to side after administration to ensure even distribution. the normal control mice were given intratracheal instillation of pbs only. bmdms were generated from c bl/ mice as previously described [ ] . briefly, femora and tibiae were flushed with cold rpmi and cells were suspended in rpmi medium containing % fetal calf serum (hyclone, logan, utah, usa). cells were then cultured in a humidified atmosphere containing % co at °c with ng/ml macrophage colony stimulating factor (m-csf, life technologies, grand island, ny, usa) at × /ml in culture medium for days. the medium was changed every days. pe-conjugated mouse anti-cd b and fitcconjugated mouse anti-f / (both from ebioscience, san diego, ca, usa) were used to confirm n % purity of bmdms by flow cytometric analysis. bmdms were primed with μg/ml lps in the presence or absence of a ( mm) for h followed by stimulation with atp ( mm) for h. following different interventions, cell supernatants were collected for detection of il- β by elisa (r&d systems, minneapolis, usa) according to the manufacturer's instructions. cells were subjected to western blot analysis of nlrp inflammasome activation. a (abcam, cambridge, uk) was diluted at mg/ml in saline. as previously described [ ] , mice were treated intraperitoneally with a ( mg/kg) every h while the first injection began at h before lps administration. groups of mice were sacrificed h after lps administration. the bronchoalveolar lavage (bal) fluid was collected for cell counts, protein quantification and enzyme-linked immunosorbent assay (elisa). lung tissues were collected for histology, immunohistochemistry, elisa and western blot analysis. another p x antagosist brilliant blue g (bbg, sigma-aldrich, st. louis, mo, usa) was also used in this study. bbg was diluted at mg/ml in vehicle (saline) solution. mice were treated intraperitoneally with bbg ( . mg/kg) every h while the first injection began at h before lps administration, as previously described [ ] . mice were sacrificed h after lps administration. bronchoalveolar lavage (bal) was performed as previously described [ ] . briefly, mice were anesthetized and sacrificed by heart puncture after opening the thoracic cavity. the trachea was exposed and an g sterile needle with blunt end was inserted into the trachea through a small semi-excision. . ml pbs was injected and withdrawn for the first lavage. the lavage procedure was repeated twice. a total volume of . ml bal fluid per mouse was collected. the bal fluid was centrifuged at g for min to pellet cells. supernatants were collected for elisa and total protein analysis. the pelleted cells were resuspended in a small volume of serum-free dmem culture medium (life technologies, grand island, ny, usa). total cell counts were determined on a grid hemocytometer. differential cell counts were enumerated on cytospin-prepared slides that were stained with wright-giemsa stain. a total of cells were counted in cross-section per sample, and the number of neutrophil was calculated as the total cell count times the percentage of neutrophil in the balf sample. total protein level was determined by using bca protein assay kit (thermo fisher scientific, rockford, il, usa) according to the manufacturers' instructions. il- β, il- , il- a and ifn-γ in lung tissues and bal fluid were measured using elisa kits for mouse il- β and il- (r&d systems, minneapolis, usa) and elisa ready-set-go kit for mouse il- a and ifn-γ (ebioscience, san diego, ca, usa), respectively, following the manufacturers' instructions. paraformaldehyde-fixed, paraffin-embedded lung tissues were sectioned for hematoxylin and eosin staining and immunohistochemical staining of f / (abcam, cambridge, uk) and il- β (santa cruz biotechnology, dallas, texas, usa). sections were counterstained with hematoxylin. the number of f / + cells in the lung was counted in consecutive fields under high power fields (hpf) (× ) and expressed as cells/hpf. proteins from lung tissues were extracted with cell lysis buffer (cell signaling technology, beverly, ma, usa) and analyzed by western blotting as described previously [ ] . the primary antibodies used in this study included: mouse anti-nlrp (adipogen, san diego, ca, usa), rabbit anti-p x antibodies (abcam, cambridge, uk), mouse anti-caspase -p (adipogen), rabbit anti-asc (adipogen) and anti-gapdh antibodies (santa cruz biotechnology). hrp conjugated antimouse and anti-rabbit igg (both from cell signaling technology) were used as secondary antibodies. signals were detected with enhanced chemiluminescence (cell signaling technology). data were presented as mean ± sem. statistical analyses were performed using one-way anova. all data were analyzed using spss software (version . ). p values less than . were considered significant. expression of the p x /nlrp inflammasome pathway was examined in the mouse model of lps-induced lung injury. enhanced protein expression of p x , nlrp , and asc was observed in the lungs from lps-induced lung injury mice compared with control mice treated by pbs (fig. ) . the active caspase -p subunit represents the highly active p /p tetrameric forms of processed caspase . expression of the p subunit of caspase was significantly elevated in the lungs from lps-induced lung injury mice compared with normal control mice (fig. ) . the effects of the selective p x inhibitor a on activation of the nlrp inflammasome were tested. expression of p x , nlrp , asc and caspase -p in lung tissue was significantly downregulated by a treatment as demonstrated by western blot analysis (fig. ) . however, expression of pro-caspase which is the precursor form of active caspase p was not influenced by a . the effects of a on lung injury induced by lps were investigated by histological examination with h&e staining and immunohistochemical staining of f / . as shown in fig. a , inflammatory cell infiltration was observed in the pulmonary interstitium of lpsinduced lung injury mice and was significantly reduced by a treatment. in addition, large number of f / + macrophages was also detected in the lps-induced injured lungs and was significantly reduced by a treatment (fig. b and c) . to further investigate the effect of a on lung inflammation, bal fluids were collected for cell counts (fig. b and c) , wright-giemsa staining (fig. a) and total protein analysis (fig. d) . in normal control mice, there were very few cells observed in the bal fluid and almost % were alveolar macrophages. however, over % cells were neutrophils observed in the bla fluids from the lps-induced lung injury mice. in contrast, a significantly reduces neutrophil and total cell number. coincident with the cell counts, the total protein level was also elevated in the lps-induced lung injury group, which was significantly reduced by a treatment (fig. d) . il- β is an important downstream molecular modulated by p x / nlrp inflammasome pathway and acts as pivotal inflammatory factor in the lps-induced lung injury. il- β expression in lung tissue and bal fluid was apparently enhanced in the lps-induced lung injury mice compared with normal control. the selective p x inhibitor a significantly inhibited il- β production as measured by elisa (fig. b and c ). similar results were confirmed by immunohistochemistry staining of il- β in lung sections as shown in fig. a. . . a reduced ifn-γ and il- a level but not il- level in both lung tissue and bal fluid ifn-γ and il- a were also examined by elisa in this study. the concentrations of ifn-γ and il- a were remarkably enhanced in the lung tissue and bal fluids of lps-induced lung injury mice compared with normal control mice. furthermore, a treatment significantly suppressed ifn-γ ( fig. a and d) and il- a ( fig. b and e) expression. il- considered as an anti-inflammatory cytokine was also remarkably upregulated in the lung tissue and bal fluids of ali mice. however, il- level was not altered by a treatment (fig. c and f) . in vitro, bone marrow derived macrophage was used to test the inhibitory effects of a (fig. ) . expression of nlrp and asc and activation of caspase as evidenced by the increase of caspase p as well as il- β production were enhanced with treatment of lps and atp. enhanced expression of nlrp and asc and activation of caspase as well as il- β production were significantly inhibited by a . in contrast, expression of pro-caspase which is the precursor form of active caspase p was not influenced by a . to further confirm that the in vivo effect of p x blockage is mediated through inhibition of nlrp pathway, we treated ali mice with another selective p x inhibitor, bbg. similar inhibitory effects on nlrp inflammasome activation and il- β production were observed as shown in fig. . inhibition of nlrp pathway resulted in reductions of inflammation (fig. a) and macrophage infiltration (fig. b) in the lungs of ali mice. the total cell count (fig. c) and protein level (fig. d) in bal fluids were also significantly reduced by bbg treatment. inflammation is the hallmark in the development of ali. however, the initiation and modification of this critical process are still beyond clear interpretation. lps-induced lung injury mouse model was used in the present study which is manifested as damage of alveolar walls, neutrophil accumulation and release of inflammatory cytokines [ ] . this study showed that blockade of p x effectively ameliorated ali via inhibiting nlrp inflammasome pathway. p x is expressed by many types of cells such as monocyte/ macrophage [ ] , dendritic cells [ ] , lymphocytes [ ] , endothelial cells [ ] , mast cells [ ] , eosinophils [ ] and alveolar type i epithelial cells [ ] . increasing data show p x is correlated with inflammation. activation of p x in human macrophages triggers the release of il- β and il- [ ] . p x null mice are protected from ali induced by lps [ ] . in this study, p x expression was significantly enhanced at the protein level in the lung tissues from ali mice, paralleled with alveolar damage and inflammatory cytokine production. to dissect the functional role of p x in ali, a selective p x antagonist a was used to treat the ali mice. we found that blockage of p x inhibited the activation of nlrp inflammasome pathway, neutrophil accumulation and production of proinflammatory cytokines, resulting in reduction of lung damage. il- β is an important mediator of the inflammatory response, which is proteolytically processed to its active from by caspase . the critical link between p x , il- β and nlrp has been brought to light recently. p x has been reported as a trigger for nlrp activation [ ] . activation of nlrp results in assembly of the nlrp /asc/caspase complex which facilitates caspase -mediated processing and release of the proinflammatory cytokine il- β [ ] . previous studies have shown that nlrp and caspase regulate the inflammatory process in many lung diseases such as ali [ , ] , chlamydia pneumoniae infection [ ] , acute allergic airway inflammation [ ] , rituximab-induced interstitial lung disease (r-ild) [ ] and late lung fibrosis [ ] . in our study, nlrp was activated in the lungs of ali mice accompanied with enhanced expression of p x . blockade of p x resulted in inhibition of nlrp /asc/caspase activation, suppression of il- β production and attenuation of lung injury, all of which indicates that the p x /nlrp pathway is involved in the pathogenesis of ali. il- is another indispensable proinflammatory cytokine produced by t-helper cells (th ) and is upregulated by il- β, il- , il- and il- [ ] . il- acts as a potent mediator in autoimmunity and neutrophil inflammation by increasing chemokine production in various tissues to recruit monocytes and neutrophils to the site of inflammation. il- a could regulate cxc chemokine and g-csf production which is necessary for neutrophil differentiation, activation and recruitment [ , ] . il- producing γδ t and th lymphocytes are reported to mediate lung inflammation in experimental silicosis while il- r-deficient animals or il- a antibody neutralization reduced neutrophil influx and lung injury [ ] . il- is also found to mediate ali induced by influenza a (h n ) virus [ ] . in our study, il- a was markedly elevated in the lung tissues and bal fluids of ali mice as demonstrated by elisa analysis. blockade of p x with a significantly reduced il- production as well as neutrophil accumulation in conjunction with inhibition of nlrp pathway. in addition, ifn-γ is a key contributor to human pulmonary injury and to the viral-storm during sars coronavirus infection and in acute respiratory distress syndrome (ards) [ ] [ ] [ ] . our data showed that blockade of p x also suppressed the production of ifn-γ. in this study, the expression of the nlrp inflammasome components is upregulated in ali mice. such an increase in inflammasome components might be simply due to the increased population of innate immune cells in the lung tissues during ali. therefore, the suppressed expression of nlrp inflammmasome components by p x treatment might reflect the reduced infiltration of innate immune cells. however, the activation of nlrp inflammasome was also significantly inhibited by p x treatment, as evidenced by reduced cleavage of caspase , suggesting that therapeutic effects of p x blockage is mediated through inhibition of nlrp activation rather than non-specific reduction of inflammatory infiltration. this was confirmed by our in vitro study using bmdms, which showed over % decline in caspase activation with the treatment of p x antagonist. activation of the nlrp pathway is proposed to require two signals. signal for priming is a cytokine or toll-like receptor ligand such as lps, which trigger the synthesis of nlrp . signal is the activation signal such as atp, nigericin or monosodium urate, which acts through the p x receptor [ , ] . in our study, bmdms were primed with lps in the presence or absence of a for h followed by stimulation with atp for h. it is surprising to note that a not only inhibited nlrp activation but also decreased the expression of nlrp and asc. this finding suggests that a also has other unspecified effects during the priming. indeed, a recent study has shown that treatment with a inhibits erk / phosphorylation [ ] . since nf-κb is required for the synthesis of nlrp induced by lps [ ] , inhibition of erk (upstream of nf-κb [ ] ) may reduce the expression of nlrp . in conclusion, the results of this study identify a new target for ali therapy. our data have shown that blockage of p x signaling effectively ameliorated ali by inhibiting nlrp inflammasome activation. therefore, targeting the p x /nlrp signaling pathway would be a potential therapeutic strategy for human ali. incidence and outcomes of acute lung injury spectrum of acute lung injury, ards, sepsis the control of adaptive immune responses by the innate immune system the inflammasome: an integrated view the machinery of nod-like receptors: refining the paths to immunity and cell death hemorrhagic shock augments nlrp inflammasome activation in the lung through impaired pyrin induction the p x receptor: a key player in il- processing and release lipopolysaccharide-induced lung injury: role of p x receptor electroporation-mediated gene transfer of the na +, k+-atpase rescues endotoxin-induced lung injury lupus nephritis: glycogen synthase kinase beta promotes renal damage through activation of nlrp inflammasome in lupus-prone mice purinergic receptor antagonist a protects against acetaminophen-induced liver injury by inhibiting p isoenzymes, not by inflammasome activation altered p x -receptor level and function in mouse models of huntington's disease and therapeutic efficacy of antagonist administration hemorrhagic shock primes for increased expression of cytokine-induced neutrophil chemoattractant in the lung: role in pulmonary inflammation following lipopolysaccharide p x blockade attenuates murine lupus nephritis by inhibiting activation of the nlrp /asc/caspase pathway nucleotide receptors: an emerging family of regulatory molecules in blood cells pharmacologic properties of p( z)/p x( )receptor characterized in murine dendritic cells: role on the induction of apoptosis purinergic receptor distribution in endothelial cells in blood vessels: a basis for selection of coronary artery grafts atp-induced pore formation in the plasma membrane of rat peritoneal mast cells p purinergic receptors of human eosinophils: characterization and coupling to oxygen radical production identification of two novel markers for alveolar epithelial type i and ii cells atp acts as an agonist to promote stimulus-induced secretion of il- beta and il- in human blood liaisons dangereuses: p x( ) and the inflammasome nonclassical il- beta secretion stimulated by p x receptors is dependent on inflammasome activation and correlated with exosome release in murine macrophages nlrp inflammasome activation is essential for paraquat-induced acute lung injury critical role for the nlrp inflammasome during acute lung injury inflammation and fibrosis during chlamydia pneumoniae infection is regulated by il- and the nlrp /asc inflammasome functional relevance of nlrp inflammasome-mediated interleukin (il)- beta during acute allergic airway inflammation involvement of nlrp inflammasome in rituximab-induced interstitial lung disease: a case report the role of the nlrp inflammasome in fibrosis directing traffic: il- and il- coordinate pulmonary immune defense the biological functions of t helper cell effector cytokines in inflammation th cell differentiation: the long and winding road il- a-producing gammadelta t and th lymphocytes mediate lung inflammation but not fibrosis in experimental silicosis il- response mediates acute lung injury induced by the pandemic influenza a (h n ) virus plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome an interferon-gammarelated cytokine storm in sars patients a probable role for ifn-gamma in the development of a lung immunopathology in sars cutting edge: nf-kappab activating pattern recognition and cytokine receptors license nlrp inflammasome activation by regulating nlrp expression cutting edge: tnf-alpha mediates sensitization to atp and silica via the nlrp inflammasome in the absence of microbial stimulation p x receptor inhibition protects against ischemic acute kidney injury in mice extracellular-regulated kinase / , jun n-terminal kinase, and c-jun are involved in nf-kappa b-dependent il- expression in human monocytes this project was supported by grants from the science and technology all authors declare no conflicts of interest in this study. key: cord- -ksrxsdpp authors: shirato, kazuya; kawase, miyuki; matsuyama, shutoku title: wild-type human coronaviruses prefer cell-surface tmprss to endosomal cathepsins for cell entry date: - - journal: virology doi: . /j.virol. . . sha: doc_id: cord_uid: ksrxsdpp human coronaviruses (hcovs) enter cells via two distinct pathways: the endosomal pathway using cathepsins to activate spike protein and the cell-surface or early endosome pathway using extracellular proteases such as transmembrane protease serine (tmprss ). we previously reported that clinical isolates of hcov- e preferred cell-surface tmprss to endosomal cathepsin for cell entry, and that they acquired the ability to use cathepsin l by repeated passage in cultured cells and were then able to enter cells via the endosomal pathway. here, we show that clinical isolates of hcov-oc and -hku preferred the cell-surface tmrrss to endosomal cathepsins for cell entry, similar to hcov- e. in addition, the cell-culture-adapted hcov-oc lost the ability to infect and replicate in air-liquid interface cultures of human bronchial tracheal epithelial cells. these results suggest that circulating hcovs in the field generally use cell-surface tmprss for cell entry, not endosomal cathepsins, in human airway epithelial cells. human coronaviruses (hcovs) are causative agents of human common colds, and most people experience initial infection during childhood (dijkman et al., ) . to date, four hcovs ( e, nl , oc and hku- ) have been identified. the first two belong to alphacoronavirus and the latter two to betacoronavirus. hcov- e was first reported in (hamre and procknow, ) and the isolate of the day is widely used as the laboratory strain of the american type culture collection (atcc, vr- ). hcov- e enters cells via two distinct pathways: the endosomal pathway using cysteine protease to activate spike (s) protein and the cell-surface or early endosome pathway using extracellular proteases for activation. studies using the atcc isolate suggest that hcov- e enters cells via the late endosome using cathepsin l to cleave s protein, although it can enter cells via the cell surface or early endosome in the presence of transmembrane protease serine (tmprss ) or trypsin (bertram et al., ; kawase et al., ) . it has been thought that this is a general feature of hcov- e entry. however, we recently reported that clinical isolates of hcov- e preferred the cell-surface or early endosome pathway to the late endosome pathway for cell entry (shirato et al., ) . the ability of a clinical isolate of hcov- e to use cathepsin l was originally low, but it became able to use cathepsin after repeated passage in hela cells (shirato et al., ) . this suggests that in natural situations hcov- e enters respiratory epithelial cells via the cellsurface or early endosome route and that the character of atcc vr- has been changed by repeated passage in cultured cells. in recent years, a culture method for human bronchial tracheal epithelial (hbte) cells that uses an air-liquid interface (ali) has been developed and has come to be used as a model of the in vivo situation of human airway epithelium (hae) (fulcher et al., ) . using this system, hcov-hku , which had previously been reported from sequence data, was first isolated (pyrc et al., ) , and it was later shown that all four hcovs could be isolated upon hbte-ali cell culture . in the present study, we found that field isolates of hcov-oc and hcov-hku could be isolated using hbte-ali cell culture, and we then used these clinical isolates to assess whether the mode of virus entry found in hcov- e was also in play in other hcovs. for isolation of hcovs, nasal swabs were collected from outpatients who showed respiratory infection as a cardinal symptom when assessed at a hospital in tokyo, japan. specimens diagnosed as containing hcovs by real-time rt-pcr were used for virus isolation. recently, the circulating hcov strain in japan has been biased toward hcov-oc (hara and takao, ; matoba et al., ; yano et al., ) . therefore, we did not obtain specimens positive for hcov- e or hcov-nl ; however, four hcov-oc and two hcov-hku isolates were finally isolated successfully (table ). the sequences of s protein were analyzed and deposited in genbank (accession nos. lc to lc ). the number of cycles of real-time rt-pcr to which virusisolated specimens were subjected ranged from . to . , and isolation was considered failed when more than cycles were used (data not shown). this suggests that at least copies of viral rna are required for successful isolation by hbte-ali culture. similar to hcov- e, hcov-oc was identified between the late s and (bruckova et al., ; mcintosh et al., a mcintosh et al., , b . the isolate at that time had been passed in suckling mouse brain and cell culture, and finally became widely used as a laboratory strain (atcc vr- ). as previously described, the entry mechanism of the clinical isolate of hcov- e differed from that of the cell-cultureadapted laboratory isolate, atcc-vr ; the clinical isolate preferred the cell-surface tmprss route to the endosomal cathepsin route preferred by the atcc isolate (shirato et al., ) . therefore, we evaluated differences in infectivity and replication in culture cells or hbte-ali culture using vr- and the clinical isolate (sgh- / ) (fig. ) . in hct- cells, vr- infected and replicated well, as expected. on the other hand, the amounts of sgh- / virus entering hct- cells were much lower than the amounts of vr- , and replication of sgh- / was about lower than that of vr- (fig. a) . in hbte-ali culture, the result was completely reversed; sgh- / infected the cells and replicated well as expected. in contrast, vr- could infect the hbte-ali culture. though the amount of entered virus was much lower compared to sgh- / , no viral rna was detected in the cell wash days after infection (fig. b) . this indicates that vr- infected the hbte-ali culture with low efficiency, but it failed to produce progeny virus. in the previous report, a -times-passaged clinical isolate of hcov- e exhibited changes that allowed it to replicate well in hela cells, but its ability to replicate in hbte-ali culture decreased relative to that of the original isolate (shirato et al., ) . the present study indicates that the phenomenon seen in hcov- e is also present for hcov-oc ; namely, cell-culture-adapted virus cannot replicate in hbte-ali culture, which mimics human airway epithelial cells. in the case of hcov- e, the clinical isolate enters cells via the cell-surface or early endosome route using tmprss , and the atcc isolate vr- tends to enter cells via the late endosome pathway using cathepsin l (bertram et al., ; kawase et al., ; shirato et al., ) . the former route can be inhibited by the serine protease inhibitor camostat, and the latter by the cysteine protease inhibitor est (e d) shirato et al., shirato et al., , . to evaluate the entry routes of clinical isolates of hcovs, viruses were inoculated onto hbte-ali in the presence of est or camostat ( μm) and the amounts virus that entered were estimated by detecting subgenomic mrnas using real-time rt-pcr (fig. ) . first, the effect of inhibitor treatment on cell viability was determined in all cells. the treatments did not affect survival of the cell cultures at this concentration (fig. a) . infection of hcov-oc , vr- , was inhibited only by est and not by camostat (fig. b ). this indicates that vr- enters cells via the late endosome pathway using cysteine proteases and it does not use tmrpss for cell entry in hbte-ali culture. in contrast, clinical isolates of hcov-oc were inhibited by camostat but were either not inhibited or were only partially inhibited by est ( fig. c-f ). in the case of sgh- / , est increased entry (fig. c) . for clinical isolates of hcov-hku (similar to hcov-oc ), entry was inhibited only by camostat and est tended to increase entry ( fig. g and h) . as shown in fig. a , treatment with inhibitors did not affect cell viability. this suggests that the reduced infection of vr- by est and clinical isolates by camostat were induced by the inhibition of proteases, not k. shirato et al. virology ( ) - inhibitor treatment. these data are identical to those derived using hcov- e (shirato et al., ) and suggest that the clinical isolates of hcovs that cause human infections enter cells via the cell-surface or early endosome pathway using cell-surface proteases such as tmrss . the data also suggest that entry of cells via the late endosome using cathepsins is the case only for cell-culture-adapted or long-term-passaged isolates of hcovs. moreover, the increased entry of some clinical isolates in the presence of est suggests that entry of cells via the late endosomal pathway induces some anti-viral host immunity. the expression of tmrss in hbte-ali and hct- cultures was confirmed by mrna quantitation (fig. a) . the expression of tmrpss mrna was found in calu- and lovo cells, which are known to be susceptible to middle east respiratory syndrome (mers) coronavirus (shirato et al., ; tao et al., ) . tmrpss expression was highly up-regulated and was prominent among cells tested in differentiated hbte-ali cultures. in hct- cells, tmrpss mrna was detected, but the expression level was lower than in immature hbte cells. almost no tmrpss was detected in hela cells. this suggests that the reduced infection and replication of hcov-oc clinical isolates in hct- cells were caused by the low level of tmprss expression relative to hbte-ali cultures. that cathepsins use hcov-oc for entry into hct- cells was confirmed by an entry inhibition assay similar to that in hbte-ali cultures ( fig. b and c) . the entry of vr- in hct- was inhibited only by est treatment at both and μm (fig. b) . in sgh- / infection, a high concentration ( μm) of est inhibited entry (fig. c) . in contrast, camostat treatment did not show any inhibition in terms of vr- and sgh- / in hct- infection; however, hct- k. shirato et al. virology ( ) - cells expressed tmprss mrna. these findings suggest that the expression of tmprss in hct- cells is insufficient for hcov-oc entry from the cell surface, and also suggest that cathepsins are used for hcov-oc entry into hct- cells. for many years, isolation of hcovs was considered difficult (peiris and poon, ; tyrrell and myint, ) . the first isolation of hcov- e and hcov-oc was reported in the mid- s to using repeated passage in cultured cells or suckling mouse brains (bruckova et al., ; hamre and procknow, ; mcintosh et al., a mcintosh et al., , b . later, some studies reported isolation of hcovs using llc-mk cells, monkey kidney cells, and human embryo fibroblasts van der hoek et al., ) . our group and others reported that addition of an external protease, such as trypsin, and a transmembrane serine protease, tmprss , enhanced the entry and replication of hcovs (bertram et al., ; kawase et al., kawase et al., , . for hcov- e, addition of these proteases resulted in successful isolation (hirokawa et al., ; matoba et al., ) . however, no reliable cell line for isolation was available, unlike (for example) the mdck cells used to isolate influenza viruses. the results of our present study show that the reported difficulty in isolating hcovs is a natural phenomenon because hcovs circulating in the natural world do not enter cells via the late endosome pathway using cathepsins. it is difficult for the viruses to grow in commonly used cultured cells, which have only the late endosomal pathway. to date, some hcovs have been isolated using cell culture as described above, but it may be that these results were rather accidental. sometimes, particular features of the cultured cells may have affected the results, and, at other times, quasispecies may have been selected because of their ability to use cathepsins for cell entry. such forced selection does indeed produce hcovs, but the character of the viruses is changed from their natural state. in this study, inhibition of the late endosomal pathway by est increased the entry of some clinical virus isolates. this suggests that cell entry via the late endosomal pathway induces some anti-viral host immunity. indeed, bertram et al. ( ) reported that expression of interferon-induced transmembrane proteins (ifitms) that inhibit diverse enveloped viruses also inhibited the entry of pseudotyped virus bearing hcov- e s protein, and that the reduced entry was rescued by co-expression of tmrpss . it is thought that ifitms exert antiviral effects at the late stage of entry (desai et al., ; li et al., ; munoz-moreno et al., ) . thus, entering cells via endosomal cathepsins seems not to afford advantages for virus survival. therefore, hcovs circulating in the field may have evolved to not use the late endosomal route with cathepsins to avoid such unfavorable reactions by host cells. in addition, hcov-oc used cathepsins for entry into the cultured cell line hct- . however, in this study est treatment could not inhibit viral entry completely, even at a high concentration ( μm). previously, we reported that the sirna-mediated gene knockdown of cathepsin l could not inhibit the entry of hcov- e completely, suggesting that other proteases are also used by hcov- e in the process of infection (kawase et al., ) . moreover, treatment with est, camostat, and leupeptin in wi- and mrc- cells (human fetal pulmonary fibroblasts without tmprss expression), did not inhibit infection by mers-cov and hcov- e. this suggested that proteases other than cysteine, serine, and threonine might be used for entry into these immature cells (shirato et al., ) . therefore, although cathepsins are used during the entry process of hcov-oc in hct- cells, proteases other than cathepsins and tmrpss are also used for the activation of s protein (like other human coronaviruses). development of the hbte-ali cell culture method allowed successful isolation of hcovs, probably in their natural state pyrc et al., ) . for the development of antiviral drugs, it is necessary to understand virus characteristics, and isolation of the virus is the first step. therefore, it is important to isolate a virus that has natural characteristics. madu et al. ( ) reported that a common motif (very highly conserved region, vhcr) is found in the s protein of coronavirus and may be a possible fusion peptide. it was thought that recognition sites for cleavable proteases might exist around the fusion peptide. in a previous study, we showed that the two amino acids that differ between laboratory and clinical isolates of hcov- e (r m and n k) were the determinants of the difference in cell entry route. these sandwich the vhcr, and were therefore not thought to be protease recognition sites. however, substitution of these two amino acids between the laboratory and clinical isolates completely changes (inverts) their cathepsin l use during cell entry (shirato et al., ) . this implies that some structural modification of s protein caused by the two amino acid mutations affects the accessibility of cathepsin l to its true recognition site. as shown in fig. , only two remarkable amino acid differences were seen between the laboratory and clinical isolates of hcov-oc (g r and l p). the g r mutation is in the furin recognition site, and it may affect sensitivity to furin. park et al. ( ) reported that mers coronavirus had different entry processes dependent on whether s protein was cleaved by furin. if s protein was cleaved by furin before cell entry, entry was sensitive to cell-surface proteases and mers entered the cell by the early endosome. yet the virus that had un-cleaved s protein was trafficked through cellsurface proteases and into the late endosome. they also reported that these un-cleaved viruses were less infectious to human airway epithelial and calu- cell cultures (park et al., ) . indeed, all clinical isolates of hcov-oc retain the furin cleavage site (fig. ) , and the data from this study show that their infectivity was dependent on the expression of tmprss . this idea supports the findings in this study because the clinical isolates of hcov-oc with cleaved s protein selected the cellsurface tmrpss route for entry and were sensitive to cleavage by cell surface proteases. thus, although the mutation in the furin site may affect the use of cell-surface proteases, these two mutations sandwich the vhcr, similar to hcov- e. as reported previously, adaptation to tmprss -negative cell cultures altered the protease sensitivity of hcov- e s protein from tmprss to cathepsin l (shirato et al., ) . additional studies are required to elucidate the role of mutations in s protein in the infection events of hcov-oc . we showed that the clinical isolates of hcov-oc and -hku preferred the cell-surface tmrrss to endosomal cathepsins for cell entry, as did hcov- e. in addition, the cell-culture-adapted hcov-oc lost the ability to infect and replicate in hbte-ali cell culture, which mimics the natural situation of hae. these results indicate that clinical isolates of hcovs from the field generally enter cells not via late endosomes with cathepsins but via the cell surface or early endosomes with tmprss . in addition, this study suggests that virus adaptation in cultured cells after isolation alters the route of cell entry and that such modified isolates might not reflect the hcovs of the natural world. the strain hcov-oc (vr ) was obtained from the atcc and propagated and titrated using rd cells (atcc ccl- ). clinical isolates of hcov-oc and hku were isolated using hbte-ali culture. nasal swabs were obtained from outpatients in a hospital in tokyo, japan between and , after approval of the national institute of infectious diseases ethics review board for human subjects. the specimens were subjected to respiratory virus testing by real-time rt-pcr (kaida et al., ) . hcov-positive specimens were inoculated onto hbte-ali cultures, and after h incubation were washed with dmem twice and incubated at °c. after incubation for days, cells were washed four times with dmem containing % (v/v) fetal calf serum, which was then used as virus stocks. virus replication was confirmed by calculating rna copy numbers using real-time rt-pcr as described earlier (dare et al., ; van elden et al., ) and comparing them to those in the washings after virus adsorption. isolated viruses and the relevant rt-pcr results are listed in table . the sequences of the s proteins were obtained using sanger's method and alignment analysis was performed using genedoc software (https:// www.nrbsc.org/gfx/genedoc/ebinet.htm). to evaluate the replication kinetics of clinical and atcc isolates of hcov-oc , copies of virus were inoculated onto hbte-ali cultures or hct- cells. after incubation for h, the cells were washed twice and incubated at °c. after day of incubation, the cells were collected and used for analysis of virus entry. cell supernatants or cell washings obtained after days of incubation were used to evaluate virus replication as described below. the amounts of replicated virus were expressed as rna copy numbers in cell supernatants or cell washings minus those remaining in cell washings after virus adsorption. for the entry inhibition assay using hbte-ali cultures, to copies of viruses were inoculated onto cells that had been pre-treated with entry inhibitors [est ( , )trans-epoxysuccinyl-l-leucylamindo- -methylbutane ethyl ester) ( : calbiochem, san diego, ca, usa)] or camostat mesylate ( : tocris bioscience, bristol, uk) for h. after incubation for h in the presence of inhibitors, cells were washed twice and incubated at °c. after day of incubation, the cells were collected. cell viability was determined using celltiter-glo and a glomax luminometer (promega, madison, wi, usa). total cellular rna was extracted using isogen (nippon gene, tokyo, japan) with ribonucleic acid from baker's yeast (r , sigma) as carrier rna. to assess virus entry, subgenomic rna containing nucleocapsid protein was amplified by real-time rt-pcr using the following primer and probe sets: for hcov-oc , forward, ′-tcactgatctcttgttagat ctttttgta- ′; reverse, ′-tttgcttgggttgagctctt- ′; and probe, ′-ggccgatcagtccgaccagttt- ′ (fam-labeled): for hcov-hku , forward, ′-tatcagcttacgatctcttgtcaga- ′; reverse, ′-tggt cagcccaagaagtttt- ′; and probe, ′-gaagctcctctggaaatcg ttcagga- ′ (fam-labeled). rna copy numbers were calculated from standard curves obtained with the aid of control rna templates. the data were statistically analyzed using the unpaired t-test and are presented as log rna copies per well. the expression level of tmprss mrna was examined by a realtime rt-pcr assay. total rnas were extracted by trizol (thermo fisher scientific, waltham, ma, usa) according to the manufacturer's instructions. real-time pcr was performed using fast virus -step master mix (thermo fisher scientific) and lightcycler (roche, basel, switzerland). commercial specific primers were used as follows: gapdh, e (thermo fisher scientific); and tmprss , hs _m (thermo fisher scientific). the data are presented as a relative value calculated based on each gapdh value. calu- cells (atcc, htb- ) and lovo cells (jcrb cell bank, japan, ifo ) were used as the positive control for tmprss expression, and hela cells (atcc, ccl . ) were used as the 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reverse-transcriptase polymerase chain reaction detection of human coronavirus oc in children with acute respiratory infections in mie we thank fumihiro taguchi (chungnam national university, daejeon, korea), for valuable suggestions. this study was supported by a grant-in-aid for scientific research (c: ) from the japan society for the promotion of science and the japan agency for medical research and development (grant nos. fk j and fk j ). key: cord- -v sb rc authors: gardin, chiara; ferroni, letizia; chachques, juan carlos; zavan, barbara title: could mesenchymal stem cell-derived exosomes be a therapeutic option for critically ill covid- patients? date: - - journal: j clin med doi: . /jcm sha: doc_id: cord_uid: v sb rc coronavirus disease (covid- ) is a pandemic viral disease originated in wuhan, china, in december , caused by the severe acute respiratory syndrome coronavirus (sars-cov- ). the severe form of the disease is often associated with acute respiratory distress syndrome (ards), and most critically ill patients require mechanical ventilation and support in intensive care units. a significant portion of covid- patients also develop complications of the cardiovascular system, primarily acute myocardial injury, arrhythmia, or heart failure. to date, no specific antiviral therapy is available for patients with sars-cov- infection. exosomes derived from mesenchymal stem cells (mscs) are being explored for the management of a number of diseases that currently have limited or no therapeutic options, thanks to their anti-inflammatory, immunomodulatory, and pro-angiogenic properties. here, we briefly introduce the pathogenesis of sars-cov- and its implications in the heart and lungs. next, we describe some of the most significant clinical evidence of the successful use of msc-derived exosomes in animal models of lung and heart injuries, which might strengthen our hypothesis in terms of their utility for also treating critically ill covid- patients. the novel coronavirus ( -ncov) has reached pandemic proportions across the world after originating in wuhan, the capital of china's hubei province, in december [ , ] . initially called -ncov, the world health organization (who) subsequently adopted the official name severe acute respiratory syndrome coronavirus (sars-cov- ) for indicating the virus, and the term coronavirus disease (covid- ) for identifying the virus-associated disease [ ] . as of may , the who has reported almost , , confirmed cases of covid- with , confirmed deaths in countries/areas/territories worldwide [ ] . the clinical spectrum of covid- is highly variable-in addition to mild, severe, and critical forms, asymptomatic or paucisymptomatic infections have been described as well [ , ] . milder clinical conditions are commonly characterized by fever, dry cough, myalgia or fatigue, headache, and mild pneumonia, whereas the severe form of the disease is associated with dyspnea, acute respiratory distress syndrome (ards), and hypoxemia (low level of oxygen in arterial blood) [ , ] . the most critical cases experience respiratory failure requiring mechanical ventilation and support in the intensive care mice was correlated with myocardial disfunction [ ] . considering the similarity with sars-cov, it has been speculated that sars-cov- infection might also down-regulate ace expression in lung and heart, thus leading to the pathological processes of lung and cardiac injuries [ ] . to date, no experimental or clinical data have evidenced that using acei/arb therapy makes patients more susceptible to the virus. therefore, several leading cardiovascular societies have strongly urged to not discontinue intake of ras inhibitors in the event the patient develops covid- [ , ] . j. clin. med. , , x for peer review of regulated after sars-cov infection in mice, and this was accompanied by increased pulmonary vascular permeability and pulmonary edema [ ] . similarly, cardiac ace down-regulation following sars-cov infection in mice was correlated with myocardial disfunction [ ] . considering the similarity with sars-cov, it has been speculated that sars-cov- infection might also downregulate ace expression in lung and heart, thus leading to the pathological processes of lung and cardiac injuries [ ] . to date, no experimental or clinical data have evidenced that using acei/arb therapy makes patients more susceptible to the virus. therefore, several leading cardiovascular societies have strongly urged to not discontinue intake of ras inhibitors in the event the patient develops covid- [ , ] . , angiotensin ( - ); at r, ang ii type receptor; aceis, angiotensin-converting enzyme inhibitors; arbs, angiotensin receptor blockers; ards, acute respiratory distress syndrome. green arrows indicate that aceis/arbs increase ace levels in the heart, therefore increasing the susceptibility of cardiac cells to sars-cov- infection [ , ] . blue dotted hammerhead indicates the hypothetical effect of sars-cov- on ace expression in lung and heart, which is based on the reported effect of sars-cov in the same body districts [ , ] . mscs are thought to prevent or reduce the cytokine storm in covid- patients, owing to their powerful anti-inflammatory and immunomodulatory functions [ ] . mscs exert these effects by directly interacting with different cells of innate and adaptive immunity, including t cells, b cells, dendritic cells (dcs), macrophages, and natural killer cells, and by indirectly releasing many types of inflammatory mediators by paracrine secretion [ ] [ ] [ ] [ ] . many studies have described a differential regulation by mscs on the different t cell subsets [ ] [ ] [ ] [ ] [ ] . mscs inhibit effector t (teff) cell proliferation induced by mitogens or alloantigens by causing cell cycle arrest at the g phase [ , ] . another explanation for this immunosuppressive capacity is the loss of cd , the alpha-chain of the il- receptor, which is cleaved from the activated t cell surface by msc-secreted matrix metalloproteinases [ ] . this leads to blockage of the il- cytokine signaling pathway required for t cells activation, expansion, and differentiation. interestingly, such t cell-suppressing properties of mscs seem to require the presence of inflammatory cytokines in the microenvironment, which provoke the production of several t cell-attracting chemokines and inducible nitric oxide synthase figure . schematic diagram showing the renin-angiotensin system (ras) cascade and the effects on the cardiovascular system and lung. ace , angiotensin-converting enzyme ; ace , angiotensin-converting enzyme ; ang i, angiotensin i; ang ii, angiotensin ii; ang ( - ), angiotensin ( - ); at r, ang ii type receptor; aceis, angiotensin-converting enzyme inhibitors; arbs, angiotensin receptor blockers; ards, acute respiratory distress syndrome. green arrows indicate that aceis/arbs increase ace levels in the heart, therefore increasing the susceptibility of cardiac cells to sars-cov- infection [ , ] . blue dotted hammerhead indicates the hypothetical effect of sars-cov- on ace expression in lung and heart, which is based on the reported effect of sars-cov in the same body districts [ , ] . mscs are thought to prevent or reduce the cytokine storm in covid- patients, owing to their powerful anti-inflammatory and immunomodulatory functions [ ] . mscs exert these effects by directly interacting with different cells of innate and adaptive immunity, including t cells, b cells, dendritic cells (dcs), macrophages, and natural killer cells, and by indirectly releasing many types of inflammatory mediators by paracrine secretion [ ] [ ] [ ] [ ] . many studies have described a differential regulation by mscs on the different t cell subsets [ ] [ ] [ ] [ ] [ ] . mscs inhibit effector t (teff) cell proliferation induced by mitogens or alloantigens by causing cell cycle arrest at the g phase [ , ] . another explanation for this immunosuppressive capacity is the loss of cd , the alpha-chain of the il- receptor, which is cleaved from the activated t cell surface by msc-secreted matrix metalloproteinases [ ] . this leads to blockage of the il- cytokine signaling pathway required for t cells activation, expansion, and differentiation. interestingly, such t cell-suppressing properties of mscs seem to require the presence of inflammatory cytokines in the microenvironment, which provoke the production of several t cell-attracting chemokines and inducible nitric oxide synthase (inos) from mscs, so that t cells migrate into proximity of these cells [ ] . at the same time, mscs have been shown to induce the survival and expansion of regulatory t (treg) cells, a subset of t cells involved in the suppression of proliferation and cytokine production by teff cells [ ] . therefore, treg cells foster the msc-mediated immunosuppressive effect. in addition to directly interacting with t cells, mscs also modulate the adaptive immune response by acting on antigen-presenting cells (apcs), such as dcs, monocytes, and macrophages, by shifting them to regulatory phenotypes characterized by t cell-suppressive properties [ , ] . the spectrum of regulatory factors secreted by mscs is collectively defined as the msc secretome, and include a complex array of soluble molecules, such as anti-inflammatory cytokines, angiogenic growth factors, antimicrobial peptides, and lipid mediators. a growing body of evidence nowadays suggests that some of these molecules are packaged into cell-secreted vesicles, known as extracellular vesicles (evs) [ ] [ ] [ ] . besides apoptotic bodies, the two main types of evs released by mscs include exosomes and microvesicles (mvs). exosomes ( - nm) are derived by fusion of multi-vesicular bodies with the plasma membrane, whereas mvs ( - nm) are formed by cellular membrane budding, and contain cellular cytoplasm. all these evs are released into the extracellular microenvironment, where they exert biological effects in a paracrine and endocrine manner, similarly to the soluble components. for this reason, a broader definition of mscs secretome encompasses the entire spectrum of bioactive factors secreted by mscs, which consists of both the soluble and the extravesicular elements. it has now been demonstrated that mscs are also able to transfer functional mitochondria or mitochondrial dna (mtdna) to target cells, thus rescuing aerobic respiration in cells with non-healthy mitochondria or regulating t cell functions [ , ] . following systemic injection, some mscs accumulate in the lung, where they release these soluble mediators, potentially recovering the pulmonary microenvironment, protecting alveolar epithelial cells, and counteracting pulmonary fibrosis, thus resulting in a final improvement of lung function [ ] . moreover, distant injured organs, such as the cardiovascular system, can also benefit from them, by virtue of the secretory abilities of these cells. to date, two studies have investigated the employment of mscs in severely affected sars-cov- patients, with both reporting remarkable reversal of symptoms within a few days [ , ] . in one of these works, the levels of biochemical indicators of liver and myocardium damage (aspartic aminotransferase, creatine kinase activity, and myoglobin) returned to reference levels days after msc treatment [ ] . the authors demonstrated that the cells expressed high levels of anti-inflammatory and angiogenic factors, such as transforming growth factor-beta (tgf-β), hepatocyte growth factor (hgf), leukemia inhibitory factor (lif), fibroblast growth factor (fgf), vascular endothelial growth factor (vegf), epidermal growth factor (egf), brain-derived neurotrophic factor (bdnf), and nerve growth factor (ngf), further demonstrating their potent immunomodulatory abilities. it has been reported that mscs are generally resistant to viral infections compared to their differentiated progeny, probably due to intrinsic expression of ifn-stimulated genes (isgs) [ ] . among these genes, those coding for proteins of the interferon-induced transmembrane (ifitm) family prevent viruses from traversing the lipid bilayer of the cell and accessing the cytoplasm, thus impairing viral infection [ ] . these antiviral proteins limit infection in cultured cells by many viruses, including sars-cov, dengue virus, ebola virus, influenza a virus, and west nile virus. however, some studies have reported that human mscs are permissive to other viruses, for example avian influenza viruses h n and h n and respiratory syncytial virus (rsv), losing vitality and compromising their immunomodulatory activities [ , ] . in the case of sars-cov- , the advantage in using mscs seems to be additionally related to the absence of ace receptors on the cell surface, which precludes their recognition by the virus. notably, in the study of leng and coworkers, the cells remained negative for ace also after transplantation in infected patients [ ] . although mscs seem to be refractory to sars-cov- infection, in order to bypass the impact of viruses on mscs, an interesting therapeutic strategy could consider the use of the msc secretome. among the bioactive factors released by mscs, evs, exosomes in particular, have gained remarkable interest in recent years because they enable more efficient communication and targeting than soluble molecules [ ] . evs, by virtue of their lipid bilayer membrane, better protect their molecular cargo of proteins and genetic material from environmental degradation (i.e., from trypsin or nuclease digestion) when compared to soluble molecules. encapsulation within evs may also facilitate delivery and targeting of these bioactive factors to distant recipient cells, mediated by binding of the ev surface proteins to cells that express appropriate receptors [ ] . msc-derived exosomes offer several advantages over traditional cell-based therapies. first, exosomes are considered safer than cells, because they are biocompatible, non-immunogenic, and lack the potential for endogenous tumors and emboli formation [ ] . in addition, exosomes are physiologically more stable than cells, because their multiple membrane adhesion proteins allow for efficient binding in the target tissues during transplantation. thanks to their resistant membrane, exosomes maintain their integrity during freezing and thawing procedures, making long-term storage without biological degradation possible [ ] . in this context, a process has recently been proposed that combines ultrafiltration and lyophilization and is able to convert msc secretome into a freeze-dried, ready-to-use powder [ , ] . the same research group also suggested the possibility to administer evs by inhalation in the treatment of respiratory diseases [ ] . this route of administration would benefit from lower invasiveness and pain, faster onset of action, and use of lower doses to achieve the same therapeutic effect when compared to oral or injection therapies. in this regard, a pilot clinical trial (nct ) will be conducted in china for exploring the safety and efficiency of aerosol inhalation of msc-derived exosomes in comparison to conventional treatment in severe patients with covid- . another advantage of msc-derived exosomes over whole-cell therapy is that, to improve their therapeutic potential, exosomes could potentially be modified with various types of cargos, including mrna, microrna (mirna), and proteins, tailored to the disease process of interest [ ] . in one pioneering work, exosomes incorporating the s protein have been explored as a novel vaccine approach against sars-cov infections [ ] . the immunogenicity and efficacy of the s-containing exosomes were tested in mice, where they induced neutralizing antibody titers. finally, from an economical point of view, msc-derived exosome therapy might enable development of cheaper treatments other than the expansion and maintenance of individualized clonal cell populations [ ] . this aspect is particularly important when a global pandemic has to be managed, as in the case of covid- . in section , we provide an overview of the currently available evidence on the effects of msc-derived exosomes in pre-clinical models of lung and heart injuries, which are the body districts most affected by sars-cov- . diabetes mellitus (dm) represents the most common inflammatory and chronic metabolic disorder worldwide, and continues to increase in number and significance-it is estimated that there will be million persons with dm by [ ] . type diabetes mellitus (t dm) accounts for - % of all cases of diabetes and results from a progressive defect in insulin production and insensitive response of the body to insulin [ ] . accumulating evidence shows that such a state of insulin resistance (ir) is closely related with obesity [ ] . obesity, mainly visceral adiposity, is, indeed, one of the most important comorbidities in diabetic patients. people with diabetes have a higher overall risk of infections that result from compromised innate cell-mediated immunity; impaired phagocytosis by neutrophils, macrophages, and monocytes; and impaired neutrophil chemotaxis and bactericidal activity [ ] . regarding covid- , it is currently unknown whether patients with diabetes have a higher susceptibility to the virus; nonetheless, there is evidence of higher risk for both infection and disease severity [ ] . as stated above, there is growing interest in the use of msc-derived evs as a therapeutic tool for the management of several diseases. however, because ev cargo usually reflects parent cell characteristics, and these are influenced by the metabolic state of source cells, it is reasonable to consider the risks associated with the employment of msc-derived evs from patients with coexisting metabolic disorders such as t dm. in effect, clinical studies have found differences in the number and composition of evs isolated from the adipose tissue of obese patients and from animal models of obesity [ ] [ ] [ ] [ ] . for example, msc evs isolated from a swine model of metabolic disorder were found to be enriched with mrnas associated with inflammation, such as those coding for the integrin family proteins, or proteins of the fgf signaling [ ] . these msc-derived evs also showed a distinctive mirnas cargo, being enriched in mirna-targeting genes involved in the development of metabolic disease and its complications, including diabetes, obesity, and insulin signaling [ ] . apart from influencing the mrna and mirna content, metabolic disorder also alters packaging of proteins into porcine msc-derived evs, promoting the inclusion of pro-inflammatory proteins, such as those involved in acute inflammatory response, cytokine production, and leukocyte transendothelial migration [ ] . the limitations of these works reside in the small sample size and short duration of metabolic disease compared to the human condition; therefore, further studies would be needed to draw clear conclusions. however, in humans also, analysis of adipose tissue-derived evs demonstrated that obesity alters their cargo of mrnas, mirnas, and proteins [ ] . in particular, the differentially expressed mirnas contained in the isolated evs stimulated up-regulation of wnt/β-catenin and tgf-β signaling pathways, which are related to inflammation, into a lung epithelial cells. overall, these observations suggest that diabetes and metabolic disorders might alter the msc-derived ev cargo, which in turn might compromise their anti-inflammatory and immunodulatory potential both in the endogenous microenvironment and after autologous transplantation. acute lung injury (ali) and ards are major causes of respiratory failure in critically ill ventilated patients, with an estimated -day mortality rate of % [ ] . ards is also one of the most common complications in severely affected covid- patients. the term ards is often used interchangeably with ali; nevertheless, ards should be reserved for the most severe form of the disease [ ] . bacterial or viral infections are the most common causes of ali and ards; however, they can also be initiated by aspiration of gastric contents, toxic inhalation, lung contusion, or trauma [ ] . the acute phase (the first - days) of the diseases is characterized by injury to both the pulmonary endothelium and the alveolar epithelium, the two barriers forming the alveolar-capillary barrier. in healthy lung microvessels, the pulmonary endothelium is maintained by vascular endothelial cadherin (ve-cadherin), an endothelial-specific adherens junction protein, whereas the alveolar epithelial barrier has e-cadherin junctions and is substantially less permeable than the endothelial counterpart [ , ] . during lung injury, ve-cadherin bonds are destabilized by increased expression of thrombin, tnf-α, vegf, and signals from leukocytes. at the same time, e-cadherin epithelial junctions are disrupted by neutrophil migration, which causes injury, apoptosis, and membrane denudation. this ultimately results in increased epithelial permeability, leading to accumulation of protein-rich edema fluid in the alveoli, and in turn to an impairment in gas exchange and to hypoxemia [ ] . dysregulated immune activation has also been implicated in the pathogenesis of ali/ards. in the air space, macrophages release pro-inflammatory cytokines and chemokines, which act locally to stimulate chemotaxis and activate neutrophils. activation of neutrophils leads to the release of numerous cytotoxic products, such as reactive oxygen species, cationic peptides, eicosanoids, and proteolytic enzymes, which may further damage the alveolar epithelium [ ] . resolution of ali/ards aims at removing alveolar edema fluid, repairing the epithelial and endothelial barriers, and removing inflammatory cells and exudate from the air spaces [ ] . to date, management of ali/ards includes lung protective ventilation, prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation. mechanical ventilation represents the mainstay treatment in ali/ards, and consists in the application of positive-end expiratory pressure for optimizing arterial oxygenation. it has been evidenced that ventilation with a low tidal volume ( ml/kg) gives better results when compared to traditional tidal volume ( ml/kg) [ ] . indeed, the use of lower tidal volumes during ventilation may reduce injurious lung stretch and the release of inflammatory mediators. prone positioning enhances arterial oxygenation by improving alveolar ventilation/perfusion matching. nevertheless, this treatment should be used with caution and should be reserved for patients with critical hypoxemia, since it does not improve survival or decrease the duration of lung ventilation. all these therapeutic options remain primarily supportive; on the other hand, alternative treatments with glucocorticoids, surfactants, inhaled nitric oxide, antioxidants, protease inhibitors, or other anti-inflammatory agents had proven unsuccessful in reducing mortality or improving ali/ards outcomes [ ] . in terms of promising novel strategies, msc-based approaches have been explored for the management of ali/ards. the benefit of msc therapy appears to be related to a decrease in pro-inflammatory cytokines and to an increase in anti-inflammatory cytokines, particularly il- [ ] . mscs release prostaglandin e , which in turn stimulates secretion of il- by monocytes and alveolar macrophages [ ] . moreover, administration of mscs seems to be effective in normalizing lung endothelial and epithelial permeability to protein, as well as in reducing pulmonary edema and increasing the rate of alveolar fluid clearance [ ] . recently, msc-derived exosomes have been demonstrated to have comparable and even greater effects than cells themselves in improving inflammation and injury in a variety of pre-clinical lung disease models, including ali/ards (table ) . for the completeness of information, we have to specify that some of these works take into account the entire spectrum of evs that, in addition to exosomes, also includes mvs. this is because as of yet there are no standardized methods for isolation, quantification, and characterization of evs, or for discriminating mvs and exosomes. consequently, in the majority of these pre-clinical studies, evs, exosomes, and mvs are collectively referred to as evs. msc-derived evs have been proven to be beneficial in both bacteria-and virus-induced ali/ards. a large number of studies have employed an endotoxin-mediated in vivo model to investigate the effects of msc-derived evs for ali/ards. in one of the first works, ali was induced in c bl/mice using the intratracheal (it) instillation of endotoxin ( mg/kg) from escherichia coli (e. coli) [ ] . mvs were isolated from the conditioned medium of human bone marrow-derived mscs with two sequential ultracentrifugations at , × g for h. then, ul of mvs, corresponding to the vesicles released by × mscs, were administrated intratracheally or intravenously in mice. after h, msc-derived mvs reduced lung inflammation and reduced edema to the same levels as mscs themselves, which were used as a positive control. furthermore, mvs also decreased the influx of neutrophils and mip- levels in the alveolar fluid, indicating a reduction in inflammation. surprisingly, the therapeutic effects of the mvs were comparable, regardless of route of administration. the authors suggested that the mechanism underlying the therapeutic effect of mvs might be in part mediated by the transfer of keratinocyte growth factor (kgf) mrna into the injured alveolar epithelium, with subsequent expression of the protein. kgf is an epithelial-specific growth factor released from mscs, which has been shown to reduce lung edema and inflammation in various ali models [ ] [ ] [ ] . in the same study, the effect of mvs was additionally evaluated in raw . cells, a mouse macrophage cell line. treatment with µl of msc-derived mvs to endotoxin-stimulated raw . cells reduced the levels of tnf-α and mip- , and concomitantly increased the production of the anti-inflammatory cytokine il- at , , and h compared with endotoxin-stimulated mouse macrophages [ ] . in the work of tang and colleagues, ali was induced in c bl/mice by the instillation of lipopolysaccharide (lps) from pseudomonas aeruginosa at mg/kg intratracheally [ ] . mvs released from human bone marrow mscs were isolated by two sequential ultracentrifugations at , × g for h, then intratracheally administrated in endotoxin-injured mice. it administration of msc-derived mvs improved the lung inflammation induced by lps in mice, including the influx of white blood cells and neutrophils, and mip- secretion. in that study, the authors found that the transfer of angiopoietin- (ang- ) mrna by mvs was essential for the reduction of inflammation and the restoration of alveolar-capillary barrier. ang- plays a key role in vascular stabilization, since it reduces endothelial permeability and suppresses leukocyte-endothelium interactions [ ] . furthermore, msc-derived mvs showed immunomodulatory effects on raw . cells in vitro by inhibiting tnf-α mrna production and promoting the mrna levels of il- after h [ ] . collectively, these two studies suggest that the beneficial immunomodulatory effect of msc-derived mvs in ali is strongly dependent on kgf and ang- mrna transfer into injured endothelial cells. it has been demonstrated that, apart from mrnas, the therapeutic effect of evs is also mediated by the transfer of functional mitochondria to target cells. mscs have been reported to naturally transfer mitochondria to recipient cells through different mechanisms-incorporated within evs, via cell-to-cell contact through tunneling nanotubes, or through direct release of naked mitochondria into the extracellular microenvironment [ , ] . in the work of phinney and colleagues, transmission electron microscopy images evidenced structures consistent with the morphology of mitochondria inside mvs over nm in size, previously isolated from the conditioned medium of human mscs after centrifugation at , × g for h [ ] . the authors found that these mitochondria were loaded in the cytoplasm into lc -containing mvs, which migrated towards the cell periphery and were incorporated into outward budding blebs in the plasma membrane. the msc-derived mvs contained functionally active mitochondria that were taken up by macrophages and resulted in improved bioenergetics after oxidative stress increment. in particular, the transfer of human msc-derived mitochondria involved fusion with mitochondria inside macrophages, suggesting that the mitochondrial membrane was not collapsed. the same study also confirmed that mitochondria were not packaged within exosomes; rather, exosomes were able to deliver mtdna, which in mammals has an average size under nm [ ] . in the context of lung injury, the group of morrison and colleagues revealed that msc-derived evs protected mice against lps-induced ali by altering alveolar macrophage (am) polarization from the pro-inflammatory m phenotype towards the m anti-inflammatory phenotype [ ] . in detail, evs were obtained from human bone marrow mscs after ultracentrifugation at , × g for h. these were used for pre-treating am, which were then intranasally administrated to lps-injured mice. the msc-derived evs increased phagocytic activity by macrophages and reduced their secretion of tnf-α and il- , two major pro-inflammatory cytokines related to ards severity [ , ] . the transfer of functional mitochondria contained in evs, associated with the promotion of oxidative phosphorylation, was supposed to be the mechanism responsible for the observed effects in macrophages [ ] . the work of monsel and colleagues was the first to evaluate the effect of msc-derived mvs in an infectious ali model [ ] . indeed, the authors instilled live e. coli bacteria into the trachea; then, they intravenously administrated ul of mvs, corresponding to the vesicles released by × human bone marrow mscs. mv injection improved survival and reduced the bacterial load, as well as the influx of white blood cells, neutrophils, and mip- levels, in the injured alveolus of c bl/mice. the authors reported that the effect was in part mediated by kgf mrna shuttled by the vesicles into target cells, as described in their previous study [ ] . in addition to testing in mice, the effect of msc-derived mvs was also investigated in human monocytes and at cells. mv treatment increased the percentage of phagocytosis of human monocytes against e. coli bacteria, thus reducing the bacterial count, and decreased tnf-α secretion. furthermore, mvs showed a beneficial effect on injured human at cell metabolism through the restoration of intracellular atp levels to control levels. in these primary in vitro cultures, the uptake of mvs was mediated by cd , which was essential for the observed therapeutic effects. cd is the hyaluronic acid receptor expressed in almost every cell type including mscs [ , ] . the results of this work suggested that mvs, similarly to their parent cells, act through different mechanisms on the basis of anti-inflammatory, anti-microbial, and metabolomic effects. msc-derived evs have also shown reparative properties on microvascular endothelial and epithelial cells, which are often severely injured in the lung during ali, and are associated with increased mortality in ards patients. hu and coworkers investigated the effects of mvs isolated from human bone marrow mscs on human lung microvascular endothelial cells (hlmvecs) in vitro [ ] . the cells were injured by cytomix, a mixture of the most biologically active cytokines found in ali pulmonary edema fluid (il- β, tnf-α, and ifn-γ at ng/ml), and simultaneously exposed to increasing doses ( or ul) of mvs using a transwell co-culture system [ ] . administration of msc-derived mvs restored protein permeability of hlmvecs by preventing the reorganization of cytoskeleton protein f-actin into "actin stress fibers" and the loss of tight and adherens junction proteins (zonula occludens- and ve-cadherin, respectively) following inflammatory injury. the internalization of mvs via cluster of differentiation (cd) receptor, as well as the subsequent transfer of ang- mrna into injured hlmvecs, were required for the observed therapeutic effects. the study of khatri and colleagues is interesting because ards was induced in pigs after infection with a mixed swine (h n , h n ) and avian (h n , h n ) influenza viruses (swiv) [ ] . pigs are often used as large animal pre-clinical models for several human diseases, including respiratory diseases, due to their close similarity in anatomy, physiology, and immunology to humans [ ] . in addition, influenza virus pathogenesis and clinical signs are similar to those observed in humans. in that work, evs ( µg/kg) isolated from swine bone marrow mscs with two ultracentrifugation steps at , rpm for min were intratracheally administrated in pigs h after swiv inoculation. msc-derived evs were found to inhibit influenza virus replication and shedding in pigs days post-infection. as in other studies, evs also modulated inflammatory cytokine and chemokine production in the lungs, as demonstrated by reduction in tnf-α and cxcl protein levels, and increase in il- protein levels. unfortunately, there are not yet pre-clinical data on the effects of msc-derived ev administration in models of coronavirus respiratory infection, mostly due to the lack of an established animal model [ ] . from the studies discussed above, it emerged that the rationale for using msc-derived exosomes, mvs, or evs in ali/ards is based on several processes, many of which are shared with those identified in the parent mscs. these include immunomodulation and anti-inflammatory properties on host tissue, reduction of the permeability of alveolar epithelium and endothelium, improvement of alveolar fluid clearance, enhancement of macrophage phagocytosis, and tissue repair through direct mitochondrial transfer with host cells (figure ). reduction in tnf-α and cxcl protein levels, and increase in il- protein levels days post-infection. [ ] potential therapy in the management of other lung diseases, such as bronchopulmonary dysplasia, pulmonary arterial hypertension, idiopathic pulmonary fibrosis, and asthma, which have been recently revised in worthington [ ] and behnke [ ] . moreover, in these lung diseases, the most common effects of msc-derived evs were decreased inflammation and restoration of the lung architecture, achieved through the reduction of fibrosis and increase of vascularization and alveolarization. [ , , , ] , reduction of the permeability of alveolar epithelium and endothelium [ ] , improvement of alveolar fluid clearance [ ] , enhancement of macrophage phagocytosis [ ] , and tissue repair through direct mitochondrial transfer with host cells [ ] . acute myocardial injury has been described as the most common cardiovascular complication in covid- patients [ ] . myocardial injury is defined as an elevation in serum levels of highsensitive cardiac troponin (ctn) above the th percentile upper reference limit, although over the years it has also been identified through an increase in different cardiac enzymes and/or electrocardiographic abnormalities [ ] . the injury is considered acute if there is a dynamic rise and/or fall of ctn values. when acute myocardial injury is caused by myocardial ischemia, it is designated as acute myocardial infarction (ami). on the contrary, myocardial injury not related to ischemic events may arise secondary to many cardiac conditions, such as myocarditis [ ] . [ , , , ] , reduction of the permeability of alveolar epithelium and endothelium [ ] , improvement of alveolar fluid clearance [ ] , enhancement of macrophage phagocytosis [ ] , and tissue repair through direct mitochondrial transfer with host cells [ ] . apart from ali/ards, there have been several investigations using msc-derived evs as a potential therapy in the management of other lung diseases, such as bronchopulmonary dysplasia, pulmonary arterial hypertension, idiopathic pulmonary fibrosis, and asthma, which have been recently revised in worthington [ ] and behnke [ ] . moreover, in these lung diseases, the most common effects of msc-derived evs were decreased inflammation and restoration of the lung architecture, achieved through the reduction of fibrosis and increase of vascularization and alveolarization. acute myocardial injury has been described as the most common cardiovascular complication in covid- patients [ ] . myocardial injury is defined as an elevation in serum levels of high-sensitive cardiac troponin (ctn) above the th percentile upper reference limit, although over the years it has also been identified through an increase in different cardiac enzymes and/or electrocardiographic abnormalities [ ] . the injury is considered acute if there is a dynamic rise and/or fall of ctn values. when acute myocardial injury is caused by myocardial ischemia, it is designated as acute myocardial infarction (ami). on the contrary, myocardial injury not related to ischemic events may arise secondary to many cardiac conditions, such as myocarditis [ ] . analyzing several reports from china, a considerable proportion of patients ( - . %) presented elevated ctn levels, and most of them required icus and showed higher in-hospital mortality [ , [ ] [ ] [ ] . the mechanisms of myocardial injury are not well established but likely involve direct or indirect processes and/or their combination (figure ) . myocardial infection by sars-cov- resulting in cardiomyocyte death and inflammation has been proposed as a possible direct mechanism, although, to date, there are no data demonstrating the presence of sars-cov- within myocardial tissue [ ] . nevertheless, a previous autopsy study in patients who died from sars identified the viral rna in % of the post-mortem human heart samples, providing evidence for direct myocardial injury by the virus [ ] . in addition, patients carrying sars-cov in their hearts died considerably earlier, suggesting that viral infiltration in the myocardium was associated with a more aggressive course of illness. systemic inflammatory response or respiratory failure and hypoxemia can represent indirect mechanisms leading to increased cardiac stress and myocardial inflammation [ , ] . in a couple of studies, biopsies taken from heart tissue of covid- patients evidenced mononuclear inflammatory infiltrates, mainly associated with regions of cardiomyocyte necrosis, which identifies myocarditis according to dallas criteria [ ] [ ] [ ] . nevertheless, acute lymphocyte infiltrates were not observed in the myocardium of sars-cov- -infected patient autopsy. died considerably earlier, suggesting that viral infiltration in the myocardium was associated with a more aggressive course of illness. systemic inflammatory response or respiratory failure and hypoxemia can represent indirect mechanisms leading to increased cardiac stress and myocardial inflammation [ , ] . in a couple of studies, biopsies taken from heart tissue of covid- patients evidenced mononuclear inflammatory infiltrates, mainly associated with regions of cardiomyocyte necrosis, which identifies myocarditis according to dallas criteria [ ] [ ] [ ] . nevertheless, acute lymphocyte infiltrates were not observed in the myocardium of sars-cov- -infected patient autopsy. other aspects of covid- in cardiac involvement include blood pressure abnormalities and arrhythmias, ranging from tachycardia and bradycardia to asystole [ ] . very recently, it has also been suggested that there is a link between sars-cov- infection and kawasaki disease (kd), especially in pediatric patients [ ] . although kd is a disease of unknown etiology, infections are considered to be one of the predisposing factors [ ] . the disease predominantly affects children under years of age and causes inflammation in the walls of medium-sized arteries, primarily the coronary arteries, those that supply blood to the heart muscle. consequences of ami are loss of cardiomyocytes and adverse remodeling of the extracellular matrix, which contribute to the reduction of pumping of the heart and further heart failure. nowadays, the best therapeutic strategy for reducing ami is timely and effective myocardial other aspects of covid- in cardiac involvement include blood pressure abnormalities and arrhythmias, ranging from tachycardia and bradycardia to asystole [ ] . very recently, it has also been suggested that there is a link between sars-cov- infection and kawasaki disease (kd), especially in pediatric patients [ ] . although kd is a disease of unknown etiology, infections are considered to be one of the predisposing factors [ ] . the disease predominantly affects children under years of age and causes inflammation in the walls of medium-sized arteries, primarily the coronary arteries, those that supply blood to the heart muscle. consequences of ami are loss of cardiomyocytes and adverse remodeling of the extracellular matrix, which contribute to the reduction of pumping of the heart and further heart failure. nowadays, the best therapeutic strategy for reducing ami is timely and effective myocardial reperfusion. however, this treatment induces oxidative stress and inflammation, thus leading to further cardiomyocyte death, myocardial remodeling, and decreased cardiac function, a phenomenon known as myocardial reperfusion injury [ ] . over the last years, management of ami using stem cell therapy was found to prevent myocardial cell apoptosis, promote local neoangiogenesis, and reduce the local inflammatory response [ ] [ ] [ ] . similarly to what was described above for lung injuries, the beneficial effect of stem cells seems to be largely attributable to the secreted evs. since the first description of the therapeutic potential of msc-derived exosomes in a mouse model of myocardial ischemia/reperfusion (i/r) injury in , several studies have subsequently reported cardio-protective effects of msc-derived evs in ami animal models (table ) [ ] . in one of these works, a single intravenous (iv) injection of msc-derived exosomes in a mouse ami model led to decreased infarct size, enhanced nicotinamide adenine dinucleotide (reduced form) (nadh) and atp levels, and reduced oxidative stress, which are hallmarks of reperfusion injury [ ] . all these events seemed to be associated with the exosome-mediated activation of the pro-survival phosphoinositide -kinase/protein kinase b (pi k/akt) signaling pathway, which resulted in an enhancement of myocardial viability and prevented adverse remodeling after myocardial i/r injury. importantly, intact but not lysed exosomes were responsible for the improved cardiac function after ami induction. another important mechanism by which msc-derived evs contribute to ischemic myocardial repair is through stimulation of neovascularization, as shown in the work of bian and colleagues [ ] . neovascularization refers to processes, such as vasculogenesis, angiogenesis, and arteriogenesis, that are associated with migration and proliferation of endothelial cells. in line with these findings, ma and coworkers also demonstrated that exosomes isolated from akt-transfected mscs accelerated angiogenesis in a rat myocardial infarction model [ ] . the authors suggested that platelet-derived growth factor d (pdgf-d), which was enriched in msc-derived vesicles, was mainly responsible for the akt exosome-mediated improvement of myocardial repair. a more recent study by xuan and colleagues identified notch as a potent modulator of angiogenesis and cardiomyocyte proliferation into ischemic mice hearts following coronary heart ligation [ ] . the role of notch signaling in inducing cardiac angiogenesis during ischemia and enhancing survival of cardiac cells is well established [ ] . the injection of msc-derived evs over-expressing notch intracellular domain (nicd) in ischemic myocardium led to decreased infarct size, improved cardiac function, and increased arteriole density in the peri-infarct area, month after ami [ ] . moreover, teng and coworkers indicated that the beneficial effect of msc-derived exosomes on infarcted rat hearts is mainly dependent on their angiogenesis-promoting activity [ ] . in their study, the authors proved that exosomes also act by restraining the inflammatory response. in agreement with the results of arslan and colleagues, they also demonstrated that fresh exosomes achieved a better therapeutic effect with respect to frozen exosome preparations. several studies agree that reduced fibrosis and apoptosis of myocardial cells are other important effects of the ev-mediated ischemic cardiac repair [ ] [ ] [ ] [ ] . in particular, zhao and coworkers showed that human umbilical cord msc-derived exosomes improved cardiac function and reduced cardiac fibrosis by preventing cardiomyocyte apoptosis and promoting cell proliferation in the border zone of infarcted rats [ ] . the effect mediated by exosomes was attributed to the up-regulation of the anti-apoptotic protein b cell lymphoma (bcl- ) in the myocardial cells. other works have proposed that specific functional mirnas contained into evs and shuttled to target injured cells are primarily responsible for the beneficial effects. for example, feng and colleagues found that mir- was up-regulated in msc-derived evs, and it possibly reduced cardiac apoptosis and fibrosis in an ami mouse model via inhibition of methyl cytosine-phosphate-guanine (cpg)-binding protein (mecp ) expression [ ] . in that study, the authors isolated evs from mscs subjected to ischemic pre-conditioning, which is an effective approach to potentiate survival and regeneration of these cells in an ischemic environment. yu and coworkers identified mir- a as the molecular mediator able to restore cardiac function and reduce infarct size in a rat model of ami [ ] . the cardio-protective role of mir- a was mediated by down-regulation of target genes, phosphatase and tensin homolog (pten), and bcl- interacting mediator of cell death (bim) in cardiomyocytes and subsequent activation of the akt and extracellular signal-regulated kinase (erk) signaling pathways. in their study, exosomes were isolated from mscs over-expressing gata binding protein (gata- ), a transcription factor able to regulate mirna expression in mscs and increase their survival in an ischemic environment [ ] . apart from observing reduced cardiac fibrosis and reduced inflammation in infarcted rat hearts days after exosome injection, shao and colleagues identified a panel of mirnas, which were similarly up-or down-regulated in mscs and the derived exosomes [ ] . on the other hand, other mirnas, such as mir- and mir- , resulted as being differentially expressed between exosomes and mscs, with this potentially explaining why msc-derived exosomes demonstrated superior beneficial effects when compared with treatment with their parent cells. several studies have demonstrated that autophagy also has an important role in mediating the therapeutic effects of msc-derived exosomes. autophagy is known to be an important mechanism in cardio-protection, and dysregulated autophagy is associated with a variety of cvd [ ] . in particular, it has been demonstrated that exosomes reduce apoptosis and the myocardial infarct size, as well as improve cardiac function by inducing cardiomyocyte autophagy both in vitro and in vivo [ , ] . collectively, the described studies documented reduction in infarct size with improved recovery of cardiac function, reduction of fibrosis and apoptosis, stimulation of angiogenesis, and decreased infiltration of macrophages and other immune cells into the injured heart regions following treatment with msc-derived evs (figure ) . when comparing the properties of exosomes recovered from different mscs sources, those isolated from adipose tissue samples exhibited the strongest cardio-protective effects [ ] . [ ] ischemia/reperfusion; left coronary artery; intravenous; glucogen synthase kinase- ; c-jun nterminal kinase; acute myocardial infarction; left anterior descending; platelet-derived growth factor d; cardiac mscs over-expressing notch intracellular domain; methyl cytosine-phosphateguanine binding protein ; b cell lymphoma ; phosphatase and tensin homolog; phosphatebuffered saline; microtubule-associated protein light chain beta. collectively, the described studies documented reduction in infarct size with improved recovery of cardiac function, reduction of fibrosis and apoptosis, stimulation of angiogenesis, and decreased infiltration of macrophages and other immune cells into the injured heart regions following treatment with msc-derived evs (figure ) . when comparing the properties of exosomes recovered from different mscs sources, those isolated from adipose tissue samples exhibited the strongest cardioprotective effects [ ] . [ , , ] , reduction of cardiac fibrosis, reduction of cardiomyocyte apoptosis [ , , , , ] , promotion of angiogenesis [ ] [ ] [ ] ] , and induction of cardiomyocytes autophagy [ ] . [ ] ischemia/reperfusion; left coronary artery; intravenous; glucogen synthase kinase- ; c-jun n-terminal kinase; acute myocardial infarction; left anterior descending; platelet-derived growth factor d; cardiac mscs over-expressing notch intracellular domain; methyl cytosine-phosphate-guanine binding protein ; b cell lymphoma ; phosphatase and tensin homolog; phosphate-buffered saline; microtubule-associated protein light chain beta. the recent coronavirus covid- global pandemic has driven the need for novel urgent therapies. mscs and their derivatives are being evaluated for the treatment of a number of diseases that currently have limited or no therapeutic options. msc-derived evs (exosomes and mvs) have recently attracted great attention because, similarly to their parent cells, they possess strong anti-inflammatory, immunomodulatory, and pro-angiogenic abilities, just to name a few. however, compared to mscs themselves, evs hold many biological and technological advantages. ev administration is considered safer than msc transplantation, lacking some of their negative side-effects, and they are more stable than mscs themselves, allowing for easier handling and storage. over the last years, a plenty of pre-clinical studies in animal models have demonstrated that the administration of msc-derived evs significantly reduced lung inflammation and pathological impairment subsequent to different types of lung injury, as well as resulted in improved cardiac function after acute myocardial injury. however, several challenges still need to be overcome to make the transition from animal models to humans possible. for example, standardized techniques for isolation, characterization, and quantification, as well as criteria for establishing dose, quality control, and storage conditions of msc-derived evs, are required before these can be advanced to the clinic. to date, it is difficult to compare and analyze studies employing msc-derived evs since there is a large degree of heterogeneity in ev preparations, and because msc-derived evs differ depending on tissues and donors from which the cells are isolated. regarding covid- , the lack of an established animal model of coronavirus-induced lung injury requires a more prudent and careful use of msc-derived evs. in this context, a significant issue is to establish under what circumstances and with what criteria to administer msc-derived evs. for example, which population among covid- patients to target and when to start ev administration. moreover, there remains the challenge to clarify the optimal route of ev administration that, in the case of lung diseases, mostly occurs through it instillation or iv injection, although the possibility of ev inhalation has recently been explored. to date, no studies have investigated the biodistribution and the in vivo metabolic fate of evs following it instillation. on the other hand, systemic iv injection has been shown to deliver evs primarily to the spleen and liver, then to the gastrointestinal tract and lungs, followed by renal and hepatic clearance in mice [ , ] . apart from the administration route, another major issue concerns the optimal ev therapeutic dose. considering that the average therapeutic dose of mscs for treating lung injuries is × cells/kg per body weight, the amount of cells required to generate enough evs to achieve the equivalent effect of mscs is generally - times higher [ ] . this necessitates large scale production of msc-derived evs. although this could be implemented with the use of bioreactors for msc expansion, different bioreactor culture conditions would result in alterations of ev content, which in turn may impact on the therapeutic efficacy. another challenge to consider for the administration of msc-derived evs to covid- patients is the need to manufacture a safe and reproducible therapeutic product. since the production of evs requires the use of living cells, these have to be cultured under good manufacturing practice (gmp)-compliant procedures to preserve the quality and safety standards criteria. therefore, ev production must follow the same rigorous scientific and ethical guidelines that apply to mscs, and any therapy based on msc-derived evs needs to be approved by the national regulatory agencies to demonstrate its safety and efficacy. in light of these observations, in our opinion, the use of msc evs could be contemplated to treat critically ill patients with ards requiring mechanical ventilation or icu support, or patients with recognized risk factors, such as pre-existing cvd, or cardiovascular complications, for whom standard therapeutic approaches have not proven resolutive. the authors declare no conflict of interest. a pneumonia outbreak associated with a new coronavirus of probable bat origin characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control 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vesicle in vivo biodistribution is determined by cell source, route of administration and targeting mesenchymal stem cell derived secretome and extracellular vesicles for acute lung injury and other inflammatory lung diseases this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -xenq xj authors: chen, hsing i title: acute lung injury and acute respiratory distress syndrome: experimental and clinical investigations date: - - journal: j geriatr cardiol doi: . /sp.j. . . sha: doc_id: cord_uid: xenq xj acute lung injury (ali) or acute respiratory distress syndrome (ards) can be associated with various disorders. recent investigation has involved clinical studies in collaboration with clinical investigators and pathologists on the pathogenetic mechanisms of ali or ards caused by various disorders. this literature review includes a brief historical retrospective of ali/ards, the neurogenic pulmonary edema due to head injury, the long-term experimental studies and clinical investigations from our laboratory, the detrimental role of no, the risk factors, and the possible pathogenetic mechanisms as well as therapeutic regimen for ali/ards. acute lung injury (ali) or acute respiratory distress syndrome (ards) is a serious clinical problem with high mortality. [ ] in animals and humans, ali can be induced by various causes such as brain injury, [ ] [ ] [ ] [ ] enterovirus, [ , ] japanese b encephalitis, [ ] and coronavirus. [ , ] the risk factors for ards included septicemia, acid aspiration, infection, traumatic injury, fat embolism, ischemia/ reperfusion, and other caused. [ , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] our cardiopulmonary laboratory has carried out experimental studies and clinical investigations on ali and ards since . [ ] [ ] [ ] , , ] the purposes of this review article are: ( ) to describe in brief the historical perspective of ards and ali; ( ) to draw attention of an important clinical issue of neurogenic ali; ( ) to present the experimental studies and clinical investigations from our laboratory from to ; ( ) to elucidate the functional role of nitric oxide (no) and other mediators involved in the pathogenesis of ards/ali; ( ) to define the risk factors for ards and ali; and ( ) to discuss the pathogenetic mechanisms and therapeutic regimen for ards/ali. ali or pulmonary embolism (pe) has been reported in humans and animals with intracranial disorders such as head trauma, brain tumor, intracranial hypertension or cerebral compression. early studies in our laboratory demonstrated that acute pe of hemorrhagic and fulminant type occurred accompanying severe hypertension and bradycardia (cushing responses) in rats following cerebral compression (cc) or intracranial hypertension (ich). the lung pathology was characterized by intravascular congestion and disruption of pulmonary large and small vessels leading to severe alveolar hemorrhage (alveolar flooding). these changes was prevented by spinal transection, sympathectomy and sympathoadrenergic blocking agents, but was not affected by decerebration, adrenalectomy, vagotomy and atropine. these results suggest that sympathetic nervous system is pivotal in the neurogenic pe. brain areas above the medulla oblongata and parasympathetic nervous system play little role. [ ] a series of studies was carried out to elucidate the hemodynamic events involved in the neurogenic pe. in anesthetized rats, we measured the aortic and pulmonary blood flow and used techniques of right and left heart bypass. the imbalance in the right and left ventricular output was characterized by a rapid and dramatic decline in aortic flow accompanying a gradual decrease in pulmonary arterial flow. in rats with a right heart bypass, ich produced severe pulmonary hypertension and pe. in the left heart-bypassed rats, ich induced systemic hypertension, http://www.jgc .com; jgc@mail.sciencep.com | journal of geriatric cardiology whereas no significant changes occurred in the lungs. [ ] in anesthetized dogs with a total heart bypass preparation, ich produced constriction of the systemic and pulmonary resistance and capacitance vessels. [ ] [ ] [ ] [ ] the implications of these findings are: ( ) central sympathetic activation elicits increase in the systemic and pulmonary vascular resistance associated with decreases in vascular capacity in both circulations; ( ) the major cause of volume and pressure loading in the pulmonary circulation is acute left ventricular failure resulting in a marked decrease in aortic flow; and ( ) systemic venous constriction causes a shift of blood from the systemic to the pulmonary circulation ( figure ) . a schematic representation summarizes the neural and hemodynamic consequence caused by cerebral compression (figure ). spectral analysis of the aortic flow and pressure wave was employed to evaluate the hemodynamics of steady and pulsatile components. in anesthetized dogs, ich caused significant increases in characteristic impedance, pulse wave reflection and total peripheral resistance with decrease in arterial compliance and cardiac output. the ventricular work was elevated. [ ] clinical study in patients with head injury of various severities, analysis of the heart rate variability with frequency analysis revealed increased low frequency percentage, and low to high frequency ratio with decrease in high frequency. the findings indicate augmented sympathetic and attenuated parasympathetic drive. these autonomic functional changes were related to the severity of brain-stem damage. [ ] these two studies further support the contention that central sympathetic activation is involved in the cushing pressor response and consequent hemodynamic and autonomic alterations. in s, my associates and i were interested in the study of chest disorders. we developed an isolated perfused rat's lung in situ preparation ( figure ). previous method involved removing the isolated lungs from the body and placing the organ on a force-displacement transducer to record the changes in lung weight and these procedures were rather complicated and unstable. our in situ preparation does not require removing the lungs. instead, the isolated lungs were left in situ. the whole rat was placed in a scale platform to measure the change in body weight (bw). since the lungs are completely isolated from the body, the changes in bw reflect the lung weight (lw) changes. the preparation can be accomplished in min. we used a digital-analogue converter to transfer the weight change from the scale platform to a recorder. the lw thus could be continuously monitored during the experiment. in this model, we can obtain the lung weight gain, lw/bw ratio, the changes in pulmonary arterial, capillary and venous pressures, the microvascular permeability (capillary filtration coefficient, k fc ), protein concentration in bronchoalveolar lavage (pcbal), dye leakage, and exhaled nitric oxide (no). the concentration of nitrate/nitrite, methyl guanidine (an index for hydroxyl radical), proinflammatory cytokines [tumor necrosis factor α (tnf α ) and interleukin- β (il- β )] and other factors in the lung perfusate can also be detected. early animal experimentations investigated the pathogenesis, modulators and mediators involved in the ali induced by phorphol, air embolism, platelets, hypoxia, ischemia/reperfusion, endotoxin [lipopolysaccharide (lps)]. the major finding is that cyclooxygenase products of arachidonic acid, thromboxane a in particular is involved in the ali and pulmonary hypertension caused by phorbol, platelets and air embolism. [ , ] furthermore, we found that l-arginine and inhaled no enhanced the lung injury caused by air embolism, while blockade of no synthase (nos) with n ω -nitro-l-arginine methyl ester (l-name) attenuated the ali. [ ] the result suggests that no is also involved. during the summers from  , we encountered a total of children suffering from hand, foot, and mouth figure . isolated and perfused lung in situ preparation. the system consists of a perfusion pump with heat exchanger and a venous reservoir. the rat is artificially ventilated. pulmonary arterial pressure (pap) and venous pressure (pvp) are monitored with transducers. the whole rat is placed on a balance platform to record the body weight change. since the lung is isolated from the whole body, the change in body weight reflects the lung weight change. disease. [ ] chest radiography on admission revealed clear lung. however, out of cases developed severe dyspnea, hyperglycemia, leukocytosis, and decreased blood oxygen tension. arterial pressure (ap) and heart rate (hr) fluctuation ensued. spectral analysis of the ap and hr variabilities showed elevation in sympathetic activity at the onset of respiratory stress. thereafter, parasympathetic drive increased with declines in ap and hr. these children died within h after the onset of ards. before death, chest radiography revealed severe lung infiltration. similar to japanese b encephalitis, destruction of the medullary depressor area caused initial sympathetic activation. reversetranscriptase polymerase chain reaction (rt-pcr) found marked inos mrna expression in the lung parenchyma, suggesting inos may also be involved in the pathogenesis of ards in patients with enterovirus infection. furthermore, we have reported ards in patients with leptospirosis. [ ] in leptospirosis-induced ards, histochemical stain demonstrated spirochetes bacteria in the alveolar space. the pathology included alveolar hemorrhage, myocarditis, portal inflammation and interstitial nephritis. antigen retrieval immunohistochemical stain disclosed inos expression in the alveolar type cells, myocardium, hepatocytes and renal tubules. spectral analysis of ap and hr variabilities indicated decreased sympathetic drive with increased parasympathetic activity. the changes in autonomic functions led to severe hypotension and bradycardia. biochemical determinations suggested multiple organ damage. the pathogenesis of lung and other organ injury might also involve inos and no production. [ , ] in subjects with scrub typhus, orientia tsutsugamushi infection caused alveolar injury. marked inos expression was found in the alveolar macrophages with increase in plasma nitrate/nitrite, suggesting that no production from the alveolar macrophages accounts for the ali. [ ] the victim from rabies was a woman bitten by a wild dog. in addition to sign of hydrophobia, hypoxia, hypercapnia, hyperglycemia and increased plasma nitrate/nitrite were observed. the woman died of alveolar hemorrhage shortly after admission. [ ] recently, we encountered five cases with long-term malignancy. these subjects displayed signs of respiratory distress following an episode of hypercalcemia. two cases died of ards after the plasma calcium was increased above mmol/l. search of literatures revealed that holmes et al. [ ] reported a patient who died of ards following a hypercalcemia crisis caused by a parathyroid adenoma. we conducted animal experiments in whole rodent and isolated perfused rat's lungs. our results indicated that hypercalcemia (calcium concentration > mmol/l) caused severe ali in conscious rats and isolated lungs. immunohistochemical staining showed inos activity in the alveolar macrophages and epithelial cells. reversetranscriptase polymerase chain reaction (rt-pcr) found marked increase in inos mrna expression in lung parenchyma. hypercalcemia also increased nitrate/nitrite, methyl guanidine, proinflammatory cytokines and procalcitonin. pretreatment with calcitonin or l-n ( iminoethyl)-lysine (l-nil, an inos inhibitor) attenuated the hypercalcemia-induced changes. we proposed that hypercalcemia produced a sepsis-like syndrome. the ali caused by hypercalcemia may involve no and inos. [ , ] in addition to the aforementioned animal experimentations and clinical observations that no production through the inos may be involved in the lung injury due to various causes, our research team demonstrated that endotoxemia produced in anesthetized rats by intravenous administration of lipopolysaccharide (lps, endotoxin) provoked systemic hypotension, endothelial damage and ali accompanied by increased plasma nitrate/nitrite and expression of inos mrna, tnf α and il- β . the lps-induced changes were abolished by nonspecific and specific inos inhibitors such as n ω -monomethyl-l-arginine (l-nmma), l-name, aminoguanine and dexamethosone. [ ] this study suggested that no/inos, tnf α and il- β were involved in the endotoxemia-induced ali. generation of no from the activated neutrophil caused alveolar injury from smoke inhalation. [ ] experiments in many laboratories using specific inos inhibitors and/or inos-knockout animals have supported the contention that no/inos is responsible for the oxidative stress and endothelial damage in the ards/ali caused by endotoxin, ozone exposure, carrageenan treatment, hypoxia, acute hyperoxia, bleomaycin administration, acid aspiration and other causes. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] our laboratory further provided evidence to suggest that the no/inos system is involved in the pathogenesis of ali caused by air embolism, [ ] fat embolism, [ ] [ ] [ ] ischemia/ reperfusion, [ ] [ ] [ ] oleic acid [ ] and phorbol myristate acetate. [ ] in these recent studies, various insults caused increase in nitrate/nitrite in plasma or lung perfusate, upregulation of inos mrna in lung parenchyma accompanied with elevation of proinflammatory cytokines such as tnf α , il- β and il- . lin et al. [ ] have suggested that an increase in inos mrna triggers the release of proinflammatory cytokines in septic and conscious rats. the inflammatory responses results in multiple organ damage including ali. inhibition of inos with s-methylisothiourea (smt) or l-nil attenuated the inflammatory changes, release of no and cytokines and prevented the organ dysfunction and ali. [ ] in animal experiments and clinical investigations, the risk factors causing ali/ards include head injury, intracranial hypertension, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] sepsis, [ , , , , , , , [ ] [ ] [ ] [ ] and infections. [ ] [ ] [ ] [ ] [ ] [ ] , , [ ] [ ] [ ] ] pulmonary embolic disorders journal of geriatric cardiology | jgc@mail.sciencep.com; http://www.jgc .com such as fat and air embolism are less common causes. [ , , , , [ ] [ ] [ ] ischemia/reperfusion lung injury may develop as a consequence of several pulmonary disorders such as pulmonary artery thromboendarterectomy, thrombolysis after pulmonary embolism and lung transplantation. [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] gastric aspiration occurs frequently in surgical patients under anesthesia and other causes such as blunt thoracic trauma, impaired glottis competency, and pregnancy. [ , , ] it is one of the major causes of acute respiratory syndrome (ards). [ , ] intratracheal instillation of hydrochloric acid (hci) or gastric particles has been employed as experimental model of acute lung injury (ali). [ , [ ] [ ] [ ] in addition, amphetamine, phorbal myristate acetate, oleic acid have been employed for the induction of ali. [ ] [ ] [ ] [ ] [ ] phorbol myristate acetate (pma, -o-tetradecanoyl-phorbol- -acetate), an ester derivative from croton oil has been used to induce ali. [ , , , ] experiments in vivo and in vitro have demonstrated that pma is a strong neutrophil activator. [ ] [ ] [ ] [ ] activation and recruitment of neutrophil that lead to release of neutrophil elastase and other mediators may play an initial role in the pathogenesis of ali. [ , ] the oleic acid-induced ali has several clinical implications. first, the blood level of oleic acid was significantly elevated in patients with ards. [ , ] second, the proportion of oleic acid incorporated into surfactant phospholipids was also increased in patients with ards and sepsis. [ , ] these observations have provided evidence to suggest that serum level of oleic acid as a prediction or prognostic factor for ards. [ , ] early studies focused on the potential toxic effects of high oxygen fractions on inspired air. [ ] ventilator-induced ali was attributed to the deleterious effects on capillary stress due to alveolar overdistension. cyclic opening and closing of atelectatic alveoli during mechanical ventilation might cause lung injury and enhance the injured alveoli. recent evidence indicated that over distension coupled with repeated collapse and reopening of alveoli initiated an inflammatory cascade of proinflammatory cytokines release. [ , [ ] [ ] [ ] in spite of the risk factors and causes, the pathophysiology of ards/ali has generally considered to be initiated by formation of alveolar edema (even hemorrhage) that is enriched with protein, inflammatory cells or red blood cells. after damage of alveolar-capillary barrier, impairment of gas exchange occurs, with decrease in lung compliance and increases in dispersion of ventilation and perfusion and intrapulmonary shunt. hypoxia, reduction in arterial oxygen partial pressure to fraction of oxygen in inspired air pao /fio , hypercapnia ensued despite ventilation with high oxygen. [ , , , , , ] in addition to the potential toxic effects of no and free radicals, certain chemokines, cytokines, neutrophil elastase, myeloperoxidase and malondialdehyde have been shown to be associated with several types of ards/ali. [ , , , , [ ] [ ] [ ] the balance between proinflammatory and anti-inflammatory mediators is regulated by transcriptional factors mainly nuclear factor-Κ b (nf-Κ b). [ ] pulmonary fluid clearance and ion transport are important factors to determine the extent of lung edema. regulator factors include cystic fibrosis transmembrane conductance regulators, sodium-and potassium-activated adenosine triphophatase (na + -k + -atpase), protein kinases, aclenylate cyclase, and cyclic adenosine monophosphate (camp). [ , , , ] the treatment of ards/ali is difficult and complex. several review articles and monographs have addressed the issue of possible therapeutic regimen. the modalities include extracorporeal membrane oxygenation, prone position, mechanical ventilation with appropriate tidal volume and respiratory pressure, fluid and hemodynamic management and permissive hypercapnic acidosis. [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] other pharmacological treatments are anti-inflammatory and/or antimicrobial agents to control infection and to abrogate sepsis, adequate nutrition, surfactant therapy, inhalation of no and other vasodilators, glucocorticoids and other nonsteroid anti-inflammatory drugs, agents that accelerate lung water resolution and ion transports. [ , , [ ] [ ] [ ] [ ] [ ] although most animal experimentations on these pharmacological options showed favorable results, the effectiveness and outcomes in clinical studies or trials were conflicting. beta agonists to facilitate water removal and ion transport have been shown to be promising. these agents may also stimulate secretion of surfactant and have no serious side effects. there were several reports on the pharmacological and molecular actions of beta agonists, surfactant and vascular endothelial growth factor and related molecules as well as angiotensin-converting enzyme (ace). [ , , ] in addition to the experimental studies and clinical investigations on the pathogenesis of ali/ards, our laboratory has carried out several experimentations on the therapeutic regimen for this serious disorder. in conscious rats, regular exercise training attenuates septic responses such as systemic hypotension, increases in plasma nitrate/nitrite, methyl guanidine, blood urea nitrogen, creatinine, amylase, lipase, asparate aminotransferase, alanine aminotransferase, creatine phosphokinase, lactic dehydrogenase, tnf α, and il β . exercise training also abrogates the cardiac, hepatic and pulmonary injuries caused by endotoxemia. [ ] insulin exerts anti-inflammatory effects on the ali and associated biochemical changes following intravenous administration of lipopolysaccharide (lps). [ ] propofol ( , -diisopropylphenol) has been commonly used for sedation in critically ill patients. [ ] this anesthetic has rapid onset, short duration and rapid elimination. [ ] propofol protects the anesthetized rats from ali caused by endotoxin. [ ] in conscious rats, oleic acid results in sepsis-like responses including ali, inflammatory reactions and increased in neutrophil-derived factors (neutrophil elastase, myeloperoxidase and malondialdehyde), nitrate/nitrite, methyl guanidine, inflammatory cytokines. it depresses the sodium-and potassium-activated atpase, but upregulates the inos mrna expression. pretreatment and posttreatment with propofol alleviates or reverses the oleic acid-induced lung pathology and associated biochemical changes. [ ] pentobarbital, an anesthetic agent commonly used in experimental studies and a hypnotic for patients improves the pulmonary and other organ functions following lps administration. it also increases the survival rate. [ ] a later study by yang et al. [ ] further revealed that pentobarbital suppressed the expression of tumor necrosis factor α , which might result from decrease in the activities of nuclear factor-κβ and activator protein and reduction in expression of p mitogen-activated protein kinase. in vivo examination of cytotoxic effects of lps disclosed that lps caused multiple organ dysfunctions. these changes were attenuated by pentobarbital. pentobarbital also reduced the cell aptosis caused by deforoxamine-induced hypoxia. nicotinamide or niacinamide (compound of soluble b complex) abrogates the ali caused by ischemic/reperfusion or endotoxin by mechanism through inhibition on poly (adp-ribose) synthase or permerase cytoxic enzyme and subsequent suppression of inos, no, free radicals and proinflammatory cytokines with restoration of adenosine triphosphate atp. [ , ] n-acetylcysteine, an antioxidant and cytoprotective agent with scavenging action on reactive oxygen species and inhibitory effects on proinflammatory cytokines ameliorated organ dysfunctions due to sepsis in conscious rats. [ , ] in a similar endotoxin-induced ali model, we found that n-acetylcysteine improved the lps-induced systemic hypotension and leukocytopenia. it also reduced the extent of ali, as evidenced by reductions in lung weight changes, exhaled no and lung pathology. in addition, n-acetylcysteine diminished the lps-induced increases in nitrate/nitrite, tnf α , and il β [ ] in isolated lungs, n-acetylcysteine attenuated the ali caused by phorbol myristate acetate. [ ] in a recent study, we reported that posttreatment with n-acetylcysteine prevented the ali caused by fat embolism. [ ] our series of experimental studies provided results in favor of n-acetylcysteine. the conflicting results and practice guidelines from clinical studies in the recommendation of n-acetylcysteine in critically ill patients [ , ] were commented and analyzed by molnár. [ ] the clinical application of results from animal studies requires further investigations. ards or ali is a serious clinical problem with high mortality. the risk factors leading to ali/ards include head injury, intracranial disorders, sepsis and infections. pulmonary embolic disorders such as fat and air embolism are less common causes. ischemia/reperfusion lung injury may develop as a consequence of several pulmonary disorders such as lung transplantation. gastric aspiration occurs frequently in several conditions such as anesthesia, trauma and pregnancy. the ventilator-induced ali has been attributed to the deleterious effects on capillary stress due to alveolar overdistension. in experimental studies, phorbol myristate acetate and oleic acid have been employed to induce ali. the pathogenesis of ards/ali is complex. experimental studies and clinical investigations from our and other laboratories have indicated the detrimental role of nitric no through inducible no synthase (inos). activation and recruitment of neutrophils that lead to release of neutrophil elastase, myeloperoxidase, malondialdehyde and proinflammatory cytokines may play an initial role in the pathogenesis of ali/ards. the possible therapeutic regimen for ali/ards include extracorporeal membrane oxygenation, prone position, fluid and hemodynamic management and permissive hypercapnic acidosis etc. other pharmacological treatments are antiinflammatory and/or antimicrobial agents, inhalation of no, glucocorticoids, surfactant therapy and agents that facilitate lung water resolution and ion transports. adrenergic beta agonists are able to accelerate lung fluid and ion removal and to stimulate surfactant secretion. there are reports on the actions of vascular endothelial growth factor and related molecules as well as angiotensin-converting enzyme. our laboratory has reported experimental studies on the effectiveness of several regimen for ali/ards. in conscious rats, regular exercise training 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response syndrome n-acetylcysteine as the magic bullet: too good to be true experimental studies and clinical investigations were supported in part by grants from the "national science council". the grant no. this fiscal year is nsc - -b- - -my . the author is grateful to ms. s. y. huang for the assistance in typing an editing. i appreciate the long-term coworkers involved this and other studies in my laboratory. key: cord- -d g toc authors: yu, feng; zhu, jing; lei, ming; wang, chuan‐jiang; xie, ke; xu, fang; lin, shi‐hui title: exploring the biomarkers associated with different host inflammation of acute respiratory distress syndrome (ards) from lung metabolomics in mice date: - - journal: rapid commun mass spectrom doi: . /rcm. sha: doc_id: cord_uid: d g toc rationale: the aim of this study was to analyze the metabolomics of lung with different host inflammation of acute respiratory distress syndrome (ards) for the identification of biomarkers for predicting severity under different inflammatory conditions. methods: cecal ligation and puncture (clp) and lipopolysaccharide (lps)‐intratracheal injection induced acute lung injury (ali). a mouse model was used to explore lung metabolomic biomarkers in ali/ards. the splenectomy model was used as an auxiliary method to distinguish between hyper‐ and hypo‐inflammatory subtypes. plasma, lung tissue and bronchoalveolar lavage fluid (balf) samples were collected from mice after clp/lps. the severity of lung injury was evaluated. expression of tumor necrosis factor‐α (tnf‐α) in mice serum and lung was tested by elisa and pcr. polymorphonuclear cells in balf were counted. the lung metabolites were detected by gc/ms, and the metabolic pathways predicted using the kegg database. results: the lps/clp‐splen group had more severe lung injury than the corresponding ali group; that in the clp‐splen group was more serious than in the lps‐splen group. tnf‐α expression was significantly elevated in the serum and lung tissue after lps or clp, and higher in the lps/clp‐splen group than in the corresponding ali group. the level of tnf‐α in the clp‐splen group was elevated significantly over that in the lps‐splen group. both these groups also showed significant neutrophil exudation within the lungs. during differential inflammation, more differential metabolites were detected in the lungs of the clp‐group ali mice than inthe lps group. a total of compounds were detected in the lungs of the clp and clp‐splen groups. contrastingly, compounds were detected in the lungs of the lps and lps‐splen groups. the lps‐splen and clp‐splen groups had significant neutrophil exudation in the lung. random forest analysis of lung‐targeted metabolomics data indicated ‐hydroxyphenylacetic acid, ‐aminocyclopentanecarboxylic acid (acpc), cis‐aconitic acid, and hydroxybenzoic acid as strong predictors of hyper‐inflammatory subgroup in the clp group. furthermore, with splenectomy, differential metabolic pathways between the clp and lps groups were revealed. conclusions: hyper‐inflammatory subgroups of ards have a greater inflammatory response and a more active lung metabolism. combined with host inflammation background, biomarkers from metabolomics could help evaluate the response severity of ards. acute respiratory distress syndrome (ards) is an acute inflammatory lung injury, associated with increased pulmonary vascular permeability, increased lung weight, and loss of aerated lung tissue [ ] . although years have passed since the first description of ards [ ] , the overall mortality is still more than % [ , ] . unfortunately, it is a clinical feature of very different mechanisms, with complex syndromes, and biological and clinical heterogeneity. because of the heterogeneity of the host response, it is difficult to found the key to curing every patient with ards. the clinical and biomarker characteristics of ards patients demonstrated hypo-inflammatory and hyper-inflammatory effects [ ] .specific subsets of critically ill patients have higher risk of disease-related outcome or differential responses to therapy [ , ] .therefore, the different inflammatory sub-phenotypes of ards may indicate varied risks related to the disease [ ] . metabolic phenotypes, which represent different pathways important to the pathophysiology of ards, could potentially be used to identify the subgroup that may benefit from certain targeted therapies [ ] . about one third of hyper-inflammatory ards patient have a higher plasma level of inflammatory biomarkers [ ] . some biological indicators, such as endocan [ ] , srage and ang- [ ] , are closely related to the hyper-inflammatory subphenotype of ards. tnf-α is an important inflammatory factor that can induce t cells to produce various inflammatory factors, and then promote the occurrence of inflammatory reactions. it has been reported that tnf-α is a potential biomarker for acute respiratory distress syndrome, as well as for mortality in patients with obesity and coronavirus (covid- ) [ ] . in addition, studies have found that -hydroxybenzoic acid has anti-catabolism and anti-inflammatory effects, and prevents the upregulation of pro-inflammatory markers, including metalloproteinases and cyclooxygenase [ ] . whether acps, as a small molecule, also has such potential needs further study. thus, classification of patients with ards into hyper-and hypoinflammatory subphenotypes using plasma biomarkers may facilitate more effective targeted therapy [ ] . metabolism has increasingly been acknowledged as a potential target for therapies aimed at modulating the immune system either to enhance or to suppress immunological responses [ ] . due to the coupling of inflammation with metabolism, the novel "inflammation-immunitymetabolism axis" may be another useful way to propose new therapeutic implications and deeper understanding for ards. metabolomics is a rapidly expanding field of systems biology that provides the ability to generate a "snapshot" measurement of all small molecules and this article is protected by copyright. all rights reserved. metabolites in a given sample [ , ] . the burgeoning field of metabolomics lies in its application to acute lung diseases, specifically pneumonia and ards [ ] . the application of untargeted metabolomics for biomarker discovery is well suited to the complexity of ards because metabolomics can detect several hundreds of metabolites, depending on the analytical platform, from a single sample, with minimal bias and no prior knowledge of the sample composition [ , , ] .we have found that specific compounds related to hypoxia may serve as early biomarkers for ards, while metabolites with significant correlations with the partial pressure of arterial oxygen (pao )/percentage of inspired oxygen (fio ) may play a role in determining its severity [ ] . however, it is difficult to find specific metabolic evidence in clinical samples related to differences in inflammatory hosts. the spleen is a site where immune responses that are deleterious to the host can be regulated [ ] . the white pulp of spleen is a secondary lymphoid organ with key functions in immune response initiation and regulation. various immune cells (macrophages, dendritic cells, subsets of b and t lymphocytes) of the white pulp trap antigens and generate an antigen specific response against invading pathogens (bacteria, viruses and fungi). patients without a spleen (resulting from traffic accidents, trauma, etc.) have severe inflammation and a high risk of death in sepsis. therefore, we think that patients without a spleen may form acute lung injury (ali) models with "all or nothing" different subtypes of inflammatory hosts. we have also confirmed that this ali lack-of-spleen model will not die quickly as a result of complete immunodeficiency [ ] . in order to explore differences of metabolism in host inflammation of ards, we established a special ali mode (with spleen or without spleen, induced by lipopolysaccharide (lps)-tracheal infusion, or cecal ligation and puncture (clp). gc/ms metabolomics was then used to determine the endogenous metabolites in the lung tissues. c bl/ mice (male, - weeks old) from the laboratory animal center of chongqing medical university (cqmu, chongqing, china) were usedthe . previous studies had shown that estrogen provides a protective effect in ards [ ] . as, however, the role of hormones is not the research aim in this study, we chose only male mice for the studies [ ] .the mice were acclimatized to the new environment for seven days at °c with a free access to water and food and with a h light/dark cycle before experiments. the study was performed according to international, national and institutional rules concerning animal experiments, clinical studies this article is protected by copyright. all rights reserved. and biodiversity rights. the study protocol was approved by the ethics committee of our institute. the abdominal incision was then closed. in a "sham" operation on mice, the abdominal wall was cut, the spleen was only slightly spit, and there was no resection. the mice were given subcutaneous injections of ceftriaxone ( ug/g) for three days after surgery. fourteen days later, these splenectomy mice were used to establish the model [ ] . lps-induced ali was performed to establish an ali mouse model, using the same anesthesia method as described above. a mg/ml solution of lps was injected into mice through intratracheal instillation, and the control group was injected with the same volume of sterile phosphate-buffered saline (pbs) [ ] . the mice were randomly divided into three groups: control group (n= ), lps group (lps, n= ), and splenectomy group (lps-splen, n= ). the nice were euthanized by carbon dioxide box anesthesia h, h, and h after being challenges with lps or pbs, and blood and lung tissues were harvested for analysis. mice were randomly divided into three groups: control group (n= ), clp group (clp, n= ), and splenectomy group (clp-splen, n= ) and the anesthesia method was followed as previously described. the mice were positioned in dorsal recumbency. after shaving and this article is protected by copyright. all rights reserved. aseptic preparation of the surgical site, a ventral midline incision ( cm) was made to allow exteriorization of the cecum. the cecum was identified and was penetrated through-andthrough with a -gauge needle with a - silk suture at % from the tip. after being punctured, the cecum was gently squeezed to extrude a small amount of feces and returned to the abdominal cavity. the abdominal incision was then closed. sham-operated control mice (sham group) were subjected to the same surgical laparotomy after anesthesia, where the cecum was exteriorized and manipulated as described but not ligated or punctured. immediately after surgery, the animals were resuscitated with ml/kg saline injected subcutaneously [ ] . at the end of the experiment, the mice were euthanized by carbon dioxide box anesthesia h, h, and h after the clp procedure to collect whole blood and lung tissues for analysis. lung tissue samples were collected h post challenge with lps or clp. the superior lobe of the right lungs was fixed with % formalin in pbs for h, dehydrated in a graded ethanol series, and embedded in paraffin. paraffin sections were then stained with hematoxylin and eosin (h&e) followed by microscopic assessment and photographic documentation. lung injury scores were estimated; the higher the score, the more severe the injury. the four following indicators of lung injury were used to arrive at this score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; and alveolar septal thickening or transparent membrane formation. the marking system was: marks, no or very slight damage; mark, mild injury; marks, moderate injury; marks, severe injury; and marks, very severe damage. the cumulative increase in the number of lesions related to the total score yields the pathological score of lung injury. the blood vessels leading to the lungs and the left bronchus were ligated h post challenge with lps or clp. μl of pbs was injected into the right lung through the trachea and, seconds later, the pbs was removed and the bronchoalveolar lavage fluid (balf) was collected. these lavages were carried out twice, and a total of μl of balf was collected. the serum, balf, spleen, and lung tissues were harvested for analysis. blood and lung tissue samples were taken h post challenge with lps or clp. blood was drawn from mice and allowed to coagulate for h at room temperature. the serum was then obtained by centrifuging the blood samples at °c for min at , ×g. the lungs were this article is protected by copyright. all rights reserved. ground in pbs ( mg tissues/ μl pbs). the serum and lung homogenate were then aliquoted and kept frozen at - °c until analysis. the tumor necrosis factor-α (tnf-α) levels were measured by elisa. total cellular rna was extracted from lung tissue using trizol reagent accompanied by dnasei digestion. quantitative real-time polymerase chain reaction (qrt-pcr) for mouse tnf-α was performed using specific primers (designed and synthesized by takara for s. gene expression normalized to gapdh was used to determine relative target gene expression by the ΔΔc(t) method. twenty mg of prepared mice lung tissue was added to fresh tubes, followed by the addition of μl of the internal standard, l-alanine- , , , -d ( mm). after adding μl of cold methanol-water ( % v/v), the tissues were homogenized and centrifuged ( , × g, min) to collect the supernatant. the lung tissue was dried using a speedvac and stored at - °c prior to derivatization. the volatilities of extracted metabolites were lowered using methyl chloroformate (mcf) derivatization, based on the protocol of smart et al [ ] . in brief, μl of sodium hydroxide ( m) was added to the speedvac-dried samples. ml of methanol and μl of pyridine were also added as the methyl group donor and catalyst, respectively. the reaction was started by adding μl mcf, followed by seconds of vortexing and subsequently adding another μl of mcf, followed by seconds of vortexing. in order to isolate derivatized metabolites from the reactive mixture, μl of chloroform and μl of sodium bicarbonate ( mm) were added and vortexed for seconds. the chloroform phase was isolated, and excess water was removed by adding anhydrous sodium sulfate. this article is protected by copyright. all rights reserved. the derivatized samples were analyzed using a gas chromatograph (agilent, santa clara, ca, usa) fitted with a zb- capillary column ( m × μm id × . μm with a -m guard column; phenomenex, torrance, ca, usa) coupled to an agilent msd single quadrupole mass spectrometer operating in electron ionization mode at ev. the gc and ms procedures followed the protocol outlined by smart et al. [ ] the isolated chloroform phase was injected at °c in pulsed splitless mode with helium carrier gas at a flow rate of ml/min. the program temperature was as follows: initial temperature of °c, ramped at °c/min to °c, then at °c/min to °c, and finally at °c/min to °c. the auxiliary temperature, quadrupole mass analyzer temperature, and source temperature were set to , , and °c, respectively. the mass range was m/z - , the scan speed . m/z units/s and the solvent delay . min. compound deconvolution and identification were performed by the automated mass spectral deconvolution and identification system (amdis; nist, gaithersburg, md, usa) software, using our internal methyl chloroformate derivatization mass spectra library of metabolite standards. the compounds were identified based on two criteria: > % match with the library spectrum and within a -min bin of the respective chromatographic retention time. the relative abundance of the metabolites was extracted via our in-house massomics software, using the peak height of the highest reference ion mass. the metabolite values were normalized by the abundance of the internal standard (l-alanine- , , , -d ) and total ion count, in order to correct for experimental variability. the metabolomics data have been deposited to the embl-ebi metabolights database (doi: https://doi.org/ . /nar/gks .) [ ] with the identifier mtbls . the abundance of identified compounds was adjusted to a gaussian distribution via log transformation prior to statistical analysis. multivariate analysis of anova followed by tukey's hsd test was performed in r. the predicted metabolic activities were determined using our pathway activity profiling r package based on the kegg online database. the relative metabolic activities were transformed to have a mean of zero and a standard deviation of one (z-score). subsequently, the metabolic pathways were classified according to their cellular processes, and only the predicted metabolic pathways with p values and q values less than . were displayed. the metabolic activities were first normalized by log transformation and pareto scaling, followed by ranking of the metabolic pathways using a random forest model to capture their contribution to the classification accuracy demonstrated in a vip plot. the metabolic network was constructed according to a pathway-based framework provided by this article is protected by copyright. all rights reserved. metscape that connected the kegg human metabolic pathways with our identified metabolites via the kamada-kawai layout, which relates the layout of metabolites to minimize metabolic reactions between metabolites within a metabolic network. all the illustrations and figures displayed were plotted using the ggplot r package, graphpad prism (graphpad software, san diego, ca, usa), and spss . (ibm, amonk, ny, usa). mice (with or without splenectomy) were euthanized h post challenge with lps or clp. the administration of lps or clp led to severe lung injury, compared with the controls ( figure a ). the lung injury scores showed that splenectomy can aggravate lung injury directly in either the lps or the clp group, p < . ( figure a ). all parts of the lung injury degree index, such as thickened alveolar wall, hemorrhage in the alveolus, alveolar collapse, and inflammatory cell infiltration in the lps-splen group or clp-splen group, were more severe than in the corresponding ali group. moreover, the lung injury in the clp-splen group is more serious than in the lps-splen group, p < . ( figure a ; supplementary table , supporting information). the concentrations of tnf-α were significantly elevated in the serum and lung tissue after lps or clp compared with the controls, p < . ( figures b and c ). in addition, the expression of tnf-α in the lps-splen or clp-splen groups was higher than in the corresponding ali (lps or clp) groups. the levels of tnf-α in the clp-splen group were significantly elevated compared with the lps-splen group, p< . ( figures b and c ). subsequently, infiltration of neutrophils was confirmed with neutrophil numbers in balf ( figure d ). after splenectomy, the lps(splen) and clp(splen) groups have significant neutrophil exudation in the lungs ( figure d ). the pls-da and leave-one-out cross-validation results are shown in figure . the supervised pls-da showed that the four different groups were well-clustered, with specific metabolic profiles for each ( figure b ). after anova was performed for the clp and clp-splen groups, a total of compounds were detected in the lungs, including metabolites from organic acids ( / , . %), amino acids ( / , . %), amino acid derivatives ( / , . %), and others ( figure a; supplementary figure , supporting information). in the random forest (rf) analysis the "mean decrease accuracy" indicates how much a certain metabolite contributes to separation of the groups, and the overall "predictive accuracy" is this article is protected by copyright. all rights reserved. indicative of the accuracy of a set of metabolites in discriminating spleen status [ ] . rf analysis of lung-targeted metabolomics data defined a set of metabolites that constitute the best predictors of differences in host inflammation status: in particular, increased hydroxyphenylacetic acid, -aminocyclopentanecarboxylic acid (acpc), and cis-aconitic acid, tridecane and hydroxybenzoic acid were strong predictors of the hyper-inflammatory subgroup in clp-induced ali ( figure b ). after anova was performed for the lps and lps-splen groups, only compounds were detected in the lungs. the organic acids ( / , %) make up the largest category among the subgroups of different inflammation in ali, which was induced by lps ( figure c ). consequently, under differential inflammation (hyper-vs hypo-), the lungs of clp-induced ali will detect more differential metabolites than the lpsinduced ali lungs ( figures a and c ). detailed information on this is displayed in supplementary table (supporting information). when clustering the hyper-inflammatory and hypo-inflammatory classes separately, difference in the lung metabolites between clp and lps were found. under splenectomybased conditions, there were differential metabolites between the clp and lps groups; the organic acids ( / , . %) and tca cycle inter mediates ( / , . %) were the two largest categories ( figure d ). under no-splenectomy-based conditions, there were only differential metabolites between the clp and lps groups and the organic acids ( / , . %) formed the largest category ( figure e ). while identifying the linked metabolic pathways, the anova test was used to extract significant pathways from them. for the clp and clp-splen groups, the kegg alignment revealed pathways that were linked to the above-detected metabolites. amino acid metabolism ( / , . %), chemical structure transformation maps ( / , . %), biosynthesis of other secondary metabolites ( / , . %), metabolism of other amino acids ( / , . %), and carbohydrate metabolism ( / , . %) were the first five largest categories ( figure a ). for the lps and lps-splen groups, however, only pathways linked to the above-detected metabolites were revealed from kegg. the benzoic acid family, bisphenol degradation, and folate biosynthesis were revealed in the h-post-lps-intervention group. in addition, tropane, piperidine and pyridine alkaloid biosynthesis, biosynthesis of phenylpropanoids, and phenylalanine metabolism were revealed in the h-post-lpsintervention group ( figure b ). the hyper-inflammatory and hypo-inflammatory groups were this article is protected by copyright. all rights reserved. then clustered to predict the metabolic pathways separately. under splenectomy-based conditions, there were differential metabolic pathways between the clp groups and the lps groups ( figure c ). however, we cannot find differential metabolic pathways between the clp groups and the lps groups under no-splenectomy-based conditions. ards is a clinically and biologically heterogeneous disorder associated with effects such as trauma, shock, infection, and sepsis. failure of clinical therapeutic trials prompted the investigation and subsequent discovery of two distinct phenotypes of ards (hyperinflammatory and hypo-inflammatory) that have different biomarker profiles and clinical courses and respond differently to management strategies [ ] . the hyper-inflammatory subgroup (about one third of all) shows t higher mortality, higher severity of illness, and worse clinical outcomes [ ] . even in covid- , the hyper-inflammatory response is closely related to the ards of critical covid- pathogenesis [ ] . a major issue is that ards is such a heterogeneous, multi-factorial, end-stage condition that the strategies for "lumping and splitting" are critical [ ] . metabolic phenotypes, representing different pathways important in the pathophysiology of ards, can be used to identify the subgroups [ ] . they can also help distinguish the subphenotypes of ards (hypo-inflammatory and hyper-inflammatory) and identify the risk of developing ards, diagnosis, risk stratification and monitoring. the use of metabolomics as a possible diagnostic tool for ards has been investigated in several studies, including exhaled breath and oedema fluid analyses. we previously found that phenylalanine, aspartic acid, and carbamic acid levels were significantly different in the plasma samples of ards patients [ ] . four metabolites (ornithine, caprylic acid, azetidine, and iminodiacetic acid) could serve as metabolic phenotypes to potentially predict the severity of ards [ ] . due to the limitations of the research conditions, it is difficult to distinguish the subtypes of inflammation solely by metabolomics. the spleen performs vital hematological and immunological functions. removal of the spleen had already been established as a routine technique to treat splenic trauma and other diseases affecting the spleen [ ] . however, splenectomized (asplenic) or hyposplenic individuals have an increased risk of infections [ ] , and this can lead to severe sepsis known as overwhelming post-splenectomy infection (opsi), which has a very high mortality rate [ ] . a previous study showed that a higher charlson comorbidity index score was significantly associated with severe sepsis/septic shock post-splenectomy [ ] . moreover, splenectomy can this article is protected by copyright. all rights reserved. alter the serum cytokine profile, exacerbating the systematic inflammatory responses and injury to multiple organs [ ] . the spleen is necessary for the recruitment of classical monocytes and neutrophil extravasation into the injured lungs [ ] , and it can play an important role in intestinal ischemia-reperfusion (iir)-induced ali [ ] . furthermore, the spleen coordinates interleukin(il)- -dependent il- production, which reduces lung injury during experimental acute kidney injury(aki) [ ] . splenic factors also exacerbate sap-associated lung injury [ ] . in animal experiments, the splenectomy model can be used as an auxiliary method to distinguish high and low inflammatory subtypes. therefore, an ali animal model of host inflammation differentiation can be established after carrying out a splenectomy. the splenectomy model, which could demonstrate the significant involvement of autoimmunity, plays an important role in the experiment. however, it has a higher mortality rate under experimental conditions, and this mortality rate is significantly increased when combined with the clp model [ ] . in our study, this mortality rate was very high after conducting the splenectomy followed by clp and the passage of time (from to h). in order to ensure the homogeneity of experimental mice in metabolic analysis, the same batch of mice was used to establish models synchronously, such that they would have an effective cluster effect. it was difficult to achieve a greater number of animals at each time point and in each group because of the extremely high mortality rate, and because of the existing limitations of these special animal models. ideally, more mice (over ) in each group would be beneficial for repetitive data collection. we found that the clp-splen and lps-splen groups had more severe lung damage than the corresponding non-splenectomy ali group ( figure a) . moreover, the lps-splen and the clp-splen groups showed significant neutrophil exudation in the lungs after splenectomy; however, the changes in the clp-splen group were more significant ( figure d ). possible causes may be that splenectomy did not affect neutrophil extravasation in the lps models of lung injury, as was confirmed by rieg et al [ ] , or because of tnf-α-induced adhesion of monocytes to endothelial cells and leukocyte transmigration in ali. we found that the expression in lps-splen or clp-splen group was higher than in the corresponding ali group, and that the levels of tnf-α in the clp-splen group were significantly elevated, compared with the lps-splen group, p< . (figures b and c) . cd receptor [ ] .the sensitizing effect of lps stimulation aggravates lung damage [ ] . lbp may play an important role in augmenting tnf-α expression by alveolar macrophages in the lung [ ] : the duration of clp action is prolonged, the stimulatory effect persists, and tnfα expression is higher than with lps-intratracheal injection (figures b and c) . on the other hand, the cholinergic anti-inflammatory pathway is completely inhibited following splenectomy [ , , ] . although, splenectomy itself was not associated with increased serum il- or lung injury, the absence of a counter anti-inflammatory response by splenic il- production results in a high proinflammatory response and lung injury [ ] . based on the difference of inflammation (after splenectomy) in ali, the metabolomic differences of lung tissues could be identified. pca and opls-da were used for discriminant analysis, and univariate statistical analysis was used to screen important differential metabolites in untreated(hypo-inflammatory) or splenectomy-treated(hyper-inflammatory) ali mice, which had been subjected to different modeling methods (lps or clp). a total of compounds and pathways were found in lungs, differing between hyper-and hypo-inflammatory in clp groups ( figures a and a) . however, only compounds and pathways were found to differ in the lungs between hyperinflammatory and hypo-inflammatory in lps (figures c and b ). this suggests that nonpulmonary ards (such as clp) has more active lung metabolomics changes that are involved in inciting the differences of host inflammatory response. moreover, continuous stimulation of clp promoted inflammation and injury to the lungs. organic acids form the largest group of differential metabolites in clp and lps under differential inflammatory conditions, and can be attributed to defects in the intermediary metabolic pathways of carbohydrates, amino acids, and fatty acid oxidation. related physiological analysis had found that sepsis experienced a highly catabolic status. many proteins decompose into amino acids to supply energy, which seemed to be relevant to poor prognosis [ , ] . thus, the concentration of amino acids and its derivatives ( / , . %) demonstrated a notable upward tendency in the clp group ( figure a ). among the metabolic pathways, this category, including amino acid metabolism ( / , . %) and metabolism of other amino acid ( / , . %), also dominates ( figure a ). interestingly, rf analysis of lung-targeted metabolomics data showed that the metabolic biomarker group with products was a strong predictor of the hyper-inflammatory subgroup in clp-induced ali ( figure b ). -hpa, as one of the major metabolites in polyphenols, is a necessary adaptive response of microbiota to the stress-induced changes in inflammation [ ] . -hpa could be a biomarker for quantifying leukocyte-mediated damage [ ] , and it has been confirmed to participate in the intermediate step of tyrosine degradation [ ] . acpc is a this article is protected by copyright. all rights reserved. nonmetabolized amino acids. amino acid transport by system "a" is sodium-dependent and results in a high intracellular-to-extracellular gradient [ ] . apac was also shown to have a high affinity for the "a" transport system of endothelial cell membranes [ ] . sepsis specifically decreases cell membrane potential and inhibits the amino acid transport system a [ ] . this is probably because of the reduction in the pulmonary absorption of amino acids in ards. hypoxia cause an imbalance of the nadph/nadp+ and nadh/nad+ ratios, accompanied by the accumulation of intermediates of the tca cycle, such as cis-aconitic acid, as was found by rf analysis. ane may be a substance derived from environmental factors. exposure to fuels and heavy metabolites ( -tridecanone, -tridecanol, and -tetradecanol) was observed only in the lung tissues, possibly indicating that metabolism occurred in the lungs [ ] . however, the aliphatic compound n-tridecane showed no cytotoxic effects on chemoattractant protein- (mcp- ) and il- production [ ] . we therefore believe that tridecane cannot be a member of metabolic biomarker group. low-molecular-weight phenolic acids (phas) are the products of the degradation of aromatic amino acids and polyphenols by the intestinal microflora [ ] , and all phas have an impact on mitochondria and neutrophils. low-molecular weight phas of microbial origin participate in the regulation of the ros production in both the circulation and tissues, thereby affecting the level of oxidative stress [ ] . therefore, there may be a group of metabolic biomarkers related to inflammation, hypoxia, infection, etc. between the hyperinflammatory and hypo-inflammatory subgroups of ards. the etiology has also become a variable. when we clustered the hyper-inflammatory and hypo-inflammatory separately, there were differential metabolites and differential metabolic pathway between the clpgroups and the lps-group in hyper-inflammatory subgroup ( figures d and c ). in the hypoinflammatory subgroup, there were only differential metabolites between the clp and the lps groups ( figure e ), although no different metabolic pathways could be found in this study. overall, the hyper-inflammatory subgroups of ards were observed to exert more abundant metabolism changes in the lung. metabolomics has the potential to improve our understanding of ards biology. from the analysis of lung metabolomics, the difference of host inflammatory response is a key link in determining the severity of ards. hyper-inflammatory subgroups of ards have a heavier inflammatory response and a more active lung metabolism. combined with host inflammation this article is protected by copyright. all rights reserved. acute respiratory distress syndrome: the berlin definition acute respiratory distress in adults acute respiratory distress syndrome current incidence and outcome of the acute respiratory distress syndrome biomarkers for acute respiratory distress syndrome and prospects for personalised medicine toward smarter lumping and this article is protected by copyright smarter splitting: rethinking strategies for sepsis and acute respiratory distress syndrome clinical trial design acute respiratory distress syndrome (ards) phenotyping coronavirus disease (covid- ): cytokine storms, hyper-inflammatory phenotypes, and acute respiratory distress syndrome genes dis. . epub ahead of print heterogeneous phenotypes of acute respiratory distress syndrome after major trauma endocan levels in peripheral blood predict outcomes of acute respiratory distress syndrome il- a and tnf-α as potential biomarkers for acute respiratory distress syndrome and mortality in patients with obesity and covid- regulation of inflammatory response in human osteoarthritic chondrocytes by novel herbal small molecules host-response subphenotypes offer prognostic enrichment in patients with or at risk for acute respiratory distress syndrome 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heparin and splenectomy on survival and plasma fibronectin levels in rat peritonitis lipopolysaccharide binding protein enhances the responsiveness of alveolar macrophages to bacterial lipopolysaccharide. implications for cytokine production in normal and injured lungs the effect of splenectomy on endotoxin-induced acute lung injury and its potential mechanism in rats. the chinese journal of trauma splenectomy inactivates the cholinergic antiinflammatory pathway during lethal endotoxemia and polymicrobial sepsis the spleen: the forgotten organ in acute kidney injury of critical illness splenectomy exacerbates lung injury after ischemic acute kidney injury in mice reprogramming of basic metabolic pathways in microbial sepsis: therapeutic targets at last? metabolomic analysis of the effects of adipose-derived mesenchymal stem cell treatment on rats with sepsis-induced acute lung injury gut-brain axis: how the microbiome influences anxiety and depression the metabolism and de-bromination of bromotyrosine in vivo association of the tyrosine/nitrotyrosine pathway with death or icu admission within days for patients with community acquired pneumonia aminocyclopentane carboxylic acid and alpha-aminoisobutyric acid: comparison to fluorodeoxyglucose and diethylenetriaminepentaacetic acid in morphologically defined tumor regions effect of sepsis on amino acid transport system a and its response to insulin in incubated rat skeletal muscle metabolites from inhalation of aerosolized s- synthetic jet fuel in rats il- from lung epithelial cells exposed to volatile organic compounds toxic effects of microbial phenolic acids on the functions of mitochondria effect of phenolic acids of microbial origin on production of reactive oxygen species in mitochondria and neutrophils we are very grateful to the laboratory of lipid & glucose metabolism at the first affiliated hospital of chongqing medical university to provide laboratory facilities. the authors declare no conflict of interest. this article is protected by copyright. all rights reserved. lungs from each experimental group were processed for histological examination after h&e staining. lps-and clp-induced mice exhibited obvious lung injury, p < . . lung injury scores were estimated by the method of mikawa, based on the following four indicators of lung injury score: alveolar congestion; bleeding; gap or vascular wall neutrophil infiltration or aggregation; alveolar septal thickening or transparent membrane formation. these were scored as marks: no or very slight damage, mark: mild injury, marks: moderate injury, marks: severe injury, marks: very severe damage. the number of lesions of the total score is the pathological score of the ali. the lung injury of clp-splen group is more serious than that of the lps-splen group, p < . .(b)(c) tnf-α in serum or lung of mice was detected by elisa or pcr. tnf-α was significantly elevated in the serum and lung tissue after lps or clp, compared with controls, p < . . expression of tnf-α in the lps-splen group or clp-splen group, was higher than in the corresponding ali (lps or clp) group. the levels of tnf-α in the clp-splen group were elevated significantly, compared with the lps-splen group, p< . .(d) infiltration of neutrophils was confirmed with neutrophil numbers in balf. after splenectomy, the lps(splen) group and the clp(splen) group have significant neutrophil exudation in the lung. ****p < . ,*p < . , by two-way anova followed by a lsd multiple comparisons test. each group n = , experiments are repeatable and most representative one was shown.this article is protected by copyright. all rights reserved. key: cord- -zjab o o authors: ali, yousaf title: self assessment questions date: - - journal: self assessment questions in rheumatology doi: . / - - - - _ sha: doc_id: cord_uid: zjab o o nan this is a common inpatient scenario. a stable patient with renal failure is admitted and undergoes a stressful procedure that triggers gout. treatment of acute gout involves high-dose nsaid, colchicine, or corticosteroids. in a patient with renal failure and diverticular inflammation, nsaids and colchicine should be avoided since they are poorly tolerated. a monoarticular presentation could be injected with local intraarticular corticosteroid assuming that the cultures are negative. in this patient with polyarticular gout a short burst of oral corticosteroid is the best option. uric acid levels fall and are often normal during an attack and should not be used as a diagnostic test. terkeltaub ra. clinical practice. gout. n engl j med. oct ; ( ) : - . this is a rare but important complication of ss. distal rta occurs due to failure to acidify urine to a ph < . . this results in anion gap acidosis, hypokalemia, nephrocalcinosis, and bone demineralization. the lymphocytes that invade the tubular epithelial cells are cd -positive, i.e., cytotoxic t cells and similar to those found in the salivary glands of patients with sjögren's syndrome. the same immunological process is probably operative in the renal tubulointerstitial tissue as in the salivary glands to induce the characteristic tissue changes of sjögren's syndrome. matsumura a -year-old patient with crohn's disease presents for evaluation of new onset arthritis. she has a -year history of colitis managed with sulfasalazine and local corticosteroid suppositories. infliximab at mg/kg was recently commenced due to persistent disease activity. she arrives with new onset polyarticular joint pain. on examination she has synovitis of the small joints of the hands and a warm knee effusion. lab work: wbc = . , hb = . g/dl, platelets = , esr = , ana = : , rf = , and renal and liver studies normal. what is the most likely diagnosis? what further tests can confirm this? how would you manage this patient? she has aggressive erosive disease with elevated markers of inflammation and hightiter ccp antibody. since she is actively symptomatic despite two diseasemodifying antirheumatic drugs (dmards) it is time to add methotrexate (mtx). triple therapy is superior to single agents although there are many pills to take weekly. a short bridge of corticosteroids may also be helpful in decreasing inflammatory symptoms. some rheumatologists will argue that aggressive early use of tumor necrosis factor (tnf) antagonists is also indicated. since these are expensive medications and the patient has not failed the "gold standard" they should be reserved unless she fails to respond to mtx. this algorithm may change as costbenefit data on the existing agents become available and as newer, safer, and more efficacious agents are introduced. a -year-old female is referred for diffuse joint pain and fatigue of years duration. her pmh is consistent with depression, posttraumatic stress disorder (ptsd), and hypothyroidism. hematologic, biochemical, and immunologic studies are negative. inflammatory markers are not elevated. on examination she has palatal hypertrophy and is markedly obese. cardiac examination reveals a loud s with rv heave. pulmonary examination is normal. she has pitting edema and multiple tender points to palpation but no synovitis. what is the most likely diagnosis? what investigations, if any, are appropriate? how would you manage her? patients with chronic sleep apnea develop pulmonary hypertension due to hypoxemia and develop subsequent right-sided pulmonary hypertension. a distinct relationship exists between poor sleep quality and pain intensity. this patient most likely has fibromyalgia, which is characterized by widespread pain, insomnia, and tender points. investigations should focus around excluding an organic cause for pain, managing sleep disturbance, and treating underlying sleep apnea and depression. physical therapy, counseling, and aerobic exercise have also been found to be useful. amyloidosis is a systemic disease characterized by deposition of insoluble beta pleated sheets of protein in various organs. the diagnosis can be confirmed by congo red staining of subcutaneous fat. although it is primary amyloidosis that usually affects the myocardium it can also occur with the secondary form. his lower extremity edema might be explained by heart failure but nephrotic syndrome also needs to be excluded. treatment of secondary amyloidosis is often unsatisfactory and primarily involves treatment of the underlying disease. a -year-old female with sle consults you for contraceptive advice. she is sexually active and has a history of serositis, leucopenia, and arthritis. current medications include azathioprine mg/daily and naprosyn mg twice daily. labs: wbc = . , hb = . , platelets = , , lupus anticoagulant present, ptt = s, and anticardiolipin (aca) antibody igm/igg strongly positive. what advice would you give her regarding contraception? answer: she should be advised to use barrier protection, progesterone only pill or iucd's this patient has a prolonged partial thromboplastin time, thrombocytopenia, strongly positive aca's, and circulating lupus anticoagulant. although the patient has not had prior thromboses she is at high risk for developing antiphospholipid antibody syndrome in view of these blood tests. these patients are hypercoagulable and should not be given estrogen-containing products due to the increased risk of thrombosis. she should be advised to use barrier protection, progesterone-only pill, or iucds. acog recommends that in sle, estrogen-containing contraceptives be avoided in patients with vascular disease, nephritis, or aca. a -year-old previously healthy student is evaluated for new onset fever, joint pain, and rash. she is sexually active but denies diarrhea, urethral discharge, or sore throat. her symptoms occurred week following her menses. there was no history of travel, tick bite, or prior joint symptoms. physical examination reveals a febrile patient with pustular lesions over the arms and tenosynovitis of the wrist. her immunologic and hematologic studies and renal parameters are normal. the laboratory calls to inform you of gram negative diplococci growing in the blood cultures. this patient has the classic arthritis-dermatitis syndrome characterized by tenosynovitis and purulent vesicles. dgi is more common in women, and menstruation appears to be an important risk factor. the diagnosis is established by culturing the blood, cervix, urethra, rectum, pharynx, and synovial fluid. the patient and her partner should also receive testing for chlamydia. treatment for dgi is an intravenous cephalosporin regimen (e.g., ceftriaxone g iv every h) until clinical improvement followed by an oral cephalosporin. a -year-old male is referred to you for the treatment of intercritical gout. his serum ua is . mg/dl and he has mild renal insufficiency with a serum creatinine of . mg/dl. allopurinol and once daily colchicine are prescribed. he calls your answering service week later with new onset of fatigue and ecchymosis. what complication has occurred? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc this condition occurs rarely and may be more common in patients with renal failure. allopurinol should be immediately discontinued and hematologic support instituted. although colchicine toxicity can also cause bone marrow failure this tends to occur with higher doses. a -year-old female is seen for follow-up of sjogrens syndrome due to persistent arthritis and parotitis. you consider starting azathioprine. she informs you that her sister took this medicine and had a "bad reaction with her blood." what tests should be ordered prior to commencing this drug? how common is this abnormality? a -year-old african american male is seen for evaluation of uveitis and bell's palsy. he has a low-grade fever, parotid gland enlargement, mild arthritis, and dyspnea on exertion. what is the most likely diagnosis? what treatment is indicated? this patient has sarcoidosis with involvement of the uveal tract and parotid gland. ninety percent of patients with sarcoidosis have pulmonary involvement, and further evaluation should include chest radiography with pulmonary function testing. the goal of treatment is to decrease inflammation and the mainstay of treatment is corticosteroids. ocular inflammation often responds to topical steroids. sarcoidosis is often a self-limiting disease. a -year-old male is referred for further management of ankylosing spondylitis. he was told he had a "bamboo spine" on routine cxr. he emphatically denies stiffness, pain, or enthesopathy. his examination shows slightly limited spine flexion, normal chest excursion, and wall-to-occiput distance of in. inflammatory markers are not elevated and he had no history of ocular disease or adolescent back pain. what is the most likely diagnosis? what other tests will facilitate the diagnosis? what treatment is advised? answer: . diffuse idiopathic hyperostosis syndrome (dish). it is very unlikely that the patient has ankylosing spondylitis (as). as typically affects young men in the second and third decade of life and is characterized by stiffness of the axial skeleton and sacroiliac joints. this elderly gentleman most likely has dish syndrome, which is a condition that involves flowing ossifications along the anterolateral aspect of four contiguous vertebra. it is of unknown etiology but usually involves the thoracic spine. a -year-old male is referred for evaluation of refractory lower extremity edema. he has had slight stiffness in the proximal areas but is more concerned about his leg swelling. he denies headaches/jaw claudication or visual changes. on examination he has small joint synovitis and + pitting peripheral edema. there are no signs of heart failure. investigations reveal normal renal function and urinalysis. hb = . g/dl, esr = mm/h. rf/ccp antibody are negative. ultrasound of the le reveals no evidence of venous occlusion. a two-dimensional echo is normal without pericardial or rightsided dysfunction. what is the most likely diagnosis? what treatment is advised? what is the prognosis? this patient has classic rs pe with peripheral edema and symmetrical swelling but no evidence of ra. this is a rare condition that mimicks pmr and ra although there are no long-term consequences of joint damage or deformity. patients often have modestly elevated inflammatory markers and respond well to oral corticosteroids. prognosis is generally excellent. a -year-old female presents with arthralgia, rash, and parasthesias. her examination reveals palpable purpura and peripheral neuropathy but no synovitis. she denies xerostomia, sicca symptoms, renal disease, or recurrent sinusitis. her laboratory tests reveal modest transaminitis with low serum albumin, prolonged prothrombin time. ana is negative but rf is moderately positive. anca tests are negative. what is the most likely diagnosis? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc this patient has evidence of chronic liver disease with decreased synthetic function. the constellation of palpable purpura, arthralgia, and neuropathy are highly suggestive of mixed croglubulinemia (mc). mc is quite common in hcv although only clinically apparent in about % of patients. it is also associated with membranoproliferative glomerulonephritis and vasculitis. hga is becoming a more commonly recognized cause of fever following a tick bite in the usa. the vector for this infection is the ixodes tick, which also carries the spirochaete responsible for lyme disease ( borrelia burgdorferi ). humans are affected when they impinge on small mammal/tick-infested areas. the common clinical presentation is fever, headache, and myalgia. arthralgias, septic shock, pancytopenia, renal failure, and acute respiratory distress syndrome (ards) are also rare complications. treatment is with oral doxycycline. coinfection with babesiosis and lyme disease can also occur and should be checked in symptomatic patients who live in endemic areas. a -year-old well-nourished caucasian female is referred for evaluation of a swollen knee. she has presented to the er on two occasions and had the knee drained. serial culture results are negative and no crystals have been observed. the effusions have been "bloody" although she denies trauma or anticoagulant use. she walks with a limp and is otherwise well without systemic complaints. on examination she has a swollen right knee with boggy synovial thickening and mild warmth. no other joints are involved. a knee radiograph is normal. lab tests including ana, rf, esr, and lyme serology are negative. hematologic studies are normal. the differential diagnosis for a hemarthrosis includes bleeding diathesis, trauma, pseudogout, charcot joint, and pvns. since her hematologic indices are normal and there has been no trauma; intraarticular hemorrhage or charcot joint seem less likely. pseudogout is typically seen in older patients with preexisting degenerative joint disease, metabolic disease, or hyperparathyroidism, and no crystals have been observed. pvns is a rare slow-growing benign tumor of the synovium with typical mri findings. treatment is arthroscopic synovectomy. her nourishment is relevant as scurvy can also cause recurrent hemarthrosis. a -year-old female is referred for treatment of osteoporosis. she has a history of breast cancer in situ without skeletal involvement, and esophageal reflux. axial t scores are − . , hip − . . on examination she has bilateral poor dentition and marked kyphoscoliosis. there is a history of hip and spine fracture. calcium, vitamin d, pth, and renal function are normal. her breast cancer is currently in remission. her gp has tried both residronate and alendronate, which caused gi distress. this patient is at a moderately high risk for future fracture. the most potent oral antiresorptive agents are bisphosphonates (bp), which have been shown to decrease incident vertebral and hip fractures. unfortunately, because of her esophageal disease she is a poor candidate for bps, which can cause gi distress. a selective estrogen modulator (serm) such as raloxifene would be a reasonable choice given her prior breast cancer although there are no data to support a reduction in hip fracture. teriperatide is an anabolic agent used in patients refractory to oral agents at high risk for fracture. it is given via the subcutaneous route and generally well tolerated in patients with esophageal issues. intravenous bisphosphonates should be avoided in this patient given the higher incidence of osteonecrosis of the jaw (onj) in patients with preexisting poor dentition, malignancy, diabetes, and nicotine use. a -year-old diabetic male has pain over the metacarpophalangeal (mcp) joints. he has about min of morning stiffness. on examination he has swollen tender second/third mcp joints but no overt synovitis. serologies are negative and radiographs reveal hook-like osteophytes at the mcp and pip joints. what is the most likely diagnosis? what tests would you order? an elevated ferritin or transferrin saturation is suggestive of hemochromatosis. patients develop osteoarthritis of the second and third mcp joint, which may be the initial clue to the diagnosis. iron deposition occurs in the pancreas causing diabetes. occasionally, chondrocalcinosis is observed. this patient most likely has a pancoast's tumor causing hypertrophic pulmonary osteoarthropathy (hpoa). bronchogenic carcinoma is one of the causes of hpoa and results in periostitis and clubbing. although the cause is unknown, abnormal expression of vascular endothelial growth factor (vegf) has been described in this condition. a -year-old female is seen due to a swollen right shoulder. she denies having trauma or prior history of crystal arthritis. her shoulder examination reveals a large warm effusion with limitation of motion in all directions. a radiograph demonstrates advanced glenohumeral destruction but no calcification. arthrocentesis reveals a bloody effusion without evidence of infection or crystals on conventional polarized microscopy; cytology is negative. what test should you order from your laboratory that will most likely yield the diagnosis? this patient most likely has milwaukee shoulder due to the presence of calcium hydroxyapatite crystals. this condition is characterized by intrarticular or periarticular hydroxyapatite crystals causing a destructive arthropathy at the glenohumeral and rotator cuff interval. the cause is unknown. a -year-old female with sle presents with a new limp and right groin pain. one week prior to presentation she received high-dose intravenous corticosteroids for class iv glomerulonephritis. on examination there are no findings apart from irritation of the right hip with rotation. a pelvic and hip radiograph are normal. what is the most likely diagnosis? how would you manage the patient? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc avn results in dead trabecular bone and marrow extending to involve the subchondral plate due to local ischemia. the highest incidence of avn of the hips occurs in patients with sle and renal transplants, who have been exposed to high-dose steroids. ultimately collapse of the femoral head occurs, which necessitates hip replacement. in early stage avn plain radiographs are normal and so a high level of suspicion needs to be maintained. the patient should be evaluated by an orthopedist and avoid weight-bearing. core decompression should be considered depending on the severity of necrosis. since the risk of avn is greater in patients with hypercoagulability, antiphospholipid syndrome should be excluded. a -year-old female is referred by the gp due to an elevated creatinine phosphokinase (cpk). she has a history of obstructive sleep apnea, type diabetes, and hypertension. there is no history of trauma, intramuscular injection, or dark urine. recently she was started on atorvastatin mg daily. a routine blood test month after the lab test revealed a cpk of iu/ml (nl < ); no baseline level is available. on examination, she is obese; weight lb. there are no rashes, nodules, or muscle tenderness. muscle strength is / throughout what additional blood tests would you like to know? what management is indicated? this patient has a slightly elevated cpk level without symptoms of muscle breakdown or inflammation. given her obesity, the most likely scenario is that the cpk reflects her high muscle mass and is normal when calibrated for her bmi. this is a common scenario in practice and in the absence of weakness, rhabdomyolysis, or pain no specific treatment is indicated. given her cardiac risk factors she should continue the statin therapy and the cpk levels should be monitored closely. a tsh should be checked to exclude hypothyroid myopathy. renal function should also be checked as rhabdomyolysis is a serious complication of muscle breakdown and needs to be excluded. the absence of arthritis, raynaud's phenomenon , or rash makes a connective tissue less likely. a -year-old female patient is referred for osteoporosis. she has a -month history of weakness, myalgia, and -lb weight gain. three months prior, she fell and fractured her pelvis. pmh is unremarkable apart from poorly controlled hypertension. her menses are normal. she takes no medications apart from atenolol. lab work reveals k = . meq, normal renal and hematologic parameters. cpk, vitamin d, and malabsorption studies including celiac antibodies are normal. a bone dexa scan reveals an axial t score of − . and hip t score of − . . corresponding z scores are both less than − . . her examination reveals a bitemporal hemianopsia, bp / , obesity, and mild muscle tenderness. what is the most likely diagnosis? what is the optimal treatment? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc excessive cortisol production from an acth-secreting tumor results in hypertension, hypokalemia, and accelerated osteoporosis. a large mass that extends into the suprasellar fossa can place pressure on the optic chiasma resulting in visual field defects. in this case prolonged exposure of cortisol has resulted in osteoporosis. a low z score below − . raises the possibility of age inappropriate low bone mass. the treatment of choice for classic cushing's disease is surgical resection of the adenoma with the goal being to relieve pressure and preserve pituitary function. a -year-old teacher presents with refractory left-sided raynaud's and left-sided neck pain. she has no other serologic or clinical stigmata of a connective tissue disease. treatment with calcium channel blockers, aspirin, nitrates, and alpha blockers is ineffective. her examination reveals a diminished left radial pulse with inspiration. blood work is normal. what is the most likely diagnosis? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc this young patient has unilateral evidence of vascular insufficiency in the upper extremity. in a nonsmoker the differential diagnosis includes raynaud's phenomenon or occlusion of the subclavian artery. usual causes of raynaud's phenomenon include idiopathic, hyperviscosity, atherosclerosis, connective tissue disease, or vasculitis. these are less likely in this case since the symptoms are unilateral. the patient has a positive adson's maneuver with a diminishing pulse on inspiration suggestive of a cervical rib or fibrous band. a cxr with apical lordotic view will show the cervical rib. you are asked to see a -year-old female from laos who is admitted as an inpatient. she presented with a febrile illness with quotidian spikes to °f. there is associated malaise and general weakness. there has been no recent travel out of the usa for over a year and no sick contacts, tick bites, or rash. on examination she is febrile, and there is diffuse shotty lymphadenopathy, splenomegaly, and bilateral warm knee effusions. her lab work reveals leukocytosis with lymphocytic predominance, normal renal function, mild transaminitis, and low serum albumin. urinalysis is normal with no proteinuria or cellular activity. microbiologic, viral, and rickettsial titers are negative. bone marrow studies are nondiagnostic and negative for mycobacteria. ct scans of abdomen, chest, and pelvis are normal. you order blood work: ana + : , rf negative, ccp negative, aso, parvovirus, and urine tests for gonorrhea are negative. esr is mm, crp = mg/dl, and ferritin = , ng/ml. what is the most likely diagnosis? how would you manage her? this patient has the classic features of aosd with spiking fever, organomegaly, derangement of liver function, leukocytosis, and a very high ferritin. this is a tricky diagnosis due to the broad differential diagnosis and potential range of infections or malignancies that can present with this type of presentation. one of the more specific clues lies in the serum ferritin, which is usually markedly elevated in aosd and often > , ng/ml. the differential diagnosis of a very high ferritin (> , ng/ ml) is limited to hemochromatosis, hemophagocytic syndrome, or aosd. once infection and malignancy have been excluded treatment with high-dose steroids should be initiated in this patient who is sick with this multisystem disease. for mild flares nonsteroidal drugs may be used. a -year-old man is referred to evaluate an elevated esr. he has had occipital and bitemporal ha of -months duration with associated jaw pain and scalp tenderness. a -cm left temporal biopsy is negative for temporal arteritis or vasculitis. on examination he appears cachectic and has diffuse tenderness over the right temporal artery. there is lymphadenopathy and right-sided diplopia with medial rectus weakness. labs reveal normocytic anemia, esr = mm/hr. hepatic and renal functions are intact. immunoelectrophoresis is without monoclonality. what tests would you order to confirm the diagnosis? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc this elderly gentleman has symptoms that are classic for temporal arteritis (ta). headaches and jaw claudication in the setting of a markedly elevated esr should raise suspicion for ta. unfortunately this is a disease of discontinuity and characterized by skip lesions on ta biopsy. ideally a - -cm biopsy should be obtained and a bilateral biopsy will increase the sensitivity, albeit marginally. in this case the -cm biopsy length is inadequate. ta responds to high-dose prednisone and untreated can cause optic arteritis resulting in blindness. opthalmoplegia has also been described. myasthenia gravis or a space-occupying lesion would not cause bilateral headaches, jaw claudication, or scalp tenderness. if the clinical suspicion is high and an inadequate biopsy is obtained it should be repeated. another option would be to obtain a color duplex ultrasound, which, in the right hands, can reveal a "halo" sign that is suggestive of active arteritis. a -year-old male is admitted with new onset of rectal bleeding after having taken , -mg ibuprofen for an acutely swollen toe. you are asked to examine him by the colorectal surgeon for acute podagra. they are considering a colectomy to arrest the bleeding, which has failed to stop by conventional means. on examination he has a red tender inflamed first toe with exquisite tenderness. lab work reveals mild anemia, mild prerenal kidney dysfunction. uric acid is normal. how would you treat his gout? this patient has acute podagra in the setting of an acute nsaid-induced gastrointestinal bleed. an ia injection of steroid is the optimal management in this patient. colchicine is contraindicated given the renal impairment and potential to further irritate the gi tract. a -year-old male presents with recurrent intermittent monoarthritis affecting the toes, knees, and ankle. arthrocentesis reveals intracellular uric acid crystals diagnostic of acute gout. lab work: cr = . mg/dl, ua = mg/dl, cbc normal. hepatic function is normal. twenty-four hour urine ua is low. what is the most likely diagnosis and cause? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc the majority of people with gout are congenital underexcretors of ua. the exact mechanism has not been elucidated but it is related to decreased secretion, increased reabsorption, or decreased filtration of ua. overproduction of ua occurs in less than % of patients. lesch nyhan syndrome secondary to hypoxanthine guanine phosphoribosyl transferase deficiency (hgprt) would cause an overproduction of ua and increased -h urinary uric acid excretion. a -year-old female with years of seropositive erosive rheumatoid arthritis presents with new onset of shortness of breath, low-grade fever, and dry cough. her current regimen includes hydroxychloroquine mg/day, prednisone mg/day, sulfasalazine g daily, and infliximab mg/kg. she has never taken methotrexate. she resides in new england, and denies recent travel. examination reveals chronic rheumatoid deformities without active synovitis. pulmonary examination reveals fine diffuse inspiratory crepitations bilaterally. a cxr describes fine perihilar reticular opacification. blood gases revealed hypoxia, and she failed to improve with broad spectrum and empiric macrolide therapy. lab work reveals normocytic anemia without leucocytosis. renal, liver function and cpk are normal. ldh is markedly elevated. peripheral smear is without hemolysis. bronchoscopy reveals negative stains for acid-and alcohol-fast bacilli on three occasions and ppd (tb) skin tests are negative. cmv, mycoplasma , legionella , q-fever, adenovirus, influenza, chlamydia , cytomegalovirus, epstein-barr virus, hepatitis b and c, and hiv titers are negative. what is the most likely diagnosis? this patient who is receiving anti-tnf therapy presents with an acute pulmonary decompensation characterized by dyspnea, hypoxemia, and an elevated ldh level. since she is significantly immunosuppressed, an opportunistic infection is the most likely scenario. rheumatoid lung disease, bronchiolitis obliterans with organizing pneumonia (boop) are also in the differential diagnosis. ra-associated lung disease tends to present more insidiously with pleural involvement and is usually not associated with elevated ldh levels. pcp is caused by a unicellular eukaryote and is a rare cause of infection in immunocompetent individuals. the diagnostic gold standard is a broncheoalveolar lavage (bal) with cytologic confirmation of induced sputum samples. ldh levels are a sensitive but poorly specific indicator of pcp. since this infection is associated with high mortality a high index of suspicion is required. a -year-old female presents with recurrent uveitis and scleritis. she has a -month history of nasal discharge and shortness of breath. cxr is reported as having cavitatory lesions. nasal mucosa is erythematous and reveals a perforation within the septum. she has no evidence of synovitis, rash, or mononeuritis on examination. microbiologic evaluation, including tb and cocaine use is negative. pfts reveal mild restriction and upper airway obstruction. what is the most likely diagnosis and how would you make this diagnosis? this patient presents with recurrent sinusitis, inflammatory eye disease, and cavitatory lung lesions typical for wg. the definitive diagnosis is made by confirming the presence of noncaseating granulomas on biopsy. serological tests include the presence of antineutrophil cytoplasmic antibodies (c-anca), which have a sensitivity of about - %. antibodies to proteinase (pr- ) are highly sensitive in active wg ( %). other features of wg include subglottic stenosis, pauciimmune glomerulonephritis, mononeuritis multiplex, and arthritis. in this patient other causes of cavitatory lung lesions such as infection need to be excluded prior to treatment. a -year-old heterosexual monogamous firefighter is referred for bloody diarrhea and new onset of knee swelling. he is stiff for about min in the morning. there is no history of travel, urethral discharge, or uveitis. on examination he has a warm left knee effusion and mild tenderness in the abdomen. spine forward flexion is slightly limited. a painful erythematous rash is noted on the anterior shins. lab work reveals mild microcytic anemia. esr is mm/h. stool cultures are negative. renal and hepatic function is preserved. what is the most likely diagnosis and how would you confirm it? how would you initially manage his joint pain? this gentleman presents with a syndrome of bloody diarrhea, erythema nodosum, and oligoarthritis. this pattern is most classic for inflammatory bowel disease (ibd). ideally his knee should be drained and fluid sent to confirm a sterile effusion. colonoscopy with biopsy will confirm the diagnosis of ibd. initial treatment of his joint disease can involve local intraarticular injections, analgesic medications, and judicious use of anti-inflammatory medications with close collaboration with the gastroenterologist. axial spondylitis may respond to physical therapy. the response to dmard therapy for peripheral and axial arthritis is often disappointing. monoclonal antibody anti-tnf agents (infliximab and adalimumab), in contrast, work well in this situation. a -year-old female with ra is referred for further management. she has multiple comorbidities including chronic renal failure, diabetes, and hypertension. she has pain in the hands, wrists, and feet with h of early morning stiffness. on examination there is polyarthritis of the small joints of the hands with significant joint margin tenderness. prednisone at mg daily and weekly methotrexate (mtx) at mg is commenced. you are called by the er week later where she is being seen for new onset odonophagia and dysphagia. on examination she has severe mucosal ulceration. lab work reveals mild hyperglycemia and stable renal dysfunction. what complication has occurred? how could this have been prevented? this patient with renal failure has developed mucositis from the methotrexate. mucosal toxicity is a well-known complication of mtx and predisposing factors include folate deficiency, renal failure, and hypoalbuminemia. there are also various genetic polymorphisms that may predict toxicity and response to mtx. the dose of mtx should be adjusted in patients with renal failure. this complication may have been prevented by the use of a lower dose and prophylactic daily folic acid. a -year-old caucasian female is referred for further management of osteoporosis. she underwent early menopause at and did not receive hormone replacement. a bone density taken years before revealed an axial t score of . and appendicular t score of − . . she was treated with calcium supplements and alendronate mg weekly. despite bisphosphonate therapy she has had several fragility fractures in the past year. a repeat dexa is performed and this confirms a slight decline in bone mass. serum electrolytes including alkaline phosphatase, calcium, celiac antibodies, pth, and -vitamin d levels are all normal. this patient has multiple risk factors for osteoporosis including early menopause, sex, race, and prior fracture. assuming that she is compliant then the fact that she is fracturing despite bisphosphonate (bp) therapy indicates that she is a true bp failure. teriperatide has an alternative mechanism of action to bp and stimulates osteoblasts as opposed to inhibiting osteoclasts. osteoporosis experts have developed a consensus opinion, published in the spring of , to help clinicians identify appropriate patients for teriperatide therapy. indications for its use were as follows: ( ) history of vertebral fracture, t score of − . or below, or age greater than years, ( ) fracture or unexplained bone loss in patients on antiresorptive therapy, and ( ) intolerance of oral bisphosphonate therapy. contraindications for teriparitide therapy listed include hypercalcemia, paget's disease, a history of irradiation to the skeleton, sarcoma, or malignancy involving bone. this patient has a classic charcot joint, which is characterized by damage secondary to loss of sensation that occurs due to the patient's underlying diabetes. the features of a charcot joint include fragmentation of bone, progressive destruction, and disorganization. although there are many causes, diabetic neuropathy is the commonest cause in the western world. lisfranc dislocation implies disruption of the joint between the rigid midfoot and more supple weight-bearing forefoot. arthrocentesis frequently yields a hemarthrosis. lisfranc was napolean's surgeon who described a technique to amputate the forefoot in soldiers suffering from frostbite. a -year-old female from the dominican republic with seropositive ra of -year duration is seen due to fever, dyspnea, and malaise. she commenced anti-tnf therapy month prior to her presentation due to poorly controlled ra. her current medications include methotrexate, folate, celecoxib, and infliximab. her examination shows a low-grade temperature of . °f. she has chronic synovitis of the mcp/pip and wrist joints with bibasilar end inspiratory crepitations on pulmonary examination. serum and urine microbiologic studies are negative. a cxr reveals chronic bibasilar fibrosis and a new apical infiltrate. what is the major clinical concern in this patient? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc this patient has developed a new apical infiltrate in the setting of anti-tnf therapy. tnf is a pleiotropic molecule important in immune surveillance and host defense. it is also important in granuloma formation and maintenance. tnf-deficient knockout mice develop fatal tb and listeriosis. reactivation of tb is a well-recognized complication of anti-tnf therapy, and patients should be screened with skin testing prior to initiation of therapy. this -year-old male presents with axial stiffness, metatarsal inflammation, enthesopathy, and sacroiliitis. this is highly suggestive of a reactive arthritis or as. gout has been effectively excluded by the absence of urate crystals and would not typically present with achilles tendonitis or sacroiliitis. other things to examine for would be digital pitting, psoriatic plaques, stigmata of inflammatory bowel disease, urogenital infection, or uveitis. the elevated uric acid is of no clinical significance and the colchicine should be stopped. reveille a -year-old female with sle is referred with new onset pleuritic chest pain. she has a history of arthritis and mucositis, which has been controlled on nsaids and antimalarial agents. she has a history of two second trimester miscarriages. on examination she has sinus tachycardia, rv heave, and a loud p . diffuse livedo reticularis is noted. lung examination is clear and cxr appears normal apart from slightly oligemic fields. she is mildly hypoxic and is using accessory muscles of respiration. what is the most likely diagnosis? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc this patient with sle most likely has antiphospholipid syndrome (aps) with a history of recurrent pregnancy loss and livedo reticularis. her examination reveals elevated right-sided pulmonary pressure consistent with right ventricular strain. pericarditis should also be considered although it would be less likely given the physical findings. aps is a disease of recurrent vascular thrombosis and fetal loss associated with antibodies to membrane phospholipids. approximately % of patients with sle have anticardiolipin antibodies but only % have aps syndrome. lockshin md. update on antiphospholipid syndrome. bull nyu hosp jt dis. ; ( - ): - . a -year-old female is admitted with malaise, fever to °f and unintentional weight loss. she has a -year history of seropositive erosive ra controlled on methotrexate and more recently intravenous anti-tnf therapy. she has known interstitial lung disease. on examination she is cachectic, with chronic deformities and low-grade synovitis. her lung examination reveals coarse bibasilar crepitations. she has axillary lymphadenopathy and bipedal edema. lab work reveals a normocytic anemia, mild eosinophilia, and hypoalbuminemia but otherwise intact renal and hepatic function. urinalysis is benign. viral, rickettsial, and microbiologic studies are normal; a ct scan reveals marked hilar lymphadenopathy and chronic fibrotic changes at the lung bases. a tuberculin skin test is negative. this patient with chronic ra presents with fever, lymphadenopathy, eosinophilia, and weight loss in the setting of a negative infectious disease evaluation. the differential diagnosis includes infection, tb, and lymphoma. the latter diagnosis is most likely given the negative microbiologic studies. patients with chronic ra are predisposed to lymphoma, and although not clear, in this risk might be further amplified by concomitant anti-tnf therapy. patients with the most severe disease activity scores have the greatest risk of developing lymphomas. when using biologic agents the benefits of treatment must be weighed against potential toxicity. a -year-old male immigrant is admitted for further evaluation of a pulmonary artery aneurysm noted on an incidental cxr. he has a prior history of uveitis and arthritis. on examination there is hypophyon, erythema nodosum, and oral ulceration. what is the most likely diagnosis? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc this patient has classic behcet's syndrome (bs), which is an hla-b -associated disease characterized by recurrent orogenital ulceration, thrombophlebitis, uveitis, and vasculitis. skin lesions include erythema nodosum, abnormal pathergy, and folliculitis. the etiology is unknown but this is a disease with racial preference along the old silk route from the mediterranean to china. pulmonary artery aneurysms are a rare cause of pulmonary hemorrhage and should raise the suspicion of bs. a -year-old african american female with dialysis-dependent renal failure is evaluated for recurrent carpal tunnel syndrome. she has had two injections in the past months, which were of temporary benefit. on examination she has atrophy of the thenar eminens and weakness of opponens muscles. phalen's sign is positive and her grip is diminished. what complication has occurred? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc a -year-old female with chronic renal failure is seen for evaluation of lower extremity edema and ankle pain. one month before she had an mri with gadolinium for low back pain. on examination a woody induration is noted over the shins bilaterally. a skin biopsy is performed, which reveals mucin deposition and a proliferation of fibroblasts and elastic fibers. what is the most likely diagnosis? discuss. yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc this patient has crest syndrome and elevated right-sided pulmonary pressures by conventional d echocardiogram. since pulmonary hypertension is a major cause of mortality in these patients, further investigations are warranted. her echocardiogram reveals pulmonary hypertension (normal mean pa pressure< mmhg) although there is a clear disconnect between her symptoms, examination, and the echo findings. there are differing opinions in this scenario but in an asymptomatic patient she could probably be observed for clinical deterioration or symptoms. a -min walk test is also helpful and if abnormal, a right heart catheterization can be considered. this patient had normal pa pressures on right heart catheterization. this case illustrates the limitations of accuracy of echocardiography in obese patients. posterior reversible encephalopathy syndrome (pres) or reversible posterior leukoencephalopathy syndrome (rpls) is an increasingly recognized neurologic disorder with characteristic computed tomographic (ct) and magnetic resonance (mr) imaging findings, and is associated with a multitude of diverse clinical entities. these include acute glomerulonephritis, preeclampsia and eclampsia, systemic lupus erythematosus, thrombotic thrombocytopenic purpura, and hemolytic-uremic syndrome, as well as drug toxicity from agents such as cyclosporine, tacrolimus, cisplatin, and erythropoietin. most, but not all, cases manifest with acute to subacute hypertension, and seizures are also frequent. two pathophysiologic mechanisms for pres have been proposed. one postulates cerebral vasospasm with resulting ischemia within the involved territories, whereas the other posits a breakdown in cerebrovascular autoregulation with ensuing interstitial extravasation of fluid. this case is particularly difficult in that a patient with sle who presents with a seizure and hypertension has to be excluded for active cerebritis or nephritis. the normal lumbar puncture and characteristic mri findings are highly suggestive of pres. treatment involves normalization of bp, removing offending agents such as cytoxan, and prevention of further seizures. a -year-old female has had low back pain for months. she has an unremarkable past history apart from one prior episode of uveitis. review of systems is negative for diarrhea, urethral discharge/travel, or constitutional symptoms. her examination reveals a positive faber sign and tenderness over the lower lumbosacral spine and sacroiliac joint. the rest of the joint and skin examination is normal apart from subungual hyperkeratosis and oil spots. what is the most likely diagnosis? yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc infections associated with tumor necrosis factor-alpha antagonists tumor necrosis factor-α is required in the protective immune response against mycobacterium tuberculosis in mice tuberculosis in the cytokine era: what rheumatologists need to know major arterial involvement and review of behcet's disease. ann vasc surg pulmonary vasculitis in behcet disease: a cumulative analysis behçet's syndrome: disease manifestations, management, and advances in treatment the clinical manifestations usually develop after several years of dialysis dependence and include carpal tunnel syndrome, destructive arthropathy, bone cysts, and fractures. risk factors for the development of dra include age, duration of dialysis treatment, use of low-flux dialysis membrane, use of low-purity dialysate, monocyte chemoattractant protein- gg genotype, and apolipoprotein e allele. surgical management is usually successful but can result in recurrence dialysis-related amyloidosis: late finding or hidden epidemic? semin dial management of carpal tunnel syndrome in renal dialysis patients using an extended carpal tunnel release procedure comparison of doppler echocardiography and right heart catheterization to assess pulmonary hypertension in systemic sclerosis pulmonary hypertension in systemic autoimmune disease a -year-old female is referred due to years of recurrent sinusitis. she has failed multiple antibiotic courses and has recently developed left-sided horizontal diplopia.her lab work is consistent with anemia of chronic disease and an elevated esr of mm/h. cultures for bacteria, fungi, and tubercles are negative. a ct scan of the sinuses confirms destructive pansinusitis with a nasopharyngeal mass. serologies are consistent with a +p-anca, −canca, and −pr /mpo antibody. renal and pulmonary functions are preserved.what is the differential diagnosis and most likely scenario? what treatment is advisable? this -year-old patient presents with a -year history of recurrent sinusitis in the setting of a positive p-anca, destructive nasopharyngeal mass, and ophthalmoplegia.the differential diagnosis includes infection with a refractory organism, such as mucormycosis or tuberculosis, malignancy, midline granuloma, or vasculitis. infection and malignancy seem less likely given the chronicity and lack of positive cultures.although most patients with wg have antibodies to proteinase- and positive c-anca antibodies, a small minority have antibodies to p-anca.this patient's inflammation has extended into the sinuses and cavernous sinus causing cranial neuropathy and ophthalmoplegia.optimal treatment involves oral corticosteroids and oral daily cyclophosphamide. prior to the introduction of cytotoxic drugs this was a disease of very high mortality. a -year-old diabetic african american male is referred for evaluation of chronic right ankle pain. there is a distant history of trauma and poorly controlled diabetes. he denies podagra, and serum uric acid is normal. on examination he has fair peripheral circulation, pes planus with mild ankle tenderness at the tibiotalar joint, and glove and stocking neuropathy is present. his radiograph shows diffuse demineralization and lisfranc dislocation of the midtarsus. the joint is tapped and hemarthrosisis noted.what is the most likely diagnosis? who was lisfranc? you are asked to see a -year-old alcoholic male to help with the management of presumed gout. he presented with acute bilateral ankle pain and swelling. he has chronic low back stiffness. the admitting physician has performed arthrocentesis of the ankle joint, which confirmed type inflammatory fluid without crystals. lab work reveals intact hematologic and renal function and ua = . mg/dl.on examination he has bilateral tenderness over the right metatarsal heads, thickening of the achilles tendon with exquisite tenderness, and warm bilateral ankle effusions. radiographs of the spine confirm right sacroiliac fusion. he is treated with colchicine without any improvement.what is the most likely diagnosis? what else would you look for on examination? nephrogenic fibrosing dermopathy/nephrogenic systemic fibrosis (nsf) is an emerging scleromyxedema-like cutaneous disorder of unknown cause that is seen in patients with renal failure; the number of reported cases has grown significantly since its first recognition. virtually all cases of nsf have been associated with the administration of gadolinium-containing contrast media. skin biopsies of affected areas reveal thickened collagen bundles, mucin deposition, and proliferation of fibroblasts and elastic fibers. the etiology is unknown but gadolinium may act as a stimulant to attract circulating fibrocytes to the dermis. ideally gadolinium should be avoided unless absolutely necessary in patients with renal failure. you are asked to see a patient with refractory erythema nodosum. he is a -yearold ex-intravenous drug abuser who has had painful raised lesions over his shins for months. he also complains of low-grade fever, arthralgia, and abdominal pain. he has lost lbs in months. on examination temperature is . °c, and there is evidence of clubbing and tender raised nodules over the anterior shins. his cardiac examination reveals a / ejection systolic murmur at the right sternal border and / early diastolic murmur. bp = / . pulmonary examination is normal, and abdominal examination reveals tenderness in the ruq. a cxr is normal.what diagnostic test should be performed?yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , humana press, a part of springer science + business media, llc this patient has endocarditis with valvular insufficiency and probable streptococcal bacteremia. streptococcus is a common cause of erythema nodosum (en). other causes include sarcoidosis, tb, yersinia, fungal infections, inflammatory bowel disease, behcet's disease, and drugs such as sulfonamides. a -year-old resident is seen for refractory foot pain of -week duration. her past medical history is significant for anorexia nervosa for which she attends monthly counseling classes. her weight is lbs, height ′ ″. on examination she has diffuse tenderness at the midfoot. she denies trauma or swelling.what is the diagnosis?yousaf ali, self assessment questions in rheumatology, doi: . / - - - - , because of anorexia nervosa and low body mass this patient is at a higher risk for osteoporosis and fragility fracture. this radiograph clearly shows two healing metatarsal fractures, which are the cause of the patient's pain. the decreased bone density in patients with anorexia nervosa (an) is due to the many effects on bone metabolism of amenorrhea, reduced levels of insulin-like growth factor- (igf- ), high cortisol levels, and weight loss. although bisphosphonates have been used the most effective treatment involves resumption of menses and weight restoration. a -year-old diabetic male has pain over the anterior hip and groin. he has recently had angina and has started a vigorous exercise regimen. on examination there is clicking of the hip with flexion but no pain with rotation. distal neurovascular examination is intact. a radiograph of the right hip and pelvis is normal.what is the most likely diagnosis? how would you treat him? the iliopsoas muscle passes anterior to the pelvic brim and hip capsule in a groove between the anterior inferior iliac spine laterally and iliopectineal eminence medially. it acts as one of the hip flexors and is occasionally injured in excessive hip flexion or trauma. patients often have an insidious onset of anterior thigh pain, which often radiates down to the knee and is associated with hip clicking.treatment involves physical therapy to alleviate pain, spasm, and swelling. a -year-old markedly obese female is referred for evaluation of scleroderma. she is asymptomatic but has limited sclerodactly and skin thickening of the face, digits. nailfold examination shows dilated capillary loops. lab and urine data are normal. she is diagnosed with crest syndrome on the basis of esophageal dysfunction and raynauds.cardiac examination reveals a normal s , and pulmonary examination is clear without rales.a baseline d echo reveals a pa pressure of mmhg (nl < ) but pfts and dlco are normal.what is the next appropriate step? a -year-old female with sle is diagnosed with class nephritis. she is started on immunosuppressive therapy with pulse cyclophosphamide, prednisone, and furosemide.three months after the initial presentation she is admitted with hypertension and has a witnessed tonic clonic seizure in the er. her bp is / and she appears post ictal when you examine her. there is no rash, synovitis, or evidence of serositis. an lp is unremarkable without evidence for infection or cerebritis.a brain mri reveals multiple hyperintensity lesions in the occipital lobe.what complication has occurred? cutaneous manifestations of psoriasis include oil spots or "oil droplets" -orangebrown patches seen through the nail plate, nail pitting, onycholysis, and subungual hyperkeratosis. psoriatic nail disease is often associated with psoriatic arthropathy. this patient also has evidence of inflammatory low back pain, uveitis,and sacroiliitis, which are all characteristic of psa. faber's test (flexion, abduction, external rotation) is a test for the sacroiliac joint and hip disease. if the patient has pain in the groin it suggests hip pathology but if the pain is in the sacroiliac area it is more consistent with sacroiliitis. a -year-old caucasian female is referred for left forearm pain for months duration. she describes pain that only occurs with activity. her pmh includes newly diagnosed pmr, cad, and spinal stenosis. her current medications include prednisone mg daily, aspirin, and atenolol. on examination she is mildly cushingoid with an absent left radial pulse. there is no peripheral synovitis, and examination of the forearm and elbow joint is unremarkable. resisted extension of the wrist fails to reproduce the pain. there is a faint left subclavian bruit.her laboratory data reveal an elevated esr of mm/h, and normal renal and biochemical parameters. she has a mild normocytic anemia.what is the most likely scenario? what is the next best step to evaluate this? this patient has a recent diagnosis of pmr and now presents with claudication of the left upper extremity in the setting of an absent pulse and markedly elevated inflammatory markers. at this point the diagnosis of exclusion is temporal arteritis (ta) with large vessel involvement. the presence of a subclavian bruit also suggests proximal occlusion. takayasu's arteritis is also a possibility but less likely given the patient's age and ethnicity. atherosclerosis needs to be considered but again should not be associated with this degree of inflammation. large-vessel involvement in giant cell arteritis occurs in over a quarter of patients with this disease. stenosis of the primary and secondary branches of the aorta may cause claudication and tissue gangrene, whereas aortitis may lead to aneurysm formation and dissection, often many years after the initial diagnosis. the important thing here is to treat the inflammation and ensure that no other organs are involved. a temporal artery biopsy is the best next step. occasionally ta can be silent and only becomes apparent when a biopsy is performed. what is the diagnosis? this is a very destructive form of psoriatic arthritis with significant periarticular bone resorption. the erosions can cause a "pencil in cup" deformity where one articular surface is eroded creating a pointed appearance; the articulating bone can be concave, resembling an upside down cup. this patient has severe hip pain.what does the x-ray show and what advice would you give to the orthopaedic surgeon? diffuse involvement of the left hemipelvis is manifested by areas of mixed sclerosis and lucency. there is also involvement of the right hemipelvis near the right sacroiliac joint, secondary hip osteoarthritis, and thickening of the iliopectineal line. antiresorptive treatment should be commenced prior to hip replacement to decrease the hypervascularity and decrease the risk of perioperative bleeding. this patient has postpartum pain.what does the x-ray show? osteitis condensans ilii (oci) is the radiologic appearance of increased sclerosis in the inferior aspect of the body of the iliac bone arising in a triangular configuration from the lateral aspect of the sacroiliac joint (si). it is seen most commonly in multiparous women, but also in some degenerative conditions. it is merely a benign reflection of bone remodeling with response to stress, but with the increased radiologic density it is indicative of sclerosis. the si joints themselves are normal or may feature some degenerative -but not inflammatory or erosive -changes. this condition may sometimes be confused with sacroiliitis, but it can be differentiated by its unilateral nature, lack of erosive or other inflammatory features, both locally and in the spine, and the general absence of clinical symptoms. this patient has refractory wrist pain. schmorl's nodes are defined as herniations of the intervertebral disc through the vertebral end-plate. schmorl's nodes are common, especially with minor degeneration of the aging spine. schmorl's nodes usually cause no symptoms, but reflect that wear and tear of the spine has occurred over time. this radiograph also shows a defect at the anterior body of l reflecting protrusion of the intervertebral disk beneath the ring apophysis of the growing vertebral body. this -year-old male is asymptomatic. a routine chest radiograph notes an abnormality in the spine. his lab work is normal.what is the most likely diagnosis based on this lateral view? what other film would be helpful? how would you manage him? a pelvic/ sacroiliac view would be helpful to exclude sacroiliitis. as is very unlikely at this age. see q .no treatment is indicated. describe these x-rays. what is the diagnosis?answer: renal osteodystrophy with rugger jersey spine. note the renal transplant.renal osteodystrophy combines features of secondary hyperparathyroidism, rickets, osteomalacia, and osteoporosis. bone resorption in renal osteodystrophy is quite complex. renal retention of phosphate results in hyperphosphatemia, which reduces serum ionized calcium levels, therefore inducing hyperparathyroidism. the hyperparathyroidism increases bone resorption, which may normalize serum calcium levels by releasing the osseous storage of calcium. the various sites of bone resorption include the subperiosteal region of the phalanges, the phalangeal tufts, proximal femur, proximal tibia, proximal humerus, distal clavicle, and calvarial trabeculae. if parathormone levels are mildly elevated over a longer period of time, its effect on bone tends to be anabolic. these effects include excessive maturation of osteoblasts leading to new bone formation and increased laying down of osteoid, which calcifies under the influence of secondarily elevated serum calcium levels. this patient has classic endplate involvement, which results in the appearance of a "rugger jersey." describe the x-ray. what is the diagnosis? characteristic radiographic findings of gout are well-defined, punched-out erosions with overhanging edges, preservation of the joint space, lack of periarticular osteopenia, asymmetrical involvement, soft tissue nodules, and intraosseous calcifications. key: cord- -juitltpi authors: babaei, fatemeh; nassiri‐asl, marjan; hosseinzadeh, hossein title: curcumin (a constituent of turmeric): new treatment option against covid‐ date: - - journal: food sci nutr doi: . /fsn . sha: doc_id: cord_uid: juitltpi in late december , the outbreak of respiratory illness emerged in wuhan, china, and spreads worldwide. world health organization (who) named this disease severe acute respiratory syndrome coronavirus (sars‐cov‐ ) caused by a new member of beta coronaviruses. several medications are prescribed to patients, and some clinical trials are underway. scientists are trying to find a specific drug against this virus. in this review, we summarize the pathogenesis, clinical features, and current treatments of coronavirus disease (covid‐ ). then, we describe the possible therapeutic effects of curcumin and its molecular mechanism against coronavirus‐ . curcumin, as an active constituent of curcuma longa (turmeric), has been studied in several experimental and clinical trial studies. curcumin has some useful clinical effects such as antiviral, antinociceptive, anti‐inflammatory, antipyretic, and antifatigue effects that could be effective to manage the symptoms of the infected patient with covid‐ . it has several molecular mechanisms including antioxidant, antiapoptotic, and antifibrotic properties with inhibitory effects on toll‐like receptors, nf‐κb, inflammatory cytokines and chemokines, and bradykinin. scientific evidence suggests that curcumin could have a potential role to treat covid‐ . thus, the use of curcumin in the clinical trial, as a new treatment option, should be considered. transmitted among humans by respiratory droplets and close contact (chan et al., ) . a recent research project revealed that sequence homology between sars-cov- and sars-cov was . % and sars-cov- belongs to the same beta coronavirus (β cov) clade as mers-cov and sars-cov (yu, du, ojcius, pan, & jiang, ) . moreover, it has been identified that sars-cov- had high homology with bat coronaviruses and likely derived from bats , but the intermediate hosts of sars-cov- have not been determined yet. the recent finding reveals that covid- has similar pathogenesis with sars-cov or mers-cov and uses the same receptor as sars-cov for entrance to human host cells wan, shang, graham, baric, & li, ) . the most important articles about covid- (from starting disease up to now) and curcumin were selected. we considered all articles of curcumin-human and animal studies-that could be effective to treat or rescue covid- -infected patients. pubmed and web of science were used as databases. as the importance of the subject, some selected papers were in the press. the keywords used for the search were as follows: coronavirus- , covid- , sars-cov- , curcumin, curcuma longa, turmeric, curcumin and antiviral, curcumin and anti-inflammatory, curcumin and antipyretic, curcumin and lung, curcumin and acute lung injury, curcumin and fatigue, curcumin and antioxidant, curcumin and ards, curcumin and bradykinin, curcumin and fibrosis, curcumin and interleukin- (il- ), curcumin and tumor necrosis factor-alpha (tnf-α), curcumin and nf-κb, curcumin and toll-like receptors (tlrs), curcumin and antiapoptotic. the sars-cov- is an enveloped nonsegmented positive-sense rna virus. two-thirds of viral rna is located in the first open reading frames that encode nonstructural proteins, whereas the remaining part of the genome encodes four essential structural proteins including spike (s) glycoprotein, envelope (e) protein, matrix (m) protein, and nucleocapsid (n) protein (cui, li, & shi, ) . s protein contributes to virus pathogenesis through binding to cell surface receptor, angiotensin-converting enzyme (ace ), and the entrance of the virus into the host cell . the possible mechanism and molecules involved in membrane invagination during virus endocytosis are still unknown. s protein is divided into the s domain that is responsible for receptor binding, and s domain that mediates cell membrane fusion (he et al., ) . recent data showed that the s protein of sars-cov- binds to ace with a higher affinity than sars-cov. for this reason, it spreads rapidly in human populations (wrapp et al., ) . the ace expressed on the surface of cells in the lung, arteries, heart, kidney, and intestine (hamming et al., ) . its concentration in the alveolar cells of men was higher than women, which may correlate with a high incidence rate of covid- among men. moreover, the expression level of ace in the alveolar cells of asians was higher than other races, which may lead to high susceptibility to disease and severe outcomes (sun, lu, xu, sun, & pan, ) . the ace is an enzyme that catalyzes vasoactive angiotensin ii to vasodilator angiotensin[ - ] (richards & raizada, ) . on the other hand, the binding of the sars-cov spike protein to ace leads to ace downregulation (kuba et al., ) . it is not clear that sars-cov- could downregulate the expression of ace or not as the homology of sars-cov- with sars-cov. ace downregulation resulted in excessive production of angiotensin by the ace, suggesting lead to pulmonary hypertension, acute lung injury (ali), and lung fibrosis (tan, liao, zhou, mei, & wong, ) . previous studies have shown the protective role of ace against various types of pulmonary illnesses such as acute respiratory distress syndrome (ards), chronic obstructive pulmonary disease (copd), pulmonary hypertension, ali, and asthma (jia, ) . it has been suggested that increasing ace levels such as using angiotensin ii receptor blocker medications could be effective to treat covid- (gurwitz, ) . however, another study showed that decreasing ace activity might be protective (zhang, penninger, li, zhong, & slutsky, ) . thus, these hypotheses might be the basis for more research to clarify new therapeutic options. the patients mainly were - years old (wu & mcgoogan, ) . a few cases were children below years old such as days old in iran (kamali aghdam, jafari, & eftekhari, ). there were one or more coexisting medical conditions, including hypertension, diabetes, and cardiovascular disease in about half the patients . these coexisting medical conditions lead to a high mortality rate in covid- patients (wu & mcgoogan, ) . there was a spectrum of clinical features ranging from asymptomatic infection to severe respiratory failure. the most prevalent manifestations include fever, fatigue, dry cough, myalgia, dyspnea, and anorexia (qin et al., ; rodriguez-morales et al., ) . the uncommon symptoms were sputum production, headache, hemoptysis, diarrhea, nausea, and vomiting qian et al., ; rodriguez-morales et al., ) . according to laboratory examination, lymphopenia, hypoalbuminemia, and high levels of c-reactive protein (crp), erythrocyte sedimentation rate (esr), and lactate dehydrogenase (ldh) were the most prevalent results in the patients (mo et al., ; qin et al., ; rodriguez-morales et al., ; talebpour, hadadi, oraii, & ashraf, ; wang, yang, li, wen, & zhang, ). patients with severe symptoms had raised levels of coagulation indexes (prothrombin time, activated partial thromboplastin time, and d-dimer), procalcitonin, il- , and serum ferritin, and multiple organ involvement, such as liver (increased lactate dehydrogenase, alanine aminotransferase, and aspartate aminotransferase levels), kidney (increased blood urea nitrogen and creatinine levels), and heart and muscle (increased creatinine kinase levels) compared with patients with mild symptoms. also, there was higher neutrophil-to-lymphocyte ratio (nlr) and lower percentages of monocytes, eosinophils, and basophils in complete blood count (qian et al., ; qin et al., ; talebpour et al., ; wan, xiang, et al., ) . the elevated levels of il- b, interleukin- receptor antagonist (il ra), il- , il- , il- , il- , basic fibroblast growth factors (fgf), granulocyte colony-stimulating factor (gcsf), granulocyte-macrophage colony-stimulating factor (gm-csf), interferon-gamma (ifnγ), interferon γ-induced protein kda (ip- ), monocyte chemoattractant protein- (mcp ), macrophage inflammatory protein- alpha (mip -α), macrophage inflammatory protein- β (mip- β), platelet-derived growth factor (pdgf), tumor necrosis factor-alpha (tnf-α), and vascular endothelial growth factor (vegf) were observed in serum sample of patients. plasma levels of il- , il- p , il- , eotaxin, and rantes (chemokine (c-c motif) ligand , ccl ) were similar between healthy adults and patients infected with sars-cov- . moreover, plasma concentrations of il , il , il , gcsf, ip , mcp , mip a, and tnf-α were higher in the intensive care unit (icu) patients compared with non-icu patients. these data suggest that the initial response of the immune system may lead to the production of cytokines and chemokines, which damage normal host organs such as lung and heart. also, hypersensitive troponin i (hs-ctni) was increased in patients with virus-related cardiac injury . the pathological features of covid- were similar to sars-cov and mers-cov infection (van den brand, smits, & haagmans, ; hui & zumla, ; nassar, bakhrebah, meo, alsuabeyl, & zaher, ) . moreover, moderate microvascular steatosis, mild lobular, and portal activity were observed in the liver biopsy samples. the heart tissue also showed interstitial mononuclear inflammatory infiltrates. the cd and cd t cells were decreased in flow cytometric analysis of peripheral blood. also, x-ray images showed a rapid progression of pneumonia in lung tissues . countries use various strategies to treat covid- patients. some protocols for the treatment are summarized in table . some clinical trials try to find an effective drug (li & de clercq, ) . organ dysfunction, including shock, ards, acute cardiac injury, acute kidney injury, liver dysfunction, and secondary inflammation are causes of death among covid- patients huang et al., ; wan, xiang, et al., ; wang, hu, et al., ) . current medical therapies are symptomatic treatment or supportive care. there is no definitive treatment yet for this ta b l e some protocols used for the treatment of covid- disease. therefore, finding effective strategies to treat infected patients and protect organs is necessary to decrease the mortality rate. curcumin, as a potential agent, could be considered to treat covid- . curcumin, as an active constituent of rhizomes of c. longa (turmeric), is a hydrophobic polyphenol ( figure ) (akbar et al., ; soleimani, sahebkar, & hosseinzadeh, ) . curcumin is used as a spice in foods and for different purposes such as cosmetic and pharmaceutical industries in world (hosseini & hosseinzadeh, ) . curcumin has several pharmacological effects such as antioxidant, anticancer, antibacterial, antiviral, and antidiabetic effects (fan et al., ; moghadamtousi et al., ; zhu et al., ) , as well as anti-inflammatory activity (cheng, yang, hu, zhu, & liu, ) . as the potential role of curcumin to treat many inflammatory disorders, at the first step we will describe all effects of curcumin that may be useful to treat covid- , and then, we explain the possible molecular mechanisms of it. curcumin prevented the replication of sars-cov and inhibited cl protease in vero e cells. also, it significantly has an inhibitory activity against the cytopathogenic effect of sars-cov in vero e cells (wen et al., ) . c. longa l, as herbal medicine, is used to treat vomiting from ancient times in asian countries . curcumin ( mg/ kg, intragastric, days) improved appetite of rats in chemotherapy induced by fluorouracil ( -fu) (yao et al., ) . it may be effective against vomiting due to covid- . in an animal study, oral administration of curcumin has an antifatigue function and improved physical function in mice (huang et al., ) . administration of curcumin ( , mg/d, days) reduced stress and fatigue in the subjects that experiencing occupational stress-related anxiety and fatigue in a randomized double-blinded placebo-controlled study (pandaran sudheeran et al., ) . curcumin ( . g, two times a day) reduced delayed-onset muscle soreness of healthy men who have a heavy eccentric exercise (nicol, rowlands, fazakerly, & kellett, ) . the use of curcumin in myalgic encephalomyelitis/ chronic fatigue syndrome as a novel therapeutic option was mentioned . curcumin inhibited sepsis-induced muscle wasting by inhibiting catabolic response in skeletal muscle via blocking nf-κb (alamdari, o'neal, & hasselgren, ). curcumin (meriva®, g, months) prevented muscle loss and improved physical performance in healthy elder subjects and delayed the onset of sarcopenia in them (franceschi et al., ) . these results suggest that curcumin may be effective to manage myalgia and fatigue symptoms induced by covid- . the antinociceptive and anti-inflammatory effects of curcumin in animal and human studies were reviewed by eke-okoro, raffa, pergolizzi, breve, & taylor, (eke-okoro et al., . about the important molecular mechanism of these effects, it will discuss later that curcumin could be effective as a novel treatment against covid- . also, in an animal study, curcumin ( mg/kg, i.p.) has an antipyretic effect in rats (haider et al., ) . it seems that curcumin overcomes the fever of covid- -infected patients. two meta-analyses of randomized controlled trials have shown that curcumin reduced circulating il- and tnf-α levels that both are the key inflammatory mediators and increase in inflammatory diseases and rantes (ferreira, nazli, dizzell, mueller, & kaushic, ) . the summary of the clinical effects of curcumin that may be useful to treat covid- is illustrated in figure . in this section, we summarize the important molecular mechanisms of curcumin that show potential ability against covid- . in severe covid- infection, pneumonia may cause hypoxemia, which, in turn, disturbs cell metabolism and reduces the energy supply, and increases anaerobic fermentation. then, acidosis happens and causes oxygen free radicals to destroy the phospholipid layer of the cell membrane . therefore, treatment with a drug that has antioxidant properties will be good for these sars-cov- : severe acute respiratory syndrome coronavirus lung injury in rats . similarly, curcumin ( mg/kg) increased sod activity and decreased mda content in the lung in acute injury induced by sepsis (xiao, yang, sun, & sun, ) . ards is a clinical syndrome and is associated with increased permeability pulmonary edema, severe arterial hypoxemia, and impaired carbon dioxide excretion, eventually resulting in respiratory failure. it may occur due to a pulmonary or extrapulmonary infectious or noninfectious insult (matthay, ware, & zimmerman, ) . major inflammatory mediators in adrs include cytokines (tnf-α, il- β, il- , il- , il- ), chemokines such as macrophage inhibitory factor (mif), and macrophage chemoattractant protein, metabolites of arachidonic acid (prostanoids and leukotrienes), and oxyradicals. until yet, mechanical ventilation is only a proven strategy for treatment to improve the survival of patients (matuschak & lechner, ) . recently, rescue therapy with high-dose vitamin c has been suggested to use for these patients (fowler et al., ) . therefore, finding new treatments to overcome these mediators and preventing respiratory failure are necessary. on the other hand, the protective effects of curcumin were studied in several pulmonary diseases such as copd, ards, pulmonary fibrosis, and asthma in animal studies (lelli, sahebkar, johnston, & pedone, ; venkatesan, punithavathi, & babu, ) . in this part of the review, we explain molecular f i g u r e possible molecular mechanisms of curcumin against covid- in the pulmonary system. aa: arachidonic acid, ali: acute lung injury, ap- : activator protein , bk: bradykinin, ace : angiotensin-converting enzyme , ang ii: angiotensin ii, ards: acute respiratory distress syndrome, cas- : caspase , cox: cyclooxygenase, cxcl: chemokine (c-x-c motif) ligand, -hpete: -hydroperoxyeicosatetraenoic acid, jnk: c-jun n-terminal kinase, lox: -lipoxygenase, mmp: matrix metalloproteinase nf-κb: nuclear factor kappa-light-chain-enhancer of activated b cells, mapk: mitogen-activated protein kinase, pai- : plasminogen activator inhibitor- , pla : phospholipase a , pg: prostaglandin, smad : mothers against decapentaplegic homolog , tgf-β : transforming growth factor-beta , tnf-α: tumor necrosis factor-α, tlr: toll-like receptor, trpa : transient receptor potential channel subfamily vanilloid member , trpv : transient receptor potential channel subfamily a member mechanisms that curcumin may be useful to prevent or treat the ards. ali is a model that is used for the ards animal study (karunaweera, raju, gyengesi, & münch, ; olivera et al., ; tian et al., ; wang, tang, duan, & yang, ) , and curcumin exhibits its effects by predominantly targeting proinflammatory nf-ĸb pathway (ahn, sethi, jain, jaiswal, & aggarwal, ; karunaweera et al., ; olivera et al., ; puar et al., ; wang, tang, et al., ) . that were stimulated with s. aureus. it has been suggested that some part of the anti-inflammatory effects of curcumin are due to regulating nf-κb activity (xu et al., ) . curcumin downregulated the production of proinflammatory cytokine (tnf-α, ifn-α, and il- ) in influenza a virus-infected human macrophages and bal fluid of infected mice. similar to other noted studies, curcumin downregulated the expression of nf-κb and increased the cytosolic iκbα and inhibited its phosphorylation in the cytoplasm in human macrophages (xu & liu, ) . in this way, curcumin reduced tnf-α, mip- , and il- in lipopolysaccharide (lps)induced ali in mice. it has been suggested that curcumin inhibits the release of cytokines by activation of ′ adenosine monophosphate (amp)-activated protein kinase (ampk) (kim et al., ) . curcumin reduced the production of tnf-α, il- β, il- , and il- , mmp- , and mmp- both in mice and in a cells infected with influenza a virus. these cytokines exacerbate the ali (dai et al., ) . the inhibitory role of curcumin on the expression of proinflammatory cytokines such as tnf-α, il- , and il- was reviewed in ali and fibrosis by gouda and bhandary ( ) . it seems that the most important molecular mechanism of curcumin on il- activities may be related to the downregulation or inhibition of il- signaling in different inflammatory diseases (ghandadi & sahebkar, ) . furthermore, curcumin has an inhibitory effect on il- a that plays a pivotal role in the inflammation of the alveolar epithelial cells in ali studies. in other words, il- by activating p causes the stabilization of the pai- , which in turn mediates the accumulation of extracellular matrix (ecm) and subsequent development of pulmonary fibrosis in alveolar type ii (atii) cells, and curcumin inhibits il- a-mediated changes in the p -fibrinolytic system (figure ) . curcumin also reduced the gene expression of chemokines such as chemokine (c-x-c motif) ligand (cxcl ), cxcl , and cxcl that is increased during inflammation in the airway epithelial cells in bleomycin-induced ali in mice (refer to figure for more details) . the inhibitory effects of curcumin on the different subtypes of tlrs including extracellular tlr and tlr and tlr and intracellular tlr have been reported, which result in the therapeutic effects of curcumin in inflammation, infection, autoimmune, and ischemic disease (boozari, butler, & sahebkar, ) . curcumin at low concentrations ( , µm) prevented apoptosis and cytokine production (tnf-α, il- ) induced by -kda mycobacterium tuberculosis protein (p ) in human macrophages. curcumin also reduced the expression of tlr /jnk that may be involved in the apoptosis of macrophages . ali/ards could be the consequence of severe influenza a virus infection with substantial morbidity and mortality. on the other hand, curcumin decreased tlr / gene expression and inhibited phosphorylation of p , jnk, and nf-κb in infected a cells with influenza a virus. it seems that curcumin regulates the tlr-mapk/nf-κb signaling pathways involved in the replication and influenza pneumonia (figure ) . however, other mechanisms have been suggested for the antiviral effects of curcumin. furthermore, curcumin increased the survival rate in infected mice with this virus (dai et al., ) . pulmonary pathology of covid- pneumonia in two patients with lung cancer showed the edema and prominent proteinaceous exudates, vascular congestion, and inflammatory clusters with fibrinoid material. also, reactive alveolar epithelial hyperplasia and fibroblastic proliferation were reported in them . on the other hand, pai- plays a key mediator role in pulmonary fibrosis. furthermore, pai- and apoptosis have an important role in the progression and pathogenesis of pulmonary fibrosis (johnson, shaikh, muneesa, rashmi, & bhandary, ) . therefore, this issue led us to point out the antiapoptotic and the antifibrotic effects of curcumin here. the antiapoptotic effects of curcumin were found in different organ injuries including diabetes, nephrotoxicity, intestinal inflammation, neurotoxicity with several mechanisms (loganes et al., ; qihui, shuntian, xin, xiaoxia, & zhongpei, ; soetikno et al., ; sun et al., ) . both antiapoptotic and antifibrotic effects of curcumin were shown on the ali model in mice. curcumin reduced the expression of p , pai- , and chemokines in bleomycin-induced ali. also, curcumin inhibited apoptosis mediated by il- and downregulated cleaved caspase- in alveolar epithelial cells. the results suggest that the cross talk between the inflammatory, fibrinolytic, and apoptotic pathways is interrupted by curcumin . similar results of curcumin were found with the bleomycin model in human alveolar basal epithelial cells (a ) (gouda, prabhu, & bhandary, ) . also, curcumin decreased pai- with cytokines and chemokines in ali induced by staphylococcus aureus (xu et al., ) . on the other hand, curcumin inhibited the expression of tgf-β and smad pathway in ali induced by sepsis in rats that may involve in the pathogenesis of ali (xu et al., ) . curcumin reduced expression fibrosis markers including smooth muscle actin (α-sma), and tenascin-c in reovirus /l-induced ali/ards in mice (avasarala et al., ) . intranasal curcumin reduced matrix metalloproteinases- (mmp- )/tissue inhibitors of metalloproteinase (timp- ) expression and increased α-sma, as a myofibroblast marker, which involves in muscle thickening in paraquat lung injury model in mice. it seems that pretreatment of curcumin prevented the early phase of pulmonary fibrosis by inhibiting inflammatory cells and producing fibrotic factors (tyagi, dash, & singh, ) (figure ). bradykinin has an important role in the inflammatory events during acute and chronic inflammatory diseases such as respiratory tract infection and asthma (broadley, blair, kidd, bugert, & ford, ; hewitt et al., ) . furthermore, it seems that bradykinin could trigger cough in these inflammatory diseases or other conditions such as in patients with cough associated with captopril and enalapril as ace inhibitors (hewitt et al., ; katsumata, sekizawa, ujiie, sasaki, & takishima, ) curcumin is an inhibitor of activated protein- (ap- ) (singh & aggarwal, ) . curcumin prevented the expression of il- induced by bradykinin in human airway smooth muscle cells via this inhibition (huang, tliba, panettieri, & amrani, ) . on the other hand, it has shown that curcumin has a greater affinity to bradykinin b receptor (bk ) with strong inhibition activity (ki value = . µg/ml) compared with bk receptor (ki value = µg/ml) (yimam et al., ) . the possible molecular mechanism of bradykinin for sensitizing cough reflex is through activation b receptors, which in turn stimulate the release of cox and cox- (rao, ) . furthermore, curcumin has shown these inhibitory effects in the airway studies (kumari, singh, dash, & singh, ; yuan, liu, ma, zhang, & xie, ) . thus, curcumin is likely to inhibit the activity of bradykinin by inhibiting the cox enzyme ( figure ). curcumin ( mg/kg, p.o.) significantly inhibits ovalbumin (ova)induced airway constriction and airway hyperreactivity to histamine in sensitized guinea pigs (ram, das, & ghosh, ) . also, curcumin ( . and mg/kg, intranasal) significantly reduced bronchoconstriction in the mouse model of asthma (subhashini et al., ) . moreover, c. longa extract ( . , mg/ml) reduced tracheal contractile response to ova and maximum response to methacholine in rats. it also decreased interstitial fibrosis (shakeri, roshan, & boskabady, ) . standard therapy with capsule curcumin mg bd daily for days in patients of bronchial asthma significantly improved forced expiratory volume one second (fev ) compared with standard therapy. however, the mean scores for cough, dyspnea, wheezing, chest tightness, and nocturnal symptoms were insignificant. curcumin is recommended to use as an add-on therapy for bronchial asthma (abidi, gupta, agarwal, bhalla, & saluja, ) . dietary administration of curcumin ( mg kg - day - , gavage during ang ii infusion) decreased the protein level of the at receptor and enhanced the expression of the at receptor/ace and results in the attenuation of myocardial fibrosis in a rat model of angiotensin ii infusion (pang et al., ) . these data suggest that similar events happen in the lung tissues to prevent fibrosis. however, this hypoth- cells, human capillary, and kidney organoids through preventing entry into host cells. however, they did not study lung organoids that are the major target organ for covid- (monteil et al., ) . advantage of curcumin over other important natural agents with reported anti-inflammatory activities such as zerumbone (prasannan et al., ) , thymoquinone (siveen, mustafa, et al., ) , honokiol (rajendran et al., ) , escin (tan et al., ) , pinitol (sethi, ahn, sung, & aggarwal, ) , and tocotrienols is that it has additional antiviral, antiemetic, antinociceptive, antifatigue, and bronchodilator effects that previously has been discussed in this review. also, it has significant protective effects in the ards model in animal studies. these mentioned effects help us to conclude that curcumin has the potential to be effective against covid- infection. to date, over clinical trials have been completed with curcumin and safety, tolerability, and outcome have been reported in all of them (kunnumakkara et al., ) . an oral dose of mg (two times a day, days) was reported safe for curcumin (soleimani et al., ) . curcumin up to , mg/day was safe, tolerable, and effective in humans, and higher doses were with toxicity (kunnumakkara et al., ; shanmugam et al., ) . curcumin has low bioavailability, but a lot of data from clinical trials showed the high efficacy of curcumin or turmeric against several diseases (kunnumakkara et al., ) . however, various strategies are used including analogs of curcumin and formulations such as adjuvants, nanoparticles, liposomes, micelles, and phospholipid complexes to improve its bioavailability (kunnumakkara et al., ) . recently, it has been shown that the encapsulation of curcumin into specific nanocarrier could enhance its therapeutic efficacy (moballegh nasery et al., ). covid- is spreading worldwide, leading a pandemic. there is no definitive treatment yet for this disease. in this review, we summarized clinical and molecular mechanisms that curcumin might be effective to treat covid- . research evidence suggests that curcumin will be useful to treat patients especially in ards cases with high mortality risk. curcumin has several therapeutic effects including antiviral, antinociceptive, anti-inflammatory, antipyretic, and antifatigue effects with several molecular mechanisms such as antioxidant, antiapoptotic, antifibrotic effects, and inhibitory effects on nf-κb, inflammatory cytokines and chemokines, toll-like receptors, and bradykinin. the importance of this review is due to the fact that curcumin is a nutraceutical that could be a new treatment option to combat the covid- pandemic. designing the best formulation with high efficacy and good bioavailability is necessary. further clinical studies should focus on curcumin against covid- infection. the authors have no conflict of interest to declare. the study did not involve any human or animal testing. marjan nassiri-asl https://orcid.org/ - - - hossein hosseinzadeh https://orcid.org/ - - - x evaluation of efficacy of curcumin as an add-on therapy in patients of bronchial asthma antioxidant effects of curcumin in models of neurodegeneration, aging, oxidative and nitrosative stress: a review genetic deletion of nad(p)h:quinone oxidoreductase abrogates activation of nuclear factor-kappab, ikappabalpha kinase, c-jun n-terminal kinase, akt, p , and p / mitogen-activated protein kinases and potentiates apoptosis critical review on curcumin as a therapeutic agent: from traditional herbal medicine to an ideal therapeutic agent curcumin and muscle wasting: a new role for an old drug? bradykinin sensitizes the cough reflex via a b( ) receptor dependent activation of trpv and trpa channels through metabolites of cyclooxygenase and -lipoxygenase curcumin modulates the inflammatory response and inhibits subsequent fibrosis in a mouse model of viral-induced acute respiratory distress syndrome impact of curcumin on toll-like receptors bradykinin-induced lung inflammation and bronchoconstriction: role in parainfluenze- virus-induced inflammation and airway hyperreactivity a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study curcumin attenuates pulmonary inflammation in lipopolysaccharide induced acute lung injury in neonatal rat model by activating peroxisome proliferator-activated receptor γ (pparγ) pathway origin and evolution of pathogenic coronaviruses inhibition of curcumin on influenza a virus infection and influenzal pneumonia via oxidative stress, tlr / , p /jnk mapk and nf-κb pathways effect of curcumin on circulating interleukin- concentrations: a systematic review and meta-analysis of randomized controlled trials curcumin in turmeric: basic and clinical evidence for a potential role in analgesia anti-inflammatory and antioxidant effects of curcumin on acute lung injury in a rodent model of intestinal ischemia reperfusion by inhibiting the pathway of nf-κb curcumin in liver diseases: a systematic review of the cellular mechanisms of oxidative stress and clinical perspective the anti-inflammatory activity of curcumin protects the genital mucosal epithelial barrier from disruption and blocks replication of hiv- and hsv- effect of vitamin c infusion on organ failure and biomarkers of inflammation and vascular injury in patients with sepsis and severe acute 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interleukin and tumor necrosis factor alpha by m macrophages from patients with behcet's disease safety, tolerance, and enhanced efficacy of a bioavailable formulation of curcumin with fenugreek dietary fiber on occupational stress: a randomized, double-blind, placebo-controlled pilot study attenuation of myocardial fibrosis with curcumin is mediated by modulating expression of angiotensin ii at /at receptors and ace in rats. drug design anti-infective properties of the golden spice curcumin key cell signaling pathways modulated by zerumbone: role in the prevention and treatment of cancer evidence for the involvement of the master transcription factor nf-κb in cancer initiation and progression epidemiologic and clinical characteristics of hospitalized patients with covid- in zhejiang, china: a retrospective, multi-centre case series protection of curcumin against streptozocin-induced pancreatic cell destruction in t d rats dysregulation of immune response in patients with covid- in wuhan. china curcumin inhibits the replication of enterovirus in vitro honokiol inhibits signal transducer and activator of transcription- signaling, proliferation, and survival of hepatocellular carcinoma cells via the protein tyrosine phosphatase shp- curcumin attenuates allergen-induced airway hyperresponsiveness in sensitized guinea pigs regulation of cox and lox by curcumin ace and pace : a pair of aces for pulmonary arterial hypertension treatment? clinical, laboratory and imaging features of covid- : a systematic review and meta-analysis curcumin downregulates human tumor necrosis factor-α levels: a systematic review and meta-analysis of randomized controlled trials pinitol targets nuclear factor-kappab activation pathway leading to inhibition of gene products associated with proliferation, apoptosis, invasion, and angiogenesis hydro-ethanolic extract of curcuma longa affects tracheal responsiveness and lung pathology in ovalbumin-sensitized rats the multifaceted role of 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associated with a new coronavirus of probable bat origin curcumin suppresses lung cancer stem cells via inhibiting wnt/β-catenin and sonic hedgehog pathways a novel coronavirus from patients with pneumonia in china coronaviruses -drug discovery and therapeutic options curcumin (a constituent of turmeric): new treatment option against covid- key: cord- -djqzgc k authors: hao, siyuan; ning, kang; kuz, cagla aksu; vorhies, kai; yan, ziying; qiu, jianming title: long period modeling sars-cov- infection of in vitro cultured polarized human airway epithelium date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: djqzgc k severe acute respiratory syndrome coronavirus (sars-cov- ) replicates throughout human airways. the polarized human airway epithelium (hae) cultured at an airway-liquid interface (hae-ali) is an in vitro model mimicking the in vivo human mucociliary airway epithelium and supports the replication of sars-cov- . however, previous studies only characterized short-period sars-cov- infection in hae. in this study, continuously monitoring the sars-cov- infection in hae-ali cultures for a long period of up to days revealed that sars-cov- infection was long lasting with recurrent replication peaks appearing between an interval of approximately - days, which was consistent in all the tested hae-ali cultures derived from lung bronchi of independent donors. we also identified that sars-cov- does not infect hae from the basolateral side, and the dominant sars-cov- permissive epithelial cells are ciliated cells and goblet cells, whereas virus replication in basal cells and club cells was not detectable. notably, virus infection immediately damaged the hae, which is demonstrated by dispersed zonula occludens- (zo- ) expression without clear tight junctions and partial loss of cilia. importantly, we identified that sars-cov- productive infection of hae requires a high viral load of . × virions per cm of epithelium. thus, our studies highlight the importance of a high viral load and that epithelial renewal initiates and maintains a recurrent infection of hae with sars-cov- . introduction observed a cytoplasmic expression and a weak junction expression of zo- at dpi (fig. a ) and dpi (sfig. a) for infected hae-ali b - and hae-ali b - , respectively. these results demonstrate that a high viral load (at least > pfu (~ . × vgc) to an epithelium of . cm , which contains with ~ × epithelial cells, is necessary to initiate a productive infection. ciliated and goblet cells are permissive to sars-cov- but not the basal and club cells. we next examined sars-cov- infection in which the inoculation of a high moi of was applied to the basolateral side in hae-ali b - . the results showed there were no detectable infectious virions released from both the apical and basolateral sides (fig. a) . the teer of the infected hae displayed no significant changes over the course of days (fig. b) , which was also evidenced for the well-preserved tight junctions (fig. c) , as well as the rich cilia expression (fig. d) . importantly, np+ cells were not detected for as long as dpi. positive anti-β-tubulin iv staining. consistent with previous imaging results (fig. ) , most of the np+ cells were also positive with anti-β-tubulin iv staining (fig. a) , whereas almost all the ckrt + basal were negative for anti-np staining (fig. c) . probing secretoglobin family a member (scgb a ) expression for club cells and mucin ac (muc ac) expression for goblet cells revealed that the secretory cells were less abundant sub-populations in the infected hae-ali cultures. while we could not locate any club cells stained positively for np expression (fig. d) , we found some np+/muc ac+ goblet cells (fig. b) . importantly, we observed that in the infected hae-ali, a subset of cykt + basal cells are found associated with the expression of ki ; however, in the mock-infected hae-ali, we did not found the cells co-expressing both ki and cykt (fig. b) . as ki is a marker of cell proliferation , this result suggested that sars-cov- infection activates basal cells towards proliferation. taking these lines of evidence together, our results confirmed sars-cov- mainly infects ciliated cells of hae, as well as the goblet cells, despite its lower abundance in hae-ali cultures. our study suggests that basal and club cells are not permissive to sars-cov- . our observation that sars-cov- was unable to infect epithelial cells from the basolateral side supports that the viral entry receptor ace is polarly expressed at the apical side , . we believe the finding that ckrt + basal cells are largely not infected by sars- cov- is important to understand the course of sars-cov- infection in hae. the airway basal cells are the epithelial cell type not presenting on the surface of the airway lumen, thus, they are not usually accessible to the virus on the apical side. however, when the infection commences and the epithelial damage occurs, the destructive mucosal lesions (and the death of the infected ciliated and goblet cells) would allow the virus to get access the basal cells (fig. c) . indeed, the detectable virus shedding to the basolateral chamber (fig. a&c , sfig. a and sfig. a) indicates a possible window to expose the basal cells to sars-cov- . notably, these time points also represent the peaks of the release of virus progeny. however, none of the ckrt + cells that was also np-positive was found from the cytospins prepared from sars-cov- infected hae at dpi when the infection appeared at the lowest level. we hypothesize that the non-permissive nature of basal cells to sars-cov- is due to the negligible expression of ace or tmpress- . the epithelial cell lining of the airways provides an efficient barrier against microbes and aggressive molecules through interdependent functions, including mechanical clearance of the mucus executed by movements of the cilia, a cellular barrier function by means of intercellular epithelial junctions formed by a set of tight junction associated proteins such as zo , and homeostasis of ion transport . at the airway epithelial cellular level, the tight junction- associated proteins, such as zo , occludin, and claudins, play a central part in the epithelial cytoprotection by maintaining a physical selective barrier between external and internal environments. the tight junction proteins are highly labile structures whose formation and structure may be very rapidly altered after airway injury, for example, airway inflammation. proinflammatory cytokines have a drastic effect on tight junction expression and barrier functions, which significantly alter the epithelial barrier permeability - . sars-cov- infection distorted the zo- expression, and thereafter causes barrier dysfunction (teer decrease). the infection not only alters the zo- expression of infected (np + cells) but also uninfected cells (np-cells) (fig. ) . this is also true for the cilia loss. we believe that sars-cov- infection produces inflammatory cytokines as an innate immunity response upon virus infection , which either disturbs zo- and tubulin expression or alters their structures. the innate immunity response also limits virus infection at the front line. in fact, sars-cov- requires a high viral load (> pfu/cm of hae) to initiate a productive infection (fig. ) . of note, the infectious titer (pfu) was determined by plaque assay in vero-e cells, which are interferon-deficient . we determined that pfu of sars-cov- in vero-e cells has a particle (viral genome copy) number of , suggesting that a load of . x particles is required to productively infect cm of the airway epithelium, which is much higher than the small dna virus parvovirus human bocavirus (hbov ) we studied . hbov can infect hae at an moi of as low as . genome copies per cell, which equals . x particles per cm of the airway epithelium. epithelial regeneration involves migration of the basal cells that neighbor the acute injured area (e.g., virus-infected area), active dividing and squamous metaplasia, rapid redifferentiation to preciliated cells, and finally ciliogenesis towards a complete pseudostratified mucociliary epithelium . airway epithelium repair is critical for the maintenance of the barrier function and the limitation of airway hyperreactivity. in a biopsy study of fresh tracheas and lungs from five deceased covid- patients, it was found that the epithelium was severely damaged in some parts of the trachea, and extensive basal cell proliferation was observed in the trachea, where ciliated cells were damaged, as well as in the intrapulmonary airways . these data support our conclusion that basal cells are not permissive to sars-cov- . as a response to these previous findings, our study observed that a subset of proliferating basal cells in the sars-cov- infected hae-ali, but not in the mock infected hae-ali (fig. b) . thus, we hypothesize that sars-cov- infection induces basal cell proliferation, which accounts for the observed long-lasting infections with recurrent peaks of viral replication, which warrants future investigation. overall, we propose a model of sars-cov- -infection of hae (fig. c) and then cytocentrifuged at , rom for min onto slides using a shandon cytospin cytocentrifuge. after cytospun, the slides were fixed overnight in % paraformaldehyde at °c. the fixed hae or dissociated cells were permeabilized with . % triton x- for min at room temperature. then, the slide was incubated with primary antibody in pbs with % fbs for h at ºc. after washing, the membrane was incubated with fluorescein isothiocyanate- and rhodamine-conjugated secondary antibodies, followed by staining of the nuclei with dapi ( ', -diamidino- -phenylindole). online ahead of print covid- : towards controlling of a pandemic genomic characterisation and epidemiology of novel coronavirus: implications for virus origins and receptor binding transmission of -ncov infection from an an 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cell death via inhibiting type i and type iii ifn restrict sars-cov- infection of human defectiveness of interferon production and of rubella virus interference in a line of african green monkey kidney cells (vero) in vitro modeling of human bocavirus infection of polarized primary human airway epithelia & trump,b.f. the respiratory epithelium epidermoid metaplasia of hamster tracheal epithelium during regeneration following mechanical injury development of cystic fibrosis and noncystic fibrosis airway cell lines distinct classes of proteasome-modulating agents cooperatively augment recombinant adeno- associated virus type and type -mediated transduction from the apical surfaces of human airway epithelia unique biologic properties of recombinant aav transduction in polarized human airway epithelia human bocavirus infection of well-differentiated human airway epithelium replication of an autonomous human parvovirus in non-dividing human airway epithelium is facilitated through the dna damage and repair pathways real-time analysis of camp-mediated regulation of ciliary motility in single primary human airway epithelial cells establishment of a replicon system for bourbon virus and identification of small molecules that efficiently inhibit virus replication confocal images were taken at a magnification of x on the indicated days post-infection (dpi) nuclei were stained with dapi (blue) three-dimensional (z-stack) imaging of sars-cov- infected primary bronchial - -infected hae-ali b - cultures at dpi were co-stained with or with anti-np and anti-β-tubulin iv antibodies (b), or co- stained anti-np and anti-zo- antibodies (b). a set of confocal images were taken at a magnification of x from the stained pierce of epithelium image as shown in each channel of fluorescence. nuclei were stained with dapi (blue) virus release kinetics and transepithelial electrical resistance (teer) measurement of hae-ali infected with sars-cov- at various viral loads (multiplicities of infection) a&c) virus release kinetics. hae-ali b - cultures were infected with sars-cov- at at the indicated days post-infection (dpi), µl of apical washes by incubation of µl of d-pbs in the apical chamber and µl of the basolateral media were taken for plaque assays transepithelial electrical resistance measurement. the teer of mock-and sars-cov- - hae-ali culture were measured using an epithelial volt-ohm meter (millipore) at the indicated dpi. the teer values were normalized to the teer measured on the day of infection, which is set as . . values represent the mean of relative teer +/-standard deviations hae-ali b - cultures were infected with sars-cov- at an moi from . to . . at dpi, both virus and mock infected hae were co-stained with anti-np and anti-zo- antibodies (a), or co-stained with anti-np and anti-β-tubulin iv antibodies (b). confocal images were taken at a magnification of x . nuclei were stained with dapi (blue). key: cord- -p q f c authors: nan title: posters_monday_ october date: - - journal: intensive care med doi: . /s - - - sha: doc_id: cord_uid: p q f c nan a huge variability in excess risk of death, ranging from to %, from ventilator associated pneumonia (vap) have been reported in the literature [ ] . this large between-study variation can be attributed to difference in definitions but also to incorrect estimation by standard statistical methods, i.e. inappropriate adjustment for informative censoring and time dependent confounders. the aim of this study was to take into account above statistical shortcomings and to assess the excess risk of vap by using an extension of a recently developed techniques from the field of causal inference [ ] . materials and methods. data was retrieved from a large longitudinal, high quality multi-centric icu database from france (outcomerea). a random sample of consecutive patients ventilated [ h from icus over a year period were included. vap was defined as clinical suspicion plus at least one positive proximal or distal sampling with quantitative count using classical thresholds. only the first vap episode was taken into account. we considered discharge from the icu as a competing risk and estimated the attributable -day icu mortality of vap by comparing the counterfactual cumulative incidence of death for the entire population under different hypothetical infection paths. baseline characteristics indicating underlying co-morbidity and longitudinal (daily measured) severity of illness indicators together with all other known confounders until vap developed were taken into account through the use of a marginal structural model [ ] . results. a total of , icu patients were included. mean (sd) age and saps ii score were ( ) and ( ), respectively. seventeen and % were admitted after scheduled and emergency surgery respectively, % were medical patients. forty-two percent had an underlying chronic illness (knaus). nine percent received dialysis in the icu. a total of ( %) patients developed vap within days of admission ( and % within and days, respectively). crude icu-mortality rates in patients with and without vap were and %, respectively. when taking into account all the confounders, we found a . % increase ( % ci . - . , p = . ) in the hazard of -day icu-death per additional day since the development of vap. provided vap could have been prevented in the whole population -day mortality would have decreased by . %. conclusion. the excess risk of death from vap estimated by marginal structural models is lower than commonly reported in the literature. this indicates that underlying comorbidities and the evolution of severity of illness until vap are insufficiently taken into account by current standard statistical methods. reference (s) . . rello et al ( ) introduction. the intensive care society (uk) has recently published national guidelines regarding many aspects of percutaneous tracheostomy management [ ] , however, these guidelines do not make any recommendations regarding antibiotic prophylaxis during the procedure. we recently audited uk practice and have established that only % of the units give prophylactic antibiotics for patients known to be colonised with methicillin-resistant staphylococcus aureus. the low rate of antibiotic use is surprising given that pneumonia and/ or bacteraemia following pt is frequently caused by organisms (non-mrsa) that colonise the patients' skin and/or airway [ , ] . objective. to establish the incidence of mrsa positive sputum and/or blood cultures following pt in patients colonised with mrsa in their nose or throat. methods. we audited all the patients who had pt performed between and who were known to be colonised with mrsa in their nose or throat, (but who had negative sputum cultures) before pt was undertaken. we wanted to find how many of these patients developed mrsa positive sputum and/or blood cultures in the first week following their pt. results. from a total of patients admitted to critical care between and only seven mrsa colonised patients required pt. all of these patients had mrsa colonised throat or nose with negative sputum and blood cultures prior to the pt. no patients were given prophylactic antibiotics during the pt as this was our standard practice. four ( %) developed mrsa positive sputum cultures in the first week following the procedure. one ( %) developed mrsa bacteraemia on day following pt. over the same period there were no case of mrsa bacteraemia in the mrsa colonised patients who did not undergo pt. microbiology bacterial biofilm has been observed in the surface of the endotracheal tube (ett) in mechanically ventilated patients and recent studies relate the presence of biofilm with the incidence of ventilator-associated pneumonia (vap). acinetobacter baumanii is a gramnegative opportunistic nosocomial pathogen involved in the production of vap, and capable of biofilm formation on abiotic surfaces. objective. to analyse the presence of biofilm in the ett by using scanning electron microscopy (sem) and to identify the microorganisms contributing to its formation in patients admitted in an icu endemic for a. baumanii. methods. from march to september , consecutive patients admitted to our unit and mechanically ventilated were included in the study. etts after extubation were (a) sent to microbiological culture, and (b) fixed with % paraformaldehyde- % glutaraldehyde for one hour, dehydrated with increasing ethanol concentrations, and processed for sem. etts were observed under sem to assess the presence and extension of the biofilm, and to recognize bacterial or fungal forms. results. there were males ( %) and females ( %) with a median age of years (range - ).the median apache ii score in the first h was (range - ). the median duration of intubation was days (range . causes of intubation were coma in patients ( %), respiratory failure in ( %), and heart failure in ( %). the microbiological isolations showed: a. baumanii ( %), staphylococcus non aureus ( %), pseudomonas spp. ( %), streptococcus viridans ( %), staphylococcus aureus ( %), enterococcus faecalis ( %), candida albicans ( %), and others ( %). under the sem, biofilm was identified in the % of all cases and was abundant in ( %), regular in ( %), and scarce in ( %). morphological identification of microorganisms observed under sem showed: cocci in ( %), bacilli in ( %), and yeast in ( % introduction. hospital-acquired infection is often linked to the standard of ward cleaning however the impact of increased quality of cleaning and deep cleans are unknown. objectives. this study aimed to determine the effect of enhanced cleaning on local contamination rates of hospital pathogens and whether this results in a reduction in patient colonisation. a cross-over one-year study was performed in the intensive care units (icu) of two teaching hospitals, which screened patients weekly for mrsa. in randomized two-month periods and in addition to conventional cleaning using detergent and mops, high contact areas were cleaned twice daily by a team of trained hygiene technicians using microfibre cloths. using contact plates, samples were taken at nine sites around the bed area and ward over bed-days per week. hand hygiene was encouraged and compliance audited. results. only . % of the planned , local samples were missed and this was equal between study phases. average hand hygiene compliance was similar between enhanced and standard phases [hospital a . % ( / ) vs. % ( / ) and hospital b . % ( / ) vs. . % ( / )]. patient characteristics were similar during standard and enhanced cleaning periods. of the sites tested, samples taken from bed rails were most likely to be contaminated with mrsa (or = . ; % ci: . - . ) followed by nurses' hands (or = . ; % ci: . - . ). analysis of these site-samples also confirmed that enhanced cleaning significantly reduced environmental contamination (or = . ; % ci: . - . ; p \ . ). the effectiveness of enhanced cleaning in removing mrsa contamination did not vary with the sample site. a sub-group analysis of samples only taken from nurses' hands showed a non-significant reduction in mrsa hand contamination associated with enhanced cleaning although associated uncertainty was large (or = . ; % ci: . - . ; p = . conclusions. this is the first prospective controlled study examining the effectiveness of enhanced cleaning in preventing spread of multiresistant pathogens within icu. although both environmental and hand contamination were reduced, enhanced cleaning of high contact surfaces was not associated with a reduction in cross infection. conclusions. isd of secretions reduces the incidence of vap in patients receiving. css alone, or in combination with isd has no significant effect on incidence of vap. hence, isd may be recommended for vap prevention, considerations other than prevention of vap should determine the choice of the suction system in a mechanically ventilated patient. to show a mortality benefit, larger, multi-center trials may be required. decreasing incidence of ventilator-acquired pneumonia (vap) is increasingly regarded as a priority in icu quality programs. subglottic secretion suctioning (sss) has been associated with a decreased risk of vap. a previous metaanalysis concluded that sss reduces the risk of vap, but it included only five randomized controlled trials (rct), and sss is still underused, perhaps considering the available evidence is insufficient. we planned a systematic review and metaanalysis of sss for vap prevention. pubmed, embase and cdsr were searched for rct studying the influence of sss on vap incidence. additional outcomes were mortality (within icu or hospital), icu and hospital stay, mechanical ventilation duration and time from intubation to vap diagnosis. additional references and sources of information were searched, and authors were contacted as necessary. rct were found, but one of them was excluded for not having enough data for analysis. rct were analysed, including , patients. quality of the rct was only moderate. qualitative outcomes were homogeneous between studies, so were analysed by a fixed effects model; quantitative outcomes were very heterogeneous, and were analysed by a random effects model. compared to control, sss decreased vap incidence (rr . ; % ci . - . ), but not mortality (rr . ; % ic . - . ). sss delayed vap onset for . days ( % ci . - . ), shortened mechanical ventilation for . days ( % ci . - . ) and decreased icu length of stay for . days (ic % . - . ). in two rct, no differences were found in the hospital length of stay. conclusion. sss reduces vap incidence and delays vap onset, shortening mechanical ventilation and icu length of stay, but not decreases icu or hospital mortality. data from rct support the use of sss as an adjunctive tool to prevent vap. introduction. in comparison to ventilator-associated pneumonia (vap), less data are available on ventilator-associated tracheobronchitis (vat). however, vat may be associated with considerable morbidity [ ] . aim. to investigate prospectively the incidence and outcomes of vat. we studied prospectively all patients who received mechanical ventilation in the general intensive care unit of a tertiary hospital in greece between september-november . vat diagnosis required temperature ([ °c) or leukocyte count [ . per ml or leukopenia \ . per ml) (at least one of these) plus new onset/change of purulent endotracheal secretions. vap diagnosis required the aforementioned criteria plus appearance of new and persistent pulmonary infiltrates on chest radiography. microbiological documentation was based on the growth of microrganisms in bronchial aspirations ([ . cfu) or bal ([ . cfu) . results. forty-six patients were included, median (iqr) age was ( . years. eleven ( %) patients presented vat, presented vap and patients presented none of these two disorders (np). there were no significant differences between vat and vap cases in terms of baseline characteristics (diagnosis, respiratory compliance, apachee, murray score), occurrence of sepsis or ards and microbiology; pseudomonas aeruginosa, acinetobacter baumannii, staphylococcus aureus and klebsiella pneum. were the most common bacteria in both vat and vap. patients who presented vat or vap had significant longer hospitalization and mechanical ventilation duration (days) compared to , ( - ) vs. ( - ), (p = . )] and [ ( - ) , ( - ), ( - ) , (p = . ), respectively]. icu mortality was , , %, for patients with vap, vat and np, respectively (p = . ). conclusions. incidence and microbiological pattern was similar in vap and vap in these case series. both vat and vap were associated with longer hospitalizations and mechanical ventilation duration. further analysis with a larger cohort of patients is required to give conclusive remarks. reference (s) . . nseir s et al ( ) nosocomial tracheobronchitis in mechanically ventilated patients: incidence, aetiology and outcome. eur respir j : - . g. c. choutas , v. g. nolas , a. kalantzi , a. moutzouri , g. k. anthopoulos intensive care unit, \ \ [ [ general air force hospital, athens, greece introduction. endotracheal suctioning is an essential part of care for patients receiving mechanical ventilation, to keep the airways free from bronchial secretions, assuring ventilation and oxygenation. there are two types of suction systems. in the open system, endotracheal suctioning requires disconnecting the patient from the ventilator and introducing a single-use sterile suctioning catheter into the endotracheal tube. closed systems are changed every and h. to determine whether ventilated patients treated with css in an intensive care unit (icu) differ as to airway bacterial colonization and colonization of the suction system based on cultivation of both bronchial secretion and suction catheter tip and if cultivation of suction catheter tip is adequate in place of bronchial aspirate cultivation. methods. patient, incubated and ventilated in the icu ward were studied in a period of one year ( to ) , on admission to the icu a css (trach care mac) was connected. closed multi-use catheters were changed daily. two-pass endotracheal suctioning was performed as needed. ba cultures were obtained on admission and the next day. radiographs taken before, during, and after ba and css cultures were graded for pneumonia and a modified score for vap. of the patients css samples ( . %) and ba samples ( . %) were sterile. airway colonization with gram-negative bacteria and fungi occurred in the majority of the patient . % and gram-positive bacteria in %. cultivation of css revealed gramnegative bacteria and fungi occurrence . % and gram-positive bacteria in . %. with the current sample no significant difference was found between the positive results of trach care tip cultivation and ba cultivation p = . . objectives. to reduce the use of sedatives and to decrease the amount of time spend on a ventilator by specific ramsay-instructions and checks for sedation-protocol-adherence. methods. in april , after introductional lessons to doctors and nurses, we started and collected data for months. a yellow reminder was attached to the medical-instructions-form and doctors were requested to fill in the ramsay-score on a daily basis. once in a week patients and records were screened to assess protocol-adherence. each nurse and each intensive care-unit received feedback on their compliance to the bundle-elements. the total amount of sedatives per month was divided by the number of ventilator days, resulting in an average dose midazolam/propofol per ventilator day. the median and interquartile range of ventilator days/patient was also calculated and all data were compared with the same period in . we accomplished a reduction in the use of sedatives and costs. introduction. ventilator associated pneumonia (vap) often occurs in patients who are mechanically ventilated. the incidence rate varies between and % for patients in the intensive care unit. it has been the second most common hospital-associated infection after that of the urinary tract. the diagnosis of vap is difficult because of different existing definitions. hypothesis. our hypothesis was, that lowering vap incidence rate, could be done by a bundle of five interventions. the purpose of introducing multiple ventilatory interventions as a bundle, was to lower vap incidence rate by %. methods. during the last months of all patients who were ventilated [ h, were investigated for vap. the diagnosis of vap was done according to the criteria supposed by the dutch working group on infection prevention. a new infiltrate on chest x-ray after h ventilatory support in combination with fever, leucocytosis, increased need for oxygen and culture of blind bronchial secretion c cfu/ml. a ventilator bundle was introduced on all icu wards as inspired by the institute of healthcare improvement. five interventions were introduced: head of bed [ °, reduction of sedatives as low as possible according to prescribed ramsay score, assessment of readiness to extubate, cuff-pressure measurement times a day with application of cuff pressures between and cm h o, and oral care with chlorhexidine . % times a day. icu nurses were trained in the first months of . the last months of were used to evaluate the bundle intervention in comparison with the last months of . results. patients were included in and in . after introduction of the ventilator bundle, the incidence per , ventilator days decreased from . % to . % per , ventilator days. introduction. continuous positive airway pressure (cpap) may improve oxygenation in patients with mild to moderate acute hypoxemic respiratory failure (ahrf) and avert further deterioration and need for intubation. objectives. aim of our study was to assess the physiologic effects produced by the addition of periodic hyperinflations (sigh) to cpap in patients with ahrf. we studied patients with non-cardiogenic ahrf. four trials of one hour each were performed at a constant fio % during ( ) spontaneous breathing (sb) via a venturi mask, ( ) cpap , ( ) cpap ? sigh/min of cm h o for s (cpap sigh ), ( ) cpap . cpap, via helmet, was maintained at cm h o troughout the whole study period. pao /fio ratio (p/f), paco , ph, respiratory rate (rr), arterial blood pressure (abp), heart rate (hr), dyspnea and patient comfort (by means of separate visual analog scales) were measured at the end of each trial. results. overall, p/f was significantly (p \ . ) improved by cpap (cpap ± mmhg, cpap sigh ± mmhg, cpap ± mmhg), as opposed to sb ( ± mmhg). overall, the sigh did not significantly improved p/f. in the six patients with bilateral infiltrates, however, the rate of improvement in p/f significantly (p \ . ) augmented with the introduction of a sigh as compared with those with monolateral infiltration ( vs. % respectively, being % the increase from venturi to cpap ). paco , ph, rr, hr, abp, dyspnea and comfort were not significantly different between trials. conclusions. in patients with ahrf, cpap improves oxygenation without affecting hemodynamics. the addition of a sigh to cpap further improved oxygenation only in patients with bilateral pulmonary infiltrates. background. adaptive support ventilation (asv) is a novel electronic ventilator protocol that incorporates the recent and sophisticated measurement tools and algorithms. the target tidal volume and respiratory rate are continually adapted to patient's respiratory physics and varying medical conditions. in injured lung, the asv should actively adjust ventilatory parameters achieving minimal work of breathing to meet the lung protective strategies. but there were little literatures describing its efficacy when applied to korean population. methods. from may to january , we observed initial mechanical ventilation parameters in patients receiving asv due to various causes ( lung injuries including community acquired pneumonia, hospital acquired pneumonia, interstitial lung diseases, pulmonary tuberculosis and idiopathic cases; without lung injury which comprise trauma cases, strokes, suicidal attempts and other cases). the mean age of studied population was . years (male:female = : ). the data were collected within the first h of mechanical ventilation. conclusion. as expected, adaptive support ventilation delivered smaller tidal volume and higher respiratory rates for injured lungs. asv efficiently operated in korean ali patients without any serious drawbacks and favorably adjusted the tidal volume and respiratory rates combination in relation with rcexp to meet lung protective strategies. introduction. neurally adjusted ventilatory assist (nava) is a mode of mechanical ventilation that uses the electrical activity of the diaphragm to control the ventilator obtaining an improved patient-ventilator synchrony and an efficient unloading of the respiratory muscles. nava is characterised by a variability of the breathing pattern and the absence of a constant flow, which makes impossible the determination of reliable data of respiratory mechanics by using the rapid occlusion method. we have previously demonstrated that the least squares fitting method (lsf) could be used during pressure support ventilation (psv), provided that the level of ps is sufficiently high to unload the inspiratory muscle. hence we made the hypothesis that ( ) reliable data of respiratory mechanics can be obtained by applying the lsf method during nava and ( ) the lsf method should work better during nava because of the characteristics of the flow and pressure traces. methods. ten patients undergoing mechanical ventilation for acute respiratory failure were enrolled. they were ventilated using randomly either psv or nava with the the same peepe and tidal volume (v t ). data of resistance (r rs ), elastance (e rs ) and total positive end expiratory pressure (peep tot ) were obtained by fitting the equation paw = r rs v ? v t / c rs ? peep tot during inspiration, because of the possible presence of expiratory flow limitation. the coefficient of determination (cd) of the applied equation was used to compare data obtained during nava and psv, the higher being the cd, the better the quality of the data. moreover patients were sedated and ventilated in volume controlled ventilation (acv) with the aim of calculating data based on rapid occlusion method and compare them with those obtained with lsf method by using the bland-altman analysis. ( ) data obtained with lsf were statistically more reliable during nava (mean cd: . ± . ) than during psv (mean cd: . ± . ; p \ . ). ( ) the cd obtained at every level of nava was always higher then . . on the contrary, the cd obtained at low level of psv was less than . due to the presence of inspiratory muscle activity. ( ) the bland-altman analysis demonstrated lower bias and higher precision between traditional data and those calculated during nava (bias: . ; limit of agreements: - . / . ) compared to psv (bias: . ; limit of agreements: - / . ). conclusion. the application of the lsf method during nava allows calculation of reliable data of rrs, ers and peeptot, which are independent of the level of nava applied. this is of clinical relevance since psv allows calculation of reliable data only at high level of pressure support. it appears that the influence of inspiratory muscle activity on respiratory mechanics is less relevant during nava ventilation, suggesting a more physiological ventilation during nava both in terms of timing and of delivering adequate level of assist throughout each breath. introduction. nava is a new spontaneous-assisted ventilatory mode based on the detection of diaphragmatic electrical activity (eadi) and its feedback to adjust ventilator settings. nava uses the eadi, an expression of the respiratory center's activity, to initiate pressurization, set the level of pressure support and cycle the ventilator into exhalation. therefore, nava should theoretically allow near-perfect synchronization between the patient and the ventilator. however there are few data documenting these effects in intensive care patients. to determine whether nava can improve patient-ventilator synchrony compared to standard pressure support (ps) in intubated intensive care patients. comparative study of patient-ventilator interaction during ps with clinician determined ventilator settings and nava with nava gain (proportionality factor between eadi and the amount of delivered inspiratory pressure) set as to obtain the same peak airway pressure as the total pressure obtained in ps. a min continuous recording with each ventilatory mode was performed allowing determination of trigger delay (t d ), patient neural inspiratory time (t in ), duration of pressurization by the ventilator (t iv ), excess duration of pressurization (t i excess = t iv -t in /t in ) and number of asynchrony events by minute: non-triggering breaths, auto-triggering, double triggering, premature and delayed cycling. results are given in mean ± sd. p is considered significant if . . . ± . . ± . . ± . n asynchrony/minute . ± . . ± . * . ± . respiratory rate (min - ) . ± . . ± . na * p \ . conclusion. compared to standard ps, nava improves patient ventilator interaction by reducing td and the overall incidence of asynchrony events. there is also a strong trend in reducing delayed cycling. this ongoing trial should provide evidence that nava can indeed improve patient-ventilator synchrony in intubated patients undergoing ps. introduction. high fio and hyperoxia may induce pulmonary injury and may increase oxidative stress. guidelines suggest a target arterial oxygen tension of kpa [ ] . a canadian questionnaire study found considerable variation in the attitudes, beliefs and practices of intensivists in the management of oxygen therapy [ ] . however, the actual response of intensivists to hyperoxia in patients has never been studied. in this retrospective database study we investigated adherence to guidelines concerning oxygen therapy in a dutch academic intensive care. all arterial blood gas (abg) data from mechanically ventilated patients from to were drawn from an electronic storage database (metavision) of a mixed -bed icu in a university hospital in amsterdam. mechanical ventilation settings at the time of the abg as well as the successive abg were retrieved. the statistical analysis was carried out with spss . . results. . abg's from mechanically ventilated patients were retrieved including corresponding ventilator settings. in . ( %) of the abg's po was[ kpa. initial ventilator settings and adjustments based on abg's of this group are shown in table [data represent median ( th/ th percentile)]. in % of the lowest fio group, fio was exactly %. in only % of cases with po [ kpa the fio was decreased. hyperoxia was accepted with no adjustments in ventilator settings if fio was % or lower. introduction. major patient ventilator asynchronies are frequent during non-invasive ventilation (niv) especially due to leaks.niv can be delivered using icu ventilators or specifically designed niv ventilators. although icu ventilators are traditionally used for invasive mechanical ventilation, specific niv modes have been recently implemented. the impact of these niv modes as well as niv ventilators on patient ventilator asynchrony is unknown. our objective was to compare the incidence of patient ventilator asynchrony between invasive or niv mode of icu ventilators and niv ventilators. patients and methods. icu patients with acute respiratory failure requiring niv were studied during three randomized consecutive min-periods of niv: icu ventilator with and without niv mode and niv ventilator. we used two icu ventilators: evita xl (dräger) and engström (general electrics) and niv ventilator: bipap vision (respironics). flow and airway pressure were continuously recorded to determine breathing pattern. to detect major patient ventilator asynchrony we used surface diaphragmatic and/or sternocleidomastoid electromyogram allowing to assess neural patient's inspiratory time and to define asynchronies: ineffective triggering, auto-triggering, double-triggering, prolonged and short cycles. asynchrony was quantified using an asynchrony index as previously described. results. these preliminary results concern ten patients ( males and female) with a mean age of ± and a saps of ± . reason for niv was acute exacerbation of copd (n = ), acute pulmonary edema (n = ) and post-extubation (n = ). at time of study, ph was . ± . , paco ± mmhg and pao ± mmhg. ventilatory settings were set by the clinician and kept constant during the three periods with a ps level of ± cm h o and a pressurization ramp of ± ms, a peep level of ± cm h o and a fio of ± %. using icu ventilators, inspiratory trigger was l/min and cycling off was % when adjustable. median asynchrony index was . % ( . - . ) using icu ventilators versus . % ( . - . ) using niv mode and . % ( . - . ) using bipap vision (p = . between invasive and niv mode, p \ . between niv mode and bipap vision). asynchrony index was greater than % in three patients using invasive mode, two patients using niv mode and no patient using bipap vision. auto-triggering was the main asynchrony. conclusion. niv ventilator (bipap vision) allowed a marked reduction in patient ventilator asynchrony during niv as compared to niv mode currently available on new generation of icu ventilators. grant acknowledgement. this study was supported by a research grant from respironics. a. armaganidis , k. stavrakaki-kallergi , c. sotiropoulou , a. koutsoukou , j. milic-emili , c. roussos athens university, athens, greece prevalence of expiratory flow limitation (efl) was estimated using the negative expiratory pressure method (nep) in anesthetized, paralyzed mechanically ventilated patients in icu. patients were studied in supine position at zero positive end-expiratory pressure (peep). a nep device especially designed and in build in an evita -draeger respirator, allowed the application of a pressure equal to- cm h o, starting at ms after the onset of expiratory flow and sustained throughout the end of the expiratory time set on the ventilator. patients were categorized in two groups: . non efl ( patients without flow limitation), in whom nep elicited an increase of expiratory flow over the entire expiratory flow-volume (v -v) curve. . efl ( patients with efl), in whom part or the expiratory v -v curve during nep was superimposed on the baseline v -v curve. half of our patients ( %) were flow-limited. no patient without pulmonary disease was found flow-limited, except of a percentage of morbidly obese patients ( %). efl was recorded in % of icu patients with pulmonary diseases: % of ards patients, % of patients with respiratory infection, % of asthmatics and % of patients with copd. time constant (s) and inspiratory flow (v insp) were found to predict the severity of flowlimitation expressed as efl % v t . objective. tidal volume (v t ) administered to ards patients can be adjusted depending on body weight. body weight may be estimated, measured or calculated for an ideal or a predicted value based on different formulas [ , ] . besides, those formulas require the measurement of height and may differ depending on gender. we hypothesized that v t value (ml/kg of body weight) may be different and show intrameasure variability depending on the method used. methods. ards patients were included prospectively in the first h after icu admission. the ventilatory parameters were selected by the attending physicians that were foreign to the study. all patients were ventilated by volume controlled-assisted mode. five independent observers estimated the weight (estw) of each patient. they also measured height with a metric tape for calculate the predicted body weight (pbw) [ ] and the ideal body weight (ibw) [ ] . after previous measurements, patients were weighed once with a calibrated scale (scaw). results were compared using analysis of variance. results. patients were studied, % women (age . ± . , saps ii . ± . ; apache ii . ± . ). ventilation parameters at inclusion: v t /scaw (ml/kg) mean ± sd . ± . . ± . . ± . . ± . . ** mean ± sd mean ± standard deviation, min minimum, max, maximum, pbw (men) or . (female) ? . (height in centimetres - . ), ibw (height in meters) , mean diff average of the intraindividual differences of calculated/estimated weight, range intraindividual difference in weight (estimated/calculated) expressed as minimum and maximum * estw versus pbw p \ . , pbw versus ibw p \ . , pbw versus scaw p = . ** v t /estw versus v t /pbw p \ . , v t /pbw versus v t /ibw p \ . , v t /pbw versus v t /scaw p \ . conclusions. our data show that there is no gold standard method for estimate or calculate body weight to adjust tidal volume in ards patients. recommendations based on pwb and ibw not guarantee that tidal volume administered is really those that we want to administrate. reference (s) . . ardsnet. nejm ; : - . . stewart te et al ( hôpital raymond poincaré ap-hp, service de réanimation médicale, garches, france, hôpital saint-louis, paris diderot university, paris, france, centre hospitalier d'etampes, etampes, france, université versailles saint quentin en yvelines, versailles, france rationale-objectives. dyspnea is a major respiratory symptom, which can reveal a severe disease. additionally, it can also result from an inappropriate ventilator setting in mechanically ventilated patients. if these patients are nowadays more and more conscious, prevalence of dyspnea and its clinical, biological and radiological correlates has never been assessed in this population. prospective cohort study conducted in two medical intensive care units (icu) during months. all patients intubated more than h and conscious have been included. the first day when the patient regained consciousness, dyspnea, anxiety and pain were assessed using a visual analogic scale (vas). if dyspnea was found, patient was asked if he experienced ''air hunger'', and/or ''excessive respiratory effort'' and if dyspnea vas was improved after ventilator setting has been changed. demographic, clinical, biological and chest x-ray data and ventilator settings have been collected. results. patients were included (age: ± years; simplified acute physiology score ii (saps ii): (iqr - ). reasons for mechanical ventilation included acute respiratory failure (n = , %), neuromuscular diseases (n = , %), coma (n = , %), and exacerbation of chronic obstructive pulmonary disease (n = , %). dyspnea was present in ( %) patients and was qualified as ''air hunger'' in patients ( %), ''excessive effort'' in ( %) and both in ( %). age, saps ii, reason for mechanical ventilation, respiratory rate, clinical examination, x-ray chest, pao /fio ratio, paco were not statistically different between patients with and without dyspnea. anxiety . ); p \ . ), assist controlled ventilation [ . ( . - . ) ] and diastolic blood pressure ; p = . ) were independently associated with dyspnea in multivariate analysis. ''air hunger'' tended to be associated with controlled ventilation (p = . ) whereas ''inspiratory excessive effort'' was significantly associated with low inspiratory flow, severe hypoxemia (median pao /fio ratio: , p = . ) and marked hypercapnia (median paco : mmhg, p = . ). in % of breathless patients, of ventilator resetting decreased dyspnea. length of icu stay was greater in patients with dyspnea (p = . ) whereas extubation within three days and icu mortality did not differ between the two groups. conclusions. dyspnea is frequent in mechanically ventilated patients and is strongly associated with anxiety, more frequently when controlled ventilation is used and is often reduced after ventilator resetting. assessment of dyspnea in conscious mechanically ventilated patients should be routinely performed in order to improve patients' comfort. methods. an experimental study design was used with a group of first year health care provider students. the students were divided into two groups related to familiarity of the location of exam. a part of students (n = ) were examined in demonstration room (dr) and the other part of students (n = ) in public place (pp) . every student received the same number of training hours ( h) and the same training method in demonstration room. during this exam the students performed a min long, single person cpr related to erc guideline. their performance was measured with calibrated ambu cpr software and the adapted point system of brendan b. spooner's scale. v and t test were used for comparison. p values less than . were considered statistically significant. we did not find difference between dr and pp groups in the correct sequence of bls steps, hand position, adequate frequency and depth of chest compression. between groups of characteristics of ventilation were not significant differences observed. it is first critical point in bls process to assess the quality of patients' spontaneous breathing; therefore it is crucial that the duration of check breathing may be sufficient long. the duration of checking for breathing was significantly (p = . ) shorter in dr groups than pr groups. in the pp groups time interval between chest-compression cycles were significantly (p = . ) longer-more than s-than in dr group. conclusions. the altered location of bls final exam shortens duration of checking for breathing which determines bls providers' decision making on starting chest compressions. the students may be full of confident in the well-known place represented by the shorter time of checking for breathing. the changed place of exam extended time interval between chestcompression cycles, therefore weaken the continuity of chest compressions, and decrease the chance of return of spontaneous circulation. aims. this paper reports an evaluation of the student experience of using a clinical competence assessment tool (ccat) in postgraduate critical care nursing education. the focus is on the perceptions of students in relation to the validity, reliability and usability of the assessment tool. the domains of competence assessed are based on five domains outlined by an bord altranais ( ) . they are: professional/ethical practice, interpersonal relationships, practical and technical skills, utilising a holistic approach to care, clinical decision making and critical thinking skills and organisation and management of care. the assessment process encompasses three clinical assessments and clinical competence is measured using the developmental process of novice, advanced beginner, and competent as described by benner ( ) . students are asked to reflect on their own learning needs prior to each assessment. the assessment includes a discussion on the knowledge that underpins practice thereby showing the integration of theoretical and practical knowledge. questionnaire was administered to all students who recently completed a graduate diploma in nursing studies (critical care) at a specific third level institution. results. the evaluation of the ccat as a mode of competence assessment in postgraduate critical care nursing education was generally positive from the students' perspective. some students considered the holistic nature of the ccat document to be a limitation, suggesting that their level of competency could have been better addressed with a tool that was more oriented toward critical care rather than being so 'broad' in nature. overall respondents considered that the ccat helped them to identify learning needs and found the use of the tool to be a positive experience and easy to use although some respondents considered that the wording of some of the sub-domains and indicators was difficult to interpret. competence assessment is about ensuring the delivery of safe and competent patient care. in order to determine competence a valid and reliable tool is needed. this small scale study presents the views of post registration critical care nursing students on using a competence assessment tool. the findings of this study cannot be generalised, however they do provide insight for educators and students using competence assessment tools in programmes preparing registered nurses for specialist nursing practice. the use of a holistic assessment process needs further explanation. students need to be encouraged to move away from the reductionist approach, which is focussed on tasks and move towards a broader understanding of competent practice. reference (s) . . an bord altranais ( ) requirements and standards for nurses registration education programmes, nd edn. . benner p ( ) from novice to expert excellence and power in clinical nursing practice. addison-wesley, california. to assess the usefulness of a web-based interactive learning package designed to supplement an undergraduate acute care course (very basic) taught to final year medical students. a web-based interactive learning package was developed to supplement a highly rated traditionally taught -day acute/critical care course consisting of pre-course reading, lectures, skill stations and interactive tutorials [ ] . the additional web-based package consisted of narrated lectures, interactive lessons, videos and animations to demonstrate practical procedures and clinical signs, self assessment quizzes and a question and answer forum. topics covered included arterial blood gas sampling and interpretation, acute metabolic disturbances, non-traumatic coma, acute respiratory failure and sepsis. both the package and the course are available to other medical schools free of charge. usefulness of the package was assessed by examining activity logs, a student questionnaire, formal focus group (conducted by an investigator not involved in course preparation or teaching), comparing the results of a post-course mcq based summative assessment with historical controls and comparing results of formative assessment included in the package with the summative assessment. results. over , student-activities were logged by students during the two week course. students completed the questionnaire. with regard to usability, [ % agreed or strongly agreed that interactive lessons and self assessment ran smoothly without faults, with a corresponding score of [ % for narrated lectures and ease of browsing. with regard to usefulness, c % agreed or strongly agreed that the question and answer forum was useful in clarifying areas of doubt and narrated lectures improved understanding of the course material; [ % agreed or strongly agreed that the content as a whole was useful in preparing the respondent to work as a doctor, interactive lessons improved their understanding of how to apply their knowledge, and their understanding of arterial blood gas interpretation and self assessment exercises improved their understanding of the course material. participants in the focus group indicated that the resources provided in the website were useful for learning, specifically the animations, narrated lectures and the question and answer forum. suggestions for improvement included improving the quality of the video and animations, increasing the range of topics covered and ensuring consistency with the printed course manual. there was no correlation between formative and summative assessments but, compared to historical controls, performance in the summative assessment improved ( vs. %, p \ . ). conclusions. the package provided a useful supplement to a traditionally delivered acute care course. introduction. faculty development refers to that broad range of activities that institutions use to renew or assist faculty in their roles. it includes activities that improve an individual's knowledge, skills and attitudes in important areas in teaching, education, research, leadership, administration and career development. in this abstract we will introduce one of the most important methods of faculty development programs. a meeting by the authors ''organizing group'' was conducted to decide on a topic for our workshop and discussed the planning and designing process. we decided on conducting a workshop on clinical teaching methods. a scientific and organizing committee was established, and accordingly work loads and assignments were distributed among them. we gave this workshop a title of ''i am the best clinical educator…are you!? our target audience was acute care management providers, with a capacity of up to participants. we gave a specific time and location of this event. venue was arranged. computers for group work, audiovisual and other logistics were provided. after summarizing the main points for the workshop the organizing committee distributed an invitation letters throughout the higher management and educational leaderships. an address remark was done through invitation from the organizing committee. hot and cold beverages and break lunch meals were provided. posters on the workshop were distributed through out the institution. folders with educational materials were provided for each candidate. pre-course registration was done. once the program for the workshop was finalized a reminder was sent out to the participants on the date and venue for the workshop. participants attended on time, folders, badges with usbs were handed out. a questionnaire was distributed to the audience to estimate their learning experiences and approaches towards teaching styles and methods which were used in their practice. certificates of attendance with cme credit hours were distributed. results. candidates attended this faculty development workshop. % were nurses and % were physicians, during this workshop, three topics were distributed over three groups, one group on how to break bad news. second group about how to conduct microteaching and the third group about how to give feedback. each group was evaluated by three members of the organizing committee, each group was ranked accordingly. all were performed by role play. at the end of the workshop an evaluation form was filled % responders. a five performance scale was used. the strength of the workshop was innovativity and ranked as strongly agree. the only weakness was the place constraint. conclusion. we concluded that a well organized workshop using role play, interactive sessions are effective modality for faculty professional development programs among acute care providers with high satisfaction rate. only of the respondents ( %) indicated they did understand the statistics they encountered in journal articles and % felt it was important to understand these concepts and that they would like to access more easily to biostatistics training. a patient is referred to a higher centre when services are needed to maintain continuity of care.there are guidelines for the safe inter and intrahospital transport of critically ill patients but no guidelines are available for the minimal mandatory content of interhospital referral notes of critically ill patients.this problem is manifold in developing countries. objective. to educate the critical care physicians regarding the deficits in the physicians referral notes with which critically ill patients are referred from one centre to another. it is a prospective observational study on out of hospital referred patients transferred to our intensive care unit (icu) over a period of year. after permission from the institutes ethical committee we reviewed the referral summaries of these patients at the time of icu admission regarding the information available of clinical details, course in the previous hospital and therapeutic interventions. patients with more than h of hospitalization before transfer were included in the study. introduction. in japan, closed icus have been gradually increasing at university hospitals. a closed icu is necessary for a university hospital not only for the hospital activity but also the education of medical students and the training of fellows. they can learn how to manage the circulation and respiration status of severely ill patients in icu. it is indispensable for effective education to ensure sufficient proper icu staffs. but the present condition of our country is that there are not so many intensivists enough to perform both of clinical duties and education of students and fellows. each icu of university hospitals is endeavoring to increase the number of intensivists. one of the popular methods is the announcement on web site to promote interest of young fellows. regrettably, the homepage of the japanese society of intensive care medicine has no such specific pages. each icu of university hospitals has to create attractive its own pages in the homepages of the hospitals. [ ] . most subjects are taught using lectures and group tutorials and the theory is applied in clinical areas to facilitate greater understanding of the newly acquired knowledge. there is no reported best practice mechanism for teaching medical ethics in a practical setting to medical students. objectives. the routine use of an ethical checklist has been proposed as a tool for the medical team to consider ethical issues on critical care [ ] . its use as a tool for teaching medical ethics within critical care has not previously been reported. the aim was to use this checklist to facilitate learning providing clinical case material for discussion in daily tutorials. one medical student (sm) undertook a one week period of study to learn about ethics in critical care practice. the checklist was used to review patient notes, guide further discussion with patients, when observing the professional behaviours and communication of the multidisciplinary team, and as a guide for case based discussions. results. the complexity and severity of patient conditions in critical care makes it the ideal setting for learning about ethics. sm considered more ethical dilemmas in this practical attachment than in the previous years of clinical placements. the checklist allowed identification of possible ethical issues relating to each patient and a deeper understanding of the patient's health care needs. it was used for daily tutorials to discuss the ethical principles and observed professional behaviours in a similar way to a discussion of clinical diagnosis and management of a patient case with a supervising doctor on a normal clinical attachment. complex issues such as capacity to consent, end of life treatments or resource allocation were seen in relation to ongoing care. on ward rounds it was observed that their conduct in an open environment could at times potentially compromise patient confidentiality. there was also a benefit from the consideration of ethics issues in a real time basis which allowed exploration and reflection on personal moral or spiritual beliefs and how they may differ from those of the patients and other medical professionals. conclusions. using an ethical checklist allowed application of theoretical lecture and workshop material to real life situations. by discussing the cases and observed behaviours with a senior critical care doctor it is possible for trainee staff to appreciate how difficult medical management decisions are made, and to improve the acquisition of the skills necessary to start to assess and discuss ethical issues surrounding a patient's care confidently. introduction. accurate data on patient's weight and height are important for management in intensive care units (icus). unfortunately, weight beds or bed scales are not available in a significant number of icus and these variables are often estimated by health care personnel. the accuracy of such estimations is poorly described. objective. to investigate the accuracy of visual estimation of weight and height in critically ill patients. methods. prospective study conducted in a -bed mixed medical and surgical icu. patients were consecutively weighed by an unblinded physician with a stretcher scale (t metric), and measured by a physical therapist using a measuring tape. the ideal weight was calculated using the ardsnet's formulas for predicted body weight. medical staff (ms), internal medicine resident (imr), nursing staff (ns), physiotherapist (pt) and nutritionist (nu) were asked to estimate patient actual weight, ideal weight and height. they were blind to the estimations during all the protocol. estimations in each healthcare group were computed as means, medians and percentage of error from actual and ideal weight and height, respectively. anova test was used to compare mean estimations between the groups. there were no significant differences between the groups in estimation of either weight (p = . ) or height (p = . ). conclusion. weight estimations from healthcare personnel are often inaccurate. there are no significant differences in accuracy between the estimations of weight and height in different healthcare groups. an effort should be made to weigh all critically ill patients. intraabdominal hypertension (iah) is often diagnosed in icu and it can lead to abdominal compartment syndrome, multiple organ failure and death [ ] . in clinical setting biochemical signals based on which iah is considered severe or detrimental on visceral tissues are scarce. currently, the only clinically relevant signal is decreasing hourly diuresis. in an attempt to find an early sign of metabolically relevant signal on clinically marked iah we investigated abdominal wall metabolite concentrations. previously high lactate/pyruvate has been detected in dialysate from rectus abdominis muscle (ram) in animal models of iah [ ] . in the present experiment we hypothesized that laparoscopic surgery which induces iah could lead to clinically significant increase of l/p ratio as a signal of anaerobic metabolism caused by iah and insufficient tissue perfusion. introduction. among the techniques proposed to assess microperfusion and oxygenation, nirs sounds to be convenient [ ] . if baseline measurements do not provide useful information for outcome of micro-vascular impairment, functional evaluation using vascular occlusion test (vot) seems to be promising [ ] . technological development of the nirs device (inspectra models and , hutchinson technology, hutchinson, minn) proposes to use a new probe measuring hemoglobin saturation at less depth than previously ( vs. mm between fiberoptic) with more data output ( value/ s vs. value/ . s) associated with an automated software to compute occlusion and reperfusion slopes. objective. to compare nirs results obtained, using the two different probes, at day of septic shock (ss) in two groups of patients having similar clinical characteristics. methods. patients (g ) and patients (g ) were included within the first h of ss. macrohemodynamic: heart rate (hr), mean arterial pressure (map), central venous pressure (cvp), cardiac output (co) and svo (mixed venous o saturation), ph, base excess, and lactate were collected as saps ii and sofa scores. baseline sto at thenar eminence was continuously monitored and a min upper arm(brachial artery) vot was performed. sto occlusion and reperfusion slopes were calculated manually in g (probe mm) using linear adjustment (r c . to be valid) or calculated by the software in g (probe mm) using the same method, p \ . was considered significant. results. median ± iqr. the two groups did not differ for macrohemodynamic nor for metabolic data (table ) . nirs data surprisingly were largely significantly different between the two groups for both baseline and slopes ( background. hypovolemia and hypovolemic shock are life-threatening conditions that occur in numerous clinical scenarios. near-infrared spectroscopy (nirs) has been widely explored, successfully and unsuccessfully, in attempt to function as an early detector of hypovolemia by measuring tissue oxygen saturation (sto ). in order to investigate the measurement site-and probe-dependence of nirs in response to hemodynamic changes, such as hypovolemia, we applied a simple cardiovascular challenge; a posture change from supine to upright, causing a decrease in stroke volume (as in hypovolemia) and a heart rate increase in combination with peripheral vasoconstriction to maintain adequate blood pressure. methods. multi-depth nirs was used in nine healthy volunteers to assess changes in peripheral vascular tone in the thenar and forearm in response to the hemodynamic changes associated with a posture change from supine to upright. a posture change from supine to upright resulted in a significant increase (***) in heart rate. thenar sto did not respond to the hemodynamic changes following the posture change, whereas forearm sto did. in the forearm, sto was significantly lower (***) in the upright position with respect to the supine position. conclusion. the primary findings in this study were that ( ) forearm sto is a more sensitive parameter to hemodynamic changes than thenar sto and ( ) cerebral hyperperfusion syndrome, caused by inflow at normal blood pressure into maximally dilated fine vessels, is a recognized complication of carotid endarterectomy (cea) strict blood pressure control in the early postoperative period can minimize the risk of cerebral hyperperfusion. until yet, diagnosis of cerebral hyperperfusion mainly relies on intermittent postoperative examinations (spect; ct angiography). non-invasive absolute cerebral oxygen saturation (scto by fore-sight technology) was validated to jugular bulb saturation (sjo ) monitoring with a constant difference of % higher for scto values. previously, sjo monitoring after severe head injury indicated cerebral hyperemia. in this study, we evaluated scto monitoring after carotid surgery as possible continuous on-line monitoring of cerebral hyperperfusion. fourteen pts scheduled for cea were monitored for h postoperatively after cea. bilateral scto monitoring was started before induction of anesthesia and maintained until h postoperatively. intra-operative eeg monitoring guided the decision to intraluminal shunt insertion. strict blood pressure control was applied at maintaining normotensive levels throughout the clamping procedure. early postoperative care focussed on strict maintenance of normotensive blood pressure. in no pt, any change in eeg was observed after carotid clamping. in all pts, ipsilateral scto significantly decreased after carotid clamping, without any scto value below %. we observed no changes in contralateral scto . mean clamping time was min ( - min). in all pts, clamp release restored ipsilateral scto to baseline values. in all pts, emergence from anesthesia was uneventful, without any new neurological deficit. in of pts, significant increases (scto [ %) in ipsilateral scto were observed in the postoperative period (m scto . %), without any changes in contralateral scto . this increase occurred at a mean of . h after carotid declamping with a mean duration . h. in these pts, we could not make any significant correlation to arterial blood pressure, as none of these pts needed more aggressive antihypertensive control. we noted that of these pts suffered from diabetes mellitus, while of pts revealed high ([ %) contralateral stenosis. further data will have to reveal the importance of these comorbide factors. non-invasive cerebral oximetry, enabling absolute cerebral oxygen saturation monitoring, could provide on-line estimation of cerebral perfusion state after cea. this could allow bedside detection (and eventual therapeutic interventions) of cerebral hyperperfusion after cea. introduction. analysis of microcirculatory alterations obtained by side-stream dark field (sdf) is time consuming. automated analysis with modern softwares could accelerate this process and help to quantify blood flow velocity. however, perfusion detection is based on the contrast between pixels and this may be influenced by image settings. objective. we aimed to compare data obtained with a new software to the traditional semi-quantitative analysis of sdf images. methods. we selected from our database six images of poor sublingual microcirculatory perfusion and six images of good microcirculatory perfusion registered by the sdf technique (microscan; microvision medical, amsterdam, the netherlands). the proportion of perfused vessels [ppv = (number of vessels with continuous flow/number of all vessels) ] \ % was used to define microcirculatory perfusion. total vessel density (tvd) was determined automatically by the software ava . (microvision medical) and also by the semi-quantitative technique, considered as the gold-standard (number of capillary crossing three equidistant vertical and horizontal lines divided by the total length of these lines). ava software was also used as default definitions or set to optimize analyses according to manufacturer instructions. vessels falsely detected (false positive = fp) or missed (false negative = fn) by the software, in comparison to the semi-quantitative evaluation, were also counted. results. tvd was significantly higher by the ava software either on default or on optimized mode than by the semi-quantitative method, and these differences were present with good or poor perfusion images (table ) . overall fp rate was %, and it was greater in poor perfusion images ( %). optimization of the ava set parameters attenuated fp rates both in poor and good perfusion images, at the expense of increasing fn rates (table) . due to intrinsic characteristics of the software, the mean total grid length was significantly lower in the ava than in the semi-quantitative analysis ( . vs. . introduction. perfusion index (pi) is the proportion of constant absorbed light compared to pulsatile absorbed light emitted from a pulse oxymeter. it ranges from a value below up to depedant of peripheral perfusion. it is measured primarily to evaluate the signal quality for the pulse oxymeter and is displayed by some pulse oxymeters to be acknowledged by the clinician. the pi changes with vasodilation and vasoconstriction. however, intubation is a stimulus able to increase endogenous catecholamines and thus leading to vasoconstriction possibly declining the perfusion index. therefore we found intubation with a double lume tube in a thoracic surgery setting as a suitable setting to evaluate changes in perfusion index as a reaction to intubation. after informed consent, we enrolled seven patients undergoing lung surgery requiring an double lume tube. they were monitored as it is standard of care in our institution with invasive blood pressure, ecg, and a pulse oxymeter displaying the pi. (radical , masimo, irvine, ca) the patients received the medication to induce anesthesia calculated adequately to their body weight. midazolam, propofol and fentanyl where used to anesthetize the patient, cisatracurium was used for muscle relaxation to facilitate intubation. pi, pulse and arterial saturation were recorded every minute from prior to induction until after successful intubation. a baseline value was recorded prior to induction and compared to the value minutes after induction. then the pi measured next to intubation was compared to the pi after induction and analysed using students t test. introduction. anticoagulation strategies for albumin dialysis suppose a difficult compromise between risk for bleeding and a high tendency to clot in the circuit. even thought the sessions are short, a premature clotting is a serious event because the lost of blood (high priming volume) and a high cost of the systems. we intended to demonstrate that the classical approach based in heparin is not adequate in these patients and should be substituted for a different strategy (mixed low dose of heparin plus epoprostenol). methods. data of a prospective registry of all cases treated in our centre (a third level, teaching hospital) with albumin dialysis (mars system). initially we used non-fractionated heparin at - u/(kg h) in patients without coagulation problems, epoprostenol [ - ng/ (kg min)] in cases with risk or thrombocytopenia and no anticoagulation when high risk for bleeding or contraindication for anticoagulation. after an intermediate analysis of our registry we detected a high number of filters clotted when heparin was used and changed our approach to use as first indication a mixed protocol with non-fractionated heparin [ u/(kg h) ] plus epoprostenol [ ng/(kg min) ]. data are presented as percentages. analysis was performed with chi-square test. to detect variables related to coagulation a stepwise backward logistic regression analysis was performed. we registered patients with a total of sessions. selecting only the first session for each patient to validate the first choice for anticoagulation, we used heparin in cases and detected the loss of filters ( . %) because clotting. after the change to mixed anticoagulation we used this as first indication in patients and in only ( . %) the sessions were prematurely ended because clotting (p ns). the rest of patients received isolated epoprostenol in cases (with - %-cases of premature clotting) and no anticoagulant in five cases (with - %-premature clotting). between the cases with heparin as first choice, three episodes of mild and one episode of severe bleeding were detected while no patients in the mixed group presented bleeding complications (p ns). in a logistic regression analysis over all registered sessions using coagulation of filters as dependent variable and type of patient, anticoagulant, arterial pressure, inr, tpta, platelets, haematocrit or bilirubin as independent variables, none of these was included in the regression model. even though more studies are necessary to validate this conclusion, a mixed protocol based in low dose heparin plus epoprostenol could be adequate as first indication for non-complicated patients submitted to a mars treatment with lower risk for bleeding than the classical approach of isolated non-fractionated heparin. optimizing oxygen delivery in critically ill patients is vital for the promotion of aerobic cellular metabolism. current practice includes the measurement of variables such as partial pressure of arterial oxygenation (pao ), cardiac index (ci) and percentage of oxygenated haemoglobin in arterial blood (sao ). these parameters reflect global oxygen delivery. the real point of interest is the end point of the oxygen cascade; oxygen utilisation in tissue mitochondria. near infrared spectroscopy (nirs) has been developed in an attempt to measure tissue oxygen saturation (sto ) in peripheral muscle microcirculation. manufacturers state normal values as ± %. it uses four wavelengths near the infrared spectrum ( - nm) to measure sto , a ratio of oxygenated haemoglobin to total haemoglobin. it is continuous and non-invasive. sto has proven efficacious in predicting oxygen delivery in trauma patients and claims to have been successfully used to guide early resuscitation [ ] . objectives. we were interested in assessing whether sto had a role in measurement of oxygen delivery in the intensive care population, and how it compared to the parameters currently used to predict oxygen delivery. we had particular interest in the usefulness of nirs in septic patients, where the pathophysiology of tissue oxygen utilization is disrupted. patients from a general, adult intensive care unit were enrolled over an month period. all patients had lidco monitoring. exclusion criteria were gtn, atrial fibrillation and patient refusal. mm sto probes were sited on the thenar eminence. serial recordings of sto , cardiac index, hr, sao , map, and pao were recorded. sto results were compared to more traditional parameters of oxygen delivery. sixteen patients were recruited, all met criteria for sirs and shock. four were excluded with incomplete data. results were analysed for individual patients and as a collective series. we found: • no statistical correlation between nirs and sao or pao . • a weak and clinically insignificant correlation between cardiac index and nirs (p \ . ). • supra normal nirs readings (normal [ %) were not infrequently gained in patients where all other parameters were indicating severe shock and poor oxygen delivery. conclusion. theoretically nirs has potential to be beneficial in measuring oxygen delivery. our results demonstrate that nirs is not accurate for our septic population. we found poor correlation with current methods used to predict oxygen delivery and it may well be more misleading than beneficial. more traditional methods of intensive care monitoring, although sometimes invasive, appear to provide a more accurate representation of a patient's oxygen delivery. background. urine output is a crucial parameter of renal function and fluid balance. conservative urine output monitoring harbors problems such as subjective reading, sampling time errors and nursing workload. an electronic urine collection device was introduced into the icu and connected to a computerized information system. this created a more reliable and accurate means for urine output monitoring and the ability to develop new calculated parameters. . to evaluate the effects of introducing an electronical urine collection device into a fully computerized icu. . to evaluate new parameters that were created by the combination of the device and a computerized data management system. patients included were all admitted to the icu at rambam medical center, haifa, israel, during the years - . urine production and flow were monitored continuously by the urinfo Ò device (med-dynamix, israel), a novel electronic urinometer, connected to a patient data management system (imdsoft, israel). graphical analysis of urine production was done and derived parameters continuously calculated. comparison was done to the conventional mechanical urine collection system. variables studied were: measurement accuracy, sampling time accuracy, nursing workload before and after the implementation process. correlation between derived parameters and conventional renal function measurements such as plasma creatinine and creatinine clearance time. results. the conservative urine output measuring system demonstrated percentage error span in range of - %, compared to a range of - % percentage error in measurement after implementation of the computerized system. before implementation, sampling time error span was found to be - min, while no sampling time error was present after implementation due to the automated recording system. time consumed by the workload of the conservative urine output monitoring system was measured at - min per nursing shift ( h). the computerized system eliminated this workload completely. derived parameters evaluated were continuous urine flow (in cc/min or cc/h), urine production acceleration rate (calculated via the slope of the ''up-rise'' in cc/min ) and the peak urine production rate (cc/min). these parameters were able to demonstrate immediate changes in renal function, hours before conventional measurements and calculations would show them. conclusion. implementation of a computerized urine monitoring system can lead to improved accuracy in renal function monitoring and eliminate a significant amount nursing workload. use of derived calculated parameters may lead to earlier detection of renal malfunction and thus lead to earlier intervention. ( g/ , ml), cica dialysate k tm (na mmol/l, k . mmol/l, mg . mmol/l, cl . mmol/l, hco mmol/l, glucose anhydrous . g/l, ph * , ) and calcium chloride mayrhofer tm cacl , mol/l. the filter was an ultraflux av s tm , the material of the bloodline tubing system was medical grade soft pvc. in three circuits used in two different patients we found an opaque white precipitation starting at the cacl side port growing along the line with the direction of the bloodflow up to a maximal mm wide and mm long stripe. to identify the composition of the white stripes we included histological examination of hematoxylin-eosin stained sections and lyophilisation with wet chemical analysis. blood samples were simultaneously taken from the venous port of the cvvhd circuit and the arterial line of one patient. results. histology showed **an organic material in form of calcific deposit, covered with coagulated blood. chemical analysis identified this deposit as calcium phosphate. the results of the blood samples are shown in table . calcium phosphate precipitates may have reached patient circulation and been deposited in the capillary bed of the lungs or other organs. no histological examinations of tissue were taken and adverse events could not be attributed to the described phenomenon. citrate anticoagulation was stopped and switched to combined heparin-epoprostenol sodium anticoagulation. conclusions. the combination of the fluids and materials used in this specific cvvhd circuit with citrate anticoagulation resulted in some patients in a detectable calcium phosphate formation in the circuit. physicians using the described setting should be aware of the phenomenon and stop citrate anticoagulation as soon as a deposit occurs. in vitro studies, using different compositions and concentrations of dialysate and substitution fluids and simulating different patient conditions (ph, ph, hb, alb,…) should clarify, which solutions could safely be used. in addition the material of the circuit should be investigated, since surface characteristics have been identified to influence the formation of a calcium phosphate layer [ ] . reference (s) objective. the aim of this study was to assess, in a medical population of critically ill patients, whether intraabdominal pressure at admission was an independent predictor for mortality and to evaluate the effects of intraabdominal hypertension on organ functions. all patients admitted to the medical icu of the hgu gregorio marañón over a period of days were studied prospectively. patients who fulfilled two or more risk factors for wsacs (diminished abdominal wall compliance, increased intra-luminal contents, increased abdominal contents and/or capillary leak /fluid resuscitation.) were included. iap was measured via a foley bladder catheter, according to the modified kron technique. data recorded on admission were the patient demographics with, acute physiology and chronic health evaluation ii score (apache ii), and type of admission; during intensive care stay, sepsis-related organ failure assessment score (sofa) and clinical concomitant factors and conditions. intraabdominal pressure were measured at least daily together with fluid balance. patients were followed throughout their hospital stay. forty-four patients were included in the study (age - , apache ii . . half were admitted for cardiopulmonary disease. twelve ( %) had pancreatic or gastrointestinal disease. twenty-two ( %) had severe sepsis or septic shock. the incidence of iah was %. mortality was %. the cause of the iah was capillary leak syndrome/fluid resuscitation in % of cases. there was no relationship between the presence of iah and the number of organ failure during admission. the only variables associated with mortality of the patients were sofa and apache ii. the presence of iah was not a factor associated with increased mortality, although these results may be confounded by sample size. conclusions. there is an unusually high incidence of iah in the population of critically ill medical patients with two or more medical risk factors for wsacs. however, unlike in other populations, our study does not demonstrate that the iap monitoring allow detecting a group at higher risk of developing multi-organ failure or death. background. drainage of ascitic fluid is a common practice in order to relief the respiratory discomfort of patients. the aim of the present study was to determine abdominal compliance after ascitic fluid removal by transcutaneous drainage. methods. twelve patients presenting with ascitic fluid were included. all patients had transcutaneous blind drainage with a wide catheter. the ascitic fluid removed was recorded, while the intraabdominal pressure (iap) was measured as proposed by wsacs. iap was measured before and min after the puncture. abdominal compliance (cabd) was calculated. results. the pre-drainage iap was . mmhg (ranging from . to . mmhg, sd . mmhg), while the post-drainage was . mmhg (ranging from . to . mmhg, sd . mmhg). the mean volume of ascitic fluid removed was ml (ranging from to , ml, sd ml). cabd after drainage was ml/mmhg (ranging from to ml/ mmhg, sd ml/mmhg). a linear correlation was found between ascitic fluid removal and iap variations. conclusion. the drainage of ascitic fluid reduces iap, facilitating in this way respiration. moreover, iap variation seems be in linear relation with the volume of ascitic fluid removed. this linear relation between iap and volume may probably predict the cabd quite accurately and vice versa. however, larger studies are necessaries in order to safely draw predicting diap-dv (cabd) diagrams, and determine the optimal ascitic fluid removal in order to achieve best comforting of the patient and slower fluid reformation. introduction. use of stroke volume variation (svv) to guide fluid therapy in preload responsive state has been studied well in patients undergoing cardiac or neurosurgery during anaesthesia. use of this dynamic monitoring variable has not been studied much in septic shock. we undertook this prospective study to evaluate utility of svv to optimize preload in patients with septic shock and ards. setting. bedded medical surgical icu of a bedded tertiary care centre in pune, india. inclusion criteria: ( ) patients with ards (po /fio b ), svv readings were taken every h with flotrac-vigileo system after confirming abolishment of spontaneous breaths by sedation or paralysis and increasing tidal volume transiently to ml/kg. fluid boluses were given to keep svv \ % for h after enrollment. attempts were made to reduce vasopressor doses keeping map c mmhg. results. patients with average age . ± . years and apache ii score . ± . were studied. each patient received an average . ± . l fluid in h after enrollment to keep svv below %. svv at h after enrollment was . ± . % improvement in microcirculation was evident as plasma lactate reduced from . ± . (at h) to . ± . mmol/l (at h) there was no worsening in pulmonary edema as po / fio increased from . ± . (at h) to ± . (at h) only out of patients needed renal replacement therapy. in patients, vasopressors could be stopped completely in . ± . h. of them survived till discharge from the icu and died of ards. in patients, vasopressors could not be weaned off completely and all of them succumbed. overall survival rate was %. conclusion. svv guided fluid therapy is a promising modality for pre load optimization in mechanically ventilated patients with septic shock and ards. introduction. cardiovascular function is an important determinant of outcome in sepsis, and heart rate (hr) has been associated with cardiovascular risk and mortality in large patient cohorts [ ] . to investigate the association between hr and or day mortality in septic shock. methods. this study is a post hoc analysis of septic shock patients who were included in the control group of a multicenter trial [ ] . demographic and clinical data, average hr and catecholamine requirements during septic shock, occurrence of acute circulatory failure, and day mortality were documented. a binary logistic regression model adjusted for the simplified acute physiology score ii (excluding hr) was used to investigate the association between mean hr and acute circulatory failure or / day mortality. a multiple logistic regression model was applied to identify independent risk factors for developing hr critical for outcome. conclusions. hr is associated with and days mortality in septic shock. hr persistently exceeding bpm during septic shock seems associated with a significant risk of death. introduction. different colloids can be used for treatment of hypovolaemia in septic pts. recently, small-volume resuscitation was introduced for initial therapy of severe hypovolaemia and shock. the concept of small-volume resuscitation encompasses the rapid infusion of a small dose of . % nacl/colloid solution [ ] . however, in septic pts hypovolaemia often associates with acute lung injury (ali). therefore in these pts great importance has influence of colloids on oxygen transport. objectives. the aim of the study was to evaluate and compare the effects of hhes and hes on oxygen transport in pts with sepsis and ss. methods. hypovolaemic pts with sepsis and ss were enrolled in the study. pts received - ml/kg ( ml) hhes ( . % nacl ? % hes) (fresenius kabi) within min and pts received hes / (voluven, fresenius kabi) ml/kg. in all pts before and after infusion the parameters of oxygen transport was measured by pulmonary arterial catheter and transpulmonary thermodilution (pulsion medical system). after infusion of hhes oxygen delivery index (ido ) increased because of increase of cardiac index (ci) despite of decrease of hemoglobin (hb) levels and absence of changes of arterial oxygen content. extravascular lung water (evlw) and shunt increased significantly immediately after hhes infusion, but this increase was not accompanied by deterioration of pao /fio . introduction. severe sepsis is characterised by a wide array of haemodynamic changes including increased capillary leak, vasodilatation, vascular hyporeactivity and myocardial depression. the resultant tissue hypoperfusion is an important catalyst of multi-organ failure [ ] . to further develop our understanding of the underlying mechanisms, we have developed and characterised a fluid-resuscitated mouse model of intraperitoneal polymicrobial sepsis. objectives. to assess alterations in cardiac performance in mice at , , and h following faecal peritonitis. methods. sepsis was induced in week old male mice (n = ) by intraperitoneal (i/p) injection of dilute faecal slurry. sham animals (n = ) received n-saline i/p. animals were fluid resuscitated at time ( ml/kg . % saline), and at and h ( ml/kg . % saline- % dextrose each time). under a minimum concentration of isoflurane to achieve light anaesthesia, peak velocity, stroke distance, heart rate and fractional shortening were measured in the short axis plane by echocardiography at the , and h timepoints. in separate sham and severe septic mice (n = per group) the cardiac response to intravenous colloid boluses was assessed at and h. results. we clinically characterised septic animals into 'mild' and 'severe'. mice with severe sepsis showed a % drop in peak velocity and cardiac output at h (vs. and % falls in the mild septic and sham-operated animals, respectively, p \ . ). while mild septic animals showed recovery by hr, cardiac output in severely ill mice remained significantly depressed (due to both low heart rate and stroke volume) compared to mild septic and sham animals [*p \ . ( fig. ) ]. stepwise . ml boluses of intravenous fluid at h in severe septic animals led to restoration of cardiac output to baseline ( h) values. however, in the h septic animals, fluid challenge produced an initial improvement in cardiac output followed by deterioration [ fig. purpose. myocardial dysfunction has been well-documented in sepsis even in hyperdynamic state, and may develop and contribute to morbidity and mortality. nicaraven, a radical scavenger, has been shown to protect the coronary endothelial and myocardial function from ischemia and reperfusion injury due to hydroxyl radical scavenging activity. the purposes of present study were to determine the effects of nicaraven on cardiac function and cytokine production in lipopolysaccharide (lps) induced sepsis. methods. this protocol was approved by our institutional committee. following arterial and venous cannulation and tracheostomy, rats ( - g) were anesthetized with pentobarbital, and mechanically ventilated with a control mode (v t = ml/kg, rr = rpm). after baseline measurements, rats (n = ) were administrated with lps ( mg/kg, intravenously) and randomly assigned to following two groups: the nicaraven group treated with nicaraven [ mg/(kg min), intravenously] and the control group treated with saline. the left ventricular pressure and volume were measured with the pressure and conductance catheter every one hour. cardiac function, including cardiac output (co), ejection fraction (ef), and maximal elastance of left ventricle (e max ) were analyzed with a computer soft. blood was collected, centrifuged ( , g, min, ) , and stored (- °c) from rats every h after operation to measure plasma concentration of tnf-a, il -b and macrophage migration inhibitory factor (mif) using enzyme-linked immunosorbent assays kits. blood lactate concentration was also measured. data were analyzed by repeated measure anova. results. the co in the nicaraven group was kept significantly higher than the control group (p \ . ). the ef and e max in the nicaraven group were also kept significantly higher than the control group (p \ . ). arterial lactate, tnf-a, il -b and mif were significantly lower in the nicaraven group versus the control group (p \ . ). conclusion. the current study indicates that the treatment with nicaraven improved cardiac dysfunction and reduced plasma concentration of cytokines, and improved lactic acidosis in septic model. methods. this protocol was approved by our institutional committee. following arterial and venous cannulation and tracheostomy, rats ( - g) were anesthetized with pentobarbital, and mechanically ventilated with a control mode (v t = ml/kg, rr = rpm). after baseline measurements, rats (n = ) were administrated with lps ( mg/kg, intravenously) and randomly assigned to following two groups: the oxytocin group treated with oxytocin ( iu/kg iv and followed by the continuous infusion of mg/(kg min), intravenously) and the control group treated with saline. the left ventricular pressure and volume were measured with the pressure and conductance catheter every h. cardiac function, including cardiac output (co), left ventricular peak pressure (lvpp), and cardiac work (cw) were analyzed with a computer soft. blood was collected from rats every h after operation to measure plasma concentration of blood lactate. data were analyzed by repeated measure anova. results. the co in the oxytocin group was kept higher than the control group but there is no significance (p \ . ). the lvpp and cw in the oxytocin group were kept significantly higher than the control group (p \ . ). arterial lactate was significantly lower in the oxytocin group versus the control group (p \ . ). conclusion. the present study indicates that the treatment with oxytocin improved cardiac dysfunction and reduced plasma concentration of lactate in septic model. introduction. conventional hemodynamic monitoring parameters like heart rate, mean arterial pressure (map), and central venous pressure may be misleading in assessment of circulating blood volume in severely septic patients. inadequate blood volume may compromise renal blood flow leading to acute kidney injury (aki). stroke volume variation (svv) is a sensitive indicator of relative preload responsiveness and has high sensitivity and specificity when compared to conventional indicators of volume status and their ability to determine fluid responsiveness. to assess the efficacy of svv guided fluid therapy in preventing aki in patients with severe sepsis on ventilatory support. mechanically ventilated patients with septic shock who had undergone resuscitation based on surviving sepsis campaign guidelines and still requiring vasopressor support were enrolled. patients with pre-existing renal failure were excluded. a total of patients were randomized to receive fluid therapy according to conventional indices or svv, in the first h after mechanical ventilation. svv was measured with flotrac vigelio after abolishing spontaneous ventilation by sedation and paralysis if required. fluid boluses were given to keep svv less than %. vasopressor therapy was optimised to maintain map [ mm hg. patients were followed during their icu course with respect to development of aki, need for renal replacement therapy (rrt), length of icu stay and icu mortality. aki was diagnosed as per the rifle criteria. primary outcome measure was development of aki. results. patients in both groups were similar with respect to age (p = . ), sex (p = . ), and admission apache ii score (p = . ). incidence of aki was / ( . %) and / ( . %) in conventional and svv groups, respectively (p = . ). there was no statistically significant difference in terms of need for rrt, icu length of stay and icu mortality ( [ ] . moreover, pnu- a (pnu), an inhibitor acting through the poreforming subunit of the channel, did not affect bp in our awake peritonitis rat model [ ] . given that vasoconstrictors, including ne, inhibit k atp channel activity [ ] , we speculate that the high sympathetic tone seen in sepsis [ ] objectives. the goal of this study was to determine if hfav improves microcirculatory alterations in ss patients. methods. by using side dark field videomicroscopy (microscan Ò , microvision medical) we evaluated sublingual microcirculation in ss patients who according to our local protocol care [ ] underwent a h-hfav as rescue therapy for refractory septic shock. hemodynamic parameters and microcirculation were assessed at baseline, after h of hfav, and h after stopping hfav. microcirculation assessments were performed at to different sublingual areas ( - s/image). images were analyzed according to recent consensus [ ] by semiquantitative scores of flow (mfi, mean flow index and ppv, proportion of perfused vessels), density (tvd, total vascular density; pvd, perfused vascular density), and heterogeneity (het mfi) of small vessels (\ lm introduction. disturbances within the microcirculation represent an important factor in the pathogenesis of multiple organ dysfunction in sepsis and septic shock [ ] . gender-specific effects may modulate the septic pathophysiology [ ] . therefore, we studied sepsis-induced changes within the intestinal microcirculation in randomly cycling and ovariectomized female rats. objectives. we hypothesized that estradiol (e ) and dehydroepiandrosterone (dhea) may have a beneficial effect on the microcirculation during experimental sepsis and resubstituted these hormones in the ovariectomized animals. methods. fifty female rats were divided in to five groups of ten animals. group received sham laparotomy without further treatment. in group - we induced experimental sepsis (colon ascendens stent peritonitis-casp model). animals of groups - were additionally ovariectomized weeks before sepsis induction. in group we administered mg/kg estradiol immediately after and h following casp surgery. the animals of group received mg/kg dhea immediately after sepsis induction. twenty-four hours after casp surgery intravital microscopy was performed to study leukocyte-endothelial interactions and functional capillary density. blood samples were taken for the measurement of estradiol, dhea and inflammatory cytokines. results. in ovariectomized rats subjected to casp the number of activated leukocytes was significantly increased in comparison to sham and not ovariectomized casp animals (p \ . ). in ovariectomized rats treated with e leukocyte adhesion was significantly reduced in comparison to untreated ovariectomized rats subjected to casp (p \ . ). the same observation was made in ovariectomized rats treated with dhea. in addition, in ovariectomized rats subjected to casp the functional capillary density was significantly decreased in comparison to sham and casp groups (p \ . ). in ovariectomized rats treated with e or dhea functional capillary density was completely restored. the results demonstrate the role of e and dhea in the sepsis-induced changes within the microcirculation. a rapid, non-genomic effect of both e and dhea is suggested [ ] . dhea may play a role through conversion to e or through direct acting on the e receptor. further investigations should be done to elucidate the underlying mechanisms. both e and dhea appear to be a promising adjunct for the prevention and treatment of sepsis-induced multiorgan failure. liver is involved in the production of no. the aim of this experimental study was to evaluate the time course of hepatic no production at the onset of hypotension occurring during septic shock. methods. male wistar rats were anesthetized with isoflurane Ò , and mechanically ventilated. a first group (sepsis group) underwent a cecal ligature and puncture (clp) peritonitis, the second one (control group) a laparotomy only. animals were euthanized at different times: h after surgery, at shock onset, and h after shock. shock was defined by systolic blood pressure lower than mmhg. each rat of sepsis group was matched with rat of control group. liver perfusion was measured using a direct laser doppler flowmetry probe. no generated in the liver was measured using a pulse voltametric method. results. rats were studied ( in each group). in sepsis group, shock occurred at ± min after clp. in sepsis group, a significant decrease of hepatic perfusion was identified h after clp ( fig. ) whereas hepatic no production was increased only at the time of shock onset (fig. ). intra hepatic no production conclusion. this study shows a time shift between hepatic perfusion disturbance, hepatic no production and shock onset in a septic animal model. introduction. microvascular blood flow alteration is a key element of severe sepsis and septic shock [ ] . one study show that microvascular alterations in septic patients could be improved with a nitric oxide donor nitroglycerin [ ] . studies in human have shown that infusion of magnesium sulphate has endothelium dependent and independent vasodilation properties, increase of red blood cells deformability in specific conditions. we hypothesized that combination of nitroglycerin with magnesium sulphate and order of priority influence microvascular improvement in patients with severe sepsis and septic shock. methods. ten septic patients who had already been fluid resuscitated randomly assigned to one of two groups. one group received magnesium sulphate infusion g/h with nitroglycerin . mg/h infusion added after min. another group received nitroglycerin . mg/h infusion with additional magnesium sulphate g/h infusion after min. if required we added crystalloids and use norepinephrine. sublingual microcirculation was evaluated using side dark field videomicroscopy (microscanÒ, microvisionmedical) . each patient's microcirculation was evaluated by examining different sublingual areas ( - s/image). in all patients measurements were obtained at baseline, at and min. images were analyzed by semiquantitative scores of flow (mfi, mean flow index; ppv, proportion of perfused vessels) and density (tvd, total vascular density; pvd, perfused vascular density). capillaries were defined as microvessels with a diameter \ lm. data are presented as median values (percentiles ; ). . the median age of the patients was ( ; ) years. in both groups we see tendency progressively increase of pvd, ppv and mfi after drug alone and combination after min, but pvd has tendency to be higher [ . ( ; . ) n/mm vs. . ( . ; . ) n/mm , p = . ] after min. in group, where magnesium sulphate infusion was given first. combination of magnesium sulphate with nitroglycerin, when magnesium sulphate is given first, has tendency to higher potential for improving of microcirculation in severe sepsis and septic shock patients, but further studies are needed to obtain more detailed results. severe sepsis remains one of the leading causes of death in critical care, with around % of patients dying within one month of diagnosis. rapid diagnosis and therapy of sepsis improves survival [ ] . in november the whittington hospital introduced a hospital severe sepsis guideline, based on the first surviving sepsis campaign guideline [ ] . the sepsis guideline was published on the hospital intranet and specified actions to be completed within the first h of the diagnosis of severe sepsis or septic shock [ ] . objectives. to assess whether publication of a sepsis guideline on the hospital intranet, coupled with departmental educational campaigns, improved the management of severe sepsis. the whittington hospital is a university associated general hospital in london. we audited the early management of severe sepsis and septic shock before and after the introduction of the new hospital sepsis guideline. the 'before' phase comprised patients with severe sepsis or septic shock admitted to critical care between november and november . the 'after' phase comprised patients with severe sepsis or septic shock admitted to critical care between january and november , after introduction of the guideline. data was retrospectively collected from case notes and observation charts. the audit tool compared immediate, , and h actions following diagnosis against the hospital guideline. the main outcome measures were compliance and day mortality. compliance was defined as the average of the percentage compliance with each of the items specified in the guideline. results were compared by chi squared. compliance with the severe sepsis guidelines was only % after publication of the hospital sepsis guideline, compared with % before publication (p. )! there was similarly no significant difference in day mortality (before %, after %, p. ). publication of a sepsis guideline on the hospital intranet, coupled with departmental teaching sessions, failed to improve compliance with surviving sepsis recommendations, perhaps because the guideline competes for attention with over other guidelines on the intranet. next we will implement an interdepartmental educational programme to try and improve guideline compliance. as guidelines proliferate it is difficult to ensure they are followed, but failure to implement a published hospital guideline may represent a significant clinical and medicolegal risk. methods. the icu is an intensivist-led bed intensive care in a bed non-academic teaching hospital. hospital mortality from sepsis in icu-patients was . % in . patients are treated under modern icu conditioning, including continuous venovenous hemofiltration and a lung-protective ventilation strategy including prone position. an intensive insulin therapy protocol for glycemic control is used. in the period between march until june , we prospectively screened all patients admitted to the icu for (severe) sepsis, without the knowledge of the nurses and most of the doctors. all severe septic patients were included in our surviving sepsis database. after h, we examined how many targets of the resuscitation and management bundles were applicable and reached. in the period between march until june , patients were admitted to the icu. twenty-two of them were suffering from severe sepsis ( . %), of which had a septic shock. focus of the sepsis was abdominal in patients ( %), pulmonary in five patients ( %), urogenital tract in five patients ( %), meningitis in one patient ( %) and catheterrelated in one patient ( %). table shows us the applicability and achievement of the bundle elements. only in one of the patients all targets were reached. however, mean individual bundle element performance was . % (sd . ). all patients received fluid resuscitation when indicated, and all patients on mechanical ventilation were ventilated in a lung-protective manner with plateau pressures \ cm h o. only percent of patients had glucose levels within the target range. scvo was never measured, though it was indicated in patients. one patient had an apache iiscore c and had no contraindications for administration of activated protein c. treatment was not considered for this patient by the attending physician. of these patients suffering from severe sepsis, three died within days after the diagnosis ( . %). introduction. the surviving sepsis campaign (ssc) guidelines give a group of interventions (''sepsis bundles'') expected to improve the outcome of patients with severe sepsis [ ] . objetives: the aim of this study was to evaluate the impact of the implementation of the ssc guidelines on the mortality in our intensive care unit (icu). methods. prospective, observational study. during one year period (january -january ) the sepsis bundles were applied to each patient with severe sepsis-septic shock and they were followed up until discharge. we considered as ''time o'' (the time of delay of the implementation of the sepsis bundles) the time of admission of the patients in the icu. for each severe septic patient the following data was registered: time delay, apache ii and sofa scores at icu admission, diagnosis, the rate of compliance with the resucitation and management bundles, microbiological data, evolution of levels of serum lactate, empiric antibiotic therapy, length of stay and mortality in icu. the application of guidelines impact on mortality was compared with historical data years before implementation in our icu ( . %) and spanish icu ( . %) [ ] . a total of severe septic patients were included in the study. ( . %) patients had severe sepsis and ( . %) septic shock. the median age was years. the mean apache ii was . (± ) and sofa was . (± . ). the main sources of infection were abdomen ( %), lungs ( %), urinary tract ( . %) and soft tissues ( . %). the most common clinical diagnosis related to an episode of severe sepsis was peritonitis ( %). a microbiological diagnosis of the infection was reached in . % and the infections were mostly caused by gram-bacilli. once the antibiogram was obtained, the initial treatment was considered appropriate in . % patients. the rate of compliance with sepsis bundles was %. the length of icu stay was . days. mortality was . %. the implementation of the sepsis bundles decreased icu mortality significantly ( . % before implementation vs. . % after implementation). non survivors were older (median age ± . ), had higher apache ii (mean . ± ) and sofa (mean ± . ), % had septic shock, . % had negative cultures and an increased on the levels of serum lactate in h. age, apache ii and sofa scores and the increased on the levels of the serum lactate were useful tools to predict mortality. conclusion. implementation of the surviving sepsis campaign guidelines was associated with a reduction in icu mortality. introduction. the objective of this before-after study is to assess the impact of a protocol of care for severe sepsis in a french emergency setting. methods. two months periods were surveyed before and after the initiation of a protocol of care for severe sepsis and septic shock. after the control period (p : november -february ), a procedure for early recognition, aggressive treatment and standardized antibiotherapy of severe sepsis was initiated. a campaign to raise medical physicians and nurses awareness concerning this new strategy of care was performed. the intervention period (p : november -february ) assessed the impact of these actions. . patients with severe sepsis or septic shock were included during p ( % of patients with a suspected infection and . % of all non trauma admissions) and during p ( . % of patients with a suspected infection and . % of admissions). the age and the proportion of patients with co-morbidities were similar during the p and the p periods ( years in median versus years, and vs. %, respectively). and % of the patients lived in long term care facilities. severe sepsis and septic shock were correctly identified by the emergency team in / ( . %) during p and in / ( . %) in p (p = . ). the delay between the admission in the emergency department and the administration of antibiotics was in median equal to h min in p and h min in p (p = . ). adequate iv fluid resuscitation was administered to % of patients in p and % of patients in p (p = . ). during p , % of patients did not qualify for admission to the intensive care unit compared to . % in p . hospital mortality did not change from . % ( / ) in p to . % ( / ) in p (p = . ). conclusion. the introduction of a standardized treatment protocol in an emergency department allowed a better recognition of severe sepsis with earlier adapted treatment . the study was not powered to demonstrate a reduction of the mortality in this elderly population. multiple studies have shown that early detection and therapy is crucial for the prognosis of a severe septic patient. many hospitals have joined the surviving sepsis campaign and its fight for the decrease of mortality in severe sepsis and have implemented the severe sepsis bundles into their daily practice. other institutions such as ours had so far not taken this step, perhaps because the process is hard and time consuming. we have tried to find an easy way to audit the implementation of severe sepsis bundles and its change in time in an institution without a set system and database for the implementation of severe sepsis bundles to help us prove, that a systemic change in clinical practice is essential. we have decided to use the first step of the resuscitation bundle-the measurement of lactate and audit the lactate requests in blood samples with elevated inflammatory markers in our hospital laboratory information system. we retrospectively audited the number of lactate requests in blood samples with c-reactive protein (crp) c mg/l and its evolvement in time between and before and after the introduction of surviving sepsis guidelines and severe sepsis bundles in our regional hospital with beds. we compared the total number of blood samples with elevated crp c over mg/l with or without procalcitonin request in our institution with the number of blood samples with crp c mg/l and lactate request (both arterial and venous) in the hospital laboratory information system. . the total number of lactate requests in samples with crp c mg/l had increased in time, the incidence widely differed between departments. the main increase was in patiens from intensive care units, the number of lactate requests in samples from general wards, emergency department and intermediate (step down) units had also increased ( lactate requests in samples with crp c mg/l- , % in and lactate requests in samples- . % in ) but still remains insufficient. surprising was that procalcitonin was in non icu patiens with crp c mg/l requested more often than lactate. although many lectures and seminars on severe sepsis bundles and the guidelines for the management of severe sepsis were organised in our intitution between the year and , it was not sufficiently effective. conclusion. retrospective audit in the hospital laboratory information system of the number of lactate requests in samples with elevated inflammatory markers appears to be a fast and a very easy first step for auditing how the surviving sepsis guidelines and severe sepsis bundles are implemented in your institution. the results help to quantify the present state, its change in time and may serve as an impulse to make systemic changes in the system of early detection and therapy of septic patients. introduction. early goal-directed therapy (egdt) is the accepted gold standard for resuscitation in septic shock [ , ] . international guidelines for the treatment of septic shock [ ] set an initial h limit to accomplish this goal. to test the hypothesis that egdt with fluids and vasopressors has better patient outcomes if each intervention is completed within h. thirty septic shock patients from the spring of and from spring were reviewed prospectively (n = ). septic shock was defined as a lactic acid c mmol/l and/or hypotension unresponsive to fluids. apache ii and sofa scores were calculated. patients were subjected to the hospital septic shock protocol according to guidelines [ ] . firstly, egdt compliance was met if the following interventions were achieved within h: lactate levels drawn, map c mmhg and cvp c mmhg; and secondly, if antibiotics were given\ h, blood cultures were taken before antibiotics and if ml/kg fluid bolus was administered prior to vasopressors. in patients / interventions were performed in time (''egdt-compliant''). the other were deemed ''egdt-noncompliant''. outcomes were mortality rate and discharge destination. fisher test was used in statistical analysis. . mr was % amongst the compliant and % amongst the noncompliant and admission to long-term care facilities (ltcf) was and %, respectively. neither one of these differences was statistically significant. a power analysis revealed that patients are required to attain statistical significance for mortality. discharge home was the same in both groups. there was no difference between groups in the number of new tracheostomies or new hemodialysis. conclusions. in a us community teaching hospital, compliance with guidelines in the treatment of septic shock had a trend towards lower mortality and higher discharge rates to ltcf but the difference was not statistically significant. larger numbers are needed for the benefits/effects of egdt-compliant therapy to reach statistical significance in the treatment of septic shock in this hospital setting. improve the survival rate of septic patients by means of education and implementation of a sepsis operative protocol including the activation of a specific consultation by an intensivist and an infectious disease specialist (i.e. sepsis team, st). aim of this study was to describe the first months activity of st, with a focus on the patients not admitted in intensive care unit (noicu). methods. the sepsis operative protocol, introduced in clinical practice in june , provides for specific instructions for the early identification and management of septic patients and for the early activation of the st for patients with severe sepsis or septic shock admitted in non-intensive departments. the st consultation ought to support the departmental health personnel in the management of septic patient and allows an early intensive care admission in case of shock or if mechanical ventilation is needed. to assess st activity, we evaluated in noicu patients the correct st activation rate, the number of st activations for each patient, the rate of central venous catheter insertion (cvc) and the days mortality. results. from june to december , the st was activated for patients ( . patients per month) whose ( %) were admitted to icu and ( %) were considered too sick to benefit. in ( %) of the remaining patients, st was properly activated: patients with severe sepsis and with septic shock. thirteen patients ( %) had no sepsis and ( %) had sepsis without organ dysfunction. % of st activations originated from medical departments (including emergency department) and % from surgical departments. the number of st activations for each single patient was ± . the days mortality was . % in patients with sepsis, % in patients with severe sepsis and % in patients with septic shock. conclusion. the rate of correct activation of st and the number of activations for each patient were acceptable considering that more than % of the activations refers to septic patients and that a mean of activations was sufficient for patient management. mortality rates observed are slightly lower than those reported by others, but further data are needed to evaluate the impact of st on patient outcome. a. estella , l. pérez fontaiña , j. i. sanchez angulo , e. moreno hospital of jerez, emergency and critical care unit, jerez, spain clinical evidence suggests that an early diagnosis and treatment of severe sepsis has been shown to improve outcome. frequently the initial management of septic patients occurs outside of the icu. objective. to describe clinical characteristics and outcome of septic shock patients admitted in icu and to compare mortality according origin prior admission in the icu (emergency department versus medical or surgical wards). consecutive patients with septic shock admitted in icu from july to november were registered. age, icu length of stay, source of infection, isolated bacteria, blood lactate concentration, apache ii score and mortality were collected. patients were classified according the origin prior admission in icu. . consecutive septic patients were admitted in icu during the time of study, global mortality was %. patients were admitted from medical or surgical wards and patients from the emergency department. mean age was years, male and female, icu length of stay was . ± . days, the mean apache ii score at admission in icu was . ± . abdominal infection, . %, was the commonest source of infection followed by pulmonary and urinary infection, . and . % respectively. patients ( %) had a positive bacterial culture, the mean baseline lactate level was . ± . mmol/l p \ . ( . ± mmol/l in the medical and surgical wards group versus . ± mmol/l in the emergency department group).there were not differences in clinical characteristics according origin prior admission in the icu except for lactate level, and mortality, . % in the medical and surgical wards group and . % in the emergency department group (p \ . ). conclusion. there were not differences in clinical characteristics, icu length of stay, source of infection, isolated bacteria and apache ii score between groups. mortality was lower in the group of patients admitted in icu from the emergency department than the group admitted from medical and surgical wards. although very high circulating concentrations are detectable in plasma, it is not known which organs actually produce the cytokines. we hypothesized that key abdominal organs affected by sepsis such as the kidney and liver produce cytokines and tested this hypothesis by measuring cytokine flux. materials and methods. pigs ( - kg) were randomised to control (n = ) and endotoxin (n = ) groups. hemodynamic measurements using picco and pulmonary arterial catheters and arterial blood gases were collected hourly. portal, hepatic and renal arterial blood flows were measured with transit time probes. arterial and venous cytokine concentrations (tnfa, il- b, il- and il- ) were measured from samples taken from each respective organ. cytokine flux was calculated as: organ blood flow (venous-arterial cytokine concentration difference). endotoxemic pigs had significant increases in heart rate (p \ . ) and mean pulmonary arterial pressure (p = . ) and decreases in cardiac output (p = . ). in contrast, these hemodynamic variables remained stable in the control animals. renal, hepatic and portal vein flows decreased significantly in all endotoxemic animals but remained stable in the control group. renal [ml/(kg min)]:control . ± . , . ± . , . ± . , . ± . versus endotoxin . ± . , . ± . , . ± . , . ± . for baseline, t = , , , respectively. portal [ml/(kg min)]: control . ± . , . ± . . . ± . , ± . versus endotoxin . ± . , . ± . , . ± . , . ± . for baseline, t = , , , respectively. hepatic [ml/(kg min)]: control . ± . , . ± . , . ± . , . ± . versus endotoxin . ± . , . ± . , . ± . , . ± . for baseline, t = , , , respectively plasma cytokines tnfa was detectable in very low concentrations (\ pg/ml) in of the endotoxemic animals, and none of the control animals. il- b, il- and il- increased significantly with time peaking at t = , and respectively in the endotoxin group. in the control group only few animals showed a cytokine response, in numbers insufficient for statistical analysis. in the endotoxin group there was a negative cytokine flux in the renal circulation, maximal at t = [- . ± . for il- b and - . ± . for il- (pg/ml), respectively]. there was a positive cytokine flux for il- reaching its peak at t = ( . ± . pg/ml). a similar pattern was seen in the hepatic ? portal circulation with maximal flux for il- b and at t = (- . ± and - . ± . pg/ml, respectively). for il- there was a positive flux peaking at -= ( . ± . pg/ml). although there was a negative il- b and il- cytokine flux in the renal, portal and hepatic circulations indicating net uptake, and vice versa for il- , none of these values reached statistical significance. conclusions. these data do not support that cytokines are produced nor consumed in the kidney and liver during endotoxemia. discussion. non-survivors show more severity at the beginning and during their icu stay, more altered biological markers and a higher mean glycemia, but do not show significant difference either at initial glycemia, history of diabetes, hypoglycemia event or insulin treatment. elevated mean glycemia appears to be a factor independently associated with higher mortality. hyperglycemia prevalence in critically ill patients is very high and the controversy whether it is a mortality marker or a mediator still remains. our results would justify starting an intensive insulin protocol and its subsequent analysis. the interest of continuous scvo was proven in the management of severe septic patients [ ] , but the place of discontinuous scvo remains unclear. objectives. to compare continuous scvo to discontinuous scvo concerning the number of therapeutic interventions in the management of severe sepsis (ss) and septic shock (ssc). methods. prospective randomized comparative study. inclusion criteria: age [ years, ss or ssc [ ] . two groups were defined: continuous scvo (c group) monitored by a central venous oximetry catheter (edwards lifescience x hs, irvine, usa), and discontinuous scvo (d group) measured on blood samples drawn every h and at the request of the treating physician. the hemodynamic management of these patients was based on the algorithm established by rivers [ ] . the primary endpoint was the number of therapeutic interventions (fluids, transfusions, inotropic drugs) triggered by a scvo \ %. non parametric tests (chi-square and mann whitney) and repeated-measures anova were used in statistical analysis (p \ . was considered significant). results. patients were included in a polyvalent intensive care unit (icu). the two groups were comparable concerning age, sex, weight, height, apache ii score, mods on admission and mechanical ventilation (mv). there were no statistical differences between the two groups concerning: mortality, duration of icu stay, duration of mv and the evolution of mods and plasma levels of lactate from day to day . the therapeutic interventions data are shown in table . introduction. the calcium activated potassium channel (bkca) exists in smooth muscle cells in most vascular beds and is believed to be important in sepsis induced hypotension and vascular hyporeactivity [ ] and also in neutrophil killing and macrophage production of proinflamatory cytokines. however the latter two roles have been disputed [ ] and we have found that bkca expression is not upregulated in aorta from septic mice using real time polymerase chain reaction. as its role in sepsis remains uncertain we sought to determine whether null mice for the bkca channel were (a) resistant to hypotension and (b) showed improved survival in a clinically relevant model of fecal peritonitis. methods. bkca null mice (based on balc) were obtained from jax Ò mice. agematched litter mates homozygous for bkca were wild types (wt). mice (age - weeks) had tethered arterial and venous lines inserted under isoflurane anesthesia. the tether enabled mice to roam cages freely whilst continuous blood pressure (bp) traces were obtained. h post surgery, echocardiogram and intraperitoneal injection of rat slurry was administered under anesthesia. fluid resuscitation of . ml/h voluven/ % dextrose ( : ) was given. at and h echo was recorded and mice culled with mesenteric arteries dissected for myography. data expressed as mean(sem) and statistical analysis anova. results. genotypic study and whole cell patch clamp recording in aortic smooth muscle cells confirmed bkca current was absent in null mice. fecal peritonitis induced equivalent hypotension in both wt (n = ) and bkca null mice (n = ) at - h (fig. a ). echocardiography at h post slurry showed no difference in cardiac output between wt- . ( . ) and bkca null mice- . ( . ) ml/min and no difference or improvement cf time (fig. b) . thus this fall in bp is due to reduction in total peripheral resistance not myocardial depression. in addition / of the bkca null mice died prior to h as opposed to / wt. hence myography was only performed on wt mesenteric arteries which were hyporeactive to norepinephrine (p = . , fig. c ). conclusion. there is no evidence from this transgenic mice study of fecal peritonitis that inhibition of the bkca channel would be beneficial for the treatment of hypotension in septic shock or would improve survival. reference(s). introduction. pro-and anti-inflammatory responses play a key role in the pathophysiology of sepsis [ ] . phosphodiesterase (pde) inhibition could play an anti-inflammatory role in this setting [ ] . previously, it was shown that among the three inhibitors of pde five currently available (sildenafil, vardenafil, tadalafil), only tadalafil could exhibit anti-inflammatory properties on endothelial cells (ec) stimulated by modified oxidized ldl or tnf alpha [ ] . to assess the potential anti-inflammatory role of tadalafil in ec stimulated by lps. methods. thp- cells ( . /ml in rpmi) were incubated alone (control group) or in the presence of either tadalafil ( lm; eli lilly, in, usa), lps from e.coli :b ( ng/ml; sigma-aldrich, inc.) or both. tnfa production, as a marker of inflammation, was measured in the supernatant (elisa assay; roche, mannheim, germany) after h of incubation ( independent experiments in quadruplet). comparisons were made by one-way anova, with bonferroni's post hoc test (mean ± sem). results. production of tnfa increased significantly after stimulation by lps alone compared to control ( . ± . -fold over the control, p \ . ) or tadalafil ( . ± . vs. . ± . -fold over control, p \ . ). levels of tnfa were significantly reduced in the lps ? tadalafil group, compared with the lps group ( . ± . vs. . ± . -fold over the control, respectively; p \ . ) (graph ). we hypothesized that daa provides varying protective effects in different organs as indicated by higher amounts of epcr in early murine sepsis. methods. sepsis was induced by cecal ligation and puncture (clp) in male nmri-mice (n = , body weight ± g). animals were randomly assigned to vehicle infusion (control), or clp sepsis with daa infusion [daa; lg/(kg hr)]. a third group received only sham operation and vehicle infusion (sham). h prior to clp all mice were given a permanent central i.v.-line and an arterial transmitter (pa-c , st. paul, mn, usa) to measure heart rate (hr) and mean arterial pressure (map). clp was adjusted to survive h. after h hearts, livers and kidneys were fixed in formalin and embedded in paraffin. immunohistochemical analysis of the paraffin sections was performed using the avidinbiotin-peroxidase complex (abc) method. for analysis an anti-mouse epcr antibody (clone , natutec, frankfurt, germany) was used (dilution : ) after heat pretreatment. anti-epcr positive cells were counted in fields in light microscopy (original magnification: . ) of each tissue and the average was recorded. data are presented as mean ± sd. *p \ . was considered significant. results. there were no significant differences in hr between the groups (sham ± per min; daa ± per min; control ± /min). map was significantly higher in sham group ( ± mmhg; p = . ) and non-significantly higher in daa group ( ± mmhg) when compared to control ( ± mmhg). anti-epcr positive cell count in heart tissue was significantly higher in sham-treated mice ( . ± . cells; p \ . ) and daa mice ( . ± . cells, p = . ) compared to controls ( . ± . cells). in kidney tissue epcr positive cells were significantly more in sham group ( . ± . ; p = . ) compared to control, but not in daa group ( . ± . ). liver samples showed no significant differences (sham . ± . ; daa . ± . ; control . ± . ). conclusion. our data showed higher amounts of epcr in murine sepsis undergoing daa therapy in heart and kidney tissues, but not in the liver when compared with control animals. this suggests that daa provides different effects in early experimental sepsis. background. caspofungin treatment is often initiated in hypovolemic shock patients, what could affect its pharmacokinetics and efficacy. the present study investigated the influence of hypovolemic shock and fluid loading on the plasma pharmacokinetic parameters and the pulmonary penetration of caspofungin in a pig model. after anesthesia and mechanical ventilation, pigs ( ± kg) were bled to induce a -h deep shock and resuscitated for h using normal saline based on hemodynamic goals. a -h perfusion of mg caspofungin was started at the beginning of the resuscitation period. lungs were removed h after the initiation of hemorrhage. sixteen animals were used as controls without hemorrhage. caspofungin concentrations were measured using high performance liquid chromatography method. in the shock group, the volume of removed blood was ± ml/kg and a volume of ± ml/kg of saline was infused through the resuscitation period. conclusion. hypovolemic shock followed by fluid loading in pig results in a significant decrease in plasma caspofungin exposition. it resulted in a decrease in the pulmonary concentration of caspofungin without affecting its diffusion to the lung. future investigations should focus on the interest for monitoring of plasma caspofungin concentrations in icu patients and on optimal dosing in these patients. objectives. the present study was designed to assess the effects of mps from septic origin on systemic hemodynamics as well as on the inflammatory, oxidative and nitrosative stresses. methods. forty healthy rats were randomly allocated to three groups: animals inoculated with mps isolated from control rats (cmps), animals inoculated with mps isolated from sham rats (shmps) and animals inoculated with mps isolated from rats with peritonitis (smps). rats were anesthetized, mechanically ventilated and were infused with the same amount of cmps or shmps or smps. we measured heart rate (hr), mean arterial pressure (map), carotid artery blood flow (cbf) and portal vein blood flow (pbf). hemodynamic parameters were recorded during h, and then animals were sacrificed. aorta and heart were harvested for further in vitro tissue analyzes. . the cellular origin (phenotype) but not the circulating concentration of mps was different in septic rats, characterized particularly by a significant increase in leukocyte derived mps. . smps but not cmps or shmps decreased mean arterial pressure without any effect on carotid artery and portal vein blood flows. all rats survived in the cmps and shmps groups whereas three rats died before the end of the experiment in the smps group. . rats inoculated with smps exhibited an increase in superoxide ion production and nf-kb activity, over-expression of inos with subsequent no overproduction and decrease in enos activation. pulse blood pressure recordings conclusions. rats with sepsis induced by peritonitis exhibited a specific phenotype of mps which could play a detrimental hemodynamic effect as a systemic vasodilatation. inoculation of smps in healthy rats decreased map likely by up-regulating nf-kb activity with subsequent inos, no and superoxide anion overproduction. these data confirm a proinflammatory detrimental role of mps in the vascular pathophysiology of septic shock. introduction. heat shock proteins (hsps) play an active part in modulating intracellular responses to stress. in the classical model for their activation de-repression of heat shock transcription factor (hsf ) occurs as a result of the titration of hsps away from hsf by misfolded proteins [ ] . however, hsps may change in many diseases without any changes in the levels of denatured proteins [ ] . objective. we propose that hsps are activated, in part, by a membrane dependent calcium channel receptor, possibly transient receptor potential vanilloid type- (trpv ). capsaicin, a known inducer of trpv , and capsazepine, a selective antagonist, were used on different mammalian epithelial cell lines. cells were pre-treated with micromolar concentrations of capsaicin or heat shock (hs) followed by treatment with capsazepine. results. capsaicin or hs induced hsf activation and the consequent accumulation of hsp , and chaperones. pre-treatment with capsazepine prior to hs or capsaicin abolished the heat shock response (hsr). capsazepine treatment prevented capsaicininduced stabilization of ikb and cell to cell adhesion and induced apoptosis. capsazepinemediated blockage of the heat shock response was reproduced with egta. moreover, treatment with trpv sirna resulted in a similar response to capsazepine. conclusion. hsr-sensing and signaling in mammalian cells depends, in part, on the transient entry of calcium by way of membrane dependent calcium channel receptor. these hsr modulators may hold promise in treating inflammation in the future. introduction. hydrogen sulphide gas, or its intravenous donor-sodium hydrogen sulphide (nahs), are promising therapeutic agents in ischaemia-reperfusion and haemorrhagic shock [ ] . we studied nahs in a short-term endotoxaemia model as relatively little is known about its effects during sepsis. methods. under isoflurane anaesthesia, male wistar rats (approx g weight) underwent left common carotid and right jugular venous cannulation for blood sampling/continuous bp monitoring and fluid administration, respectively. animals were kept normothermic on a heating mat. tissue oxygen tension (tpo ) was monitored using oxylite probes (oxford optronix, oxford uk) placed in thigh muscle. after a -min stabilization period, fluidresuscitated rats [ ml/(kg h)] were subjected to iv lps ( mg/kg over min). comparisons were made against animals receiving nahs ( . mg/kg bolus given immediately after lps, followed by a mg/(kg h) infusion). echocardiography (vivid , ge healthcare, bedford) and blood gas analysis were sequentially performed. sham-operated, non-septic animals also received nahs (n = ) or placebo (n = ). at the doses given, nahs had no effect on either sham-operated animals (data not shown), nor on the endotoxic rats (table ) . data shown as mean (±se). timepoints chosen reflect the biphasic response to endotoxin: = baseline, = initial hypotensive phase, = maximal recovery, = end of experiment. conclusion. nahs does not improve haemodynamics, tissue oxygenation nor shockrelated biochemical parameters in a severe model of fluid-resuscitated endotoxaemia. we will further investigate the effects of dose and time of therapeutic intervention in this model, in addition to testing it in a long-term septic model. intestinal endothelial and epithelial barrier dysfunction remain severe clinical problems as they may contribute to the development of sepsis and multiorgan failure. we have recently established an isolated rat small intestine model with access to vasculature, lumen and lymphatics for study of inflammatory changes in fluid balance [ ] stable for min, rendering it less suitable for examination of changes in gene and protein expression profile. the aim of this study was to assess the long term functional and metabolic stability of this model. adult female wistar rats were anaesthetized, small intestines cannulated and perfused vascularly ( . ml/min) and luminally ( . ml/min) and placed in a warm humidified chamber for up to h. arterial, venous and luminal pressures as well as venous, luminal and lymphatic effluent flows and intestinal weight were recorded continuously. as measures of metabolic integrity, oxygen consumption, lactate/pyruvate ratio and galactose uptake from luminally administered lactose were analysed every min. structural and barrier integrity were assessed as histostability score (mesenteric and antimesenteric fraction of fully epitheliated villi), wet/dry weight ratio and translocation of vascularly applied fitc albumin to lumen and lymphatics. data were compared using paired t tests. ± . / . ± . ml/(min g) dry weight (**)) as well as galactose uptake ( . ± . / . ± . mg/(min g) dry weight (n.s.)) were very stable with time pointing towards high metabolic stability. during the whole experiment, luminal effluent flow was slightly lower than applied ( . ± . ml/min, min) resulting in net liquid absorption over the whole time period ( . ± . / . ± . ml/min (n.s.)), and lymph production stayed in the physiologic range ( . ± . / . ± . ml/min (n.s.)). the organ weight did not change with time which, together with the balanced luminal fluid flow and end experimental wet/dry weight ratio of . ± . (compared to . ± . at the beginning of the experiment (**)), indicate absence of edema. minimal leakage of vascular fitc albumin to the lumen ( . ± . %) and a histostability score of . ± . show integrity of the vascular-luminal barrier until the end of the experiment. the isolated small intestine model presented earlier [ ] displays excellent long term physiologic, metabolic and histologic stability and opens up a wide field of applications including inflammatory gene transcription and protein expression. introduction. mitochondria play a major role during ischemia-reperfusion as well on cytotoxic pathways as protective such as ischemic preconditioning. the aim of this study is a better understanding of the mitochondrial pathophysiologic response to several oxygen regimens in an isolated mitochondria model. mitochondria were isolated from rat heart. enriched mitochondrial pellets were conditioned in presence of glutamate ( mm) and malate ( mm) inside the oxygraph chamber during min. oxygen partial pressures were: mmhg for control group; to mmhg for hypoxia group and mmhg for anoxia group. then, after a min oxygenation period, several measurements were realized: oxygen consumption (vo ) were measured with or without adp ( mm) (state and of mitochondrial respiration); calcium retention capacity (crc); mitochondrial membrane potentiel (dwm). to explore the involvement of reactive oxygen species (ros), mitochondrial vo were measured in presence of a specific mitochondrial antioxidant drugs (xbj). all results were expressed in percent of variation in comparison to control group [median (minimum-maximum)]. the different groups were analyzed using a kruskal-wallis, a mann-whitney with a bonferroni correction or a sign test when necessary. after hypoxia and reoxygenation the mitochondrial function was altered. this impairment of mitochondrial function was not found after anoxia and reoxygenation. this difference in mitochondrial function between hypoxia and anoxia suggests the involvement of ros. this hypothesis was confirmed by the effect of the antioxidant xbj that reestablished after hypoxia the same level of vo than after anoxia. [ ] . superoxide dismutase (sod) catalyses the dismutation of superoxide oxygen free radicals to oxygen and hydrogen peroxide (h o ). the therapeutic potential of exogenous sod administration in ards is evidenced by demonstrations of efficacy in acute lung injury models [ ] . anti-oxidant defenses, particularly the extracellular sod isoform, extracellular sod (ec-sod), are downregulated by endotoxin [ ] . we proposed that ec-sod delivered via a novel viral vector would ameliorate lung injury caused by lipo-polysaccharide (lps) pulmonary instillation. methods. three groups with nine rats per group were randomised to receive either adenoassociated virus expressing ec-sod (aav-ec-sod), adeno-associated virus coding for no product (aav-null), or vehicle control, days prior to planned lps instillation. a model of lipo-polysaccharide (lps) induced acute lung injury by pulmonary instillation was established in male sprague dawley rats. twenty-four hours following lps delivery, animals were anaesthetized and mechanically ventilated and their baseline compliance and oxygenation recorded. there was a statistically significant improvement in the oxygenation of animals recieving aav-ec sod as compared to aav-null or vehicle control (mean pao = . vs. . and . , respectively). there was a significant increase in amount of ec-sod as determined by real time pcr in the group who were administered aav-ec sod. no significant differences in static compliance or bronchoalveolar lavage cells counted were noted. conclusion. aav delivered ecsod is protective in a animal model of lps induced acute lung injury. the down regulation of the ec sod system seen in the systemic inflammatory response [ ] and its subsequent replacement exogenously may explain our findings. further work will focus on other components of cellular anti-oxidant pathways and confirmation of down regulation of ec sod in our injury model. aims. the endothelial specific angiopoietin (ang)-tie ligand-receptor system has been identified as a non-redundant mediator of endothelial activation in experimental sepsis. binding of circulating ang- to the tie receptor physiologically protects the vasculature from leakage, whereas binding of ang- antagonizes tie signaling and disrupts endothelial barrier function. we tested whether administration of exogenous recombinant ang- improves survival and attenuates multi organ failure in a lethal murine sepsis model. to induce septic acute kidney injury and to evaluate survival time cecal ligation and puncture (clp) was performed in twenty sv mice. half of the mice received an intravenous application of recombinant human ang- ( lg) immediately before clp and every h thereafter. in the other half, saline was administered in the same fashion. for tissue assessment (western blot, immunohistological) clp was induced in versus (ang- vs. saline) additional mice; animals were sacrificed after h. laparotomy served as sham control (n = ). further, a panel of cytokines has been assessed with a cytometric bead array (cba) system after h. . ± . mmol/l, p \ . ) were lower in ang- treated septic mice compared to controls. similar results were obtained at h after clp. renal tissue revealed that saline treated mice exhibit a marked loss of expression of vascular endothelial (ve)-cadherin, a major component of endothelial adherens junctions. in contrast, loss of ve-cadherin expression was prevented by ang- (pre-) treatment (wb densitometry: ang- : . ± . ; saline: . ± . ; p = . ). however, contrary to previous reports, intravenous injection of exogenous ang- enhanced not only the expression of adhesion molecules (icam- , vcam- ) in renal vasculature, but also circulating cytokine levels (tnfa, mcp- , il- , il- ). conclusions. our study demonstrates that administration of exogenous recombinant ang- improves survival time in a lethal experimental sepsis model. enhanced survival was accompanied by an improvement in microcirculatory function, probably via stabilization of adherens junctions. however, ang- injection deteriorated expression of vascular adhesion molecules and raised plasma cytokine levels. although ang- may have utility as an adjunctive agent for the treatment of septic multi-organ failure, additional dose-finding and efficacy studies are required. adaptive immune responses to infection. in contrast to neutrophils, macrophages or lymphocytes, there are virtually no data on the time course of circulating dcs in septic shock (ss). using a novel specific and sensitive assay, we analyzed the evolution of circulating myeloid (mdcs) and plasmacytoid (pdcs) dcs in ss. we enrolled immunocompetent adult patients with ss (n = ), shock from other etiologies (nss, n = ) and with sepsis without organ dysfunction (s, n = ). age-matched healthy controls (hc) served as reference for mdcs and pdcs. blood samples ( ll) were drawn on the day of shock, then after and days. dcs were counted using the dc-labelling kit trucount Ò assay (bd biosciences). cd c? cd -(mdc) and cd c-cd ? (pdc) cells were selected by flow cytometry (facscanto tm , bd biosciences). hla-dr mean fluorescence index (mfi) was measured. age, sex ratio, saps ii, sofa score, nosocomial infection (ni) and mortality rates did not statistically differ between ss and nss pts. at day , mdcs and pdcs counts were significantly lower in ss and nss pts as compared to hc and s ( fig. ). pts with ss had significantly lower mdcs and pdc counts than nss at days and . hla-dr mfi of mdcs and pdcs was lower in ss pts compared to hc (p = . and . , respectively). interestingly, of the ss pts developed ni after a median time of ( . - ) days in the icu. whereas mdcs increased in pts without ni, mdcs counts remained low at day in pts who developed ni: mdcs counts and their relative variation between day and were significantly lower in pts who developed ni than in those who did not (p \ . ). logistic regression analysis indicate that a negative mdcs relative variation is associated with an increased risk of nosocomial infection with an or ( . - ) (p = . ). figure conclusion . ss is associated with quantitative and qualitative abnormalities of circulating mdcs and pdcs as early as day , independently of the haemodynamic injury. the persistence of low counts of mdcs after ss is associated with the advent of nosocomial infection during the icu stay, suggesting that dcs play a role in the development of sepsisinduced immunosupression. introduction. liver dysfunction is common in sepsis but its mechanisms are unclear. the aim of the study was to evaluate the effects of lps on cultured primary human hepatocyte respiration over time. methods. human hepatocytes were isolated and cultivated from human liver resection specimens. cultivated cells were exposed to lps ( lg/ml) for , and h. after incubation, cells were trypsinized and respiration rates were measured using a high-resolution oxygraph (oxygraph- k, oroboros instruments, innsbruck, austria). glutamate ? malate (g ? m), succinate (s) or ascorbate/tmpd (a/t) were used as substrates to test the function of complex i, ii and iv, respectively. human hepatocyte mitochondrial function in the cells treated with lps for h exhibited a significant reduction in the maximal complex ii-dependent mitochondrial respiration [control: ± vs. lps: ± pmol/(s million cells) ( table ) ]. after and h of lps incubation no significant reduction in cellular respiration was observed ( and h: n = and h: n = ). statistics: paired t test, *p = . control vs. lps ( h incubation). introduction. acute kidney injury (aki) in critically ill patients is a frequent clinical problem and a rising incidence has been reported over the past several years. recently two consensus definition for aki have been developed: rifle [ ] in by the acute dialysis quality initiative workgroup (second conference) and akin [ ] in . insofar akin and rifle criteria have been applied in large retrospective studies, limited to the initial days of icu. nefroint is an italian initiative for an observational prospective multicenter study to evaluate epidemiology of aki in italian icus employing rifle and akin classifications. a pilot study has been performed in one of the centers enrolled. objectives. primary endpoints of nefroint are: application and comparison of rifle and akin criteria for aki definition in a prospective observational study; estimate, along such criteria, of aki incidence in critically ill patients; correlation of aki stages with prognosis. method. an observational prospective multicenter study has been designed, in italian adult icus (medical and surgical). all incident icu patients have been enrolled over a month period. exclusion criteria was age \ years, or icu stay \ h. data collection about patients was performed on a web-based electronic case report form. data included icu admission diagnosis, daily urine output ( h interval), daily laboratory data. sepsis events diagnosed on clinical and/or microbiological basis where as well marked for each patient. severity scores have been calculated at admission and daily. aki patients had higher severity of illness scores and higher serum creatinine values on admission. they also were older and more likely to have a respiratory diagnosis as reason for icu admission. conclusions. nefroint is an initiative aimed at comparing rifle and akin scores to promote a uniform use of a single definition of aki that will render subsequent studies comparable. early aki recognition could potentially allow implementation of timely corrective interventions, and hopefully prevent progression to more severe stages. aim. sepsis and septic shock remain the most important causes of acute kidney injury (aki) in critically ill patients and account for more than % of cases of acute renal failure (arf) in intensive care units (icu). its mortality varies with the severity of sepsis from % to %. the aim of this preliminary study was to investigate the differences in the course and prognosis of aki that was induced by community and hospital acquired sepsis. method. patients with sepsis induced aki were included in the study. rifle criteria were used to define aki. clinical and laboratory characteristics of the patients were compared with student t test and chi square tests. results. forty-one patients were included in the study and of them had community acquired septic aki (akic). ninety percent of the patients received mechanical ventilation (mv). etiologies of sepsis were mostly community acquired pneumonia and ventilator associated pneumonia. age, gender, admission apache ii scores and sofa scores at the time of aki diagnosis were similar across the groups (p [ . ). hospital acquired septic aki (akih) developed later when compared to community acquired septic aki ( th and rd days of sepsis respectively, p . ). akih was significantly and more frequently associated with oliguria ( vs. %, p . ), bacteremia ( vs. %, p . ), nephrotoxic antibiotic usage ( vs. %, p . ) and tend to progress more frequently to acute renal failure( vs. %, p . ) compared to akic. akic episodes were more frequently ( vs. %, p . ) and rapidly ( vs. days, p . ) reversible. mean blood pressure and scvo % were significantly lower and more vasopressor and steroid therapies were required during akih episodes compared to akic (p \ . ). while length of mv and mortality rates were similar, duration of hospitalization was significantly longer in the akih group ( vs. days, p . ). conclusion. these results suggest that, akih has worse clinic and prognosis than the akic so further and larger studies are necessary to investigate the preventive and therapeutic approaches. introduction. severity-of-illness or organ dysfunction scores are inaccurate to predict outcomes in patients with acute kidney injury (aki), even when specific aki scores are used. in recent years, the third versions of simplified acute and physiology score (saps ) [ ] and of mortality probability model (mpm -iii) [ ] scores were developed, and information on their use in patients with aki is scarce. objectives. to validate the use of saps and mpm -iii at the start of renal replacement therapy (rrt) in patients with aki. prospective cohort study conducted in the icus of three tertiary-care hospitals. data used to calculate the scores were collected at start of rrt. discrimination was assessed by area under receiver operating characteristic (aroc) curves and calibration by hosmer-lemeshow goodness-of-fit test. a total of consecutive patients were included between january and july . the mean age was . ± . years. the main contributing factors for aki were ischemia/shock ( %), sepsis ( %), contrast/nephrotoxins ( %), rhabdomyolysis ( %) and urinary tract obstruction ( %) (a patient could have more than one contributing factor). eightnine ( %) patients received rrt on the first day of rrt and ( %) thereafter; continuous rrt was used as first indication in ( %) patients. the icu and hospital mortality rates were and %, respectively. the mean saps score at the start of rrt was . ± . points. both the standard equation of saps and mpm -iii scores tended to underestimate mortality. discrimination was better for saps [aroc = . ( % ci, . - . )] than for mpm -iii [aroc = . ( % ci, . - . )], as was the calibration. however, mortality prediction and calibration improved when the customized equation of saps for countries from central and south america was used. in multivariate analyses, both higher prognostic scores and length of icu stay prior to rrt were the main predictive factors for hospital mortality. conclusions. the saps score at the start of rrt was accurate in our cohort of patients and seems a promising instrument for predicting hospital mortality critically ill patients with aki. objectives. the aim of this study was to investigate the effect of hes administration on kidney function compared with other colloids or crystalloids. methods. systematic review and meta-analysis of the effects of hes administration on kidney function. inclusion criteria for the study were prospective randomized trials comparing hes to control with reporting on variables of kidney function. aims. during the initiation phase of experimental acute kidney injury (aki), subtle but devastating changes, such as loss of brush borders, disruption of tubular cell polarity and cytoskeletal changes are detectable only to a certain extent by routine histologic methods. for this reason, subjective and moderate reproducible semi-quantitative scoring of tubular changes (e.g. vacuolization, detachment, cast formation, and necrosis) still remains the method of choice to quantify the extent of experimental aki. lectins are glycoproteins which are able to bind carbohydrate structures specifically. it has previously been shown that immunolabeling of the lectin phaseolus vulgaris erythroagglutinin (pha-e) is highly specific to the brush border of proximal tubular epithelial cells of rats, mice, and humans. the aim of this study was to ( ) develop a simple and fast lectin (pha-e) based staining protocol ( ) to objectively quantify, and ( ) to analyze brush border loss in a murine model of septic aki. methods. septic aki in mice (n = ) was induced by cecal ligation and puncture (clp). animals were sacrificed h after clp.sham operated (n = ) and healthy animals (n = ) served as controls. in order to specifically stain the tubular brush border, binding of biotinylated lectin pha-e was visualized by the biotin-avidin-complex (abc) glucose-oxidase (go) method coupled to tetranitroblue tetrazolium (tnbt) in -lm paraffin sections of renal tissue. the mean brush border area of five randomly chosen, non-overlapping cortical highpower fields was analyzed by planimetric software. lectin pha-e staining was highly selective for brush border of proximal tubules (black colour). virtually no staining was present in glomeroli and medulla. the xx software reliably identified lectin-positive areas, as confirmed by image overlay controls. we found a significant difference between sepsis induced aki, sham operated animals, and healthy mice (clp: . ± . ; sham: . ± . ; healthy controls: . ± . pixel ratio; p \ . ). our findings with the pha-e staining protocol correlated significantly with the conventional semi-quantitative scoring system (r = . , p \ . ). conclusion. the here presented lectin pha-e staining method followed by computerassisted planimetric quantification of brush border area is a highly reproducible and objective tool to analyze early histological changes during septic aki in mice. when an imbalance between oxygen supply and demand exist, anaerobic respiration commences and a metabolic acidosis develops. base excess and lactate have been used to identify a higher risk group of patients who should be admitted in icu prior to development of multiple organ failure. and at a time when appropiated therapy may previne the decline to death. acute kidney injury failure is a common complication in critically ill patients and it always difficult separate the acid base effects of critical illness per se from those of aki. the aim of this study was to examine wheter values of base excess or lactate taken on admission of patients with aki to a intensive care unit indicate prognosis and if wheter this can be used as screening tool for future intensive care admissions. we restropectively examinated data from patients with aki. to define the unique acid base characteristics of aki patients, we used a control group. the matched group consisted of icu patients wihtout aki matched for apache ii score. the base excess and lactate were collected at admission and then at h. a total of patients were enrolled at study over a month-period. there were no difference with respect age, sex and apache score between groups. the icu survival rates were % to the aki group and % to control group. the value of base excess with the best predictive prognosis ability was - mmol/l to the aki group and- . (p \ . ) to the matched group and the corresponding value for lactate was higher than . to both groups. the combination of these two markers on admission to the intensive care unit led to a sensitivity of % and specifity of % for mortality. conclusion. both base excess and lactate, or the combination of the two, can be used to predict day mortality in patients admitted to the intensive care unit. in patients with aki a different cut off of base excess should be used.these variables could be utilized to identify patients who have a higher risk for mortality to whom resources could be better directed. nonthyroidal disease (ntd) is a common finding in patients who are critically ill or on dialysis or with cardiovascular disease. its presence has been associated with inflamatory conditions. the aim of this study was to analyse the posible association of ntd with the development of acute kidney injury (aki). secondary targets where to estimate the incidence of ntd in a polyvalent icu and observe the realationship between the levels of t and some inflamatory markers: c reactive protein (crp), albumin and cortisol. during months in , after approval of the local ethical committee, we prospectively determined the following parameters in every patient admited to the icu: t , t , tsh, serum creatinine (scr), crp, albumin and cortisol. after excluding patients who died or were discharged before h, patients were studied. the degree of aki was calculated using the rifle scale. at admission the values of the analysed parameters were (mean ± sd): t . ± . pg/ml; t . ± . ng/dl; tsh . ± . liu/dl; scr [ ] . its incidence ( - %) is rising due to increasing numbers of ct scans and contrast studies conducted, and the higher prevalence of risk factors such as chronic renal impairment, diabetes mellitus and old age. although usually selflimiting, cin can be associated with a need for ongoing dialysis or increased mortality [ ] . to highlight the problem of contrast induced nephropathy and the difficulties in interpreting the current evidence for possible prevention strategies. we present the case of a year old man admitted to intensive care with acute pancreatitis. he underwent eight contrast-enhanced abdominal ct scans and received nacetylcysteine (nac) for all but one of these, after which he developed acute renal failure which did not recover. we also present a review of evidence for various proposed strategies. results. several studies have examined possible renal protective strategies around contrast administration. saline and bicarbonate have been shown to be beneficial when given pre-contrast [ , ] . theophylline has been shown in meta-analysis to have a significant beneficial effect, but heterogeneity of methodology between studies makes it difficult to clarify the degree of benefit achieved [ ] . nac has shown benefit in of trials. twelve meta-analyses showed inconsistent results, with showing nac to be beneficial. none showed harm. we analysed the heterogeneity of methods, endpoints and patient groups that makes these studies difficult to compare. critically ill patients may be considered at even greater risk of cin. only one study has specifically looked at this group. strategies such as volume loading may be inappropriate in some patients and there may not be time for nac for h pre-contrast. we were unable to find specific guidelines for the prevention of cin in critically ill patients. conclusion. the evidence for strategies to prevent cin specifically in critically ill patients is unclear. we review the current literature and propose renal protective strategies including hydration, nac and theophylline for this patient group based on the evidence available. objectives. the present study addresses the issue of how the different modes of rrt are currently used and performed. we conducted a prospective observational study in three portuguese intensive care units (icu). patient demographics, type of rrt used and outcomes were collected. we studied patients who were treated with rrt for rf, with a median age of years and a saps-ii score of . ± . , a sofa score of . ± . at admission; patients ( . %) were treated with continuous replacement therapy (crrt), patients ( . %) with sustained low-efficiency dialysis (sled)and patients ( . %) were initially treated with crrt and latter with sled. using the rifle criteria for the stratification of acute renal dysfunction at the beginning of the rrt we observed: risk- ( . %), injury- ( . %), failure ( . %), loss- ( . %), esrd- ( . %). we used anticoagulation in almost all patients ( . %). among patients who received anticoagulation, heparin was the most common choice ( . %), followed by low molecular weight heparin ( . %), and by sodium citrate ( introduction. in the intensive care unit (icu), severe sepsis and multiple organ failure are frequently associated with renal failure. continuous veno venous hemofiltration (cvvh), which is used as renal replacement therapy, also removes circulating inflammatory mediators. standard cvvh is currently prescribed with a substitution flow of ml/(kg min). theoretically, when hemofiltration is performed with higher volumes, buffer balance will be restored more rapidly, while also more inflammatory mediators will be removed. this may result in faster stabilisation from septic shock. indeed, animal-and some human studies show promising results, but have several (methodical) limitations. to evaluate hemodynamic and metabolic changes during hv-cvvh in patients with septic shock in comparison to (standard) cvvh. we performed a retrospective, observational, single-center study. all patients admitted with septic shock who were treated with cvvh in the period until were included. cvvh was defined as a substitution-flow b , ml/h, hv-cvvh as[ , ml/h. the decision to start with lv-cvvh or hv-cvvh was made by the attending icu-physician on an intention-to-treat basis. statistical analyses were performed with spss . introduction. haemostatic changes in critically ill patients are complex due to simultaneous pro-and anticoagulant processes. routine ptt and aptt assays monitoring clot formation poorly reflect hypo-or hypercoagulant state, especially during anticoagulation. endogenous thrombin potential (etp) comprises an in-vitro system for measuring thrombin generation beyond clot formation and may be more informative. objective. to assess whether etp has a role in monitoring systemic anticoagulation and predicting circuit clotting in critically ill patients receiving cvvh. methods. in a prospective study in an -bed general icu, we included patients with acute renal failure (arf) requiring cvvh (postdilution, - l/u). patients received a bolus of , iu of nadroparin followed by iu/h. samples of arterial and postfilter blood were taken at baseline and , , and h after start of cvvh to measure aptt, ptt, anti-xa and etp. we compared patients with early circuit clotting (circuit life £ lower quartile) and those with normal circuit life. median baseline arterial etp-area under the curve (auc) was ma (iqr - ma) (normal values - ma). baseline etp-auc was positively related to antithrombin and inversely to ptt, aptt, anti-xa (p \ . ) and sofa score (p = . ). median circuit life was . h (iqr - h). at baseline, the four patients with early filter clotting (£ h) had prolonged ptt and aptt, higher sofa score and a tendency to lower etp (table ) . during cvvh and nadroparin infusion, arterial and postfilter ptt and aptt were prolonged (p \ . ), antixa lower (p = . ) and etp-maximal concentration (cmax) lower (p \ . ) when circuits clotted early. while arterial etp-auc tended to be lower (p = . ), postfilter etp-auc was not different between groups. in critically ill patients with arf requiring cvvh with nadroparin anticoagulation, baseline etp is lower than normal and inversely related to organ failure and (a)ptt, probably reflecting consumption of coagulation factors. within the cvvh circuit, etp-auc and anti-xa show opposing patterns. the concurrence of early filter clotting with prolonged (a)ptt, lower antixa, lower etp and higher sofa score emphasizes the role of severity of disease and associated coagulation activation and heparin resistance in circuit clotting. introduction. nadroparin is a low-molecular-weight heparin (lmwh) used to prevent clotting in the extracorporeal circuit during cvvh. in renal failure lmwh accumulates and is associated with more bleeding (ref) . whether nadroparin is removed by hemofiltration and whether the anticoagulant activity accumulates during continuous infusion is controversial. objective. to study the kinetics and removal of anti-xa activity during continuous infusion of nadroparin in patients requiring cvvh using a cellulose tri-acetate filter. methods. in a randomized crossover trial in an -bed general icu, patients with acute renal failure (arf) were randomized. in group , postdilution cvvh was initiated at filtrate flow of l/h (blood flow (bf) ml/min), which was converted to l/h (bf ml/min) after min; in group , l/h was converted to l/h. patients (\ kg) received a bolus of , iu nadoparin followed by iu/h. samples of arterial blood, postfilter blood and ultrafiltrate were taken at baseline, h after the start and min, , and h after the conversion to measure anti-xa activity. results. fourteen patients with arf were equally randomized. patients in group had higher median sofa scores ( vs. , p = . ), baseline coagulation markers were not significantly different. arterial and postfilter anti-xa values are presented in fig. . during cvvh arterial anti-xa tended to decrease in time (p = . ). the median ratio of postfilter to arterial anti-xa was . (iqr . to . ). there were large differences between patients; differences between groups were not significant, except for postfilter anti-xa at h, which was significantly higher in group ( l/h) (p = . ) . anti-xa activity was not detectable in the ultrafiltrate. conclusions. critically ill patients receiving nadroparin during cvvh showed no signs of accumulation of anticoagulant activity, although extracorporeal removal of anticoagulant activity could not be demonstrated. apparantly, nadroparin is cleared by these patients despite renal failure. the differences in anti-xa between patients may be related to severity of disease. introduction. unfractionated heparin (ufh) is used as the first-line agent for anticoagulation of the extracorporeal circuit during continuous renal replacement therapy (crrt) in % of icus in the uk (uk) [ ] . its use is monitored with serial measurements of activated partial thromboplastin time (aptt) or its ratio (apttr) in % of icus [ ] . there is, however, considerable variation in practice [ ] . anticoagulation is useful for prolonging haemofilter life and facilitates the provision of continuous therapy, but must be balanced against the risk of haemorrhage, which has been correlated with increasing apttr [ ] . most icus in the uk use an apttr target of . - . [ ] , despite recent guidance that a target range of . - . provides adequate filter life with less risk of bleeding [ ] . objectives. to investigate the adherence to our local target range for ufh therapy (apttr . - . ) and the occurrence of over-anticoagulation in our patients. ]. there were apttrs ( %) which were above our target range, and incidences ( %) where the apttr was greater than or equal to . . the apttr was greater than . on occasions ( %). conclusions. this study was conducted in an icu which delivers crrt at a higher than average frequency [ ] , and which consistently has a standardized mortality rate below the national average. despite this, there was wide deviation from our target apttr range and a considerable incidence of significant over-anticoagulation, which may place our patients at risk of haemorrhage. the vast majority of apttrs were in excess of recent guidance [ ] . regional citrate anticoagulation (rca) may provide longer filter life with a lower incidence of bleeding [ ] . its use is increasing worldwide [ ] , though it is not commonly used in the uk [ ] . we are investigating the possibility of introducing rca in our icu. in the meantime, we will set a lower apttr target for our patients. we prospectively studied patients who received cvvh from july to december . age, gender, admission diagnosis, and apache-ii were obtained and the patients were divided into three groups: low dose heparin group, low molecular weight heparin group (lmwh), and no anticoagulation group (normal saline washing) based on assessment of coagulation status. for each circuit, circuit life, bleeding, platelet count, pt, inr, aptt, creatinine and urea were collected before and after crrt. results. seventy-seven critically ill patients with acute renal failure were treated with crrt and circuits were observed. among these circuits, received unfractionated heparin (ufh) anticoagulation, received lmwh anticoagulation and received no anticoagulation. the mean circuit life ( . ± . h) in low dose ufh group, was significantly longer than in lmwh ( . ± . h) and in no anticoagulation group ( . ± . h). there was no significant difference in baseline patient pre-crrt hb, creatinine and urea among three groups. the inr and pt and aptt in baseline were significantly higher in no anticoagulation group compared to the other two groups (p \ . ). the platelet count was significantly lower in the no anticoagulation group compared to ufh group and lmwh group in baseline and during crrt. there was no significant difference in the filter pt, aptt, among the three groups during crrt. the clearance of creatinine and urea during crrt were no significant difference among the three groups. bleeding complication secondary to crrt were no significant difference among the three groups. objectives. the purpose of the study was to assess the duration of time spent off therapy during the first five days of crrt in post-traumatic arf, and to identify the reasons for this. ullevaal between january and december , were retrospectively reviewed. the hospital is the regional trauma referral centre for approximately . million adult ([ years) persons. according to the local treatment protocol, dialysis filters were routinely changed after h due to time-out. individuals were identified and data collected using several institutional registries. patients were grouped according to presence of rhabdomyolysis based on peak serum creatine kinase levels exceeding , u/l or not. categorical data were compared employing two-sided pearson chi-square test, whereas continuous data were analyzed utilizing two-tailed mann-whitney u test. results. patients were included during the study period. during the first five days of therapy there was a total of dialysis days, and the total number of pauses was . the median duration of crrt was . h per day, giving a downtime of . h per day. the number of pauses per day was significantly larger in patients with rhabdomyolysis compared to patients without rhabdomyolysis ( pauses in dialysis days vs. pauses in dialysis days, p \ . ). this resulted in a shorter duration of crrt in rhabdomyolytic compared to non-rhabdomyolytic persons ( . vs. . h per day, p \ . ). overall the reasons for pauses during crrt were filter clotting ( %), therapeutic procedures ( %), catheter problems ( %), filter time-out ( %) and diagnostic examinations ( %). patients with rhabdomyolysis had more pauses due to therapeutic procedures ( vs. %, p = . ), whereas non-rhabdomyolytic persons had more pauses due to catheter problems ( vs. %, p = . ) and filter time-out ( vs. %, p \ . ). the number of pauses per day stayed relatively stable during the first five days of crrt, but the reasons for pauses changed during the study period. conclusions. this study indicates that trauma patients with rhabdomyolysis had more frequent dialysis pauses during the first days of crrt than those without rhabdomyolysis, resulting in shorter duration of dialysis therapy. the reason for this was more frequent use of therapeutic procedures, i.e. surgery and radiological interventions, in rhabdomyolytic compared to non-rhabdomyolytic persons. grant acknowledgement. the author is supported by institutional grants. introduction. treatment of acute pancreatitis is aimed at correcting any underlying predisposing factor and at the pancreatic inflammation itself. hypertriglyceridemia is an uncommon cause of pancreatitis. a serum triglyceride level of more then , to , mg/ dl is an identifiable risk factor. interestingly, serum pancreatic enzyme levels may be normal or only minimally elevated in such cases. severe necrotizing pancreatitis is associated with a high rate of complications and significant mortality. the reduction of triglyceride level to below , mg/dl effectively prevents further episodes of pancreatitis. this study aimed to determine the effectiveness of plasma exchange (pe) in reducing triglyceride levels during an acute attack of hyperlipidemic pancreatitis (hlp). methods. prospective, observational study including six patients hospitalized with hyperlipemic pancreatitis treated with plasmapheresis between and in the medical icu of a teaching hospital in malaga. demographic data, apache ii score, organ support needed and prognosis were prospectively collected. a total of hypertriglyceridemic patients with the complication of acute pancreatitis received one or two consecutive sessions. mean age was ± years and mean apache ii was ± . icu mortality was %. we performed sessions. the development of multiorgan failure in patients with hyperlipemic necrotizing pancreatitis was associated with grave prognosis ( %), needed mechanical ventilation, vasoactive agent and renal replacement therapy. however, we had a good outcome in the majority ( %) with a effective reduction of triglycerides after the session of plasmapheresis (pe). four of six patients ( %) recovered completely in a single session. two patients developed intraabdominal abscess, requiring more than one consecutive session and surgical debridement of infected necrosis and died due to both septic shock and multi-organ failure. the respective mean removal rates during a single pe for triglyceride were %. conclusions. the best treatment of hypertriglyceridemic pa is a drastic reduction of tg-s to normal. experiences with plasmapheresis are limited. we report six patients of hypertriglyceridemic necrotizing pancreatitis with mildly elevated amylase and lipase, treated successfully with plasmapheresis. in summary, pe treatment is an effective method to clear lipids and enzymes from plasma in a single session for most hlp patients. the presence of multisystem organ failure appears to be a more important indicator of outcome than does the presence of infection. results. sixteen ( %) patients were female and ten ( %) were male. the median age was years old. the median apache ii score was . . mechanical ventilation ( %), vasoactive agents ( %) and renal replacement therapy ( %) were the most common forms of organ support needed. sessions of plasmapheresis were performed. ( %) patients had been diagnosed with thrombotic thrombocytopenic purpura (ttp), six ( %) patients had hyperlipemic pancreatitis, five ( %) patients had pulmonary-renal syndrome (prs), three ( %) patients had guillain-barré syndrome (gbs) and two ( %) had myasthenia gravis. we obtained a decreased in the values of apache ii score following the plasmapheresis performed. there were six death ( % mortality) due of the severity of the disease. the number of complications were minimal and commonly described in the literature and there was a low mortality as a result. conclusion. results indicate that the performance of plasmapheresis was on a heterogeneous sample of patients with neuroimmunological diseases, rheumatology diseases and hyperlipemic pancreatitis. we conclude that plasmapheresis is a safe treatment which can be made by the staff trained in intensive care in any moment with a wide spectrum of clinical indications and with a minimum adverse effect. the aim of the study is to evaluate that early treatment of septic shock with cpfa may improve patient outcome. methods. twenty septic patients who were admitted to the icu have been enrolled in this study. cpfa treatment was performed immediately after septic shock was diagnosed (early group h after diagnosis). every patient had - cpfa treatments for h with q blood = ml/h, q ultrafiltration = ml/(kg h) and q plasma = % of q blood. we measured the plasma concentration of procalcytonin (pct), blood lactic acid levels, crp, serum creatynine, wbc and pao /fio ratio. the apache ii score, hemodynamic parameters, norepinephrine dosage were evaluated before cpfa (t ), t (after first cycle), t (after second), t (after third cycle) and t (after h). introduction. the development of electrolyte disturbances in intensive care patients could be prevented by the use of better adapted dialysis fluids. a common problem is hypophosphatemia which has been shown to occur in up to % of the patients. correction by intravenous phosphate supplementation is known to improve respiratory muscles, cardiac index, oxygen delivery to tissues and insulin resistance. lately it has been reported that phosphate can be added directly to the dialysis fluid. this facilitates phosphate handling, but there is a risk of precipitation with calcium. an additional problem is that the amount of phosphate required to correct total body deficit varies and repeated serum measurements are needed to establish phosphate insufficiency. the process is time consuming and leads to treatment delay and excessive cost. objectives. this study evaluated the possibility to achieve and maintain normal phosphate balance over time by using a new phosphate-containing dialysis fluid. objective. the purpose of this study was to evaluate the impact of different dialysate and replacement flows in the acid-base balance of the blood. furthermore we tried to assess the way partial pressure of oxygen (po ) in the blood is affected by high flow crrt. methods. this was a prospective observational study. thirty consecutive critically ill patients that were admitted in our icu and required crrt during their course were enrolled in the study. for each patient, blood flow, dialysate and replacement flow as well as ultrafiltration adjustments were performed by the responsible intensivist. any time that the clinical condition required a modification in any of these parameters, and after a period of time of no less than h, a simultaneous blood sample was drawn from both the arterial and the venous part of the circuit and the samples were analysed by a blood gas analyzer. arterial and venus samples were then compared for differences in ph, po and pco concentration. results. in total we performed measurements in patients. mean patient age was . years, mean apache ii score was , mean icu stay was days and mean crrt days was days. overall, ph in the venous line of the circuit was higher, pco was lower and po was lower as well compared to the respective values in the arterial line of the circuit, with no difference reaching a statistical significance. concerning the blood flow, we observed that when using high hemodiafiltration flows the difference in oxygen partial pressure between the arterial and the venous line of the circuit was greater, but again it did not reach statistical significance. conclusion. the use of crrt may influence the po in the returning blood. although we did not reach statistical significance in our study, there was a definite trend towards lower po in the venous line of the circuit when high flow crrt was applied. introduction. renal failure (rf) is a common complication in critically ill patient and is associated with high mortality and has a separate independent effect on risk of death. the continuous renal replacement therapy (crrt) is physiologically superior; however, there is lack of strong evidence to prove a clinical benefit. hybrid therapies (sled) that combine the benefits of intermittent haemodialysis and continuous therapies have emerged in the past few years. objectives. the aim of this study was to assess what type of renal replacement therapy (rrt) used and relate them to severity of the illness and outcome we conducted a prospective observational study in three portuguese intensive care units (icu). patient demographics, type of rrt used, saps ii and sofa score at admission and when we started the rrt and outcomes were collected. we studied patients who were treated with rrt for rf, with a median age of years and a median saps-ii score of ; patients ( . %) were treated with continuous replacement therapy (crrt), patients ( . %) with sustained low-efficiency dialysis (sled) and patients ( . %) were initially treated with crrt and latter with sled. aim. tetanus is traditionally treated with very high doses of diazepam and morphine. it often required prolonged periods of paralysis and was associated with very high mortality and prolonged periods of ventilation. magnesium sulphate (mgso ), due to its effects on neuromuscular and autonomic system should be effective in controlling muscle rigidity, spasm and autonomic instability in patients affected with tetanus. we introduced an icu protocol using mgso as first line treatment. we wanted to evaluate our patient outcome following the introduction of our protocol. we retrospectively analysed the effects of introduction of mgso in our intensive care for management of tetanus. aim. electrolyte disturbances were often seen in patients in intensive care unit (icu). hypomagnesemia is not enough described but can be contributed in icu mortality. the aim of this study was to define the prevalence of hypomagnesemia in critically ill patients and to evaluate its relationship with duration of mechanical ventilation day, length of icu stay and mortality. a prospective study was done on patients with respiratory failure admitted to the icu between . . and . . . total serum magnesium level, electrolyte levels, albumin, total protein, and lactate levels were evaluated at the admission. patients demographic features, accompanying neurological and cardiac diseases, apache ii score, duration of mechanical ventilation, and the length of icu stay and mortality were recorded. at admission % of patients had hypomagnesemia. a positive correlation was found between serum magnesium and calcium level (p = . ), but there was no relationship between other laboratory tests. also there was no relationship determined between hypomagnesemia and duration of mechanical ventilation, and the length of icu stay and mortality (p [ . ). conclusion. electrolyte levels are important in critically ill patients. however routine monitoring of serum magnesium level is not necessary. so we should increase the case number and also evaluate the serum magnesium level with urine magnesium level to see the effects of hypomagnesemia. method. medical records of copd patients who underwent invasive mechanical ventilation (imv) were reviewed. the patients' age, sex, body mass index (bmi), apache ii scores at admission, previous diagnosis of hypothyroidism or hyperthyroidism, history of thyroid replacement therapy or antithyroid medications, and the serum thyroid stimulating hormone (tsh), free triiodothyronine (ft ), and free thyroxine (ft ) at admission were recorded. the primary outcome measure was prolonged mv (pmv), which was defined as dependence on mv for [ days. the outcome and the relation between the serum thyroid levels were evaluated. results. ninety-five copd patients were included, % were male, with a mean age of . ± . years. bmi's of the patients were . ± . and the mean value of apache ii score was . ± . . only two patients ( %) had a history of hypothyroidism. two more patient were diagnosed hypothyroidism at admission and treated with thyroid medications. the patients treated with thyroid replacement therapy were liberated from mv successfully. patients ( . %) could not be weaned. serum ft level ( . ± . ) of the patients, who could not be weaned, was statistically lower than other group who could be liberated (p = . ).however there was no statistical difference between serum ft and tsh levels and two groups. hypothyroidism is an uncommon cause of ventilator dependent respiratory failure with an incidence of %, but it is treatable, so it should be considered in patients who can not be liberated.more prospective studies are also needed to evaluate the significance of hypothyroidism in patients with respiratory failure and failure to wean. smoke inhalation injury represents an important prognostic factor in patients admitted in the hospital after smoke exposition. objectives. we determined whether initial antithrombin (at) levels help in diagnosis and prognosis of sepsis after smoke inhalation. smoke inhalation was diagnosed according to classical clinical and laboratory findings in patients admitted in the hospital with suspected inhalation after smoke exposition. at levels, coagulation parameters (fibrinogen levels, prothrombin time (pt), activated partial thromboplastin time (aptt) and liver function tests were determined on admission and correlated each other and with outcome of the patients. . initial at and fibrinogen levels were significantly lower in patients with severe smoke inhalation compared to control (p \ . ). initial at levels were lower in the ones who developed septic complications with disseminated intravascular coagulation (dic) compared to those without dic (p \ . ). initial at levels were significantly lower in patients who died as compared to survivors (p \ . introduction. x-ray finding of pleural effusion is fairly common in icus. this may vary from mild to massive effusions and of different etiologies. epidemiological and outcome data for this icu problem are scarce in literature. the objective of this study was to find how common this finding is in our icu, their respective etiologies and any bearing on icu mortality. a single centre, prospective, observational study conducted in two mixed medical and surgical icus in kolkata, india. over six month period (october to march ) all consecutive patient admissions to these two icus were screened for a x-ray evidence of pleural effusion, either on admission or during their icu stay. as per icu protocol apache ii scoring were done in all patients. those with effusions were grouped according to etiology. finally in icu mortality were observed for those with or without an effusion. a total of icu admissions were studied. among these patients were found to have x-ray evidence of pleural effusion. median apache ii score was (iqr - ) among the study population with predominant ( . %) medical admissions. incidence of bilateral effusions were a total of ( %). the common causes of pleural effusion include chronic kidney disease (n - %), heart failure (n - %), pneumonia (n - %), post operative (n - %), chronic liver disease (n - %) and rest others (e.g. trauma, pancreatitis, pte, malignancy). the overall icu mortality was ( . %) and ( . %) in groups with and without effusion respectively with a p value of . , showing number of deaths in pleural effusion group were significantly higher. our study showed x-ray finding of pleural effusion quite common in icu patient population even many a times being bilateral. in this small study the overall icu mortality were also higher in pleural effusion group, but a wider multicentric study is needed. introduction. acute lung injury (ali) is a clinical manifestation of respiratory failure caused by lung inflammation and the disruption of the alveolar-capillary barrier. to prevent alveolar edema, it is of critical importance to preserve the physical integrity of the alveolar epithelial monolayer which is regulated by the balance between centripetal forces arising from cytoskeletal tension and cell-cell and cell-matrix tethering forces [ ] . intercellular junctions, such as tight junctions are closely related to actin cytoskeleton-related barrier regulation. proteins of the coagulation cascade such as thrombin (thr)-that stiffens [ ] and contracts [ ] alveolar epithelial cells (aec)-or activated protein c (apc)-an endothelial barrierprotective agent [ ] -could modulate this balance of forces in the epithelial monolayer. to study the combined effects of thr and apc on the barrier integrity through the tight junction zo- of aec by western blotting and immunofluorescence. methods. aec (a ) were incubated for h with apc ( lg/ml) or vehicle (control). subsequently, thr ( nm) or medium was added to the cell culture. for zo- western blotting, cell lysates were first ultracentrifuged ( , g, min, °c) to obtain membrane and cytosol fractions. then the samples were subjected to western blotting and the amount of zo- fractions was calculated by densitometry. for zo- immunofluorescence, aec were grown on glass coverslips and fixed in . % formaldehyde solution. zo- antibody was used to localize the tight junction and the zo- integrated optical intensity was then measured. . treatment with apc did not induce significant changes in any zo- amount of fraction protein analyzed by western blot. thr induced a *fivefold increase ( ± % of control values) in zo- membrane fraction while no changes were detected in zo- cytoplasm protein content ( ± % of control values). by contrast, apc concentration of lg/ml showed a clear tendency to reduce the effects induced by thr on zo- membrane fraction ( ± % of control values). for zo- inmunofluorescence, apc and thr treatments resulted in different patterns of zo- in the cell-cell contacts. after thr challenge cells showed discontinuous staining of zo- compared to untreated cells indicating a disruption of alveolar monolayer. conclusions. the increase in zo- amount of membrane fraction after thr challenge lends support to a protective mechanism avoiding cell-cell contacts disruption. treatment with apc reduced the increased zo- amount of membrane protein induced by thr suggesting an improvement of the barrier integrity in this model. ( ) interleukin- (il- ) is said to be involved in organ injury. we investigated the il- values of septic acute lung injury (ali) and acute respiratory distress syndrome (ards) patients. the subjects were patients during the -year period from to from whom it was possible to collect a blood specimen within approximately h of the onset of septic ali or ards. their mean age was years, and their mean apache ii score was . their sofa score was , and their mean pao /fio (p/f) ratio was . the p/f ratio was in the ali group and in the ards group. there were cases ( . %) in the -day mortality group, and cases ( . %) in the -day mortality group. the value of il- in died group was significantly higher than in survived group ( , ± , vs. , ± , pg/ml; p \ . ), and in the ards group also significantly higher than in ali group ( , ± , vs. , ± pg/ml; p \ . ). these results suggested that il- may play an major role in progression of ards in respiratory disorder as multiple organ failure (mof). [ ] . it is well-known that the pathophysiological mechanisms and factors involved in the liberation of no and the activation of inflammatory responses differ between aud and non-aud patients. objectives. the main hypothesis of this study is that ards patients with aud and non-aud differ in their response to the application of evidence based algorithms with respect to no response (aud patients are more frequent non-responders). patients with ards (meeting aecc criteria) were included in this ethically approved study. patients with severe chronic lung fibrosis and/or bridging for lung transplant were not included. patients were allocated to aud and non-aud patients. the auddetection was performed by the published algorithm [ ] . statistical analysis: wilcoxon-mann-whitney and chi-quadrat test was used. results. so far, patients with ards were included. prevalence of aud was % in our ards patients. baseline characteristics are given in table . frequencies of no nonresponse, extracorporeal lung support and mortality are given in table . frequency of no non-response was in tendency different: % in aud patients versus % in non-aud. overall mortality was % in aud patients versus % in non-aud patients. introduction. acute lung injury (ali) is a critical illness characterized by increased vascular permeability and impaired gas exchange leading to death in some cases. inflammation plays a pivotal role in the induction and maintenance of ali and is therefore therapeutic target to treat ali. rho, a small gtpase, is involved in the regulation of inflammation through the activation of recruitment of neutrophils to the site of inflammation and through activation of transcription factors such as nf-kb. we hypothesized that a rho kinase (rock) inhibitor, y- may be beneficial to dampen the inflammatory response in ali. male sd rats were intravenously pre-treated with either saline or rock inhibitor (y- , mg/kg). ali was induced by intratracheal instillation of mg/kg e. coli lipopolysaccharide (lps). control rats received saline intratracheally. h after the induction of ali, lungs were harvested and analyzed for myeloperoxidase (mpo) activity and expression of the proteins ijb, inos and enos. bronchoalveolar lavage fluid (balf) was used to assess total protein concentration as a measure of vascular permeability. pre-treatment with the rock-inhibitor resulted in significantly decreased levels of lps-induced mpo expression and prevented the upregulation of both lps-induced inos and enos expression. furthermore, lps-induced degradation of ikb was attenuated by pretreatment with y- . finally, y- improved vascular permeability by decreasing the lps-induced protein concentration in the balf. conclusion. inhibition of rho-kinase decreases lung inflammation and vascular permeability in acute lung injury and may therefore be a good approach to treat patients suffering from ali. we hypothesized that due to the cyclic changes of pulmonary air content there are po oscillations also in the mixed venous blood (pvo ), potentially influencing pao oscillations. in each of three healthy pigs of kg, anesthetized and ventilated with constant minute volume we studied three different tidal volume settings ( , and ml/kg) resulting in different respiratory rates. a calibrated oxygen probe (fiber optic, fluorescence-quenching probe, foxy-al ; ocean optics, dunedin, fl, usa) was inserted into the pulmonary artery through a fr catheter. the catheter position was previously controlled by pressure tracing. pvo was sampled with temperature compensation at hz with a multi frequency phase fluorometer (mfpf , tau theta, fort collins, co, usa) after a generated timestamp to synchronize with the electric impedance tomography (eit) signal (goettingen goemf ii, viasys healthcare, the netherlands) sampled at hz. eit and pvo were simultaneously recorded for min during each tidal volume setting and analysed with and without low pass filtering at the heart rate. we obtained pvo oscillations with amplitudes between to mmhg with the main frequencies matching the respiratory rate. ventilation with tidal volumes of ml/kg provided higher pvo amplitudes than ventilation with ml/kg. these results are preliminary and the source of the measured pvo oscillations is not clear. alternate backflow from the superior and inferior vena cava due to changes in intrathoracic pressures during mechanical ventilation may be responsible for these oxygen partial pressure oscillations in the mixed venous blood. conclusion. mixed venous oxygen partial pressure oscillates in accordance to the respiratory rate. whether arterial po oscillations are due to cyclic recruitment and derecruitment of the lung or to corresponding mixed venous oscillations remains to be evaluated. [ ] and in neonates [ ] . to our knowledge this is the first validation of the model using a large cohort of samples from intensive care patients. aim. to assess the ability of the severinghaus equations [ ] to estimate values for po and so in critically ill adult patients. methods. , sequential blood gas samples were analysed to validate the severinghaus oxygen dissociation curve, of these , measurements had a so b . % and were included in subsequent analyses. bland-altman plots were used to examine the agreement between measured po and that calculated from the severinghaus equations, and between measured and calculated so , both with and without correction for ph. the differences between measured and estimated values were analysed using paired t tests with a p value \ . considered significant. results. the severinghaus oxygen dissociation model accurately reflects the relationship between po and so observed in clinical samples. there is reasonable agreement between the measured and calculated values for po and so , with the majority of values falling between the lines of % agreement. there was a statistically significant difference between observed and calculated values of po even when adjustment for ph was made (p \ . ), however the mean difference between the groups was not clinical significant ( . mmhg when ph adjusted). there was also a statistical difference between measured and calculated values of so (p \ . ), again, however, this difference may not be considered clinically significant ( . %). patient data and severinghaus oxygen dissociation conclusions. the severinghaus equations accurately reflect the oxygen dissociation curve in critically ill adult patients and whilst they provide values for po introduction. zinc (zn) is an essential trace element, which plays a role in many biological functions including immune function. development of respiratory infections and changes in respiratory tract cells may be affected by low zn levels. in critically ill children mortality of septic shock and degree of organ dysfunction were associated to low blood zn levels , . our aim was to study serum zn in the beginning of acute respiratory failure (arf) and its association to development of organ failures and day mortality. during an -week study period (from april to june ) adult patients with arf were treated in intensive care units (= finnali-cohort). after consent blood sample for zn analysis was drawn at baseline). samples were taken in zn-free tubes, freezen and stored in - °c for analysis. all samples were analyzed with an atomic absorption spectrophotometry in the oulu university hospital laboratory. the range of normal values is - lmol/l. organ failures were assessed by daily maximal sequential organ failure assessment (sofamax) score. results. serum zn samples were obtained during h after the baseline with median time of h. only zn values were within and two over the normal range. median (iqr) serum zn levels were . ( . - . ) and . ( . - . ) lmol/l for survivors (n = ) and nonsurvivors (n = ), respectively, with no significant difference (p = . ). in patients with or without infection (pneumonia, respiratory infection or sepsis) during h prior to arf, zn levels were . ( . - . ) and . ( . - . ) lmol/l, respectively (p = . ). zn levels were significantly lower (p \ . ) in patients with cardiovascular sofa - than - , . ( . - . ) and . ( . - . ) lmol/l, respectively. a significant correlation of zn level and daily sofamax (spearman's q - . , p \ . ) was found (fig. ) . conclusions. low serum zn levels were detected in almost all patients with arf. no association to day mortality was detected to support the earlier findings with pediatric critically ill patients. however, we found a significant correlation to organ failure development in adult patients with arf. mountaineering is closely related to a range of adverse influences. the overriding factor that affects a climber may be the hypobaric hypoxia, which is compensated by hyperventilation and other adaptive changes in the pulmonary and systemic circulation. west ( ) theoretically predicted hypoxemia combined with respiratory alkalosis [ ] , and low oxygen saturation ( … %) has been observed on peak broad, karakorum [ ] . lack of adaptation is known as mountain sickness (occurrence … % [ , ] ), which may be alleviated by acetazolamide. the importance of understanding pathophysiology of mountaineering is dictated by the gradual expansion of western consumer-oriented society to higher altitudes. the goal of our study was to obtain precise information on changes in arterial blood gas composition, acid-base status, and degree of hemoglobin desaturation relative to altitude. materials and methods. experienced athletes-four males between and years and female years attempted to ascend mt. makalu ( , m) in april-may . acetazolamide , bid was used from april till may . femoral arterial blood rather than radial arterial blood was analyzed before reaching base camp ( background. ventilator associated lung injury is a complication of mechanically ventilated patients. knowledge about pathological pathways comes from animal studies, which are necessary to generate hypotheses to be tested in humans. various experimental methods of inducing acute lung injury (ali) have been used in animal models. the results of animal studies and human research appear to be conflicting; however, this may be a consequence from the different animal models used as such for comparison. we hypothesized that effects on gas exchange, respiratory mechanics, histo-pathologic lung damage and systemic inflammation are depending on the model of ali used. in five groups of pentothal anesthetized rats acute lung injury was induced by either lung lavage or hydrocloric acid aspiration. rats were then ventilated with lung protective settings in pressure controlled mode with positive endexpiratory pressure (peep) of cm h o or breathing spontaneously with continuous positive airway pressure (cpap) = cm h o for h. blood pressures, cardiac output, pulmonary mechanics and gas exchange were measured. results. the tidal volume was . ± . ml/kg in ventilated and . ± . ml/kg in cpap groups. respiratory rate and minute ventilation were constant in ali animals and controls, but showed variability in spontaneous breathing animals. only half of the cpap animals with ali survived [ h. no significant differences were found for pco , cardiac output or blood pressure between models, but mean arterial pressure decreased in ali. in the lavage and aspiration model, pao was lower after induction of ali ( ± and ± mmhg, respectively) than controls, and increased in lavage ( ± mmhg) but not the aspiration model ( ± mmhg) after h (p \ . ). dynamic compliance of the respiratory system decreased permanently after induction of ali to . ± . ml/cm h o (lavage) and . ± . ml/cm h o (aspiration) as compared to controls, which maintained at . ± . ml/cm h o after h. the lungs from five additional anesthetized, unassisted breathing animals, taken directly after induction, showed significant atelectasis, neutrophil infiltration and interstitial and alveolar edema (diffuse alveolar damage (dad) score . ± . ), as compared to control animals without ali (dad . ± . in ventilated, . ± . in cpap, respectively). the dad was higher in aspiration ( . ± . ) than in lavage ( . ± . ) induced ali, with no significant differences between ventilated and cpap animals. no hyaline membranes were observed. conclusions. anesthesia induces significant alveolar inflammation, which is partially reversible by use of peep. the ali model of acid aspiration induces persistent changes in gas exchange, respiratory mechanics and alveolar damage, which are more severe and consistent than those induced by the lavage model. background. acute lung injury (ali) is characterized by exaggerated inflammation and a high metabolic demand. mechanical ventilation can contribute to ali, resulting in ventilator induced lung injury (vili). a suspended animation-like state induced by hydrogen sulfide (h s) may reduce metabolism and co production, allowing for a lower minute ventilation to maintain gas exchange, thereby decreasing vili. h s may also limit lung injury via reduction of inflammation. the effect of h s-induced suspended animation on myocardial function is unknown. methods. in rats, vili was induced using a peak inspiratory pressure (pip) of mmhg and zero peep. controls were ventilated with a pip of and peep of mmhg. respiratory rate was adjusted to maintain normocapnia. suspended animation was induced by infusion of a h s donor, controls received saline. blood gases were drawn, bronchoalveolar lavage fluid (balf) was collected, lungs were removed. aortic flow was measured. statistics include kruskal-wallis and mann-whitney u. introduction. alveolar oedema is a hallmark of ards and ali. fluid clearance and the influence of anaesthetics on oedema resolution are poorly understood on a molecular level in the injured lung. oedema resolution is mediated by osmotic water reabsorption, following active sodium reabsorption via the apically located epithelial sodium channel (enac), driven by sodium-potassium-adenosin-triphosphatase (na ? /k ? -atpase). objectives. our aim was to investigate the influence of mac (= . vol%) sevoflurane on mrna and protein levels of enac and na ? /k ? -atpase in injured alveolar epithelial cells (aec). methods. primary culture of aec was stimulated with lipopolysaccharide (lps, lg/ ml) and exposed to normal air containing % co with or without sevoflurane. mrna levels were measured at h using the taq-man real-time pcr method. additionally, proteins for western blotting were analyzed at , and h (n = ). in the presence of sevoflurane mrna level of the a -subunit mrna of na ? /k ? -atpase in control cells was downregulated by % (p \ . ). a-subunit na ? /k ? -atpase protein expression, however, was not influenced by lps or sevoflurane at all time points. mrna of c-enac was decreased by % in the presence of sevoflurane and by % upon stimulation with lps. in the lps-sevoflurane group downregulation was even more pronounced with % (p \ . ) after h, but not statistically different from the lps group. on the protein level of c-enac protein expression a first change was observed at h with a downregulation of % upon lps exposure (p \ . ). sevoflurane did not have an effect of this transporter protein. previous studies have shown that halothane decreases na ? /k ? -atpaseand sodium channel activities in alveolar epithelial type ii cells [ ] . despite this finding for halothane, we could not see similar effects for the volatile anaesthetic sevoflurane. our results suggest that neither the driving force of alveolar oedema resolution, the sodium potassium atpase, nor c-enac, which is considered the rate limiting step in sodium coupled water reabsorption are influenced by sevoflurane and lps in an in vitro model of ards. to further characterize the impact of sevoflurane on water transport, functional analysis of these two transporters have to be performed. grant acknowledgement. objectives. we evaluated the effects of two nebulised sfa perfluorohexyloctane (f h ) and perfluorobutylpentane (f h ) at different dosages ( ml/kg vs. . ml/kg) on pulmonary mechanics and gas exchange in healthy lungs. design. after approval by the local animal care committee, prospective, randomized animal study. subjects. thirty-five new zealand white rabbits. interventions: tracheotomised and ventilated juvenile rabbits were nebulised intratracheally with either a high or a low dose of two different sfa (f h low/high and f h low/high ) or saline (nacl). ventilated healthy animals served as controls (sham). arterial blood gases, lung mechanics, heart rate and blood pressure were recorded prior to nebulisation and in min intervals during the -h-study period. results. immediately after starting aerosol therapy p a o /f i o -ratio and dynamic lung compliance decreased in all groups, with the exception of the f h low group which behaved like the sham group. although p a o /f i o -ratio showed a continuous improvement in the other groups over time respiratory mechanics still remained impaired. high dose groups with nebulisation of liquid perfluorohexyloctane (f h high ), perfluorobutylpentane (f h high ) or saline (nacl) showed no significant differences neither in oxygenation, blood pressure nor in pulmonary compliance and resistance. in contrast to f h high , there were no residues of f h high detectable in bronchoalveolar lavage. regarding f h low we were not able to detect any adverse effects on gas exchange or pulmonary mechanics. additionally, wet-dry-ratio of apical lung tissue samples revealed no significant edema. conclusions. high dose aerosolized sfa ( ml/kg), either f h or f h , equals effects of high dose inhalation of saline. when comparing the low-dose sfa-groups, there is a convincing discrepancy in favour of f h . f h low impairs pulmonary function, whereas a low dose application of f h (low) shows no interference. this may be due to the faster evaporation of f h . a new sfa-based pulmonary drug delivery system for lipophilic or water-insoluble substances could be developed on the basis of a low-dose application of f h . objects. hypertonic exposure reduces cell volume and thereby creates a relative excess of plasma membrane (pm). as a result the lipid bilayer of the pm can simply unfold with a minimal increase in lateral tension when an externally imposed shape change demands it. to test this hypothesis, we determined the effects of osmotic pressure on the susceptibility of deformation injury and pm wound repair. we measured deformation injury and repair responses of a . cell culture media were consisted of x hmem and mannitol (v/v / ) with osmolarity of (iso), (hyper) and mosm (hypo). cells conditioned with media were either stretched or deliberately injured with a scalpel. the fraction of wounded and healed cells was measured using a dual label method. . ( ) exposure to a hypertonic environment tends to lower the susceptibility of a to deformation injury ( . ± . % for iso, . ± . % for hyper), while exposure to a hypotonic environment uniformly increases it ( . ± . % for hypo) in stretch injury. introduction. the migration of polymorphonuclear leukocytes (pmns) into the lung plays a critical role in the development of acute lung injury (ali). adenosine receptor a (a ar) is one of four g protein-coupled adenosine receptors that has been demonstrated to modulate pmn trafficking in various models of inflammatory disorders including sepsis and asthma. however, the role of a ar in ali has not been investigated systematically yet. the objective of this study was to determine the role of the a ar in a murine model of lpsinduced lung injury and in an in vitro transmigration system with human cells. methods. the migration of pmns into the different compartments of the lung was determined by flow cytometry in adult male c bl/ mice (wildtype [wt] ) and homozygous a receptor knockout (a ko) mice. we used chimeric mice that were generated by transferring bone marrow between wild-type and a ko mice to differentiate the role of a on hemopoietic and nonhemopoietic cells. furthermore, microvascular permeability was assessed by the extravasation of evans blue and the release of chemotactic cytokines into the alveolar airspace was determined by elisa. paraffin-embedded sections of the lung were stained for pmns after lps inhalation to illustrate their accumulation in the lung. in a human in vitro assay, we quantified neutrophil transmigration across an epithelial monolayer (a cell line). in all murine in vivo experiments and in the in vitro transmigration assay, we assessed the effectivity of the specific a -agonist cl-ib-meca. all statistical analyses were performed by using anova. p \ . was considered statistically significant. results. inhalation of lps significantly increased the number of pmns in wt and a ko mice in all lung compartments. no differences in pmn counts were observed between wt, a ko, and chimeric mice. pretreatment with cl-ib-meca led to a significant decrease of pmns in all lung compartments of wt mice but not in a ko mice. pharmacological activation of a ar diminished the lps-induced microvascular permeability in wt mice but not in a ko mice. upon lps-inhalation, a ko mice exhibited significantly higher levels of the cytokines cxcl und cxcl / in the alveolar airspace than wt mice. in wt mice, pretreatment with cl-ib-meca reduced levels of tnfa and il- significantly. transmigratory activity of human pmns across an epithelial monolayer was reduced when a was activated in pmns. in contrast, pretreatment of the epithelial cells did not inhibit migration of pmns. introduction. lung overdistention during mechanical ventilation causes an increase in pulmonary vascular permeability, which is characterized by interstitial and alveolar edema secondary to a diffuse endothelial and epithelial injury. thrombopoietin (tpo), a humoral growth factor that stimulates the proliferation of megakaryocytes, has also been identified as a pro-inflammatory mediator in various clinical conditions. the receptor of tpo, c-mpl, is constitutively expressed on endothelial cells and may modulate the permeability of the endothelium. we investigated the contribution of tpo in the development of acute alveolar edema formation by mechanical stretch. in an ex-vivo model of mechanical ventilation (mv), lungs of c bl mice were ventilated for h with high stress pressure cycled ventilation (end inspiratory pressure = cm h o, peep = cm h o, i:e ratio = : ) and perfused with % bovine serum albumin rpmi medium at a rate of ml/min, in the presence or absence of tpo ( mg/ml). following ventilation, lung elastance was measured and protein concentration was analyzed in the bronchoalveolar lavage. data are mean ± se. mechanical ventilation (mv) to treat patients with ards or acute lung injury (ali) has the end objective to increase the dynamic functional residual capacity (dfrc), thus increasing overall functional residual capacity (frc). simple methods to estimate dfrc at the end of expiration for a given positive end expiratory pressure (peep) would provide a valuable metric to track and modulate therapy. however, such methods do not exist and current methods are time-consuming and relatively invasive. methods. this study utilizes a constant stress strain ratio for an individual patient's volume responsiveness to peep to estimate dfrc at any peep. the estimation model identifies two population parameters from clinical data to estimate a patient-specific dfrc, b and mb, where b captures physiological parameters of frc, lung and respiratory elastance and varies depending on the peep level used, and mb is the gradient of b versus peep. dfrc was estimated at different peep values ( , , , , ) cm h o, and compared to the measured dfrc for ali/ards patients to validate the model. patients and methods. in a years period patients ( males, females) with haematological malignancies were admitted in icu. malignancy type, reason for admission, haematological profile, requirement for invasive ventilation, bronchial and blood cultures and survival rate were recorded. results. patients suffered from: hodgkin's lymphoma ( ), non-hodgkin's lymphoma ( ), chronical lymphocytic leukaemia ( ), acute myelogenous leukaemia ( ) and multiple myeloma ( ) . admission to icu was precipitated by: emergency surgical procedure ( ), respiratory failure ( ), sepsis ( ), pulmonary oedema ( ) and coma ( ) . pulmonary infiltrates was the main finding in chest x-ray. bronchial secretions cultures were positives in patients while blood cultures were positives in patients. apache ii score ranged from to (average . ) and the icu days ranged from to (average . ). all the patients required invasive ventilation. all the patients with sepsis and serious neutropenia were died, while the total mortality was / ( . %). conclusion. the admission of patients suffering from haematological malignancies in icu is associated with high mortality. immunosupression that renders them susceptible to infections, thrombocytopenia, and invasive ventilation are factors that contribute to this. early recovery of bone marrow and non invasive ventilation could improve the outcome in these patients. in liver transplanted patients, immunosuppressive therapy can increase the risk of infections and post-operative arf. nimv has been proposed as an alternative technique to reduce complications related to endotracheal intubation. the aim of our study was to evaluate nimv in liver transplanted patients, developing arf in the post-operative period. materials and methods. in this study we evaluated liver transplanted patients, developing postoperative arf. measurements of respiratory and haemodynamic parameters were performed at baseline, after h and at the end of the treatment. we evaluated intubation rate, nimv tolerance, length of stay in the icu (los), icu and hospital mortality. results. ( %) out of patients were successfully treated with nimv, while ( %) failed and were intubated. we observed no significant differences among groups in gas exchange, but rr was significantly reduced in the success group during treatment (p \ . ). in both groups we found no significant differences in pao /fio initial improvement, but the success group showed a significantly higher rate of pao /fio sustained improvement (p \ . ). no significant differences between the two groups were found in terms of hours and days of nimv. success and failure groups were significantly different in saps ii (p \ . ) los (p \ . ), icu and hospital mortality ( vs. %, p \ . , vs. %, p \ . ). reasons for nimv failure were not related to respiratory causes, but acute systemic causes such as septic shock and mods. conclusions. nimv can represent a valid alternative to invasive mechanical ventilation for the treatment of postoperative arf in liver transplanted patients; in nimv success patients reduced los and mortality can be expected. the influence of body posture on expiratory flow-limitation (efl) was estimated in flowlimited, mechanically ventilated patients using the negative expiratory pressure (nep) method. a device especially designed and in build in an evita -draeger respirator allowed the application of a pressure equal to- cm h o, starting at ms after the onset of expiratory flow and sustained throughout the end of expiration. patients were considered flowlimited, if despite the application of nep part or the expiratory flow-volume curve was superimposed on the baseline curve. patients were studied in supine and in semi-seated position ( ) at baseline and then min after administration of bronchodilators ( mg of inhaled salbutamol) with a nebulizer connected to the inspiratory port of the ventilator. supine position was significantly related to the occurrence of efl (p = . ). efl was abolished in % of our patients when changing from supine to semi-seated position, while in general a significant improvement of efl was noticed (from to % of v t , p = . ). significant improvement of efl was achieved as well (p = . ) after bronchodilative therapy. peepi was the only variable significantly related to efl improvement when changing body posture from supine to semi-seated, while for bronchodilative therapy, none of the variables studied was significantly related to efl improvement. l. c. woodson , university of texas medical branch, anesthesiology, galveston, usa, shriners hospital for children, anesthesiology, galveston, usa aims. laryngeal injuries are common among burn patients and can result in long term functional deficits. we have included careful laryngeal examination with our initial fiberoptic bronchoscopic evaluation of burn patients. the goal has been to allow early identification of laryngeal injuries and to facilitate laryngology consultations. methods. digital video recordings were made of upper airway endoscopies performed during airway management on admission or at the time of anesthesia for initial wound excision. these recordings were used to identify laryngeal injuries and to facilitate laryngology consultations. a wide variety of laryngeal injuries were identified and the digital recordings (which can be communicated by email) greatly facilitated laryngology consultations. in many cases these recordings guided therapeutic interventions and were often sufficient to avoid a separate exam under anesthesia. diagnosis of thermal necrosis provided an indication for early tracheostomy. identification of the mechanism of mechanical airway obstruction (e.g. supraglottic edema, fibrinous exudates, granulomas, vocal fold dysmotility) resulting in failure of a trial of extubation frequently guided therapy. early identification of posterior glottic damage provided more timely corrective laryngological interventions. educational use of these videos helps increase awareness of risks of laryngeal injury in thermally injured patients. aims. the most used weaning predictor f/v t ratio, is not a consensual predictor. when it was reported on the first time, this ratio was considered highly sensitive and specific. but others papers seems to disagree with it, suggesting other cutoff values to determine weaning failure in specific populations, as the elders. advanced age is thought to be an import associated factor in the intensive care unit (icu), but its effect on the weaning process is unclear. no studies have found strong evidence that conventional weaning parameters are reliable for this population. the widest used weaning criteria, f/v t ratio, does not seems to keep the same performance in this kind of population. the main purposes of this study were to identify the possible differences of the f/v t ratio measured in a spontaneous breathing trial, between an adult and an elderly group. we designed a protocol to study the variation, sensibility and specificity of the frequency-to-tidal volume ratio between an adult group (ag; up to years) and an elderly group (eg; older than years) in a daily weaning screening trial. methods. the study cohort comprised patients ready to undergo weaning trial. the parameters studied were: weaning success ( h of spontaneous ventilation after extubation), respiratory rate (f), tidal volume (v t ), frequency/tidal volume ratio (f/v t ), gasometric and ventilatory parameters. the weaning method was spontaneous breathing trial (sbt). measurements were made in the beginning of sbt (t ) and min after (t ). we analyze possible differences in the sensibility and specificity of the f/v t ratio between elderly and adults and compare with previous values already published. the chi-square test, anova and the t test were used in the statistical analysis. weaning success was . % in eg and . % in ag (p = . ). the baseline characteristics were similar. comparisons of ag and eg at t and t showed statistical differences in weaning criteria: f, v t and f/v t ratio. conclusion. weaning success in our study was low, but similar to the described in other trials. elderly patients showed higher f and lower v t . consequently, f/v t ratio was lower too. the area under the roc curve for f/v t ratio was smaller than already published. results. / ( %) were successfully extubated and patients required re-intubation. the demographic data showed no differences in age, bmi, apache ii, icu admission diagnosis or sex distribution between groups (table ) . patients who failed extubation had small but statistically significant differences in vital capacity (vc), peak negative inspiratory pressures (pnip), pao /fio ratios (pf) and were ventilated longer prior to extubation. paradoxically, patients failing extubation had positive end expiratory pressures that were statistically but not clinically significant higher. the ratio of respiratory rate to tidal volume (f/vt) was not significantly different. patients failing extubation were also more likely to have weaker cough, gag, level of consciousness as measured by glasgow coma scale and more secretions ( table ). having no cuff leak did not predict failure of extubation. the most common reasons for reintubation were secretion retention and/or absence of cough ( %). pressure support ventilation (psv), a widely used assisted mode, has the purpose to avoid diaphragm disuse allowing the patient to generate spontaneous inspiratory efforts optimizing comfort and work of breathing. however, still little is known about the individual response of respiratory muscles under these conditions. we hypothesized that respiratory muscles of patients ventilated with clinic psv might result, at least sometimes, excessively unloaded. we performed an observational study in the intensive care unit on patients ventilated with psv set by the clinician in charge. twenty intubated, mechanically ventilated patients ( ± years old) during the weaning phase entered the study. the patients had no sedation at least for the last h. respiratory timing, tidal volume (v t ), peak airway pressure (paw peak ), electrical activity of diaphragm expressed as percentage of its maximum (edi/ edi max ), inspiratory (ptpes) and diaphragm (ptpdi) muscle effort were measured during min of clinic psv. results. we found that seven out of twenty patients generated a negative pes swing only during the psv inspiratory triggering phase (psv t ) in comparison with the remaining patients in whom pes was negative throughout most of the mechanical breath (psv n ). in the psv t group, pes swing was either flat or positive after inspiratory triggering. therefore, in the psv t group both ptpes /min and ptpdi /min were fivefold lower than normal values. v t / predicted body weight (pbw) was significantly higher in the psv t versus psv n group (see table ). during weaning with psv: ( ) a significant number of patients ( %) showed a pes shape similar to that observed during pressure assist/control modes, and inspiratory muscle effort abundantly lower than normal, both indicating excessive inspiratory muscle unloading; ( ) among the variables used to set psv, only a high v t /pbw (higher than ml/kg) hallmarked excessive unloading; ( ) due to the ample prevalence of the phenomenon, the question whether high levels of inspiratory muscle unloading can cause detraining and prolonged mechanical ventilation merits an answer from further research. introduction. the liberation from mechanical ventilation (mv) should be done as soon as possible in order to avoid complications and the risks associated with prolonged unnecessary mv, such as ventilator-associated pneumonia, ventilator induced lung injury, and increased icu and hospital stay. this procedure should be carried out properly and safely. objective. evaluate the extubation success rate, mv time and weaning time using a daily weaning screen followed by a spontaneous breathing trial (sbt). patients who were ventilated for more than h were subject to this procedure, which was carried out by respiratory therapists. methods. in our icu, between february and august of , all intubated patients who were ventilated for more than h underwent a daily weaning screen, which contained variables such as hemodynamic, gas exchange, consciousness and resolving the need for mv. if these variables were stable, these patients were submitted to a sbt and were extubated if they did not show any signs of respiratory discomfort or hemodynamic changes for at least min. conclusion. the use of a daily weaning screen followed by a sbt was associated with a high extubation success rate and a very short weaning duration with % of unsuccessfully extubations. c. chatt , d. pandit , g. raghuraman birmingham heartlands hospital, critical care, birmingham, uk, birmingham heartlands hospital, birmingham, uk aim. the aim of the study was to assess the impact of pmv on the course of weaning in mechanically ventilated patients. we wanted to assess the optimal pressure support at which pmv can be initiated which would enable prolonged use of pmv without affecting the duration of respiratory support. method. data on all patients who were mechanically ventilated for greater than eight days were obtained from icnarc database, who underwent tracheostomy as part of their weaning process. satisfactory level of pressure support was achieved (between and cm h o) pmv was introduced into the patient's breathing circuit and spontaneous ventilation was attempted. we applied mann whitney u tests for parametric data, fisher exact tests for non-parametric data and anova was used to compare the three groups with different pressure supports at initiation of pmv. a p value \ . be statistically significant. results. patients who were ventilated for greater than eight days identified. of these, patients were excluded because they did not have a tracheostomy during their period of ventilation. of the remaining patients, pmv was used in patients ( %). there were no significant differences between the demographic data (sex, age) or the data on admission to intensive care (apache ii score, ratio of medical to surgical patients) and duration of mechanical ventilation between the two groups. however, there were significant differences in the mortality, total respiratory support days after tracheostomy and length of stay in intensive care and length of hospital stay between the two groups. in the group on pmv, no record of aspirations was found documented on the intensive care charts. in patients in whom pmv was used (n = ), pmv was initiated at cpap (continuous positive airway pressure) in patients ( %). patients ( %) had pmv initiated at a pressure support of b cm h o and in patients ( %) pmv was initiated at a pressure support of [ cm h o. there was a significant difference in the duration of mechanical ventilation post tracheostomy (p = . ) and the length of hospital stay (p = . ) between the two groups, with the cpap group being ventilated for a shorter duration but with a longer stay in hospital the same difference was shown when comparing three groups of pressure support when pmv was commenced (cpap, pressure support b cm h o and pressure support [ cm h o). however, in the pressure support [ cm h o group we observed that the duration of use of pmv was lower than in the other two groups despite longer duration of mechanical ventilation and total respiratory support days. although this was not statistically significant, it could be clinically significant. our study suggests that use of pmv at pressure support b cm h o could increase the duration of its use without affecting the length of mechanical ventilation. we would therefore recommend weaning to a pressure support b cm h o before pmv is commenced in the acute setting. no data are available concerning the oxygenation target to aim during the weaning phase from mechanical ventilation. also, in opposition to ards patients there is no clear recommendation for the upper limit of spo to maintain during weaning. this study is part of a research project on peep and fio settings automation during mechanical ventilation. methods. this observational study was designed to assess the spo target aimed during the weaning phase of invasive mechanical ventilation (fio b . and peep b cm h o). patients were recruited in icus from several countries (canada, france, italy, tunisia, argentina). the following data were prospectively collected by the respiratory therapists at each round during a months period: spo , fio , peep level, ventilatory mode, anatomic site of the pulse oxymetry sensor, quality of the spo signal. results. data from centers ( icus) from quebec city, canada, and center from créteil, france are available. patients were prospectively included. , observations were performed. the mean level of fio was . ± . with fio c . and . in . % and . % of observation times respectively. the mean level of peep was . ± . cm h o and was below, equal or above cm h o in . , . and . % of the cases respectively. the most frequent ventilatory modes were pressure support ( %), simv ( %) and acv ( %). the pulse oxymetry sensor was applied on a finger of the hand in . % of the cases and was deemed of good quality in % of the time. the mean spo was . ± . % for the whole population and was . ± . % for patients with fio c . . spo was higher than % in % the observations. desaturation with spo below % were recorded in . %. the spo signal was deemed available by the bedside nurse in . ± . % of the time. conclusion. this study demonstrates that spo levels may be maintained at high levels unduly. this may have an impact on the weaning phase of mechanical ventilation. this study also shows that the spo signal availability was high enough to be used in a closed-loop oxygenation system. introduction. automated weaning systems are viewed as a challenge to weaning decision-making autonomy by some clinicians. clinician perceptions of the utility of such systems may influence uptake in to practice. to assess the perceived utility of the automated weaning system, smartcare/ ps. a survey was generated based on comprehensive literature review and -year's experience using smartcare/ps. survey pilot testing was conducted with senior clinicians experienced in smartcare/ps weaning in an independent icu. questions addressed perceived system usability and appropriateness of automated weaning, system benefits and disadvantages, as well as patient indications deemed suitable and unsuitable for smartcare/ps weaning. participants were also asked to indicate if they would continue using smartcare/ps on trial completion. the survey was administered to clinicians on completion of a randomized controlled trial conducted to compare smartcare/ps to non-protocolized weaning . of staff surveyed, surveys were returned by nurses and doctors (response rate %). eight respondents had no experience with smartcare/ps despite the year trial duration, leaving surveys with evaluable responses. the majority of respondents perceived smartcare/ps was easy to activate ( / , %) and to use once activated ( / , %). the system was observed to wean appropriately by / ( %) respondents; experienced smartcare/ps to wean inappropriately. comments on inappropriate weaning identified clinically unacceptably increases of pressure support (ps) for patients with profound tachypnea and complicated lung pathology. smartcare/ps' ability to reduce the overall duration of weaning was questioned by all but / ( %) respondents. ps adjustment according to patient requirements was the most frequently perceived benefit ( / , %). most respondents did not perceive any advantage of smartcare/ps for patient comfort / ( %), assessment frequency ( / , %) and automated control of weaning ( / , %). less control over weaning was the most regularly cited disadvantage of smartcare/ps ( / , %). system issues such as program abortion without identifiable reason and mandatory peep reduction prior to a spontaneous breathing trial to assess readiness for separation were less frequently cited disadvantages [ / ( %) and / ( %) respondents respectively]. most respondents ( / , %) felt smartcare/ps was best suited for weaning postoperative patients and should be avoided for patients with neurological dysfunction ( / , %). only / ( %) respondents stated they would not continue to use smartcare/ps. clinicians demonstrated moderate acceptance of smartcare/ps. more work is needed to identify those patients more likely to benefit and confirm the overall utility of smartcare/ps as a weaning tool. introduction. physician approaches to ventilation withdrawn varies among physicians whereas the prompt recognition of respiratory failure reversal and usefulness of weaning protocols in reducing duration of mechanical ventilation (mv) have been largely demostrated as nursing staff attend patients h a day its leadership in this process can be effective and safe. objective. to demonstrate that a nurse-directed protocol to withdraw mv could reduce a % its duration. prospective sequential study performed in two periods. during de first period ( months) data concerning weaning definite criteria appearance, duration of mv, reintubation or need for nonninvasive ventilation (nimv) and demographic data were collected to all mechanically ventilated patients blinded by attending nurses and physician. after a three months phase of staff training there was a second months period where weaning criteria were checked al each nurse working shift during the first days of mv. when criteria were fullfilled a min of spontaneous breathing trial was perfomed and tracheal tube removed if there were no intolerance criteria. same data as the first phase were collected. we used mann-whitney s u test to compare mv duration, time to reach weaning criteria (trwc) and extubation delay (mv duration minus trwc). weaning failure was compared using x square. data are presented as median ( - percentile). results. patients were screened ( in the first period and in the second) but only patient reached weaning criteria in the first days ( in the first period in the second), . % men, aged ( - ) years. aim of this study is present our experience about elective bedside pdt with the blue-rhino kit over an year period, in order evaluate its efficacy in terms of intraoperative and postoperative complications. patients and methods. the study included a total of consecutive icu patients requiring tracheostomy. all pdt were performed by icu staff physicians at patients' bedsides, using a blue rhino kit. the following data were recorded: age, sex, simplified acute physiology score (saps) ii, fraction of inspired oxygen (fio ) before the tracheostomy, days on mechanical ventilation before the tracheostomy, bleeding, tracheal tear, subcutaneous emphysema, pneumothorax, wound infection, hypotension, lowering sao during the procedure, inability to complete the procedure, and procedural mortality. distance follow-up included fiberoptic bronchoscopy to evaluate tracheal stenosis. results. there were a total of ( . %) complications (tracheo oesophagel fistula and bleeding). forty -one patients died in the icu ( %), although none of these deaths were related to technique complications. mean duration of the procedure was . ± . minutes. the pdt performed at bedside in the icu, using the blue rhino kit is a simple and safe procedure that offers many advantages in terms of safety and efficacy. objectives. questioning the need for several specialized physicians or extra assistance to perform a single percutaneous tracheostomy using fibrescopic tracheoscopy, we performed a prospective study into the complication rate of percutaneous tracheostomy without tracheoscopy on our mixed medical and surgical icu. , consecutive patients were included after having received a percutaneous tracheostomy. indication for tracheostomy was always a long anticipated duration of mechanical ventilation. if no contra indications were present, percutaneous tracheostomy was performed. if contra indications against the use of percutaneous tracheostomy without tracheoscopic control were present, tracheoscopy was performed to ensure maximum patient safety. the mean age at the time of receiving a tracheostomy was . ( - ) years. the cohort consists of male patients en female patients. only ttwo percutaneous tracheostomy were performed under fibrescopic control due to contra-indications for an uncontrolled procedure. in procedure, sixteen minor, and no major complications were encountered. this resulted in a . % minor complication rate. conclusions. the number of complications in our group is approximately the same as those which are suggested in international literature where tracheoscopy was performed during percutaneous tracheostomy. none of the complications encountered could have been prevented by the use of tracheoscopy. therefore we postulate that in the hands of an experienced team and in adherence to strict guidelines, percutaneous tracheostomy can safely and successfully be performed without tracheoscopy. objective. to assess the risks and complications associated with the bedside pdt in our years experience of over pdts in icu. pdt is a relatively newer technique and has been introduced as an alternative to open tracheostomy as a safer and convenient procedure. however, the risks and complications of the pdt have not been highlighted in the icu of a developing country. a retrospective analysis of the data gathered from patients undergoing pdt was done in a -bedded tertiary level multidisciplinary icu of a teaching hospital. the data was collected between april and march . all intubated patients with indications for elective tracheostomy, as well as patients who required emergency tracheostomy were included in the study. demographic and other clinical details of the patients who underwent pdt were collected. griggs [ ] technique was most commonly adopted while other adopted techniques were ciaglia [ ] , white tusk/blue rhino tapered dilator and percutwist technique. a total of , pdts were done in , patients, over a period of years. of the , patients ( %) were males and ( %) were females. the mean age of patients was . years. the average duration of intubation before pdt was . days. ( %) pdt were done bedside in icu while ( %) were done in wards, coronary care unit, high-dependancy unit and liver transplant unit. griggs technique was adopted in ( . %), ciaglia in ( . %), white tusk/blue rhino tapered dilator technique in ( . %) and percutwist technique in ( . %) patients. long-term ventilation was the most common indication in ( . %) followed by airway protection in ( . %), facilitation of weaning in ( . %) while airway obstruction/difficult intubation was observed in ( . %) patients. pre-procedure coagulopathy was observed in ( . %) patients, ( . %) were morbidly obese while ( . %) required emergency tracheostomy. no complications were observed in ( . %) patients. procedural complications were seen in ( . %) patients. bleeding from the site was the leading complication affecting ( . %) patients. difficult tube placement was seen in ( . %) patients, premature extubation in ( . %), false passage in ( . %), guidewire dislodgement in ( . %), subcutaneous emphysema in ( . %), arrhythmia in ( . %) and bleeding requiring transfusion was seen in ( . %) patients. no procedure related mortality was observed. conclusion. on the basis of this large single centric study we found that pdt is a safe, reliable and convenient procedure which can be easily performed bedside by experienced intensivists. results. of the recipients who underwent pdt, were liver, kidney and heart transplant recipients. the respective mean values for age, weight and apache ii score were . ± . years, . ± . kg, and ± . all pdts were performed at bedside by experienced staff anesthesiologists with direct bronchoscopic guidance. in all cases, the indication for pdt was prolonged mechanical ventilation due to acute respiratory failure. the mean time from transplant to pdt was ± months and the mean duration of endotracheal intubation before pdt was ± days. twelve patients had coagulopathies. the calculated lung compliance and pao :fio ratio improved after pdt ( . ± . vs. . ± . ml/cm h o, p = . and ± vs. ± , p = . respectively). transient hypoxemia (n = ) and mild extratracheal bleeding (n = ) were the only early complications. there were no procedural failures and no pdt-related late complications and deaths. conclusion. the results suggest that percutaneous dilational tracheotomy is an efficacious and safe technique for prolonged airway management with improved ventilatory mechanics in solid organ transplant recipients. a. vianna , g. cabral , r. azambuja , g. carleti , t. balbi , g. pereira clinica são vicente, rio de janeiro, brazil aims. we studied diferent aspects of tracheostomy procedures performed in intensive care units (icus) located in the municipality of rio de janeiro and compared them with the medical literature. a questionnaire was elaborated and sent through email to the coordinators of every icu in the city of rio de janeiro in the period of july to august . the questionnaire was sent to the coordinators of the icus located in rio, and was answered by ( . %) of them. among the studied icus, ( . %) are public, ( . %) are private, and ( . %) are part of university hospitals. ( . %) are medical/ surgical, ( . %) are medical, and ( . %) is a surgical unit. the average number of beds is ± . . the decision to perform the procedure is taken by the icu team in ( . %), by the patient's primary team in ( . %), and by both in ( . %). tracheostomy is performed by a surgeon in ( . %) units, by an intensivist in ( . %), and by both in ( . %). the procedure is performed at the bedside in ( . %) of the icus. the most frequent indications for tracheostomy are: prospect of prolonged mechanical ventilation, coma, and airway protection. . % of are performed between the first and second week of mechanical ventilation, and % between the second and third week. control chest x-ray is performed in . % of the units. surgical tracheostomy is available in all the studied units. only ( . %) units perform percutaneous tracheostomy. the reasons given for the preference for surgical tracheostomy were the lack of a qualified team for performing the percutaneous tracheostomy or material needed for this procedure. all icus that perform the percutaneous procedure use the ciaglia technique with bronchoscopic guidance. late followup is performed in ( . %) of the studied units. the study showed great differences between the tracheostomy protocols used in the hospitals of rio de janeiro and those found in the medical literature. in particular, the use of percutaneous tracheostomy is still infrequent in the icus of rio. on all the patients, aged at least years or more, admitted to our postoperative icu since january through december , we collected demographic profiles, operative data and short and long-term outcomes. spss . was used for statistical analysis and p \ . was considered the level of significance. a total of patients ( . %), . % males and with a median (iqr) age of ( - ) were admitted to our post-operative icu over the study period. iddm was recorded in the . % of the population, copd in the . %, hypertension in the . %, chronic renal failure in the . % and arteriopathy in the . %. out the total population, . % of patients, with a median (iqr) pre-operative crs of ( - ) underwent a coronary-artery bypass grafting (cabg) surgery, whereas . % of them, with a preoperative median (iqr) nyha of ( . - ) needed a valve replacement (vr) and . % of them combined (cabg ? vr) operations; moreover, . % of patients underwent other type of cardiac and aortic surgery. overall median (iqr) post-operative mechanical ventilation length was ( . - ) hrs. while no statistically significant difference was recorded in terms of mv duration among the four surgical groups. overall recorded mortality rate was %, with the lower . % for cabg and the higher . % for vr (p = . ). kaplan meier curves showed no differences in survival likelihood at th (log rank = . , p = . ) th (log rank = . , p = . ) and th (log rank = . , p = . ) days after surgery among the different surgical groups. conclusions. the outcome after heart surgery in octogenarians is excellent; the operative risk is acceptable and the late survival rate is good. therefore, cardiac surgery should not be withheld on the basis of age alone. introduction. re-do cardiac operations have been reported to be increasing in incidence and are associated with a higher operative risk [ ] . this study aimed to determine the impact on intensive care provision. methods. data from , procedures spanning twelve years (april -march ) was examined. the re-do operations were further analysed by gender, age, pathology (new, progressive, combined) , duration between procedure, theatre time, length of stay, complications and mortality. as the number of cardiac operations performed has increased over the twelve year period, the relative incidence of re-do procedures have remained stable at . %. operative length at re-do was significantly longer (mean min vs. ) however anaesthetic time pre surgical incision was not significantly increased. subsequent length of stay on the intensive care or high dependency unit increased by % (mean . vs. . days), with higher complication rates affecting all systems (except post operative myocardial infarction). renal and pulmonary complications showed the most significant increases. renal related complications occurred in % and pulmonary in . % of cases which represents an and % increase on first operation rates. infection rates were also significantly increased at double that of the initial procedure. the total hospital stay was found to be % longer (by . vs. . days, respectively) while in hospital mortality increased from . % at initial procedure to . % at re-do. mortality rates were further elevated in the presence of renal failure post operatively, as re-do valve mortality increased from . to % and re-do cabg from . to % in this subgroup. conclusion. these results, combined with the stability of percentage re-do surgery over the twelve year period, enable specific planning and management of intensive care provisions. the knowledge of extended theatre times and subsequent stay in intensive care/high dependency units has a further impact on the throughput of routine cases. the data also highlights the increased costs associated with these patients, as they not only require longer hospital stays but also suffer increased complications requiring more investigations and interventions. specific costing therefore applies to this subgroup of intensive care patients. objectives. we aimed to assess hrqol at days after surgery in relation to preoperative hrqol. we compared patients with decreased hrqol to patients with unchanged or increased hrqol to identify disparities between these two groups. a prospective cohort study including patient scheduled for cardiac, vascular, abdominal and orthopedic surgeries in a tertiary hospital was performed. patients filled-out a hrqol questionnaire (sf- ) the day before surgery and days after. preoperative, intraoperative, postoperative data were collected. changes of pre-and postoperative physical component summary ( simultaneus kidney-pancreas transplantation is the best treatment option for type diabetic patients with chronic kidney disease. currently, the medical and surgical complications have decreased significantly, although these represent a high risk of morbidity and mortality in the short term. objectives. this study sought to investigate the incidence of medical and surgical complications, the clinical characteristics and prognostic factors influencing graft and patient's survival in a recent cohort of pancreas-kidney recipients. patients and methods. the present study included patients who received simultaneous pancreas-kidney transplantation in our center from january to february . we studied demographic, clinical and immunological characteristics of patients, and surgical and medical complications during his admission to intensive care unit. results. the average age of recipients was . years and mean age of donors was . years. the median cold ischemia time was . h ( % confidence interval - ). the average stays on the waiting list was . days. % of patients were extubated within the first h. % of patients required transfusion during their icu admission, amine infusion was started at % patients in the early hours. during follow-up, surgical reintervention in the immediate postoperative occurred in % of the patients. major surgery complications reported in the literature are graft thrombosis, although in our serie there have been only kidney graft thrombosis and pancreas graft thrombosis. only % of patients died within the first months posttransplantation surgery. conclusion. surgical complications after pancreatic transplantation remain a significant concern. hence we our results add further evidence to support the notion that the double and simultaneous pancreas-kidney transplant is in fact the treatment of choice in selected patients with end-stage renal failure due to type diabetes mellitus. a. shono , t. mihara , y. murakami , j. ota , f. kono , y. saito shimane universiy hospital, anesthesiology, izumo, japan pulmonary catheter is widely used for cardiac surgery. the complications of indwelling pulmonary catheter, such as perforation of pulmonary artery, pulmonary embolism, are well known. however, the thrombosis associated with introducer sheaths has received much less attention. we evaluated the incidence and risk factors for internal jugular vein thrombosis (ijvt) associated with introducer sheaths for pulmonary catheter after cardiac surgery. methods. the patients who underwent cardiac surgery and insertion of introducer sheaths ( . f) at right internal jugular vein (ijv) were included. ultrasonographic evaluations of ijvt were performed prior to insertion and daily until introducer sheaths removal. we investigated demographic data, underlying disease, length of surgery, use of cpb and iabp, complications during cannulation and duration of catheterization. coagulation status (pt, aptt, platelet count, d-dimer) and cardiac index at before and after surgery were also recorded. the student's t test, v test, and fisher's exact test were used for statistics and p value of . was considered significant. results. patients were included in this study. mean age of patients was ± years (range - ), mean duration of catheterization was ± days. ( . %) patients developed ijvt which occurred only one day after insertion. the incidence of ijvt was related to presence of underlying disease (relative risk, . ; % confidence interval, . to . ) and was unrelated to emergency operation, the use of iabp and cpb, number of insertion attempts. there were significant differences between patients with or without ijvt in duration of catheterization ( . ± . vs. . ± . days, p = . ), cardiac index at day after surgery ( . ± . l/(min m ) vs. . ± . l/(min m ), p = . ), the value of d-dimer at day after surgery ( . ± . vs. . ± . lg/ml, p = . ). no clinical symptoms related to ijvt were found in observation period. our results demonstrated that ijvt associated with introducer sheaths was a frequent complication and cardiac index was significantly lower in patients with ijvt. though the incidence of ijvt was higher in patients with prolonged catheterization, it developed even on day after surgery and was usually asymptomatic. this risk should be carefully considered when the insertion of pulmonary catheter is chosen for cardiac surgery. methodology. it is a prospective study of patients admitted to our icu after undergoing robotic radical prostatectomy (da vinci) in the time interval going from january up to april . we analyzed clinical and demographic data, the length of stay in the icu and hospital, the need for blood transfusion, surgical times and the complications suffered during hospital stay. data are expressed as mean, median or percentage, using the student's t test and chi-square to compare averages and detect possible associations between variables. results. seventy three patients underwent surgery with a median of years. mean surgical time was min. in recent months this time is reduced to min. the mean haemoglobin at admission ( . g/dl) was significantly higher than when dismissed ( . g/ dl), p \ . . an average of two units of concentrated red blood cells was transfused in the surgery room in . % of patients. only one patient required transfusion at the icu. cardiac or renal mild complications appeared in . % of patients. this could not be associated with age. the median of mechanical ventilation length was h. one patient required conversion to open surgery due to profuse bleeding. there was no hospital mortality and no need for reoperation. mean stay at icu was day, significantly less than those patients who suffered complications (p \ . ). the median stay in ward was days. conclusion. robotic radical prostatectomy (da vinci) has a very low associated morbidity, minimal blood transfusion requirement and short is the stay at the icu and hospital, in contrast to published data with open surgery. there was no hospital mortality. objectives. to evaluate short-and long-term outcome in patients undergoing coronary artery bypass grafting (cabg), who received an intra-aortic balloon pump (iabp) prior to surgery. methods. between january and june , all patients (n = ) who received an iabp prior to on-pump cabg in our center were included. patients received the intra-aortic balloonpump for vital indications (i.e. either unstable angina refractory to medical therapy or cardiogenic shock; group ; n = ) or for prophylactic reasons (group ; n = ). a cox proportional hazards model was used to identify predictors of long-term all-cause mortality. compared with the euroscore predictive model, observed -day mortality in group ( . %) was not significantly higher than predicted ( . %). a dramatic decrease in -day mortality occurred in group (median predicted mortality was . % and observed was %, p \ . ; fig. introduction. physiological abnormality is associated with adverse outcome and a high percentage of patients admitted to icu have abnormal physiology in the hours prior to admission [ ] . track and trigger systems are used to enable timely intervention to a deteriorating patient. the mews system is used in our level care general wards with more intensive monitoring in our level care facilities. objectives. we hypothesised that the mews for patients admitted from the general wards were being inadequately documented and that this be may influencing outcome. we therefore also hypothesised that patients admitted from the level care general surgical and medical wards in our hospital had a poorer outcome following icu admission than those admitted from sites of level care, such as the high dependency unit (hdu), emergency department and theatre recovery. we undertook a prospective week audit of all patients admitted to our district general icu. all patients' case notes and monitoring charts were reviewed by an icu consultant with additional data (e.g. apache ii scores) obtained from the ward watcher icu database. day mortality data was obtained from the hospital's sci patient database. we then compared the data with that available for patients admitted over the previous year ( ) background and aim of study. aorto-femoral bypass (afb) is widely used for the patients with peripheral vascular disease (pvd). nevertheless, there is no consensus about the type of anesthesia for this difficult group of patients. we hypothesized that continuous spinal anesthesia (csa) will be more secure and suitable for the patients with pvd combined with pulmonary and cardio-vascular co-morbidities. the aim of our study was to compare alterations of the mean arterial pressure (map) and delivery of oxygen (do ) during afb and in the early postoperative period under the influence of ga and csa. after approval of our hospital helsinki committee prospective randomized study was performed between and : male patients with pvd were included in our work. risk of anesthesia was equal to the third degree according to asa scale. in the first group of patients (n = ) ga with mechanical ventilation was employed. in the second group of patients (n = ) csa was used. both groups of patients were similar with respect to age and co-morbidities (copd % in both groups, ischemic heart disease and arterial hypertension). for ga we used propofol, midazolam, fentanyl, and isoflurane. bupivacaine was used for csa. in combination with mental sedation by intravenous midazolam. map was measured directly through radial artery catheter and do with the help of tetrapolar rheovasography. both parameters were measured during fixed points: before the operation, at the end of induction of anesthesia, after cross clamping of the aorta, after release of aortic clamp, first hour after operation, h after operation and h after operation. mann-whitney test was used for statistical analysis of our results. in the patients with csa during the operation and in the early postoperative period, map was lower but statistically non significant. map was lower statistically significant only during the cross clamping of the aorta and in the first postoperative hour, most probably due to the influence of the sympathetic block. at the same time do had almost no difference in both groups. only in induction stage it was lower in the ga group that most probably was connected with negative influence of propofol on cardiac output. conclusions. both methods of anesthesia ga and csa gave us opportunity to preserve stable map and do during afb that we performed in this difficult pvd patients with copd and cardiovascular co morbidities. over the last few decades, several scoring system have been developed for use in critically ill patients, not only to assist therapeutic decision making but also to guide resource allocation and quality of care. to evaluate the tiss- in surgical intensive care unit (icu) patients and the possible relationship between tiss- and the type of surgery, severity of illness, and outcome in these patients. prospectively collected data from all patients admitted to a postoperative icu between st march and th june were analyzed retrospectively. a-priori subgroups were defined according to gender, age, saps ii score, sofa score, surgical procedures, and the occurrence of major morbidity or death in the icu or in-hospital. a total of , patients were admitted during the study period ( . % male, mean age . years) constituting , observation days. the highest tiss- scores were observed on the day of admission. the highest tiss- was observed in patients who underwent cardiothoracic surgery, the lowest in neurosurgical patients. during the first week in the icu, tiss- was correlated to the severity of sepsis syndrome; however tiss- scores remained elevated only in patients with severe sepsis/septic shock. tiss- score was correlated to saps ii (r = . , p \ . ) and sofa score (r = . , p \ . ) throughout the icu stay and was consistently higher in non-survivors than survivors during the first weeks in the icu. conclusions. the highest tiss- scores are observed on the day of admission to the icu with marked variations according to the type of surgery. tiss- correlates well with the severity of sepsis syndrome and outcome in these patients. our data could be helpful in icu planning, risk stratification, and resource allocation in the surgical icu setting. introduction. pain and opioids for treatment of pain can affect immune function in cancer patients, which may in turn influence metastatic capability of a primary tumour during and after surgical excision. it is also been shown morphine has a direct effect on cancer cells, but results of these studies have been conflicting. we therefore aimed to determine effect of morphine, commonly used in anaestehsia and intensive care, on in vitro breast cancer cell migration using two breast cancer cell lines. we used two cell lines: mcf is er positive breast cancer cell line while mda-mb- is er negative, less differentiated and more invasive. cells were incubated with or without morphine (concentrations - ng/ml) for , and h, corresponding to clinically relevant concentrations and exposure times. cell proliferation was determined using an mts (promega inc.). h cell migration was determined using a -well flourescent kit (chemicon). results were compared using independent sample t test for differences between the groups. morphine had positive effect on cell proliferation, which was greater in mda-mb- cells. proliferation of mda-mb- was increased the most at h incubation and higher concentrations ( and ng/ml caused and % increase in proliferation at h incubation and up to % increase at h incubation). proliferation of mcf cells was increased by % in and h incubation periods. morphine caused an increase in migration of both cell lines, which was again more evident with mda-mb- cells at higher concentrations of morphine ( , % and % increase with , and ng/ml respectively). our experiments have shown morphine has potential to directly stimulate breast cancer cell proliferation and migration in vitro, especially in less differentiated breast cancer cell line. further studies are needed to determine its effect on other metastatic mechanisms such as invasion and gene expression as well as the implication of these results for clinical practice. objectives. aim of this study was to evaluate the predictive value of nt-probnp levels and inflammatory markers (crp, il- , tnf) on late mortality in patients who underwent thoracic surgery for lung cancer. methods. patients median age ( ± years) without history of heart disease or renal failure. the blood tests for nt-probnp, crp, il- and tnf analyses were drawn one day preoperatively, h, h and days postoperatively. patients' demographic data, laboratory results and mortality were collected and assessed. results. nt-probnp at h was significant higher in non-survivors ( , ± , pg/ ml) compared to survivors ( ± pg/ml, p = , ). furthermore, nt-probnp at h was associated with survival in the cox-regression analysis (p = . , hr = , , % ci: , - , , units: pg/ml). crp preoperatively was significant higher in non-survivors ( ± mg/dl) versus survivors ( ± mg/dl, p = , ). il- preoperatively was significant higher in non-survivors ( ± pg/dl) compared to survivors ( ± pg/dl, p = . ). conclusions. high nt-probnp levels at h postoperatively, associated with increased mortality in patients undergoing thoracic surgery but there was no relation ship between crp, il- and mortality. introduction. the occurrence of post-operative delirium in elderly orthopaedic patients is associated with neurological complications and cognitive decline [ ] . although the etiology of the decline is less understood, cerebral ischemic events may be involved [ ] . high plasma concentration of n-methyl-d-aspartate (nmda) receptor antibodies (nr ab) has been proven highly predictable for occurrence of the postoperative neurological events in cardiac surgery [ ] . objectives. the aim of the present study was to investigate the predictive value of blood levels of nr ab for postoperative delirium, cognitive dysfunction or any other neurological complications after hip and knee replacement surgery. methods. the study enrolled consecutive patients, aged over , requiring acute or elective knee or hip replacement surgery. cognitive impairment was evaluated by minimental state evaluation (mmse) test administered before and after surgery. daily postoperative delirium was evaluated by confusion assessment method for intensive care unit (cam-icu). plasma levels of nr ab were recorded before surgery, at the moment of hospital discharge ( - days postoperative) and weeks after discharge. all other possible risk factors for postoperative delirium were also recorded. cognitive decline was present in patients ( %) before surgery and in patients ( %) at the moment of hospital discharge (p \ . ). plasma levels of nr ab were . ± . ng/ml preoperatively, . ± . ng/ml at the moment of hospital discharge and . ± . ng/ml weeks postoperatively, with no significant differences. conclusions. the incidence of cognitive decline in elderly patients after othopaedic surgery was significantly higher when compared with the preoperative status but there was no correlation between the cognitive decline and the plasma levels of nr ab. methods. patients older than years, whose performed endoscopic, colonoscopic or both procedures, under sedation performed by the intensive care deparment of the hospital del tajo. data were collected for months. demographic characteristics, medical history, asa (american society of anesthesiologists classification), drugs bolus and total dosages, respiratory and hemodynamic data, the length of procedure and recovery, and complications were collected. tolerance was assessed by endoscopist, with a (very bad) to (very good) scale. quantitative data are expressed with mean and standar desviation, and qualitative data with percentage. results. procedures were included. table shows main characteristics. tolerance and complications are referred in table . the . % of the procedures were appropriate ( or ) . the main complications were vomiting ( . %) and hallucinations ( . %). there were only incidences of respiratory depression and of hypotension. background. the authors hypothesized that the efficacy and quality of a remifentanil (r)-based regimen versus a piritramide (p)-based analgosedation in major post-surgical patients with renal and hepatic impairments still more potent even if prevention of narcotic induced hyperalgesia (nih) [ ] was done. the nih was made by sulphate magnesium (m), ketamine(k) or clonidine(c). methods. patients were randomly allocated to receive a blinded infusion of either r at a rate of . l/(kg min) (± . ) (g : n = ) or p at . mg/(kg h) (± . ) (g : n = ) coupled to an hypnotic sedation of propofol. r and p were titrated in icu after surgery, to achieve an optimal sedation as defined by a sedation conclusions. the remifentanil-based regimen allowed a more rapid emergence from sedation and facilitated earlier extubation diminishing total icu hospitalisation time and cost. even if we prevent the narcotic induced hyperalgesia by used of magnesium, ketamine or clonidine, needs of tramadol in rescue still lower in the remifentanil group due to its high power coupled with its high fexibility compared to piritramide. its reducing, by the way, risks of tramadol's metabolites accumulation in case of renal or leaver impairment. a ''fast track'' approach to cardiac surgery has significantly shortened the length of icu stay. however, quick awakening from anesthesia and subsequent extubation after discontinuation of sedative drug sometimes cause instability of hemodynamics, such as increase of bp or hr. dexmedetomidine (dex), a agonist, is a sedative drug that can be continuously infused during weaning and extubation. the aim of this double-blind study was to evaluate the effect of dex on time to extubation and hemodynamics during weaning from mechanical ventilation after cardiac surgery. with irb approval and informed consent, the patients undergoing cardiac surgery were randomly divided into two groups, dex group [infusion of . lg/(kg h) of dex] and saline group. drug administration was started at sternal closure and continued h. ramsay sedation score, times to extubation, systolic blood pressure, heart rate, respiratory rate, pulmonary artery pressure, central venous pressure, cardiac index were examined. we analyzed these parameters on icu admission, when the patients opened their eyes on order, at immediately before extubation, min, h, and h after extubation. unpaired t test was used for statistics and p value less than . was considered significant. results. patients were included in this study (n = in the dex group, n = in the saline group). there were no significant differences between two groups in age, length of surgery, length of anesthesia, and total dose of propofol and fentanyl. time to extubation was ± min in the dex group and ± min in the saline group (mean ± sd), which were also no significant differences. ramsay sedation score were maintained and no patients needed additional sedative drug during assisted ventilation in the dex group. althought mean systolic bp and mean pa, mean cvp, rr were similar in both groups during infusion. hr at eye opening, immediately before and after extubation were significantly lower in the dex group than in the saline group. conclusion. our results demonstrated that the infusion of . lg/(kg h) of dex decreased hr during weaning from mechanical ventilation. dex could not only provide adequate sedation but also suppress the stress response after cardiac surgery. dex is a useful sedative drug for preventing instability of hemodynamics on fast track approach. however, most anxiolytics impair intellectual function. dexmedetomidine (dex) is an alpha agonist that may offer sedation without overt cognitive decline. we performed a comparison between dex and propofol (pro), a drug often used for icu sedation. methods. prospective, randomized, double-blinded, cross-over study of awake and intubated brain-injured (bi, n = ) and non-bi ( ) icu patients, each receiving pro and dex using a cross-over design with periods of baseline (analgesic use fentanyl only), drug a, interval washout (fentanyl only), drug b. sedation was titrated to a score of or - (calm, cooperative) on the rass and hopkins nics scale. cognitive testing was performed at each study period using the validated -pt hopkins ace cognitive battery. objectives. we evaluated the effect on final outcome of a treatment regimen with lowdose haloperidol initiated when a positive cam-icu occurred as a quality improvement project. methods. the cam-icu was previously implemented in daily care in all patients who stayed[ h in our bed medical-surgical icu. in the first months of the study (period ), the cam-icu was used as an adjunct to daily care and the treatment of delirium was left to the physician on an intention to treat basis. subsequently, a month study pause was defined. thereafter, in the second months of the study (period ), the cam-icu was judged to indicate the presence of delirium and haloperidol was directly started with a loading dose of mg iv with subsequent daily dosing of . - . - mg iv (age \ ) or - oversedation is still common in many intensive care units (icu) despite the demonstrated benefits associated with sedation sparing strategies, including shorter duration of mechanical ventilation, and shorter length of stay in the icu and hospital. our aim was to observe whether a minimal sedation policy could be feasible in a multidisciplinary department of intensive care. prospective observational study over a two month period (december , to january , ) in a -bed medico-surgical department of intensive care of a university tertiary hospital. all adult patients who stayed in the icu for more than h were included. data were collected on duration, type, dose, and indication for sedative and opiate analgesic agents. self extubation was used as a safety surrogate. disease severity was assessed by the apache ii score within the first h of admission. statistical analysis was performed with spss software (spss incorporation, chicago, il, usa). a total of patients (male %) with a median age of years were included; ( . %) received some sedation, the majority [ ( . %)] during mechanical ventilation. midazolam ( %) and propofol ( %) were the most frequently used sedative agents. the most common indications for sedation were: early postoperative ( %), severe respiratory failure ( . %), short term procedures ( . %), and withdrawal syndrome ( . %). the median percentage of time during which patients received mechanical ventilation without sedation was . %, and was not related to severity as assessed by the apache ii score (rho . -p = . ). in the group of patients who required sedation for longer than just short procedures or uncomplicated postoperative care ([ h), the median percentage of time during which patients received mechanical ventilation without sedation was . %. analgesic opiates were often required ( %), predominantly by continuous infusion ( %). morphine was the most frequently used agent ( %). self-extubation occurred in patients, but only needed re-intubation. conclusion. in a mixed medical-surgical population of critically ill patients, a strategy of minimal or no sedation (''sedationless'') is feasible and without major adverse effects. we propose that comfort, hemodynamic instability, and mechanical ventilation should be abandoned as usual indications for sedation. grant acknowledgement. drs received grants from the doctoral fellowship program of capes/ brazilian ministry of education and from the federal university of rio de janeiro. r. russai the royal free hospital, anesthetics, northwood, uk postoperative cognitive dysfunction (pocd) is reported to occur frequently after cardiac surgery, even in low-risk patients. predictors of neurocognitive deficits can suggest the potential etiology and outcome of patient that has developed pocd. there is a wide range of neurological manifestations from subtle cognitive impairment to deadly stroke. over a period of weeks a population of patients underwent cardiac surgery in our hospital. we have looked for any signs of pocd in correlation with the possible etiology. data have been collected prospectively focusing on past medical history (pmh), possible contributors, manifestation, complications and treatment. pocd has occurred in patients ( male, female) with age range of to years (median age years), of whom % has had pmh of neurological impairment (predementia; cerebrovascular disease). multifactorial etiology was found: respiratory failure %, morphine %, tramadol %, renal failure %, remifentanyl %. in patients no related causes were recognised. all patients showed confusion as leading manifestation, although in patients confusion presented in combination with aggressive behaviour ( ), cognitive dysfunction ( ), paranoia ( ). in occasions pocd resulted in major complications such as difficulties in airway management ( ), removal of cvp line ( ), removal of arterial line ( ). the majority of patients ( ) required pharmacological treatment with single or multiple drugs therapy, the most common used was haloperidol ( % pts). the average length of stay in itu was . days, and the average length of hospital stay was . ( - ) days. conclusion. pocd is a common and potentially devastating complication with a complex and broad etiology, which may affect the rehabilitation process and the final outcome. early diagnose is essential for personalise treatments and therefore preserve in both life quality and life expectancy. introduction. sedation and analgesia is given to the icu patient for adaptation to the intensive care environment. however, side-effects of drugs used are increasingly acknowledged as negative factors increasing the risk of delirium, vasopressor therapy, prolonged ventilation and length of stay. objectives. the purpose of the proposed study was to study the practice of sedation in norwegian icu's and the challenges experienced by nurses and physicians. a national postal survey for clinicians in all norwegian icu's was conducted in september and october . all intensive care units treating mechanically ventilated patients for more than h (n = ) were included. two respondents from each unit ( intensive care nurse and icu physician) were invited to answer the questionnaire. the survey was based on two previous danish surveys. questions on practice and perceived problems were scored on a numeric rating scale and a lickert scale, as well as a few questions with response categories based on theme. results. the response rate was % (n = ). all icus were represented with nurses with formal education in intensive care and physicians specialized in anesthesiology as respondents. written protocols are not routine in norwegian icus, but half of the departments titrated sedation according to a scoring system, most commonly maas. the most commonly used sedatives were propofol and midzolam, while fentanyl and morphine were the most used analgesics. the main indication for sedation was to achieve tolerance to ventilation and to treat other bothersome symptoms. side-effects were reported to be frequent with both sedatives and analgesics. the most frequent side effects (% reported as often present or always present) with sedative agents were circulatory instability ( %), delayed awakening ( %) and sleep disturbances ( %), while the most frequent side effects experienced with analgesics were gastrointestinal problems ( %), circulatory instability ( %) and delayed awakening ( %). the main indication for tracheostomy was reported as longterm ventilation and the wish to reduce sedation. discussion/conclusion. written protocols were not routinely used. side-effects of sedation are perceived as a problem by the majority of clinicians, leading to circulatory instability and delayed awakening. tracheostomy is used first of all to be able to reduce longterm ventilation and sedation. these results indicate a potential for new sedative agents and analgesics with fewer side-effects and more focus on the use of sedation protocols. [ ] and has been associated with gaba agonist use [ ] . delirious patients may not be overtly agitated, so signs of delirium must be actively sought. the confusion assessment method for the intensive care unit (cam-icu) is a validated and easy to use screening tool [ ] . in a recent study on this bed medical and surgical intensive care unit (icu), more patients who had received gaba agonists were delirious compared with those who were sedative free [ ] . however, the percentage of patients having at least one episode of delirium was lower than expected ( %), perhaps because they were screened only once daily and in the daytime. we repeated this study with twice daily assessments (morning and after dark) and a larger number of patients in order to obtain a more accurate prevalence of delirium and confirm an association with gaba agonist use. methods. two doctors attached to the icu received a min tutorial on using the richmond agitation and sedation score (rass) and the cam-icu assessment tools. the cam-icu was performed on all rousable patients (rass score [ - ) twice daily (morning and after dark). the following information was also noted: ( conclusion. this study shows that the prevalence of delirium on this unit is comparable with published research [ , ] and higher ( . vs. %) [ ] when patients were screened after dark as well as in the daytime. the study shows that any sedating drug was associated with significantly increased prevalence of delirium. the unexpected higher prevalence of delirium in the patients receiving non-gaba agonists versus gaba agonists cannot be explained by haloperidol use to treat delirium. results. there were patients in the icus. the mean age was . years old with a predominance of chinese ( . %) and a slight male predominance of . %. forty-six per cent of the patients were mechanically ventilated and . % had tracheostomy done. there were an average number of devices per patient. sedation was administered in . % of the patients with no sedation scales used in a quarter of these patients. only . % of the sedated patients were on sedation protocol. the majority of patients ( . %) were monitored hourly and on propofol ( %) and midazolam ( . %). up to . % of sedated patients did not have daily interruption of sedation. there were no significant difference noted in the use of sedation between medical and surgical icus. slightly more than a third of patients were given analgesia (n = ) with no analgesia scales used in a third of these patients. one third of them were administered with paracetamol and about a third with morphine. patients in surgical icus were more likely to receive analgesia compared to medical icus patients. most of these patients ( . %) were monitored hourly. only patients were on neuromuscular blockade. there was no usage of any formal delirium assessment tools at all with . % of the patients being assessed for delirium based on clinical judgement of the caring team. only % of the patients had some form of sleep promotion in the icu. conclusions. this national multi-center study reveals several deficits in the adult icu with regards to sedation, analgesia and delirium assessment and management. several initiatives should be implemented to improve patients' safety and quality of care in the icu. methods. this study was conducted in patients who visited emergency care center following caustic ingestion during a period ranging from january of and august of , in whom a retrospective analysis of medical records was performed. findings for the esophageal lesion were classified according to the change of the esophageal wall and the infiltration of periesophageal soft tissue. also, clinical, laboratory, and endoscopic data from this patients were reviewed. the correlation between the degree of esophageal damage seen on ct scans and esophageal constriction seen on esophagography were then evaluated. a total of cases of caustic ingestion were identified (age range, - years). the most common caustic agent ingested was acid ( %). the most frequent cause for ingestion was attempt of suicide ( %) as opposed to accidental ( %). the findings of thoracic ct in patients were follows: first-degree esophageal injury in ( . %), seconddegree in ( . %), third-degree in ( . %), fourth-degree in ( . %). fourteen patients ( . %) developed caustic esophageal stricture. the degree of esophageal damage got closer to grade iv, the more prevalent esophageal constriction became. this correlation was statistically significant (p \ . ). of the total patients, underwent endoscopy in the early stage after they visited emergency care center. an analysis of the correlation between the degree of esophageal damage seen on endoscopy and that seen on ct scans was performed. this revealed a significant correlation (p = . , r = . ). conclusions. thoracic ct grading suggesting periesophageal soft tissue infiltration and fluid collection (grade iii to iv) rather than only edema (grade i) may be associated with stricture formation. early ct grading was very safe and useful for predicting the development of stricture induced by caustic ingestion. conclusion. in our area critical care transport teams provided safe transfers for critically ill patients.adequate preparation, strict adherence to checklists and adequate personal are the key of optimal solving of problems. introduction. although endovascular repair of traumatic aortic injury (ertai) has revolutionized the practice of vascular surgery, many questions still remain unanswered. endoleaks, coverage of the left subclavian artery, stent fold/collapse, access complications and durability are the most important complications associated with the procedure. we describe our experience with stent fold/collapse after endovascular repair of blunt aortic injury in otherwise healthy and young patients. from january to december , patients (mean age years, mean apache score , mean length of stay days) who underwent endovascular repair of a blunt aortic injury were admitted in our icu. every day clinical examination and invasive blood pressure monitoring were employed for all our patients. when persistent hypertension was detected, transthoracic (tte) and transoesophageal echocardiography (tee) were initially used, followed by spiral computed tomography (ct) and angiography as confirmatory methods. of the patients, ( %) developed a pressure gradient of [ mmhg at the level of the stent that was initially investigated with continuous wave doppler at the descending thoracic aorta (suprasternal view). the complication presented with refractory hypertension (requiring more than two classes of antihypertensives in high doses) and difficult weaning. the cause of hypertension in of those patients was a stent collapse, while in the other patients the stent appeared folded but not collapsed. endograft revision by open surgery was necessary in of the patients. conclusion. the absence of especially designed grafts for the treatment of blunt aortic injury and the subsequent use of oversized grafts are associated with severe complications. refractory hypertension after ertai can be a manifestation of poor stent alignment and/or stent collapse. echocardiographic monitoring proved to be a useful tool in the early diagnosis of this kind of stent-graft complication. as far as we know, it is the first time that echocardiography is described in the relative literature as an early diagnostic technique for this serious complicationction. facing an aging population, the number of interventions of the french emergency medical service (ems) among very elderly is increasing. a previous retrospective study showed that except from out-of-hospital cardiac arrest survival to discharge was remarkably high after ems intervention for life-threatening pathology [ ] . the aim of the present study was to evaluate prospectively outcomes of very elderly patients managed by ems. methods. after irb approval, we conducted a prospective study over year, including all patients aged years or more managed by our physician staffed ems department. characteristics of patients including previous medical status (mccabe and knaus scoring systems), functional independence (katz adl scale), clinical conditions, the index de gravité simplifié ambulatoire (igsa) severity score were recorded. patients were followed until their hospital discharge. the -month mortality was recorded as well as the adl score. data are expressed as mean ± sd, median [iqr] or percentage of patients and compared using univariate and multivariate analysis. a p \ . was considered the threshold for significance (*). results. of the patients included, died on-scene, were transferred to the hospital and patients were left on scene because of significant improvement in medical status making hospitalization unnecessary, or on the contrary in near-death situations. mean age was ± years ( men). their adl was ( - ) and % of patients were living at home. main conditions were pneumonia (n = ), acute coronary syndrome or chest pain (n = ) and acute pulmonary oedema (n = ). at months, survival rate was % (n = ). the proportion of patients living at home was % and adl among survivors was ( - ) (vs. ( - ) initially for this subpopulation, p = . ]. when compared with deceased patients, survivors were significantly younger ( ± vs. ± years*), had lower adl penetrating anterior chest wounds causing cardiac injury are typically fatal, with only % of patients surviving to reach hospital. while the majority of patients with thoracic trauma can be managed conservatively or with simple intercostals catheter, a small but significant number of blunt ( %) and penetrating ( - %) injuries, require emergency resuscitative mediam sternotomy as a component of initial resuscitation. case report. a years old men, fall from meters high, while working in a truss. he was immobilized with semirigid cervical collar and backboard in the scene and transport to our trauma center, witch was a h car-distance. anesthesiologist team was present since the initial management in emergengy room (er) and act according to advanced trauma life support principles. in primary survey, patient was paraplegic, had a gcs of / , a normal respiratory rate, a slight hypotension and a slight tachycardia. when surgeon places a chest drainage, it drains immediately more than , ml blood, and the patient vital signs started to fade, to extreme bradicardia. the patient was then intubated with a rapid sequence intubation, with a single lumen endobronquial tube, and ventilated with protective lung ventilation. hypotension postinduction was promptly treated with vasoactive drugs (nor-adrenaline and dobutamine) and ongoing volume resuscitation. an emergency resuscitative mediam sternotomy incision was perfomed in the er and reveled a clavicule and sternum fracture and laceration in the braquiocephalic artery which has repaired. maintenance was performed with total intravenous anesthesia with fentanyl and nondepolarizing muscle relaxant. monitoring include standard monitoring plus direct arterial and central venous pressures. during the surgical procedure we treat massive blood loss, with multiple transfusions of units of red blood cells (unmatched type-specific), seven units fresh plasma, and pools of plaquets, fibrinogen and cryoprecipitate. at the end of the surgery, still ongoing blood loses, made us suspect of coagulopathy, and to use octaplex Ò . it was also performed a nasal tamponade, to stop severe epistaxis and suture a major scalp wound with evidence of basal skull fracture. patient was transferred to an intensive care unit (icu) ventilated.we was extubated at the th day post-operative. after days, he still remains in icu, because he is recovering from lumbar spine fixation for a total fracture-dislocation of d -d . discussion. although he remains paraplegic, we think emergency sternotomy have had a significantly impact in this life-threating situation. the use of cell-saver Ò would have been beneficial, but it was unavailable in er. we included only patients attended in this unit by icu personnel. these patients belonged to the area assigned to cruces which has been reference centre of the northern area of spain until december . we collected all the information needed from the clinical history and the treatment sheet, and used the spss . programme to perform the statistic analysis. results. we found patients that meet the severely burned patients criteria and that were attended by the icu personnel in colaboration with the plastic surgey unit. their medium age was . ± . years, % of those patients were men, and the medium burned body surface was ± . %. these patients remained hospitalized in this unit during a medium time of . ± . days. during their stay, the % of them needed mechanical ventilation, % presented acute renal failure, % had a pao /fio less than , and . % suffered some kind of infection. methods. prospective and observational study developed in a burn unit, in which were included all patients with total surface body area burn (tsba) [ % and/or inhalation syndrome who were admitted in our unit from march to december . we used transpulmonar thermodilution by means of monitor picco Ò in a total of measurements per patient (admission and every h). we collected measurements of cardiac index (ci), intrathoracic blood volume (itbv), extravascular lung water (evlw), inhalation syndrome or not (it was diagnosed by broncoschopy), percentage of tsba and abbreviated burn severity injury score (absi) in a total of patients.the average change of measurements of ci, itbv and evlw was studied in the following determinations and their association with few factors with a general and lineal model of mixed effects longitudinal data unbalanced. results. the evolution of thermodilution measurements was the following (graphic ) cardiac index: ci = . , ci = . , ci = . , ci = . , ci = . , ci = . , ci = . , ci = . , ci = . , ci = . . intrathoracic blood volume: itbv = , itbv = , itbv = , itbv = , itbv = , itbv = , itbv = , itbv = , itbv = , itbv = . extravascular lung water: evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . , evlw = . . in our serie, % of patients were male and the average age was . ( - ). nine out of all the patients ( . %) suffered inhalation syndrome, the average absi was . ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) and the average of tsba was % ( - %). mortality in our serie was % ( patients).ci and itbv measurements increased significantly while the reanimation advanced (ci p . ) (vsit p . ). in evlw we only find significantly differences in post hoc study between first measurements and fourth one (p . ), th (p . ), th (p . ), th (p . ), th (p . ) and th (p . ).in the evlw/itbv ratio (permeability index = pi) we did not observe significantly changes in the evolution.the inhalation factor did not change ci outcomes neither magnitude nor in the measurements evolution (p . and p . respectively), the same form, itbv (p . and p . respectively), but inhalation modified evlw (p . ) and the permeability index was in the signification stadistic limit (p . ).mortality was higher in patients who ci was lower and evlw was in higher values. conclusions. thermodilution in the reanimation period in critically ill burn patient shows significantly haemodynamic changes in the evolution that can help to adapt the treatment.the inhalation syndrome only modified the measurements of evlw significantly in this period but it influenced neither ci, itbv nor pi. introduction. one major issue in trauma management is to get every patient directly from the scene to the appropriate hospital for the injury he sustained. patterns of interfacility transfers have been thouroughly investigated in trauma system settings, but scarce data are available about transfers in non trauma system settings. objectives. this study aims to assess interfacility transfers that eventuate in the abscence of a formal trauma system and to estimate the potential benefits from the implementation of a more organized process. the 'report of the epidemiology and management of trauma in greece' is a one year project of trauma patient reporting throughout the country. it provided data concerning the patterns of interfacility transfers. in greece there is no formal trauma system employed and to our knowledge, all available data concerning the epidemiology of trauma in the country are either extrapolations of relevant data from other countries or based on police reports and individual hospital reports. in this study, we attempted to evaluate the paterns of interfacility transfers, information reviewed included patient and injury characteristics, need for an operation, intensive care unit (icu) admittance and mortality. trauma patients were devided in two groups, the transfer group was compared to the non-transfer group. analysis employed descriptive statistics and chi-square test. interfacility transfers were furthermore assessed according to each health care facility's availability of five requirements; computed tomography scanner, icu, neurosurgeon, orthopedic and vascular surgeon. results. data on , patients were analyzed; . % were treated at the same facility, whereas . % were transferred. in transferred group there were more male, the mean age was lower than that of the non transferred group and the injury severity score was higher. transferred patients were admitted to icu more often, had a higher mortality rate but were less operated on compared to non-transferred. the transfer rate from facilities with none of the five requirements was . %, whereas the rate of those with at least one requirement was . %. facilities with at least three requirements transferred . % of their transfer volume to units of equal resources. conclusions. the assessment of interfacility transfers can reflect current trends in a nontrauma system setting and could indicate points for substantial improvement. results. , patients included, , injuries analyzed. average age was . , . % men. . % were car accidents, % falls, . % motorcycle, . % run over and . % bicycle. . % had one injury, . % two and . % three. most frequent injury was tbi ( . %), thoracic traumatism ( . %) and ortophaedic ( . %); severe tbi was . %. ctrate according to marshall classification was . % ii, . % v and . % iii. iss averaged , higher in dying patients than in the survivors ( ± . vs. . ± . ; p \ . ). of the non mechanical-ventilated patients, . % were so in the first h following admittance. during this, . % patients were given blood transfusions, platelets . %, plasma . % and prothrombinic complexes . %. in the first h . % underwent surgery, most frequent was neurosurgery ( . %). complications: nosocomial pneumonia ( . %), catheter related bloodstream infection ( . %), acute kidney injury ( . %), ards ( . %), cns infection ( . %). . % renal replacement therapy. invasive ventilation was used in . % with . ± . days, non invasive ventilation in . %. average stay in the icu was days. . % of the patients were transferred to a ward. . % were transferred to another hospital. gos on discharge was higher than on . %. % died in icu, % brain death. tbi as a main injury showed a . % mortality rate. depending on trauma type, mortality was higher in fall ( %) and run over ( . %). if due to car accident ( . %), motorcycle ( . %) or bicycle ( . %), mortality in icu was lower (p \ . ). prehospitalary variables related to mortality were age, gcs \ and a motor component \ , pupil alteration, shock, respiratory failure, prehospitalisation intubation and iss (p \ . ). on arrival to hospital, the variables were: haemodynamic instability in the first h, transfusions need and number, marshall iv-vi, mechanical ventilation (p \ . ) and initial fibrinogen (p \ . ). evolutive variables related to a higher mortality rate were days of stay, invasive ventilation, tracheostomy and the show up of complications (catheter related sepsis -p \ . -, nosocomial pneumonia, acute kidney injury, ards, renal replacement therapy (p \ . ). in a logistic regression model, prehospitalisation variables having an influence on icu mortality rate were age (or . ; p \ . ), mydriasis (or . ; p \ . ), gcs-motor component (or . ; p \ . ), shock (or ; p \ . ) and iss (or . ; p \ . ). conclusions. multiple trauma patients show a high need of resources, with many peaks of treatment involving a high monitorization and handling. many of the variables are related with a higher mortality rate in icu: iss, mydriasis, gcs motor component and shock. introduction. trauma systems are multifactorial modules that incorporate any aspect of traumatic injury from the very moment of the injury to the final outcome. a significant prerequisite for the development of a trauma system is the trauma registry. trauma registries are the actual core of any trauma system since they provide valuable information about the standard of care offered to the patients and are amenable to quality control and statistical evaluations, which in turn allow improvements and amendments in the definite care. contrary to what is common practice in the usa, trauma registries in european countries are in embryonic stage. in our country with no actual trauma system, the epidemiology of trauma and the reports on care outcomes are based on police reports and national emergency service reports. objectives. the purpose of this study was to assess the possibility of a national trauma registry in greece and to provide accurate data on the epidemiology of trauma in the country. methods. the project, entitled ''report of the epidemiology and management of trauma in greece'', was initiated in october and lasted for twelve months. all the national representatives of the hellenic society of trauma were invited to participate. the representatives are certified surgeons employed in hospitals receiving trauma. data presented here are those reported from two tertiary care facilities in athens and twenty eight other primary and secondary hospitals around the country. inclusion criteria were defined as trauma patients with documented need for admission in the hospital, patients that arrived dead or died in the emergency department of the receiving hospital and patients that required transfer to a higher level center. in total . trauma patients were included in the study in twelve months time. of them . % (n = , ) were male, aged . ± . (mean ± sd) and . % were female (n = ), aged . ± . (mean ± sd). as expected and reported in most trauma registries, males are leading in all subcategories of iss. the age group - years incorporates . % of the total injuries, in accordance to the axiom that trauma is the disease of the young. conclusions. trauma registries are the cornerstone of any trauma system. even in a non-trauma system setting, registries are a valuable tool for quality control of the provided health care and for further development of the health care system. objectives. determine the impact of rurality in epidemiology, injury severity, health care facilities, length of stay (los), mortality, functional outcome and quality of life in trauma patients. retrospective study in trauma patients that were admitted in our emergency room(er) between and . data was collected from the prospective trauma registry and follow-up registry months after the accident. we classified patients according to statistical national institute classification: urban areas-areas with more than inhab/km or have a place with more than , inhabitants; semi-urban areas-areas with more than inhab/km and less than inhab/km or have a place with more than , inhabitants and less than , inhabitants; rural areas-areas that were not classified as semi-urban or urban areas.patients were divided in three groups according to residence area: r (rural), su (semi-urban), u (urban). we studied several variables in order to find a relation with rurality: sex, age, type of injury, los in hospital and intensive care (icu), anatomic severity (ais), politrauma severity (iss), physiologic severity (rts), surveillance probability (triss index), pre-hospital care, previous admission in other hospital, icu admission and mortality. we report two outcome measures: euroqol to evaluate quality of life and extended glasgow outcome scale for functional outcome. we used qui-square test, t test, mann-whitney test, kruskal-wallis test for statistic analysis. results. , patients were admitted in the er. patients ( . %) were excluded with missing data related to residence area. we studied patients, where patients were from rural areas ( . %), from semi-urban areas ( %) and from urban areas ( . %). we find a statistical significant relation between rurality and pre-hospital care, previous admission in other hospital and icu admission. urban area patients had a higher incidence of pre-hospital care(r: . %; su: . %; u: . %, p \ . ). semi-urban and rural patients were admitted more frequently in other hospitals before admission in er (r: . %; su: . %; u: . %, p \ . ) and also had higher admissions in icu (r: . %; su: . %; u: . %, p \ . ). there were no statistical differences in the other variables studied. conclusions. rural trauma patients are similar from those that live in urban areas concerning epidemiology, injury severity and outcome. despite lack of pre-hospital care and higher previous admission in other hospital in rural patients, mortality between groups did not differed in our trauma centre. introduction. metformin-associated lactic acidosis (malta) is a rare but severe complication ( . / , patients/year) of metformin treatment in type-ii diabetes. metformin impairs neoglucogenesis and liver lactate clearance in the presence of a disease that enhances its production. although frequently used, there is no recommendations regarding hemodialysis in this poisoning. to study the prognostic factors of malta and the interests of blood metformin measurement. . on admission, patients presented profound lactic acidosis with arterial ph . ( . - . ), serum bicarbonate . mmol/l ( . - . ) and plasma lactate . mmol/l ( . - . ). early symptoms associated coma ( %), asthenia ( %), vomiting ( %), abdominal pain ( %), and diarrhoea ( %). renal function was significantly altered [creatinine clearance: ml/ min ( - ); p \ . ). all patients received massive alkalinization, / ( %) were hemodialyzed while / ( %) were mechanically ventilated and received catecholamines. six patients ( %) died in the icu. duration of icu stay was days [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . there was no significant differences regarding malta severity and treatments between suicidal and accidental poisonings. neither lactic acidosis severity nor acute renal failure were predictive of death. there was no correlation between prognosis and the time-course of plasma metformin concentrations, with or without dialysis. toxicokinetics showed significant tissue distribution when the patient was early admitted or plateaued concentrations if he was later admitted and survived, even though his situation improved and his lactates decreased. metformin dialysance suggested an interest for extra-renal elimination enhancement although its impact on survival could not be analysed based on this limited study. our study showed that malta is severe with elevated mortality in icu whatever the poisoning is accidental or intentional. metformin toxicokinetics are useful case by case to better understand the patient's outcome. the most important guidelines [ ] for trauma care recommend (us)-fast as the first step investigation to rule out major bleedings. however, us is less sensitive and accurate than multislice computed tomography (ct). the spreading concept that ''moderrn cts require little time'' often brings surgeons to ask for total body ct also in haemodynamically unstable patients. to understand how long it really takes to perform a total-body ct in patients suffering from major trauma (mt) we analysed the data of the ritg project, a pilot multicenter study to define the national standards for trauma care and establish a national trauma registry. methods. italian level trauma centers were involved into the first stage of the ritg project. data of all major trauma patients (iss [ ) who were admitted to either one of the three hospitals during a months period of time ( july - june ) were prospectively entered into the ritg database. time between hospital admission and the first scan were recorded for all patients. patients who, for any reason, were submitted to a ct with a delay of °or more were excluded. the time elapsed between the first scan and the patient's exit from the ct room was measured in a subset of pts from a single center equipped with a new generation ct next to the emergency room. during the study period mt patients were admitted to the three trauma center. patients were submitted to an emergency total body ct scan within . patients died before arrival in the ed. more died soon after admission and before the secondary survey. the interval times are shown in table . seven patients died in the ct room. the average interval between hospital admission and the first available scan was °. however, even where a new generation ct next to the er was used, the average time needed to stabilize the patients, get a correct position on the ct and start the scanning process was °as an average. in the most severely injured patients, who are frequently artificially ventilated, the time required to stabilize the patient and perform a total body ct scan is longer than expected and to a certain extent, independent from the ct scanner itself unless the very last technology as the sliding ct scanners are employed [ ] , thus ct should be considered with extreme caution in the unstable patients. in univariate analyses, survival to discharge was significantly lower with two of acute conditions (acute coronary syndrome and acute inflammation), and with five of chronic conditions (chronic heart failure, diabetes mellitus, kidney failure, hepatic cirrhosis and malignancy). recent surgery was strongly associated with higher odds of survival. the most consistent multivariate predictors of survival to discharge were liver cirrhosis (or . ; % confidence interval . - . ) and malignancy (or . ; . - . ). malignancy was not predictive for outcome after cpr attempts, whereas liver cirrhosis was predictive both in all dispatches and in dispatches involving cpr. recent surgery was strongly associated with higher multivariate odds of survival (or . ; . - . ) after cardiac arrest. in dispatches without cpr, absence of chronic conditions was associated with higher likelihood of survival (hr . ; % ci . - . ). increasing numbers of chronic conditions were significantly and continuously associated with lower survival (p for trend \ . ). advanced age only weakly predicted survival, but age c years was, along with malignancy, the strongest predictor of not attempting cpr in patients with cardiac arrest. conclusions. comorbidities are important determinants of survival after in-hospital met dispatches, with and without cardiac arrest. survival odds are lowest with malignancy and liver cirrhosis. recent surgery increases odds of survival by exclusion of those most severely ill. advanced age at best weakly predicts worse survival, but strongly predicts not attempting cpr. design. retrospective, cohort study. setting. emergency department (ed) and intensive care unit in a university hospital. measurements and main results. the study subjects included of consecutive severe trauma patients. a systematic review of the computer-based medical records of the patients was conducted to provide the base line characteristics and dic-related variables. the worst data of these variables were obtained at time points within h after arrival to the ed; time point , immediately after arrival at the ed to h after the arrival; time point , to h after the arrival; time point , to h after the arrival; time point , to h after the arrival. one hundred and forty one patients ( / , . %) diagnosed as jaam dic showed significant differences in the prevalence of multiple organ dysfunction syndrome (mods) and the outcome in comparison to the non-dic patients. a stepwise logistic regression analysis showed that the maximum jaam dic scores during the study period independently predicted the patient death (odds ratio . , % confidence interval . - . ). all of the patients who developed isth overt dic during the study period could be identified by the jaam dic criteria at early time points. the mortality rate and the incidence of mods of the patients with the isth overt dic were higher than those only met the jaam dic criteria. stepwise increases in the isth overt dic scores and the incidence of the isth overt dic were observed in accordance with the increases in the jaam dic scores. while the mortality rates were identical, there were marked differences in the incidence of mods and sequential organ failure assessment scores between the dic patients associated with trauma and sepsis. conclusions. the jaam scoring system has acceptable validity for the diagnosis of dic at an early phase of trauma. the jaam dic further exists in a dependent continuum to the isth overt dic. in addition to mods, other factors may affect the prognosis of the trauma patients associated with dic. introduction. in the uk, more than , units of fresh frozen plasma (ffp) are transfused every year. since there has been a reduction of % in its use, but it has been suggested that as many as % of transfusions in critical care may be inappropriate. there is significant morbidity associated with the transfusion of ffp. guidelines for the use of ffp do exist, but the indications for its use are limited. coagulation studies, such as prothrombin time (pt), abnormalities are often assumed to be a risk factor for bleeding prior to invasive procedures, but evidence suggests that ffp may not have a prophylactic role. in addition to this the pt or international normalised ratio (inr) were not intended to assess haemostasis in patients without a history of bleeding. review of the blood bank database between st january and st december revealed all prescriptions of ffp for patients on intensive care (itu). the case notes were analysed to find the indication and timings of administration and weight of the patient. the pathology database was examined to find the clotting studies immediately before transfusion. in patients received ffp; this was only . % of the total admissions to the itu. there were prescriptions and a total of units transfused. the mean prescription of ffp was . units and overall each patient received a mean of . units. the mean dosage of ffp was . ml/kg. the pt pre-transfusion mean was . ± . s with a median of . s. the aptt pre-transfusion mean was . ± . s with a median of . s. only . % of transfusions had not had a clotting screen done prior to administration of ffp. % of administrations were given prior to procedures being undertaken on the itu and a further % were given in preparation of the patient for the operating theatre. a significant number of patients are receiving ffp outside international guidelines. a third of transfusions were given for prophylactic correction of coagulopathy prior to an invasive procedure where there is least evidence for using ffp. most patients received a sub-therapeutic dose of ffp; there is ongoing debate on the correct dosage required to normalise coagulopathy, but it is likely to be greater than ml/kg. [ ] . little data exists on the demographics of mt and subsequent demand on a hospital's blood bank. we examined the mt requirements of a bedded tertiary referral hospital over a month period. objectives. to establish the mt demographic, speciality distribution, prbc demand and associated mortality; within a tertiary referral hospital over a month period. to assist with future mt logistics, planning, implementation and audit. methods. the hospital blood bank database was reviewed for cases of mt from jan to aug . inclusion criteria were the administration of c units of (prbc) within a h period. cross referencing with the laboratory records and medical notes was undertaken to establish patient demographics, hospital specialty, diagnosis, outcome and number of prbc transfused. results. patients received mt over a -month period; male ( . %) female ( . %). median age years (range - ). median mt of prbc was (range - ) units. units of prbc were transfused in the treatment of mt during the study period, accounting for a hospital expenditure of over € , . the main specialties associated with mt were the: emergency department ( patients, . %), cardiothoracic surgery ( patients, . %), and general surgery ( patients, . %). of the patients receiving massive transfusions ( . %), did not survive to hospital discharge. % of those patients who died, did so in the first h with a further % dying in the next h. % of the further deaths occurred within and % after thirty days. conclusions. mt in our establishment is associated with a high mortality and predominantly early deaths. recipients were generally elderly with significant co-morbidity. provision of prbcs and blood components for massive transfusion recipients, is challenging for blood bank services [ ] . the demand of mt, within our establishment, was predominantly within the acute specialties; emphasizing the need for close communication between them and the laboratory services. in light of this data we propose the implementation of a mt protocol together with continuous audit, to assess its effect on outcome. in the department of health updated the 'better blood transfusion' circular, a drive to decrease the use of blood products which have become increasingly scarce and expensive [ ] . there is evidence that blood product transfusions in icu patients are associated with an increase in morbidity, length of stay and mortality. there has been concern over the increasing use blood products and despite guidelines [ ] for their use, both national and local audits have demonstrated a high degree of inappropriate transfusion [ ] . derriford hospital is a -bedded tertiary referral centre. the blood bank database was examined for the use of blood products on icu from st january to st december . there was a steady rise in icu admissions from , patients in to , patients in . during this time there was a marked decline in both the transfusion of fresh frozen plasma (ffp) and cryoprecipitate. the decreased use of these blood products has occurred with only a very modest introduction of new pro-coagulant therapies, prothrombin complex concentrate (pcc) and recombinant factor viia (rfviia). usage of rfviia and pcc conclusions. our usage of blood products does not reflect the national trends of increasing use of cryoprecipitate and pcc with a small reduction in ffp transfusion, and is more in line with the hsc requirements for better use of blood products. this has been achieved with little use of the newer rfviia and pcc. the evidence for the use of all these blood products is not strong, particularly in critically ill patients. national guidelines exist for their use, but these are poorly adhered to. to test the hypothesis that transfusion of prbcs has a deleterious effect on clinically relevant outcomes in patients with septic shock receiving early goal directed therapy (egdt). retrospective cohort study of patients who presented in an academic center in septic shock and received egdt. data was collected on patients identified via the surviving sepsis campaign chart review database and linked to the project impact database. pearson chi square and fisher's exact test were used to test for clinical significance. primary outcome was mortality and secondary outcomes included mechanical ventilation days, intensive care unit (icu) length of stay, and hospital length of stay. . / patients presented via the emergency department. / patients received at least one prbc transfusion during their hospital stay. the two groups were balanced with respect to age, gender, apache ii, and baseline lactate levels. the prbc group had a mortality of . vs. . % in the no prbc transfusion group (p = ns). the prbc group also had more mechanical ventilation days ( . vs. . days, p = \ . ), longer hospital length of stay ( . vs. . days, p = \ . ), and longer icu length of stay ( . vs. . days, p = \ . ). conclusions. in this study, transfusion of prbc was associated with worsened clinical outcomes in patients with septic shock treated with egdt. this trial is limited by its small sample size and retrospective nature. however, the results are consistent with data from previous trials pointing to a deleterious effect associated with prbc transfusion. further studies are needed to determine the impact of transfusion of prbc within the context of early resuscitation of patients with septic shock, as the beneficial effects gained by an early and goal oriented approach to resuscitation may be lost by the negative effects associated with prbc transfusion. introduction. blood transfusion therapy (btt) is thought as one of transplantation of living cell, that means btt includes several risk such as infection and btt should be thought to derived from precious material by courtesy of donors. patients with traumatic cardiopulmonary arrest on arrival on the hospital (t-cpa) usually suffered from lethal hemorrhage and required rapid supplement of red blood cells for resuscitation of circulation and oxygen transport, that is to say btt. however, the prognosis of t-cpa patients is well known hopeless. the aim of this study is to evaluate the propriety of our strategy concerning btt for t-cpa patients. we retrospectively examined the medical records of t-cpa patients for the past years. we do btt until (the first period) for t-cpa patients regardless of rosc without any restriction. after then (the second period), we do btt case by case but only after rosc in principle. the rate of rosc, admission to icu, survive to discharge were compared between these two period, and were compared within the first period between the patients group who underwent btt (btt group) and the group who did not underwent btt (non-btt group). in blunt t-cpa and penetrating t-cpa patients, and % achieved rosc, and % admitted to icu, and and % were survive to discharge. in penetrating t-cpa in the first period, units of packed red cells (prc) were used before rosc for non-survivors. in the second period, no prc was used for non-survivor before rosc. in blunt t-cpa in the first period, prcs were used for non-survivors before rosc. in the second period no prc was used for non-survivors before rosc. concerning the effect of btt on the prognosis of t-cpa in all cases, the rate of rosc and admission to icu were statistically higher in the first period than in the second period (p = . and . ). however, there was no statistical difference in the rate of survive-to-discharge between these periods. there was a same tendency in witnessed cases. in cases with electrical rhythm on the scene, only the rate of rosc were higher in the first period (p = . ). restricted in the first period, only the rate of rosc was statistically higher in non-btt group than btt group in all cases, in witnessed cases, and in cases with electrical rhythm on the scene (p = . , . , and . ). however, there was no statistical difference in the rate of admission to icu and survive-to-discharge between these groups.. our retrospective serial study showed a possibility that btt before rosc for t-cpa improves the success rate of rosc but add no effect on the improvement of survival rate. btt is thought to be futile for t-cpa before rosc. management of refractory coagulopathy due to adult onset acquired autoimmune haemophilia. d. hendron , g. allen , m. brady , g. benson belfast city hospital, intensive care, belfast, uk, belfast city hospital, department of haematology, belfast, uk we report a case of life-threatening haemorrhage occurring as a result of a rare acquired condition caused by the production of an antibody to clotting factor viii. this necessitated administration of recombinant activated factor viia (novoseven) to bypass this step of the clotting cascade. a -year-old man presented to intensive care following ogd for acute upper gastro-intestinal haemorrhage, with recent haemoptysis and haematuria. ogd had demonstrated a large clot obstructing the oesophagus and extending through stomach into duodenum. this could not be removed and no bleeding points were identified. a coagulopathy was detected which failed to correct with administration of appropriate amounts of fresh frozen plasma, cryoprecipitate and activated prothrombin complex concentrate (apcc), necessitating clotting factor studies. this demonstrated a factor viii level of % with a detectable antibody inhibitor. acquired haemophilia was diagnosed and activated factor viia was administered resulting in rapid correction of coagulation studies and arrest of haemorrhage. it was necessary to continue daily activated factor viia at a dose of mg a day in addition to anti-inhibitor coagulant complex (feiba-vh)-an activated prothrombin complex with factor viii inhibitor bypassing activity. definitive treatment of the coagulopathy was chemotherapy with cyclophosphamide, vincristine and rituximab. this destroyed the factor viii inhibitor and returned his factor viii levels to almost %. laparotomy and gastrotomy were required to relieve the oesophageal obstruction from the accumulated clot. he was eventually discharged from hospital and remains well. acquired haemophilia is a rare haematological condition that presents with refractory haemorrhage and coagulopathy and these patients are likely to be referred to critical care services for ongoing support and management. it has an incidence of approximately . cases per million per year [ ] . underlying medical conditions can be identified in up to % of patients and include autoimmune disease, solid tumours, lymphoproliferative malignancies and pregnancy [ ] . international recommendations on the diagnosis and treatment of patients with this condition have recently been published and advise recombinant activated factor viia to control bleeding followed by a combination of corticosteroid and chemotherapy [ ] . the paucity of cases presents an obstacle for randomised controlled trials and therefore these recommendations are based on anecdotal evidence and expert opinion. reference (s) objective. to analyze the application of blood transfusion in critically ill trauma patients after wenchuan earthquake. a retrospective study was made in icu of huaxi hospital on patients who had received transfusion at least once during month after the earthquake. their primary diagnosis and clinical features and apacheii score were obtained at admission. non-active bleeding patients were classified into s group if operation was done during his icu stay, otherwise n group. the function of liver and kidney, and the state of circulation and oxgenation were compared between groups, as well as the hemoglobin level before each transfusion were investigated. a total of patients ( . %) had received transfusion at least once, among which were non-active bleeding. the average frequency was . ± . and . ± . , amount was . ± . ml and . ± . ml, the incidence of transfusion-related complication was . %( / ) and . %( / ) in active and non-active bleeding patients respectively. the apacheii score, mean arterial pressure, ast, serum creatinine, oxgenation index and hemoglobin level on day , , after admission to icu showed no statistically significant difference between s and n group. the frequency and amount of transfusion were similar also, while the hb level before each transfusion was significantly lower in n group ( . ± . g/l)than in s group ( . ± . g/l) (p \ . ). the incidence of transfusion-related and infectious complications, time with ventilator and the -day mortality were similar. conclusion. transfusion strategy is more strict in icu doctors than surgeons, while the similar result on organ function, incidence of complications and outcome raises the need for a more wide-accepted transfusion trigger. keywords. earthquake trauma transfusion trigger. extracorporeal life support (ecls) represents an ultimate rescue technique in poisonings. the optimal anticoagulation protocol remains unclear. objectives. we aimed to investigate the coagulation status at ecls decision in order to validate the best heparin protocol to administer. [ packs ( - ) ] and fresh plasma [ packs ( - ) ] transfusions were required within the first h. hemorrhages ( / ), thrombosis ( / ) or lower limb ischemia ( / ) seemed equivalent to previous series using more complicated anticoagulation protocols. conclusions. poisoned patients present at ecls time with important alteration in their clotting tests, associated with various degrees of hepatocellular failure, dic, defibrination, as well as dilution. a simple heparin protocol appears optimal to reduce complications in these critical situations. henna is the dried and powdered leaves of the henna plant. the plant is lawsonia alba and the powdered leaves are used to apply decorative designs over the skin. henna application is widely practiced in the arab and asian communities. they create fascinating designs over the skin, especially over the hands and feet. it is widely practiced during wedding ceremonies and at childbirth. g pd deficiency is common in the community of the arab world. lawsone, the chemical compound in the henna leaves, is capable of inducing severe acute hemolysis in g pd deficient cases. the compound is chemically related to naphtha. we report a case of acute sever hemolysis in a young girl who presented with dizziness and jaundice and diagnosed to have acute severe hemolysis. her symptoms had started while preparing for her wedding by henna application. the girl was g pd deficient, and found to have severe hemolysis resulting from henna application on her skin. very few cases have been reported of similar nature. the matter is also of tremendous practical implication in areas of g pd deficiency. the relevant literature is reviewed as well. background. lactate has prognostic use in critically ill medical and trauma patients, and is a core component in identification of early sepsis. elevated lactate levels in these patients prior to icu admission, e.g. in an a&e setting or pre hospital setting identify patients at risk of death and can trigger an earlier optimization of triage decisions and earlier targeted treatment. a range of poc methodologies for lactate measurement are available but there is little standardization between methodologies. stat sensor lactate is a new poc lactate meter based on a patented multiwell and multilayer electrochemical technology that incorporates control wells that measure and correct for common interfering substances. the electrochemistry technology is layered onto a gold platform providing a stable and robust surface for the electrochemical reaction kinetics. the aim of this study was to assess the performance and functionality of stat sensor lactate. whole blood venous samples were collected from adult patients admitted to a&e. samples were tested for lactate using statsensor lactate (nova biomedical) and the omni b bga (roche) routinely used for lactate measurement. precision was assessed using donated whole blood and spiked with a concentrated lactate solution. results. within run precision was acceptable at all levels tested. for the lowest level sample (mean . mmol/l) %cv for the two meters tested was ( . and . %) at the three other levels tested (mean . , . , . mmol/l) % cv precision was \ %. lactate values during the method validation ranged from . to . mmol/l by the reference method (nova . to . mmol/l background and objectives. this research work's intention is to describe the epidemiology in patients suffering from anemia who were interned into emergency room (er). a preliminary work will be conducted in which three days in june will be randomly chosen. during these days, all patients satisfying certain criteria will be registered. the criteria fitted to this work are the following: be using the emergency channel of the hospital, score any diagnostic and be over years old. paediatric, gynaecologic and traumatic cases fall out of this research. anemia was diagnosed according to who criteria. outcome. patients were interned through the aforementioned er channel. . % were subject of blood analysis using classification proceedings. from the latter, . % were diagnosed with anemia. age, intake of clopidogrel and/or aspirin, admission and place of admission resulted statistically significant among anemia patients versus non-anemia. anemia was to be found in . % of patients younger than years old, % of the times in patients between and and . % among patients above years old. according to vcm, . % were microcitic-anemia, % were normocític-anemia and . % resulted macrocític ones. conclusion. anemia is among a large share of patients coming into the hospital through emergency proceedings. its likelihood increases accordingly to the risk group analysed and dominating among the elderly population and among patients suffering from renal disfunction and non aggregated. most common are normocitic and macrocitic anemia-types. early identification and valuation could bear prognostic consequences. a. s. omar tawam hospital, critical care medicine, al ain, united arab emirates introduction. an elevated serum creatinin phosphokinae (cpk) and the presence of myoglobin in the urine characterize rhabdomyolysis. rhabdomyolysis had been described in various traumatic and non-traumatic conditions [ ] , there are few reports of its association with anaphylaxis. in this paper, we report cases of anaphylaxis both complicated with rhabdomyolysis. aim of the work. to discus the association between rhabdomyolysis and anaphylaxis and the value of early screening of cpk in such cases. setting. two patients were included in this review in multidisciplinary intensive care unit of tawam hospital/uae. the two patients survived, both developed rhabdomyolysis shortly after admission, evidenced by fivefold or greater increase in serum cpk [ ] . both patients had transient hypotension through the presentation, but none of them had persistent shock requiring vasopressors or complicated with acute renal failure. conclusion. we observed rapid increase in serum cpk in our two cases suggesting the potential benefits of early assessment of cpk in such patients which may amplify early goal guided management and avoiding logistic organ dysfunction. keywords. rhabdomyolysis, anaphylaxis. the blood oxygen and carbon dioxide levels are a direct measure of the effectiveness of ventilatory support in patients on mechanical ventilation. head injury patients require strict control of the cerebral homeostatic state. these patients also need careful management of sedation, maintaining a fine balance between patient comfort, hemodynamic instability and ability to assess conscious levels. biphasic intermittent positive airway pressure (bipap) ventilation is thought to be better tolerated by the patient allowing for spontaneous breathing at any point, thus reducing the amount of sedatives and muscle relaxants used. but the effectiveness of this ventilatory mode in achieving stable blood oxygen and carbon dioxide levels in this group of patients is not known. we hypothesised that bipap is more labour intensive to adapt to the target blood gas parameters as the volume delivery is not constant and that the blood gases may be more unstable in the initial resuscitation phase of head injury patients without conferring much advantages in terms of usage of sedatives and muscle relaxants. retrospective data collected from case record review of head injury patients with no primary respiratory insult, requiring mechanical ventilation with volume controlled synchronised intermittent mandatory ventilation (simv) was compared to the data from similar patients treated with bipap ventilation. both the data groups specifically looked at two time periods, the first h and - h after intensive care admission. blood gas parameters classified as hypocarbic, hypercarbic and/or hypoxic, use of muscle relaxants, number of episodes of raised intracranial pressure (icp) above mmhg as recorded in the intensive care chart every hour, number of episodes of cerebral perfusion pressure (cpp) below mmhg as recorded in the chart every hour was noted. need for muscle relaxant in the first h of admission was noted. the outcome was recorded as either ''alive'' or ''dead'' at the end of itu stay. the data was checked for normality of distribution and compared using non parametric tests (spss for windows). results. baseline characters were comparable between the groups. increased episodes of hypoxia ( . ± . vs. . ± % p = . ) and hypocarbia ( . ± . % vs. . ± . % p = . ) in bipap mode, compared to simv volume control mode. all measurements being percentages of total blood gases for that patient in the first h. there was no difference in the usage of muscle relaxant ( . vs. . % p = . ), raised icp, reduced cpp or mortality between the groups. conclusion. bipap mode of ventilation requires more intensive monitoring and changes in ventilatory settings before adapting to the target blood gas parameters in the first h of admission. at the same time the quoted advantage of using less sedatives and muscle relaxants is not significant. acute post-traumatic brain swelling is one variety of the pathological forms, which needs emergent treatment following traumatic brain injuries. we investigated the effects of clinical effects of decompressive craniectomy (dc) in patients with acute post-traumatic brain swelling (bs). seventy-four patients of acute post-traumatic bs with midline shifting more than mm were divided randomly into two groups: dc group (n = ) and routine temporoparietal craniectomy group (control group, n = ). the vital sign, the intracranial pressure (icp), the glasgow outcome scale (gos), the mortality rate and the complications were prospectively analysed. the mean icp values of patients in dc group at , , and h after injury were much lower than those of routine temporoparietal craniectomy group ( . ± . , . ± . , . ± . and . ± . mmhg vs. . ± . , . ± . , . ± . and . ± . mmhg, respectively). the mortality rates at month after treatment were % in the dc group and % in the control group (p \ . ). good neurological outcome (gos score of to ) rates year after injury for the groups were . and . %, respectively (p = . ). the incidences of delayed intracranial hematoma and subdural effusion were and %, respectively (p \ . ). in conclusion, dc has superiority in lowering icp, reducing the mortality rate and improving neurological outcomes over routine temporoparietal craniectomy. however, it increases the incidence of delayed intracranial hematomas and subdural effusion, some of which need secondary surgical intervention. therefore, the effects of dc in patients with acute post-traumatic bs should be further evaluated. we analyze among others variables: age, injury severity score (iss), abbreviated injury score (ais); admission and discharge glasgow coma score (gcs), extended glasgow outcome score (gose), complications, icu and hospital mortality. differences between groups were tested with students t test and v testing for statistical analysis. results. fourteen patients with intracranial hypertension were treated with decompressive craniectomy . compared with control group, patients with dc had a better gcs ( ± g ; ± g p = , ) and gose index not only at icu discharge ( ± g ; ± g p = , ) but also at hospital discharge ( ± g ; ± g p = . ). the mortality rate was lower in the craniectomy group (g : %, g ; % p = . ). conclusions. in our center, the use of dc for treat patients with severe tbi and refractory cranial hypertension (gcs b and pic c ) improved outcome and mortality significantly compared with medical conventional approach. method. in this retrospective study we present patients who underwent decompressive craniectomy following traumatic brain injury at king's college hospital between and . results. % of these patients presented at a&e with a glasgow coma scale of or below whereas the remaining % presented with gcs above and deteriorated following admission. the patients underwent decompressive craniectomy to reduce raised icp resistant to medical treatment (barbiturate coma excluded). the procedure resulted in significant decrease in icp. out of patients had the operation within h following their injury. we also found that dc in younger patients (\ years) was correlated with lower icp following the operation compared to older patients ([ ) . our study also showed that early dc (\ h) is correlated with a shorter stay in itu. conclusions. the findings of the present study are limited by its retrospective nature and small sample size which does not permit any definitive conclusions from these results. however, they form the basis for further investigation. we present the study with a review of the recent literature. introduction. the objective is to study the correlation of secondary icp indices with ct findings and outcome in tbi. a cerebrovascular pressure reactivity index (prx) can be determined as the moving correlation coefficient between mean icp and mean arterial blood pressure . it is a surrogate marker of cerebrovascular reactivity. the rap coefficient was calculated as the running correlation coefficient (r) between slow changes in pulse amplitude (a) and mean icp (p). it is a surrogate marker of pressure-volume compensatory reserve. all components of the icp waveform that have a spectral representation within the frequency limits of . to . hz can be classified as slow waves. methods. prospective observational study of patients with tbi at the royal london hospital. all patients were managed according to the local guidelines for the management of tbi . secondary indices derived from the icp waveform were analyzed by icm ? software. an initial ct was performed in all patients before admission to icu. marshall classification has been shown to predict mortality in tbi. we found a strong association between all these secondary indices and the initial ct findings. all these markers of cerebral haemodynamics correlate significantly with outcome in headinjured patients. conclusions. surrogate markers of cerebrovascular reactivity and pressure-volume compensatory reserve correlates with ct findings and outcome in tbi. these secondary icp indices may be used in the management of tbi. introduction. following the introduction of national guidance [ ] on the management of patients with head injury, the use computed tomography (ct) imaging of the head has increased markedly. the impact on anaesthetic and critical care services is unknown. . determine the impact of national guidelines on ct scanning in the head injured patient upon anaesthetic and critical care services in a university teaching hospital. . determine the incidence of acutely abnormal ct appearances in patients referred for ct scanning under the guidelines. . estimate in-hospital mortality in this population and its sub-groups. a case-note analysis was performed in october of consecutive emergency department (ed) patients who were recorded as having a ct head. of the cases, did not actually have ct head. details of the analysed subjects, indications for the scan and day mortality rates are reported in table . in patients with severe traumatic brain injury pro-and anti-inflammatory mediators are released into the systemic circulation. however, the relationship between the inflammatory response and the kind and duration of secondary insults remains unclear. objectives. the aim of this study was to investigate in severe traumatic brain injured patients the relationship between the systemic concentrations of pro-and anti-inflammatory mediators and the total duration of secondary insults occurring during the icu stay. methods. ten consecutive traumatic brain injury patients admitted to the icu were included. physiological variables were continuously recorded and analyzed minute-by-minute to identify the occurrence of secondary insults (intracranial hypertension, systemic hypotension, hypoxemia and hyperthermia) according to the edinburgh university secondary insult grading scale. serum samples were obtained at admission, , and h, in which pro-and anti-inflammatory mediators were analyzed by a bioplex assay. results. ten male patients were enrolled, mean age ± , gcs ± , apache ii ± , iss ± . patients were monitored for . days (median value, range - ; , total minutes recorded); intracranial hypertension occurred for , min ( . % of total period recorded, range . - %), hypotension occurred for , min ( . % of total period recorded, range . - %), hypoxemia occurred for min ( . % of total period recorded), not enough data were validated for fever. interleukin (il)- , il- beta, il- , il- and il- ra were in the detectable range. a significant correlation was found between the total duration of intracranial hypertension and the median value of il- (p \ . , r = . ), il- beta (p \ . , r = . ), il- (p \ . , r = . ), il- (p \ . , r = . ), and il- ra (p \ . , r = . ) measured during the period of observation. no correlation was found between these inflammatory mediators and the occurrence of hypotension or hypoxemia. no significant correlation was present between the baseline values of these inflammatory mediators and the severity indexes (gcs, iss and apache ii). conclusions. these results suggest that the duration of secondary insults such as intracranial hypertension was associated with a systemic inflammatory reaction, while the severity of injury on admission was not related to the initial concentrations of these inflammatory mediators. grant acknowledgement. aim. assessing behavioral responses to pain is difficult in severely brain-injured patients recovering from coma. we here propose a new scale developed for assessing pain in vegetative (vs) and minimally conscious (mcs) coma survivors: the coma pain scale (cps) and explore its concurrent validity, inter-rater agreement and sensitivity. methods. concurrent validity was assessed by analyzing behavioral responses of postcoma patients to a noxious stimulation (pressure applied to the fingernail) ( vs. and mcs; age range to years; non-traumatic and of traumatic origin). patients' were assessed using the cps and four other 'pain scales' employed in non-communicative patients: the 'neonatal infant pain scale' (nips) and the 'faces, legs, activity, cry, consolability' (flacc) used in newborns; and the 'pain assessment in advanced dementia scale' (pa-inad) and the 'checklist of nonverbal pain indicators' (cnpi) used in dementia. for the establishment of inter-rater agreement, fifteen patients were concurrently assessed by two examiners. results. concurrent validity assessed by spearman rank order correlations between the cps and the four other validated pain scales was good. cohen's kappa analyses revealed a good to excellent inter-rater reliability for the cps total and subscore measures, indicating that the scale yields reproducible findings across examiners. finally, a significant difference between cps total scores was observed as a function of diagnosis (i.e., vs or mcs). conclusion. the cps constitutes a sensitive clinical tool for assessing pain in severely brain injured patients with disorders of consciousness. this scale constitutes the first step to a better management and understanding of pain in patients recovering from coma. methods. study group: consecutive patients with cervical spinal cord injury admitted to icu. mean age: , years. patients asia a, asia b, asia c. the more frequent neurological level was c ( %). the requirement of mechanical ventilation was considered the key sign for establishing the diagnosis of severe respiratory failure. the blood gas values (po , pco , and pao /fio ) before and after connection to mechanical ventilation [mv(if needed)], were used to estimate the more probably mechanism of respiratory insufficiency. the increase of pco levels was considerate as a sign of neuromuscular weakness; the low po level before ventilation, and the persistence of pao /fio below normal values was considered a sign of v/q mismatch. for this purpose statistic analysis (mean values comparison using student t test) comparing blood gases before and after mechanical ventilation treatment was performed. results. ( %) patients developed severe respiratory failure. mean delay between admission and mechanical ventilation was h. previously to mechanical ventilation patients developed pulmonary atelectasis, and four pneumonia. the incidence en respiratory failure was significantly higher in patients with neurological level above c (p \ . ). conclusions. the incidence of respiratory failure is related with the severity of neurological deficit (relationship between incidence of respiratory failure and neurological deficit level). in addition, our data support that, besides the neuromuscular weakness (moderate increase of co levels), a significant v/q mismatch with shunting phenomena associated (significant hypoxemia no completely solved after mv) is involved in the respiratory failure of cervical spinal cord injured patients. . moderate and severe traumatic brain injury is more likely in middle aged men; more than one third present other major trauma and intensive first level medical treatment is required in most of them. . the most frequent complications found were infectious diseases like ventriculitis and vap. . independent mortality risk factors in moderate and severe trauma brain injury were age, high apache ii score, neuromuscular blocking drugs and icu los. . outcome was significantly improved after six months, and most of the patients only present mild disability and good recovery. nosocomial infections are leading causes of increased morbidity and mortality of severe brain injured patients [ ] . the mechanism underlying the susceptibility to the infections is a subject of great scientific interest and still to be clarified [ ] . it has been recently recognized that injury of brain induces a disturbance of balance between the central nervous and immune system [ ] . objective. the aim of this study was to investigate changes in frequency of lymphocytes subpopulation in peripheral blood of patients with severe brain injury during the course of intensive care treatment. human peripheral blood samples were taken from the severe brain-injured patients at day , and and peripheral blood mononuclear cells (pbmc) were immediately isolated by gradient density centrifugation. the percentage of lymphocytes subpopulation were analyzed by simultaneous detection of surface antigens using fluorochrome conjugated monoclonal antibodies directed toward cd , cd , cd , cd , cd , cd , cd . t lymphocytes were distinguished from the other lymphocyte subpopulation as cells labeled with anti-cd monoclonal antibody but negative for cd staining (cd ? cd - patients. eighty-seven patients with head injury, glasgow coma scale \ . measurements and main results. clinical and demographic data, and head ct scan were taken at admission. patients underwent advanced neuromonitoring and were treated according to brain trauma foundation guidelines. s b concentration was quantified at admission and , and h post-tbi (days , , and ). outcome was assessed months after discharge using glasgow outcome score. significant negative correlations were found between -year gos and s b concentrations on days - , but not on day (day , p = . ; day , p = . ; day , p \ . ; day , p \ . ). patients who deceased showed higher s b concentration than survivals for all the samples. good versus poor outcome (gos = - ) differed significantly on days and . logistic regression analysis showed that samples , and h post-tbi sample predicted death outcome. roc curve analysis showed -h sample was the strongest predictor for decease. poor outcome was only predicted by the -h sample. conclusions. s b levels h post-tbi was the strongest predictor for poor and fatal -year outcome, whereas levels at admission do not. a temporal profile of s b release from admission to h post-tbi is strongly recommended for use in identifying the subset of patients liable of developing a worse outcome. according to our results, s b protein might be an early, sensitive, accurate and useful biomarker for predicting long-term outcome in patients with acute severe tbi. grant acknowledgement. this research was made possible in part by the generous donation of protein s b electrochemiluminescence assay kits by roche diagnostics, mannheim, germany. introduction. brain intercellular fluid glycerol concentration as measured by microdialysis catheters has been recognized as an index of glial and neuronal cellular destruction. we present a data analysis correlating glycerol levels with intracranial pressure (icp), cerebral perfusion pressure (cpp), brain tissue oxygen partial pressure (pbtio ), lactate to pyruvate concentration ratio (l/p) and outcome. methods. data of head injured patients is presented. all had simultaneous monitoring of icp, pbtio and metabolic biochemistry by three brain intraparenchymal bolt catheters inserted via the same one burrhole (icp codman or camino, pbtio licox and microdialysis-cma). there was not a clear straight correlation of raised glycerol levels with bad outcome. however, glycerol elevation seemed to be a predictor of intracranial hypertension together with l/p raise. in subarachnoid hemorrhage patients glycerol elevation was an early sign of secondary ischemic insult. conclusion. multimodal monitoring with intracranial catheters is a useful clinical tool for management of critical neurosurgical patients. metabolic biochemistry as measured by microdialysis, and specially l/p and glycerol levels, can early predict incoming intracranial hypertension as well as secondary ischemia. the pulsatility index (pi), a parameter derived from the blood velocities along the cardiac cycle, has been used as an indirect way to evaluate intracranial pressure. the aim of this research has been to evaluate the accuracy of transcranial doppler sonography (through pulsatility index) in the inference of intracranial pressure. methods. population of the study group (high-pi-group): severe head injured patients (gcs at admission \ ; mean age . years; patients with diffuse injury (traumatic coma data bank) type ii ( %) and iii ( %)) who presented episodes of increase of pulsatility index (pi [ . ) in the acute phase of head injury. control group (normal-pi-group): severe head injured patients, with tcd recordings of normal pi (pi b . ). in all the patients the intracranial pressure (icp) was continuously monitored using a intraparenchymal device. all the tcd recordings are referred to the middle cerebral artery of the cerebral hemisphere were icp catheter was inserted. in the transcranial doppler recording, the pulsatility index was automatically calculated derived from the formulae: pulsatility index = (systolic velocity -diastolic velocity)/mean velocity. transcranial doppler sonography recordings of with pulsatility index c . (high normal value of pulsatility index) were correlated with the simultaneous icp value. the incidence of intracranial hypertension (icp [ mmhg) was analyzed in the high-pi-group, and compared with the incidence of intracranial hypertension in the normal-pi-group. methods. in a double-blind, randomized, placebo-controlled clinical trial, patients scheduled for elective cabg was randomly assigned into two groups. after matching inclusion and exclusion criteria and induction of general anesthesia, one group received intrathecal sufentanil (s) and the other group received the same dose of sufentanil plus supplemental bupivacaine (sb). except for this, all the cases were similar regarding anesthesia and surgery. mean arterial blood pressures were measured before and after induction of anesthesia, during the bypass time and after weaning from bypass were checked. also, the need of the patients for administration of inotropic agents after weaning was compared. results. there was more stable mean arterial blood pressure and less inotropic need after weaning from cardiopulmonary bypass in the sb group. also, the sb patients had a more stable hemodynamic profile during the bypass period; especially after the initiation of the bypass. less inotropic agents were needed after weaning in the sb patients. there was no difference between the two groups regarding the extubation time. discussion. the administration of intrathecal sufentanil plus bupivacaine seems to keep the hemodynamic status of the patients more stable than intrathecal sufentanil alone. methods. this study was approved by the hospital s ethics committee. prospective observational study including consecutive patients. preoperatory and postoperatory data were collected. interventions included blood samples for nt-pro bnp taken prior to operation, and and h in postoperative. troponin-i was taken and h postoperatively. blood obtained was processed for nt-probnp with cobas h system Ò point of care (poc) by roche diagnostics, with range from to , pg/ml. the serum nt-probnp level was also correlated with the logistic euroscore and ejection fraction (ef). serum ntpro-bnp and troponin i values were compared between patients with and without postoperative length of stay in the intensive cardiac unit (icu) [ h. and hospital [ days. all results are in median ± sd * p \ . , **p \ . tables ??? and ??? conclusions. preoperative euroscore and nt-probnp levels were higher in patients with ef \ %. the troponin i after surgery increased more in patients whose length stay in icu was longer. after surgery nt-probnp levels increased significantly,and they differ significantly between patients with length stay in icu for more than h and days at hospital. our data collection confirmed that measurement of nt-probnp is useful and helpful during postoperative period and it also predicted a higher possibility for a long stay in icu and a later hospital discharge. however, owing to the small size sample, these results must be regarded as preliminary. conclusions. in spite of the limitations of our trial, percutaneous aortic valve implantation appears to be safety. a high rate of maccv events were observed, essentially due to a disruption of the a-v conduction, in most cases transitional. despite the definition of ''inoperability'' is difficult, less-invasive aortic valve procedures will undoubtedly find a place within current cardiac surgical practice. objective. to describe the evolution of cardiac transplant patients, presenting clinical low cardiac output in the immediate postoperative period, and after handling routine, they are treated with levosimendán (lv). descriptive, prospective and observational in a postoperative care unit for cardiac surgery from a terciary hospital. study period: january -december . lv was used when the patient had inotropic dependence over h, to try to remove the amines or added to them in those cases that do not get these drugs with an adequate hemodynamics. bolus was used in occasions and then infusion of . - . mcg/ (kg min). we analyzed demographic variables, hemodynamic response to the input of the drug if you can reduce or discontinue other medications, clinical tolerance and side effects, overall development, the icu and hospital stay. we studied patients ( women and men). presented a mean age of . . before surgery, all of whom were in nyha functional class iii-iv. three patients were transplanted in emergency. in this series, there is a case without pulmonary hypertension (pah) pre-transplant, patients with mild htp and htp moderate to severe, with a transpulmonary gradient(gtp) between and mmhg. the patients with gtp [ mmhg had a positive reversibility test with sildenafil. ischemia time of surgery was . . in the immediate post, all the patients studied had low cardiac output syndrome by graft postoperative ventricular dysfunction, cardiac index measured by pulmonary artery catheter. in all patients echocardiography was performed to rule out a pericardial effusion with hemodynamic deterioration in cardiac cavities and showed ventricular dysfunction, right dominance in patients. in all patients we observed a good tolerance to the drug. in lv cases facilitated the withdrawal of the remaining. patients were used lv only after the withdrawal of treatment with inotropic dependence on it. in the remaining cases to be associated with other drugs. only two cases could not withdraw inotropic treatment after the lv infusion. in five patients with pulmonary arterial hypertension and prevalence of right ventricular failure, to reduce poscarga also added pulmonary arterial vasodilators. patients have a stay in icu between and days. one patient mortality. . the primary graft failure is a severe potential complication of post-cardiac, which is associated with a worse prognosis. . lv shows good tolerance, without serious adverse effects attributable to the drug, and facilitated the removal of amines and clinical recovery. . it is necessary to expand the case to confirm the results, and to establish the most appropriate indications and patterns of use of this drug. post-infarction ventricular septal defect (infarctvsd) is a rare but serious complication of myocardial infarction, usually quickly followed by low cardiac output. repair of infarctvsd is still a challenging procedure with a high risk of mortality. improvement of surgical outcome depends on results of large studies in this setting. the aim of this retrospective study was the evaluation of preoperative and surgical parameters influencing the -day mortality following surgical repair. conclusions. in this large study, pre-operative left ventricular function and troponin level were found to be the best predictors identifying patients at high risk for -day mortality following surgical closure of infarctvsd. both parameters may be helpful in deciding on the time of the operation and preoperative preparation. in contrast to other findings, in our cohort the location of the vsd (anterior vs. posterior) did not affect mortality. this may be due to improvement of surgical technique and perioperative management over time. adequate fluid therapy is the first step of hemodynamic optimization after cardiac surgery [ ] . cardiac surgery exposes patients to ischemia and reperfusion, which are well known risk factors for a systemic inflammatory response and increased capillary permeability in the lungs [ ] . it is still unclear what type of fluid should be given in the presence of increased pulmonary vascular permeability at hypovolemic status. objectives. aim of this study was estimate the optimal type of fluid for intravascular volume deficit treating without evoking pulmonary oedema. a prospective clinical study at the intensive care unit was performed on mechanically ventilated patients within h after elective cardiac surgery involving cardiopulmonary bypass. patients, divided into four groups, were subjected to fluid challenge according to the global end-diastolic volume index (gedvi) measurements with normal saline , ml or the colloids % gelatin, % hes / . or % albumin ml in min. hemodynamic and extravascular lung water index (evlwi), gedvi measurements were performed exactly before fluid challenge, afterwards and min after challenge. results. the change in evlwi did not differ between saline and colloid fluid challenge. gedvi increased by % in saline group, by % in % gelatine, in % hes / . and in % albumin. conclusions. all colloid fluid infusion leads to the greater increase in cardiac preload compare to normal saline (saline in four times larger volume). the change in evlwi did not differ between saline and colloid fluid groups and did not increase pulmonary oedema despite in the presence of increased pulmonary vascular permeability, when fluid overloading is prevented. introduction. the annual incidence of prosthetic valve thrombosis is up to - % (patients-year) despite the anticoagulant therapy. conventionally, the treatment of choice for this event was the surgical valve replacement. however, fibrinolytic therapy has become a valid alternative for the treatment of this serious complication, especially in high-risk surgery patients. to analyze the clinical factors, diagnosis and treatment management of patients with prosthetic valve thrombosis admitted to the acute cardiac care unit. we designed an observational-descriptive study, including patients admitted between and . clinical factors were analyzed: sex, age, prosthetic valve position, time from valve replacement, inr at admission, clinical features, diagnostic technique and treatment used. results. patients were included. . % were women, . % men. mean age was . ± . years. the highest incidence was at the tricuspid prosthetic valve position ( . %), followed by the mitral ( . %) and the aortic position ( . %). when a triple valve replacement was performed, the tricuspid position was the most often affected. mean time from the first valve replacement surgery was . ± . years. clinical features which led to the diagnostic were: acute heart failure ( . %), peripheral embolization ( . %), chest pain ( . %) and syncope ( . %). the diagnostic techniques used were transesophageal echocardiography (tee) and cinefluoroscopy in all the patients. inr at admission time was lower than adecuate anticoagulation recommendations in . % of patients. the most widely used treatment was the systemic fibrinolytic therapy ( . %), followed by surgery ( . %) and conservative treatment with heparin alone ( . %). the most widely used thrombolytic was rtpa in . % of patients, with a mean dosage of . ± . mg. one patient was treated with . mil. ui of streptokinase. unfractionated heparin was added to all patients whom received fibrinolytic therapy, with a mean dose of ± ui/h. a . % incidence of minor bleeding was found in the fibrinolytic group. there were no major complications due to fibrinolytic. total mortality rate was . %. our experience, suggests that systemic fibrinolytic therapy is safe and effective in patients with prosthetic valve thrombosis. objective. to describe the outcomes of patients with acute, refractory, non-ischaemic and not postcardiotomy, cardiogenic shock treated with extracorporeal membrane oxygenation (ecmo) and to evaluate whether survivors and non-survivors differed with respect to clinical characteristics, pre-ecmo treatment and laboratory values. design. in this retrospective cohort study, information is collected from a database with additional review of medical records. patients. consecutive adult patients, males, mean age . ± . year, presenting to hospital with non-ischaemic acute severe, refractory cardiogenic shock, supported by central or peripheral venoarterial (va) ecmo. measurements and main results. characteristics of survivors and non-survivors were compared using chi square test. twelve patients ( %) were transported to our institution on ecmo. eleven patients ( %) were weaned from ecmo, seven ( %) bridged to ventricular assist devices. in two patients ( %) ecmo support was withdrawn. mean duration of ecmo support was . ± . h. overall survival was %, and did not differ between patients with myocarditis (n = ), cardiomyopathy (n = ) and acute on chronic non-ischaemic cardiogenic shock (n = ). a larger proportion of the three patients with or more complications died as compared to the seventeen patients with less than complications ( % versus %, p = . ). pre-ecmo intra-aortic balloon counterpulsation (iabp) was used in patients, % survived, as compared to % of those who did not receive iabp (p = . ). we have not identified any other significant differences between survivors and non-survivors. conclusion. the survival of patients on ecmo in this unique heterogeneous patient cohort is similar to the survival of ecmo support for fulminant myocarditis in the literature. we recommend to institute ecmo early in all medical patients with acute non-ischaemic cardiogenic shock, refractory to conventional therapy, or to refer these patients in time to an ecmo centre. introduction. human parvo b virus is associated with a broad spectrum of clinical manifestations, mostly in children or immune-compromised patients. in adults, severe myocarditis due to this viral agent is a rare disorder, presenting as acute congestive heart failure. we describe a patient with rapidly progressive heart failure, needing circulatory support by extracorporeal membrane oxygenation (ecmo). methods. case report. a -year-old previous healthy female was admitted to our icu with nausea, vomiting, bradycardia and hypotension with a blood pressure of / mmhg. two weeks before admission, patient had signs of erythema infectiosum. on physical examination the patient was pale, with venous congestion, third heart sound and hepatomegaly. the initial electrocardiogram showed a slow, regular, ventricular rhythm. admission chest x-ray showed normal heart size with bilateral pleural effusion. echocardiography revealed dilated ventricles (rv and lv) with depressed systolic function and a thrombus in the rv apex. patient was initially treated with intravenous medical therapy, but unfortunately developed progressive cardiogenic shock. troponin levels, serum transaminases and bun were extremely elevated. it was therefore decided to implant a percutaneous ecmo by femoro-femoral cannulation which permitted to stabilize hemodynamic conditions while peripheral organ functions returned to normal range. progressive cardiac recovery was observed after days with a circulatory assistance with a mean flow rate of . l/min. as myocardial function improved, ecmo was gradually weaned and removed after days of support. however, atrioventricular conduction did not recover, necessitating implantation of temporary vvi-pacemaker, which was later replaced by a permanent ddd pacemaker system. pathology of the endomyocardial biopsy showed extensive lymphocytary infiltration with destruction of myocytes. parvo b dna-pcr was positive in both the biopsy and serum. these findings suggest that this patient developed severe myocarditis induced by parvo b viral infection. to our knowledge, parvo b viral infection is an uncommon cause of severe myocarditis in adult patients. sparse literature is available describing the use of ecmo in these adult patients. conclusion. this case report shows that parvo b virus should be recognised as a potential infective agent in adult patients presenting with severe myocarditis. furthermore, ecmo can be safely used to stabilize hemodynamics and peripheral organ perfusion in expectation of myocardial recovery in these patients. copeptin is easier to measure than vasopressin, and could be used as a marker of vasopressin release [ ] . the aim of the study was to compare plasma concentration of avp and cop during cardiac surgery, and specifically during post cardiac surgery vasodilatory syndrome (pcsvs). methods. two-month consecutive patients scheduled for cardiac surgery with cardiopulmonary bypass (cpb) were included in the study except patients suffering from chronic renal failure and under dialysis. blood samples were obtained from blood withdrawals routinely operated before cpb, during cpb and after surgery, at the postoperative hour (h ). these samples were used for avp and cop measurements. pcsvs, assessed as hypotension unresponsive to volume replacement therapy and without cardiogenic shock features, was treated with norepinephrine (ne). patients treated with ne have been compared to the others. statistical test consisted of variance analysis, non parametric test (mann whitney or wilcoxon) and linear regression. a p value of less than . (p \ . ) was considered statistically significant. results. patients have been included, out of which have been treated with ne. correlation between avp and cop plasma concentrations is significant (r = . , p \ . ). avp and copt concentrations increased significantly at h but the increase is less pronounced in ne-treated patients (fig. ) . ne-treated patients had lower preoperative left ventricle ef ( . ± . vs. . ± . %, p = . ), longer cpb ( . ± . vs. . ± . min, p \ . ) and clamping times ( . ± . vs. . ± . min, p \ . ), higher incidence of low output syndrome ( / vs. / , p \ . ) longer extubation time ( . ± . vs. . ± . h, p \ . ) and higher plasma cop before (t ) and during cpb (fig. ) . avp (ng/ml) et copeptin (cop, pmol/l), in patients. *p \ . ne versus others discussion. correlation between avp and cop is similar to that observed in other studies [ ] . the correlation coefficient is rather weak that is possibly related to avp dosage limitations (binding of avp to blood platelets, lack of antibody-specificity). increased cop plasma concentrations before and during cpb is observed in sicker patients undergoing more complex surgery, which seems to expose them to relative postoperative vasopressin deficiency and pcsvs. background. waiting list for heart transplantation has been growing up. high doses of cathecolamines has been an exclusion criterion for heart donation and norepinephrine use is still controversial. to assess if norepinephrine used on heart donors modify receptors outcome. methods. historical cohorts study from april to march . patients were divided in two groups: group : patients with local donors treated with norepinephrine (n = ). group : patients with local donors managed with other cathecolamines (n = ).cathecolamines were used at least for h and doses were between . and mcg/(kg min) if norepinephrine and between and mcg/(kg min) if dopamine or dobutamine. mortality risk factors published on the last international society for lung and heart transplantation guidelines were recorded. graft dysfunction risk factors were also collected. heart transplant outcome was measured by -day mortality, mortality rate at first, second, fifth and tenth years; and graft dysfunction incidence. chi-squared and t student test was used. multivariate logistic regression was used to evaluate norepinephrine impact on the outcome. mortality in group was . and % in group . no differences in mortality or graft dysfunction incidence were found in multivariate analysis. conclusions. norepinephrine used for donors management compared with dopamine and dobutamine does not increase mortality or graft rejection incidence in heart transplantation. groups were not uniform so further studies may be made to determine this association. introduction. coronary artery bypass surgery on cardiopulmonary bypass is associated with significant morbidity and mortality. with present technology, all arteries on the heart can be bypassed off-pump. the benefit of this technique is higher for patients whom are at increased risk of complications from cardiopulmonary bypass, such as those who have heavy aortic calcification, carotid artery stenosis, prior stroke, and compromised pulmonary or renal function. to evaluate the short-term follow up results of off-pump coronary artery bypass (opcab) and postoperative management of these patients admitted to our coronary care unit. we designed an observational study that included patients who underwent opcab from july to december . data were collected on preoperative age, sex, major cardiovascular risk factors, history of prior ami, number of affected vessels and ventricular function. after the surgery we evaluated: the extubation time, postoperative bleeding, troponin maximum level, need for blood transfusion, use of vasoactive drugs and intra-aortic balloon pump, development of renal failure, atrial fibrillation, neurological complications and reintervention. results. patients were included. . % were men and . % women. mean age was . ± . years. % of patients had one or more cardiovascular risk factor: hypertension was present in . %, smoking . %; diabetes mellitus . % and dyslipidemia in . %. there was prior myocardial infarction in . % of patients. prior coronary angiography showed . % of patients with vessels disease and . % of vessels disease. mean lvef was %. mean number of grafts was . . mean extubation time was . h. mean postoperative bleeding was estimated in cc. . % of patients needed blood transfusion; . % vasoactive drugs; and . % needed an intra-aortic balloon pump. . % of patients developed troponin t elevation with a mean level of . ng/ml. . % of patients developed atrial fibrilation, and . % renal dysfunction (two patients needed hemodialysis). there was no neurological complications. patient needed a reintervention. mean of intensive care unit stay was . ± . days. total mortality rate was . %. our experience shows that the off-pump coronary artery bypass graft surgery is a safe and effective technique for coronary revascularization, with low mortality and morbidity rates and reduced postoperative complications. objectives. to assess if deterioration of left atrial function in patients with severe sepsis and septic shock could predict mortality. we studied patients with severe sepsis or septic shock with mean age of . ± . . underlying echocardiographic parameters were measured on admission, th and th day, which comprised left ventricular ejection fraction (ef), and atrial function which is expressed as atrial ejection force (aef), with aef defined as the force that the atrium exerts to propel blood into the left ventricle (lv). all patients were subjected to bnp assay well. multivariate analyses adjusted for acute physiology and chronic health evaluation score ii (apache ii score) was used for mortality prediction. results. underlying source of sepsis was lung in patient ( %), blood in seven patient ( . %), abdomen in seven patients ( . %), while three patient ( %) had urinary tract infection (uti) as a cause of sepsis. only one patient had cns infection. severe sepsis was admission diagnosis for patients, patients were labeled as septic shock. look for days mortality. in-hospital mortality was . % ( patients) . admission ef showed significant difference between survivors and non-survivors . ± . versus . ± . % (p \ . ), on the other hand admission aef showed insignificant changes between the same groups . ± . versus . ± . k/dynes p = . , while bnp was significantly higher in the non-survivors , ± . versus . ± . pg/ml (p \ . ). multivariate logistic regression, the predictable variables for mortality was apache ii score, bnp then ef. conclusion. in septic patients, left atrial function unlike the ventricular function and bnp levels cannot be used as independent predictor of mortality. objectives. to analyse the relationship between plasma levels of nt-probnp and lcd diagnosed by echocardiograph during ss. methods. prospective observational cohort study. inclusion criteria: consecutive patients with ss [ ] . non inclusion criteria: creatinine clearance \ ml/min, years \ age \ years, cardiac surgery patients, pre existing coronary or cardiac insufficiency, neoplasia and systemic diseases. the evaluation of the left ventricular function was realised by a trans-thoracic or a trans-oesophageal echocardiograph on day . the lcd was defined by a left ventricular fraction of ejection \ % evaluated by teicholtz. the blood tests for nt-probnp analyses were drawn on days , , and . serum nt-probnp measurements were made automatically by elecsys analyser with the truss nt-probnp (roche diagnostics, myelan, france) by the electrochemiluminescence immunoassay method (eclia). data are expressed as mean ± sd and percentages. statistical analysis was performed by repeatedmeasures anova and roc curves (p \ . indicated statistical significance). . patients were included in a period of months (medical patients n = , surgical patients n = and trauma patients n = ), age = ± years, bmi = ± kg/m , apache ii = ± , igs ii = ± , duration of intensive care unit stay = ± days, mortality = %. lcd was observed in patients. the statistical analysis showed a significant elevation of nt-probnp in patients with lcd (table ) . on day , the area under roc curve was . , and the cut off value of nt-probnp predictive of lcd was , pg/ml (sensibility = %, specificity = %). introduction. fluid responsiveness can be predicted by the respiratory variation of arterial pulse pressure (ppv) or of pulse contour-derived stroke volume (svv) as well as by the changes in pulse contour-derived cardiac index during a passive leg raising manoeuvre (plr) or a tele-expiratory occlusion (teo). we evaluated the ability of an infrared photoplethysmography arterial waveform (cnap device) to estimate ppv. we also tested the ability of this non invasive estimate of ppv to predict fluid responsiveness compared to the invasive measure of ppv, to svv and to the plr and teo tests. in patients with septic shock ( ± years of age, receiving norepinephrine, saps = ± , lactate = . ± . mmol/l), we measured the response of cardiac index (pulse contour analysis, picco device) to fluid administration ( ml saline over min). before fluid administration, we recorded the ppv directly calculated from the non invasive arterial pressure signal (ppv ni ), the ppv directly calculated from the invasive arterial pressure signal (ppv i ), the ppv automatically provided by the picco device (ppv picco ), the svv automatically provided by the picco device, the changes in cardiac index induced by a plr test and the changes in cardiac index induced by a -s teo. results. five patients were excluded because the arterial curve could not be obtained by the cnap device due to excessive vasoconstriction. in the remaining patients, fluid administration increased cardiac index by more than % ( ± %) in ''responders''. the fluid-induced changes in invasive (? ± %) and non invasive (? ± %) mean arterial pressure were correlated (r = . , p \ . ). at bland-altman analysis, ppvni accurately reflected ppvi (bias %, limits of agreement ± %). for predicting fluid responsiveness in the patients, the receiver operating characteristics (roc) curves for ppv ni , ppv i , ppv picco , svv, plr and teo were . ± . , . ± . , . ± . , . ± . , . ± . , . ± . (all non significantly different). when considering only the patients ventilated with a tidal volume b ml/kg predicted body weight, were falsely classified as non responders by ppv ni , ppv i and two others by ppv picco and svv, but all four were well classified by plr or teo. in septic shock patients, provided that vasoconstriction is not excessive, the non invasive assessment of arterial pulse pressure seems valuable for predicting fluid responsiveness. introduction. mechanical ventilated patients often require inotropic support. however, the role of mechanical ventilation (mv) in myocardial depression is not well understood. septic patients often have impaired cardiac function and are in need of mechanical ventilation. we hypothesized that mv enhances sepsis-induced myocardial depression. objectives. in this study we investigated the influence of mechanical ventilation on cardiac function in an acute sepsis model. sepsis was induced in male wistar rats using ip injection of lps. healthy and septic rats were randomized to one of three ventilation groups; ( ) non-injurious ventilation with a tidal volume of ml/kg and cm h o peep (low tidal volume, ltv), ( ) injurious ventilation with a tidal volume of ml/kg and cm h o peep (high tidal volume, htv) and ( ) spontaneous breathing. arterial pressure was kept at least at mm hg. cardiac output (co, thermodilution method), central venous pressure (cvp) and mean airway pressure were measured in vivo. after h of ventilation, animals were sacrificed and cardiac function was measured ex vivo in a langendorff setup and expressed as developed pressure and ?dp/dt. cardiac wet to dry weight ratio was calculated. results. cardiac output in vivo was lower during htv ventilation than during ltv ventilation (p \ . ). cvp did not differ between ventilation strategies while mean airway pressure was higher in htv ventilation than in ltv ventilation (p \ . ). ex vivo, cardiac function of septic animals was depressed compared to healthy controls (p \ . ) in septic animals, cardiac function was better in htv ventilated animals than in non ventilated animals (p \ . ). ventilation lowered cardiac wet/dry ratio (p \ . ). developed pressure (p \ . ) and ?dp/dt (p \ . ) correlated inversely with cardiac wet/dry ratio. [ ] . perfusion may be also evaluated by other parameters such as lactate or venous-arterial pco gradient (delta pco ). objectives. to evaluate if early normalization of scvo after emergency intubation in septic patients persists over time and if it is associated with similar trends in lactate and delta pco . methods. ten septic patients subjected to emergency intubation for respiratory or circulatory failure and in whom scvo increased to [ % after the procedure. these patients were included in a large prospective study published elsewhere [ ] . patients used a common intubation protocol and we evaluated several perfusion related parameters before, min and h after emergency intubation. statistical analysis included friedman and wilcoxon tests. results. evolution of perfusion parameters after intubation is presented in table . five patients died during icu stay. as a whole, scvo remained stable in pts and decreased dramatically at h by[ % in non-survivor patients (lowest %). only pts had a high lactate before intubation that did not normalize at h (both non-survivors). delta pco exhibited erratic changes over time with no correlation with scvo changes and with mortality ( fig. ). introduction. venous to arterial carbon dioxide difference (pv-aco ) could reflect the sufficiency of blood flow in shock states. time evolution of pv-aco during early phases of resuscitation in septic shock has not been widely characterized. we proposed to describe the association between time course of pv-aco during the initial resuscitation and outcomes in septic shock. methods. patients with a new septic shock episode admitted to icu were included. general management was guided according surviving sepsis campaign recommendations. time (t ) was set when a central venous catheter was inserted to guide reanimation. simultaneous measurements of lactate and arterial-venous gases were obtained at t and h after (t ). pv-aco was calculated as the difference between venous co (blood samples drawn from a central catheter) and arterial co . a value of pv-aco [ was considered as high. survival at day was described for four groups: persisting high pv-aco (high at t and t ), increasing pv-aco (normal at t , high at t ), decreasing pv-aco (high at t , normal at t ) and persistently low (normal at t and t ). survival probabilities were estimated using kaplan-meier method. log-rank test was use to estimate a two-tailed p value for the differences in survival among groups. results. sixty septic shock patients were analyzed. mortality rate was . %. no demographic differences at baseline between survivor (s) and non-survivors (ns) were found. there were no differences in the amount of fluids administered at t and t . no significant differences in scvo at t for s introduction. septic shock (ss) has been defined as sepsis related hypotension despite adequate fluid resuscitation ? perfusion abnormalities such as lactic acidosis [ ] . despite this, an operationally simplified definition overlooking perfusion parameters, has been utilized in several landmark studies during the last decades [ ] [ ] [ ] [ ] . more recently, a new consensus reemphasized the pivotal role of hypoperfusion in ss definition and added low svo as a surrogate [ ] . several problems emerge from these apparently interchangeable definitions, including pathophysiologic and epidemiologic (incidence, outcome) issues. objectives. our aim was to evaluate if applicating different commonly used ss definitions to vasopressor-requiring septic patients leads to distinct outcomes. methods. we applied the two most utilized ss definitions to hypotensive septic patients managed with a ne-based algorithm [ ] for years, generating two major subgroups for analysis (fig. ) . statistical analysis included chi-square test. (fig. ) . pts of subgroup , exhibited persistent normal lactate levels with a mortality of . % which was similar regardless of svo [ or \ : p = . . (fig. ) . conclusions. commonly used ss definitions are not interchangeable and when applied to the same vasopressor requiring septic patients lead to statistically different mortalities. our data suggest that lactate and svo cannot be used indistinctly to define shock condition. a reappraisal of clinical septic shock definition appears to be necessary. objectives. to assess intra-and inter-observer agreement of ecg interpretation in adults with septic shock (vasst, nejm ; : ) . methods. patients were randomised to receive a blinded infusion of low-dose vasopressin or norepinephrine in addition to open-label vasopressors. eight icus participated in this ecg sub-study; and -lead ecgs were recorded at baseline (prior to study drug infusion), and h, and days after initiation of study drug. an intensivist (reader ) and a cardiologist (reader ), blinded to patient data and randomization group, interpreted all of the ecgs in duplicate, using a checklist. prior to ecg interpretation, a calibration exercise was performed to refine definitions and maximize inter-observer agreement; both readers reviewed ecgs (from the current study) representing the spectrum of normal to abnormal. cohen s kappa statistic was used to assess intra-and inter-rater reliability. methods. the model consists of eight elastic chambers including the heart and circulations. identification of the parameters is made only from measured pressures in the aorta and pulmonary artery, and the volume in the right ventricle. septic shock was induced in (n = ) healthy pigs with endotoxin infusion over min. right ventricular pressure-volume loops were recorded by conductance catheter and end-systolic ventricular elastance was assessed by varying right ventricular preload. consent was obtained from the university of liege medical ethics committee. errors for the identified model are within % when the model is identified from data, re-simulated and then compared to the clinically measured data. even with a limited amount of available experimental data to identify the parameters of the model, all simulated parameters trends match physiologically expected changes during endotoxic shock. in particular, a close match of the trends of the right ventricular end-systolic elastances are obtained, when compared to previously reported experimental results [ ] , including capturing of the peak after min and a decaying oscillation after min. conclusions. pig-specific parameters for the cvs model were accurately identified using a significantly reduced data set. this research shows the ability of the model to adequately and realistically capture the impact of pressure-volume changes during endotoxic shock. in particular, the model is able to aggregate diverse measured data into a clear, clinically and physiologically relevant diagnostic picture as the condition develops. this research thus increases confidence in the clinical applicability and validity of this overall diagnostic monitoring approach. background. conflicting data exist concerning the effects on the microcirculation of increasing mean arterial pressure (map) with norepinephrine (ne) in septic shock. nearinfrared spectroscopy (nirs) has been proposed as a tool to quantify microvascular dysfunction in patients with sepsis. by inducing a vaso-occlusive test (vot), a variety of nirsderived variables can be measured to assess local metabolic demand and microvascular dysfunction. this trial was conducted to test the effects of increasing map by ne on microvascular reactivity in patients with septic shock. after local ethical committee approval and informed consent, we enrolled patients in septic shock with an arterial pressure stabilized by ne. in addition to hemodynamic measurements, svo and blood lactate level, we measured thenar muscle oxygen saturation (sto ) and muscle tissue hemoglobin index (thi) by a tissue spectrometer (inspectra tm model , hutchinson technology inc, mn). serial vot (upper limb ischemia induced by a rapid pneumatic cuff inflation around the upper arm) were performed. we also recorded during the vot: basal sto , thi, the slope of the decrease in sto during the occlusion (desc slope; %/min) and the slope of the increase in sto following the ischemic period (asc slope; %/s). muscle oxygen consumption (nirvo i) was calculated as the product of the inverse of the slope value by the mean of thi over the first minute of arterial occlusion and is expressed in arbitrary units (u) (skarda shock ). all these data were obtained at different times: baseline and with map of mmhg, then at mmhg and mmhg of map by increasing the ne doses and finally to baseline . we report here data corresponding to the mean and sd of baseline and versus map mmhg analyzed by repeated measures analysis of variance (at % level) with bonferroni adjustment to account for multiple comparisons. increasing ne dose induced an increase in cardiac output (from . ± . to . ± . l/min, p \ . ) without any changes in heart rate and an increase of svo (from . ± . to . ± . %, p \ . objectives. to investigate: . the effects of ''successful'' protocolised resuscitation (egdt) on microvessel perfusion (particularly density). . whether there is different effects of egdt on the microcirculation of septic compared to critically ill non-septic patients and . whether there is a difference in the behaviour of ''true'' capillaries (i.e - lm) compared to larger microvessels ( - lm) at baseline or after resuscitation. prospective observational study in the emergency and intensive care departments of an urban teaching hospital. subjects: septic and critically ill control patients requiring shock resuscitation (map less than mmhg, ±cvp less than mmhg, ±central venous saturations less than %). all patients had invasive monitoring and identical cardiovascular targets. patients with known cardiogenic shock or pre-stabilised trauma were excluded. we performed sidestream dark field (sdf) videomicroscopy of sublingual microcirculation at the point of egdt initiation and again on attainment of at least out of cardiovascular goals. three sites were imaged for s and the clips were analysed randomly off-line to provide an average value for capillary density (total length and count per mm) and a semi-quantitative description of microvessel flow (continuous, intermittent or stopped) as previously described. vessels were grouped according to diameter as small ( - lm) and medium ( - lm). non parametric analysis was used for all within or between group comparisons, data is displayed as median values with [range]. *p \ . was considered significant. ( ) ( ) ( ) ( ) duration of occlusion (min), mean ± sd . ± . . ± . . ± . . ± . minimal sto (%), mean ± sd ± ± ± ± as expected, all septic shock patients, except one (for the vot fa % ) and two (for the vot a % ) had a recovery slope lower than normal when sto decreased to % during arterial occlusion. by contrast, when occlusion lasted min, many patients including patients who eventually died, were misclassified since their recovery slopes were in the normal range. these results could be due to the smaller decrease of sto and in turn a less strong hyperemic response when ischemia lasted only min. additionally, a significantly (p \ . ) shorter time to reach % was required when arm (compared to forearm) occlusion was performed. conclusion. when a vot is required for assessing microcirculatory disturbances in septic shock, we recommend performing it using an arm occlusion until sto reach %. aims. to analyze the correlation between sto (and its changes derived from a transient ischemic challenge) and global oxygen delivery (do ) parameters measured invasively using a pulmonary artery catheter (pac). observational study, performed in a -bed medical-surgical icu, at a university hospital. we recruited adult patients with cardiovascular insufficiency that required a pac placement for hemodynamic monitoring and resuscitation. we collected demographic data, and hemodynamic and oxymetric data derived from the pac. simultaneously, we measured sto and its changes derived from a vascular occlusion test (vot). results. twenty-two patients were studied. all the patients had a mean arterial pressure (map) above mmhg. the do index (ido ) range in the studied population was - mlo /(min m ). the mean svo value was ± %, mean cardiac index (ci) . ± l/ (min m ), and blood lactate . ± . mmol/l. the correlations found between sto and invasive oxygen delivery-related variables are shown in table . the sto -deoxygenation slope (deox) during the vot showed a significant correlation with svo (r . , p . ). we did not find any correlation between sto and global flow measurements, such as cardiac index (ci), but we found a correlation between sto and ido . this correlation seems related to the arterial oxygen content, and not to global flow. normal sto values could not rule out low ido and low ic states. therefore, sto seems to be poorly sensitive to exclude hypoperfusion states. in clinical practice there remains issues over the appropriate prescribing of antibiotics in patients with unproven sepsis. the prescribing of antibiotics is not without risk and creates a selective pressure on existing bacterial flora resulting in the emergence of virulent and resistant organisms [ ] . there is also a cost issue from the inappropriate prescription of antimicrobials [ ] . the diagnosis of sirs can be made with confidence [ ] , sepsis cannot and requires confirmation from microbial tests. empirical usage of antibiotic therapy is commonplace but not ideal. rapidly detectable, reliable markers of sepsis would help in directing antimicrobial therapy. objectives. the aims of this study are to determine the significance of % band forms in sirs patients suspected to have sepsis. can they be used as a diagnostic tool in conjunction with procalcitonin in order to direct antimicrobial therapy? methods. this is an observational study aiming to assess the ability of serum procalcitonin and percentage band forms in identifying nosocomial sepsis in patients with sirs. patients were recruited over an month period in a mixed medical-surgical university teaching icu. all patients had suspected sepsis arising 'de novo' and had not received prior antimicrobial therapy. patients had a septic screen performed along with baseline, and hpct and % band form count. introduction. pneumonia is the most frequent infectious complication after successfully resuscitated cardiac arrest (ca). however, diagnosis is difficult because of many clinical, biological and radiological confounding factors as well as the widespread use of therapeutic hypothermia. this could lead to a broad antibiotic prescription. to assess the utility of plasma procalcitonin (pct) measurements for diagnosis of early-onset pneumonia in successfully resuscitated ca. monocentric study (july -march with retrospective review of a prospectively acquired icu database focusing on all consecutive patients admitted for ca and surviving more than h. patients with an infection prior to ca or with an extra-pulmonary infection developing within days following admission were not studied. all files were reviewed to assess the diagnosis of early-onset pneumonia p(?), or not p(-) during the first days of icu stay. p(?) was defined by the presence of a new pulmonary infiltrate on chest radiography, persistent for at least h, associated with either positive quantitative culture of the endotracheal aspirates, either, in case of lack of bacteriological sample, conjunction of purulent sputum and hypoxemia (p/f \ ). pct was measured at admission, days (d) , and (brahms kryptor Ò ). among patients admitted for ca, were studied ( death before h, evolutive infections and incomplete samples). pneumonia was diagnosed in patients ( %), and antibiotics were prescribed in during the first days of icu stay. characteristics of p(?) and p(-) patients were (median, iqr): age ( - ) versus ( - ) (p = . ), ''no flow'' ( - ) versus ( - ) min (p = . ), ''low flow '' ( - ) versus ( - ) min (p = . ), shockable rhythm versus % (p = . ), cardiac etiology versus % (p = . ), therapeutic hypothermia versus % (p = . ), post-resuscitation shock versus % (p = . ) and icu mortality versus % (p = . ). using a threshold value of . ng/ml, negative predictive values were % at admission, % at d , % at d , whereas positive predictive values were , and %, respectively. patients with post-resuscitation shock had higher pct levels than those that did not require vasopressors: . versus . ng/ml at d (p \ . ), . versus . at d (p \ . ) and . versus . at d (p = . ). conclusion. diagnosic value of pct is poor in survivors of ca and pct should not be recommended to assess early-onset pneumonia. post-resuscitation disease could play a major role in the lack of specificity and predictive values. in acute community respiratory infection, low levels of procalcitonin (pct) have been shown to allow a marked reduction of antibiotic use. the aim of the study was to look for the same efficacy in case of suspicion of infection during icu stay. method. from april to december , patients hospitalized in the five intensive care units (icu) of the university hospital of liège in belgium, were prospectively randomized to either a procalcitonin guided approach to antibiotic therapy (pct group, n = ) or to a standard approach (ctrl group, n = ) when they were suspected of developing an infection. for pct group guided therapy only, the use of antibiotics was more or less strongly discouraged (pct level . or. lg/ml, respectively) and more or less recommended (pct level [ or[ . lg/ml, respectively) . number and duration of antibiotic treatments were recorded. diagnosis and treatment decisions were reviewed by infectious disease (id) specialists at the end of icu stay. results. there were no differences between groups in terms of age ( vs. ), saps ii score ( . ± . vs. . ± . ), type of patients (medical: vs. %, scheduled surgery: vs. %, emergency surgery: vs. %, trauma: vs. %), icu length of stay [ (iqr - ) vs. days (iqr - )] for pct and ctrl group respectively. suspicion of infection was either evoked on admission (in and %) or during icu stay (in and %) in pct group and ctrl group respectively. at the time of suspicion, pct levels was. lg/ l in . % of the infectious episodes in pct group and . % in ctrl group. episodes of suspected infection with pct level . lg/ml were recorded. clinicians decided not to treat % of these episodes (n = ). the remaining episodes were treated, of which % were eventually considered as probable or confirmed infections by id specialist (n = ). at the end of icu stay, id specialists classified infectious episodes of both groups as confirmed (n = ; . %), probable (n = ; %), possible (n = ; %) or absent (n = ; . %). for confirmed episodes of infection, pct levels were . lg/ml in as much as . % and above lg/ml in . %; for absence of infection, pct levels were . lg/ml in only . % and above lg/ml in . %. the ability of pct to discriminate between confirmed and probable infections on the one hand and possible or absent infection on the other hand, was tested by the measurement of the surface under the roc curve, which was . , which is too low to recognize pct as a valuable marker of infection. there were no difference in the number of treated patients ( vs. %) nor in the number of antibiotic days ( vs. %) between pct and ctrl group respectively. conclusions. procalcitonin level as an aid for the decision to treat infection in icu patients appeared not to be helpful. antibiotic consumption was not reduced using this tool in our study. introduction. respiratory infections, pneumonias in particular are a common cause of mortality in the intensive care unit (icu) patients worldwide. early identification and prompt management of these patients especially with associated sepsis is crucial in reducing the mortality. many clinical and laboratory markers have been studied extensively to predict the outcomes in them. there have been numerous studies on the clinical utility of serum procalcitonin (pct) in the past decade, in systemic inflammation, infection and sepsis. objective. to evaluate the role of serum procalcitonin, in predicting the outcomes of patients admitted in the icu with respiratory infections associated with sepsis. setting: bedded icu of a tertiary referral hospital. study design: prospective observational study. subjects: adult ([ years) patients admitted in the icu with lower respiratory tract infections with associated sepsis during the period july to january were prospectively followed up. primary outcome measure: day mortality. we measured pct levels using the brahms immunochromatographic technique(semiquantitative estimation) on the first day of admission into the icu . normal pct was taken as . ng/ml. patients were grouped into four groups-group a (pct \ . ng/ml), group b (pct [ . - ng/ml), group c (pct [ - ng/ml),group d (pct [ ng/ml). sepsis, severe sepsis, septic shock are defined according to the accp/sccm criteria. results. the overall mortality was . % with mortality of . , . , , and % in groups a, b, c and d, respectively. there is a statistically significant difference (p \ . ) in the mortality rates of groups c and d as compared with group a and b, but no difference was observed in the mortality rates between groups a and b and groups c and d .also significant statistically are the apache ii scores, septic shock and multiorgan failure incidence in the groups c and d as compared to groups a and b. conclusions. serum procalcitonin level [ ng/ml on the first day of admission in icu appears to be a good predictor of mortality in patients admitted with lower respiratory tract infections and associated sepsis. methods. in a retrospective study we assessed acutely ill patients investigated for pct and treated by a physician blinded for pct value. for each patient we also calculated new simplified acute physiology score (saps ii). we evaluated many clinical and instrumental parameters and diagnosis was done upon our usually clinical practice results. the mean age of patients (pt) was . yeats, shock was found in patients ( . %),median value of saps ii score was (iqr - ), and median estimated mortality from saps ii was % (iqr - ). bacterial infection was found in . % (septic shock . %, pneumonia . %, cholecystitis . %, pleural empyema . %, other infections . %) non infective disease in . % (pulmonary embolism . %, acute coronary syndrome . % heart failure . % other disease . %. a pct value [ . ng/ml was considered positive: so pct was elevated in . % of bacterial infection patients and in . % of non infective disease patients. we also compared pct values with antibiotic therapy and considered appropriate the administration if pct [ . ng/ml: there was discrepancy in . %. the review of these cases found medical decision wrong in cases versus ( . %); pt with pct \ . ng/ml had antibiotic therapy without bi and cases with pct [ . ng/ml did not have antibiotic therapy but had a bacterial infection. subsequent to this review discrepancy felt to . % (ci % . - . ) and was found especially in pt with pct \ . ng/ml. at cut off point of . the sensitivity was . (ci %: . - . ) specificity . (ci %: . - . ) or . and at point . the sensitivity was . (ci %: . - . ) specificity . (ci %: . - . ) or . , with high predictive positive value. all-causes mortality was . %. mortality if pct \ . ng/ml was . %, if pct . - . ng/ml was . %; if pct . - . lg/ml was . % and if pct [ ng/ml was . % without significant difference between bacterial infection and non infective disease group. comparing pct with saps ii score, area under roc-curve was not significantly different (pct . -ci %: . - . ) (saps ii . -ci %: . - . ). conclusions. pct in acutely ill patients is a useful marker to discriminate bacterial infections with high sensibility but low specificity and it may be useful to guide the therapy also with values higher than . ng/ml. our data suggest a real prognostic utility of pct in these patients, regardless of bacterial infections, but our efforts to elaborate a mathematical predictive model aren't still satisfying and further data are required in this setting. h. taniuchi , t. ikeda , k. ikeda , s. suda tokyo medical university, hachioji medical center, division of critical care medicine, tokyo, japan introduction. its apparent that detection of the causative bacteria is useful for the therapeutic strategy. however, conventional tests for the detection of the causative bacteria are not high sensibility. in order to diagnose sepsis or septic shock and start appropriate therapy rapidly, it's also important to know whether the infection is cause of gram negative bacteria, that is to say, whether the infection is cause of endotoxin. in this study, we investigate the severity level of sepsis and initiation criteria of direct hemoperfusion with polymixin b immobilized fiber column (pmx-dhp) treatment from the result of severity level by using endotoxin activity assay (eaa) and using measurement of procalcitonin (pct). subjects and methods. patients who developed severe sepsis or septic shock and admitted to icu were included. on the day of icu admission, a general blood biochemistry, eaa and pct levels, and apache ii and sofa score were measured. patients were evaluated retrospectively the relationship between the severity of sepsis and each measurements and investigated the relationship between the measurements and pmx-dhp. serum eaa level was measured using smart line eaa luminometers. serum pct level was measured using immune luminometric assay. results. the average age of the patients is ± , apacheii score was . ± . , sofa score was . ± . , the median pct was . ng/ml (range - ), eaa was . ± . . the underlying diseases of the enrolled patients were the abdominal infection ( patients), the urinary tract infection ( ), pneumonia ( ), the meningitis ( ), the soft tissue infection ( ) and other infection ( ) . the causative bacteria were gram positive bacteria ( ), gram negative bacteria ( ), virus ( ), and unknown ( ). there was no statistical correlations between eaa or pct level and apacheiiscore. there was no statistical correlations between eaa level and sofa score. although there was no statistical correlation between pct level and sofa score, the pct level tended to rise as pct level rises. we investigated the relationship between eaa and pct levels. there was also no statistical correlations between eaa and pct. we investigated the relationship between the causative bacteria (gram positive bacteria, gram negative bacteria and the others) and eaa or pct level. there was no statistical correlations between the causative bacteria and eaa level nor pct, that was contrary to our expectation that eaa level should be high for gram negative bacterial infection. we further investigated the relationship between whether or not the pmx-dhp was implemented and eaa or pct level. there was no statistical relationships. conclusion. high levels of the eaa and pct would not indicate the severe infection with gram negative bacteria, and the initiation of pmx-dhp. further study is needed, in which more patients will be enrolled and evaluated. introduction. sepsis still the major cause of death in the late post traumatic period in patients with major burns. early diagnosis of sepsis is crucial for management and outcome of critically burn patients. attempted in this study to assess whether plasma procalcitonin (pct) level was related to diagnostic and prognostic of sepsis in burned patients. patients and methods. pct was measured over the entire course of stay in patients with predictive signs of sepsis according to american college of chest physician. the patients were assigned to two groups depending on the clinical course and outcome: a = no septic patients, b = septic patients. optimum sensitivity, predictive values, and area under the receiver operating characteristic (roc) curve were evaluated. results. over a month period starting from july to december , patients were admitted. were investigated. in group a et in group b. procalcitonin was significantly higher in septic group . ± ng/ml compared to no septic group . ± . ng/ml. area under the curve was . on the day of sepsis diagnostic. pct cut-off value of . ng/ ml was associated with the optimal combination of sensitivity ( %), specificity ( %), positive predictive value ( %), and negative predictive value ( %). in survived septic patient the pct value was significantly lower than in deceased septic patients . ± . versus . ± . ng/ml. pct cut-off value for optimum prediction of outcome in septic patients was . ng/ml with sensitivity ( %), specificity ( %), positive predictive value ( %), and negative predictive value ( %). conclusion. procalcitonin appears to be a powerful marker of sepsis in burn patients. it is sensitive, specific, reliable and easy to measure. a high pct concentration ([ . ng/ml) would indicate poor outcome in septic patients. n. v. beloborodova , a. s. khodakova , a. y. olenin , s. t. ovseenko bakulev scientific center for cardiovascular surgery, moscow, russian federation objectives. accurate and timely diagnosis of sepsis remains challenging for clinicians. the diagnosis of sepsis is defined as typical symptoms of systemic inflammation (temperature, tachycardia, respiratory rate, leukocytosis) with clinical evidence of an infection site, but the criteria are met by a large number of intensive care unit (icu) patients. among studied biomarkers, serum procalcitonin (pct) has been described as one of the most promising predictors of bacterial sepsis, but in some clinical situations it is not enough. the search of reliable markers of sepsis is still in progress. in present study the significance of raised levels microbial phenylcarboxylic acids in serum of patients with sepsis are assessed. methods. the present study evaluated serum samples of patients (pts) with documentary sepsis, according to well known consensus criteria. the comparison groups were: no. - clinically healthy volunteers, no. - pts. with acquired heart diseases, no. - pts with ventilator-associated pneumonia. blood concentrations of phenylcarboxylic acids were determined by gas chromatography-mass spectrometry (gc-ms). results are presented as median and range of th and th percentiles. the statistically significant differences between the various groups were calculated using mann-whitney test. results. increased levels of phenyllactic (pla), p-hydroxyphenylacetic (hpaa), p-hydroxyphenyllactic (hpla) acids were observed in group of pts with sepsis. the level of hpaa was increased up to two orders in comparison with groups no. and [ . ( . - . ) vs. . ( . - . ) and . ( . - . ) lm, p \ . ). the levels of hpla and pla were increased up to one order [( . [ . - . ] table for illustration of importance of phenylcarboxylic acids blood level monitoring. introduction. acute kidney injury (aki) is a frequent complication of sepsis, and is associated with high mortality and morbidity rates. routinely used measures of renal function, such as levels of blood urea nitrogen (bun) and serum creatinine, increase only after substantial kidney injury occurs, resulting in delayed diagnosis of aki. therefore biomarkers, which enable early diagnosis, are needed. objectives. this clinical study was designed to investigate whether human interleukin- (il- ) and neutrophil gelatinase-associated lipocalin (ngal) are early predictive markers for sepsis-induced aki. urine and blood samples have been collected prospectively from icu patients, who met defined clinical criteria of severe sepsis. aki was defined by rifle criteria. urinary and serum levels of n-gal and il- have been quantified by elisa in patients with sepsis without aki (n = ) and in patients with sepsis induced aki (n = ). results. both, urinary il- and serum il- considerably increased (respectively, . and . -fold over the baseline) two days before the patients reached rifle risk. urinary ngal raised significantly ( . -fold over the baseline) one day before occurrence of aki, whereas serum ngal did not show any prior elevation. no increase in the levels of any of these markers could be found in patients who did not develop aki. conclusions. both urinary and serum il- seem to be sensitive early biomarkers for sepsis associated aki, while urinary ngal has less accuracy for aki prediction. objectives. to define a biomarker panel able to predict infection in case of severe acute dyspnea in emergency situations. we designed a prospective observational study of patients admitted in the emergency department (ed) and in medical polyvalent intensive care unit (icu) in a university hospital. inclusion criteria were acute dyspnea with spo b % and/or respiratory rate (rr) c b/min. patients with an immediate need of coronarography or with obvious spontaneous pneumothorax were excluded. five biomarkers were measured from blood sample at admission on ed or icu: nt b type natriuretic peptide (nt probnp), cardiac troponin i (ctni), ddimeres (dd), c-reactive protein (crp) and procalcitonin (pct). all clinical and biological data were recorded. an independent blinded data monitoring committee classified the patients according to all the available data including response to treatment and outcomes but blindly to biomarkers. the roles of biomarkers were assessed quantitatively and then using terciles of the distribution. the contribution of the biomarkers in the diagnosis was assessed using multiple logistic regression taking into account other clinical and biological explanatory variables. . patients were enrolled consecutively. the final diagnosis was: severe sepsis (n = ), acute heart failure (n = ), pulmonary embolism (n = ), copd (n = ), other causes (n = ). the days mortality was %. there was no significant association between infection diagnosis and dd, ctni, nt probnp. interestingly, a crp value of less than mg/l was not discriminant in predicting infection. adjusted on clinico-biological covariates selected, both pct with cutpoints of . and . ng/ ml (discrimination auc . ; p = . ) and crp with cutpoints of and mg/l (discrimination auc . ; p . ) were significantly associated with the diagnosis of sepsis. both biomarkers used simultaneously lead to a discrimination of the model (auc . ). conclusion. both crp and pct are able to predict the diagnosis of infection in case of severe acute dyspnea independently of clinico-biological variables. in this particular subpopulation, the best threshold for crp is higher than the standard one. an external validation is needed to prospectively validate the clinical utility of these findings. t. trefzer , i. nachtigall , a. weimann , c. de grahl , c. spies charite universitaetsmedizin berlin, campus virchow, department of anesthesiology and operative intensive care medicine, berlin, germany, charite universitaetsmedizin berlin, campus virchow, zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany aims. infections are the most relevant icu-admission complication. crp and pct are labvalues used for diagnosis of infections. however, their use is often not evidence based. this study aimed to access whether the adherence rate increased after introducing an evidencebased standard operating procedure (sop). in an evidence-based sop was approved by experts of our department. in july it was made available to icu-physicians via intranet, which is accessible from every work station. altogether, we assessed sop-adherence rates of patients: in june (pre-sop), patients in august (one month post-sop) and in january ( months post sop). every crp and pct measurement was assessed for adherence to the standard operating procedure (sop). at first, the three periods were assessed for significant differences concerning the adherence. according to the percentage of sop-conform measurements the patients were then divided into two groups: the sop-group (c % of measurements sop conform) and the non-sop (nsop) group (\ % conform) in a second step, patients in the sop-and nsop-group were compared concerning icu scores (sofa, tiss, apacheii, saps) and outcome parameters (length of icu-stay, length of hospital stay, duration of mechanical ventilation, hospital mortality). statistics: p b . was considered as statistically significant; hospital mortality was assessed by a v test, icu scores and outcome parameters were compared using the mann-whitney u test. all parameters with p \ . were included into a logistic regression analysis. no change was observed concerning the implementation of the sop pre and postintroduction: . % in june , . % in august and . % in january . the non-conform pct-and crp-measurements resulted in additional costs of approximately . euros/year. the univariate analysis revealed significant differences in the sop-and nsop-group: the nsop-group had higher saps-, sofa-and tiss-scores, as well as increased length of icu-stay, length of hospital stay and duration of mechanical ventilation. logistic regression analysis revealed tiss score and length of hospital stay as an independent predictor for low sop adherence. conclusion. distribution of an evidence based sop without further education did not lead to a significant increase in adherence rates, but tiss score and length of hospital stay have shown to be independent predictors for low adherence to the sop. the significant higher tiss-scores in the nsop group might be a indicator for actionism of clinicians in the face of more severely ill patients. objetives. to asses the evolution of the risk-adjusted mortality rates of sepsis and septic shock in our icu in a ten years period. patients and method. analisys of prospectively recorded data of all pacients admitted with severe sepsis and septic shock in a bed icu during a period of years. patients were followed up until death or discharge from the hospital, excluding those with unknown outcome. mortality prediction was made using apache ii model with % confidence intervals. statistical analisys was made with spss . using anova test or t test to compare means and chi square test to compare categorical variables. results. from january to december a total of patients with sepsis were admitted, with an anual increase to reach % of all icu admissions. age and severity of illness increased anually as did sofa in the first h (sofa ) thus rising up calculated risk of death. from to mortality rate was between % ics of calculated risk of death, falling below inferior ic from and after . (fig. ). hospital mortality versus risk of death per year mortality was . % in the pre- period and . % from and on (p = . ) with non significant differences in apache ii, risk of death nor sofa , but with significantly greater age in the post- period ( . vs. . years p = . ). this non significant difference between the two periods of the study became significant when we analized the outcome in both sex. being significant in women (mortality . % in pre- period vs. . % in post- p = . ) but not in men ( . vs. . % p = . ). overall sepsis moratlity is lower in female without significant differences in age, apache ii score nor risk of death (table ) , being the only signifficant difference found in sofa ( . in male vs. . in female p = . ). introduction. low-grade systemic inflammation has been shown to play a key role in the pathophysiology of several chronic noncommunicable diseases [ , ] and may be attenuated by anti-inflammatory treatments such as administration of statins [ ] . so far, the association between acute systemic inflammation experienced during critical illness and long-term mortality after hospital discharge has not been investigated in intensive care unit (icu) patients. objectives. to assess the association between acute systemic inflammation, assessed by crp levels, and post-hospital mortality in non-surgical icu patients. methods. the study was performed as a prospective, observational follow-up study and included non-surgical critically ill patients with an icu length of stay [ h. patients who died during the icu or hospital stay, were \ years or pregnant, as well as patients discharged from the hospital with the plan to limit life support were excluded. demographics, chronic diseases, admission diagnosis, the simplified acute physiology score ii, length of icu stay, maximum crp levels during the icu stay (crpmax) and crp levels at icu discharge (crpdis) were documented. after a mean ± sd follow-up time of . ± . years, mortality and causes of death were determined. adjusted cox models were calculated to investigate the association of crpmax and crpdis with post-hospital mortality. a receiver operating characteristic analysis was used to identify optimal cut-off levels to predict post-hospital mortality. background. the prevalence of hiv infection is increasing worldwide as a public health problem. survival of hiv/aids patients has improved since highly active antiretroviral therapy, but sepsis has grown as an important cause of icu admission in this population. an international conference has set a system composed of specific risk factors, site and microbiology of severe infections and host response and organ dysfunctions (piro) to help identify patients at risk for sepsis. piro factors have not been classified for hiv/aids population yet. objectives. to identify predisposing factors, microbiology of infections, host clinical response and incidence of early organ dysfunctions of severe sepsis on hiv/aids patients, admitted to a specialized infectious diseases icu; to analyze long-term survival of hiv/aids critically ill patients. a prospective case-control study of septic and non-septic hiv/aids patients admitted between june and may was performed. demographic data, causes of admission, time since aids defining condition, cd cell count, and opportunistic infections were evaluated as predisposing factors to sepsis. microbiology and site of infections were registered. clinical response to severe infections was evaluated by ali/ards and shock incidence on day of icu admission. organ dysfunctions (sofa score) were reported soon after icu admission. icu length of stay, hospital and -month mortality were compared between septic and non-septic groups. a multivariate regression analysis was done to identify risk factors for icu mortality. kaplan-meyer survival curve was built. . icu admissions of hiv-infected patients were studied. half ( ) fulfilled criteria for severe sepsis diagnosis. septic group was younger ( . ± . vs. . ± . years, p \ . ) and had more female patients ( vs. %, p \ . ). time since aids diagnosis, cd cell count and opportunistic infections prevalence were not different. sites of infection were predominantly pulmonary ( %) and catheter-related ( %). ninety percent of infections were nosocomial. forty-three percent of septic patients presented bacteremia. pseudomonas sp, s aureus and enterobacteriacae were commonly identified, but five patients had mycobacterium tuberculosis isolated ( on blood cultures). multiple organ dysfunction syndrome was frequent, and incidence of cardiovascular, respiratory and hematological dysfunctions was significantly higher in septic group. longer length of icu stay ( . ± . vs. . ± . days, p \ . ) and icu mortality ( vs. %, p \ . ) was observed for septic patients. severe sepsis also influenced long-term survival, as mortality continues significantly higher after months (log rank . , p \ . ). conclusions. piro system is applied to septic hiv/aids patients. shock, ali/ards and hematological dysfunctions are prominent for septic hiv/aids population. septic hiv/ aids patients are at severe risk of short and long-term mortality. international guidelines for management of severe sepsis and septic shock suggest the use of recombinant human activated protein c (rhapc) in adult patients with high risk of death (apache ii c or multiple organ failure). the objective of this study is to analyse the characteristics and outcome of patients treated with rhapc in our medical intensive care unit. retrospective study of patients with severe sepsis/septic shock treated with rhapc between january to december . all of them were c years, with apache ii c and two or more organ dysfunction, and were treated on basis of a bundle for severe sepsis management: complete early goal-directed therapy, early administration of broadspectrum antibiotics; corticosteroids in vasopressors unresponsive patients and monitor for lactate clearance. chi-square analysis were used to compare categorical data. continuous data were compared using student's t test. prognostic factors of mortality were studied by means of multivariable logistic regression analysis. results. forty-one patients were studied. % were male. their mean age was ± years. % had comorbidities ( % immune pathology). severity scores. apache ii ± , sofa ± , % of patients had three o more organ dysfunction. % had septic shock. serum lactate level was . ± . mmol/l. the primary location of infections was: respiratory %, abdominal %, urinary %. . % were positive blood culture. % of patients needed mechanical ventilation ( ± days). % of rhapc infusions were not completed, mainly for bleeding risk ( %) and death ( %). . % of patients had bleeding event. at the end of the infusion % of patients remained with two or more organ dysfunction and % were vasopressors dependent. mean hospital stay was days and days in icu . days mortality was %, icu mortality . % and hospital mortality . %. analyzed data included age, comorbidities, primary location of infections, severity scores and serum lactate level. univariable analysis showed that statistically significant factors related to mortality were: apache ii ( ± vs. ± , p = . ), organ dysfunction number: vs. [ ( vs. %, p = . ) and primary location of infections: pneumonia versus others ( vs. %, p \ . ). a multivariable logistic regression analysis showed that age (or . , % ci . - . , p = . ), organ dysfunction number (or . , % ci . - . , p = . ) and serum lactate levels (or . , % ci . - . , p = . ) had statistically significant relationship to mortality. conclusion. in our study the patients with severe sepsis and septic shock remained with high vasopressors dependency and organ dysfunction at the end of the rhapc infusion. despite of rhapc therapy the mortality of patients was very high. the age and the severity at icu admission were independent prognostic factors of mortality. a higher incidence of severe sepsis in blacks compared to whites is well documented, however prior analyses do not discriminate whether this is due to a higher incidence of infections, a higher risk of developing organ dysfunction once infected, or both. objectives. we sought to understand whether higher severe sepsis incidence in blacks is due to higher infection susceptibility, higher risk of organ dysfunction once infected, or a combination of both. we analyzed , , hospitalizations from hospital discharge records of us states ( % of us population). we linked these records to us census data to generate age and sex-standardized incidence rates. we identified infections of bacterial and fungal etiology based on icd- cm criteria, including characterization by site and type of infection (gram negative vs. gram positive). we defined severe sepsis as documented infection plus acute organ dysfunction based on previous work by angus et al we estimated the risk of organ dysfunction among those hospitalized with infections using logistic regression, adjusting for age, sex and comorbidities (charlson score). fig. b ]. the combination of both events led to a % higher severe sepsis hospitalization rate for blacks ( . vs. . per , population, irr: . - . ). these differences persisted when stratified by sex, comorbidities, site and type of infection. infection incidence and severe sepsis risk conclusion. the higher incidence of severe sepsis among blacks is due to a higher hospitalization rate for infections, as well as a greater likelihood of organ dysfunction once infected. future interventions to reduce racial disparities in severe sepsis incidence should target both distinct events. grant acknowledgement. dr. mayr was supported by t hl - . objective. to describe recent epidemiological data and mortality risk factors of patients admitted to icu for severe pneumococcal pneumonia (pp). multicentric retrospective study (january -june ). prospective acquired data from patients admitted in french medical icu for severe pp were considered. patients with concurrent meningitis, severe copd with known sp colonization, hiv or aspiration pneumonia were not included. pp was defined by the combination of a suggestive clinical context, the presence of a new pulmonary infiltrate on chest radiography and a s.pneumoniae positive bacteriological sample (pulmonary quantitative culture, pleural fluid, blood culture or urinary antigen assay). all files were reviewed and approved by two independent investigators (nm, am). . patients were included. median age was ± . hospital survivors were significantly younger ( ± vs. ± , p = . ). sex ratio m/f was / , but male sex was associated with higher risk of death (male: vs. %, p = . ). active tabagism ( %) or alcohol abuse ( %) were more common than asplenia ( %). organ dysfunctions were mainly respiratory ( %), haemodynamic ( %) and renal failures ( %). low doses steroids were prescribed in % of patients with septic shock. icu mortality rate reached % ( % in the first days); hospital mortality rate was %. univariate analysis demonstrated that age, male sex, cirrhosis and organ failure support were strong predictors for icu mortality. multivariate analysis only highlighted age [or . ( . - . )], cirrhosis [ . ( . - . ) ] and renal replacement therapy [ . ( . - . )] as independent mortality predictors. activated protein c treatment was associated with decreased mortality [or . ( . - . )]. bacteremia had no impact on outcome. conclusion. this is the most important cohort of pp requiring icu admission. despite adequate antibiotherapy, mortality is still preoccupant. determination of factors related to the bacteria (virulence) or to the host (genetic susceptibility) could allow a better understanding of this important health problem. introduction. to identify the risk factors of mortality for patients with severe community-acquired bacteremic pneumococcal pneumonia. retrospective study realised in the intensive care units of two hospital medical centers. the studied population was patients with serious community-acquired bacteremic pneumococcal pneumonia. all the patients entered the intensive care units between january of and december of . study variables were: age, sex, concomitant pathology, toxic habits, pre-vaccinal ( - ) and postvaccinal periods ( ) ( ) ( ) ( ) ( ) ( ) ( ) , serotype and sensitivity of streptococcus pneumoniae to penicillin, the initial use of the non-invasive mechanical ventilation, the development of empyema pleural, apache ii and sofa scores during the first h after admission. results. the age average was of years. forty one percent of our patients required mechanical ventilation, and % had acute renal failure that required hemofiltration. average values of apache ii and sofa were . and . respectively. in hospital mortality of the series was of %. in patients with severe community-acquired bacteremic pneumococcal pneumonia: ( ) the presence of empyema pleural is an independent risk factor for mortality. introduction. procalcitonin (pct) is an interesting marker of pulmonary infection [ ] . it is useful as an help for infection diagnosis but also for treatment follow-up [ ] . besides, initiation of effective antimicrobial therapy is the strongest predictor of outcome in patients with septic shock [ ] . the aim of the study was to analyse whether kinetics of pct decline may reflect sensitivity of identified infectious agents to initial antimicrobial therapy (at). patients with diagnosis of severe pneumonia following major cardio-thoracic or vascular surgery were retrospectively included in the study. severe pneumonia was suspected as a combination of several manifestations including fever or hypothermia, hyperleucocytosis or leucopenia, new radiological infiltrate, and/or a clinical pulmonary infection score [ , pct [ ng/ml and pao /fio \ . initial antimicrobial treatment was chosen according to the guidelines in use in our institution for community-acquired or nosocomial infections. microorganism identification from endotracheal aspiration or bronchoalveolar lavage, and antibiotic susceptibility testing, allowed to classify patients according to appropriate (aat) versus inappropriate initial at(iat). pct was measured daily over days and its kinetics compared between both groups. data are expressed as median (extremes) or mean ± sd (decrease rate). results. patients aged ± ( - ), operated on vascular (n = ), thoracic (n = ), or cardiac surgery (n = ) have been studied from october to july . pneumonia occurred within the st to the st postoperative day (median . days), with a septic shock in cases and deaths at day . initial at was appropriate in % ( / ) patients. pct peak was not statistically different between aat versus iat patients ( . ± . ng/ml vs. . ± . , respectively) but pct decrease was significantly steeper and constant in iat patients (fig. ). pct decrease (%) from peak value over days discussion: the results suggest that absence of early decrease in pct within days may reflect failure of the at. conversely, an average decrease in pct plasma concentration of % in days seems to be a good marker of sensitivity of the causative infectious agent to the initial at. in case of unchanged pct within days at change should be considered. icu mortality was % for pts with rb early vap while it was % ( of cases) in those with sb or negative cultures (p = . ). mortality was higher than the predicted according to apache ii score in pts with rb vap ( vs. ± %, p \ . ). however, it was lower than predicted in those with negative or sensible isolates ( vs. ± %, p = . ). conclusions. rb were the most common cause of early vap among our patients. the burden of illness, los in icu before intubation and previous use of antibiotics were associated with early vap due to rb. inappropriate empiric therapy and mortality were higher among patients with early vap due to rb. to evaluate the performance of the saps piro model in patients with severe community acquired pneumonia (cap), over a period of years ( - ) , in a general icu in a central hospital. material and methods. we analysed data prospectively registered in an informatic data base, which contains information referring to all patients admitted in this unit. analysed were patients. discrimination was accessed by the area under the roc curve (aroc). calibration was evaluated by the by the hosmer-lemeshow Ĉ test. conclusion. implementation of a sedation protocol requires constant follow up and regular adaptation to prove efficient over time. constant feed back information to both the medical and nursing staff is mandatory. treatment of hyperactive delirium. hal haloperidol, bzd benzodiazepine, pro propofol, aa atypical antipsychotic, nd no drugs, na not answered discussion. there tends to be a general pessimism regarding obese patients within the intensive care community. our data indicates that this opinion could be misplaced. reduced ventilator days may reflect a reluctance to invasively ventilate obese patients. the apache ii scoring does not take into account the bmi which would eliminate any severity scoring bias. high bmi alone should not be a consideration in the decision regarding suitability for admission to critical care. during the last decades, a growing medical knowledges have changed the clinical approach to elderly patient diseases. they receive major surgery or intensive treatment for acute medical illness but often the recover is condictioned by the previous chonical diseases. this determines a long period to stay in intensive care unit (icu) because the slow improvement and cause an occupation of bed places. in our hospital, after a period of training performed by an intensivist (bc) and an internist (ag), icu patients who need a non invasive ventilation (niv) or tracheostomized elderly patients who have difficult weaning were admitted in a dedicated area in a medical department (md). this study desribes the results of one year of observation. in the last year, forty nine patients (age . ± . ; m f ) were transferred from icu to md. twenty three patients were treated with niv (age . ± . ; m f ), fourteen tracheostomized patients (age . ± . ; m f ) receive positive pressure ventilation because the difficult weaning in icu while twelve don't need any respiratory support. at the admission was performed a multidisciplinary plan and many specialists were involved (dietist,physiotherapist, pneumologist) and in invasively ventilated patients (ivp) was done a program of weaning. we follow all the patients until the discharge at home where someone need oxigenotheraphy, niv or mechanical ventilation. for the invasive ventilated patients we try to identificate significative differences beetween patients discharged at home and patients who died in hospital. data are given as mean ± sd and statistical analisis t test was performed results. patients underwent niv stay in hospital for . ± . days ( . ± . days in icu- . ± . in md) and ventilation was performed for the entire period in icu while for . ± . days in md. all the patients were discharged at home: twelve with niv, fourteen with oxygen. the lengh to stay in hospital for the ivp in wich weaning was failed in icu was . ± . days ( . ± . days in icu- . ± . in md). in md they continue the invasive ventilation for . ± . days. seven were weaned from ventilation after . ± . days, one was discharged at home with the ventilator while six died in hospital. patients who died were older ( . ± . vs. . ± . years-p . ), have more chronical diseases ( . ± . vs. . ± . -p . ), longer hospitalization ( . ± . vs. . ± . days-p . ), glascow coma scale ( . ± . vs. ). elderly patients often require a long period of recovery from acute ilness. in selected patients md could be a useful place where continue the treatment started in icu. in our study ivp who died had more chronical diseases and a more significative cognitive compromission. aim. documenting the qualitative and quantitative properties of administered and lost fluids is a common critical care monitoring practice. these nurse-registered fluid balances (fb) are used to optimize patient care and in clinical decision-making. this ''good clinical practice'' has also found application in research: recent studies reporting superior outcomes expressly refer to (negative) fb. we prospectively assessed the accuracy (review of all fluid balance charts and correction of arithmetic errors) and consistency (gold standard: body weight changes [bwc] registered with standardized measurements of body weight on admission and discharge [precision ± g]) of nurse-registered cumulative fb. total (tfb) and daily fb (dfb = total fb/los) were calculated. we analysed the unadjusted cumulative fb (unafb: without considering additional losses, i.e. perspiration/fever/liquid faeces) and the adjusted cumulative fb (adjfb: considering the above as proposed in the literature) in all patients (all) and in three subgroups (cardiaccerebral:card; septic:septic; others). exclusion criteria: lack of admission/discharge weight, incomplete fb data. we calculated l = kg. among patients admitted during the study period were eligible and analyzed. fb were inaccurate in cases ( %) (error range: - . to ? . l, mean arithmetic error ± sd: ? . ± . l, mean absolute error: . ± . l). the body weights at admission and discharge were . ± . kg and . ± . kg, with a bwc of . ± . kg ( . ± . kg per day). unatfb were . ± . l, unadfb . ± . l. adjtfb was . ± . l, adjdfb . ± . l. correlation (r ) and bland and altman was poor between bwc and unatfb ( . and - . ± . kg) and slightly better between bwc and adjtfb ( . and ? . ± . kg). the sd of the difference between bwc and fb per day of the icu stay was always [ kg. a multiple regression model including unatfb, duration of intubation, maximum temperature, estimation of liquid faeces, age and the calculated caloric deficit during the icu stay, only modestly improved correlation (r . ). compared to the two other groups, septic were significantly more severely ill, had a higher and longer fever, a longer los, larger bwc and cumulative fb, and presented larger differences between bwc and cumulative fb (poor correlation and bland and altman). though, consistency betwenn bwc and cumulative fb in card and other was still scarce. conversely, another multiple regression model (including only unatfb and the maximal temperature) in septic yielded an r of . . conclusion. fb are often inaccurate and they are not consistent with the gold standard of bwc. the correlation and the agreement with bwc of both adjtfb and unatfb are poor, with sd per icu day-stay[ kg or l. multiple regression models including several variables slightly improve correlation, yet remaining disappointing. consequently, clinical decisions should rather be based on other methods than fb. a prolonged hdu los was associated with a high sofa score for respiratory, hepatic and coagulation variables, preoperative ecg alterations, an increased urea and bmi and important bleeding. sofa score should be use in the first h to assess organ failure and a possible icu transfer for patients with an elevated score. evaluation of procalcitonin, neopterin, c-reactive protein, il- and il- as a diagnostic marker of infection in patients with febrile neutropenia financed by the following fellowships: rd / / from retics, fiss pi and fijc p-ef- reference(s). . bohoun c ( ) a brief history of procalcitonin biomarkers of sepsis: is procalcitonin ready for prime time? definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis procalcitonin assay in systemic inflammation, infection, and sepsis. clinical utility and limitations usefulness of procalcitonin for diagnosing complicating sepsis in patients with cardiogenic shock patients at our er, icu and wards of internal medicine and surgery meeting the ssc criteria were included. blood cultures were taken before administration of antibiotics, other cultures when appropriate. laboratory tests included wbc, crp, lactate, and pct. we categorized patients using bacteriological criteria group bacteriological proven infection (negative blood cultures but any other culture(s) positive) using chi square test there was no difference in survival, pct and findings on chest x-ray between the groups. with the mann whitney u test we found no differences in wbc, crp and lactate between survivors en nonsurvivors in a cohort of patients meeting the ssc criteria only % met bacteriological criteria for sepsis. wbc, crp, lactate and pct did not differ between patients with and without bacteriological proof of infection nor between survivors and non-survivors il rn and tnfr in the severity and outcome of community-acquired pneumonia to investigate whether polymorphisms within genes encoding for inflammatory or anti-inflammatory molecules are associated with susceptibility design. prospective observational, cohort study a cohort of , spanish caucasians with cap and subjects were genotyped for the following polymorphisms: tnfa - and - , lta ? sequential kaplan-meier survival analysis of tnfrsf b ? g/t polymorphism showed a protective role of the gt genotype. cox regression analysis adjusted for age, gender, hospital of origin and comorbidities showed that patients with gt genotypes had lower mortality rates compared with those patients with gg or tt genotypes (p = . our study does not support a role for the studied polymorphisms of the tnfa, lta, il and il rn genes in the susceptibility or outcome of cap. a protective role of heterozygousity for the functionally relevant tnfrsf b ? variability in genes involved in the inflammatory response in patients with community-acquired pneumonia to investigate whether polymorphisms within genes encoding for inflammatory or anti-inflammatory molecules are associated with susceptibility design. prospective observational, cohort study a cohort of , spanish caucasians with cap and , controlsinterventions: subjects were genotyped for the following polymorphisms: tnfa - and - , lta ? sequential kaplan-meier survival analysis of tnfrsf b ? tt versus gg/gt genotypes suggested a detrimental role of the tt genotype. longrank c tests at and days yielded p = . and . , respectively; cox regression for -and -day survival, adjusted for age, gender ghent university hospital, intensive care we aimed to describe the incidence and characteristics of healthcare-associated pneumonia (hcap) diagnosed at the emergency department of our university hospital compared to cap, hcap occurs in more debilitated or at-risk patients, is more frequently caused by nosocomial pathogens, and has worse outcome. nursing-home pneumonia is included within the definition of hcap but could have characteristics different from the other categories of hcap ) with a diagnosis of 'pneumonia'. episodes were categorized in cap and hcap according to the definition of the american thoracic society/infectious diseases society of america. within hcap, distinction was made between nursing-home pneumonia (nhp) and non-nhp hcap. severity of the pneumonia was assessed using curb- during the study period, episodes of pneumonia were diagnosed in patients; episodes ( %) were categorized as cap, and ( %) as hcap. within hcap, ( %), respectively ( %) episodes were further classified as respectively nhp and non-nhp hcap. median age of the patients was years ( - ) and % of patients were male median curb- pneumonia severity score in patients with cap and hcap was ( - ) and ( - ) respectively (p = . ); in nhp and non-nhp hcap, median curb in bivariate logistic regression analysis, both increasing curb- (or . , ci . - . ) and categorization as nhp (or . , ci . - half of the episodes of pneumonia diagnosed at our emergency department could be classified as hcap. severity of the pneumonia was higher in patients with hcap as compared to cap. categorization as nhp, but not as non-nhp hcap was independently associated with hospital mortality after adjustment for severity of the pneumonia medical intensive care unit community-acquired pneumonia (cap) is the leading cause of infectious death, severe sepsis and the seventh leading cause of overall death. severe cap (scap) is defined as need of aggresive intensive care unit (icu) management due to shock to describe the episodes of severe community-acquired pneumonia (scap) in a multicentric european study and to assess management practices and outcome of scap patients admitted to icu observational, prospective, multi-centre study conducted in icus of consecutive patients requiring invasive mechanical ventilation for an admission diagnosis of pneumonia or mv for [ h were recruited in each icu. statistic analysis was performed using spss years p \ . ) and presented a higher saps ii score at admission ( . sd . vs. . sd . p \ . ). patients were treated with monotherapy in . % and combination therapy . %. empirical antibiotic treatment was in accordance with idsa guidelines in ( . %) patients. combination was prescribed with macrolides in . % and quinolones in . %. in patients receiving combination therapy in accordance with idsa guidelines, a cox regression analysis adjusted by saps ii and age identified that macrolides use was associated with lower icu mortality when compared to the use of quinolones in patients with severe community acquired pneumonia who had therapy in accordance with the idsa guidelines, only combination therapy with macrolides was associated with better outcomes the eu-vap/cap project was endorsed by eccrn hospital de mataró, critical care, mataró, spain, hospital universitario germans trias i pujol, microbiology, badalona, spain, hospital de mataró, microbiology, mataró, spain, hospital universitario germans trias i pujol this leads to improved survival in a sterile acute lung injury model in wild-type mice, compared to mmp- knockout mice. we recently showed that mmp- deficient mice had better survival in a cecal ligation and puncture (clp) sepsis model than wild-type mice. in humans, functional genetic variations of the mmp- gene exist, but their relation to outcomes of severe infections, such as cap, is unknown. objectives. we hypothesized that functional human single nucleotide polymorphisms (snps) leading to increased mmp- levels are associated with worse survival and higher incidence of severe sepsis in patients with cap. methods. we examined data from genims, a multicenter prospective cohort study of patients with cap and analyzed potentially functional snps (rs , rs , rs ) in the mmp- gene in caucasians by polymerase chain reaction (pcr) the overall incidence of severe sepsis was . % (n = ), and . % of patients (n = ) died within days. the rs genotype distribution was significantly associated with -day mortality by armitage's trend test failure plot for rs conclusions. the non-synonymous rs snp is associated with -day survival in patients with cap. our findings suggests a trend towards a transcriptome analysis of ventilator associated pneumonia in trauma patients the sepsichip project the diagnosis of acute infection in the critically ill remains a challenge. transcriptional profiles (tp) of circulating leukocyte can be used to monitor the host response to infection. ventilator-associated pneumonia (vap) is a frequent complication of major trauma, raising morbidity and mortality data files were analyzed with r and bioconductor. unsupervised analysis was conducted using the dbf-mcl algorithm. supervised analysis was conducted using the significance analysis of microarray algorithm, using siggenes library. all statistical analysis used corrections for multiple comparisons. results. vap occurred in of the trauma patients ( . %). one hundred and fifteen samples were hybridized on husg k microarray ( sirs and sepsis samples for the patients who developed a vap, and sirs samples for those who did not). whereas clinical parameters (iss, chest trauma) discriminated trauma patients with or without vap, admission samples transcriptome analysis did not lead to the identification of prognostic markers. analysis of paired samples of the patients who developed a vap identified a transcriptional signature. these genes were involved in transcriptional regulation, cell survival, hemostasis and endocrine regulation. conclusions. by comparing whole blood admission samples, we were not able to identify transcriptional prognosis markers in trauma patients who did or did not develop vap. however, using vap as a model of sepsis in trauma patients, we identified a set of genes which may serve to diagnose vap in trauma patients to investigate the correlation between transfusion practice and the development of ventilator associated pneumonia (vap) in patients with traumatic brain injury (tbi) we analyzed which tbi individuals developed vap in regard to the transfusion practice and if the number of transfused prbcs increases the risk of pneumonia development. we counted the total amount of prbcs units received by each patient during icu stay, as well as those given before vap development. patient's data included: demographics, apache ii, iss, gcs, vap characteristics, duration of mechanical ventilation (mv), length of stay (los) and outcome. cpis and mods were calculated on the day of vap detection.statistical evaluation was performed using univariate and multivariate logistic regression, students t-test and pearson's chi square test %) tbi patients who developed vap received on average units of prbcs during icu stay, compared with non vap individuals who were transfused on average with two units of prbcs (p \ . ). vap patients received on average four units prbcs before vap development. after correcting for age, apache ii, gcs and iss, transfusion was independently associated with vap. the odds ratio for vap aspiration pneumonia (ap) in comatose patients (pts): clinical and microbiological findings ap is a common complication in comatose pts. we aimed to update data on their incidence standard guidelines were used for diagnosis and treatment of ap. daily chest x-ray were retrospectively reviewed. ap was diagnosed if pts met following criteria: persistent radiographic infiltrate within days following ti, and at least two of the following: purulent sputum, fever/hypothermia duration of mv was days ( - ) and length of stay in icu days ( - ) on the day of ap diagnosis, main pts characteristics were: saps ii %) received empirical antimicrobial therapy. main empirical antibiotics were coamoxiclav ( %) and third generation cephalosporin ( %) ) and mv duration ( vs. days, p \ . ), even considering only non-cardiac arrest pts. gcs h after ti and ap were associated with a [ days duration of mv in multivariate analysis ap was associated with higher overall -day mortality in univariate analysis ( vs. %, p = . ) but no longer in multivariate analysis mortality in the icu was . % with a corresponding hospital mortality of . %. a microbiological documentation was obtained in . % of the patients, with streptococus pneumonia being the most frequent ( % of the isolates). the cap was classified as localized in . %, unilateral mean (±sd) saps piro score was . ± . points, with a corresponding predicted mortality of . ± . % (standardized mortality ratio . ). the aroc was . ( . - . ). the value of the hosmer-lemeshow Ĉ test was saps piro presented a discrimination similar to the originally described. however, it significantly overestimated mortality sepsis mortality prediction based on predisposition, infection and response the piro-cap score was proposed earlier this year to stratify patients with severe community acquired pneumonia (cap) to evaluate piro-cap score in patients with severe cap, over a period of years we analysed data prospectively registered in an informatic data base, which contains information referring to all patients admitted in this unit survival curves were built as proposed by the original authors. outcome was evaluated at icu discharge overall, it was a severe population: . % of the patients presented at least one chronic disease, saps : . ± . points (predicted mortality . % ± . ), length of stay in the icu . ± . , icu mortality was . %. a microbiological documentation was obtained in . % of the patients, with s. pneumoniae being the most frequent ( % of the isolates). the cap was classified as localized in . %, unilateral icu mortality was piro-cap presented an excellent discrimination. however, mortality rates were greater than the ones described by the original authors in all groups (except group ), with the system significantly under-predicting mortality. consequently, we recommend caution in their widespread use. cumulative survival reference(s). clinical and biological assessments in icu patients delirium is a life-threatening, acute organ dysfunction with an incidence of % in uk mechanically haloperidol is recommended as treatment [ ] despite limited evidence base. objective. a national postal survey of consultant members of the uk intensive care society (ics) was performed to determine the current management of delirium in the ) drug treatment of hypoactive and hyperactive delirium as described by two clinical vignettes and ( ) level of agreement with five statements regarding delirium. results. six hundred and eighty one replies were received from , questionnaires senta response rate of %. twenty five percent of respondents routinely screen for delirium. only % use a validated screening tool, most ( %) of whom use the confusion assessment method, icu. hyperactive delirium is treated pharmacologically by %, the majority using haloperidol. hypoactive delirium is treated pharmacologically by %, with haloperidol again the most common treatment ( %) grant acknowledgement. intensive care foundation, intensive care society a practical algorithm to diagnose delirium in critical care-validity and reliability delirium occurs in up to % of critical care patients [ ], but often remains undiagnosed because standardized delirium monitoring is often dismissed as being too time-consuming or too complicated [ ]. the 'harvard flowsheet', derived from the 'confusion assessment method for intensive care unit' (cam-icu) [ ], provides a practical, algorithm-type handling advice to assess the four dsm-iv delirium criteria in a standardized fashion in intubated patients. it mostly allows for truncation of assessments to save time after approval from our institution's ethics committee, patients of a -bed surgical icu-department were screened in five sessions for delirium ( ) by a psychiatrist as the reference rater using the dsm-iv delirium criteria, and ( ) by two 'harvard flowsheet'-investigators, each unaware of other's ratings. motoric delirium subtypes were classified according to the richmond agitation sedation scale (rass) [ ], which was rated for the feature ('altered level of consciousness') of the 'harvard flowsheet'. patients were deemed as having hypoactive delirium if they were dsm positive by the reference rater and had rass - to , or having hyperactive delirium if their rass was between ? and ? . for interrater reliability the median time to complete the 'harvard flowsheet' in delirious patients was s (iqr, - s) vs. s ( - s)] in non-delirious patients. conclusions. the 'harvard flowsheet' has high sensitivity, high specificity and very high interrater reliability. false-negative ratings can occur infrequently and likely reflect the fluctuating course of delirium with intermittent lucid states a decrease of the overall cost of sedation and of sedation/day of mv followed protocol implementation and has been pursued each year: sedation cost which was greater than €/day in / has decreased to less than €/day in acute renal failure in critically ill patients: a multinational, multicenter study to evaluate current transfusion practice and the association between the age of red blood cells (rbcs) and outcome in critically ill patients design. prospective, multicenter observational study patients: critically ill adult patients receiving at least one unit of rbcs %)] revealed an unadjusted absolute reduction rate (arr) in mortality of % ( % ci - %). after adjustment for disease severity, patient age, other product transfusions, number of transfusions, pre-transfusion haemoglobin concentration, pre-icu transfusions, and cardiac surgery the odds ratio (or) for hospital mortality in critically ill patients in australia and new zealand transfusion of rbcs is delivered within current international recommendations. however, within such a practice patients enrolled included men and women, with mean age of ± years. there were ( %) liver transplantation, ( . %) renal transplantation and ( . %) pulmonary transplantation and ( . %) renal-pancreas transplantation. the days mortality for liver, renal, pulmonary and renal-pancreas was: ( . %), ( . %), ( . %) and ( . %). the mean saps score for liver, renal ic apache ii auc . % . - . , ic % . - . . conclusions. in these study, no differences were observed comparing saps and apache ii in the mortality prediction from liver, renal and pulmonary transplantation current opinion in critical care introduction of a rapid response team: why we are glad we met dew ma and members of the medical emergency response improvement team (merit) committee ( ) mature rapid response system and potentially avoidable cardiopulmonary arrest in hospital the effect of a rapid response team implementation in a private hospital adult patients often exhibit physiological deterioration hours before cardiopulmonary arrest to determine the effect of a rapid response team on the rate of in-hospital cardiac arrests, total and unplanned intensive care unit admissions, and icu and hospital mortality before and after implementation of a rapid response team standard criteria were used to activate the rrt and included acute changes in the patient's mental status, respiratory rate, heart rate, oxygenation, or blood pressure and hypoxia, chest pain, or worry from clinical staff. we measured: admitting diagnosis after rrt were a total the activations. the most common reasons for rrt activation were ventilator dysfunction ( %), cardiac changes ( %) and acute neurological changes ( %). % were transferred to icu and the main reasons were cardiac changes ( %), ventilatory dysfunction ( %) and acute neurological changes ( %) the rrt implementation was associated with decreases in rates of inhospital cardiac arrest, but was not associated with reductions in hospital or icu mortality the . lives campaign: setting a goal and a deadline for improving health care quality gender difference in critical care response team activations impact on outcome saudi arabia, king saud bin abdulaziz university for health sciences, riyadh, saudi arabia introduction. gender-related differences in outcome of ccrt intervention has been documented in the literature indicating that more men were admitted to the intensive care unit our center is the only center in the kingdome of saudi arabia which implements an intensivest-lead ccrt services h/ . the team is leaded by in house board certified in critical care medicine. ccrt services started in chest pain unit-viable option when risk of cardiac etiology is modest. multitudinous patient population brought together for structured survey and care at appropriate level what alters physicians' decisions to admit to the coronary care unit? m. camara , g. silva , s. silva , c. dias , j. nóbrega , e. maul hospital central do funchal, funchal, portugal introduction. procalcitonin (pct) and c reactive protein (crp) are markers of sepsis and the levels correlate with the severity of illness.aims. to evaluate the relationship of procalcitonin (pct) and c-reactive protein (crp) kinetics within the first days of sepsis with the appropriateness of antibiotic therapy and the outcome. a prospective cohort study, over months including patients with documented sepsis in our -bed intensive care unit. crp and pct were simultaneously measured four times (m -m ) during the first days of antimicrobial treatment. the pct and crp time course were analysed according to the appropriateness of the empirical antibiotic therapy as well as according to the patient outcome.results. between january and march of , patients were admitted to the icu. patients presented with sepsis on admission or during their stay. the most common infection site was the lung ( . %) followed by primary bacteraemia ( . %). gram-negative and gram-positive bacteria were isolated in the following proportion: . and . %, respectively. enterobacter, acinetobacter and escherichia were the most frequently isolated ( . % each). gram-positive sepsis was mainly caused by haemophylus influenzae ( . %). sepsis was polimicrobial in . % of cases. . % of the patients were given inappropriate antibiotics. the proportion of gram-negative bacteria isolated was significantly higher in patients who did not receive appropriate antibiotics. the magnitude of the pct and crp elevation was not associated with the appropriateness of antibiotic therapy. logistical regression analysis showed that infection without agent was an independent predictor of inappropriateness of antibiotic therapy.age, saps ii, apache ii and sofa were not associated with an unsuccessful treatment. regarding the absolute value of crp and pct there was no significant difference between successful or unsuccessful. multivariate analysis showed that dpct was not associated with antibiotic appropriateness and mortality.conclusions. although the sample is small, our study suggests that crp and pct kinetics are not associated with the appropriateness of antibiotic therapy and outcome. introduction. patients with hematological malignancy who need advanced life support in the icu because of a life-threatening complication may have a poor prognosis. that's why it is necessary to identify clinical, analytical and biological factors that can help doctors with the decision to admit these patients into the icu.objective. the aim of this study was to assess the utility of procalcitonin serum levels (pct) in predicting the outcome of patients with hematological malignancies admitted to the icu. a total of patients with hematological malignancy were admitted to the icu from january until march . epidemiological data were collected before admission, and patients were followed up clinically and analytically during icu stay. serum samples were collected from icu admission until a maximum period of days. pct values were measured by an immunofluorescent assay based on trace (time-resolved amplified cryptate emission) technology (kryptor pct, brahms ag, hennigsdorf, germany). mean age: (sd ); men/ women. among the patients included, hematological diseases were: non-hodgkin lymphoma ( patients), acute myeloblastic leukemia ( ), acute lymphoblastic leukemia ( ) , multiple myeloma ( ), chronic lymphoproliferative disorder ( ) , others ( ). twenty patients ( %) had previously received hematopoietic stem cell transplantation. thirty patients ( %) presented neutropenia at the moment of icu admission. the main causes for icu admission were respiratory failure in patients ( %) and septic shock in ( %). pct levels were not significantly higher in those patients that required mechanical ventilation. pct levels were significantly higher (p = , ) in those patients admitted because of septic shock. pct levels were lower in days , and in the patients who survived with respect to those who died: day : . ng/ml (sd . ) versus . (sd ); day : . (sd . ) versus . (sd . ) ; day : . (sd . ) versus . (sd . ). the differences were significant in days (p = . ) and (p = , ). there was a trend to have higher pct levels in those patients who had microbiologically documented infection respect to the rest; day : . ng/ml (sd . ) versus . (sd: . ); day : . (sd . ) versus . (sd . ) and day : . (sd . ) versus . (sd . ) .conclusions. serum pct levels are higher in patients with septic shock. serum pct measurement might be useful for predicting mortality in patients with hematological malignancy who require advanced life support. introduction. sepsis is a major cause of mortality in the intensive care unit (icu). efforts have been made to reduce the time needed to diagnose sepsis in order to reduce mortality from sepsis-related multiple organ dysfunction. procalcitonin (pct) has been reported elevated levels at the onset of bacterial infections and seemingly correlated to severity of infection. several clinical trials have detected a high pct level in patients with evidence of systemic bacterial infections, whereas relatively low pct levels occur in patients with only localized bacterial infections.objective. the aim of the present study was to assessed the ability of pct through sensitivity, specificity, positive and negative predictive value (ppv, npv) in patients with suspected sepsis, septic shock, inflammatory systemic response syndrome (sirs) and compared it with variables like crp, mortality, band%, renal failure, active cancer and an isolated bacterial cultures. finally we wanted to evaluate if exists a no infectious correlation in patients who received blood transfusions. we conducted an observational study including all patients admitted to the multidisciplinary icu of the abc medical center (tertiary reference hospital) to whom requested pct at admission in the suspect of sepsis and we followed their outcomes. total populations was patients (p). % were females and % were males. median age was years. of the total of pct sample % were positive and % were negative. the sensitivity and specificity in septic patients were and %. ppv and npv were and %, respectively. we did not found any statistical difference between positive value of pct and sepsis, septic shock, sirs, mortality, crp, band%, acute renal failure, acute lung injury, ards (acute respiratory distress syndrome), blood transfusions and active cancer. the mortality in the populations was %.conclusions. the pct has a wide range of diagnostic in the septic patients. in our study the rate of false positive was % and limited the use for sepsis diagnosis. we suggest that the better utility is for outcome biomarkers more than diagnosis biomarkers of sepsis. y. jin , c. guolong , iit study group of zhejiang province in china zhejiang hospital, hangzhou, china introduction. the use of intensive insulin therapy (iit) in severe sepsis and septic shock has been shown to decrease morbidity and mortality rates significantly when given to high risk surgical patients.objectives. the aim of this study was to assess the efficacy of iit in severe sepsis and septic shock patients in intensive care unit.methods. this is a muticentre, prospective, randomized and controlled study. we randomly assigned patients who admission to icu with severe sepsis or septic shock into three groups: a group (target range for blood glucose is - mg/dl); b group (target range for blood glucose is - mg/dl); c group (target range for blood glucose is - mg/dl as a control). primary end point ( -day mortality for any cause) and secondary end points (icu stay days, mv duration, apacheii scores and mods scores) were obtained serially for days and compared between the three groups. of the enrolled patients, were randomly assigned to group a and to group b and to group c; there were no significant differences between the groups with respect to base-line characteristics. -day mortality was percent in the group a and . percent in the group b assigned to iit, as compared with . percent in the group c assigned to conventional therapy (p = . ).during the interval from first hour to -day stay in icu, the patients assigned to group a and group b had a significantly lower apache ii scores( . ± . and . ± . vs. . ± . , p = . ) and mods scores( . ± . and . ± . vs. . ± . , p = . ) than those assigned to conventional therapy, there were no differences in icu stay days( . ± . , . ± . , . ± . , p = . )and mv duration( . ± . , . ± . , . ± . , p = . ) between the three groups. compared with the conventional therapy group, the group a had a higher rate of severe hypoglycemia [blood glucose level b mg/dl ( . mmol/l); . . vs. . %; p \ . ]. intensive insulin therapy provides significant benefits with respect to outcome and scores in patients with severe sepsis and septic shock in icu, on the other hand, intensive insulin therapy brings a higher rate of severe hypoglycemia. to determine the prognosis factors in elderly patients (c years) with severe sepsis admitted to an intensive care unit (icu).method. an observational, prospective and multicenter study was realized. it includes all the patients of the database edusepsis study (adults with severe sepsis admitted to spanish medical-surgical icus). the clinical and demographic characteristics of all patients including age, sex, origin of the infection, location of the patient at the moment of diagnosis of sepsis, apache ii modified score (apache ii score age excluded), number of organic failures, initial therapeutic strategy (measures of resucitación and measures of treatment), icu length of stay and hospital mortality were registered. the patient were classified in young cohort (\ years) and elderly cohort (c years). elderly cohort patients were also classified in young-old patients ( - years) and very-old patients (c years). descriptive comparative study of both cohorts was realized and multivariate logistic regression for the two subgroups of elderly patients was performed to study the risk factors of hospital mortality. a total of , patients wer enrolled ( . ± . years, apache ii modified score of . ± . , . ± . organic failures, hospital mortality . %). the elderly cohort (n = ; . %) presented a lower apache ii modified score ( . ± . vs. . ± . , p . ), higher abdominal infection as origin of the sepsis ( . vs. . %, p \ . ), higher nosocomial infection ( . vs. . %, p . ) and a lower application of measures at initial treatment ( . vs. . %, p . ) than the young cohort. there were not significant differences in the number of organic failures and days of stay in uci between both cohorts. the apache ii modified score (or . ; % ic . - . ; p \ . ), the nosocomial infection (or . ; % ic . - . ; p \ . ), the thrombocytopenia (or . ; % ic . - . ; p . ) and the acute renal failure (or . ; % ic . - . ; p . ) were associated independently to mortality in the subgroup of young-old patients. in the very-old patients only the apache ii modified score (or . ; % ic . - . ; p \ . ) was independently associated with higher mortality and in this population subgroup the application of measures of initial resuscitation was a protective factor (or . ; % ic . - . ; p . ).conclusions. the elderly patients (c years) admitted in the icu whith severe sepsis have higher mortality, more abdominal infections as origin of the sepsis and fewer application of measures of initial treatment than the young patients (\ years). nevertheless, in the subgroup of very-old patients (c years) the aggressive initial treatment decreases the mortality. objectives. the aims of this study were to determine the crude and related to bacteremia mortality rates in icu patients with bacteremia who receive appropriate empirical antibiotic therapy and to describe the factors associated to mortality in this appropriated treated patients material and methods. during a twelve years and a half period, from to , icu-patients with clinically significant bacteremia were prospectively evaluated. for purposes of this investigation, appropriate empirical antimicrobial treatment of a bloodstream infection (aeat) was defined as the microbiological documentation of infection that was effectively treated based on its antibiotic susceptibility at the time the causative microorganism were suspected. clinical and microbiological variables were recorded. logistic regression analysis was performed to determine the risk factors associated to global and associated to infection mortality. results. among icu-bacteremic patients, aeat was applied in patients ( . %). apache ii and sofa score were . ± . and . ± . , respectively and the incidence of septic shock was . % in this aeat patients. global and associated to infection mortality rates were . and . %, respectively in aeat patients. logistic regression analysis confirmed copd (or . ; % ci: . - . ) and age (or . ; % ci: . - ) as factors independently associated to global mortality and diabetes mellitus (or . ; % ci: . - . ) presentation as septic shock (or . ; % ci: . - . ) and serum levels of albumin (or . ; % ci: . - . ) as a protective factors for global mortality whereas factors as nosocomial origin (or . ; % ci: . - . ) and again serum levels of albumin (or . ; % ci: . - . ) were considered protective for related mortality to bacteremia conclusions. mortality rates remains excessively high in aeat bacteremic-icu patients. different factors were identified as predictive factors for global and associated to mortality in aeat patients. only serum levels of albumin seems to be an independent protective factor for both global and associated to infection mortalities. introduction. severe sepsis is hallmarked by organ hypoperfusion or dysfunction. the transition from severe sepsis to septic shock carries with it an increase not only in morbidity but also in mortality [ , ] . objectives. the aim of the study was to demonstrate the effect of shock at admission in sepsis comparing severe sepsis and septic shock admission diagnoses.methods. single center retrospective study in a bed mixed icu of a tertiary university hospital. during a -years period of study patients were unplanned admitted in the unit: the median was age of ( - ), the males were . % and the mean of sapsii was ± . we randomly select two groups: severe sepsis ( patients) or septic shock ( patients) at admission. statistical analysis of variables: v , mann-whitney test, unpaired t student test, cox regression. no statistical significant differences were found about age and sex between groups. about the origin of infection no statistical significant differences were found between groups, meanwhile the diagnosis respiratory infection appears to be more frequent in the severe sepsis group ( . vs. . %, p . ). the proportion of post-operative admissions (in surgical related conditions) was not different between groups. the sapss ii and sofa at h were higher in the septic shock group [ ( - ) vs. ( - ) , p \ . ]; ( - ) vs. ( ) ( ) ( ) ( ) ( ) , p \ , , respectively]. sofa at discharge appears to be higher in the shock septic group (excluding deaths) [ ( - ) vs. ( - ) (p . )]. the mortality and length of stay (excluding deaths) was higher in the shock septic [ . vs. . % (p \ . ); ( - ) vs. ( - ) (. ), respectively]. the ventilator associated pneumonia was not significantly different between groups. the probability of discharge, across an initial period of days, was lower in the septic shock group [hazard ratio . ( % ci: . - . )], mainly between the th and th days, as shown in the kaplan-meier plot (see graph ) .admission diagnosis: probability of discharge conclusions. septic shock at admission patients had a poorer outcome. the difference in the probability of discharge between groups was higher when mechanical ventilation related events are likely to occur [ , ] . we emphasize the importance of the institution of early goal-directed therapy in the wards and emergency departments prior to admission in an intensive care unit [ , ] .introduction. it is not clear whether patients with community acquired severe sepsis (cass) or hospital acquired severe sepsis (hass) have a same presentation. objectives. to evaluate the characteristics of a severe sepsis (ss) population admitted through the er (cass) and those coming from the ward (hass). all patients were treated by the same team of intensivists and er doctors in a shock room, so we could minimise the differences due to management. methods. all adult patients admitted to the medical icu were eligible if they met the criteria for ss. we collected demographic characteristics, apache ii and sofa score, comorbidities and immuno-compromised conditions. scvo or svo (if possible), lactate concentrations. the milestones of the surviving sepsis campaign (ssc) were measured regularly during the first h of treatment. the data collection went on in the icu stay too. treatment for septic shock was conformed to the recommendations of ssc. results. we enrolled pts with ss, including with cass and with hass. there was no difference in demographic features and comorbidities, including immuno-compromised conditions, haematological malignancy and chronic respiratory diseases .there were no significant differences in hemodynamic variables or indices of tissue perfusion like scvo (or svo ) and blood lactate levels, or in amounts of fluids infused or needs of vasopressor agents. the need for mechanical ventilation (mv) after the first has greater for hass than for cass patients, but during the icu stay the need for mv was the same for both groups; similarly, during the icu stay there was no difference in the need for extracorporeal renal support or need for adrenergic agents. at the beginning the scvo was around % for the entire population. after the first h both groups reached the target of %. at the admission % of patients had a scvo less than % ( . % for hass patients and . % for cass, without any difference between groups) and % of patients had a scvo higher than %. the mean svo for both groups was higher than % already at the beginning of the observational period. conclusion. only a half of pts with ss or sho had fever. the presence of fever is often associated with a positive microbiological diagnosis, but better prognosis. while hypothermia was often viewed in severe ill pts and was associated with a worse prognosis. to investigate the possible differences in characteristics and outcome between early and late-onset severe sepsis in surgical intensive care unit (icu) patients. we conducted a retrospective analysis of prospectively collected data from all adult patients ([ years) admitted to our -bed surgical icu between st march and th july .results. of , patients admitted to our icu during the study period, patients ( . %) had severe sepsis; ( . %) had early-onset and ( . %) late-onset severe sepsis. respiratory infections ( . vs. . %, p = . ) and infections of unknown origin ( . vs. . %, p = . ) were more frequently recorded in patients with late-onset than those with early-onset severe sepsis, whereas abdominal infections were more frequent in early-onset than in late-onset severe sepsis ( . vs. . %, p = . ). gram-positive infections were more frequent in late-onset than in early-onset severe sepsis ( . vs. . %, p = . ). the time of onset of severe sepsis was not independently associated with an increased risk of in-hospital death (early vs. late: or . % ci . - . , p = . ).conclusions. respiratory infections and infections of unknown origin were more frequently recorded in patients with late-onset than in those with early-onset severe sepsis, whereas abdominal infections were more frequent in early-onset than in late-onset severe sepsis. the time of onset of severe sepsis has no impact on mortality. objectives. to describe the causes, microbia spectrum, and prognosis of pregnancyassociated sepsis treated in icu in france along the last years. we conducted a retrospective study in a medico-surgical icu of beds in a non-teaching hospital in france where a high risk maternity unit was opened in . patients admitted between and for sepsis occurring during pregnancy or the post-partum period were included. the patients were excluded if the sepsis was due to a nosocomial icuacquired infection. charts were reviewed to collect data on sources of infection, microbia, maternal and fetal prognosis. data are shown as median (extremes) or percentage. data before and after were compared using non parametric tests.results. patients were admitted for pregnancy-associated sepsis ( % of total pregnancy-related icu admissions). included patients had the following characteristics on admission: age: ( - ) years, gravidity: ( - ) pregnancies, parity: ( - ) children, , . vasopressors, mechanical ventilation, and hemodialysis were required in respectively , , and % of cases.characteristics of infections are shown in table . microbiological data about bacterial infections, and specially infections of pelvic origin (chorioamniotitis, endometritis, septic thrombophlebitis), are shown in table .all urinary infections were due to e. coli. lung infections were most often documented clinically but not microbiologically.maternal mortality rate was % ( deaths before and deaths after ). for those infections that occurred in the pre-partum period, fetal mortality was %. after exclusion of fetal deaths that had occurred before icu admission, pregnancy was interrupted during icu stay in % of cases, resulting in fetal mortality of %.conclusions. despite of disappearance of post-abortum sepsis in france, sepsis remains a significant cause of icu admission during or after pregnancy, and a significant cause of maternal and fetal mortality.grant acknowledgement. none. introduction. the glasgow coma scale (gcs), universally used for assessing comatose states, has the advantage of ease of use making it accessible to all levels of clinical competence. there are, however, significant drawbacks. its relative subjectivity in the interpretation of verbal and eye responses and its mesencephalic limit in the rostro-caudal assessment of brain vitality. these two drawbacks limit its use particularly in icu intubated patients. we propose a new score, the sousse coma score (scs) that overcome the eye and verbal responses and explore the brain vitality up to brain death. the score ranges from (normal consciousness) to (brain death). the performance of this score was compared to a modified gcs (gcsm; gcs reduced to its only eye and motor components) and four score. our study interested prospective and consecutive comatose patients who were admitted to a medical icu, intubated and under ventilatory support. the level of consciousness was assessed at admission by physicians of different levels of competence. the inter-observer reliability was assessed by measuring the spearman correlation between the responses of different observers to the scs, gcsm, four score and their respective components. the prognostic predictive value of the three studied scores was assessed by the analysis of possible correlation with mortality and the roc curves. inter-observer reliability was excellent (spearman rho [ . ) for the three studied scores, but with better performance for the scs ( . , p \ . ) compared to the gcs ( . , p \ . ) and four ( . , p \ . ). the level of overall inter-observer reliability for gcs and four was paradoxically higher than that of their respective motor components. this is probably the result of a summation effect of their respective components.regarding the relationship between mortality and the studied coma scores, there was for all three scores a threshold below which mortality was %; / for scs, / for the gcs and / for the four. beyond this threshold, only the scs provides a highly significant correlation with the risk of death (spearman = - . , p = . ). a similar correlation was observed with the motor components of the gcs and four. a better correlation was found between mortality and the scs. the area under the roc curve, however, was poor for all three scores. in evaluating the minimally consciousness states and from eight value of scs, the gcs and four scores provided a wider and more subtle level of consciousness assessment. the scs provides a better inter-observer reliability and a better prediction of death while being easier to achieve. the apparently good inter-observer reliability of gcsm and four was simply the result of summation effect. however, scs was not very sensitive in detecting variations of the minimal consciousness states. the results of our study should be confirmed in larger multicenter studies for the external validation. introduction. the use of a sedation assessment scale and a sedation goal is recommended in critically ill adults [ ] . several studies have found a reduction of icu length of stay (los), of mechanical ventilation (mv) duration, and of cost [ , ] . but, durability and efficiency of such procedures over time have not been evaluated. a -bed medical icu in a university hospital. a sedation and analgesia protocol has been implemented since - . the sedation goal is prescribed each day by the intensivist; the ramsay sedation score is evaluated by the nurses every h and doses of drugs are adapted accordingly. we conducted an annual survey from to to evaluate the quantity of sedative drugs utilized, the impact on icu los and on mv duration, and cost of sedation; both the medical and nurse staff were regularly informed and the protocol was modified if necessary. u. guenther , j. weykam , u. andorfer , t. muders , h. wrigge , c. putensen university of bonn, anaesthesiology and intensive care, bonn, germany background. acute brain dysfunction (delirium and coma) is reported to occur in up to % [ ] , and to be associated with longer mechanical ventilation and stay in the icu, and increased -months mortality rates up to % [ ] . such outcome data, to the best of our knowledge, are predominantly given on medical patients with delirium incidences and mortalities much higher than we expected in surgical patients. this study assessed incidence and impact of acute brain dysfunction on length of stay on the ventilator and in the icu, and mortality in cardiac surgery patients. after approval from our local ethics committee, every patient admitted to our cardiac surgery -beds icu from october through november was daily monitored for delirium with the ''confusion assessment method for the intensive care unit (cam-icu)'' [ ] , level of consciousness was assessed with the richmond-agitation-sedation scale (rass) [ ] . acute brain dysfunction was diagnosed if patients were comatose without sedative medication or delirious. patients were contacted months later to obtain information about their further clinical course.results. patients were eligible for analysis [male , female , age, mean (iqr), ( - ) years]. % had acute brain dysfunction while in icu, these had significantly higher apache-scores on admission, higher tiss-and saps-scores, were longer mechanically ventilated [ ( - ) vs. ( - ) days, p \ . , mann-whitney test) and had longer stay in icu [ ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) vs. ( - ) days, p = . , mann-whitney test). patients were lost to follow-up; -months mortality in patients with acute brain dysfunction in icu was vs. % (p = . , log-rank test).discussion. incidence of acute brain dysfunction and mortality found here in cardiac surgery patients are lower compared to reports on medical patients. even though, duration of mechanical ventilation, length of stay in icu, and -months mortality were increased in patients with acute brain dysfunction in our icu.conclusion. these data emphasize the need for routinely monitoring of consciousness and delirium and to develop strategies to reduce incidence of acute brain dysfunction. introduction. the incidence of obesity is increasing globally, with billion overweight people (body mass index (bmi) [ kg/m ), and million of them obese [ ] (bmi [ kg/m ). the number of obese individuals presenting to critical care is likely to increase, but data on effect of obesity on outcome is conflicting [ ] . there is a general perception that obese patients are likely to have a higher incidence of adverse outcome in critical care. to investigate the effect of bmi on length of stay and mortality in patients admitted to critical care in city hospital, birmingham, uk.methods. an observational study was performed over a month period from february-april . pre-morbid data on bmi was collected from medical records and direct questioning of patients. apache ii score at h, critical care length of stay (los) and survival data to hospital discharge were collected. the figures were compared against their predicted mortality. readmissions, patients with a los \ h, patients \ years old and those without complete apache ii data were excluded. a total of patients were admitted. met the exclusion criteria, bmi data was unavailable for patients and apache data was unavailable for patients. patients were included in the final analysis. . % were female and . % were male. % of patients were above their ideal body weight, and % were obese. the obese cohort had a mean apache ii score of . with a mean los of . days and a mean hospital mortality rate of . %. the corresponding figures for the non-obese group (bmi \ ) was . , . days and . % (table ) . obese patients had reduced hospital mortality in comparison to predicted rates from apache ii scores. statistically, there was no increase in mortality of obese patients. the los and ventilated days were also comparable to the nonobese patients. introduction. managing glucose levels in critically ill hospitalised patients has been shown to play a role in improving clinical outcomes. as a result glycaemic control protocols are widely used in critical care settings and require rapid and frequent testing of patient glucose levels. poc glucose meters have migrated from ambulatory testing into hospital.there is increasing recognition that the clinical accuracy of nearly all commonly used glucose meters are affected by components or substances often present in the blood matrix of critical care patients giving rise to an increase risk of adverse incident. the aim of this study was to challenge the accuracy of a glucose meter designed to correct for these interferences.methods. paired random arterial whole blood samples were collected from icu patients admitted for [ h, samples were tested for glucose using stat strip glucose (nova biomedical) and the omni b bga (roche) routinely used for blood gas analysis.statistical methods: spearman rank correlation/regression, bland-altman analysis. results were compared to iso standard for glucose measurements. omni bga: correlation coefficient r = . , slope = . with intercept - . .bland altman plot for absolute glucose concentration showed that mean bias compared to reference was - . ± . mmol/l with limits of agreement - . - . mmol/l. study aim. to assess predictors of t and its impact on icu-, hospital-length of stay and costs in a cardiac surgical patient cohort admitted to our eight bedded icu, since through june .methods. all the pre-, intra-and post-operative variables were prospectively put into an electronic database. patients were divided into: ( ) ntg group, not needing a tracheostomy;( ) tg group, undergoing a tracheostomy. p values \ . were considered significant. out of a total of , patients with a median (iqr) age of years ( - ) ( ) from post-operative icu to the cardiac surgical ward and ( ) from the cardiac surgical ward to the rehabilitative one, increased significantly higher in the ntg group than in tg group (respectively: log-rank = . , p = . and log-rank = . , p = . ,). tg group showed a lower mortality ( . vs. . %, p = . ) than ntg one.conclusion. this study allowed us ( ) to define a predictive model for identifying patients that are likely to undergo a tracheostomy ( ) introduction. patients admitted to itu increasingly have significant medical comorbidities that require chronic therapy for adequate control . on admission to itu, acute medical problems take precedence and many long term medications, such as thyroxine, may be withheld or ceased. in chronic hypothroidism the patient is physiologically dependent upon ongoing administration of thyroxine. the optimal management of chronic medical conditions such as hypothyroidism within the itu may be essential to patient recovery and should be a quality assurance issue. to assess the prescription of thyroxine and the thyroid function in patients admitted to itu with previously diagnosed chronic hypothyroidism. a six year retrospective review of the electronic records of patients with hypothyroidism who were admitted to a bed tertiary referral hospital itu from to was performed. patients were included if they were admitted to the itu for a period of more than days and were on thyroid replacement therapy prior to itu admission. patient demographics, daily thyroid replacement dose/route, thyroid function tests (tsh and free t ) and rate and type of nutrition were obtained. patients were grouped by their worst recorded tsh according to predefined ranges . conclusions. patients did not receive their thyroid replacement for a significant proportion of their admission. this was predominantly due to either lack of prescription or lack of tolerance of enteral feed. the tsh was appropriate for the free t level. a significant proportion of patients ( %) did not have their tsh measured at all. of those that did, abnormal tests were inconsistently repeated or acted upon. having processes in place to ensure the appropriate prescription and adjustment of relevant chronic medications is essential in the provision of high quality care in itu.background. cell-free dna has been investigated as a diagnostic marker in many diseases, including acute conditions such as stroke, myocardial infarction, burns, sepsis etc. its serum and plasma levels have been shown to correlate with disease severity in all those. free circulating dna is released from dead cells (necrotic or apoptotic) and activated inflammatory cells. our hypothesis was that in acute pancreatitis free serum dna correlates with the extent of pancreatic necrosis and that it may be an early marker of severity. free dna was measured in sera from patients with acute pancreatitis at admission, on the first, fourth and seventh day following admission. severetiy of illness was assessed with atlanta criteria. on the first day following admission patients who would develop severe pancreatitis had significantly higher serum dna levels than those with mild disease (median . vs. . ng/ml respectively; p \ . ). this parameter showed very good characteristics as a potential predictor (area under roc curve . ). free serum dna was in correlation with the extent of pancreatic necrosis.conclusions. free serum dna correlates with the extent of pancreatic necrosis and is a potential early marker of severe acute pancreatitis.keywords. acute pancreatitis, cell-free dna, prognostic marker, pancreatic necrosis. introduction. the performance of general prognostic models in patients with transplantation in need for intensive care unit (icu) admission is poor, showing a tendency towards significant underestimation of the risk of dying. the objective of our study is to evaluate the acute physiology and chronic health score ii (apache ii) and simplified acute physiology score (saps ) and their days mortality prediction after liver, renal and pulmonary transplantation [ ] [ ] [ ] .methods. this is a prospective cohort study in a transplantation icu in porto alegre, brazil, during the period of may -december . clinical data of pos transplantation patients admitted at icu were collected at admission and saps and apache ii calculated with respective estimated mortality rates. the area under receiver operating characteristic curve (auroc) was obtained for both scores. objectives. to validate the saps model, over a period of one year, in a general icu. material and methods. we analysed data prospectively registered in an informatic data base (icdoc), which contain information referring to all patients admitted in this unit.we studied all the patients admitted in the year ( patients). excluded from the analysis were readmissions and one patient still in the hospital. analysed patients.results. discrimination was accessed by the area under the roc curve (aroc) and calibration by the hosmer-lemeshow ĉ test for the general equation and for regional equations (southern europe and mediterranean countries).the mean age of the patients was . ± . years. from the total of the patients, were medical ( . %), were scheduled surgical ( . %) and were emergency surgical ( . %). the mean icu and hospital mortality was . and . %. the mean saps score was . points ( - ). discrimination was good with an aroc of . ( . - . ).there was a statistical significant difference between the mortality predicted by the general equation and the observed mortality (ĉ = . ; p = . ); this discrepancy was not significant by using the regional equation (ĉ = . and p = . ). the saps overestimated hospital mortality with the predicted mortality by the regional equation getting closer to the observed mortality [standardized mortality ratio (smr) = . ] than the predicted by the general equation (smr = . ).conclusion. the saps model, particularly using the regional equation introduction. saps has been previously validated in our icu and it has been routinely used in hospital mortality prediction. as we have shown before, saps had a good accuracy regarding discrimination and calibration, with better predictions done by north american and western europe customized equations than the south american one [ ] . in a larger sample we have been observed deterioration in calibration model, especially among groups of lower probability of death, regardless of the equation in use. therefore we tested saps accuracy considering days mortality in comparison to hospital mortality. we considered consecutive admissions in a medical-surgical icu in a private tertiary hospital in sao paulo -brazil, in the period from january to november of . probability of death was derived from given equations of the original study [ ] . hospital and days mortality were considered as end point. discrimination was performed by the area under the roc curve (auroc) and calibration by the hosmer-lemeshow (hl) statistic. observed to expected (o/e) mortality ratio was also calculated. to assess factors concerning prognosis of patients older than years admitted to the icu: group a, to years old and group b, older than years. both groups were compared for the apache ii, admission group, lenght of stay, mortality and usual intensive care procedures (arterial and venous catheters, mechanical ventilation). statistical anlysis: quantitaive variables were expressed as mean and standard deviation (sd). student t test was employed for these variables. categorical variables were compared by the chi-square. p \ . was considered statistically significant. a total of patients were included in group a (mean age . , sd . ) and in group b (mean age . , sd . ). apache ii score was . for group a and . for group b (p = , ); predicted mortality was . and . % respectively (p = . ). ther were no differences for admission group or procedures among groups. mortality was significantly higher in group b ( . vs. . %, p = . ). when mortality was analyzed for admission groups, it was higher just in cardiological group, wich included ischemic cardiopathy, cardiac failure and arrhythmia ( . vs. . %, p \ . ). the investigation of the association between a differential access to intensive care services and patient or hospital outcomes is increasing markedly [ ] [ ] [ ] [ ] . objectives. the aim of this study was to compare demographic, clinical characteristics, and outcomes of patients admitted to tertiary-level intensive care units from a tertiary hospital ward (intrahospital transfer) to patients transferred from a secondary hospital ward (interhospital transfer). single centre retrospective study in a bed mixed icu of a tertiary university hospital. during the study period ( ) ( ) conclusions. the interhospital transferred patients are younger, but at admission severity of the disease is comparable.these findings, within this case mix of patients, suggest there are not significant differences in mortality, length of stay, icu-nosocomial respiratory infection or physiological disability at discharge between intrahospital and interhospital transferred patients to our unit. in this study we did not find a different impact in outcome considering these differential sources of admission. we prospectively analysed data of all patients (pts) undergoing cardiac surgery between january and june , and discharged from our icu by h from surgery. on all patients the following was collected:(i) demographics, risk factors and gravity scores anamnestic illnesses (ii) intra-operative variables [i.e. type of operation, cardiopulmonary by-pass (cpb) and aortic cross clamp (acc) times] (iii) icu-related variables. one-way anova test was used for continuous variables whereas, differences in proportions were compared using chi-squared test.a binary logistic regression model was used to estimate the effect of each considered risk factor on discharging from cardiac surgical to rehabilitative ward, considered as a dycotomous outcome (yes = early b days/no = late [ days). statistic analyses were performed using spss software. p values less than . were considered significant. on all the patients, aged c years, admitted to our post-operative icu since january through december , we collected demographic profiles, operative data and outcomes. a logistic regression model was set up to assess predictors of hospital outcome. a total of patients ( . %), . % males and with a median (iqr) age of ( - ) were admitted. the below table shows the outcome predictors (see table ). objectives. the purpose of this study was to evaluate prospectively in our medium the capacity of apache iii score to stratify prognostically critically-ill-patients upon their admission to the icu, not only with regard to hospital mortality, but also to hospital length of stay. study aim. to assess if cardiopulmonary by-pass (cpb), aortic cross clamp (acc) time and duration of mechanical ventilation (mv) may impact on icu and hospital length of stay in a cardiac surgical patient cohort admitted to our bedded icu, since through june . all the patient pre-, intra-and post-operative variable were prospectively put into an electronic database. on all patients the following was collected:(i) demographics, risk factors and gravity scores anamnestic illnesses (ii) intra-operative variables [i.e. type of operation, (cpb) and (acc) times] (iii) icu-related variables (i.e. duration of mechanical ventilation, use and type of inotropes. statistic analyses were performed using spss software. p values \ . were considered statistically significant. a total of , patients with a median (iqr) age of years ( - ) were admitted through the study period. . % underwent a cabg operation, whereas . % valve surgery and . % aortic and lung surgery. a bivariate analysis was performed considering as independents variables respectively the natural logarytm (nl) of ( ) cpb time, ( ) acc time, ( ) mv duration, whereas dependent variable was considered the nl of the total hospital stay. we showed that a linear correlation exists between total hospital stay (ln) and ( ) conclusion. this audit allowed us to assess that the longer is the cpb and acc time and mv duration the longer is likely to be the total hospital length of stay of the patients undergoing heart surgery. introduction. high-dependency units (hdu) were designed as a bridge between the operating theatre and the surgical ward for postoperative patients demanding a higher than standard level of care. the aim of our study was to determine the risk factors as well as the predictive value of four severity scores for a prolonged hdu length of stay (los). three hundred fifty-eight consecutive adult patients were included in the study for a period of months. asa, saps ii, possum and sofa scores were calculated for the first h following admission. the demographic and the scores variables were subjected to a univariate and, consecutively, a multivariate logistic regression analysis. a receiver operating curve (roc) model was used to determine the predictive value of the scores for a prolonged los. the presence of a patient for three or more days in the hdu was defined as prolonged stay.results. the median los was ( - ) days, patients were transferred to the intensive care unit and the in-hospital mortality was . % ( patients). the univariate logistic regression revealed the following variables as significant for a prolonged los (p \ . ): asa, possum preoperative, possum postoperative, possum total, possum cardiac, possum ecg, possum type of surgery, possum blood loss, sofa, sofa respiratory, sofa cardiovascular, sofa liver, sofa coagulation, igs ii, igs respiratory, igs urinary output, igs urea, igs potassium, igs bicarbonate, and bmi. seven variables were identified as having a statistically significant association with the los (table ) . according to the roc model, sofa score was the best predictor for a prolonged los, with an area under the roc (auroc) of . .warning systems and rapid response team: - s. saxena , s. jafrey , j. zwaal kingston hospital, anaesthetics, kingston, uk, kingston hospital, kingston, uk background. published data suggests that the patient group with the highest mortality in icus comprises those patients admitted from the hospital wards [ ] . studies have shown that in-hospital cardiac arrests are commonly preceded by physiological abnormalities [ ] . if admission to icu, is preceded by specific physiological derangement, then early identification of these high risk hospital in-patients may be possible. this may improve survival of patients. objectives. to determine . the effectiveness of new track and trigger pathway in identifying patients requiring icu admissions. . the impact of new system on outcome of icu admissions method. . retrospective case notes survey of all icu admissions from the ward over a month period. . the pathway is triggered when abnormalities are present in two or more of the following parameters: response to painful stimuli, respiratory rate, oxygen saturation, systolic blood pressure, and heart rate. . .triggering steps progress through involvement of junior medical staff and outreach teams at step , to more senior staff at step , to consultant involvement at step , depending on the level of deterioration of the patient. . forms were collected over a period of months. icu mortality: patients with abnormality at any time prior to icu admission: / ( %) icu mortality: patients with c abnormalities any time prior to icu admission: / ( . %) mortality of patients who were pathway followers: / ( %) mortality of patients who were pathway non-followers: / ( . %) average length of stay in icu who were survivors from pathway followers: days average length of stay in icu who were survivors from pathway non-followers: . days discussion. . there was low sensitivity of pathway for identifying icu admissions. . poor documentation of triggering events . pathway followed inadequately in majority of patients due to combinations of delay in, or absence, of triggering when indicated . lack of consultant involvement at step . no patients with chronic kidney disease admitted to intensive care have poor outcomes [ , ] . in % of cardiac arrest calls in our hospital were from the renal unit (personal communication.) at this time critical care outreach teams were recommended as means of improving intensive care outcomes through earlier ward assessment of critically ill patients [ ] [ ] [ ] . in modified early warning system (mews) charts to wards and a dedicated seven-day ward-based consultant led service ( - ) were introduced on our renal unit [ , ] . aims and methods. the impact of these change interventions was analysed. primary outcomes were the incidence of cardiac arrests calls to the renal unit, admission apache ii scores and icu mortality. secondary outcomes were age, sex, intensive care and hospital length of stay, in-hospital mortality, cpr prior to icu admission and emergency admissions to the renal unit. cardiac arrests, mortality rates and emergency admissions were compared with the v test; other outcomes via mann-whitney u test. a p value \ . was regarded as significant.results. the results are outlined in the table [ ] ; this group has a high mortality [ ] . deterioration in vital signs often precedes referral to critical care and this is evidenced by a rise in the patient at risk score (pars). higher pars may be associated with worse patient outcomes [ ] . most pars systems have a trigger value at which critical care input should be sought. we hypothesised that the duration of physiological deterioration prior to critical care admission would be associated with mortality and used the delay between pars trigger and admission as an estimate of this.methods. we collected data on over consecutive patients that had deteriorated on the ward and required admission to general critical care (hdu and icu) at both acute hospitals in sheffield. patients admitted to specialist facilities such as cardiac and neurosurgical units were excluded. those already triggering at time of hospital admission were also excluded. to assess if any of the ccrt activation criteria was associated with higher incidence of icu or hospital mortality. our hospital is bed tertiary care center. cohort analysis of prospectively collected data of each ccrt activation including demographic data of the patients and their outcome in terms of icu and hospital mortality and ccrt activation criteria. ccrt activation from st january to th september .the activation criteria for ccrt includes: threatened airway,tachypnea defined as respiratory rate more than or less than breath/min, hypoxemia defined as oxygen saturation less than % on oxygen flow l/min, arrhythmias defined as heart rate less than or more than beat per minute, hemodynamic instability if systolic blood pressure less than mmhg or more than mmhg, decrease level of consciousness defined as drop of gcs = or more points from baseline, seizure and serious concern about the patient.we analyzed each factor separately as independent predictor of icu and hospital mortality. [ ] . with this in mind efforts have been made to develop physiological early warning scoring systems which have been shown to predict subsequent outcome [ ] . we have recently introduced an early warning system (ews) chart for all patients in our hospital and we wanted to assess its impact on our icu admissions. to assess the calculation of the ews, the scores of patients admitted to icu and the compliance with guidelines regarding further intervention for patients who were ultimately admitted to icu.methods. chart review of twenty five consecutive emergency icu admissions, examining the ews in the h prior to admsission.results. ews charts were completed for % of emergency icu admissions; of these % of scores were calculated correctly. only % of ews had all parameters completed for all set of observations. the mean peak ews prior to icu admission was . with a range from to . higher peak ews was strongly associated with increased icu mortality: a ews of - was associated with mortality of . %, whereas a ews of - was associated with a mortality of % (see below). for each ews recorded specific action was required to be triggered according to the protocol. in % of cases appropriate action was taken, however, in % the required action was not taken and a number of patients were thought to have delayed referral to critical care as a result of this.conclusions. following this audit we have introduced a critical care outreach team and have embarked on an educational programme for staff with emphasis both on the complete and accurate recording of early warning scores and the necessity for appropriate action to be taken on the basis of these scores. aim. the quality of care prior to icu admission has been a focus of attention [ ] . mews had been chosen by the trust as a trigger device to identify deteriorating (sick) patients in the general wards. this retrospective study looked at the clinical characteristics of unplanned admissions to our icu and assessed the mews as a predictive tool to trigger early intervention in such cases.methodology. all patients who were non-electively admitted to our icu from the medical wards were included in the study (january-march [ ] score and standardised mortality ratio(smr) had occurred since the introduction of our nurse lead outreach serrvice and high dependency unit. this seemed counterintuitive so we decided to look at in futher detail and whether the phenomena of lead time bias occurred.objectives. primary end point was to assess if the apache ii scores and smr were different if assessed from the point of contact with outreach or hdu for patients admiitted to general intensive care. secondary endpoints looked at which physiological scores were most altered by these systems. a cohort prospective study was setup with ethics committee approval. all patients seen by outreach (group ) or on hdu (group ) prior to admission to general icu were included over a six month period. two sets of apache ii scores and mortality prediction were generated for each group, a 'pre' and 'post' score. the pre score was a h scoring period started from up to h prior to admission to icu on point of contact on hdu or by outreach. the post score was a period for scoring taken h from the point of admission to icu, ie the conventional apache ii score .therefore each patient had two sets of scores for apache ii and predicted mortality. the apache ii and predicted mortality scores were then compared using a two tail paired t test, the individual physiology scores were compared via a wilcoxon rank sum score. in total patients from hdu were included and patients from outreach were included.the primary question was answered as a significant difference in apache ii and smr was found in both groups. introduction. unplanned admissions to intensive care units (icu) are associated with an increased mortality. in order to identify in-hospital patients at risk of deterioration, several scores based on physiological parameters have been published. however, routine application of these parameters is not common in all european hospitals yet. the goal of this prospective study was to evaluate the efficiency of the current practice of handing over ward patients at risk for decompensation by physicians and nurses. furthermore, factors associated with admission to the icu or alarming of the physician on duty should be identified. the study was conducted at the university hospital of regensburg, germany on wards with predominantly gastroenterological and general medical patients ( beds). over a time period of months, the daily routine report of patients at risk to the physician on call after hours was recorded. in addition, the nurse assessment of patients at risk and the documentation of the decompensation defined by calling the physician on duty during the night were registered.results. patients were treated during the surveillance period. in total, patients ( women, men) with a mean age of ± years were either judged by the attending physicians or the nurses at risk for deterioration. patients suffered from decompensation during the night shift. of those, patients were correctly identified by physicians and patients by nurses, respectively. in patients ( %), an icu admission was necessary.discussion. only a small portion of patients reported at risk experienced a severe decompensation at night, defined as icu admission. interestingly, those were only in part correctly identified by the physician and nurse reports. a further evaluation of the correlation of those reports with the previously published ''early warning score'', and physiological parameters associated with decompensation are currently being performed in order to estimate the value of standardized patient assessment, and will be presented at the meeting. introduction. the need to implement a patient follow-up program after icu discharge arises from several facts: ( ) at icu discharge patients are now more fragile (aged, chronic comorbidities, complex). ( ) the demand for intensive care exceeds its availability. as much as % of patients die after discharge from the icu, many of them in spite of a low predicted mortality, perhaps due to premature icu discharge. ( ) compared to nursing care in the icu, the level of that received upon transfer to the floor, as measured by tiss- , may be reduced up to more than %. we believe that, in order to change icu behavior towards focus on long term outcomes, we need to increase global awareness of disability post-icu discharges, and expand the involvement of the icu team in key decision management outside the icu. we propose an alternative model of care for the critically ill patient. this involves an expanded role for clinicians with expertise in critical illness at several points along the continuum of care.objectives. due to lack of adequate clinical resources to care for some recoverable patients when are discharged to hospital wards after a long time in icu, we have planned a follow-up program focused in detecting risk factors associated to bad prognosis and, decreasing adverse events in general hospital wards. qualitative, prospective and interventional study realised during seven months (from march to september ), in the medical uci of a teaching hospital in malaga. we determined demographic data, icu admission reason, comorbidity index (charlson scale), follow-up reason (polineuropathy, tracheostomy, analgesia), family support in ward, difference in nursing activities score between icu and ward (tiss- ), intervention done out of icu with patient; satisfaction of patient, icu readmissions, reason to end follow-up and mortality at day after icu discharge. we enrolled patients in this analysis. comorbidity was charlson scale (very high) in . % of patients, apache-ii mean score points and mean expected mortality rate %. more than % of patients had five or more risk factors (age [ years, icu stay [ days, transfusions, inotropic drugs, mechanical ventilation, tracheostomy, kidney failure, parenteral nutrition, polineuropathy). nursing activities score in icu was . before discharge versus . in ward ( . % decreasement). mean follow-up were . (range - ).in hospital mortality rate was . %, rest of the patients were discharged at home. our study found the implementation of continue follow-up program from icu staff is associated with an important decreased of the mortality. encouraging clinical results and a non excesive workload for icu staff justify to continue this follow-up program in cases in which is going to be an important decrease in nursing care after icu discharge, and have bad prognosis risk factors. objectives. we examined the prevalence of adverse events (ae), suboptimal assessments of vital signs and whether there were advance directives prior to icu admission from the general wards among patients who died within days of icu admission. the patients were those admitted to the general icu from the general wards at the university hospital, lund in and who died within days after icu admission. there were patients with a mean age of years and a mean apache ii score of . we used the global trigger tool model for measuring ae (http://www.ihi.org). the frequency of vital functions assessments, and which parameters were controlled were studied in relation to patient status and the local routine for frequency of modified early warning scoring (mews).the patient records were also controlled for descisions to forgo treatment before admission to the icu. . patients ( %) suffered from at least one ae prior to icu admission. patients had an ae contributing to death, among those patients suffered from an ae that with a probability greater than % was deemed avoidable. seven of those patients suffered from a most likely avoidable ae contributing to death.vital signs were recorded inadequately in % of the patients in the h before admission to the icu. the vital signs most often recorded were blood pressure, heart rate and oxygen saturation, whereas consciousness and breathing frequency were the least recorded parameters.descisions to forgo resuscitation, or some other limitations due to ethical considerations were found only in % of the patients before admission to the icu.conclusions. patients admitted to the icu who died within days suffer from a considerable proportion of avoidable ae contributing to death. vital signs are not recorded in a satisfactory way during the h before admission to the icu in this most severely ill population. there are very rarely documented descisions to forgo treatment in this group of patients before icu admission. thus, poor control of vital signs in the general wards leads to severe and avoidable ae contributing to death. the lack of descisions to forgo treatment before icu admission in this group of patients most probably contributes to prolonged dying and suffering, and unnecessary intensive care. results. a significant correlation (p \ . ) was detected between the type of prehospital care and the early outcome among the patients. the majority of the patients was transported by ambulance services ( . %) from which half of the patients ( . % of the total) were seen by a paramedic and the rest by a physician beforehand. a relevant proportion of the patients visited the cpu without having been seen by medical personnel ( . %) before. a smaller group of patients was referred by an attending hospital physician to the cpu ( . %). . % of all patients were admitted to a cardiologic ward, . % to icu and . % underwent immediate cardiac catheterization. the rest was referred to other departments within the hospital, other hospitals or was discharged and no one died within h after admission to the cpu. almost half of the patients ( . %) who underwent immediate cardiac catheterization was transported by emergency physician car whereas half of the rest ( . %) visited the cpu as out-patient (p = . ). this very similar ratio can be seen within the patient admitted to icu ( . %). conclusion. the detection of the early symptoms of chest pain is an important prevention strategy for lay people because they can immediately turn to a chest pain unit ( . %) where almost half of them might have life threatening situation ( . %) requiring acute intervention (cardiac catherization or icu-treatment). the adequate in-time treatment can reduce the length of hospitalization and secure quicker recovery. key: cord- -p c fneh authors: bosma, karen j.; taneja, ravi; lewis, james f. title: pharmacotherapy for prevention and treatment of acute respiratory distress syndrome: current and experimental approaches date: - - journal: drugs doi: . / - - sha: doc_id: cord_uid: p c fneh the acute respiratory distress syndrome (ards) arises from direct and indirect injury to the lungs and results in a life-threatening form of respiratory failure in a heterogeneous, critically ill patient population. critical care technologies used to support patients with ards, including strategies for mechanical ventilation, have resulted in improved outcomes in the last decade. however, there is still a need for effective pharmacotherapies to treat ards, as mortality rates remain high. to date, no single pharmacotherapy has proven effective in decreasing mortality in adult patients with ards, although exogenous surfactant replacement has been shown to reduce mortality in the paediatric population with ards from direct causes. several promising therapies are currently being investigated in preclinical and clinical trials for treatment of ards in its acute and subacute, exudative phases. these include exogenous surfactant therapy, β( )-adrenergic receptor agonists, antioxidants, immunomodulating agents and hmg-coa reductase inhibitors (statins). recent research has also focused on prevention of acute lung injury and acute respiratory distress in patients at risk. drugs such as captopril, rosiglitazone and incyclinide (col- ), a tetracycline derivative, have shown promising results in animal models, but have not yet been tested clinically. further research is needed to discover therapies to treat ards in its late, fibroproliferative phase. given the vast number of negative clinical trials to date, it is unlikely that a single pharmacotherapy will effectively treat all patients with ards from differing causes. future randomized controlled trials should target specific, more homogeneous subgroups of patients for single or combination therapy. the acute respiratory distress syndrome (ards) arises from direct and indirect injury to the lungs and results in a life-threatening form of respiratory failure in a heterogeneous, critically ill patient population. critical care technologies used to support patients with ards, including strategies for mechanical ventilation, have resulted in improved outcomes in the last decade. however, there is still a need for effective pharmacotherapies to treat ards, as mortality rates remain high. to date, no single pharmacotherapy has proven effective in decreasing mortality in adult patients with ards, although exogenous surfactant replacement has been shown to reduce mortality in the paediatric population with ards from direct causes. several promising therapies are currently being investigated in preclinical and clinical trials for treatment of ards in its acute and subacute, exudative phases. these include exogenous surfactant therapy, b -adrenergic receptor agonists, antioxidants, immunomodulating agents and hmg-coa reductase inhibitors (statins). recent research has also focused on prevention of acute lung injury and acute respiratory distress in patients at risk. drugs such as captopril, rosiglitazone and incyclinide (col- ), a tetracycline derivative, have shown promising results in animal models, but have not yet been tested clinically. further research is needed to discover therapies to treat ards in its late, fibroproliferative phase. given the vast number of negative clinical trials to date, it is unlikely that a single pharmacotherapy will effectively treat all patients with ards from differing causes. future randomized controlled trials should target specific, more homogeneous subgroups of patients for single or combination therapy. acute lung injury (ali) and the acute respiratory distress syndrome (ards) arise from direct or indirect injury to the lungs, and results in a life-threatening form of respiratory failure. ali/ards is both common and serious: . - . % of patients admitted to an intensive care unit (icu) will be diagnosed with ali or ards, [ ] [ ] [ ] and approximately one-quarter to one-half of these patients will succumb to this disease process. [ , [ ] [ ] [ ] over the past years, ards has been the focus of extensive basic science and clinical research, although no single pharmacotherapy has been shown to reduce mortality in a large, randomized, controlled, multicentre trial of adult patients. the reasons for this are manifold, and include issues of dosing, route of administration and timing of the various interventions tested. more importantly, however, may be the nature of the disorder itself: the diagnosis of ards envelops a heterogeneous group of patients with varying causes and pathophysiological mechanisms at work. the notion that a therapeutic agent that can successfully alter a single biological target in an animal model of ali will reduce mortality in all patients with ards may be unrealistic. nonetheless, there is reason for hope on the scientific horizon. recent advances have been made in our understanding of the pathophysiological mechanisms underlying ali/ards, leading to the identification of potential novel targets for pharmacological intervention. some therapies are best aimed at preventing the development of ards, while others treat the syndrome as it unfolds or aid in its resolution. the challenge lays in identifying the subgroup of patients most likely to benefit from such focused therapy. this paper reviews the current experimental and existing approaches to managing ards, highlighting the pathophysiological basis for their use and potential for future clinical development. ali may occur following a direct insult to the pulmonary system such as aspiration of gastric contents, bacterial pneumonia or viral pneumonitis (e.g. h n influenza virus), or an indirect insult such as the systemic inflammatory response associated with pancreatitis, sepsis or multiple trauma. table i shows common direct and indirect causes of ali/ards. whether this 'first hit' to the lung is direct or indirect, a pulmonary inflammatory response may occur, which often is adaptive and self-limited. however, when coupled with repeated 'hits' to the lung from insults such as injurious mechanical ventilation or other secondary processes such as hypotension, a cycle of intense inflammation and worsening pulmonary injury ensues. the 'multiple hit' theory of ards progression also provides a framework for studying the disease process (figure ). clinically, ali manifests as bilateral airspace disease observed on chest radiograph and hypoxaemia, such that the ratio of partial pressure of arterial oxygen to fraction of inspired oxygen (pao /fio ) is greatly reduced. according to the american european consensus conference (aecc) definition, a chest radiograph consistent with pulmonary oedema and a pao /fio ratio < is sufficient to diagnose ali in the setting of an inciting pulmonary insult and the absence of congestive heart failure. the aforementioned criteria but with a pao /fio ratio < is classified as ards. [ ] although differentiated by the aecc definition, ali and ards are often grouped together for the purpose of clinical trial enrolment and are treated as a single entity throughout this review. although not all patients follow the same clinical course, progression of ali/ards may be considered along a pathophysiological timeline of early, mid and late phases, with considerable overlap between these phases. table ii summarizes the pathogenetic mechanisms at work during each phase, linking each biological pathway to a potential drug therapy. a general overview of the pathophysiology of ards is provided here, with more detailed descriptions of the specific biologic pathways discussed in sections . - . as they pertain to each potential pharmacological therapy. the early phase, within the first hours of the inciting lung injury, is characterized by inflammatory damage to the alveolar-capillary barrier. this results in increased vascular permeability, leading to interstitial and alveolar oedema as proteinaceous fluid fills the alveolar space. this inflammation-induced pulmonary oedema disrupts normal gas exchange and increases the work of breathing, leading to respiratory failure and the need for mechanical ventilation. mechanical ventilation itself may cause secondary insult to the already inflamed oedematous alveoli. during each tidal breath induced by mechanical ventilation, unstable alveoli undergo cyclical collapse and shearing open, termed 'atelectrauma'. furthermore, the non-collapsed alveolar units may receive a greater proportion of the delivered tidal volume, leading to damage due to overdistention or 'volutrauma'. further breakdown of the endothelial-epithelial barrier may occur with atelectrauma and volutrauma, along with the release of local proinflammatory mediators which further b see text for details. aa = arachidonic acid; arb = angiotensin receptor antagonist (blocker); fa = fatty acid; gm-csf = granulocyte macrophage colonystimulating factor; hne = human neutrophil elastase; icam- = intercellular adhesion molecule- ; il- = interleukin- ; mmps = matrix metalloproteinases; nf-kb = nuclear factor-kb; paf = platelet-activating factor; ppar-g = peroxisome proliferator activated receptor-g; rhpaf = recombinant human paf; tnfa = tumour necrosis factor-a. propagate this cycle of ventilator-exacerbated lung injury. [ ] as inflammation ensues, neutrophils are recruited to the lung. damaged endothelial cells exhibit increased activity of the transcription factor nuclear factor-kb (nf-kb), which upregulates the surface expression of intercellular adhesion molecule (icam)- . icam- mediates leukocyte adhesion and migration across the endothelium to the alveolar epithelium. activated neutrophils release proteases, such as matrix metalloproteinases (mmp) and neutrophil elastase (ne), which further damage the alveolarcapillary membrane. [ ] activated neutrophils also contain high levels of arachidonic acid, [ ] which is metabolized into leukotrienes, prostaglandins and thromboxanes. leukotrienes attract more neutrophils, prostaglandins are proinflammatory mediators, and thromboxanes play a role in vasoconstriction and platelet and leukocyte aggregation. neutrophil recruitment and activation may be an adaptive physiological response to injury, or may incite a vicious cycle of inflammation and further damage. [ ] at this stage, patients may recover from the initial insult, with clearance of the pulmonary oedema and restoration of the barrier between capillary endothelial and alveolar epithelial cells, or may progress to the exudative or mid phase of ards. it is not fully understood why two patients exposed to the same insult may have completely different clinical courses; however, genetic factors, [ ] co-morbid illnesses such as diabetes mellitus and alcohol addiction, [ ] nutritional status, medications and exposure to further insults are all likely to play a role. understanding the host and environmental factors that place a patient at high risk of progressing to the exudative phase of ards will facilitate identification of targets for earlier intervention. the exudative or subacute phase typically occurs over the - days following the initial insult. pathologically, this mid phase is characterized by formation of intra-alveolar hyaline membranes rich in plasma proteins, fibrin and cellular debris. [ ] a biopsy of the lungs at this stage will show diffuse alveolar damage and, clinically, the lungs have poor compliance with ongoing gas exchange problems including hypoxaemia and elevated dead space fraction. the inflammatory milieu within the alveoli, coupled with the cyclical opening, stretching and collapsing of alveoli via mechanical ventilation, initiates a number of pathogenic pathways in concert or in series. these include disruption of surfactant function and metabolism, ongoing neutrophil recruitment and activation, along with increased expression and release of inflammatory mediators, imbalance of oxidant and antioxidant activity, and activation of complement and coagulation cascades. each of these pathways is further discussed to provide context for the drugs or therapies aimed at ameliorating these various mechanisms (see section ). interestingly, only a minority of patients will succumb to severe hypoxaemia or hypercarbia, as the major source of mortality is not the pulmonary injury per se, but rather the occurrence of multiple organ failure. in this setting, the injured lung may represent a rich source of inflammatory mediators that could contribute to the development of multi-organ failure. for example, stress failure and necrosis of the endothelial-epithelial barrier may allow various inflammatory mediators, bacteria and endotoxins to quickly spread from the lungs into the systemic circulation. indeed, it is this de-compartmentalization of inflammatory mediators from the lungs into the circulation that is felt to lead to cell apoptosis in distal organs, [ ] and ultimately multiple organ dysfunction syndrome (mods) [ figure ]. [ ] once mods develops, disease is often irreversible and mortality may increase significantly to - %, the latter occurring when three or more organs are involved for a period of more than days. [ ] [ ] [ ] thus, a key to developing novel therapies that will reduce mortality in ards will be identification of the cellular and molecular mechanisms by which ards leads to mods. survivors of the first week of ali/ards may enter the late phase of the disorder, known as the fibroproliferative phase. during days - , the exudates and hyaline membranes become organized, and fibrosis may become apparent. type ii alveolar cells proliferate and line the alveolar walls, fibroblasts migrate and differentiate into myofibroblasts in the interstitial and alveolar spaces, and a collagen-rich extracellular matrix is laid down in the interstitium. [ ] alveoli may be destroyed, pulmonary vascular area may be reduced and chronic inflammation is generally present. patients in the fibroproliferative phase of ards may slowly recover, or may fail to wean from mechanical ventilation and succumb to complications of a lengthy critical illness or pre-existing co-morbid illnesses. pharmaceutical interventions for late ards must interrupt the fibrosing alveolitis and aid in resolution, remodelling and repair of injured lungs. [ ] often, therapies that might be beneficial during the early phase of lung injury are started too late in the course of the disease, when fibrosis is already established, muting their potential efficacy. when tested specifically for the late fibroproliferative phase of ards, anti-inflammatory therapies have yielded disappointing results. basic science research examining mechanisms of idiopathic pulmonary fibrosis may illuminate therapeutic pathways for fibroproliferative ards, but further work is required in this area. although no pharmacological therapies have been proven to reduce mortality in large, randomized controlled trials (rcts) involving adult patients, it appears that improvements in supportive care have reduced mortality to some extent. for example, mortality estimates ranged progression of acute respiratory distress syndrome (ards) to multi-organ failure (mof). initially, inflammatory damage to the alveolar-capillary barrier results in increased vascular permeability, leading to interstitial and alveolar oedema as proteinaceous fluid fills the alveolar space. there, the proteinaceous fluid interferes with the function and metabolism of the endogenous surfactant system. coupled with this, neutrophils that infiltrate lungs are subsequently activated and represent an important source of inflammatory mediators and oxygen free radicals, inducing further epithelial and endothelial cell damage and an altered host immune response. newly secreted mediators and/or spillover of inflammatory mediators from the lung into the systemic circulation ultimately contribute to the development of mof. inflammatory mediators released from organs such as the liver, heart and kidney return to the lung via the systemic circulation and may contribute to further pulmonary inflammation. thus, each new insult to the pulmonary system accelerates the acute lung injury cycle (reproduced from bosma et al. [ ] [ ], with permission). from % to % as reported in the literature in the s and early s [ ] [ ] [ ] [ ] to more recent estimates of - % in observational epidemiological studies [ , ] and - % in large clinical trials. [ , ] although this mortality reduction may in part reflect differences in diagnostic criteria used post publication of the aecc definition, undoubtedly the largest impact has been the move to more 'protective' strategies of mechanical ventilation. in , the national institutes of healthsponsored ards network (ardsnet) trial involving low tidal volume ventilation was published, and now constitutes the standard of care for patients with ali and ards. this trial compared a traditional tidal volume of ml/kg with a lower tidal volume of ml/kg in patients and reported a mortality reduction from % in the control arm to % in the treatment arm. [ ] these results definitively ended the debate fuelled by three previous inconclusive smaller trials regarding lower versus conventional tidal volumes. in terms of furthering ali/ards research, several lessons have been learned from this landmark study. first, ardsnet was set up to conduct well designed, large, phase iii studies with a concerted effort to optimize patient enrolment through involvement of many centres in an organized and cohesive group. [ ] this enabled a study sufficiently powered to realize a mortality difference to be conducted within a reasonable timeframe, and pointed the way for other similarly structured ards research networks to become established. second, the treatment arm was associated with lower oxygenation values than the conventional arm, highlighting the potential danger of relying on oxygenation or other physiological parameters as surrogates for mortality. third, this study demonstrated that a nonpharmacological intervention could alter mortality, indicating that future rcts need to be carefully standardized in all aspects of supportive care in both treatment and control arms. one potential caveat ensuing from this study has been the assumption that any additional proven therapy would reduce mortality across a population as heterogeneous and diverse as that enrolled in the ardsnet low tidal volume trial. this approach may be misguided, as subsequent studies have demonstrated differences between patients with direct and indirect causes of ali/ards in responsiveness to specific therapies. [ , ] research is ongoing to determine whether newer modes of mechanical ventilation, such as high-frequency oscillation (hfo), can further improve outcomes in ards relative to the ardsnet low tidal volume strategy. [ ] in addition, other aspects of supportive care have been evaluated in large clinical trials, some conducted by ardsnet, and have proven effective in reducing morbidity associated with critical illness. these include cautious fluid management, [ , ] adequate nutrition, [ ] prevention of ventilator associated pneumonia, [ ] [ ] [ ] [ ] prophylaxis for deep venous thrombosis [ ] and gastric ulcers, [ ] weaning of sedation and mechanical ventilation as early as possible, [ ] and physiotherapy and rehabilitation. [ ] a recent review of all patients enrolled in ardsnet studies between and showed that these advancements in critical care (aside from lower tidal volume ventilation) are likely responsible for the improved survival in ali/ards patients in clinical trials noted over the last decade. [ ] additional modalities used as 'rescue therapies' for the ards patient at risk of succumbing to severe hypoxaemia or respiratory acidosis have also been tested, including nitric oxide, prone positioning, hfo and extracorporeal membrane oxygenation (ecmo). nitric oxide [ ] and prone positioning [ , ] have not been shown to reduce mortality or duration of mechanical ventilation in patients with ali/ards, and are therefore not recommended for routine use. however, combined together, these therapies may provide a sustained improvement in oxygenation for patients with severe hypoxaemia and a mortality benefit for patients who are failing conventional mechanical ventilation strategies. [ ] [ ] [ ] a clinical trial of hfo for routine care of patients with ards is currently underway, but existing evidence supports its use as salvage therapy if instituted early for patients failing conventional ventilation, [ ] and may have additive benefits when combined with nitric oxide and prone positioning. [ ] finally, ecmo has recently been studied in the cesar trial (see table iii for a list of trial acronyms). [ ] this study showed that transferring adult patients with severe but potentially reversible respiratory failure, whose murray score exceeds . or who have a ph of < . on optimum conventional management, to a centre with an ecmo-based management protocol, significantly improved survival without severe disability. recent evidence suggests ecmo is also useful for rescue therapy for adults with severe ards due to h n -influenza a virus infection. [ ] pharmacological treatments for ali/ards may be employed prior to the onset of ards or in the early, mid or late phases of ards (table iv) . accordingly, their purpose may be to prevent ali in those at risk, mitigate the pathogenic mechanisms responsible for the cycle of lung injury and systemic inflammation in established ards, or aid in lung healing and repair. some therapies, such as corticosteroids, have been studied for prevention of ards, treatment of early ards and treatment of late ards, and are discussed within each context. the concept that ards may be prevented in those at high risk after an inciting insult is not new, but is one that is garnering greater attention in the scientific literature in recent years. since no pharmacological agent has proven effective in treating established ards in adults, attention has turned to prophylactic treatment to prevent the development of ards in those at highest risk. of course, any pharmacotherapy that is initiated prior to the diagnosis of disease must have a very high benefit to risk ratio and be cost effective. as such, it should have the following attributes: (i) be low risk, without serious adverse effects; (ii) be easily and widely applicable; and (iii) be relatively inexpensive. drug classes studied for ards prevention include imidazoles (e.g. ketoconazole), ace inhibitors, thiazolidinediones (e.g. rosiglitazone), chemically modified tetracycline derivatives, antioxidants, and corticosteroids and other immunomodulating agents. over years ago, the first clinical trial examining prophylactic use of ketoconazole to prevent ards in patients at risk was published. [ ] the rationale for using ketoconazole, an antifungal drug with anti-inflammatory properties, was as follows. as mentioned in section , patients with ards have increased levels of arachidonic acid metabolites in their bronchoalveolar fluid. [ , ] metabolism of arachidonic acid leads to the production of leukotrienes, prostaglandins and thromboxanes. thromboxane a is a potent vasoconstrictor, and is involved with platelet and leukocyte aggregation, while leukotrienes act as powerful chemokines to attract neutrophils. ketoconazole is an antifungal agent of the imidazole class which selectively blocks thromboxane synthetase. ketoconazole also inhibits -lipoxygenase, the enzyme necessary to generate leukotrienes, and inhibits procoagulant activity. [ ] in addition to showing promise in preclinical animal studies, when given prophylactically to patients at risk of developing ards, ketoconazole has been shown to reduce the incidence of severe ards in three small trials. a study of patients admitted to a surgical icu showed that in the group treated prophylactically with oral ketoconazole mg/day, of patients ( %) ultimately developed ards, whereas of ( %) patients in the control group developed ards (p < . ). [ ] similar results followed in a study of patients with septic shock admitted to a surgical icu, where the incidence of ards in the group treated with ketoconazole mg/day was % ( of patients) compared with % ( of patients) in the control group (p = . ), and mortality was % versus %, respectively (p = . ). [ ] although both of these studies were conducted prior to the aecc definition, ards was strictly defined in the aforementioned studies, including a pao /fio ratio < or intrapulmonary shunt > % in patients requiring mechanical ventilation and who had diffuse infiltrates on chest radiograph without clinical evidence of heart failure as pulmonary arterial occlusion pressures were < mmhg. building on the results of these two studies, sinuff and colleagues [ ] developed practice guidelines for prophylactic ketoconazole use, and tested the implementation and efficacy of these guidelines in two icus (one control and one active comparator). they reported a significantly decreased incidence of ards in the icu population receiving ketoconazole prophylaxis, although mortality was equivalent within the two units. [ ] in , ardsnet published the karma study evaluating oral ketoconazole versus placebo for patients within hours of an established diagnosis of ali or ards according to the aecc definition. [ ] the study was stopped early after enrolment of patients for failing to show a difference in mortality or ventilator-free days. of note, this study was designed to look at early treatment of ali/ards rather than prevention of ards in patients at risk, and therefore did not necessarily negate the findings of the three previous smaller studies. furthermore, a problem identified in the karma study was that even though blood ketoconazole concentrations were adequate, urinary metabolites of thromboxane were not affected, raising the possibility that the proper dose to achieve an anti-inflammatory effect was not given. however, since the karma f mortality reduction in subgroup of patients with ards, septic shock and relative adrenal insufficiency. g no mortality reduction in larger study, n = (lasrs). il- = interleukin- ; ma = meta-analysis; mc = multicentre; ppar-c = peroxisome proliferator activated receptor-g; rhpaf = recombinant human platelet-activating factor; rsp-c = recombinant surfactant protein-c; sc = single-centre. study showed no difference in mortality, widely considered the most important endpoint to achieve, further research on ketoconazole for ali/ards ceased. [ ] additionally, ketoconazole has numerous drug interactions and requires an acidic milieu to be absorbed via the enteral route, making routine use in the icu complicated. further research should examine whether other drugs in the imidazole class given intravenously have similar anti-inflammatory properties, and also establish the inflammatory dose-response curve for ali/ards. in addition, although the concept that prevention of ards will definitely lead to decreased mortality is intuitive, this still has to be proven in large multicentre clinical trials. the authors are unaware of any studies being conducted in this area presently. angiotensin-converting enzyme (ace) is produced in the lungs and is responsible for converting angiotensin i into angiotensin ii, a peptide active in vasoconstriction and sodiumfluid balance to maintain blood pressure homeostasis. ace inhibitors and angiotensin ii receptor antagonists (blockers; arbs) are classes of drugs commonly used to treat hypertension, and prevent progression of diabetic nephropathy in patients with diabetes. ace inhibitors also help to preserve vascular structure and function, by exerting a protective effect on endothelial cells. endothelial cell damage is the catalyst for the inflammatory and coagulation cascades activated in ali/ards. thus, the protection of endothelial cells offered by ace inhibitors may have a beneficial role in ards. [ ] studies in transgenic mice have shown that ace, angiotensin ii and angiotensin ii receptor type a may promote lung injury, whereas ace , a close homologue of ace, and angiotensin ii receptor type may protect against severe lung dysfunction in models of ards. [ ] the ace inhibitor captopril has been shown to prevent severe lung injury in an oleic acid-induced model in rats. in this model, captopril reduced expression of icam- in lung tissue, indicating a protective effect on endothelial cells, diminished activity of tissue plasminogen activator, involved in coagulation, and blocked nf-kb, the major signal transduction pathway that regulates the expression of multiple early-response genes related to inflammation. [ ] in humans, two small cohort studies have demonstrated that polymorphism of the ace gene increases susceptibility to the development of ards and its outcome. [ , ] two additional studies, published only in abstract form to date, have examined the association between ace inhibitor use and ards. a retrospective cohort study of adult critically ill patients found that . % of patients developed ards after hospital admission, and that preexisting, long-term use of an ace inhibitor or arb was associated with decreased risk of ards development, after adjusting for predisposing conditions (odds ratio [or] . ; % ci . , . ; p = . ). [ ] the second abstract, a case-control study nested within a prospective cohort of critically ill patients at risk for ards, reported that patients on ace inhibitors had a lower prevalence of respiratory failure on admission to icu, but not lower incidence of ards after adjusting for confounders on multivariate analysis. however, among patients who developed ards, ace inhibitor use was associated with lower mortality (adjusted hazard ratio . ; % ci . , . ). [ ] the associations observed in these clinical studies is consistent with preclinical animal data, but requires further research prior to being applicable clinically. [ ] peroxisome proliferator activated receptors (ppars) are ligand-activated transcription factors related to thyroid hormone, steroid and retinoid receptors. [ ] there are three isoforms: ppar-g, ppar-a and ppar-b/d. ppar-g plays a central role in glucose homeostasis. thiazolidinediones, a class of oral antidiabetic drugs, are synthetic ligands for ppar-g. synthetic ppar-g agonists also have anti-inflammatory properties, inhibiting proinflammatory cytokine production and macrophage activation in vitro. [ , ] this action is mediated in part by antagonizing the activity of transcription factor nf-kb. when activated, nf-kb induces overexpression of inflammatory cytokines such as tumour necrosis factor (tnf)-a, which in turn induces upregulation of icam- expression, as well as recruitment and activation of immune cells. icam- , expressed on the surface of endothelial cells, mediates leukocyte adhesion and migration through endothelium into tissues. the anti-inflammatory properties of thiazolidinediones have been demonstrated in vivo in murine models of inflammatory bowel disease [ ] and rheumatoid arthritis. [ ] rosiglitazone is the most potent selective ppar-g of the thiazolidinediones. prophylactic administration of rosiglitazone has been shown to attenuate ali in an animal model of pancreatitis-associated ali. [ ] in this study, rosiglitazone was dissolved and given intravenously to rats minutes prior to induction of acute pancreatitis by sodium taurocholate. compared with control group rats with acute pancreatitis and its associated lung injury, prophylactic administration of rosiglitazone resulted in a significantly lower histological pulmonary injury score, reduced pulmonary expression of tnfa and icam- messenger rna, and decreased lung tissue myeloperoxidase activity, a measure of neutrophil infiltration in the lung. [ ] this suggests that prophylactic rosiglitazone mitigates the ali associated with acute pancreatitis by its anti-inflammatory effect. unfortunately, the safety of rosiglitazone has recently been questioned due to its augmentation of sodium and water retention, leading to increased incidence of congestive heart failure in diabetic patients placed on this drug long-term. [ , ] thus, further animal studies are needed to confirm the effects of rosiglitazone in acute pancreatitis and evaluate potential complications related to its use, prior to proceeding to human studies. during the early phase of lung injury, neutrophils are recruited into the pulmonary vasculature and activated to release proteases, such as mmps and ne, which damage the alveolarcapillary membrane, [ ] resulting in further release of inflammatory mediators. a single laboratory in the state university of new york (new york, ny, usa) has demonstrated in various animal models that blocking the proteases ne, mmp- and mmp- with a unique modified tetracycline can prevent the increased pulmonary vascular permeability that ultimately leads to ards. the same group has developed a 'two-hit' porcine model of sepsis plus gut ischaemiareperfusion injury that parallels the insidious onset of sepsis-induced ards in humans. in this model, anaesthetized yorkshire pigs undergo cross-clamping of the superior mesenteric artery for minutes to induce intestinal ischaemia, followed by intraperitoneal placement of a faecal blood clot. pigs are then awakened, extubated and taken to an animal icu for hours of continuous observation, where they receive intravenous fluids, broad-spectrum antibacterials and pain control medications. when the pao /fio ratio falls below , pigs are anaesthetized and placed back on mechanical ventilation with tidal volumes of ml/kg. in this model, they demonstrated that prophylactic administration of a synthetic, nonantimicrobial derivative of tetracycline called incyclinide (col- ; collagenex pharmaceuticals), prevented the development of both ards and septic shock. [ ] incyclinide has not yet been tested in any human studies of ards prevention; however, the complex model developed by this group contains all the elements of a clinically relevant animal model and, therefore, these results show potential for phase ii studies. oxidative stress is associated with development of ards and mods via direct tissue injury. nathens and colleagues [ ] examined the effect of antioxidant supplementation using atocopherol and ascorbic acid in critically ill surgical patients. in a prospective rct of surgical icu patients (mainly victims of trauma), they found antioxidants did not reduce the risk of developing ards, but did decrease the risk of developing mods, and shortened duration of mechanical ventilation and length of icu stay. [ ] antioxidants supplementation and nutritional strategies are now being studied for critically ill patients with early signs of mods, [ ] but not specifically for ards prevention. antioxidants and nutrition have also been studied for treatment of ards, and are further discussed in this context in section . . . given that excessive and protracted inflammation is the overriding principle responsible for the various pathophysiological mechanisms leading to ards, broad and potent anti-inflammatory drugs, such as corticosteroids, would seem to be a rational choice for prevention. four rcts, published between and , have examined the use of corticosteroids to prevent the onset of ards in patients at risk. a recent meta-analysis of these studies demonstrated that preventive corticosteroids may actually increase the risk of developing ards in critically ill adults. [ ] furthermore, the meta-analysis suggested a weakly increased risk of death associated with preventive corticosteroid therapy in those patients who ultimately developed ards. thus, corticosteroid therapy is not recommended for preventing ards in those at risk. corticosteroid therapy has also been extensively studied for the treatment of established disease in the early and late phases, and is discussed further in these contexts (see the corticosteroids subsection of section . . and section . . ). platelet-activating factor (paf) is a potent proinflammatory mediator that is degraded by the enzyme paf acetylhydrolase. recombinant human paf acetylhydrolase (rhpaf-ah; epafipase) was studied in a phase iib rct to prevent ards in septic patients. [ ] patients with severe sepsis were randomized to receive rhpaf-ah mg/kg, rhpaf-ah mg/kg or placebo. the incidence of ards was not different amongst the three groups, but -day all-cause mortality was significantly decreased in the mg/kg treatment group compared with placebo ( % vs %; p = . ). therefore, although rhpaf-ah does not appear to be an effective prophylactic treatment for ards, it may hold promise for treatment of severe sepsis. the majority of research to date has focused on treating ards once the diagnosis is established. although many studies are designed to treat 'early ards', with randomization occurring within hours of diagnosis, these studies also likely capture many patients in the exudative phase of ards with intra-alveolar hyaline membranes and histological diffuse alveolar damage at the time of enrolment. this problem arises in part because the diagnostic criteria for ards are subjective and lack sensitivity and specificity when compared with pathological diagnosis. [ ] thus, timing an intervention at a certain point after 'diagnosis' could result in the patient receiving treatment in the early, mid or even late pathophysiological stage of ali/ards. some more recent studies are now targeting time after intubation rather than time after diagnosis to achieve more uniform timing of intervention. however, since the acute and exudative phases occur along a continuum and are not generally distinguished clinically, therapies targeting these phases will be considered concomitantly. therapies currently under investigation for early and/or exudative ards include those targeting the disrupted surfactant system, oxidative stress and antioxidant activity, neutrophil recruitment and activation, expression and release of inflammatory mediators, activation of the coagulation cascade, and microvascular injury and leak. treatment of the overall inflammatory response with agents such as corticosteroids has also been studied and is discussed. finally, the only drugs specifically targeting resolution of the alveolar oedema of the acute phase are b -adrenergic receptor agonists (b -agonists). clearance of alveolar oedema depends on the balance between oedema formation and reabsorption. the rate of fluid reabsorption depends on the active transport of sodium and electrolytes; water follows in the direction of the transported electrolytes. the active transport of salt and water occurs via epithelial sodium channels induced via na + /k + adenosine triphosphatase (atpase). [ ] b -agonists are thought to increase alveolar fluid clearance via two possible mechanisms: (i) increasing the levels of intracellular cyclic adenosine monophosphate, which in turn upregulates na + /k + atpase, causing increased sodium transport across alveolar type ii cells; and (ii) reducing alveolar-capillary permeability, thereby decreasing oedema formation. preliminary animal and ex vivo studies demonstrated the potential of b -agonists to accelerate the rate of alveolar fluid clearance. [ , ] a small, single-centre rct randomized patients with ali/ards to receive intravenous salbutamol (albuterol) mg/kg/h or placebo for days. [ ] the primary endpoint of balti- was extravascular lung water measured by the singleindicator transpulmonary thermodilution system (picco Ò ; pulsion medical systems) at day . patients in the salbutamol group had lower extravascular lung water and plateau pressures, although oxygenation did not differ between the treatment and placebo groups. this latter finding was perhaps due to the vasodilatory effects of b -agonists contributing to shunting of oxygen in the capillary bed. there was no difference in -day mortality or ventilator-free days, although the study was not sufficiently powered to detect a difference in these endpoints. [ ] funded by the medical research council, the same investigators in the uk are now conducting balti- , using the same intravenous salbutamol protocol as in balti- , but powered to detect clinically important outcomes. [ ] it will be interesting to determine if the physiological benefits observed in balti- confer a reduction in -day allcause mortality in balti- . aerosolized b -agonists have fewer systemic adverse effects than intravenous preparations. the national heart, lung and blood institute (nhlbi), in conjunction with ardsnet, conducted a study of an aerosolized b -agonist, the alta study. [ ] the study was stopped for futility at the first interim analysis after enrolling patients. [ ] there was no difference in the primary outcome of ventilator-free days to day . this study may have been negative for the following reasons: (i) delivery of nebulized drug to lung injury sites may have been suboptimal, as was the case with aerosolized surfactant; and/or (ii) less severely ill patients with ali (rather than ards with more severe hypoxaemia) may retain adequate alveolar fluid clearance without the need for upregulation with b -agonists. sixty-day mortality in the alta study was . % compared with a -day mortality of % in the severely ill group of patients who received physiological benefit from intravenous salbutamol in balti- . [ ] exogenous surfactant administration has been very successful in treating and preventing neonatal respiratory distress syndrome (nrds). given the physiological and pathological similarities between nrds and ards, exogenous surfactant therapy has been under investigation for treatment of ali/ards for over a decade. although clinical trial results have been largely disappointing, recent studies show promise. the strong scientific rationale for targeting the disrupted surfactant system, as well as lessons learnt from previous trials, therefore merit further attention. endogenous surfactant is composed of % lipids (mainly phosphatidylcholine and phosphatidylglycerol) and % proteins. the role of endogenous surfactant in the healthy lung is to decrease surface tension and thereby prevent alveolar collapse. in addition, surfactant plays a role in suppressing inflammation and scavenging free oxygen radicals. four apoproteins have been identified, termed surfactant protein (sp)-a, -b, -c and -d. whereas the presence of either or both of the hydrophobic surfactant proteins sp-b and -c are important for the biophysical function of surfactant, the hydrophilic proteins sp-a and -d perform the various host defence roles, including modulation of leukocytes, enhancement of the function of phagocytic cells [ ] and regulation of the host's immune system. [ , ] in ali, disruption of the endogenous surfactant system occurs by a number of mechanisms: injury to alveolar type ii cells results in abnormal synthesis and secretion of surfactant, serum proteins that leak into the airspace interfere with surfactant function, serine endopeptidase and phospholipase a cause degradation of surfactant, and, finally, mechanical ventilation, particularly with high tidal volumes, causes conversion of functional surfactant aggregate forms into dysfunctional forms. without optimal surfactant function, there is high surface tension at the alveolar surface in a non-uniform pattern within the lung leading to alveolar instability and collapse. the presence of bacteria within the airspace may also release and activate endotoxins, a process that is augmented in the presence of an abnormal surfactant system. based on the functional importance of the endogenous surfactant system in the normal lung and, more importantly, the consequences of an altered surfactant system in ali/ards, there is good rationale to consider exogenous surfactant administration as a therapeutic intervention in these patients. [ ] in , a phase iii, double-blind rct tested an aerosolized, synthetic surfactant called exosurf Ò (glaxo wellcome) in patients with sepsis-induced ards. [ ] this study showed no significant difference in overall survival, duration of mechanical ventilation or oxygenation between the treatment groups and standard care. it was postulated that this lack of efficacy was due to a low level of alveolar deposition of the aerosolized preparation and/or due to the absence of surfactant proteins in the preparation. [ ] currently, this surfactant preparation is not being evaluated for patients with ali/ards and is no longer marketed in the us. shortly afterwards, a smaller, open-label phase ii clinical trial evaluated tracheal instillation of a liquid bolus of the natural bovine extract surfactant, survanta Ò (ross laboratories), in patients with severe ards. [ ] there was a trend toward decreased mortality in the group of patients receiving up to four doses of phospholipids mg/kg surfactant compared with the patients in the control group ( . % vs . %; p = . ), and no safety concerns were identified. however, survanta Ò contains only very small amounts of sp-b. coupled with concerns regarding resource limitations, no further clinical trials of this exogenous surfactant preparation for adults with ards have been performed. recognizing the importance of surfactantspecific proteins brought progress to clinical surfactant research. in , results were published for two phase iii clinical trials evaluating effect of a liquid, recombinant sp-c (rsp-c) surfactant, venticute Ò (nycomed), instilled intratracheally in patients with established ards. [ ] the two studies enrolled a total of patients within hours of diagnosis of ards and were powered to show a difference in ventilator-free days. although oxygenation was significantly better during the -hour treatment period in the surfactant group, there were no significant differences noted in the number of ventilator-free days or in -day survival. [ ] a post hoc analysis demonstrated that patients with 'direct' causes of ards (i.e. pneumonia, witnessed aspiration of gastric contents or both) had a mortality benefit with surfactant treatment compared with standard care. a followup meta-analysis pooling results of five multicentre studies of rsp-c confirmed this finding: the subgroup of patients with severe ards due to pneumonia or aspiration had decreased mortality when treated with rsp-c ( . % vs . % in the usual care group; p = . ). [ ] subsequently, a prospective phase iii rct evaluating effect of venticute Ò in patients with pneumonia or aspiration of gastric contents was conducted. the study was terminated at patients due to futility. neither these results nor the potential reasons for futility have been published to date. [ ] calfactant (infasurf Ò , ony inc.) is a modified natural surfactant produced by extracting the phospholipids, neutral lipids and surfactantspecific proteins sp-b and sp-c from newborn calf lungs. in in vivo animal lung studies, calfactant has shown greater surface activity than exosurf Ò and survanta Ò , [ ] [ ] [ ] [ ] and the highest level of resistance to inactivation due to its high ratio of protein sp-b to phospholipids. [ ] [ ] [ ] from to , calfactant was used in a multicentre study of ali/ards in the paediatric population week (full-term infants) to years of age. overall, calfactant significantly improved oxygenation and reduced mortality ( % vs %; p = . ), although the greatest impact was observed in the subgroup of patients with direct ali/ards while calfactant had little effect in patients with indirect ali or ards. [ ] indeed, calfactant is the first and only pharmacological agent to demonstrate a mortality benefit for treatment of ali/ards. it is of note, however, that this study differs from other adult studies in that the majority of paediatric patients had direct causes of ards and the most common cause of death was respiratory failure, whereas adult studies have included a larger proportion of patients with indirect causes, such as sepsis, wherein the most common cause of death is multi-organ failure. based on those encouraging results, pneuma pharmaceuticals began conducting a large phase iii multicentre rct of calfactant for direct ards (origin of ards must be infectious pneumonia, aspiration, near drowning, smoke inhalation without pulmonary burn or inhaled industrial gas) in adults and children. a total of patients in two consecutive studies of patients under and over years of age was planned. however, after the first interim analysis in january , the paediatric arm of the study was stopped for futility due to an unexpectedly low mortality rate. recruitment in the adult arm (ages - years) is continuing as the interim analysis did not reveal futility or any safety concerns (wilson d, university of virginia health sciences center, charlottesville, va, usa, personal communication). [ ] since reactive oxygen species also contribute to the tissue damage incurred in ali, antioxidant therapies have also been investigated as therapeutic options for established disease. n-acetylcysteine (nac) is a commercially available antioxidant approved for the treatment of paracetamol (acetaminophen) toxicity. nac is a precursor for glutathione, an antioxidant present in normal lungs and deficient in bronchoalveolar lavage fluid from ali/ards patients. additionally, because of its thiol group, nac can scavenge reactive oxygen species such as hydrogen peroxide and superoxide anion. in an rct of patients, nac and oxothiazolidine carboxylate (procysteine Ò , clintec technologies inc.), another glutathione precursor, were studied for their combined effect in ali/ards but failed to reduce mortality compared with placebo, [ ] negating promising results of three prior small studies. [ ] [ ] [ ] interestingly, recent evidence suggests that genetic diversity may explain variable responsiveness to nac. glutathione-s-transferases (gsts) are enzymes from a complex, multigene family with important roles in oxidative stress pathways. a study by moradi and co-workers [ ] demonstrated that deletion of specific gst gene polymorphisms correlated with mortality and that treatment with nac significantly lowered mortality in these subgroups of patients. these results suggest that patients with gst gene deletions are more vulnerable to oxidative stress contributing to ards and may be in greater need of antioxidant therapy. [ ] antioxidant supplementation to enteral nutrition rich in omega- fatty acids has also been investigated for patients with ali/ards. while the rationale for nutritional antioxidants such as vitamins e and c is to reduce the oxidative stress present in ali, the purpose of the omega- fatty acids is to reduce production of proinflammatory mediators. eicosanoids, such as prostaglandins, thromboxanes and leukotrienes, derived from omega- fatty acids are generally much less proinflammatory than those derived from omega- fatty acids. since omega- fatty acids compete with omega- fatty acids for the same rate-limiting enzymes in the production of eicosanoids, diets with a high proportion of omega- fats are thought to be proinflammatory and prothrombotic. examples of polyunsaturated omega- fatty acids are a-linolenic acid, eicosapentaenoic acid and docosahexaenoic acid. [ ] a phase ii study enrolling patients with ali compared an antioxidant enteral feeding formula containing eicosapentaenoic acid, g-linolenic acid and antioxidant vitamins with placebo, and observed improved oxygenation, reduced pulmonary inflammation, fewer days of mechanical ventilation and fewer non-pulmonary organ failures in the treatment arm, although there was no difference in mortality between this approach and the control group. [ ] ardsnet proceeded to conduct the omega study, a phase iii study examining efficacy of omega- and antioxidant supplementation to enteral nutrition. the study was stopped for futility, but results have not yet been published. [ , ] several therapies aimed at modulating neutrophil activity have been studied. to understand why previous clinical trials have been negative and highlight potential targets for novel therapies, it is important to understand the role of neutrophils in propagating lung injury and mods. polymorphonuclear neutrophils (pmns) form the first line of defence against invading pathogens, and neutropenia or defective neutrophil function predisposes the host to increased morbidity. extensive clinical and experimental data support the role of the activated neutrophil in the pathogenesis of organ injury in sepsis. the lung is particularly vulnerable. postmortem studies of patients with ards show massive pulmonary accumulation of neutrophils, with the highest counts in non-survivors. [ ] the pathological impact of neutrophils may be due to their activation, transmigration or delayed apoptosis. however, neutrophil-independent mechanisms of ali must also exist, since ards has been described in neutropenic patients. neutrophil kinetics in the pulmonary circulation differ substantially from that of microvascular beds in the systemic circulation. the pulmonary circulation harbours a large intravascular reservoir of leukocytes, mainly neutrophils, referred to as the 'marginated pool'. [ ] this marginated pool may equal or even exceed the pool of circulating neutrophils and exchanges with the latter as an ongoing phenomenon. thus, it is important to appreciate that circulating neutrophils, when isolated for experimental analysis, may not represent the characteristics of the entire population of neutrophils in the bloodstream. intravital microscopic studies have revealed that, in contrast to the systemic circulation where neutrophil sequestration is almost exclusively confined to the venular compartment, the major site of neutrophil retention in the lung is the alveolar capillary bed. [ ] neutrophil activation can also lead to cytoskeletal changes that reduce cell deformability and slow their transit time through the alveolar capillaries. since one of the earliest manifestations of ards is accumulation of large numbers of neutrophils in the alveolar capillaries, it is possible that the accumulation of neutrophils may initiate selective capillary blockade and arteriovenous shunting leading to hypoxia seen in ards. activated neutrophils also produce human ne (hne), a protease capable of producing tissue damage by means of its degradation of elastin, fibronectin, laminin, collagen and proteoglycans. normally, protease inhibitors impede ne, but in the setting of an overwhelming inflammatory response, neutrophils generate reactive oxidants that inactivate endogenous protease inhibitors, leaving the activity of hne unchecked. this may lead to increased pulmonary inflammation and endothelial cell permeability. [ ] sivelestat (elaspol Ò , ono pharmaceuticals) is a competitive inhibitor of ne. it was launched in japan after a phase iii study demonstrated reduced icu stay and improved pulmonary function in patients with ali associated with the systemic inflammatory response syndrome (sirs). [ ] however, the strive study [ ] was terminated early after randomizing patients from sites in six countries, when the data and safety monitoring board found a trend to increased mortality at days. final analysis revealed no difference in -day all-cause mortality ( % in both groups) or number of ventilator-free days between the treatment group and controls. epi-hne- or depelestat (debiopharm s.a.) is another hne inhibitor currently under development for treatment of inflammatory pulmonary diseases, including ali. in a repeated lung injury rat model depelestat administration afforded a significant protective effect on lung compliance and alveolar inflammation at day compared with the control group. [ ] a phase ii study examining safety and efficacy of intravenous depelestat for patients with ards has been completed, but results have not yet been published. [ ] neutrophil transmigration neutrophil margination allows for a molecular interaction between the cell surfaces of the neutrophil and endothelial cell to occur. subsequently, as a consequence of cell surface integrins and their ligands, neutrophils undergo adhesion with endothelial cells. following adherence, neutrophils must pass through the endothelial monolayer, interstitial tissue and alveolar epithelium to reach the alveolar space. passage of large numbers of activated neutrophils can cause epithelial damage, sloughing and increased permeability both due to mechanical force exerted by neutrophil pseudopodia as well as due to release of toxic substances such as proteinases (e.g. elastases, cationic peptides, defensins, oxidants and mmps). [ ] while there are conflicting reports on the effects of elastase on increased epithelial permeability, cationic peptides such as defensins can cause both epithelial and endothelial cell injury. defensin levels have been found to be greatly elevated in patients with ards and their levels correlate with the severity of lung injury. [ ] neutralizing its effects could be important in the management of ards. ongoing research is examining if defensins can be used to identify patients with ali at an early stage. [ ] delayed apoptosis of neutrophils once egressed into the extravascular space, neutrophils cannot return to the circulation and their elimination is dependant upon their clearance by apoptosis and subsequent recognition and elimination by macrophages and other phagocytic cells. normally, neutrophils are terminally differentiated cells with a terminal half-life of - hours in vivo. upon completion of their lifespan, neutrophils institute a programme of cell death known as 'apoptosis' and are then removed from the circulation by the liver and spleen. apoptosis, as opposed to necrosis, is believed to be crucial for resolution of inflammation as it does not result in loss of cell membrane integrity and bystander tissue damage by release of intracellular enzymes, proteases and reactive oxygen species. [ ] expression of neutrophil apoptosis is delayed in ards. [ ] this is not an unexpected finding, especially since pmn apoptosis is delayed in other critically ill patients with sepsis, trauma and burns. [ , ] apoptosis of neutrophils may be an important consequence in determining the extent of lung injury. for example, it has been shown that the induction of neutrophil apoptosis by the administration of dead escherichia coli prior to reperfusion resulted in significant improvement in lung injury. [ ] induction of neutrophil apoptosis in the alveolar space has the potential for resolution of inflammation in ards, and can be carried out in a number of ways that could include multiple strategies such as ligation of fas, activation of proapoptotic caspases and modulation of mitogen-activated protein kinases or transcription factors such as nf-kb. hastening neutrophil apoptosis in the alveolar space may also decrease the probability of secondary necrosis and further tissue damage in ards. it is intriguing that no significant differences were found between the expression of neutrophil apoptosis in patients at risk and those with established ards, nor did the extent of apoptotic inhibition correlate with overall outcome in ards. [ ] therefore, while it is well established that ards is associated with accumulation of large numbers of neutrophils in alveolar spaces, their contribution to the severity of ards in humans remains uncertain. in summary, targeting neutrophil responses in ards may have therapeutic potential. however, as has been learnt from various ali and sepsis trials in the past, simple strategies to control dysregulated neutrophil behaviour may not be effective. rather, key stages of neutrophil function and kinetics may need to be identified in different clinical phases of ards, and selective immunomodulation strategies may need to be identified for individual patients. in addition to modulation of neutrophil function, there are other facets of the immune and inflammatory response currently under investigation as potential therapeutic targets for treatment of ards. these include modulation of macrophage activity with granulocyte macrophage colony-stimulating factor (gm-csf), inhibition of inflammatory mediators and broad suppression of the inflammatory response with corticosteroids. although most prostaglandins are proinflammatory mediators, prostaglandin e (pge ) has potential beneficial effects in ali, specifically due to its ability to modulate neutrophil activation. however, exogenous pge is associated with several adverse effects and patient intolerance due to haemodynamic instability has been observed. tlc-c- (ventusÔ; the liposome company) is a liposomal dispersion of pge . the development of pge in liposomal form may potentiate its role in neutrophil downregulation, improve peripheral delivery of the drug to the lung and decrease systemic adverse effects, thus providing a good rationale for testing in humans. [ ] a phase iii trial of patients with ards randomized to intravenous tlc-c- at escalating doses for days versus placebo found no difference in duration of mechanical ventilation or -day mortality between the treatment and control groups, although treatment was associated with accelerated improvement in oxygenation. [ ] however, more than % of patients required a dose reduction due to hypotension or hypoxaemia. interestingly, those patients who tolerated and received at least % of the full dose had a shorter duration of mechanical ventilation. a subsequent multicentre phase iii trial of tlc-c- in ards patients [ ] demonstrated no differences in time to liberation from the ventilator or -day mortality; the trend to shorter duration of hypoxaemia in the treatment group failed to reach statistical significance. gm-csf has been shown to stimulate phagocytosis and oxidative functions of host defence neutrophils, monocytes and macrophages. [ ] in addition to its systemic actions, gm-csf may also influence pulmonary host defence by modulating alveolar macrophage function and surfactant metabolism. as noted, apoptosis of neutrophils is an important mechanism by which these cells are cleared from inflamed lung regions, thereby facilitating resolution of inflammation. although both granulocyte colony-stimulating factor and gm-csf are thought to inhibit neutrophil apoptosis, in animal models of lung injury, gm-csf has been shown to help restore capillary barrier integrity, [ ] preserve alveolar epithelial function and improve alveolar fluid clearance. [ ] a pilot study of patients with ards undergoing serial bronchoalveolar lavage found that patients who survived ards had higher concentrations of gm-csf in the bronchoalveolar lavage fluid on day than patients who died. [ ] the authors speculated that gm-csf might improve survival by prolonging the neutrophil lifespan in the alveoli and/or inducing proliferation of alveolar macrophages, thereby improving host defence and reducing infectious complications in this setting. in a phase ii trial, molgramostim (schering-plough), a recombinant human gm-csf, was given intravenously at a low dose ( mg/kg) for days to ten patients with severe sepsis and sepsis-related pulmonary dysfunction (defined as a pao /fio ratio of < with a pulmonary infiltrate on chest radiograph). [ ] the primary outcome was -day survival, and secondary outcomes included oxygenation, occurrence of ards and degree of organ dysfunction at day . there was no difference in -day survival between the treatment and placebo groups, but oxygenation improved in the gm-csf group. ards was present in four of ten patients in the gm-csf group on study entry, but resolved in two of these patients by day , whereas in the placebo group ards was present in three patients on study entry and five patients on day . organ dysfunction was similar between the two groups, with no change between study entry and day . from july to july , the nhlbi enrolled patients who had been diagnosed with ali/ards for at least days into a phase ii rct of recombinant gm-csf (sargramostim [leukine Ò ], genzyme corporation) versus placebo. [ ] the primary outcome was the number of ventilator-free days during days - . secondary outcomes included measures of lung epithelial cell integrity, alveolar macrophage function, changes in severity of respiratory gas exchange, non-respiratory organ failure and incidence of ventilator-associated pneumonia. this study has been completed, but results have not yet been published. [ ] cytokine inhibitors cytokines are glycoproteins that act as messengers to cell surface receptors to promote or diminish the inflammatory cascade. specific cytokines are observed in high amounts in the bronchoalveolar lavage fluid of patients with ards, and are thought to play an important role in propagating lung injury. unsaturated phosphatidic acid plays an important role in intracellular signalling leading to neutrophil accumulation within the lungs, as well as proinflammatory cytokine expression and cell membrane oxidation, all of which leads to lung tissue damage. [ ] lisofylline (cell therapeutics) is a cytokine inhibitor that impedes synthesis of phosphatidic acid- a and, therefore, was thought to hold potential for treatment of ards. however, ardsnet stopped a phase ii/iii trial, the larma study, for futility after the first interim analysis failed to demonstrate any difference in -day mortality, ventilator-free days, organ failures or levels of circulating free fatty acids. [ ] interleukin (il)- is another potent chemoattractant for neutrophils, observed in high levels in patients with early ards [ ] and associated with increased mortality. [ ] anti-il- monoclonal antibody has been shown to reduce pulmonary oedema and neutrophil accumulation in animal models of ards [ , ] but has not yet been tested in humans. finally, tnfa has long been recognized as an important proinflammatory cytokine in ards, but more recent evidence suggests that it actually plays a dichotomous role in both contributing to permeability oedema but also increasing alveolar fluid clearance capacity. monoclonal anti-tnfa antibodies have been tested in patients with sepsis with disappointing results. [ ] given its dual role in alveolar oedema formation and resorbtion, a more sophisticated approach than simply blocking all tnfa activity is likely to be required in ards. studies examining the efficacy of corticosteroids for acute exudative ards have shown conflicting results. in , bernard et al. [ ] published results of a study of patients with ards randomized to high-dose pulse methylprednisolone ( mg/kg every hours for hours) or placebo. there was no difference in -day mortality ( % vs %; p = nonsignificant) but the confidence intervals were wide, suggesting that the study may have been underpowered to detect a small difference in a population with heterogenous outcomes. in , meduri and colleagues [ ] published their results of patients with severe early ards (< hours) from five hospitals randomized to methylprednisolone mg/kg/day for days versus placebo. they found corticosteroids significantly reduced icu mortality ( % vs %; p = . ), duration of mechanical ventilation and length of icu stay. [ ] annane et al. [ ] published a post hoc analysis of ards patients enrolled in an rct of low-dose corticosteroids in septic shock. patients in the treatment group received hydrocortisone mg every hours plus fludrocortisone mg/day for days. although there was no mortality difference for ards patients overall, ards patients with relative adrenal insufficiency and septic shock had significantly reduced mortality when treated with low-dose hydrocortisone ( % vs % in the placebo group; p = . ). [ ] the use of corticosteroids for acute exudative ards remains controversial, although the evidence is more definitive for corticosteroid treatment initiated late for fibroproliferative ards (see section . . ). a study examining low doses of corticosteroids as adjuvant therapy for lung injury associated with h n influenza virus (cortiflu) is planned. [ ] . . activated protein c microvascular injury and coagulation play critical roles in the pathogenesis of ali. plasma protein c levels are decreased in patients with ali, and are associated with higher mortality and fewer ventilator-free days. [ ] recombinant human activated protein c (rhapc; drotrecogin alfa; eli lilly) was tested in a phase iii clinical trial of patients and demonstrated a significant mortality reduction from % to % in patients with severe sepsis. [ ] a phase ii study was sponsored by the nhlbi to determine if drotrecogin alfa increased ventilator-free days in patients with ali (patients with severe sepsis were excluded). the study was terminated by the data safety monitoring board. although drotrecogin alfa significantly increased plasma protein c levels and decreased pulmonary dead space fraction, there was no significant difference in the number of ventilator-free days or in -day mortality ( of vs of patients, respectively; p = . ). [ ] . . hmg-coa reductase inhibitors (statins) hmg-coa reductase inhibitors, commonly known as statins, have recently been proposed as a treatment for ali/ards. the rationale for this is based on animal models suggesting that statins can attenuate organ dysfunction by reducing vascular leak and inflammation. [ ] a prospective cohort study in ireland showed a nonsignificant trend towards lower odds of death in ards patients receiving a statin during their icu admission (or . , % ci . , . ; p = . ). [ ] however, a recently published retrospective cohort study from the mayo clinic (rochester, mn, usa) showed no difference in mortality or organ dysfunction in ards patients treated with statins. [ ] stip is currently enrolling patients admitted to an icu with respiratory distress and a pao /fio ratio < due to the h n pandemic strain of influenza. [ ] patients in this trial will be randomized to receive rosuvastatin mg/day or placebo for days. since this is a specific subpopulation of patients with ali, findings from this study may not be generalizable to other ali subgroups. the sails trial (also rosuvastatin mg/day vs placebo) is also planned but not yet open for recruitment. [ ] patients who survive the early and exudative phases of ards generally enter a period from week to consisting of fibroproliferation and organization of exudative debris within the airspace. this fibroproliferative relatively 'late' phase either slowly resolves or progresses to fibrosis. during this phase, patients are at risk of dying from other complications such as mods, or may fail to wean from mechanical ventilation due to severely impaired respiratory muscle and lung function. those who successfully wean off mechanical ventilation may have residual pulmonary fibrosis and reduced exercise capacity. for resolution to occur, removal of inflammatory cells, cellular debris, and soluble and insoluble proteins needs to take place. as noted in section . . , apoptosis of neutrophils facilitates resolution of inflammation. monocyte and macrophage phagocytic clearance of apoptotic cells appears to be an important mechanism by which neutrophils are cleared from inflamed lung regions. soluble proteins are likely to be primarily removed via paracellular diffusion, but removal of insoluble proteins appears to depend on the function of alveolar macrophages. mechanisms involved in remodelling of hyaline membranes and restoration of a functional alveolar-capillary barrier are incompletely understood at present, but therapeutic interventions aimed at modulation of phagocytosis/apoptosis are being evaluated. to date, far less research has targeted this later phase of the disease, as most trials have focused on earlier preventative processes. fibroproliferative ards is characterized by ongoing inflammation. in addition to being tested for prevention of ards, and treatment of the early and mid exudative phases, corticosteroids have also been tested for efficacy in reversing the fibrosing alveolitis of the late phase of ards. a study by meduri and colleagues [ ] examined the effect of prolonged methylprednisolone therapy ( mg/kg/day for days) on patients with severe ards that was unresolved after days of respiratory failure. although this study demonstrated a significant hospital mortality benefit ( of patients [ %] in the corticosteroid group died vs of [ %] in the placebo group), the significance of these findings was controversial for two reasons: the calculated sample size to demonstrate a % absolute difference in mortality was patients but the study was terminated early after enrolment of patients, and the mortality in the placebo group was slightly higher than anticipated. [ ] to shed further light on this issue, ardsnet specifically designed a study to focus on the late fibrotic stage of the disease, called lasrs. [ ] this study examined the role of corticosteroids in patients in the late phase (> days from onset) of persistent ards. methylprednisolone, dosed at mg/kg/day for days followed by tapering doses until day , was compared with placebo. there was no difference in -or -day mortality rates. methylprednisolone improved oxygenation, respiratory system compliance and blood pressure, resulting in an increased number of ventilator-free and shock-free days; however, a higher rate of neuromuscular weakness and, if initiated more than days after the onset of ards, a significant increased mortality was observed in the methylprednisolone group. therefore, despite the improvement in cardiopulmonary physiology, methylprednisolone does not improve overall mortality in ards and is not recommended for treatment of late ards. given these results, the convincing lack of efficacy for prevention of ali prior to diagnosis and the lack of evidence of benefit in the early phase, corticosteroids cannot be recommended for routine treatment of ali/ards at any stage, at this time. furthermore, it may prove to be exceedingly difficult to determine which individual patient might benefit from corticosteroids and at what specific point to intervene. clearly, the current status of treatment options for patients with ali/ards is suboptimal. at this time, the clinical management of patients with ali/ards involves supportive therapy only. this primarily includes low stretch or 'lung protective' mechanical ventilation, conservative fluid management and adequate nutritional support. although the term 'supportive' may sound somewhat discouraging, these are important observations, not only because they impact on the outcome of patients with ali/ards but also because they should be embraced and implemented as 'standard care' for this patient population. furthermore, any new therapy being tested should be compared with optimal 'standard care'. other methods proposed to offer greater protection to the lungs while providing mechanical support to respiration include hfo and ecmo. studies into these modes are ongoing. although supportive therapies have reduced mortality, there is still significant need for improvements. previous studies have provided important insight into the pathophysiology of ali/ards. research is ongoing into therapies to prevent ali/ards in those at risk, treat it early in its course or aid in its resolution. each of these goals is associated with specific challenges. demonstrating that a prophylactic intervention reduces mortality, morbidity and is cost effective is challenging at best. this is most likely to occur when the risk of acquiring the disease is high, the outcome of the disease is uniformly devastating and treatment for the disease is nonexistent. for some critically ill patients at risk for ards, this may be the case. however, the diagnosis of ali/ards encompasses a very heterogeneous population, with incompletely understood risk factors and non-uniform, diverse outcomes. the greatest likelihood of success for prophylactic therapy will come when we have further delineated the subgroups at highest risk of dying from ali/ards and have accurate diagnostic tests to identify these patients. for ali/ards, specifically targeting the pathogenic mechanisms responsible for the increased risk of death in these patient subgroups would theoretically be high yield. basic science research identifying genetic polymorphisms of patients with highest mortality or greatest need for specific therapies shows great promise in this regard, but is not yet clinically applicable. until then, validating biomarkers and clinical indicators for poor prognosis in ali/ards should remain a primary research goal. finding therapies to treat ards in its late fibroproliferative phase is also in great need. too often patients survive the early and mid phase of ards only to succumb to complications in the late phase or undergo withdrawal of life support as they are unable to be weaned from mechanical ventilation. research into mechanisms of idiopathic pulmonary fibrosis may help identify common pathways to target for therapy. to date, the majority of research has focused on treating ali/ards in its earlier stages, in the hope that the disease process may be reversed prior to the patient entering the fibroproliferative phase. progress in finding therapies to treat established ards has been slow and hampered by a long series of negative clinical trials. however, there are several lessons to be learned from these rcts. first, a 'one-size-fits-all' approach has not worked for pharmacotherapy for ards. in this sense, the syndrome of ali/ards may be likened to cancer. cancer as a broad term signifies the uncontrolled replication of abnormal cells, but there are specific chemotherapeutic treatments for specific types of cancer, depending on its origin. some treatments may be effective for more than one type of cancer, but not for other types, and the magnitude of the benefit might vary according to the type and stage of disease. oncologists would not design a trial enrolling all patients with differing types of cancer and expect to find a single drug that shows a survival benefit. yet, that is what has been attempted with several large ards trials. recent studies have demonstrated that direct ards is likely to respond differently than indirect ards, and in fact within these broad categories, pathogenesis may differ. therefore, different therapies may need to be developed for specific aetiologies such as sepsis-related ards, sirs-related ards and various direct causes of ards. second, a well designed negative rct does not necessarily mean that the therapy tested should be abandoned. it means that the therapy is likely to not be appropriate for widespread application. however, just because a drug does not work for every ards patient does not necessarily mean it should not be used for anyone with ards. for example, there is no evidence for treating all patients with acute ards with corticosteroids, but there is evidence that treating ards patients with relative adrenal insufficiency and septic shock with low doses of hydrocortisone is likely to be beneficial. similarly, nitric oxide should not routinely be applied to all patients with ali/ards, but may be useful in refractory hypoxaemia, particularly in conjunction with other ventilation rescue strategies. third, a negative rct should potentially lead to further research so that we can gain further insight as to why the therapy failed to yield a clinical benefit. thomas edison, when asked why he pursued his quest to invent a functional and practical light bulb after innumerable failed attempts, is reported to have replied, ''i have not failed. i've just found ways that won't work''. ards research should take us from bench to bedside and back to the bench again. basic science can help us understand basic mechanisms of disease, discover why a therapy failed, then provide new ideas to apply to the clinical realm. rcts are necessary to prove benefit and quantify risk prior to changing clinical practice. since we are in urgent need of therapies to treat ali/ ards, it is necessary that rcts continue to advance our clinical care. however, these rcts need to be well founded in basic biology and physiology research, and focused on specific hypotheses regarding mechanisms of disease. continuing to conduct large clinical trials on heterogeneous patients with ali/ards from multiple aetiologies will not only prove ineffective but also add enormous cost to the healthcare system. the most significant and promising finding from an rct to date is that calfactant, the natural bovine surfactant rich in sp-b and -c, reduces mortality in ali from % in the control arm to % in the paediatric population. indeed, calfactant is the first and only pharmacological agent to demonstrate a mortality benefit for treatment of ali/ards. the ongoing cards study will attempt to reproduce that finding in adults with direct causes of ards. 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directly relevant to the content of this review.